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DHA Ameliorates Cognitive Ability, Reduces Amyloid Deposition, and Nerve Fiber Production in Alzheimer’s Disease

Background The etiology of Alzheimer’s disease (AD) is very complex. Docosahexaenoic acid (DHA) is important in cognitive ability and nervous system development. A limited number of studies have evaluated the efficacy of DHA in the treatment of AD. Introduction We detected neurofibrillary tangles (NFT) in the hippocampus and cortex of transgenic mice brain through silver glycine staining. We determined the activity of neurons by staining Nissl bodies, used liquid NMR to detect metabolites in the brain, and functional magnetic resonance imaging results to observe the connection signal value between brain regions. Materials and Methods We fed 3-month-old APP/PS1 double transgenic mice with DHA mixed feeds for 4 months to assess the effects of DHA on cognitive ability in AD mice through the Morris water maze and open field tests. To evaluate its effects with AD pathology, continuous feeding was done until the mice reached 9 months of age. Results Compared to AD mice, escape latency significantly decreased on the fifth day while swimming speed, target quadrant stay time, and the crossing number of platforms increased by varying degrees after DHA treatment. Brain tissue section staining revealed that DHA significantly reduced Aβ and nerve fibers in the brain of AD mice. Conclusion DHA significantly reduced the deposition of Aβ in the brain and inhibited the production of nerve fibers, thereby increasing cognitive abilities in AD mice. In addition, DHA suppressed blood lipid levels, and restored uric acid and urea levels, implying that DHA is a potential therapeutic option for early AD.

INTRODUCTION
Alzheimer's disease (AD) is a progressive neurodegenerative disease that is characterized by cognitive difficulties, reduced language abilities, weakened learning and memory abilities, and motor dysfunctions (1). Not only does it cause great harm to the patients, it is also associated with a heavy socio-economic burden on the family and society. Currently, senile plaques (SP) formed by Amyloid β-peptide (Aβ) deposition and nerve fiber tangles (NFT) formed by aggregation of hyperphosphorylated tau proteins are two key pathological factors associated with AD (2). As the aging population increases, so will the number of AD patients in the world. However, there is no effective therapeutic option for AD.
The etiologies for most AD cases is unknown, except for 1-5% of cases where genetic differences have been identified. Several competing hypotheses have been proposed to explain the etiology of this disease. They include genetic, cholinergic hypothesis, amyloid hypothesis, and Tau hypothesis among others. In the last decade, more than 50 drug candidates have successfully passed phase II clinical trials, but none has passed phase III (3). The main challenge in the fight against Alzheimer's disease is the absence of drugs that are capable of slowing disease progression. Since 2003, no new drugs have been approved for the treatment of Alzheimer's disease. There are only limited studies of some dietary components improving AD conditions. Long chain omega-3 polyunsaturated fatty acids (n-3 LC PUFAs) are long-chain fatty acids (20 or more carbons) whose first double bond is located at the methyl end after the third carbon. Docosahexaenoic acid (DHA), an omega-3 essential fatty acid, is a polyunsaturated fatty acid (C 22 H 32 O 2 ) with six double bonds at positions 4, 7, 10, 13, 16, and 19. DHA and its derivatives have been shown to regulate gene expression levels of inflammatory mediators, as well as enzymes involved in lipid metabolism and Aβ processing. They are also important in preventing or retarding inflammatory aspects of AD pathology (4). Twenty-four week supplementation with 900 mg/day DHA was found to improve learning and memory functions in agerelated cognitive decline. Therefore, it is a beneficial supplement for improving cognitive health with aging (5). Studies have reported potential therapeutic effects of DHA in preventing, regulating or improving AD progression.
Studies on the effects of DHA on AD pathology focused on behavioral aspects, which could not be associated with ADrelated pathological indicators, and could not further explore the underlying mechanisms. Based on previous research findings, we used various comprehensive research methods to study the effects of DHA-rich mixed feeds on PS1/APP double transgenic AD mice models through behavioral experiments, in vivo NMR detection, liquid NMR detection and other methods. Learning and memory abilities, anxiety, Aβ formation and Tau hyperphosphorylation were evaluated.
The purpose of this study is to explore how DHA improves the symptoms of AD and try to discover the mechanism of treatment. The results of this study have certain guiding significance for the follow-up in-depth research on effective means of treating AD.

Animals and Treatment
Double transgenic AD mice models (2 × Tg-AD) carrying human gene mutants APPswePSEN1dE9/Nju and B6C3F1 WT mice were purchased from GemPharma Tech Co., Ltd. (Nanjing, China). Animals were raised in an animal facility under the conditions of 12-h light/12-h dark cycle at 22 ± 2 • C with free access to food and water. The SPF grade standard feed and docosahexaenoic acid mixed feed were provided by CABIO Biotech (Wuhan) Co., Ltd. The 3-month-old 2 × Tg-AD male mice were randomly allocated into three groups: (i) AD control group; (ii) The 50 mg/kg DHA-treated AD group; (iii) The 300 mg/kg DHA-treated AD group, while age/gender-matched WT mice were allocated into two groups, which were the WT control and the 300 mg/kg DHA-treated WT groups. There were 12 mice in each group. The AD control and WT control mice groups were fed with the standard feed. For behavioral testing, all mice were fed to 7 months of age. Feeding was continued until 9 months of age (6 months of dosing), some mice were obtained for liquid nuclear magnetic resonance and functional magnetic resonance experiments while the remaining mice were euthanized and brain tissues as well as serum obtained for various biochemical and pathological experiments. Ethical approval was obtained from the Animal Ethical and Welfare Committee of Shenzhen University, China. All animal experiments were performed according to the animal experiment guidelines and regulations of Shenzhen University, China.
The low concentration of DHA is determined with reference to the new resource food 2010 No. 3 announcement issued by the China Food and Drug Administration, and the high concentration is determined with reference to the Chinese health food functional evaluation method (2020).

Morris Water Maze
The Morris water maze task was performed as previously described with some modifications (6) to assess spatial memory performance of the 9-month-old mice. The water maze was a blue tank (160 cm diameter, 50 cm height) filled with water (21-23 • C) to a depth of 26 cm. At this temperature, mice are eager to find an opening through which they can escape from the water because they do not like swimming. The circular water pool was artificially divided into four equal areas, the first to fourth quadrant, and separated using an invisible platform (12 cm diameter, 1-2 cm below the water surface) in the middle of the third quadrant. During the five consecutive days of training, mice were gently released into the water in the first quadrant to start the test and were allowed up to 1 min to find the submerged platform. In case they could not mount the platform within 1 min, they would be guided to the platform for 10 s before being returned back to the cage. The escape latency and swimming speeds for the mice were recorded. After 24 and 72 h of training sessions, probe trials were performed in which the platform was removed. Each mouse was gently released into the first quadrant and searched freely for 2 m. Time that mice spent in the target quadrant (third quadrant) and the number of mice crossing the previous platform position were recorded. The trials were recorded using a video monitor connected to a computer. The test was run using a water maze software (Water Maze MT-200, Chengdu, China).

Open Field Test
The open field equipment was a 1.2 m × 1.2 m square PVC box, and the bottom of it was divided into 25 grids (5 × 5). The test was performed as previously described with some modifications (6). Briefly, a single mouse was placed in the middle grid of the box and timed for 3 mins. The number of grids that the mouse crossed and the number of rearing (the two front legs were raised at the same time) were recorded. To avoid affecting the experimental results, the smells were wiped off using ethanol before the next mouse experiment.

Tissue and Serum Preparation
Mice were anesthetized using isoflurane and their brain tissues rapidly collected. The left hemisphere was fixed in 4% phosphate-buffered paraformaldehyde, while the right one was dissected into the hippocampus and cortex and stored at −80 • C for the subsequent biochemical analyses. Blood samples were obtained by removing eyeballs of the mice. Blood was left to stand for 3-4 h and centrifuged at 15 × rpm for 30 min. Then, the supernatant was aspirated to obtain mice serum.

Serum Biochemical Analysis
Biochemical analyses were performed using a fully automatic biochemical analyzer (iMagic-M7, ICUBIO). Test kits were manufactured by ICUBIO (Shenzhen, China), include Glutamate Pyruvate Transaminase (ALT) Assay Kit, Glutamate Oxaloacetate Transaminase (AST) Assay Kit, Low density lipoprotein cholesterol (LDL-C) Assay Kit, High density lipoprotein cholesterol (HDL-C) Assay Kit, Urea Assay Kit, Triglycerides (TG) Assay Kit. All procedures were performed according to the manufacturer's instructions.

Thioflavin T Staining
Brain sections were washed several times using ddH 2 O after which they were immersed in 0.001 g/mL ThT working solution (Sigma Aldrich, St. Louis, MO, United States) and stained at room temperature for 30 mins. Subsequently, sections were washed using ddH 2 O for 5 s and mounted with the antifluorescence quenching medium. Finally, sections were imaged using a confocal microscope (OLYMPUS FV1000).

Silver Staining
After being washed several times using ddH 2 O, brain sections were treated with formic acid for 5 mins and washed three times again. Then, sections were placed in silver glycine solution (preheated at 37 • C in advance) for 3-5 min, removed and the residual glycine silver solution on the sections quickly shaken off. Sections were put into reducing solution I (warmed to 45 • C in advance), removed after a few seconds, quickly shaken, placed in reducing solution II for a few seconds (preheated to 45 • C in advance) and cleaned using ddH 2 O. In case the staining background was too deep, it was treated using sodium thiosulfate solution and washed three times using ddH 2 O. The slides were mounted with anti-fluorescence quenching sealers and imaged by confocal microscopy (OLYMPUS FV1000, Olympus Corporation, Made in Japan).

Statistical Analysis
Data were analyzed using GraphPad Prism software and presented as mean ± standard error of the mean (S.E.M.). Statistical significance were considered at * p < 0.05; * * p < 0.01; * * * p < 0.001. Statistical analyses were performed by one-way ANOVA followed by the Student's t-test.

DHA Treatment Ameliorated Cognitive Impairment and Depression/Anxiety Behaviors in Alzheimer's Disease Mice
The Morris water maze (MWM) was used to evaluate the effects of DHA treatment on spatial memory and learning abilities in 2 × Tg AD mice (6). After 4 months of DHA treatment, some mice groups exhibited progressively shorter escape latencies, while the WT mice treated with high DHA reached the shortest escape latency significantly on the fifth day ( Figure 1A). Swimming speeds of mice in the WT, AD + DHA-L, and AD + DHA-L groups were significantly different from those of the AD group ( Figure 1B), implying that DHA partially restored exercise and spatial learning abilities of AD mice. After 24 h of the detection period, target quadrant retention time of the WT, AD + DHA-L, and AD + DHA-L groups were significantly higher than those of the AD group, while there were no significant differences in the other groups ( Figure 1C). Platform crossing numbers of DHA -treated mice group were not significantly higher than those of the other groups after 24 and 72 h. Similarly, DHA did not result in a significant increase in crossing numbers of mice in other groups ( Figures 1D,F), implying that DHA could partially improve short-term memory of AD mice, but not their long-term memories. These findings show that feeding DHA for 4 months partially restored motor functions of AD mice and improved some of their learning and memory impairments. The open field test (OFT) was performed to investigate the effects of DHA on depression-and anxiety-related behaviors of AD mice (6). After 4 months of DHA feeding, compared to WT mice, AD mice exhibited typical depression/anxiety behaviors, including significant reductions in the frequencies of grid crossing and rearing (Figures 2A,B). Compared to AD mice, the DHA treatment mice group exhibited significantly improved depression/anxiety behaviors, which was associated with a significant increase in grid crossing and feeding frequencies (Figures 2A,B). These behavioral MWM and OFT findings  ( Figures 1, 2) show that DHA treatment (including moderate or high DHA concentration) effectively improved some cognitive decline and depression/anxiety behaviors in AD mice.

DHA Significantly Suppressed the Pathological Hallmarks of Alzheimer's Disease Mice
Western blot was used to detect the expression levels of Aβ oligomers and APP protein in the hippocampus of mice. It can be seen from the results that the expression levels of Aβ protein and APP protein in AD group mice were significantly increased compared with WT group mice, and DHA administration for 4 months significantly decreased the AD + DHA-L and AD + DHA-H groups. The Aβ protein expression level and APP protein expression level of mice were not significantly different (Figure 3).
Interactions between Aβ and tau may be central to the development of Alzheimer's disease (7) while APP/PS1 transgenic mice develop age-related accumulation of plaques and tangles in the brain (8). Detection of plaques in the hippocampus and cortex of mouse brain through thioflavin T staining, to some extent, reflects the accumulation and distribution of Aβ in the brain. From the findings, the areas occupied by Aβ plaques of WT mice were all reduced significantly in both the cortex and hippocampus, compared to AD mice. Compared to AD mice as control group, low or high DHA treatments reduced Aβ plaque aggregations in CA3, CA1, DG, and Cor of AD mice (Figure 4).
The results of Western blot showed that total Tau (Tau5) and Tau phosphorylation sites (S202, S404, and S422) had no significant changes in all groups of mice ( Figure 5).
Glycine silver staining was used to detect neurofibrillary tangles (NFTs) in the hippocampus and cortex of mouse brains. Formation of NFT was due to excessive phosphorylation of the tau protein, which caused it to entangle with each other after falling off the microtubule tangle of nerve fibers (9,10). From the results, the NFTs of WT and DHA treated mice were significantly less than those of the AD group in CA3, CA1, DG, and Cor. However, DHA treated mice exhibited slightly worse outcomes than the WT group (Figure 6).

DHA Had No Obvious Improvement Effect on Liver Functions, Kidney Functions, and Blood Lipid Levels in Experimental Mice
Apolipoprotein A1 (ApoA1) and apolipoprotein B (ApoB) are lipid-related indicators (11, 12). ApoA1 is a high-density lipoprotein whose main role is to transport lipids from tissues and organs to the liver, and the liver decomposes the lipids (13). ApoA1 is negatively correlated with atherosclerotic cardiovascular disease. ApoB exists on the surface of low-density lipoprotein and is used to detect and take up low-density lipoproteins, increasing coronary atherosclerotic heart disease incidences even at low levels (12). Triglyceride (TG), also known as neutral fat, is the main source of energy in the body. Its content increases with age, especially for middle-aged and elderly people whose weight exceeds the standard. High triglyceride levels can easily result in thick blood and thrombosis leading to high blood pressure, gallstones, pancreatitis, and Alzheimer's. Apolipoprotein A1 (ApoA1) is mainly present in form of HDL and is the main apolipoprotein of HDL. It is mainly synthesized in the liver and small intestines. Under normal circumstances, high levels of HDL are better than low levels, which helps prevent hyperlipidemia, fat deposition and arterial Atherosclerosis. As the main apolipoprotein of LDL, Apolipoprotein B (ApoB) has various physiological functions, including the synthesis, assembly and secretion of VLDL rich in triglycerides. Clinically prescribed ApoB refers to ApoB100, which is better at low levels under normal conditions. We found that DHA had no significant effects on the levels of triglycerides, HDL, and LDL in each group of mice.
Aspartate transferase (AST) and alanine aminotransferase (ALT) are used to evaluate liver functions (14). Alanine aminotransferase (ALT) is an enzyme involved in the metabolism of human proteins. When liver cells are damaged, ALT is released from cells. We found that liver functions of AD mice in the WT group were significantly impaired. Cell damage can cause serum ALT activity to double, therefore, determination of ALT activity is one of the most sensitive indicators of liver cell damage. After adding DHA, serum ALT activities of AD + DHA-L and AD + DHA-H groups were significantly suppressed, which was basically the same as that of the WT group. Serum ALT activities of the WT + DHA-H and WT mice groups were significantly altered. Therefore, high-dose DHA had no toxic effects on WT mice, while low-dose DHA had a certain protective effect on the livers of AD mice (Figure 5A). Aspartate aminotransferase (AST) was formerly known as glutamic oxaloacetic transaminase (GOT). AST has two isoenzymes, ASTs and ASTm, which exist in soluble cytoplasm and mitochondria, respectively. When liver cells are mildly damaged, ASTs increase significantly, and in severe injuries ASTm appear in the serum in large quantities. We found that there were no significant differences in AST levels among the WT, AD, and DHA treatment groups, implying that drug treatment had no toxic and side effects on mice livers (Figures 7A,B).
For kidney functions, uric acid (UA) and urea are very important. UA is the final product of purine metabolism. Alterations in UA levels can fully reflect the body's metabolic, immune and other functions (15). Urea is a product of human protein catabolism (16). Excessive uric acid and urea levels  indicate liver and kidney damage, while an increase in UA levels but within the normal range can hinder the onset of AD (17). In this study, there were no significant differences in UA and urea levels among the WT, AD, and DHA groups, implying that drug treatment had no toxic or protective effects on kidney functions (Figure 8).
From the analysis of serum biochemical indicators, DHA did not improve liver as well as kidney functions and neither did it affect blood lipid levels of experimental mice. In addition, it had no toxic effects on experimental mice.

DHA Had Little Effects on Inositol, Glycine, and Taurine
Inositol, glycine, and taurine are common small molecules in the brain. They are associated with brain development, neurotransmission among others (18)(19)(20). The results of the concentration of Myo-instal in different brain regions showed that the WT group did not change significantly compared with the WT + DHA-H group, and the concentration of Myo-instal in the WT group showed an upward trend compared with the AD group. For mice in the AD + DHA-L, AD + DHA-H, and AD groups, they were concentration of Myo-instal down-regulated in the striatum, cortex and epithalamus, with significant differences. Since the specific role of concentration of Myo-instal is not yet clear, the result needs further verification.
Concentrations of glycine in different brain regions and the ratio of WT group to WT + DHA-H were all down-regulated. Moreover, the concentration of glycine in AD mice were also down-regulated. There was almost no change in glycine levels in the AD + DHA-L and AD groups, but the content of AD + DHA-H group has changed. These findings indicate that high-dose DHA may not have much effect on normal mice, but it can improve glycine levels in the brains of AD mice.
Compared to the WT group, the WT + DHA-H group exhibited significantly decreased concentrations of taurine in the striatum. The concentration of taurine results in other brain regions were different from the wild type group. WT group compared to the AD group, taurine in the HYP area was significantly up-regulated. After feeding with DHA, mice in the AD + DHA-L group exhibited significantly down-regulated taurine levels in the cortical area, while significant effects were not observed in mice in the AD + DHA-H group (Figure 9). Detection of neurofibrillary tangles (NFTs) in the hippocampus and cortex of mouse brain by silver glycinate staining. The NFT positive areas were evaluated using Image-Pro Plus Software. Western blot detection of Aβ protein expression in hippocampus of mouse brain. The pictures represent NFT level (Figure 10) and Aβ level (Figure 11) in the mice brain.

DISCUSSION
Alzheimer's disease is a progressive neurodegenerative disorder that is commonly associated with the onset of dementia in elderly people. The main clinical symptoms for AD include progressive memory loss and cognitive impairments (21). Typical pathological features include accumulation of Aβ plaques in brain tissues, nerve fiber tangles in brain cells, and neuronal loss among others, resulting in inflammation, oxidative stress, impairments in vasorelaxation, and progressive brain atrophy (1,7,22,23). DHA has been shown to enhance brain development, cognition, and learning behaviors (24). After DHA digestion, it can bypass tight junctions of the blood brain barrier (BBB) and enter the brain through inner membrane lobules. Therefore, improving Alzheimer's disease outcomes through dietary intake of DHA is safe and effective. Free DHA in dietary is transported across the outer membrane leaflet of the blood-brain barrier (BBB) via passive diffusion, attenuates the brain inflammatory response that leads to AD. A limited number of studies have evaluated the mechanisms through which DHA interferes with Alzheimer's disease progression. In this study, we used APP/PS1 transgenic mice model that developed age-related accumulation of plaques and tangles in the brain, showing some relatable symptoms of Alzheimer's disease to test the hypothesis that DHA is effective in treating Alzheimer's disease. Compared to AD control mice group, DHA improved spatial learning, memory ability and anxiety of mice. It also suppressed the accumulation of some Aβ oligomers and the formation of neurofibrillary tangles. In mice models, DHA did not interfere with liver and kidney functions, and it improved AD symptoms by improving blood lipid functions and preventing cardiovascular and cerebrovascular diseases.
Not only is DHA an essential factor for the healthy growth and development of infants and young children, it is also involved in improving cardiovascular health, reducing the symptoms associated with rheumatoid arthritis and depression, and improving human cognition. Large amounts of DHA are contained in the cerebral cortex and retina, especially in the gray matter and photoreceptor cells of the cerebral cortex. These DHAs are mainly in the form of phospholipids (25). DHA is very important in brain development (26). With cell membrane formation, neurons continue to form axons and dendrites. The growing cell membrane must be relatively fluid, and DHA is very fluid. Moreover, synapses of the most important functional units in the brain circuit are preferentially composed of DHArich cell membranes (27). DHA is an essential factor for the differentiation and migration of neurons as well as glial cells during brain development. It is also important in stimulating myelination and synapse. In addition, DHA is involved in neural cell differentiation (28), neural development (29) and in preventing synapse loss (30). Alzheimer's disease is prevalent in Western countries. Due to the association between DHA and brain development, and the relatively low intake of omega-3 fatty acids in these countries, there is a need to determine whether increasing DHA intake can reduce AD risks and improve cognitive repair in patients with mild AD. After death, in death patients, DHA levels decrease in specific areas of the brain, such as the pons, white matter, and especially in the frontal gray matter and hippocampus (31). In addition, since DHA contains six double bonds, it is very sensitive to lipid peroxidation. Elevations in peroxidation products of DHA in the brains of AD patients assumes that the loss of DHA can be attributed to the increase in oxidative stress (32). This lipid change occurs in the latter stages of AD, but also in the early stages of the disease (33). PUFA plays important roles in the prevention of AD, other types of dementia, and cognitive impairment (34). The prevention of AD by DHA is mostly based on the amyloid hypothesis (3,35). Dietary DHA was shown to be involved in improving learning-related memory deficiencies in Aβ peptideinfused AD rat models. DHA pre-administration ameliorated the deterioration of learning ability-related memory in AD rat models (36). Moreover, dietary DHA is protective against   Aβ production, accumulation, and its potential downstream toxic effects (37). Cell cultivation experiments revealed that DHA down regulates the secretion of Aβ peptides in aging human neural cells (38). Dietary DHA was associated with significantly increased levels of memory in the hippocampus and frontal cortex, which was associated with the protection of synaptic proteins. Both parameters were strongly correlated with learning as well as memory capacities and were implicated in the prevention of Aβ oligomer-induced impairments. Therefore, DHA is important in AD prevention (39). DHA was shown to protect against and ameliorate learning ability impairments in Aβ1-40 infused AD rat models, with concurrent increases in DHA levels and decreases in lipid peroxide levels and reactive oxygen species in the cortico-hippocampal tissues (40).
The mechanisms through which DHA affects AD symptoms may involve indirectly influencing blood vessels to prevent Aβ production. A diet rich in DHA altered the behaviors and cognitive abilities, cerebral hemodynamics [relative cerebral blood volume (rCBV)] as well as Aβ deposition levels in APPswe/PS1dE9 mice at 8 and 15 months of age (41). The study found that DHA altered rCBV in 8-month-old APP/PS1 and wild-type mice [AU1]. In 15-month-old APP/PS1 mice, DHA supplementation improved spatial memory, reduced Aβ deposition, and slightly increased rCBV, implying that a diet rich in DHA can reduce the pathological symptoms associated with AD. Therefore, long-term consumption of DHA-rich foods can improve AD symptoms. The Framingham Heart study showed that high plasma DHA levels can reduce the risk of AD development (42).
Moreover, adequate DHA intakes can reduce the risk of AD development (43)(44)(45) while dietary intake of n-3 PUFA may suppress cognitive declines (46,47). Freund-Levi (48) performed a trial and proved the positive effects of DHA supplementation on cognition of very mild AD patients. In APP transgenic mice, brain Aβ levels were found to be decreased after dietary DHA supplementation (37,49). It has been postulated that DHA supplementation increases the amounts of neuron sorting protein LR11, which regulates the processing of APP and the subsequent reduction in Aβ production (50). In addition, DHA supplementation in tg2576 AD mice depleted of DHA improved memory acquisition (30). The study by Hooijmans corroborates these findings (51). In their study, they used openair experiments to determine exploratory behaviors and used the Morris water maze (MWM) and 8-and 15-month-old wild-type and double-transgenic APPswe/PS1dE9 mice. The 12 circular orifice plates were used for spatial learning and memory assessments (41).
The water maze experiment is the most classic experiment to test spatial learning and memory abilities of mice (51). The escape latency of the 5 days' spatial training, swimming speed, time in target quadrant and the number of crossing platform reflects learning abilities, movement abilities, and spatio-memory abilities (52). In this study, the escape latency for mice in each group was shortened day by day. However, some mice in the same cage were injured in a fight, mice in the AD + DHA-H group took more time to find the platform on the 5th day than in the previous 4 days due to injuries in some mice. At 24 and 72 h after training, the time spent in the platform quadrant of the mice in each DHA treatment group was significantly increased compared to WT and AD mice. Compared to 72 h, residence time of mice in the 24 h treatment group and the AD group was significantly increased. The WT + DHA-H group exhibited an increasing trend compared to the WT group, however, the difference was not significant. The number of times mice in each treatment group crossed the platform was also better at 24 h than at 72 h. Compared to the AD group, the number of times mice in the WT group crossed the platform was significantly high. Finally, 24 and 72 h after training, the number of times mice in each treatment group crossed the platform was also better at 24 h than at 72 h. Results for mice in the WT group were more significant than those in the AD group. Compared to AD group mice, the number of times that mice in the AD + DHA-L group crossed the platform was significantly increased. In this study, low-dose DHA improved memory and learning abilities of AD mice, without significant changes in other groups.
The open field experiment, which exploits mice innate avoidance to open fields and exploration of new things, is used to evaluate spontaneous activities and anxiety in mice (6). In this study, we found that long-term DHA supplementation increased the number of rearing and crossing grids of AD mice, implying that the spontaneous activity and anxiety of AD mice were improved by DHA treatment. Combining the results from the water maze experiment and the open field experiment, longterm DHA supplementation had a certain therapeutic effect on impaired cognitive and motor abilities of AD mice.
As earlier mentioned, Aβ and hyperphosphorylated tau are two major toxic proteins of Alzheimer's disease (7). Aβ is attributed to continuous shearing of APP proteins by β-amylase and γ-secretase. Large amounts of Aβ precipitate outside neurons to form senile plaques (SP) (7,22,53). Tau promotes the assembly of microtubules and maintains cytoskeleton functions. Overphosphorylated tau not only loses its biological functions but also dissociates from the microtubules and aggregates with each other, eventually forming a large number of NFTs (9,54). Aβ has strong toxic effects and induces tau hyperphosphorylation, which leads to neuronal degeneration, dysfunction, and death (55,56). In this study, through brain section thioflavin and silver staining, we found that the number or area of beta amyloid in DHA-fed AD mice was reduced in certain brain regions, showing the Aβ clearance effect of DHA. The number of NFTs in AD mice was also decreased with increasing DHA concentrations. Neuronal damage and death, especially in brain learning and memoryrelated areas, are pathological features of AD (1). Therefore, DHA suppressed neuronal damage by reducing Aβ and inhibiting tau protein hyperphosphorylation, which improved cognitive abilities and mobility of AD mice.
Obesity, hypertension, cardiovascular and cerebrovascular diseases increase the risk of AD (57)(58)(59). Lipoproteins are complexes of different lipids and proteins that act on the transport and clearance of lipids or lipid-related molecules from circulation (60). Lipoproteins also play a vital role in brain functions. High-density lipoproteins (HDL) and their main protein component, ApoA1, are directly involved in the outflow of cholesterol from the brain (12,61). Overexpression of human ApoA1 in circulation was shown to prevent learning and memory impairments in APP/PS1 transgenic mice, partly by suppressing neuroinflammation and cerebral amyloid angiopathy (12). In addition, ApoB was found to be elevated in AD plasma and serum, and over expression of ApoB in transgenic mice triggered apoptosis and neurodegeneration in the brain (62)(63)(64). In this study, there were no significant differences in ApoA1 and ApoB levels between the WT and AD groups. Moreover, after DHA supplementation, differences in ApoA1 and ApoB levels were not significant, implying that DHA had no effect on the apolipoproteins.
Analysis of serum biochemical indicators showed that DHA had no effects on blood lipid levels, kidney functions and liver functions of experimental mice. AST and ALT are the most sensitive indicators of liver cell damage. When liver cells are severely damaged, AST and ALT levels are significantly elevated in serum (14). In this study, neither DHA supplemented AD mice nor WT mice had significantly elevated AST and ALT levels, and there were no differences between the groups, implying that DHA did not impair liver functions.
Among the possible markers for age-related cognitive decline, UA is controversial because it has antioxidant properties.
However, elevated UA levels increases the risk of gout and cardiovascular disease and affects kidney functions. Serum UA levels in AD patients were found to be significantly low than in healthy controls (14,65). In this study, there were no significant differences in renal function indices in WT, AD and DHA treatment groups, implying that DHA had no toxic effects on kidney functions, and no obvious protective effects.
Inositol is associated with signal transduction, and its imbalance leads to neurological disorders (18). Inositol was shown to improve pathological characteristics of Alzheimer's disease animal models and reversed cognitive impairments (66,67). However, we found that inositol was stably expressed in different groups. Glycine is a neurotransmitter with antiinflammatory, signaling, and cellular immunity functions (19). Inhibition of glycine transporter-1 (GlyT1) elevated glycine levels, thereby inhibiting cognitive impairments in Alzheimer's disease animal models (68). We also found that high-dose DHA may not have a significant effect in normal mice, but it improved glycine levels in the brains of AD mice, which may be a good phenomenon. Due to aging, taurine levels in mammalian brain decrease, affecting memory ability (69).
In this study, taurine levels in the brains of AD mice fed with DHA did not significantly improve.

CONCLUSION
(1) DHA suppressed neuronal damage by reducing Aβ and inhibiting tau protein hyperphosphorylation, which improved cognitive abilities and mobility of AD mice.
(2) DHA has a certain effect on lowering blood lipid levels and may have a positive effect on the prevention of vascular diseases, which may improve Alzheimer's disease outcomes.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Ethical and Welfare Committee of Shenzhen University.

AUTHOR CONTRIBUTIONS
MX: conceptualization, methodology, project administration, and writing -review and editing. WX: data curation, writing -original draft preparation, and formal analysis. YC: writing -review and editing and investigation. NP, SL, and YZ: writing -review and editing. XD: resources, data curation, and formal analysis. BL: project administration. YH and XL: supervision. All authors contributed to the article and approved the submitted version.


s2fieldsofstudy: Biology Medicine

text:
Circular RNA 0010117 promotes aggressive glioblastoma behavior by regulating the miRNA-6779-5p/SPEN axis

Highlights • Circ-0010117 is down-regulated in glioblastoma.• Circ-0010117 regulates aggressiveness via miRNA-6779-5p in glioblastoma.• SPEN contributed to regulate miRNA-6779-5p in glioblastoma.• Upregulated Circ-0010117 inhibited in vivo tumor growth of human glioblastoma xenograft.

Introduction
Glioblastoma is the most common primary brain cancer of glial origin worldwide and is the leading cause of neurologic cancer-related death worldwide [1]. High-grade glioblastoma in particular has a very poor prognosis and poses a serious threat to human health. In recent years, although advances have been made in surgery, chemotherapy and molecular targeted therapies, glioblastoma still has poor morbidity and mortality. Given the poor strategies for diagnosis and treatment, a better understanding of glioblastoma pathogenesis is critical for advancing and providing potential biomarker-driven treatments.
Noncoding RNAs (ncRNAs) are a class of nonprotein translation RNAs that include long noncoding RNAs (lncRNAs) more than 200 nucleotides in length, microRNAs (miRNAs) 20-22 nucleotides in length, and circular RNAs (circRNAs), which are characterized by covalent closed loops. Similar to other ncRNAs, circRNAs are expressed in multiple tissues with great variability. CircRNAs are a special class of noncoding RNAs that originate from exons, introns, intergenic regions or untranslated regions by a noncanonical splicing process termed back splicing [2]. Importantly, circRNAs participate in the occurrence of many kinds of tumors and regulate tumor development. Circular RNA hsa_circ_0001785 has been reported to inhibit the proliferation, migration and invasion of breast cancer cells in vitro and in vivo by sponging miR-942 to upregulate SOCS3 (suppressors of cytokine signaling 3) [3]. The circular RNA hsa_circ_0079,929 has also been validated to inhibit tumor growth in hepatocellular carcinoma [4]. Another novel circular RNA, circENTPD7, has been confirmed to contribute to glioblastoma progression by targeting ROS1 (ROS proto-oncogene 1) [5]. However, the potentially important functional role of circ-0010117 in the progression of glioblastoma is still unclear.
In this study, we first investigated the expression of circ-0010117 in human glioblastoma tissues. We subsequently investigated the effects of circ-0010117 on the aggressiveness of human glioblastoma cells. Furthermore, we also explored the molecular mechanisms underlying these effects.

Human osteosarcoma clinical specimens
A total of 6 paired osteosarcoma and corresponding paratumoural tissues were collected at the First Affiliated Hospital of Nanchang University from November 2021 to January 2022. All tissue specimens were resected during surgery and directly snap frozen in liquid nitrogen. The study protocol was approved by the ethics committee of the First Affiliated Hospital of Nanchang University. All written informed consent was received from patients who underwent surgical resection.

Quantitative RT-PCR
Total RNA from tissue samples and cells was isolated using the mirVana TM miRNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. RT-qPCR analysis was performed with SYBR green in a 7500 Fast Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA). Circ-0010117, microRNA-6779-5p (miR-669-5p) and SPEN were measured by qRT-PCR. U6 was used as an internal reference for circ-0010117 and miR-669-5p, as previously described [6]. β-Actin was used as an internal control for SPEN. All primers were commercially available from Sangon Biotech (Shanghai, China). The amount of circRNA, miRNA or each gene relative to the internal control was calculated as previously described [7].
For the circ-0010117 overexpression plasmid (circ-0010117), the sequence was cloned into GV248. The backbone of pLCDH-ciR (Geneseed, Guangzhou, China) was used to construct the circ-0010117 overexpression plasmid. The linear sequence of circ-0010117 together with the flanking circ-0010117-inducing sequence was amplified and subcloned into the EcoRI and BamHI sites of pLCDH-ciR.

MiR-6779-5p/SPEN overexpression and inhibition
For SPEN overexpression in vitro, we used an SPEN vector construct (OriGene Technologies, Inc., Rockville, MD). U251 and U87 cells were seeded in a 6-well plate (Corning, Cambridge, MA, USA) (2 × 10 5 cells/ well) with complete medium. The cells were transfected with 2 μg of SPEN vector or the vector control (OriGene Technologies) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) with Opti-MEM (Gibco) for 4 h. Then, we replaced the Opti-MEM with complete medium. After 48 h, the cells were collected for the next determination. MiR-6779-5p mimics are commercially available from life technologies. Transfection of miRNA mimics was performed using RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. miRNA vector control (NC mimic) was used as a vector control. The shSPEN and miR-6779-5p inhibitor are also commercially available from GeneCopoeia (Rockville, MD).

Cell Counting Kit-8 (CCK8) assay
A CCK-8 assay was performed to assess the proliferation of U251 and U87 cells. For the CCK-8 assay, mock and infected cells (1 × 10 5 cells/ well) were seeded into 96-well plates (Corning, Cambridge, MA, USA) after 24 h of transfection and cultured for 24 h, 48 h and 72 h. The absorbance at 450 nm was measured using CCK-8 (Dojindo Laboratories, Kumamoto, Japan) at the indicated times according to the manufacturer's protocols.

Colony formation assay
For the colony formation assay, we melted 1.4% (v/v) agarose (Sigma-Aldrich, Shanghai, China) in a microwave and cooled it to room temperature. Then, we mixed equal volumes of the 1.4% melted agarose with the complete cell culture medium. After that, 2 ml of 0.7% (v/v) low melt agar was added into each well of the 6-well plate (Corning, Cambridge, MA, USA) and set aside to allow agarose to solidify. Cells (5 × 10 3 cells/well) were mixed with 1.4% agarose in complete culture medium and plated on top of the solidified layer to form colonies at 1-3 weeks. Cells were fed complete culture medium every 3 d.
The colony is required to consist of at least 50 cells [8]. Clones were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (Millipore, Billerica, MA, USA). Visible colonies were manually counted.

Apoptosis assay
Flow cytometry was performed for the apoptosis analysis. U251 and U87 cells were digested with trypsin (Invitrogen, Carlsbad, CA, USA) and washed with PBS. After blocking with 10% FBS, the cells were stained with 5 μl FITC annexin-V and 1 μl propidium iodide (Becton Dickinson, Heidelberg, Germany) and incubated at room temperature for 15 min in the dark. Then, flow cytometry was performed (Life Technologies, Darmstadt, Germany). The cells were then analyzed with CellQuest software (BD Biosciences, San Jose, CA). The dead cells and the relative ratio of apoptotic cells were compared in each condition.
Wound scratch assay analysis U251 and U87 cells were first transfected with shcirc-0010117 or circ-0010117 or their related control for 24 h. A wound scratch assay was performed as previously described [9][10][11][12]. Then, the cells were seeded into 12-well plates (5 × 10 5 cells/well) with medium and cultured for 24 h to reach ~90% confluence. A 10 μl pipette tip was used to make a scratch line. The cells were washed with PBS 3 times to remove the scratched cells. Then, the cells were cultured in medium containing 0.5% FBS. The cells were incubated at 37 • C, and images were captured at 0 and 48 h using a light microscope (Olympus Corporation, Tokyo, Japan).

Cellular invasion
A Boyden chamber assay with a 24-well collagen-based cell invasion assay kit (Millipore, Bedford, MA, USA) was used to perform the transwell assay. U251 and U87 cells were first transfected with shcirc-0010117 or circ-0010117 or their related control for 24 h. Then, 200 μl of serum-free medium containing 5 × 10 5 cells was added to the filter.
Then, 750 μl of complete cell culture medium was added to the bottom chamber, in which FBS was used as a chemoattractant. After incubation for the indicated time, the noninvasive cells were cleaned by scrubbing with a cotton swab. The cells that adhered to the outside of the membrane were fixed and dyed with Cristal Violet solution (Millipore, Billerica, MA, USA). An Olympus optical microscope (Olympus Corporation, Tokyo, Japan) was used to observe invasive cells.

Dual-luciferase reporter assay
The binding sites of circ-0010117 for miR-6779-5p of its 3'-UTR and mutant were commercially available at GeneCopoeia (Rockville, MD). U251 and U87 cells were seeded in a 6-well plate (Corning, Cambridge, MA, USA) at a density of 2.5 × 10 5 cells/well with complete medium. The cells were cotransfected with 1 µg circ-0010117 3'-UTR (circ0010117-WT) or mutant luciferase reporter construct (circ0010117-Mut) with 20 nM miRNA mimic or NC mimic using Lipofectamine 2000 (Carlsbad, CA, USA) with Opti-MEM (Gibco, Waltham, MA, USA) for 48 h. Luciferase assays were performed using the dualluciferase reporter assay reagent from GeneCopoeia. Data are expressed as the ratio of firefly luciferase activity to Renilla luciferase activity.

Western blotting
Total protein was extracted from cells with and without circ-0010117 with T-PER Tissue Protein Extraction Reagent (Thermo Pierce, Rockford, IL, USA) with 1% Halt Protease and Phosphatase Inhibitor (Thermo Pierce, Rockford, IL, USA). Then, the extracted protein was loaded and run on sodium dodecyl sulfate-polyacrylamide gels (8-12% separating gel, 5% concentrated gel). The protein in the gel was transferred onto a PVDF membrane (Millipore, Bedford, MA, USA) for antibody detection. The PVDF membrane was blocked for 1 h at room temperature in 5% nonfat milk in Tris-buffered saline with 0.1% Tween-20 detergent (TBST). The membrane was washed with TBST 3 times for 10 min each at room temperature and incubated with primary antibodies (anti-truncated-cas-3, full-cas-3, cas-8, cas-9, BCL-2, BAX, P53, and JNK1) overnight at 4 • C. β-actin served as an internal control (Abcam, Cambridge, MA, USA). Then, the membrane was washed with TBST 3 times for 10 min each at room temperature and incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG or goat antirabbit IgG secondary antibody (Thermo Pierce, Rockford, IL, USA) for 1 h at room temperature.wAfter the membrane was washed with TBST 3 times for 10 min each at room temperature, proteins were visualized by using ECL western blotting detection reagents (SuperSignal® West Dura Extended Duration Substrate, Thermo Pierce, Rockford, IL, USA). The relative expression of the target protein was quantitatively analyzed by ImageJ (NIH, Bethesda, MD, USA).

Mouse xenograft assays
Female nude mice (BALB/c, SPF grade, weighing 16-20 g and 4 weeks old) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, China). Transfected cells were digested with trypsin and washed twice with PBS. Cells were resuspended in 200 µL solution containing 100 µL PBS and 100 µL Matrigel substrate at a final density of 2 × 10 7 cells/0.2 mL. Four-week-old female BALB/c nude mice were subcutaneously injected with 2 × 10 7 tumor cells. Mice were sacrificed at Day 35 after tumor cells had been injected, and tumor volume was measured.

Statistical analysis
Statistical analyzes were performed using GraphPad Prism, and all data are presented as the mean ± standard deviation (SD) from triplicate experiments. Differences between groups were analyzed by unpaired t test. A p value < 0.05 was considered significant.

Circ-0010117 is downregulated in glioblastoma
In this study, we first determined the mRNA expression levels of circ-0010117 in 6 paired glioblastoma and corresponding paratumoural tissues with qRT-PCR. Circ-0010117 mRNA levels were significantly downregulated compared with those in normal tissues (P < 0.05, Fig. 1A). Subsequently, we tested circ-0010117 expression in U251 and U87 cells. Synthesized interfering oligonucleotides targeting circ-0010117 significantly decreased the expression of circ-0010117 in U251 and U87 cell lines ( Fig. 1B and C). Circ-0010117 overexpression strongly increased the expression of circ-0010117 in U251 and U87 cell lines (Fig. 1D).

Circ-0010117 regulates aggressiveness in glioblastoma
The silencing of circ-0010117 in U251 and U87 cell lines remarkably enhanced cell proliferation compared with the control ( Fig. 2A and B). However, the overexpression of circ-0010117 in U251 and U87 cell lines significantly suppressed cell proliferation ( Fig. 2C and D). We next sought to establish whether alteration of circ-0010117 could affect U251 and U87 clonogenicity. As shown in Fig. 2E-H, circ-0010117 silencing significantly enhanced the anchorage-dependent growth of MSTO-211H cells ( Fig. 2E and F). In addition, circ-0010117 overexpression strongly decreased the anchorage-dependent growth of U251 and U87 cells ( Fig. 2G and H). Thus, circ-0010117 can regulate growth activities in glioblastoma cells.
Enhanced migration and invasion are another key feature across the metastatic cascade. To assess the effect of circ-0010117 on the migration and invasion of MPM cells, wound scratch and transwell assays were performed, respectively. As shown in Fig. 2I, the rate of gap closure was greatly enhanced after circ-0010117 silencing in U251 and U87 cells compared with the control. However, the rate of gap closure was significantly decreased after circ-0010117 overexpression in U251 and U87 cells compared with the control (Fig. 2J).
Similarly, a markedly increased cell invasion was observed in U251 and U87 cells with circ-0010117 silencing compared with the control by transwell assay (Fig. 2K). In contrast, we observed significantly decreased cell invasion in U251 and U87 cells overexpressing circ-0010117 compared with the control (Fig. 2L). We also assessed apoptosis alteration by circ-0010117 regulation through flow cytometry. U251 and U87 cells exhibited a reduced percentage of apoptotic cells after circ-0010117 silencing (Fig. 2M). For U251 and U87 cells overexpressing circ-0010117, the percentage of cells exhibiting apoptosis was significantly increased (Fig. 2N). These results suggest that circ-0010117 regulates aggressiveness in glioblastoma.

Circ-0010117 regulates aggressiveness via miRNA-6779-5p
To unravel the relevant mechanism underlying circ-0010117 contributing to the malignant phenotype of glioblastoma. We searched for a potential regulatory mechanism of circ-0010117. We next asked whether miRNAs play important roles in the regulation of circ-0010117 related to the malignant phenotype of glioblastoma. We found that miR-6779-5p significantly reduced the luciferase activities of circ-0010117 WT but not Mut in U251 and U87 cells (Fig. 3A). We next investigated the effect of inhibiting or overexpressing miR-6779-5p in U251 and U87 cells with circ-0010117 silencing or circ-0010117 overexpression. The miR-6779-5p expression level was confirmed after inhibiting or overexpressing miR-677-5p (Fig. 3B).
Shcirc-0010117 and miR-6779-5p inhibitor were transfected into U251 and U87 cells to knockdown circ-0010117 and miR-6779-5p expression. The effect of the miR-6779-5p inhibitor on U251 and U87 cells was consistent with the effect of circ-0010117 on cell proliferation detected with CCK-8 (Fig. 3C). In contrast, circ-0010117 and miR-6779-5p mimic were transfected into U251 and U87 cells to overexpress circ-0010117 and miR-6779-5p. We found that miR-6779-5p overexpression enhanced U251 and U87 cell proliferation, which reversed the effect of circ-0010117 overexpression on cell proliferation (Fig. 3D). Similar results were found for invasion and apoptosis. The effect of the miR-6779-5p inhibitor on U251 and U87 cells was consistent with the effect of circ-0010117 on cell invasion and apoptosis detected with transwell assays and flow cytometry ( Fig. 3E and G). In addition, we found that miR-6779-5p overexpression enhanced U251 and U87 cell invasion and apoptosis, which reversed the effect of circ-0010117 overexpression on cell invasion and apoptosis in cells overexpressing circ-0010117 and miR-6779-5p ( Fig. 3F and H).

SPEN contributed to miRNA-6779-5p regulation
To explore the regulatory mechanism underlying circ-0010117 and miR-6779-5p for the malignant phenotype of glioblastoma. We subsequently identified a potential target of miR-6779-5p. We found that miR-6779-5p significantly reduced the luciferase activities of SPEN WT but not Mut in U251 and U87 cells (Fig. 4A). The SPEN mRNA level was also found to be significantly decreased after miR-6779-5p inhibition in U251 and U87 cells (Fig. 4B) but markedly increased after miR-6779-5p overexpression (Fig. 4C).
The miR-6779-5p mimic and SPEN overexpression plasmid were transfected into U251 and U87 cells to increase miR-6779-5p and SPEN expression. SPEN overexpression in U251 and U87 cells reversed the effect of miR-6779-5p overexpression on cell proliferation detected with CCK-8 (Fig. 4D). We also transfected U251 and U87 cells with miR-6779-5p inhibitor and shSPEN to knock down miR-6779-5p and SPEN. As shown in Fig. 4D, SPEN knockdown blocked the effect of miR-6779-5p inhibition on cell proliferation suppression.
Consistent with cell proliferation, SPEN knockdown reversed the effect of miR-6997-5p silencing on invasion and apoptosis in U251 and U87 cells (Fig. 4E and G). However, SPEN overexpression repressed U251 and U87 cell invasion and apoptosis caused by miR-6779-5p overexpression ( Fig. 4F and H).

Circ-0010117 upregulation inhibited tumor growth in vivo
To further assess whether overexpression of circ-0010117 could suppress tumor formation in vivo, we inoculated stable overexpressed circ-0010117 with the U251 cell line (Fig. 5A) subcutaneously into 4week-old nude mice. The results showed that tumor size and weight were remarkably reduced when transplanted with tumor cells overexpressing U251 (Fig. 5B-D).

Discussion
Currently, glioblastoma is a common malignant tumor worldwide [13]. In recent years, with research on biomarkers of glioblastoma, the targeted diagnosis, treatment and prognosis of glioblastoma have achieved preliminary results, but more research is needed to combat glioblastoma. Therefore, the identification of key biomarkers in glioblastoma is an effective strategy for the treatment of glioblastoma.
The roles of circRNAs in cancer progression have attracted much attention due to their key roles in human cancers. In this study, our goal was to explore the potential important roles of circ-0010117 during glioblastoma progression. This examination confirmed that circ-    0010117 participated in the glioblastoma process via miRNA-6779-5p and SPEN, revealing that circ-0010117 served as a novo biomarker for glioblastoma.
CircRNAs represent a new class of endogenous noncoding RNAs that have recently been recognized as important regulators of glioblastoma.
MiRNAs are a group of 19-22 nucleotide noncoding, single-stranded RNA molecules that participate in sequence-specific posttranscriptional regulation of gene expression [14]. Recently, emerging evidence has confirmed that circRNAs can regulate the expression of tumor-suppressive or oncogenic genes through the circRNA-miRNA- mRNA axis. For instance, circular RNA circ-0001946 has been validated to act as a competing endogenous RNA to inhibit glioblastoma progression by modulating miR-671-5p and CDR1 [15]. Circular RNA circMTO1 inhibits the proliferation of glioblastoma cells via the miR-92/WWOX signaling pathway [16]. Because their expression and function in glioblastoma development are still largely elusive, we screened the differentially expressed circRNAs between glioblastoma and matched nontumor liver tissues and focused on the role and underlying mechanism of the decreased circ-0010117 in glioblastoma progression. In this study, we found that circ-0010117 is downregulated in glioblastoma. In addition, circ-0010117 can regulate aggressiveness in glioblastoma. This invention expanded knowledge on the role of circ-0010117 in the modulation of glioblastoma, which may be helpful to better elucidate the pathological mechanism of glioblastoma.
Many researchers have illustrated that circRNAs regulate the process of diseases via miRNAs [17]. Previous researchers found that many miRNAs were involved in glioblastoma; for instance, miRNA-381 inhibited EMT in glioblastoma [18]. miRNA-124-3p was reported to suppress the growth of glioblastoma [19]. In contrast, miRNA-1908 promoted glioblastoma [20]. Here, experimental proof identified miRNA-6779-5p as downstream of circ-0010117, as they have confirmed connection points. We found that the level of miRNA-6779-5p was clearly upregulated and was in contrast with the level of circ-0010117 in glioblastoma.
Split ends (SPEN) is an ERα corepressor that we have identified as a tumor suppressor protein in ERα-positive breast cancer cells [21]. SPEN has been reported to play important roles in cancer progression. For instance, SPEN has been found to induce miR-4652-3p to target HIPK2 in nasopharyngeal carcinoma [22]. SPEN has also been identified as a crucial factor for Xist function through forward genetic screening in haploid embryonic stem cells [23]. However, it is still unclear whether SPEN contributes to the circ-0010117 and miR-6779-5p regulatory network. In this study, we found that SPEN is a direct target of miR-6779-5p and that SPEN contributes to the miRNA-6779-5p regulatory network.

Conclusion
In summary, we determined that circ-0010117 is linked to aggressiveness in glioblastoma through miRNA-6779-5p and SPEN. Our results provide the rationale for the use of circ-0001946 as a novel potential therapeutic target in glioblastoma.

Data availability
All the data from the current study are included in the article or uploaded as supplementary information.

Funding
The study was supported by Science and technology project of Health Commission of Jiangxi Province (No: 202210394).

Ethics approval and consent to participate
The experimental protocols were approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University. This paper has not been published elsewhere in whole or in part. All authors have read and approved the content and agree to submit it for consideration for publication in your journal. There are no ethical/legal conflicts involved in the article. Informed consent was obtained from all individual participants included in the study.

Declaration of Competing Interest
The authors declare that they have no conflicts of interest.


Pathogenicity and Metabolites of Purpureocillium lavendulum YMF1.00683 against Meloidogyne incognita

Purpureocillium lavendulum is a biological control agent with several registered products that can parasitize the eggs and larvae of various pathogenic nematodes. In this study, the pathogenicity and secondary metabolites of the fungus P. lavendulum YMF1.00683 were investigated. The strain YMF1.00683 had infection efficiency against the plant root-knot nematode Meloidogyne incognita. The strain’s process of infecting nematodes was observed under a microscope. Moreover, seven metabolites, including a new sterol (1), were isolated and identified from cultures of YMF1.0068 in Sabouraud’s dextrose agar. A bioassay showed that 5-methoxymethyl-1H-pyrrole-2-carboxaldehyde (7) is toxic to M. incognita and affects the egg hatching. It caused 98.23% mortality in M. incognita and could inhibit 80.78% of the hatching eggs at 400 μg/mL over a period of 96 h. Furthermore, 5-methoxymethyl-1H-pyrrole-2-carboxaldehyde (7) showed a strong avoidance effect at 40 ppm, and its chemotactic index value was −0.37. The results indicate that P. lavendulum could produce active metabolites against M. incognita.

Introduction
The damage caused by plant-parasitic nematodes to agriculture reaches USD157 billion every year, and the most serious damage is caused by the plant root-knot nematode belonging to the genus Meloidogyne [1,2]. Chemicals are still the most widely used pesticides in agriculture and forestry. However, long-term use of chemical nematicides may lead to the development of resistance among pathogens and may have detrimental effects on the environment and human health [3]; for example, methyl bromide is a broad-spectrum fumigant that is toxic to nematodes and has thus been banned. Currently, there is an urgent need for environmentally friendly biopesticide formulations, but their successful application is often affected by factors such as environmental conditions, storage time, and the degradation of the biocontrol strain. Therefore, one key measure to improve the effect and stability of biological pesticides is to analyze the control mechanism.
With the rapid development of whole-genome sequencing technology and informatics biology, the sequencing of numerous fungal genomes shows that most fungal metabolic biosynthetic potential has not been fully exploited. By analyzing the genomic data of P. lavendulum YMF1.00683 (which have not been released yet), it was found that this strain strain has the potential to produce abundant secondary metabolites. In general, the metabolites of microorganisms often play an important role in biological control of nematode. For example, 5-hydroxymethylfuran-2-carboxylic acid was isolated from Drechmeria coniospora and possessed nematocidal activity against M. incognita [10], and leucinostatins obtained from Paecilomyces lilacinus showed nematocidal activity [11], while other compounds such as phenolic acids and sesquiterpenoids were obtained from Paecilomyces lilacinus and exhibited nematocidal activity [11,12]. Therefore, it is necessary to carry out research on active metabolites from biocontrol potential fungus P. lavendulum YMF1.00683. In the present work, we observed the nematodes infected by P. lavendulum YMF1.00683 under a microscope and found that lethality gradually increased over time ( Figure 1) during co-cultivation. We speculate that P. lavendulum YMF1.00683 produced small molecule compounds to kill the nematodes. Therefore, in this study, the metabolites were purified from culture extracts of P. lavendulum YMF1.00683, and we tested the compounds isolated for their activity against M. incognita. The results indicated that the metabolites from P. lavendulum YMF1.00683 showed multiple activities against nematodes.

Pathogenicity of P. lavendulum YMF1.00683 against M. incognita
Many reports on fungi of the genus Purpureocillium (formerly Paecilomyces) have shown that they can control and antagonize root-knot nematodes of M. incognita [6,7]. Here we provide the microscopic observation of P. lavendulum YMF1.00683 infestations and the decomposition process of M. incognita in the laboratory. As shown in Figure 1, it can be seen that after three days of spore germination, the hyphae had overgrown the plate. At this time, approximately 50 M. incognita worms were added to each plate. After 24 h of M. incognita addition, the nematodes still had good viability. After 48 h, the vigor had been significantly reduced, and 20% of the M. incognita had died. At 72 h, 34% of M. incognita had died, and the body walls of the dead nematodes had ruptured. At 96 h, 41% of the nematodes had died, and the bodies of the dead nematodes had been degraded.

Pathogenicity of P. lavendulum YMF1.00683 against M. incognita
Many reports on fungi of the genus Purpureocillium (formerly Paecilomyces) have shown that they can control and antagonize root-knot nematodes of M. incognita [6,7]. Here we provide the microscopic observation of P. lavendulum YMF1.00683 infestations and the decomposition process of M. incognita in the laboratory. As shown in Figure 1, it can be seen that after three days of spore germination, the hyphae had overgrown the plate. At this time, approximately 50 M. incognita worms were added to each plate. After 24 h of M. incognita addition, the nematodes still had good viability. After 48 h, the vigor had been significantly reduced, and 20% of the M. incognita had died. At 72 h, 34% of M. incognita had died, and the body walls of the dead nematodes had ruptured. At 96 h, 41% of the nematodes had died, and the bodies of the dead nematodes had been degraded.

Isolation and Structural Identification of Compounds
In order to investigate the metabolites and their function of P. lavendulum YMF1.00683, a systematic isolation of the extract of the strain with different chromatographic techniques is achieved. The extract (200.5 g) from the SD solid fermentation of YMF1.00683 was Pathogens 2022, 11, 795 3 of 11 chromatographed on various columns (such as RP-18, Sephadex LH-20, and silica gel), and seven compounds (1-7) were obtained ( Figure 2). Their structures were identified by NMR and MS data.

Isolation and Structural Identification of Compounds
In order to investigate the metabolites and their function of P. lavendulum YMF1.00683, a systematic isolation of the extract of the strain with different chromatographic techniques is achieved. The extract (200.5 g) from the SD solid fermentation of YMF1.00683 was chromatographed on various columns (such as RP-18, Sephadex LH-20, and silica gel), and seven compounds (1-7) were obtained ( Figure 2). Their structures were identified by NMR and MS data. Compound 1 was obtained as a colorless solid, and its molecular formula was found to be C29H44O4 using high-resolution mass spectrometry 457.3311 ([M + H] + , Calcd. 457.3312); it contained eight unsaturation degrees. According to the nuclear magnetic resonance (NMR) spectrum data of compound 1 (Table 1), it contained 29 carbon signals, including 5 quaternary carbons, 11 methines, 7 methylenes, and 6 methyls.  Compound 1 was obtained as a colorless solid, and its molecular formula was found to be C 29 H 44 O 4 using high-resolution mass spectrometry 457.3311 ([M + H] + , Calcd. 457.3312); it contained eight unsaturation degrees. According to the nuclear magnetic resonance (NMR) spectrum data of compound 1 (Table 1), it contained 29 carbon signals, including 5 quaternary carbons, 11 methines, 7 methylenes, and 6 methyls.

Evaluation of the Nematocidal Activity of Compounds against M. incognita
As shown in Table 3, at 400 ppm, the nematocidal activities of 5-(methoxymethyl)-1H-pyrrole-2-carboxaldehyde (7) were gradually enhanced with the increase in time, and compound 7 showed strong activity at 96 h (corrected mortality rate > 90%). We found, using multiple comparisons in SPSS (Table 3), that other compounds were not significantly different at the 0.05 level during all observation periods, and they showed moderate activity (30-40%) at 96 h. Meanwhile, the adjusted mortality of avermectin was 81% at a concentration of 100 µg/mL over a period of 48 h.  (7) showed obvious activity in inhibiting egg germination and nematocidal activity against M. incognita J2. Therefore, it was selected for the analysis of its chemotactic effect on M. incognita. The results showed that when the compound's concentration was 40 ppm (Figure 3A), it exhibited a strong avoidance effect towards root-knot nematodes of M. incognita (CI < 0). When the compound's concentration decreased to 5 ppm ( Figure 3B), it showed a weaker attractive effect (0 < CI < 0.2).  (7) showed obvious activity in inhibiting egg germination and nematocidal activity against M. incognita J2. Therefore, it was selected for the analysis of its chemotactic effect on M. incognita. The results showed that when the compound's concentration was 40 ppm (Figure 3A), it exhibited a strong avoidance effect towards root-knot nematodes of M. incognita (CI < 0). When the compound's concentration decreased to 5 ppm ( Figure 3B), it showed a weaker attractive effect (0 < CI < 0.2).

Discussion
Root-knot disease caused by M. incognita is a serious concern because it affects several economically important crops globally. Researchers found that Paecilomyces lilacinus (currently valid name: Purpureocillium lilacinum) showed a parasitic effect on root-knot nematodes [6][7][8]. Several experts have conducted numerous studies on the fungus and have confirmed that Paecilomyces lilacinus has control effects on various plant-parasitic nematodes [3,21]. Paecilomyces lilacinus can parasitize the eggs and larvae of important pathogenic nematodes, including root-knot nematodes and cyst nematodes. Paecilomyces lilacinus was subsequently found to disrupt the lipid and chitin layers of nematode eggshells [22]. Additionally, Paecilomyces lilacinus can also produce a variety of biologically active secondary metabolites, including polyketides and non-ribosomally synthesized peptides, such as leucinostatins, which are nematocidal, antiviral, and phytotoxic and possess a series of biological activities [11,23,24]. By comparing the 18S rRNA gene, internal transcribed spacer, and partial translation elongation factor 1-a of the isolates of P. lilacinus from the environment and organisms, the genus Purpureocillium was proposed [4], which includes P. lilacinum, P. lavendulum, and P. takamizusanense [4,25].
While studying the infection of M. incognita by P. lavendulum, a certain percentage of nematodes died gradually over time (Figure 1) after the co-cultivation of Pur. lavendulum with M. incognita. Through the isolation, purification, and activity assaying of the compounds from the solid fermentation medium, two compounds showed activity against M. incognita. Among them, 7,8-dimethylalloxazine (6) exhibited weak egg hatching inhibitory activity; this is the main photodegradation product of riboflavin under neutral or acidic conditions and is known as an effective photosensitizer [26][27][28]. Some microorganisms and plants also produce 7,8-dimethylalloxazine [29,30] and show various types of activity: 7,8-dimethylalloxazine was isolated from culture filtrates of Chlamydomonas as a quorumsensing signal-mimicking compound capable of activating Pseudomonas aeruginosa LasR receptor [31], and it was also purified and identified from a culture of the bacterium Sinorhizobium meliloti and found to be responsible for stimulating root respiration and plant

Discussion
Root-knot disease caused by M. incognita is a serious concern because it affects several economically important crops globally. Researchers found that Paecilomyces lilacinus (currently valid name: Purpureocillium lilacinum) showed a parasitic effect on root-knot nematodes [6][7][8]. Several experts have conducted numerous studies on the fungus and have confirmed that Paecilomyces lilacinus has control effects on various plant-parasitic nematodes [3,21]. Paecilomyces lilacinus can parasitize the eggs and larvae of important pathogenic nematodes, including root-knot nematodes and cyst nematodes. Paecilomyces lilacinus was subsequently found to disrupt the lipid and chitin layers of nematode eggshells [22]. Additionally, Paecilomyces lilacinus can also produce a variety of biologically active secondary metabolites, including polyketides and non-ribosomally synthesized peptides, such as leucinostatins, which are nematocidal, antiviral, and phytotoxic and possess a series of biological activities [11,23,24]. By comparing the 18S rRNA gene, internal transcribed spacer, and partial translation elongation factor 1-a of the isolates of P. lilacinus from the environment and organisms, the genus Purpureocillium was proposed [4], which includes P. lilacinum, P. lavendulum, and P. takamizusanense [4,25].
While studying the infection of M. incognita by P. lavendulum, a certain percentage of nematodes died gradually over time (Figure 1) after the co-cultivation of Pur. lavendulum with M. incognita. Through the isolation, purification, and activity assaying of the compounds from the solid fermentation medium, two compounds showed activity against M. incognita. Among them, 7,8-dimethylalloxazine (6) exhibited weak egg hatching inhibitory activity; this is the main photodegradation product of riboflavin under neutral or acidic conditions and is known as an effective photosensitizer [26][27][28]. Some microorganisms and plants also produce 7,8-dimethylalloxazine [29,30] and show various types of activity: 7,8-dimethylalloxazine was isolated from culture filtrates of Chlamydomonas as a quorum-sensing signal-mimicking compound capable of activating Pseudomonas aeruginosa LasR receptor [31], and it was also purified and identified from a culture of the bacterium Sinorhizobium meliloti and found to be responsible for stimulating root respiration and plant growth. Moreover, 5-methoxymethyl-1H-pyrrole-2-carboxaldehyde (7), which was isolated from plants and other fungi [32,33], showed multiple activities in the present work: at a low concentration of 5 ppm, the metabolite showed a certain attraction to M. incognita, while at 40 ppm, it had a strong avoidance effect. Lastly, this compound showed nematocidal activity and inhibited egg hatching activity in M. incognita at a concentration of 400 ppm.

Materials
Optical rotations were measured with a Jasco DIP-370 digital polarimeter (JASCO, Tokyo, Japan). Ultraviolet (UV) spectra were recorded on a Shimadzu UV-2401PC spectrophotometer. The nuclear magnetic resonance (NMR) spectra were recorded on Avance III-600 spectrometers, with tetramethylsilane (TMS) as an internal standard. The electrospray ionization mass spectra (ESI-MS) and high-resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on a high-resolution Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).
P. lavendulum YMF1.00683 was isolated from a soil sample in Yunnan. After 8 days of growth on PDA at 28 • C, the hyphae of P. lavendulum YMF1.00683 have covered 6 cm plates, and it is no growth at 35 • C. Colonies consist of dense basal mats composed of many conidiophores and sparse aerial hyphae, and they fold radially toward the periphery. Conidiophores grow from the mycelium 3-4 days at 28 • C, are long stem chains, unicellular, subglobose, apically or lemon-colored at the base, and the clumps are pinkish-purple [34]. P. lavendulum YMF1.00683 was stored in glycerol at −80 • C at the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, China. The YMF1.00683 strain was inoculated in PDA medium and incubated at 28 • C for 6 days, and then was transferred into a fresh medium for 5 days as a seed strain. Meloidogyne incognita was obtained from the roots of tomatoes grown in E'shan County in Yunnan Province.

4.2.
Infection of M. incognita by P. lavendulum P. lavendulum grown on PDA plates for seven days, was harvested by cutting the sample into pieces, shaking it at 100 RPM in 0.1% Tween−80 for 30 min, and filtering it through sterilized four-layer filter paper to obtain a spore suspension. The spores were counted, and their concentration was adjusted to 10 6 mL −1 . A 200 µL aliquot of the spore suspension was dispersed on a cellophane-covered water agar plate, which was then cultured at 28 • C for three days to germinate spores. M. incognita was washed with sterile ddH 2 O, and then approximately 50 M. incognita worms were added to the germinated spore plates. The infection process and number of dead nematodes were observed and recorded at 24, 48, 72, and 96 h.

Extraction and Separation of Metabolites
The mycelium of Pur. lavendulum was cultivated in SD solid medium (10 g tryptone, 40 g glucose, 15 g agar, 1 L water) for 21 days at 28 • C. The culture (30 L) was cut into small pieces and extracted by EtOAc/MeOH/AcOH (80:15:5, v/v/v) 3 times, and then concentrated under reduced pressure to obtain a crude extract. The crude extract was dissolved with 2.5 L of pure water, extracted with EtOAc to obtain the EtOAc crude extract (100.2 g), and then extracted with n-butanol to obtain the n-butanol extract (100.3 g). After LC-MS analysis, both extracts were combined.
The whole crude extract (200.5 g) was placed on a reversed-phase silica gel column    Assays were performed in 24-well cell culture plates. M. incognita egg acquisition [10]: oocysts were picked from the root knots of susceptible tomato and washed of impurities with ddH 2 O, shaken with 0.5% NaClO for 3 min, and then filtered with a 500-mesh sieve to remove NaClO, rinsed with sterile water 3 times, shaken with 2% NaClO solution for 3 min to obtain eggs, and subjected to 10 µL microscopic examinations after completion to prevent excessive cracking. For the second stage juveniles of M. incognita (J2), the obtained eggs were hatched in a 25 • C incubator, and M. incognita (J2) were collected for later use.

Spectroscopic Data
The experimental operation of the effect on the egg hatching of M. incognita [35]: we used sterile water to prepare the egg solution; we prepared a compound solution with methanol sterile water, and the final concentration of methanol was 1%; egg-containing solution and compound solution were added to each well for a final total volume of 500 µL, containing approximately 150 eggs, and the tested compound working concentration was 400 ppm. Sterile water containing 1% methanol was used as a control, and each treatment was repeated thrice.

Assay of Nematocidal Activity against M. incognita
The tested compounds were dispersed in MeOH. Two hundred M. incognita J2s (100 µL) were added to each sample, and the final concentration of the tested compounds was set at 400 ppm. The total and dead nematode numbers were determined every 24 h [36]. The worms were considered dead if they were flat or cracked, and then the nematode mortality was calculated. According to the nematocidal activity strength classification standard, the five-level grading criteria are as follows: inactive, adjusted mortality rate ≤10%; weak activity, adjusted mortality rate of 10% to 30%; moderate activity, adjusted mortality rate of 30% to 70%; strong activity, adjusted mortality > 70%. Avermectin was used as a positive control; the test solution without compound was used as a negative control. Three replicates were conducted for each test.

Assay of Chemotaxis Activity against M. incognita
The nematode chemotaxis assay was conducted for compound 7 using the fourpoint plate method [37]. The 90 mm plate contained 1% agarose for the chemotaxis assay. Compound 7's working concentration was set to 40, 20, 10, 5, 1, 0.5, and 0.01 ppm using 1% methanol sterile water, and sterile water containing 1% methanol was used as a control.