extfieldsofstudy: Chemistry Medicine

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Effect of Sanguisorba Minor on Scopolamine-induced Memory Loss in Rat: Involvement of Oxidative Stress and Acetylcholinesterase

Sanguisorba minor (S. minor) has neuroprotective and antioxidant activities; nevertheless, its potential benets in ameliorating learning and memory functions have not been explored yet. So, in this study, rats were treated with S. minor hydro-ethanolic extract (50, 100, and 200 mg/kg, intraperitoneal (i.p.)) as well as rivastigmine (0.5 mg/kg, i.p.) for 21 consecutive days. Thereafter, their behavioral performance was assessed using Morris water maze (MWM) and passive avoidance (PA) tasks. Scopolamine was also injected 30 min before conducting the tasks. Finally, oxidative stress biomarkers and acetyl cholinesterase (AChE) activity were determined in the brain. The extract characterization was presented using liquid chromatography-mass spectrometry (LC-MS), which conrmed the presence of quercetin, myricetin, kaempferol, catechin, ellagic acid, and gallic acid derivatives. According to the results, the extract at all doses could signicantly recover the impairment of cognitive performance of the scopolamine-treated rats. In the MWM test, the extract and rivastigmine reduced escape latency and travelled distance, compared to the scopolamine group. Moreover, in the PA test, the latency to enter the dark chamber was signicantly increased by the extract and rivastigmine, compared to the scopolamine group (p<0.05-p<0.001). Similar to rivastigmine, the extract attenuated both AChE activity and oxidative injury in the brain as evidenced by the increased antioxidant enzymes and total thiol content, but it decreased malondialdehyde level (p<0.05-p<0.001). In conclusion, the results suggested the effectiveness of S. minor on preventing cognitive dysfunction induced by scopolamine. Accordingly, these protective effects might be produced through the regulation of cholinergic activity and oxidative stress. The LC-MS analysis was performed in an AB SCIEX QTRAP (Shimadzu) liquid chromatography coupled with a triple-quadrupole mass spectrometer. MS analysis was conducted in both negative and positive modes of ionization, in order to maximize the number of the monitored metabolites ions. Liquid chromatography was performed on a Supelco C18 (15 mm×2.1 mm×3 µm) column at a ow-rate of 0.7 ml/min. The gradient analysis started with 90% of 0.1% aqueous formic acid, isocratic conditions were maintained for 15 min, and then a 20-min linear gradient to 30% methnol along with 0.1% formic acid was applied. From the time of 35 min to 80 min, the acidied methanol was increased to 100%, followed by 10 min of 100% acidied methanol, and nally 10 min of 90% of aqueous formic acid to re-equilibrate the column. Finally, the mass spectra were acquired in a range of 200 to 1200 after the 100-minute scan time. Mass feature’s extraction of the acquired LC-MS data and the maximum detection of peaks were done using MZmine analysis software package, version 2.3.

Introduction
Dementia and neurodegenerative diseases are accompanied with cognitive impairments (Terry et al. 2011). Functional de cits in cholinergic neurotransmission have been found to be directly linked to cognitive impairments. Hence, many studies have recently focused on the cholinergic system (Par tt et al. 2012; Giovannini et al. 2015). The function of acetyl choline (ACh), a major neurotransmitter in the central cholinergic system, is important in the process of perception, attention, learning, and memory. ACh function in the hippocampus and brain cortex is terminated by acetyl cholinesterase (AChE) (Hasselmo 2006).
Acetyl cholinesterase inhibitors (AChEIs), including rivastigmine, galantamine, and donepezil, are currently used as the most effective pharmacotherapeutic agents. Correspondingly, these can ameliorate However, alternative or adjuvant anti-amnestic therapies are required in this regard because these drugs produce some adverse effects (Ng et al. 2006).
The roles of oxidative stress and cholinergic dysfunction in cognitive decline have been explored in some studies . Scopolamine, as a muscarinic cholinergic receptor antagonist, was observed to impair short-term memory and learning ability (Klinkenberg and Blokland 2010). As well, the induction of AChE activity by scopolamine contributes to mitochondria dysfunction and subsequently to oxidative stress (Melo et al. 2003;Leuner et al. 2012).
Sanguisorba minor (S. minor) is a member of the Rosaceae family that has been traditionally used for the treatment of some diseases such as bleeding, eczema, and diarrhea (Zhao et al. 2017). A variety of biological activities, including anti-in ammatory, antibacterial, antiviral, anti-oxidant, neuroprotective, and anticancer effects were proposed for S. minor extracts (Zhao et al. 2017;Cirovic et al. 2020;Finimundy et al. 2020). The ethanol extract of S. minor could also suppress cyclooxygenase-1 and AChE enzymes activity in vitro (Cirovic et al. 2020;Finimundy et al. 2020). Additionally, some in vitro protective properties of S. minor against β-amyloid neurotoxicity were found (Nguyen et

Liquid chromatography-mass spectrometry (LC-MS) apparatus
The LC-MS analysis was performed in an AB SCIEX QTRAP (Shimadzu) liquid chromatography coupled with a triple-quadrupole mass spectrometer. MS analysis was conducted in both negative and positive modes of ionization, in order to maximize the number of the monitored metabolites ions. Liquid chromatography was performed on a Supelco C18 (15 mm×2.1 mm×3 µm) column at a ow-rate of 0.7 ml/min. The gradient analysis started with 90% of 0.1% aqueous formic acid, isocratic conditions were maintained for 15 min, and then a 20-min linear gradient to 30% methnol along with 0.1% formic acid was applied. From the time of 35 min to 80 min, the acidi ed methanol was increased to 100%, followed by 10 min of 100% acidi ed methanol, and nally 10 min of 90% of aqueous formic acid to re-equilibrate the column. Finally, the mass spectra were acquired in a range of 200 to 1200 after the 100-minute scan time. Mass feature's extraction of the acquired LC-MS data and the maximum detection of peaks were done using MZmine analysis software package, version 2.3.

Animals
Male Wistar rats (aged 6-8 weeks, weighted 200 ± 20 g) were obtained from the animal house of the The animals were housed under the standard conditions (at 22 ± 2°C, 12 h light-dark cycles: light on from 7:00 to 19:00), with free access to food and water ad libitum.

Drug administration and experimental design
After the acclimatization for 1 week, the rats were randomly allocated into the following ve groups (n = 10 per group): Control group (group I) received saline (intraperitoneal (i.p.) injection, 1 mL/kg, daily); scopolamine group (group II) received saline (i.p.) for two weeks and then the treatment with scopolamine was performed (2 mg/kg dissolved in saline, i.

Behavioral assessments
Morris water maze (MWM) test Spatial learning and memory were examined using MWM task (Morris 1984), which is a black circular tank (136 cm in diameter and 60 cm in height) containing water (depth of 30 cm, at 24 ± 1°C). A circular platform (10 cm in diameter) was set 2 cm under the surface of the water in the center of the southwest quadrant of the pool. Swimming behaviors were recorded by a camera that was connected to a computer and the parameters, including latency to nd the hidden platform and traveled distance were calculated.
At the rst day of performing the MWM test, each one of the rats was acclimatized to the maze for 30 sec in the absence of the platform. At each one of the 4-day training period, 4 trials was performed for each animal. For each trial, the rat was separately placed (facing the wall) into each one of four starting points (North (N), East (E), South (S), and West (W)) and then, it was practiced to nd the platform within 60 sec. If the rat found the platform, it was allowed to stay there for 15 sec, otherwise, it was then guided to the platform gently. After each trial, the rat was transferred to its cage and allowed to be dried. By passing 24 hours from the acquisition phase, spatial probe test was performed while removing the platform from the pool and the rat was allowed to seek out the platform in 60 sec (Norouzi et al. 2019).
Passive avoidance (PA) test A passive avoidance (PA) learning test was performed to evaluate memory retention (Norouzi et al. 2019). The apparatus used for PA test was an acrylic shuttle box consisting of a white illuminated compartment and a dark one (30 cm × 20 cm × 20 cm) that were connected to each other via a guillotine door. In a habituation session, the rats were individually put into the light chamber and then allowed to move freely between the two chambers for 5 min. For performing the acquisition trial, an inescapable foot-shock (2 mA for 2 sec) was delivered to the animals through the grid oor by passing 5 sec from entering the dark compartment. The retention trials were done 3h, 24, 48h, and 72h later. In these trails, the rats were placed in the light chamber and an entering delay of the rat to the dark chamber was then measured as the latency time. Additionally, the time spent by the animal in the dark and light compartments was recorded (Bavarsad et al. 2020). Notably, in retention trails, the shock punishment was not applied.

Biochemical measurements Tissue preparation
Following conducting the behavioral assessments, the rats were euthanized under deep anesthesia (using i.p. injection of ketamine and xylazine at 100 mg/kg and 15 mg/kg, respectively) and the wholebrain tissues were quickly isolated afterward. A tissue homogenate of cerebral cortex and hippocampus (10 % w/v) was prepared in ice-cold 0.1 M PBS (with pH level of 7.4), which was then stored at -80 o C for biochemical assessments (Norouzi et al. 2019).

Measurement of oxidative stress biomarkers
Assay of lipid peroxidation Lipid peroxidation was measured on the basis of formation of thiobarbituric acid reactive substances (TBARS) following MDA reaction with TBA (Norouzi et al. 2019). In this regard, MDA was regarded as the main product of lipid peroxidation and TBARS (red color) was then expressed as MDA equivalent. In brief, the reaction mixture was prepared by adding TBA/TCA/HCl reagent (2 mL) to the homogenates (1 mL).
Subsequently, the obtained mixture was heated in boiling water and after 40 min, the samples were cooled and the absorbance was spectrophotometrically measured at 412 nm. The following equation was used to calculate the concentration of MDA (Bavarsad et al. 2020).
Assessment of total thiol content Estimation of total thiol content was done based on the reaction of the thiol groups with DTNB. Firstly, the homogenates were centrifuged at 4°C and 1000×g, and the supernatants were also collected.
Thereafter, Tris-EDTA buffer (1 ml, pH 8.6) was mixed with the supernatant obtained from each sample (50 ml) and the absorbance was then recorded at 240 nm using a UV-vis spectrophotometer (A1). After adding the DTNB solution (20 µl, 10 mM in methanol) to each sample, the absorbance was recorded again (A2). The following equation was used to calculate the total thiol concentration (Bavarsad et al. 2020): Where B is the absorbance of the blank.

Assessment of antioxidant enzymes activity
Superoxide dismutase (SOD) activity in the homogenates was assessed using the previously described procedure (M). The amount of SOD inhibiting 50% of pyrogallol auto-oxidation was de ned as one enzyme unit (Bavarsad et al. 2020).
The estimation of catalase (CAT) activity was also done based on its ability in converting hydrogen peroxide to water. The absorbance was read at 240 nm using the UV-vis spectrophotometer. In this method, the amount of hydrogen peroxide consumed per each milligram of the protein sample was considered as one unit of CAT activity. Finally, the obtained results were shown as unit per gram of tissue (Norouzi et al. 2019).

Assessment of AChE activity
The AChE activity was assessed using the method of Ellman (Ellman et al. 1961). The supernatant obtained from tissue homogenate (40 µl) was added to a solution containing PBS (2.55 ml) and DTNB 10 mM (0.1 ml). The absorbance of the resultant mixture was determined after the incubation for 5 min at 37°C. Then, the reaction mixture was further incubated (for 5 min at 37°C) after adding 0.02 ml of acetylthiocholine. The absorbance of the sample was spectrophotometrically recorded at 412 nm and AChE activity was estimated as µmol/g tissue/min.

Statistical analysis
All the obtained results were presented as mean ± SEM (standard error of mean) and then analyzed by GraphPad Prism software 8.0 (GraphPad Software, La Jolla, CA, USA). P < 0.05 was considered as statistically signi cant. For the data of the acquisition part of the MWM, including the time and distance during the 4 days, the experimental design included treatment and day as a repeated measures. Therefore, two-way analysis of the variance (ANOVA) with repeated measures was used, and interactions were also reported (treatment x day). The remained data were analyzed using one-way ANOVA followed by Tukey's post hoc multiple comparison test.

LC-MS analysis of S. minor hydro-ethanolic extract
In total, 26 compounds were identi ed in the hydro-ethanolic extract of S. minor using LC-MS analysis in its negative mode. These compounds were phenolic compounds, including quercetin, myricetin, kaempferol, kaempferol-3-glucuronide, quercetin − 3-glucuronide ellagic acid, catechin, and various gallic acid derivatives (consisting of galloyl glucoside, galloylquinic acid, methylgallate hexoside, and catechin gallate). Moreover, Ayoub (2003) isolated unique phenolic carboxylic acids, 4, 8-dimethoxy-7-hydroxy-2oxo-2H-1-benzopyran-5,6-dicarboxylic acid, and 2-(4-carboxy-3-methoxystyryl)-2-methoxysuccinic acid in relatively high amounts in the hydro-ethanolic extract of S. minor. This extract also contained a high level of glucogallin, which is a phenolic compound formed from β-D-glucose and gallic acid. The compounds identi ed in the extract characterization are shown in Table 1 and total ion chromatograms obtained in ESI-mode are also shown in Fig. 1. The MS spectral data were compared with those that were available in the literature. The results also show that both the treatment and day affected the distance during the 4-day period of learning (F (5, 936) = 167.09; p < 0.001 for the treatment and F (3, 936) = 25.87; P < 0.001 for the day). As well, an interaction was found between the treatment and the day (F (15,936) = 3.07, p < 0.001). Correspondingly, escape latency time and traveled path were observed to be longer in the scopolamine-injected animals compared to the control group (p < 0.01-p < 0.001). However, the animals treated with both 50 and 100 mg/kg of the extract at the rst day showed shorter latency time compared to the scopolamine group (p < 0.05-p < 0.001). Notably, no signi cant difference was found in terms of the latency time between the 200 mg/kg extract group or rivastigmine-treated group and the scopolamine group at the rst day. Besides, the treatment with the extract (at all doses) and rivastigmine signi cantly reduced the escape latency time compared to that of the scopolamine group, on days 2-4 (p < 0.001). Additionally, it was observed that the administration of the extract (at all doses) and rivastigmine signi cantly decreased the traveled path compared to that of the scopolamine group (p < 0.001). Therefore, it can be stated that the treatment with rivastigmine and the extract signi cantly improved memory performance compared to the scopolamine group (p < 0.001).
According to the results of the probe test (Figs. 3a and b), both the time spent and the distance traveled in the target quadrant signi cantly decreased in the scopolamine group compared to the control group (p < 0.001). Moreover, the animals treated with rivastigmine and the extract (at all doses) exhibited longer time spent and distance traveled in the target quadrant than those of the scopolamine group (p < 0.001). Some signi cant differences were also found in terms of the time spent in the quadrant between the groups treated with 50 and 100 mg/kg of the extract as well as between the groups treated with 100 and 200 mg/kg of the extract (p < 0.001). Similarly, a signi cant difference was found in terms of the distance traveled in the quadrant between the two groups treated with 50 and 100 mg/kg of the extract (p < 0.001).

Passive avoidance test
As illustrated in Figs. 4a and b, in comparison to the control group, the scopolamine-injected animals exhibited lower time latency to enter the dark chamber 3, 24, and 48 h after the shock (p < 0.01, p < 0.001).
Besides, no signi cant difference was observed in terms of the time latency 72 h after the shock between the scopolamine group and the control group.
In contrast, higher time latencies were observed 3, 24, 48, and 72h after the shock in those rats that were treated with rivastigmine compared to the rats in the scopolamine group (p < 0.05, p < 0.001). Moreover, the administration of the extract at both 100 and 200 mg/kg was found to be associated with a signi cant increase in the time latency 3, 24, 48, and 72h after the shock compared to the scopolamine group (p < 0.001). As well, some signi cant differences were observed in the time latency 3, 24, 48, and 72h after the shock between the two groups treated with 50 and 100 mg/kg of the extract as well as between the two groups treated with 50 and 200 mg/kg of the extract (p < 0.001).
On other hand, the animals in the scopolamine group were observed to spend longer times in the dark chamber than the control group at 3, 24, and 48 h time points (p < 0.001). However, no signi cant differences were observed in this regard between the scopolamine and control groups 72 h after the shock. Besides, the animals treated with rivastigmine were found to spend shorter times in the dark compartment 3, 24, 48, and 72 h after the shock compared to the scopolamine group (p < 0.01, 0.001). Furthermore, the administration of the extract (at all doses) caused a signi cant increase in the time spent in the dark at 3, 24, 48, and 72 h after the shock compared to the scopolamine group (p < 0.05 to p < 0.001). Moreover, the animals treated with the extract (50 mg/kg) indicated no signi cant difference in the time spent in the dark, 48h after the shock compared to the scopolamine group. However, some signi cant differences were reported in the time spent in the dark 3, 24, 48, and 72 h after the shock between the two groups treated with 50 and 100 mg/kg of the extract as well as between the two groups treated with 100 and 200 mg/kg of the extract (p < 0.001).
As illustrated in Fig. 4c, the time spent in the light chamber signi cantly decreased following the injection of scopolamine (p < 0.001) in comparison to the control group 3, 24, and 48h after the shock (p < 0.001).
However, no signi cant difference was observed in this regard between the scopolamine and the control groups 72 h after the shock. However, a longer time was spent in the light room by the rats that received rivastigmine compared to the scopolamine group 3, 24, 48, and 72h after the shock (p < 0.01, p < 0.001).
Notably, the spent time in the light chamber increased after the treatment with the extract (at all doses) 3, 24, 48, and 72 h after the shock compared to the scopolamine group (p < 0.05-p < 0.001). The animals treated with 50 mg/kg of the extract indicated no signi cant difference in terms of the time spent in the light 48 h after the shock compared to the scopolamine group. However, there were some signi cant differences in the time spent in the light 3, 24, 48, and 72 h after the shock between the two groups treated with 50 and 100 mg/kg of the extract and also between the two groups treated with 100 and 200 mg/kg of the extract (p < 0.001).
According to the results illustrated in Fig. 4d, although the frequencies of entry to the dark chamber were higher in the scopolamine group compared to that of the control group 3, 24, and 48h post-shock delivery, the differences were not signi cant. The groups that received the extract at 100 and 200 mg/kg demonstrated a signi cant decrease in the frequencies of dark chamber entry compared to those of the scopolamine group 24 (p < 0.05, p < 0.01) and 72 h (p < 0.05, p < 0.001) post-shock delivery. However, no signi cant decrease was found in the frequency of entry to the dark chamber in the rivastigmine group at either time point compared to the scopolamine group.
Biochemical estimation S. minor restored MDA and thiol concentration in the brain According to Fig. 5a, scopolamine-injected rats exhibited the elevated levels of MDA in their hippocampal and cortical tissues in comparison to the control group (p < 0.001). However, the rivastigmine administration signi cantly restored the elevated levels of MDA induced by scopolamine in the rats' hippocampus and cortex, compared to the rats of the scopolamine group (p < 0.001). It is notable that the treatment with all doses of the extract signi cantly counteracted scopolamine-induced increase in MDA level in both the hippocampus (p < 0.001) and cortex (p < 0.01, p < 0.001) compared to the scopolamine group.
By determining thiol content in the hippocampal and cortical tissues, the non-enzymatic defense potential of the extract against the oxidative stress was indicated. Accordingly, the thiol concentrations in both the hippocampus and cortex of scopolamine-injected rats were signi cantly lower than those of the control group (p < 0.001). As well, the administration of rivastigmine effectively restored the scopolamine-induced decreases in thiol contents in both tissues compared to the scopolamine group (p < 0.001). All doses of the extract signi cantly restored the decreases in the concentrations of thiol caused by scopolamine in the hippocampus and cortex compared to the scopolamine group (p < 0.01, p < 0.001) (Fig. 4b).
S. minor enhanced the antioxidant defense in the brain As shown in Figs. 5a and b, the antioxidant enzymes (SOD and CAT) activities were signi cantly attenuated in the hippocampus and cortex of the rats of the scopolamine group in comparison to those of the controls (p < 0.001). The treatment of these animals with rivastigmine and all doses of the administered extract resulted in a signi cant increase in the activities of the enzymes in the hippocampus and cortex (p < 0.001). More importantly, 200 mg/kg of the extract had the most marked effects on restoring the scopolamine-induced suppression of antioxidant enzymes (p < 0.001).
S. minor suppressed the activity of AChE in the brain As shown in Fig. 7, AChE activity signi cantly increased in the hippocampal and cortical tissues of the scopolamine-injected rats compared to the control group (p < 0.005). However, the rivastigmine administration signi cantly reduced the AChE activity in the hippocampal and cortical tissues compared to the scopolamine group (p < 0.05, p < 0.001). An ameliorative effect on the AChE activity in the hippocampus was found in the animals treated with different doses (50, 100, and 200 mg/kg, p < 0.001) of the extract. Meanwhile, only the highest dose of the extract (i.e. 200 mg/kg) was observed to reduce the AChE activity in the cortex signi cantly compared to the scopolamine group (p < 0.05).

Discussion
The present study was conducted on a scopolamine-induced amnesia rat model and demonstrated the bene cial effects of S. minor on cognitive decline. As shown by performing the passive avoidance and MWM tasks, scopolamine induced a noticeable decline in both learning and memory of the experimental rats compared to the controls. During 4 days of training in the current study, scopolamine prolonged the escape latency and distance to reach the platform. Additionally, the rats of the scopolamine group did not remember the location of the platform in probe trial and the time spent in the target quadrant decreased following the scopolamine injection. In PA task, scopolamine reduced the escape latency in the dark chamber as well as the time in the light chamber, while it prolonged the time in the dark chamber.
Consistent with our ndings, some previous investigations have also reported the cognition-impairing effects of scopolamine using behavioral tests (Marefati et al. 2019;Brinza et al. 2021). Accordingly, Hoang showed that the mean latencies and swimming distances increased by the administration of scopolamine (Hoang et al. 2020). Additionally, scopolamine reduced the latency to enter the darkness after shock (Ishola et al. 2020).
On the other hand, it was observed that the extract of S. minor effectively reversed the behavioral changes induced by scopolamine. By performing the MWM test, the ability of the rats to recall the platform location increased following the treatment with the extract, especially at the dose of 200 mg/kg. Since the MWM task generally indicated spatial memory ability of rodents (Crawley 2008; D'Hooge and De Deyn 2001), so these ndings suggested that S. minor extract boosted the rats' learning and spatial memory abilities in this study. Furthermore, in PA test, the administration of the extract improved the indices of memory in the scopolamine-treated animals. Similar results were also observed in the animals that received rivastigmine, which is a standard anti-amnesia drug considered as an AChE inhibitor. In the current research, the extract restored oxidative stress parameters levels disturbed by the administered scopolamine. The extract and rivastigmine signi cantly reduced lipid peroxidation while, they enhanced total thiol content, SOD, and CAT in the cortex and hippocampus. Additionally, AChE activity was attenuated after the treatment with both the extract and rivastigmine. Accordingly, rivastigmine also exhibited antioxidant activities in an animal model of Alzheimer's disease (Mahdy et al. 2012). Moreover, it was previously shown that rivastigmine causes memory consolidation and acquisition. The compounds with AChE inhibiting properties such as rivastigmine were found to reduce cognitive impairments induced by scopolamine (Howes and Houghton 2003).
Our ndings suggested that the inhibition of oxidative stress may contribute to memory enhancing effects of the extract in the rats, which was also reported in previous studies (Soodi et

Conclusions
The present study provided some evidence that the hydro-ethanolic extract of S. minor could ameliorate the scopolamine-induced memory de cits in rats as shown by the data obtained from the MWM and PA tasks data. In addition, the neuroprotective potential of S. minor extract may be partly linked to its anti-AChE and antioxidant activities in the brain tissue. Therefore, S. minor may be a candidate for the prevention or treatment of the cognitive disorders. However, further in vivo and clinical studies should be done before application of this herbal extract for treatment of human amnesia. (Bunse et al. 2020 Figure 1 The total ion chromatogram of S. minor hydro-ethanolic extract using LC-MS in the positive mode.    Effect of S. minor hydro-ethanolic extract on SOD (a, b) and catalase activities (c, d) in hipocampus and cortex of scopolamine-treated rats. Values were expressed as mean ± SEM (n = 10). ***p<0.001


Early puberty in short-haired Guinea pigs kept in laboratory animal facilities

Abstract Lab animals, such as Guinea pigs (Cavia porcellus), are crucial for scientific development, as they play an important role in the development and quality control chain of vaccines and drugs distributed by the Brazilian public health system. Investigating their biological and physiological parameters is fundamental to raise and keep these animals, so the handling of the facilities that hold them can be updated whenever new information comes up, with the well-being of the animals and alignment with the 3 Rs in mind. In the search for understanding reproductive aspects of Guinea pigs, the present study had the main goal of studying puberty by means of estrous cycle analysis in short-haired Guinea pigs. Guinea pigs have a vaginal occlusive membrane that covers the vaginal orifice. Its rupture takes place gradually and naturally, moments before labor and during estrus. The present study followed 42 females as for the presentation of the vaginal occlusive membrane. Once the membranes ruptured spontaneously, a swab was collected to study vaginal cytology. Membrane rupture was observed in 39 females; six females showed membrane rupture with less than 21 days of age (17 to 21 days). Twenty-three females were characterized as being in estrus due to cytology showing a prevalence of anucleated superficial cells. One of these females was younger than 21 days old. The opening of the vaginal occlusive membrane took place most frequently in intervals between 17 and 18 days, and the membrane remained open between one and three consecutive days. It was possible to follow three cycles of membrane opening on six females. The present study showed the need to adapt handling guidelines for C. porcellus kept in research animal facilities. The early age of puberty imposes the need of separate the female daughters from their fathers at 16 days old.

Introduction
Guinea pigs (Cavia porcellus) are very important in the field of science, especially to the quality control of vaccines and drugs distributed via the Brazilian public health service (Couto, 2002). In addition to providing blood and blood products for the development of diagnostic methods for diseases.
From the anatomical point of view, these animals have a membrane occluding the vaginal canal. That disappears and regenerates every reproductive cycle. This occurs due to hormonal influence, throughout the life of the females (Stockard and Papanicolaou, 1919). In the estrous cycle, the vaginal canal opens remains open between three and seven days, coincinding with the proestrus, estrus and metaestrus phases (Luna et al., 2003). In mature females, the membrane remains open for one to three days during estrus, but in juvenile females this phase can last longer (Araníbar and Echevarría, 2014). According to Li and Shen (2015), vulvar morphological characteristics in Guinea pigs suffer small alterations in between the different phases of the estrous cycle: swelling, redness, and secretion during proestrus and estrus, and partially closed, light-colored vaginal membrane during the metaestrus.
Pregnancy in Guinea pigs lasts 68 days on average and the development is precocious from embryonic life. The first coat are still observed at this stage, at 50 days of gestation, and at 60 days the coat already presents the characteristic colour throughout the body (Viana et al., 2019). In a study on embryonic development, it was observed that, after 30 days of gestation, it is possible to distinguish males and females, observing the formation of urogenital folds. At 45 days, the folds show characteristics of the future labia and, in the final of the pregnancy, the labia and the vaginal occlusion membrane are formed (Santos et al., 2016).
Guinea pigs are early developing animals. The newborns weigh between 60 and 100 g, with fully haired, ears and eyes open, teeth, eating solid foods and drink water (Hubrecht, 2010). It is known that puberty occurs between 55 and 70 days of age; the first estrus usually takes place at the age of 68+21 days, on average (Andrade et al., 2002;Lapchik et al., 2017). However, females can reach sexual maturity with less than one month of age, with a body mass of 300 g (Kaliste, 2007;Hubrecht, 2010). Corroborating this information, a unique study reported the rare occurrence of estrus at only 20 days of age (Rood and Weir, 1970).
According to guides and manuals to care this species, the weaning should take place around 21 days old (Andrade et al., 2002;Trillmich et al., 2006;Hubrecht, 2010;Lapchik et al., 2017) or when the animals are weighing ≥180 grams (Brasil, 2016). However, if the Guinea pigs present an estrus when they are younger than 21 days, period during which they are still in the family group, this makes it possible for them to be impregnated by their fathers. The risk of providing pregnant animals is an ethical concern and an important experimental variable. In addition, establishing a proper reproductive management helps reduce an excessive number of animals in the facilities.
The premise of the present study was therefore the existance of early puberty in shorthaired Guinea pigs, allowing for unplanned pregnancies while the animals remain in their family groups at a research facility. To confirm this hypothesis, females were monitored via vaginal cytology and observation of the vaginal occlusive membrane.

Animal facilities and handling
The study was carried out at the Service of Raising of Rodents and Lagomorphs of the Institute of Science and Technology in Biomodels -ICTB of the Oswaldo Cruz Foundation (Rio de Janeiro, Brazil).
The animal facilities in which the Guinea pigs were held into autoclavable polypropylene cages measuring 90 x 60 x 30 (length x width x height), in family groups with 6 animals (females, breeding males and puppies upon completing 21 days of age).
The environment was kept at temperatures between 19 and 21 °C, relative air humidity between 40 and 60%, and light-dark cycles of 12:12 hours. All food and materials offered to the animals were submitted to chemical and/or physical decontamination processes. Nutritional handling consisted of specific Guinea pig feed (Quimtia®), supplemented with vitamin C (Vetec Quimica Fina LTDA), a multivitamin (Organnact®), and autoclaved water.

Experiment outline
At 14 days old the puppies received microchips in the dorsal scapular region for identification.
Observation of vaginal membranes began when the animals turned 15 days old. Every day, between 9 and 10 a.m., the animals were physically restrained, weighed, and had their genital area observed, with a macroscopic evaluation of the vaginal occlusive membrane classified in three stages: Partially open (VMPO) -when had opening of the membrane but not enough for a swab to be introduced; Closed (VMC) -when had vaginal canal completely covered by the membrane. From 30 to 60 days of age, the membrane was inspected every two days. When it was seen opened, cytological samples were collected daily, until observed closed. The macroscopic aspects of the genitalia (color of the vagina, presence of mucus or fluids, swellingo f the vulva) were also recorded daily.

Vaginal cytology
The vaginal material was collected with an extra-fine cylinder-shaped interdental brush, 3 mm of diameter, soft bristles, damp with saline solution and introduced in the vaginal canal, then rotated to gently exfoliate the vaginal epithelium.
Following the collection, the material was smeared on two microscopy slides, which were identified (sample ID) with a combination of a random number and a letter corresponding to the staining technique to be used (A for rapid panoptic and B for Papanicolaou).
The Romanowsky staining technique (panoptic staining) was made by a commercial kit (LABORCLIN). The Papanicolaou (1933) staining also used a commercial kit (RENYLAB) and was performed according to the author, with adaptations (Kugelmeier et al., 2011). Both were carried out at the SCPRIM/ICTB Reproduction Laboratory at ICTB/Fiocruz.
The quantitative evaluation considered the counting of cell types in absolute numbers, for a total of 200 cells, which was performed by optical microscopy (100 and 400 x zoom). After the counting, the percentage of cell types contained in the vaginal epithelium was calculated. According to the classification used by Guimarães et al. (1997), into the following cells: basal, parabasal, intermediate, nucleated superficial, and enucleated superficial. The qualitative evaluation observed the presence of mucus, leukocytes, fungi, bacteria, cleanliness, and quantity of material on the slide. The presence of a finding was qualified according to the follow criteria, and then recorded in the spreadsheet (vaginal material samples collection): Lightobserved in few fields; Moderate-observed in some fields; High-Observed in all fields. The macroscopic evaluation of the vulva, of the distribution of cell types and of the material on the slide was used to characterize the phases of the estrous cycle. Characterization of diestrus considered the analysis of slides collected beginning on the fifth day of membrane opening.

Analysis
For the description of the results, the following criteria were considered: opening of the occlusive membrane, age, time the membrane remained open and phase of the cycle during which the membrane opened. For the vaginal cytology figures, descriptive statistic, average and standard deviation, standard error, and variation of original data were considered. Variance analysis (ANOVA) was used to compare the number of cell types present in the three cycles observed and the T Test used to compare the averages obtained in the first and second cycles, using the Bioestat free software (version 5.0).
The level of significance used to reject H 0 (hypothesis of nullity) was 5%, that is, lower than 0.05.

Ethical aspects
This study was approved by the Ethics Committee for Use of Animals -CEUA Fiocruz, under licence number LW-12/15 and the additive term P39/14.4 for the inclusion of the activity "sampling for vaginal cytology".
Observation was finished when the animals turned 61 days old. The animals' management general routine was not modified, except for the observations and sample collections.

Results
Evaluation of estrous cycle between 15 and 60 days of age.
The macroscopic evaluation of the vaginal membrane was carried out in all 42 animals of the study (total of 1274 observations). We observed that in 73% of evaluations, the status found was that of closed vaginal membrane ( Figure 1A, 1B), while in 13% the membrane was partially open ( Figure 1C). The vaginal membrane was found to be open in 14% of observations ( Figure 1D). The vaginal occlusive membrane broke at least once in 39 of the 42 females (92.85%) under evaluation. Of the total number of females with open vaginal membrane, 23 (58.97%) had a second opening, with an interval between openings from 7 to 21 days, while six females (15.38%) had a third opening, at intervals between 15 to 18 days. We observed a variation in the period during which the vaginal membrane remained open, from 1 to 12 days. The duration of the interval in between membrane openings varied between 7 and 21 days, with a higher frequency at 17 and 18 days, totalling 44.81% of the intervals observed (Table 1).

Vaginal exfoliative cytology of Cavia porcellus
Overall, 164 slides were prepared and evaluated, to analyze the average distribution of the cell types found. The average distribution of cell types of the vaginal epithelium shows a predominance of enucleated and intermediate superficial cells, according to the description (Figure 2).

Puberty prior to 21 days of age
In 39 females that presented vaginal occlusive membrane opening, six were younger than 21 days (17 to 21 days) corresponding to 15.38% of females in the first opening of the membrane. For the females of this age group, the time membrane remained open varied between 1 and 5 days.
As for the distribution of total superficial cells in vaginal smears, in animals younger than 21 days (n = 6). One 21 days old female showed a counting above 140 superficial cells (nucleated and enucleated), low presence of intermediate, parabasal and basal cells, as observed in Figure 4. In one of the samples, the material showed high concentration of mucus and formation of lumps, making it impossible to differentiate and count the cell types. The study observed a significant difference (p=0.02) between average body weight values between the six females with vaginal membrane opening before 21 days of age (299 ± 45.42 g) and the 33 females whose vaginal membrane opened at more than 21 days of age (264 ± 32.03 g) ( Figure 5).

Discussion
In this study it was possible to prove, via vaginal cytology analysis and observation of vaginal occlusive membrane, the occurrence of early puberty in C. porcellus. Of the 42 females under study, six had vaginal occlusive membrane opening before 21 days of age. Trillmich et al. (2006) observed that the first opening of the vaginal occlusion membrane in a group of guinea pigs under similar conditions occurs around 26 days of life. These investigators considered that the first day of opening the vaginal occlusion membrane corresponds to the beginning of the estrus phase, based on studies by Young (1937) and Touma et al. (2001). However, they did not perform serological or cytological investigations to conclude this finding in their studies. Stockard and Papanicolaou (1919) described vaginal occlusive membrane rupture as a reliable sign of estrus. However, this condition is not the only mark of estrus. Therefore, beginning with the examination of vaginal exfoliative cytology, it was possible to characterize estrus in at least one of the females younger than 21 days, in which we observed a higher prevalence of superficial cells, a marker of this phase of the cycle in Guinea pigs (Kühnel and Mendoza, 1992;Lilley et al., 1997).
It should be highlighted that the fact that evidence of estrus was observed on the vaginal smear in only one female younger than 21 days does not rule out the possibility of occurrance in the other five females that presented early opening of the vaginal occlusive membrane. A revision of literature showed a duration of the estrous phase between 8 and 24 hours, according to Lilley et al. (1997), and between 8 and 11 hours, according to Shomer et al. (2015). The study by Alkhalaf et al. (1992) shows that the estrous phase corresponds to the second day of vaginal membrane opening, with ovulation taking place between 28 and 36 hours after opening, usually at night, between 6 p.m. and 6 a.m. (Lilley et al., 1997). Therefore, for a better following of estrus, it would be necessary to use a smaller interval in between cytological samplings, as already previously suggested by Lilley et al. (1997). However, we chose not to collect more than one sample per day, as this would mean more frequent handling of the animals and consequently higher stress levels, which may compromise the study, especially as these were very young females. We therefore chose to adopt a methodology that caused less disturbances in the daily management routine of the animals under study, to preserve their well-being and aligning with the Three Rs in animal research according to Russel and Burch (1959). In addition, unlike Lilley et al. (1997), in the present study the rupture of the vaginal occlusive membrane was not mechanically forced. All material samples for vaginal cytology in this study were collected only during natural rupture intervals, considering the fact that the study was carried out on very young females, up to 60 days old.
The significant difference observed in the average values of body weight at 20 days of age between females with vaginal occlusive membrane rupture before and after 21 days of age corroborates the study developed by Bauer et al. (2008), in which they show that animals with different weight gain were younger when their first estrous cycle took place.
The results, in the conditions reported herein, showed a wide range in the age of spontaneously opening of vaginal occlusive membrane, between 15 and 60 days old. Trillmich et al. (2006) suggest that the presence of the mother and the breeding male that present a courtship vocalization behavior can induce the precocity of estrus in young guinea pigs, as they observed in their studies that, in the family groups of guinea pigs kept the males of the family together with the young females, until what they consider as the 1st estrus, there was a complete opening of the vaginal membrane around 26 days. However, they could not clearly distinguish the effect or confirm it as an inducer of the opening of the vaginal membrane in young females.
The time during which the vaginal membrane remained opened for the first time varied greatly (1-12 successive days) and was higher than that reported by other authors (Alkhalaf et al., 1992;Luna et al., 2003), who described a period of membrane opening closer to what was observed during the second opening verified in the current study (1-7 days). It should be emphasized, however, that only two females had opening periods higher than that described by other authors (8 and 12 days). In addition, the higher frequency of membrane opening period (between 1 and 3 days) corresponded to the study by Araníbar and Echevarría (2014). Macroscopic observation of the vulva showed morphological differences, such as swelling, redness and secretion, like those described by Li and Shen (2015).
The duration of the interval in openings showed a variation between 7 and 21 days, occurring with a higher frequency to 17 and 18 days, higher than those presented in literature, which describe cycles between 15 and 17 days (Lilley et al., 1997;Stockard and Papanicolaou, 1919;Hubrecht, 2010), or between 14 and 16 days (Kühnel and Mendoza, 1992;Wang et al., 2010;Li and Shen, 2015).
Vaginal cytology proved to be reliable for the characterization of the estrous cycle. There was distribution of cell types of the vaginal epithelium in each phase, as in the study by Li and Shen (2015). During proestrus, intermediary cells were observed in a higher number, with moderate presence of mucus. This aspect was also considered a characteristic of the proestrus phase by Kühnel and Mendoza (1992). During estrous, the predominant cells were superficial ones, mostly enucleateed, while Kühnel and Mendoza (1992) and Lilley et al. (1997) observed a more effective presence of nucleated superficial cells in this phase. During metoestrous and diestrous, cell types observed were like those described in the work of Kühnel and Mendoza (1992), with a high number of leukocytes and increased number of parabasal cells. For differentiation, the slides characterized as diestrous were those collected from the fifth day of vaginal membrane opening on, period that Alkhalaf et al. (1992) and Shomer et al. (2015) consider the beginning of this phase.
Thus, observation of the rupture of vaginal occlusive membrane, together with the characterization and incidence of cell types through vaginal cytology, made it possible to detect estrous in Guinea pigs, supporting previous reports in studies by Li and Shen (2015).
This study provides important and novel information on the reproductive biology of young C. porcellus females with the evidence of early estrous (before 21 days) and the characterization of at least three estrous cycles before 60 days old.

Conclusion
The Guinea pig females kept in laboratory animal facilities conditions showed that the rupture of the occlusive membrane occurs gradually, in some cases, with a partially open phase that makes cytological sampling impossible. Differences in body development (weight) of the females with early puberty is one of the possible factors in the distribution of age in the opening of the vaginal membrane.
In the conditions in which the study was carried out, Guinea pigs showed vaginal occlusive membrane rupture and vaginal cytology characteristic of estrous at an age below 21 days, pointing to puberty before the standard weaning period. Because of this observation, in laboratory animal facilities is necessary to modify the Guinea pigs management practices aiming to adapt the separation of female daughters from their fathers at 16 days old.


s2fieldsofstudy: Biology Medicine

text:
Circulating cell‐free circRNA panel predicted tumorigenesis and development of colorectal cancer

Abstract Background Colorectal cancer (CRC) is reported with high morbidity and mortality. Currently, the sensitivity of diagnostic markers for colorectal cancer is low. Therefore, further exploration of new plasma diagnostic markers for early detection of colorectal cancer is of great value. We aimed to explore potential circRNAs in plasma as biomarkers for early diagnosis of CRC. Methods We employed the circRNA microarray to investigate dysregulated circRNAs in plasma samples of CRC patients, colorectal adenoma patients (CRA), and healthy controls. Through in‐depth analysis, significantly differentially expressed circRNAs were screened as candidate targets. Results Eight circRNAs (hsa_circ_104885, hsa_circ_100185, hsa_circ_103171, hsa_circ_001978, hsa_circ_105039, hsa_circ_103627, hsa_circ_101717, and hsa_circ_104192) were obtained as candidate circRNAs with upregulation in CRC comparing with both CRA and healthy control. Through detecting the plasma expression levels of eight candidate targets, we identified three circRNA (hsa_circ_001978, hsa_circ_105039, and hsa_circ_103627) with increased level which were consistent with the microarray results in training set. Further validation found the circRNA panel was consistent with training set. The ROC curve also revealed a high diagnostic ability of hsa_circ_001978, hsa_circ_105039, and hsa_circ_103627 in predicted the CRC from CRA patients (AUC = 0.966) as well as healthy controls (AUC = 0.969). Conclusion Our data suggest that hsa_circ_001978, hsa_circ_105039, and hsa_circ_103627 might be a CRC‐specific biomarker for early diagnosis.

developed countries has been ranked in the third and second malignant tumors. In China, with economic development, the incidence of colorectal cancer is increasing year by year. 5 Most patients have been diagnosed with local advanced or distant metastasis, especially liver metastasis, with poor treatment effect and poor prognosis. 2 In recent decades, researchers have developed many effective methods for the diagnosis and treatment of colorectal cancer, which significantly improve the survival of colorectal cancer patients. However, the overall prognosis of colorectal cancer patients is still poor. 6 Due to the lack of specific clinical manifestations, simple, and fast diagnostic methods and accurate evaluation methods, diagnosis, and treatment of colorectal cancer is still difficult. There is a long way to go in the diagnosis and treatment of colorectal cancer in China. Therefore, to improve the prognosis of patients with colorectal cancer, the key point is to improve the efficiency of early diagnosis of colorectal cancer. Developing effective minimally invasive diagnostic markers to detect colorectal cancer at an earlier stage, patients with colorectal cancer can receive timely treatment, effectively improve the survival of patients, which has important clinical research value.
Recently discovered circRNAs consist mainly of exon-derived transcripts produced by non-collinear reverse splicing. 7 Based on whether they can be translated, circRNAs can be divided into noncoding circRNAs and coding circRNAs. [7][8][9] The circRNA is widely expressed in a variety of human cells and plays a key role in the regulation of post-transcriptional gene expression. 10 Different from linear RNA, circRNA harbored a closed-loop structure, stable expression, is not easy to degrade, and is affected by RNA exonucleases. 11 The circRNA has miRNA binding sites and acts as a miRNA sponge, blocking or reducing miRNA expression and promoting the expression of target mRNA. 12 There is an increasing evidence that circRNA can be used as biomarkers for early diagnosis and prognostic prediction in various diseases. 13 Abnormal circRNA expression in peripheral blood can be used as a biomarker of rectal cancer. 14 Similarly, circRNA can also be used as an early diagnostic marker for some non-neoplastic diseases, such as Parkinson's disease, and can be used to monitor the severity of the disease. 15 Besides, recent colorectal cancer tumor markers, such as CEA and CA19-9, have been confirmed to have obvious limitations in their diagnostic sensitivity and specificity, especially in patients with early colorectal cancer, whose diagnostic efficacy is weak. However, it can predict postoperative metastasis and recurrence of colorectal cancer. 16 Based on this, it is necessary to explore novel biomarker for the early diagnosis of colorectal cancer.
In this project, we anchored circRNA as the research object and combined with the characteristics of stable expression of circRNA, we believe that circRNA might be used as a potential biomarker. We

| Study design
The circRNA microarray was employed by using Arraystar Circular Next, we conducted an in-depth analysis of the differentially expressed circRNA detected by microarray. Intersection analysis was conducted for circRNA with high differential expression compared with CRA group and circRNA with elevated expression compared with healthy people, and the final differential circRNA was obtained as a candidate. Subsequently, plasma samples from 20 CRC patients and 20 healthy controls were randomly selected for preliminary validation to obtain circRNA with differential expression in a small sample. Based on the above results, a large sample (80 cases in each group) was used for revalidation. Finally, the sensitivity and specificity of candidate patients and their combinations in CRC diagnosis were calculated by setting training set and validation set. The receiver operating characteristic (ROC) curve analysis was applied.

| RNA isolation and qRT-PCR
All blood samples were stored by EDTA anticoagulant tube, and plasma samples were obtained by 1000 g centrifugation for 10 min within half an hour after blood samples were taken. All plasma samples were stored in the −80°C refrigerator. Total RNA was extracted by Trizol method, and circRNA was enriched by RNase treatment after RNA quality control detection. The RNase-treated RNA were retrotranscribed using random primers method, and the reverse transcribed cDNA was amplified using a SYBR ® Premix Ex Taq™ kit (Takara). Finally, the amplification curve was compared with the internal reference to obtain the relative expression level, and triplicates were used to detect each samples. ABI7900 (ABI) was used for amplification.

| Statistical analysis
In this study, continuous variables were analyzed using the student t test, and categorical variables were analyzed using the chi-square test. All the experiment assay was repeated three times in triplicate.
We have also tested the homoscedasticity in all the data obtained.
After confirmed the homoscedasticity, after that, a parametric test (t test, ANOVA) was applied. In analyzing the sensitivity and specificity of diagnostic markers, we quantified the area under the receiver operating characteristic (ROC) curve. Specific methods of risk stratification analysis are described in previous reports. STATA14.0 was used for statistical analysis, and GraphPad Prism 8.0 was used for graph presentation. In all statistical analyses, p < 0.05 was considered statistically significant.

| High-throughput microarray detection of plasma circRNAs
Totally, 100 patients diagnosed with CRC were enrolled with 100 paired healthy controls. 100 patients diagnosed with colorectal adenoma was also participated. The detailed clinical information is listed in Table 1. Based on clinical information analysis, it was found that there were no significant statistical differences in age and gender among the groups in this study. We further presented subgroup information based on clinical characteristics of tumor patients, including tumor size, tumor differentiation degree, and tumor TNM analysis.
After obtained the dysregulated circRNA, the following parameters were used for difference analysis for all circRNA: (1) p < 0.05, (2) the absolute value of CT detection <35, and (3) 75% detectable in all the samples. After the above screening, we obtained 134 circRNA with high expression in CRC compared with the CRA group.120 cir-cRNA were elevated in CRC compared with healthy subjects. Further, the enrichment of differentially expressed circRNA in each group was presented by clustering map, and the general distribution of differentially expressed circRNA was shown by volcanic map (Figure 1A-C).
Further cross comparison was conducted for the above two groups of differentially expressed circRNAs, and 8 circRNAs with the highest expression in CRC were obtained ( Figure 1D). We further selected the eight circRNAs as candidate biomarkers.
We next employed an independent cohort to further validate

| Risk score analysis for colorectal cancer biomarkers
The panel of three circRNAs (hsa_circ_001978, hsa_circ_105039 and hsa_circ_103627) was further validated in an independent cohort including 80 samples in each group. Comparing with both CRA group and healthy control group, we found the three circRNA panel was evaluated in the plasma samples of CRC patients ( Figure 3).
As mentioned above, based on the above study and multi-step verification, we preliminarily found three circRNA panel might be potential biomarkers of CRC. We next employed the risk score analysis Data was presented as mean ± SD, **indicated p < 0.01. *indicated p < 0.05, n.s, indicated no significance obtained the sum of sensitivity and specificity as the node of the maximum risk score, regarding as the cutoff. We classified the population as high-risk group and low-risk group. The high-risk group was suggested to be colorectal cancer, and the low-risk group was the control group (value = 4.31). We found the positive predicting value for CRC from healthy was 1.00 and the negative predicting value was 1.00.
The same method was applied in validation set, and the obtained the positive predictive value and negative predictive value were 0.90 and 0.91, respectively, based on a cutoff value of 8.72 ( Table 2).
The specific diagnostic efficacy of the above fingerprints was presented by ROC curves. As shown in Figure 4, the AUC of hsa_

| Expression stability validation
To verify the stable presence of the three circRNAs in human plasma, plasma samples from randomly selected healthy patients were treated in different ways to test the stability of circRNA expression in different environments. The samples were placed at room temperature for 12 and 24 h. In addition, the plasma samples were subjected to three consecutive freeze-thaw treatments. It was found through detection that the expression levels of these three circRNAs in the above plasma did not altered significantly indicating that the three circRNA panel could be stable expressed in human plasma samples ( Figure 6).

| DISCUSS ION
At present, the diagnosis of CRC mainly relies on colonoscopy, imaging and tumor biomarker detection. However, due to the unsatisfactory results of these methods, patients with CRC are usually diagnosed as intermediate to late stage. In addition, as the gold standard, colonoscopy has not been widely promoted due to the high cost of examination, the painful process of examination, and the poor health awareness of the public. 17 Similarly, imaging examination is difficult to be used for early diagnosis of colorectal cancer due to its poor identification. In terms of diagnostic markers, carcinoembryonic antigen and CA19-9 has been widely applied in the diagnosis of tumor, but previous studies show that CEA is more suitable for being used in the detection of for tumor recurrence and metastasis; in addition, the CEA levels in intestinal inflammation and adenoma has increased in the tumor, for early colorectal cancer its poor sensitivity and specific degree, CA19-9 is a good predictor of tumor metastasis. 18 Therefore, finding new tumor markers with high sensitivity and specificity is very important for the diagnosis of CRC.
It is worth noting that in the current research, more and more researchers have found that circRNAs play an important role in many human diseases. In addition, many circRNAs can be expressed in plasma. For example, circ_0124554 blocked the ubiquitination of AKT promoting the skip lymphovascular invasion on hepatic metastasis in colorectal cancer. 19 Due to the circRNA specific circular structure, they can be stably expressed in human body fluids, including plasma and urine, and thus have the ability to serve as biomarkers. 20 It has been shown that circRNA can be used as an important marker of early tumor occurrence and predict the occurrence of gastric cancer in gastrointestinal tumors. 21

This work was supported by the Science Foundation of Anhui
Medical University project (2020xkj038).

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.

AUTH O R CO NTR I B UTI O N S
XC conceived and designed the experiments; LQ and YP performed the experiments and contributed to reagents/materials/analysis tools; LQ AND MT analyzed the data; All authors contributed to the article and approved the submitted version.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.


miR-210-3p protects against osteoarthritis through inhibiting subchondral angiogenesis by targeting the expression of TGFBR1 and ID4

Excessive subchondral angiogenesis is a key pathological feature of osteoarthritis (OA), as it alters the balance of subchondral bone remodeling and causes progressive cartilage degradation. We previously found that miR-210-3p correlates negatively with angiogenesis, though the specific mechanism of miR-210-3p-related angiogenesis in subchondral bone during OA progression remains unclear. This study was conducted to identify the miR-210-3p-modulating subchondral angiogenesis mechanism in OA and investigate its therapeutic effect. We found that miR-210-3p expression correlated negatively with subchondral endomucin positive (Emcn+) vasculature in the knee joints of OA mice. miR-210-3p overexpression regulated the angiogenic ability of endothelial cells (ECs) under hypoxic conditions in vitro. Mechanistically, miR-210-3p inhibited ECs angiogenesis by suppressing transforming growth factor beta receptor 1 (TGFBR1) mRNA translation and degrading DNA-binding inhibitor 4 (ID4) mRNA. In addition, TGFBR1 downregulated the expression of ID4. Reduced ID4 levels led to a negative feedback regulation of TGFBR1, enhancing the inhibitory effect of miR-210-3p on angiogenesis. In OA mice, miR-210-3p overexpression in ECs via adeno-associated virus (AAV) alleviated cartilage degradation, suppressed the type 17 immune response and relieved symptoms by attenuating subchondral Emcn+ vasculature and subchondral bone remodeling. In conclusion, we identified a miR-210-3p/TGFBR1/ID4 axis in subchondral ECs that modulates OA progression via subchondral angiogenesis, representing a potential OA therapy target.

Introduction
Osteoarthritis (OA) is a chronic degenerative disease that causes high morbidity and disability rates, endangers the health and quality of life of patients and increases medical care system burden and socioeconomic costs (1,2). The pathogenic factors of OA include aging (3), mechanical alteration (4), dysfunction of synthesis and metabolism (5), inflammation (6) and immune abnormalities (7). All these complex factors result in joint tissue lesions mainly characterized by cartilage damage, subchondral bone remodeling and osteophyte formation (8)(9)(10)(11)(12)(13). In recent years, substantial progress has been achieved in understanding the role of subchondral bone remodeling in the pathogenesis of OA (14)(15)(16). As subchondral bone provides mechanical stress support and is the main source of nutrients for cartilage, microenvironmental and structural changes in subchondral bone might affect cartilage metabolism directly or indirectly and are presumed to be one of the factors initiating OA (14). Specifically, aberrant angiogenesis in subchondral bone alters the balance of bone remodeling and results in sclerosis of subchondral bone in pre-OA lesions, which changes the mechanical stress characteristics of the subchondral bone and the nutrition supply pattern of cartilage and increases cartilage vulnerability (14,17). In advanced OA, increased neurovascular coupling causes the most significant clinical symptom of OA, pain (18). Meanwhile, the subchondral vasculature gradually invades the cartilage through the tidemark and causes more serious degradation of the cartilage matrix (19). Thus, inhibition of subchondral angiogenesis is proposed to be crucial in preventing the pathological progression of OA, allowing cartilage to maintain a "healthy" state. zone of articular cartilage are also less than 1% (22). Thus, vascular invasion from subchondral bone to cartilage occurs under hypoxic conditions in vivo. Notably, miR-210-3p, called the "master miRNA of hypoxia", exerts multiple and complex effects on different diseases and tissues (23)(24)(25). We previously reported significantly increased expression of miR-210-3p in tissues with avascular necrosis of the femoral head (26), indicating that miR-210-3p may be negatively correlated with angiogenesis in subchondral bone. In particular, miR-210-3p manifests antiangiogenic properties in diseases with aberrant vascular formation. For example, miR-210-3p significantly inhibits the angiogenic ability of human retinal vascular endothelial cells by directly targeting insulin-like growth factor 2 (IGF2) in the retina (27). In preeclampsia, overexpression of miR-210-3p impairs extravillous trophoblast formation of endothelial-like networks (28). Therefore, we assume that the decrease in miR-210-3p expression may be attributed to angiogenesis in subchondral bone during the development of OA.
Here, we aimed to study the mechanism by which miR-210-3p affects subchondral angiogenesis and its therapeutic effect on OA. As a method to achieve our goal, we first overexpressed and inhibited miR-210-3p expression in endothelial cells (ECs) to determine its effect on the angiogenic ability of ECs in vitro. RNA sequencing and subsequent molecular experiments were implemented to determine the targets and downstream pathways of miR-210-3p. In vivo, we selectively overexpressed miR-210-3p in ECs of mice with anterior cruciate ligament transection (ACLT) induced OA to verify its role in the progression of OA using an adeno-associated virus (AAV) containing the tyrosine kinase with immunoglobulin like and EGF like domains 2 (TIE2) promotor (miR-210-3p/TIE2/AAV). We then performed CatWalk, microCT, histological staining, flow cytometry and immunochemistry experiments to evaluate the progression of OA. The regulatory mechanisms employed by miR-210-3p were also investigated in our study. We highlight a novel role of the miR-210-3p/transforming growth factor beta receptor 1 (TGFBR1)/ DNA-binding inhibitor 4 (ID4) axis in subchondral ECs that modulates the progression of OA via subchondral angiogenesis, thereby suggesting a potential target for OA therapy.

Cell culture and transduction
Human umbilical vein endothelial cells (HUVECs) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HUVECs were cultured in Dulbecco's modified Eagle's medium (BI, Israel) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 50 U/ml penicillin and 50 mg/ml streptomycin in the presence of 5% CO2 and 1% O2 at 37°C in a humidified incubator. All culture media were renewed every 3 days. The miR-210-3p mimic, inhibitor and each negative control were synthesized by Ribobio (Guangzhou, China) and transfected into HUVECs using Lipo3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. In addition, we constructed shRNAs to knock down the expression of TGFBR1 and ID4 using the pLKO.1-puro vector. Scrambled shRNA was used as shRNA negative control. Then, we packaged lentiviral particles in HEK293T cells using a packaging system including GAG, TAT, Rev, and VSVG vectors and constructed plasmids. We collected culture medium containing lentiviral particles at 48 and 72 hours and filtered it with a 0.45 mm filter. HUVECs at a 60-80% density were infected for 24 hours and selected with blasticidin, puromycin or neomycin for 7 days to establish overexpression or KD cell lines. The shRNA sequences are listed in Table S1.

Transwell assay
HUVECs cultured under different intervention conditions were digested with 0.25% trypsin and adjusted to a density of 2 × 10 5 cells/ml with serum-free DMEM. Then, 100 mL of the resuspended cells were added to the upper chamber of transwell plates (8 mm pore size) (Corning, NY, USA). Then, 600 mL of DMEM containing 10% FBS were added to the lower chamber. After 18 h of incubation at 37°C in a 5% CO2 incubator, the cells were fixed with methanol for 10 min and stained with a 0.1% crystal violet staining solution for 5 min. Next, cotton swabs were used to gently remove cells remaining on the top of the filter. Images of migrated cells were captured using an inverted microscope. Five visual fields were randomly chosen, and stained cells were counted by ImageJ software.

EdU staining
An EdU staining kit was purchased from Ribobio (Guangzhou, China). According to the manual, we diluted the EdU solution at a ratio of 1000:1 with complete culture medium to reach a concentration of 50 mM. Then, we added 100 mL of the diluted EdU solution to each well and incubated the samples for 2 hours. After the incubation, we fixed the cells in each well with 4% paraformaldehyde for 30 min at room temperature, added 50 mL of 2 mg/mL glycine to each well, and incubated the samples for 5 minutes. Next, 100 mL of penetrant (0.5% Triton X-100 in PBS) were added to each well, and the samples were incubated for 10 minutes. Then, we applied Apollo ® 567 staining solution and Hoechst for EdU and nuclear staining, respectively. The reaction solution was incubated in the dark at room temperature for 30 minutes. Finally, each well was washed with 100 mL of PBS 1~3 times. Images were captured immediately using a fluorescence microscope after the staining was completed.

Tube formation
Ninety-six-well plates were precoated with 50 mL of Matrigel (Corning, NY, USA) and incubated at 37°C for 30-60 minutes. A total of 50 mL of the HUVEC suspension (4×10 5 cells/ml) was seeded in Matrigel-coated 96-well plates and incubated with 5% CO2 at 37°C in a humidified incubator for 6 hours. After the incubation, the formation of tube-like structures was assessed using a phase contrast microscope and ImageJ software.

Western blotting
Adherent cells were digested with RIPA buffer (Biotime, Shanghai, China) containing PMSF (Yeasen, Shanghai, China) after two washes with ice-cold PBS. Twenty micrograms of total protein from each sample were separated on SDS-PAGE gels and transferred onto NC membranes (Millipore, Bedford, MA, USA). Then, we used 8% nonfat milk to block nonspecific sites for 1 hour at room temperature. The membranes were then incubated with primary antibodies against vascular endothelial growth factor (VEGF; Servicebio, Wuhan, China), TGFBR1 (Abcam, Cambridge, UK), ID4 (Santa Cruz, Dallas, Texas, USA), C-C motif chemokine ligand 2 (CCL2; Huabio, Hangzhou, China), Smad2 (Santa Cruz, Dallas, Texas, USA), pSmad2 (CST, Danvers, Massachusetts, USA), Smad3 (CST, Danvers, Massachusetts, USA), pSmad3 (CST, Danvers, Massachusetts, USA) and GAPDH (CST, Danvers, Massachusetts, USA) at 4°C overnight. The membranes were washed with TBST 3 times for 10 minutes each and then incubated with a horseradish peroxidase-conjugated goat antirabbit or goat anti-mouse IgG secondary antibody (CST, Danvers, Massachusetts, USA) at room temperature for 2 hours according to the species of primary antibody. Finally, the ECL Detection Kit (NCM Biotech, Suzhou, China) was used for detection and photography. Images of protein bands were analyzed using ImageJ software.

Dual-luciferase reporter gene assay
The 500 bp UTRs of TGFBR1 and ID4 containing the predicted binding sites or mutant predicted binding sites of miR-210-3p were subcloned downstream in the pmirGLO miReport vector. We cotransfected the vector and miR-210-3p mimic or scrambled negative control with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Forty-eight hours after transfection, we lysed the cells and measured firefly and Renilla luciferase activity with the Dual-Luciferase Reporter Assay System (Solarbio, Peking, China).

Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from each sample using TRIzol reagent (Thermo, Waltham, Massachusetts, USA), separated using chloroform and precipitated with isopropanol according to the manufacturer's instructions. Then, reverse transcription was performed using the PrimeScript RT Master Mix Kit (Takara, Tokyo, Japan) to acquire cDNAs. RT-qPCR was performed in a 20 µl reaction system using TB Green Premix Ex Taq (Takara, Tokyo, Japan) according to the manufacturer's instructions. miR-210-3p and U6 were reverse-transcribed using Bulge-Loop miRNA qRT-PCR Primers (Ribobio, Guangzhou, China). GAPDH and U6 were used as internal references for mRNA and miRNA quantification, respectively. The primer sequences are listed in Table S2. Relative gene expression was quantified using the 2 −DDCT method.

Animal experiments
Animal experiments were performed according to the protocol approved by the Department Laboratory Animal Science of Fudan University. We used 6-week-old C57BL/6 mice and assigned 6 mice to each group. All C57BL/6 mice other than those in the control and sham groups received intra-articular (i.a.) or intravenous (i.v.) injection of AAV-control or miR-210-3p/TIE2/AAV 3 weeks before ACLT. For the i.a. injection, the mice were immobilized in the supine position, the left hindlimb was straightened, and the knee joint and surrounding hair were shaved. We used an insulin syringe and vertically injected 10 µl of AAV (1.5×10 12 v.g./mL) into the knee joint. In addition, 100 µl of AAV (1.5×10 12 v.g./mL) were used for the i.v. injection. For ACLT, C57BL/6 mice were anesthetized with 0.3% sodium pentobarbital. A 0.5 cm longitudinal incision was made along the left knee joint of the mice. The fascia above the patellar ligament was bluntly separated. A longitudinal incision was made at the medial side of the patellar ligament to open the joint cavity. Then, we pulled the patellar ligament laterally to expose the joint cavity. After blunt separation of the infrapatellar fat pad, we transected the anterior cruciate ligament. Notably, we did not transect the anterior cruciate ligament of the sham group at this step. Then, the joint cavity was washed with saline, and the patellar ligament, fascia and skin were stitched with absorbable sutures after repositioning the ligament.

In situ hybridization (ISH)
ISH staining for miR-210-3p in subchondral bone of normal mice and mice was performed 4 weeks after ACLT. The miR-210-3p probes and ISH kits were purchased from Servicebio (Wuhan, China) and used according to the manufacturer's instructions. Briefly, paraffin sections of mouse knees were subjected to dewaxing, repair and prehybridization. For hybridization, 50 mL of the probe-containing hybridization solution were added to cover the tissue, and samples were incubated in a humid chamber at 37°C overnight. After the incubation, sections were washed with 37°C prewarmed 2×SSC solution once for 10 minutes and 37°C prewarmed 1×SSC solution twice for 5 minutes each. DAPI was used for nuclear staining. Images were captured using a fluorescence microscope.

Histology and immunohistochemical analysis
We harvested the left hind knees of mice after euthanasia at 2 or 4 weeks after ACLT and fixed them with 4% formaldehyde for 48 hours. Then, the knees were immersed in 1.5 M EDTA for 1-1.5 weeks for decalcification. Following dehydration in a gradient of alcohol solutions, the knees were embedded in OCT compound for sectioning (6 mm). For safranin-O & fast green staining, sections were dewaxed in water and immersed in fast green staining solution for 6 minutes. After washing with distilled water for 1 minute, the sections were immersed in safranin O solution for 4 minutes, rinsed with distilled water for 1 minute and differentiated in glacial acetic acid for 1 minute. The sections were sealed with neutral resin after dehydration. For immunohistochemical staining, sections were dewaxed in water and repaired in boiled antigen retrieval solution for 8 minutes. After blocking with a 3% BSA/PBS solution for 1 hour at room temperature, sections were incubated with antiendomucin (Emcn; Santa Cruz, Dallas, Texas, USA), anti-matrix metalloproteinase 13 (MMP13; Abcam, Cambridge, UK) and anti-Runt-related transcription factor 2 (RUNX2; Servicebio, Wuhan, China) antibodies, followed by fluorescent dye-conjugated secondary antibodies (Abcam, Cambridge, UK) for fluorescence imaging and HRP-conjugated secondary antibodies (Servicebio, Wuhan, China) for DAB staining.

Flow cytometry
We harvested inguinal lymph nodes at 2 weeks after ACLT and digested the lymph nodes with a mixture of type II collagenase (Yeasen, Shanghai, China) and DNase I (Yeasen, Shanghai, China) for 20 min at room temperature. The digest was filtered through a 70 mm cell strainer (Miltenyi, Germany) and then washed with 1× PBS. For staining of cytokine interleukin 17a (IL-17a), cells were stimulated with the leucocyte activation cocktail BD Pharmingen (BD Biosciences, Franklin Lakes, New Jersey, U.S.) for 4 hours at 37°C. Then, the cells were incubated with antibodies against surface markers on ice for 30 minutes in the dark. After fixation and permeabilization with a BD CytoFix/CytoPerm Kit (BD Biosciences, Franklin Lakes, New Jersey, U.S.), cells were stained with IL-17a fluorescent antibodies on ice for an additional 30 minutes in the dark. The T cell panel included the following antibodies: Zombie NTR Fixable Viability APC-Cy7 (Biolegend, San Diego, California, U.S), CD25 PE-Cy7 (Biolegend), CD3 FITC (Biolegend) and IL-17a PE (Biolegend). Analyses were performed using FlowJo software.

MicroCT analysis
The isolated knee joints were fixed overnight with 4% formaldehyde and analyzed using microCT (Sky-scan 1174, Bruker MicroCT) (voltage, 65 kVp; current, 153 mA; and resolution, 9 mm/pixel). We used image reconstruction software (NRecon v1.6, Bruker), data analysis software (CTAn v1.9, Bruker), and 3-dimensional model visualization software (mCTVol v2.0, Bruker) to analyze the parameters of the tibia subchondral bone. The whole subchondral bone medial compartment was the region of interest, and 100 consecutive images from the medial tibial plateau were used for 3-dimensional reconstruction and analysis. The bone mineral density BMD and bone volume/tissue volume (BV/TV) were measured in three-dimensional structures.

CatWalk analysis
The CatWalk gait analysis system (Noldus Information Technology) was used to measure the disability of mice in this study. Mice were placed individually in the walkway and allowed to walk freely. When the mouse traversed from one side of the walkway to the other, a high-speed color video camera recorded mouse movements and footprints simultaneously. The software automatically identified all contacted areas and assigned them to the respective paws. The durations and left hind limb duty cycle were analyzed.

Statistical analysis
We performed statistical analyses using GraphPad Prism 9 software. Numerical data are presented as the mean ± SD. Unpaired two-tailed Student's t test was used to compare the results from two groups, and one-way ANOVA was used to compare variables between more than two groups. Statistical significance was defined as *P < 0.05 and **P < 0.01.

miR-210-3p expression is negatively related to aberrant subchondral angiogenesis in OA
We applied ACLT to induce OA in mice as a method to verify the potential correlation between miR-210-3p expression and aberrant vascularization of subchondral bone. At 4 weeks after ACLT, the cartilage was significantly degenerated in ACLT mice, as shown by safranin-O & fast green staining ( Figure 1A), and the Osteoarthritis Research Society International (OARSI) scores were also significantly increased compared with those of control mice ( Figure 1E). Meanwhile, the expression of MMP13, a molecule directly involved in cartilage degradation, was significantly increased in the cartilage of ACLT mice compared with control mice (Figures 1B, F). Next, we stained for Emcn, a specific marker of capillaries and sinusoids in the metaphysis and diaphysis (29)(30)(31), to assess the vasculature in the subchondral bone of mouse knees using immunohistochemical staining. We observed abnormally proliferating Emcn + blood vessels in subchondral bone, and some vessels even invaded the cartilage through the tidemark in ACLT mice ( Figures 1C, G). We next validated the expression of miR-210-3p in subchondral bone by performing an ISH assay. The expression of miR-210-3p was significantly decreased in subchondral bone of ACLT mice compared with control mice (Figures 1D, H). Based on these findings, we speculated that miR-210-3p may be involved in the abnormal vascularization of subchondral bone during the development of OA.
miR-210-3p regulates the angiogenic ability of HUVECs in vitro under hypoxia We transfected the miR-210-3p mimic or inhibitor into HUVECs in vitro to investigate the role of miR-210-3p in aberrant angiogenesis of subchondral bone in OA. The miR-210-3p level was substantially increased after the miR-210-3p mimic transfection, as evidenced by the qPCR results ( Figure 2D). Briefly, proliferation, migration and tube formation were assessed as measures of the angiogenic ability of ECs. Notably, we found that miR-210-3p had no effect on the angiogenesis ability of HUVECs under normoxic conditions ( Figure S1). Then, we evaluated the effect of miR-210-3p on the angiogenic ability of HUVECs grown in the presence of 1% oxygen. Our results revealed that miR-210-3p did not affect the proliferation of HUVECs (Figures 2A, F)  RNA sequencing of miR-210-3poverexpressing HUVECs and miR-210-3p target prediction We performed RNA sequencing of miR-210-3p mimictransfected and control HUVECs under hypoxic conditions to evaluate the transcriptional changes in the miR-210-3poverexpressing (OE) HUVECs. The Benjamini-Hochberg false discovery rate (FDR) method was applied to obtain FDRadjusted p-values (q-values). Based on our results, forced miR-210-3p OE downregulated 43 genes and upregulated 36 genes (FC > 2, q< 0.05) ( Figure 3A). These differentially expressed genes were also displayed in a heatmap ( Figure 3B). The Gene Ontology (GO) enrichment results showed that miR-210-3p mainly affected the process of cellular responses to external stimulation such as cytokines and peptides, which are critical in the angiogenic behavior of ECs ( Figure 3C). Furthermore, The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that VEGF-independent angiogenic signaling pathways, such as the TGF-b signaling pathway and pathways related to cytokine-cytokine receptor interactions, were significantly enriched in miR-210-3p-OE HUVECs ( Figure 3D). Subsequently, we used the miRWalk database to predict the target genes of miR-210-3p and intersected them with the genes downregulated in miR-210-3p-OE cells identified by RNA sequencing (Figures 3E, F). The results showed 4 overlapping genes. Among these genes, ID4 and TGFBR1 were of particular interest because of their proangiogenic properties. ID4 ranked the highest, and ID4 has been reported to promote EC-related angiogenesis by affecting the stability of chemokine mRNAs. TGFBR1 is an important membrane receptor in the TGF-b signaling pathway that is closely related to the angiogenesis of ECs. Therefore, we sought to verify ID4 and TGFBR1 as target genes of miR-210-3p.  According to our results, the ID4 mRNA and protein levels were significantly decreased in the miR-210-3p mimic group but were significantly increased in the miR-210-3p inhibitor group compared with the control group ( Figure 4A), which provided indirect evidence that ID4 is a target gene of miR-210-3p. Consistent with the ID4 expression level, the levels of the proangiogenic chemokines C-X-C motif chemokine ligand 1 (CXCL1) and CCL2 showed similar trends ( Figures 4C, D), indicating that CXCL1 and CCL2 may be downstream molecules of ID4. Although TGFBR1 mRNA expression level showed no differences among the groups ( Figure 4B), TGFBR1 protein expression was significantly inhibited by the miR-210-3p mimic and was significantly increased in the miR-210-3p inhibitor group (Figures 4D, F) mainly inhibits the translation of TGFBR1 instead of degrading the TGFBR1 mRNA. Regarding the activation of downstream molecules in the TGF-b signaling pathway, levels of the phosphorylated forms of Smad2/3 showed a similar trend to TGFBR1 (Figures 4D, F), suggesting that miR-210-3p inhibits the activation of the TGF-b signaling pathway by inhibiting TGFBR1 expression. Importantly, the VEGF protein level did not differ among the groups (Figures 4D,  E), which further illustrated that miR-210-3p regulates the angiogenic ability of ECs through VEGF-independent signaling pathways. We then validated the predicted sites by which miR-210-3p binds to ID4 and TGFBR1 using a dual-luciferase reporter assay ( Figures 4G-I), providing direct evidence that ID4 and TGFBR1 are target genes of miR-210-3p.

Knock down of TGFBR1 and ID4 regulates the angiogenic ability of ECs
Without interfering with the expression of miR-210-3p, we used shRNAs to knock down the expression of TGFBR1 and ID4 to observe whether this approach was sufficient to inhibit the angiogenesis of ECs. We constructed three shRNA plasmids each for TGFBR1 and ID4 (Table S1) and selected one shRNA plasmid each for TGFBR1 and ID4 with satisfactory knockdown (KD) efficiency and no effect on the survival of ECs. Unexpectedly, our results showed that TGFBR1 KD significantly suppressed the expression of ID4 (Figures 5A, C), while ID4 KD increased the expression of TGFBR1 ( Figures 5B, D), suggesting that ID4 is also downstream of TGFBR1 and regulates the expression of TGFBR1 expression through negative feedback. Regarding the angiogenesis of ECs, the proliferation, migration and tube formation ability of the TGFBR1 KD group were significantly decreased. Although the proliferation of the ID4 KD group was increased, the migration and tube formation ability were significantly reduced ( Figures 5E-J). The effects of TGFBR1 and ID4 KD largely explain the regulation of the angiogenic ability of ECs by miR-210-3p.

miR-210-3p overexpression induced by AAV attenuates OA progression
Based on the results described above, we used miR-210-3p/ TIE2/AAV to selectively overexpress miR-210-3p in ECs and observed whether miR-210-3p/TIE2/AAV exerted a therapeutic effect on the OA model. Briefly, we i.a. or i.v. injected miR-210-3p/ TIE2/AAV, and ACLT was applied to induce posttraumatic OA 3 weeks after AAV administration. Biological and behavioral tests were performed at 2 and 4 weeks after ACLT ( Figure 6A). Before ACLT, we assessed Zsgreen fluorescence to determine the efficiency of miR-210-3p/TIE2/AAV infection in each group. Our results illustrated that i.a. administration alone was sufficient to infect the entire knee joint tissue, and i.v. administration induced more obvious infection of blood vessels in subchondral bone, while the i.a.+i.v. administration showed the most pronounced infection intensity ( Figure 6B). In further therapeutic efficacy assessments, we found that the ACLT+miR-210-3p i.a. group, the ACLT+miR-210-3p i.v. group and the ACLT+miR-210-3p i.a.+i.v. group exhibited alleviated cartilage degradation at 2 and 4 weeks compared with the ACLT group, as evidenced by safranin-O & fast green staining and OARSI scores (Figures 6C, F). In addition, the expression of MMP13 in cartilage was also significantly reduced in the three treatment groups compared with the ACLT group ( Figure 6D). The proportion of IL-17 + cells among T lymphocytes (CD3 + ) in inguinal lymph nodes was doubled at 2 weeks after ACLT and significantly decreased in ACLT+miR-210-3p i.a.+i.v. group (Figures 6E, G). Regarding behavioral testing, the CatWalk analysis results showed that the time it took for mice to pass through the test channel was significantly prolonged in the ACLT group and significantly decreased in the three treatment groups, especially in the ACLT +miR-210-3p i.v. group and ACLT+miR-210-3p i.a.+i.v. group ( Figure 6H), while the duty cycle of the left hind limb of mice was significantly reduced in the ACLT group and returned to normal levels in all treatment groups ( Figure 6I). Based on these results, miR-210-3p/TIE2/AAV had surprising therapeutic efficacy in attenuating OA progression with respect to cartilage protection, immune change as well as symptom relief.

OA alleviation by miR-210-3p overexpression is attributable to the inhibition of subchondral angiogenesis
We next assessed subchondral bone remodeling and angiogenesis in each group. The microCT results showed that subchondral bone sclerosis occurred in the ACLT group at 2 weeks and was more severe at 4 weeks, which was manifested as higher intensities in the reconstructed 3D images ( Figure 7A), increased bone mineral density (BMD) ( Figure 7D) and increased relative bone volume fractions ( Figure 7E). However, subchondral bone sclerosis was significantly relieved in the treatment groups, especially in the ACLT+miR-210-3p i.a.+i.v. group. Meanwhile, the expression of RUNX2, a key transcription factor associated with osteoblast differentiation, was significantly increased in subchondral bone of the ACLT group, with a positive zone adjacent to the tidemark (dotted lines), and was significantly decreased in the treatment groups ( Figure 7B). Notably, Emcn + blood vessels in subchondral bone were abundant and invaded the cartilage layer at 4 weeks after ACLT, as evident from Emcn staining (Figures 7C, F). Impressively, the growth of abnormal Emcn + blood vessels in subchondral bone was effectively inhibited in ACLT+miR-210-3p i.a. group and ACLT+miR-210-3p i.v. group and was particularly reduced in the ACLT+miR-210-3p i.a.+i.v. group. In conclusion, gene therapy with miR-210-3p/TIE2/ AAV significantly alleviated the process of OA by inhibiting angiogenesis in subchondral bone.

Discussion
As subchondral bone serves as mechanical support and a dominant source of nutrition for cartilage, alterations in the subchondral bone microenvironment are intimately involved in the progression of OA. These alterations are caused by biological uncoupling and coupling interactions among osteocytes, osteoblasts (OBs), osteoclasts (OCs), ECs and sensory neurons in subchondral bone (15,32). In the early stage of OA, the balance of subchondral bone remodeling is skewed toward bone All data are presented as the means ± standard deviation. Statistical significance was determined using unpaired, 2-tailed Student's t test.
resorption in response to altered mechanical stress and inflammation. An increased ratio of receptor activator of nuclear factor kB ligand (RANKL)/osteoprotegerin (OPG) in osteocytes and excessive levels of interleukin (IL)-6, prostaglandin E2 (PGE2) and MMPs produced by OBs promote osteoclastogenesis (33-36). Subsequently, OCs produce proangiogenic cytokines, such as platelet-derived growth factor-BB (PDGF-BB), VEGF and TGF-b, and promote excessive subchondral neurovascularization and bone formation, causing worsening of joint pain and progressive OA (37,38). We found that miR-210-3p deficiency is responsible for angiogenesis in subchondral bone in an OA model, and selective overexpression of miR-210-3p in ECs effectively attenuated OA progression, as evidenced by cartilage protection, immune changes and improved mobility. In our opinion, the improvement in mobility is a direct reflection of the treatment effect on OA. We found that the mobility of the treatment groups was almost indistinguishable from that of normal mice, which is fairly rare in previous studies (16,37), providing evidence that miR-210-3p is an effective treatment target for OA. A substantial number of epigenetic variations and the expression of noncoding RNAs, such as miRNAs, circular RNAs and long noncoding RNAs, are known to dynamically regulate changes in gene expression in normal and OA articular joints (5,(39)(40)(41)(42)(43). However, recent studies have mainly focused on functional noncoding RNAs in cartilage and assessed the efficacy of treatments targeting chondrocytes (44-47), and few reports of noncoding RNAs associated with pathological changes in subchondral bone, including subchondral angiogenesis, in OA are available. We found that miR-210-3p expression is significantly downregulated in subchondral bone in an OA model in parallel with aberrant subchondral angiogenesis. Indeed, miR-210-3p is widely considered a proangiogenic miRNA involved in ischemia-reperfusion diseases and cancer (48)(49)(50). However, we found that selective miR-210-3p overexpression in ECs induced antiangiogenic effects in vitro and in vivo. This discrepancy may be due to the differences in the biological effects of different miR-210-3p levels on tissues and ECs. Germana Zaccagnini et al. reported that macrophages with elevated miR-210-3p levels in samples with mismatched miR-210-3p levels in ischemic tissue display dysfunctional angiogenesis, leading to impaired tissue repair (51). This finding further illustrates the heterogeneity of miR-210-3p function in cells and tissues. Consistent with our study,   (52,53) and preeclampsia (28). In summary, we provide the first evidence that miR-210-3p may be exploited as an effective target to inhibit aberrant subchondral angiogenesis in OA. However, we still must explore the molecular mechanism underlying miR-210-3p deficiency in subchondral bone in OA and its effects on processes other than angiogenesis in the subchondral microenvironment in future studies. AAVs are small (≈25 nm in diameter), nonenveloped, single-stranded DNA (ssDNA) viruses that serve as promising vectors for gene therapy in animal experiments and clinical trials (54)(55)(56)(57)(58)(59). In this study, we applied AAV vectors to overexpress miR-210-3p in subchondral bone vascular ECs. However, more than ten AAV serotypes have been isolated from human and nonhuman primate tissues, and different serotypes have preferences for distinct cells and tissues (60). For i.a. injection, we selected AAV5 for its cartilage permeability and affinity for blood vessels (61)(62)(63)(64). For i.v. injection, we chose AAV9 for its extensive ability to invade the circulatory system and affinity for bone tissue (65). In particular, we added TIE2, an endothelial cell-specific promoter, upstream of the miR-210-3p sequence to specifically overexpress miR-210-3p in vascular ECs. Finally, our study showed that miR-210-3p/TIE2/AAV effectively infected subchondral vascular and inhibited aberrant subchondral bone angiogenesis in vivo. Apart from its cartilage-protecting effect, miR-210-3p also showed strong inhibition of subchondral bone sclerosis, similar to therapies targeting PDGF-BB (37) or prostaglandin E receptor 4 (EP4) (34) in OCs. However, the group that received i.a. and i.v. injection of miR-210-3p/TIE2/ AAV exhibited a reduced BMD of subchondral bone compared with that of the control group, indicating that high levels of miR-210-3p might impair the subchondral vasculature and result in decreased bone formation.
During the development of OA, the inflammatory environment together with complex multicellular interactions affects subchondral angiogenesis, and the molecular mechanisms remain to be elucidated. In the cartilage layer, hypertrophic chondrocytes express high levels of VEGF (66), and cartilage with high mechanical stress secretes excess TGF-b (67). These molecules further promote vascular invasion of cartilage and disrupt cartilage homeostasis during OA development. In OA subchondral bone, TGF-b1 produced by OBs is mainly responsible for subchondral angiogenesis in early OA (11). Our study showed that miR-210-3p inhibits the angiogenic ability of ECs by directly inhibiting the translation of the TGFBR1 mRNA and subsequently affecting Smad2/Smad3 phosphorylation under hypoxia. In addition, we also found that miR-210-3p degrades the ID4 mRNA in ECs. ID4 is a member of a protein family that functions as a negative regulator of helix-loop-helix transcription factors. Giulia Fontemaggi et al. reported that ID4 binds and stabilizes mRNAs encoding the proangiogenic factors IL8 and CXCL1 to promote neuroangiogenesis (68). In our study, we found that ID4 may promote angiogenesis by stabilizing the CXCL1 and CCL2 mRNAs. Interestingly, we identified ID4 as a possible downstream molecule of TGFBR1 that regulates TGFBR1 through negative feedback, producing a strong inhibitory effect on cells overexpressing miR-210-3p. In summary, we verified that miR-210-3p modulates TGFBR1 and ID4 expression and attenuates OA progression by inhibiting subchondral angiogenesis, providing a new therapeutic target for OA treatment.
Nonetheless, this study still has some limitations. First, we did not assess changes in the expression of miR-210-3p in normal and OA human knee joints. Furthermore, the therapeutic effect of miR-210-3p on human OA remains to be further validated. Second, we tested the treatment efficacy of miR-210-3p at 2 weeks and 4 weeks after ACLT in mice, but its long-term therapeutic effects and side effects related to the cardiovascular system and vital organs still require further investigation. Finally, this study only preliminarily suggested that ID4 is downstream of TGFBR1, and we are working to elucidate the specific underlying molecular mechanism.

Conclusion
In conclusion, our study provides the first evidence that miR-210-3p inhibits the angiogenic ability of ECs by directly targeting TGFBR1 and ID4, and the miR-210-3p/TGFBR1/ID4 axis in subchondral ECs modulates the progression of OA via subchondral angiogenesis, which might represent a potential target for OA therapy.

Data availability statement
The RNA-seq data of effect of overexpression of miR-210-3p on gene expression of human umbilical vein endothelial cells in hypoxic conditions is available in the GEO database with accession number GSE214170.

Ethics statement
The animal study was reviewed and approved by The Department Laboratory Animal Science Fudan University.

Author contributions
HT, WZ, LC, CG, and DM developed the concept for the study. HT and LC designed the research plan. HT and WZ performed the experiments and analyzed data. HT and WZ wrote and edited the manuscript. LC, JZ, and JL revised the manuscript. All authors read and approved the final version of the manuscript.