Estrogen Mediates an Atherosclerotic-Protective Action via Estrogen Receptor Alpha/SREBP-1 Signaling

Menopause is associated with dyslipidemia and an increased risk of cardiovascular disease, the underlying mechanism of dyslipidemia is attributed to an insufficiency of estrogen. In this study, we find that estrogen mediates an atherosclerotic-protective action via estrogen receptor alpha/SREBP-1 signaling. Increased lipid accumulation and low-density lipoprotein (LDL)-uptake in HepG2 cells and THP-1 macrophages were induced by treatment of mixed hyperlipidemic serum from postmenopausal women; 17β-estradiol [estrogen (E2)] (10 nM) administration significantly improved hyperlipidemic profiles, relieved fatty-liver damage and attenuated the plaque area in the heart chamber of high-fat diet (HFD)-fed ovariectomized (OVX) ApoE–/– mice. Expression of sterol regulatory element-binding protein (SREBP)-1 mRNA of circulating leukocytes in postmenopausal women was strongly correlated to the serum E2 level. Exploration of data from the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that expression of SREBP-1 protein correlated to expression of estrogen receptor (ESR)α protein in the liver, blood and in normal tissue. Genetic overexpression/inhibition of ESRα resulted in increased/decreased SREBP-1 expression as well as attenuated/deteriorated lipid deposition in vitro. An inhibitor of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway, AZD8055, abolished ESRα-induced SREBP-1 expression in HepG2 cells. Moreover, E2 and statin co-treatment significantly reduced lipid accumulation in vitro and hindered the progression of atherosclerosis and fatty-liver damage in OVX ApoE–/– mice. Collectively, our results suggest that estrogen could exerted its atherosclerotic-protective action via ESRα/SREBP-1 signaling. E2 might enhance the cellular sensitivity of statins and could be used as a novel therapeutic strategy against atherosclerotic disorders in postmenopausal women.

INTRODUCTION
The menopause is an age-related loss of ovarian functions and a decline in gonadal steroids. In premenopausal women, 17βestradiol (E2) is the predominant endogenous estrogen primarily and variably synthesized in the ovaries during the menstrual cycle. Depletion of ovarian follicles in the perimenopausal period leads to a steady decline in E2 production. Compared with premenopausal women, postmenopausal women are more prone to obesity and carry a higher risk of atherosclerotic cardiovascular disease (ASCVD) and have an atherogenic plasma lipid profile (1,2). Specifically, they have greater circulating concentrations of total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), production of very low-density lipoprotein (VLDL) triglyceride, and greater ability to remove VLDL triglyceride (2). Levels of the independent predictors of cardiovascular disease risk, apolipoprotein (Apo)B, ApoA-I and ApoAII, are also increased during menopause (3)(4)(5). These differences in concentrations and subclass distributions of lipids are likely to account (at least in part) for the cardioprotective effect of E2, which was reported be esterified via Lecithin Cholesterol Acyl Transferase (LCAT) function. E2 esters can be carried by HDL particles and could be physiologically relevant with the pathogenesis of atherosclerosis (6).
The effects of menopause are also supported by ovariectomy models in animals. They show that a depletion in circulating E2 levels results in enhanced hepatic lipid accumulation, attenuated inhibition of lipogenesis and inflammation of adipose tissue (7,8), and these effects can be reversed by exogenous administration of E2 (9,10). Understanding E2-caused differences in lipid metabolism and the factors involved in the regulation of lipid kinetics and plasma lipid profile (a major risk factor for cardiovascular disease) is important because there are significant differences in the ways premenopausal and postmenopausal women experience ASCVD. For decades, hormone replacement therapy has been used for the relief of menopausal symptoms for women. Conventional E2 therapy might beneficially affect adiposity and the risk of diabetes mellitus but also a perceived increased risk of breast cancer, as well as an established risk of stroke and venous thromboembolism.
The effects of E2 are mediated by the two nuclear estrogen receptors (ESRs): α and β (11). E2 activates ESRs in target cells, which act as transcription factors to regulate expression of target genes, ultimately controlling cell growth, cell differentiation and homeostasis (12). The liver impacts lipid metabolism in response to E2 signaling. More than 1000 genes display a sex bias in their expression in the liver (13) and the top biological pathways are in lipid homeostasis related to ASCVD (14). Studies using chromatin immunoprecipitation assays have revealed 43 lipid genes to be regulated by ESRα (15,16). However, the relative contributions of E2 signaling through ESRs with regard to lipid metabolism, lipoprotein metabolism and ASCVD were not well defined.
Membrane-bound, basic helix-loop-helix leucine zipper transcription factors are referred to as "sterol regulatory elementbinding proteins" (SREBPs). The latter have a central role in regulating the genes that are important for the biosynthesis and uptake of lipids (17,18). Previously, we found that SREBP-1 expression in circulating leukocytes was downregulated significantly in patients with ASCVD (19). After re-stratification and consultation with participants after that study, we discovered that the decrease in SREBP-1 expression showed an age-related, menopause stage-dependent pattern.
Here, we evaluated the lipid profiles and expression of gonadal hormones and SREBPs in premenopausal, perimenopausal and postmenopausal women. Furthermore, to investigated if E2 signaling regulates lipid metabolism and is protective against the early stages of atherosclerosis, lipid metabolism and arterial plaques were also evaluated in a foam-cell model and in ovariectomized (OVX) ApoE −/− mice. We demonstrated that E2/ESR ligands reduced LDL uptake and lipid accumulation in hepatocytes and foam cells in vitro through a novel non-classical E2/ESR/SREBP-1 signaling pathway, and that E2 treatment enhanced cellular statin sensitivity by perturbing cholesterol metabolism in OVX ApoE −/− mice.
The human hepatic carcinoma cell line HepG2 and monocytic THP-1 were obtained from Shanghai Institute of Cell Bank (Shanghai, China). Human monocytic (THP-1) cells were cultured in phenol red-free medium supplemented with 10% FBS and incubated with 50 ng/mL of phorbol esters (PMA) for 48 h for differentiation into macrophages, THP-1 macrophages and HepG2 cells were maintained in tissue culture dishes (diameter 100 mm) in phenol red-free Dulbecco's Modified Eagle Medium (Fisher Scientific, Waltham, MA, United States) that was supplemented with 10% (v/v) heat-inactivated and charcoalstripped FBS (Fisher Scientific), 1% antibiotics of 50 U/mL penicillin, and 50 µg/mL streptomycin (Invitrogen, Grand Island, NY, United States) at 37 • C and 5% CO2. When the initial cells (1 × 10 5 /mL) became ∼70% confluent, the cells were starved with medium low in serum (0.1% v/v FBS) for 24 h before treatments. HepG2 Cells and THP-1 macrophages were cultured with 10 nM E2 or its vehicle in the presence of 10% hyperlipidemic serum (HS) for 24 h for further staining and LDL-uptake experiments.

Participants
A total of 231 healthy women (21-70 years) were recruited in Taihe Hospital from August 2018 to April 2020. After an overnight fast, participants provided a blood sample, underwent medical examinations and were instructed to complete a self-reported questionnaire which included detailed questions regarding menopausal status. We enrolled women who experienced natural menstrual states. The inclusion criteria were identical to those described previously (20) (Supplementary Data). Determination of menopausal status was based on the responses to questions regarding menstrual irregularity and amenorrhea in the self-reported questionnaire. "Perimenopause" was defined as the presence of menses within the previous 3 months with a reduction in cycle predictability in the year preceding examination, or 3-11 months of amenorrhea. All blood biochemical parameters were detected by standard methods, which were undertaken in the core laboratory of Taihe Hospital.
The study protocol was approved by the Medical Ethics Committee of Taihe Hospital of the Hubei University of Medicine (Hubei, China). Our study was undertaken in accordance with the ethical guidelines of Declaration of Helsinki 1964 and its later amendments.

Preparation of Mixed Hyperlipidemic Serum
We enrolled 26 postmenopausal study participants with hypercholesterolemia. Blood samples were obtained from these women. Serum samples were mixed together and passed through a 22-nm filter (Millipore). The final concentration of biochemicals and hormones was measured and is illustrated in Supplementary Table S1. "hypercholesterolemia" was defined as TC ≥ 6.2 mmol/L and/or LDL-C ≥ 4.1 mmol/L (21).

Transfection of ESRα, SREBP-1
Transient transfection was performed with Lipofectamine 3000 (Lipo3000; Invitrogen, United States) transfection kits. HepG2 cells were cultured with 5% FBS and 10 nM E2 and transfected with ESRα-cloned vector (ESRα Overexpression), controlled vector (pcDNA), and short-hairpin RNAs (shRNAs) according to the manufacturer's instructions. All transfection reagents were synthesized by VectorBuilder Inc. (Guangzhou, China), The specific shRNA sequences used are listed in the Supplementary  Table S3. After 48 h of transfection with lipo3000, transfection effectiveness was evaluated in qRT-PCR and Western Blots.

qPCR and Western Blotting
Total RNA was extracted from cells using TRIzol R Reagent (Invitrogen, CA, United States) according to manufacturer instructions. mRNA expression was quantified by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), ChamQ SYBR qPCR Master Mix (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was regarded as an endogenous reference. PCR were performed by using Bio-Rad CFX Manger system (Bio-Rad Laboratories, Hercules, CA, United States).
Relative gene expression was normalized to that of the housekeeping gene GAPDH and calculated using the formula 2 − CT . The sequences of gene-specific primers are listed in Supplementary Table S2.
To determine the expression of SREBP-1, SREBP-2, ESRα, mTOR, and p-mTOR in THP-1 and HepG2 under different treatment were detected by western blot. The cells were lysed in the cold lysis buffer and the proteins were separated on SDS-PAGE and immunoblotted with indicated antibodies as described previously (22). An enhanced chemiluminescence (ECL) detection system was used to detect immunocomplexes and images were acquired using the ChemiDoc TM Touch imaging system (Bio-Rad Laboratories, Hercules, CA, United States). Detailed qPCR and WB methods were described in Supplementary Method.

Oil Red O Staining
HepG2 Cells and THP-1 macrophages were pretreated with 10 nM E2 or its vehicle in the presence of 10% HS for 24 h. The cells were washed with PBS and then applied to Oil Red O (ORO) staining assay according to the protocol of a commercial kit (Abcam, #133102). Briefly, cells were first washed with PBS and fixed with formalin solution for 15 min. The fixed lipid droplets were then stained with Oil Red O solution for 30 min at room temperature. After washed with PBS for third times, microscope images were taken to visualize red oil droplets staining in differentiated cells (Leica, Wetzlar, Germany). ImageJ (National Institutes of Health) was used to measure the mean staining area intensity (23).

Low-Density Lipoprotein-Cholesterol Uptake Assay
Low-density lipoprotein-cholesterol uptake was determined using an LDL Uptake Assay Kit according to manufacturer (133127; Abcam) instructions as described previously (22) and detailed methods were described in Supplementary Method S1.4.

Immunofluorescence Staining of Cells
Cells were seeded into six-well plates. The next day, cells underwent different treatments for 24 h followed by fluorescence staining according to standard methods as described previously. Immunofluorescence images were visualized and photographed using a laser scanning confocal microscope (FV3000RS; Olympus, Tokyo, Japan). ImageJ (National Institutes of Health) was used to measure the mean fluorescence intensity (23) (Supplementary Method S1.5).

Surgical Model of Menopause and Atherogenesis in Mice
Female ApoE −/− mice (5 weeks) were purchased from Biocytogen (Nanjing, China). Mice were maintained in a temperature-controlled room and exposed to a 12-h light-dark cycle. After 3 weeks of acclimatization, mice were randomized to undergo bilateral ovariectomy after the induction of anesthesia (1% pentobarbitone). Seven days after surgery, mice were stratified into three groups of five in the Part 1 and stratified into four groups of eight in Part 2. E2 and Rosuvastatin were dissolved in physiologic saline and administered into mice by intragastric administration. The diagrams of animal research in part 1 and part 2 were shown in the Supplementary Method S1.6. Animal experiments followed the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, United States) and their conduct was approved by the Animal Care and Use Committee of Hubei University of Medicine.

Assessment of Aortic Atherosclerotic Lesions
After the treatment, mice were anesthetized with Pentobarbital sodium salt (1% pentobarbitone). The liver, heart, and arterial tree were dissected carefully and were incubated with physiologic saline and fixed in 4% formalin, then dehydrated with sucrose gradient. Tissues were embedded in Tissue Freezing Medium and serially sectioned at 8 µm using a Thermo cryostat (Thermo Scientific, Waltham, MA, United States). To quantify the dimensions of atherosclerotic lesions, 10-12 sections were stained with ORO and counterstained with hematoxylin. Images were captured with Leica CS2 and lesion areas were quantified with ImageJ (23).

Statistical Analyses
The continuous variables are expressed as mean ± standard deviation (SD) or as median (inter-quartile range), as appropriate, based on distributions. Continuous variables were compared by the student's t-test or Mann-Whitney U test. Correlation analysis between blood lipids levels and E2 levels was performed using the non-parametric Spearman's rank correlation test. Student's t-test was used to evaluate the significance of pairwise in differences. All statistical significance levels were set at p < 0.05 (two-sided) and all statistical analyses were performed using SPSS version 23.0 (SPSS Inc., Chicago, IL, United States).

The Serum E2 Level Is Negatively Correlated With Levels of Total Cholesterol and Low-Density Lipoprotein-Cholesterol in Postmenopausal Women
The basic clinical characteristics of 231 women (80 premenopausal, 20 perimenopausal, 131 postmenopausal) are shown in Supplementary Table S4. As shown in Figure 1, serum E2 levels were negatively correlated with TC (r = −0.240, P = 0.006) ( Figure 1AIII) and LDL-C (r = -0.239, P = 0.006) levels ( Figure 1BIII) in postmenopausal women. Moreover, the serum level of E2 was negatively correlated with the LDL-C level (r = -0.134, P = 0.042) ( Figure 1BIV) in the total study cohort.

Effects of E2 on Hyperlipidemic Serum-Treated Cells and Ovariectomized ApoE −/− Mice
In postmenopausal females, circulating levels of hormones change dramatically because ovarian function begins to decline; E2 secretion decreases gradually and the homeostasis of lipid kinetics is disrupted (24). We wished to build an in vitro foamcell environment to mimic the postmenopausal state. Hence, we used mixed HS from postmenopausal women to treat THP-1 cells. As shown in Figures 1C-E, 10% HS treatment increased intracellular lipid deposition (P < 0.001), LDL-uptake (P < 0.001), LDL-receptor expression (P < 0.001) in HepG2 cells and THP-1 macrophages, compared with those in the control groups. Furthermore, these effects were attenuated by treatment with E2 (10 nM).
We used HFD-fed ApoE −/− mice that had undergone ovariectomy as a comorbidity model of postmenopausal status and atherosclerosis in women on the basis that E2 deprivation by OVX and HFD consumption in ApoE −/− mice induces dyslipidemia and atherosclerosis. HFD consumption increased the bodyweight (Supplementary Figure S1), serum lipid level (Supplementary Figure S2), hepatic lipid accumulation ( Figure 1F) and plaque area in heart chambers ( Figure 1G) in OVX mice compared with that in OVX mice fed a normal-fat diet. Administration of E2 (10 nM) had no effect on bodyweight or serum levels of LDL-C, TG, or TC (Supplementary Figure S1). However, ORO staining in the liver and hematoxylin and eosin (H&E) staining in the heart revealed that E2 (10 nM) attenuated the effect of HFD induced-fatty liver damage and atheroscleroticplaque formation in OVX mice (Figures 1F,G).

SREBP-1 Mediates the Effect of E2 on Foam-Cell Formation
Accumulating evidence suggests that reprogramming of fattyacid metabolism modulates lipid disorders in cardiovascular disease. To investigate if E2 influenced lipogenesis during foamcell formation, we examined the effects of HS on expression of SREBP-1 (the master transcriptional regulator of fatty-acid biosynthesis). First, we examined the correlation between serum E2 levels and SREBP-1 mRNA expression in leukocytes in female participants with different physiological status (Figure 2A). Expression of SREBP-1 mRNA was positively correlated with the E2 level in postmenopausal women (R = 0.279, P = 0.001) as well as in all female participants (R = 0.353, P < 0.001). Second, we used different concentrations of HS to treat THP-1 cells. HS pretreatment for 24 h increased expression of the cleaved form of SREBP-1 (n-SREBP-1) (Supplementary Figure S2). Interestingly, expression of the pre-form of SREBP-1 (pre-SREBP-1) was not affected by pretreatment with HS, which suggest that selective SREBPs signaling was involved in foam-cell formation. Notably, treatment with 10% HS led to maximal foam-cell formation and SREBP-1 expression (Supplementary Figure S2).
Furthermore, given that n-SREBP-1 is the mature form of SREBP, which can regulate gene expression, we next investigated the effect of E2 on n-SREBP-1 expression. We found that E2  The cells were then subjected to microscopic analysis for detection of LDLR expression, which are indicated by green light. Total area of LDLR expression normalized to cell number (n = 10 cells/field/slice, n = 3 slices/group) was determined using Image J software and shown in (EII). (F) Eight-week-old ApoE −/− mice were subjected to ovariectomy. Seven days later, they were randomized to three groups: normal diet (NF) group (n = 5), high-fat diet (HFD) group (n = 5), or HFD + E2 (1 µg/kg/day) administration group (n = 5). Treatments continued for 20 weeks until euthanasia. After euthanasia, liver tissue were prepared as rapid freezing section and subjected to ORO staining for detection of lipids accumulation. Representative images of liver ORO staining are shown (FI) (n = 3 sections/mouse/group, n = 5 mice/group). Densitometric quantification was conducted using Image J and values are expressed as the fold change compared with control and are presented as mean ± SEM (n = 5 mice/group). (G) Heart tissue were prepared as rapid freezing section and subjected to H&E staining for detection of plaque area of the three groups of mice (n = 3 sections/mouse/group, n = 5 mice/group). Densitometric quantification was conducted using Image J and values are expressed as the fold change compared with control and are presented as mean ± SEM (n = 5 mice/group). Student's t-test was used to evaluate the significance in differences between two groups of observations, * P < 0.05; * * P < 0.01; * * * P < 0.001. E2, 17β-estradiol; HS, hyperlipidemic serum from postmenopausal women; LDL, low-density lipoprotein; LDLR, LDL receptor; OVX, ovariectomized; TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; ORO, Oil red O; OVX, ovariectomy.
promoted n-SREBP-1 expression which was induced by HS pretreatment in HepG2 and THP-1 macrophages (Figures 2B-E). Moreover, we examined the cellular localization of SREBP-1 by immunofluorescence assay before and after HS treatment (Figures 2F,G). At baseline, most SREBP-1 was localized to the nucleus in HePG2 cells while its immunoreactivity was very weak in THP-1 cells. Upon addition of 10% HS, we observed a significant increase of immunoreactivity in HePG2 cells and THP-1 cells; most SREBP-1 was localized to the cytoplasm in HePG2 cells while most SREBP-1 was localized to the nucleus in THP-1 cells under HS treatment. Furthermore, supplementation with E2 enhanced the overall intracellular expression of SREBP-1 (Figures 2F,G). Interestingly, variation in the nucleus-tocytoplasmic immunoreactivity (NC ratio) of SREBP-1 which was induced by E2 in the two cell types was not consistent (Figures 2F,G). The NC ratio of SREBP-1 was decreased in HepG2 cells upon E2 treatment, but the NC ratio was increased in THP-1 cells upon E2 treatment. These findings uncovered the dynamically integrated and finely tuned lipid-metabolism signaling of E2.

E2 Influenced ESRα Expression
ESRα is a nuclear hormone receptor that governs expression of the target genes involved in numerous metabolic pathways, including those guiding mitochondrial oxidative phosphorylation and lipid metabolism (25). Postmenopausal women had reduced E2 levels and decreased ESRα expression, so we speculated that E2 supplementation may restore expression of ESRs and potentially mediate the regulatory effects of E2. Treatment with 10% HS enhanced the immunoreactivity of ESRα in THP-1 cells and HepG2 cells, in particular the mean fluorescence intensity in the nucleus was increased dramatically (Supplementary Figure S3), which implied that E2 transcription activated ESRα. Furthermore, E2 treatment increased expression of ESRα protein in HepG2 cells and decreased expression of ESRα protein slightly in THP-1 cells.

SREBP-1 Mediates the Lipid Regulatory Effect of E2 via ESRα
ESRα is a targeted receptor of E2, treatment with 10% HS enhanced the immunoreactivity of ESRα in THP-1 cells and HepG2 cells, in particular the mean fluorescence intensity in the nucleus was increased dramatically (Supplementary Figure S3), we postulated that E2/ESRα might regulates SREBP-1 expression. Based on the GEPIA database, we found that SREBP-1 expression was strongly correlated with ESRα expression in the liver, blood as well as in TCGA (The Cancer Genome Atlas) normal tissue (Supplementary Figure S4). We explored the pattern of SREBP-1-expression variation by manipulating ESRα expression. ESRα overexpression caused a significant increase in mRNA ( Figure 3A) and protein expression of ESRα and n-SREBP-1 ( Figure 3B). Correspondingly, knockdown of ESRα expression with shRNA resulted in downregulated mRNA and protein expression of ESRα and SREBP-1. The immunofluorescence analysis of SREBP-1 by manipulating ESRα expression in THP-1 and HepG2 were consistent with the WB results ( Figure 3C). Moreover, to assess the role of SREBP-1 in E2/ESRα-mediated LDL uptake and lipid accumulation in foam cells, E2 + HStreated HepG2 cells were overexpressed and/or depleted of ESRα/SREBP-1 expression. ESRα overexpression significantly attenuated lipid accumulation and LDL-uptake effects in HepG2 cells that had been stimulated by HS treatment; depletion of ESRα with shRNA restored those effects (Figures 3D,E). Silencing SREBP-1 expression also abolished the inhibition of lipidaccumulation effects of ESRα overexpression, which suggested that the E2/ESRα/SREBP-1 regulatory pathway was involved in the E2-related lipid-metabolism mechanism.

ESRα Regulated SREBP-1 Activity Partly via the mTOR/p-mTOR Pathway in HepG2 Cells
Over recent years, several studies have revealed that mammalian target of rapamycin complex 1 (mTORC1) has a crucial role in promoting lipid biosynthesis by activating SREBP-1 (26,27). To assess if E2/ESRα-activated SREBP-1 expression was mediated by mTORC1, we treated ESRα-overexpressed cells with a mTORC1 inhibitor (AZD8055) for 24 h. ESRα overexpression did not affect expression of mTOR or p-mTOR in two cell lines (Figures 4F,G). AZD8055 treatment decreased p-mTOR protein expression in HepG2 cells and THP-1 foam cells, and it decreased n-SREBP-1 expression in HepG2 cells (Figures 3F,G and Supplementary Figure S5), which suggested that ESRα . Expression and location of SREBP-1 and nuclear SREBP-1 detected by immunofluorescence (FI,GI) (n = 1 cells/field, n = 5∼8 fields/slice, n = 3 slices/group), the relative expression intensity in nucleus vs in cytoplasm were illustrated in (FII,GII). Data illustrated on the bar graph are the mean ± SD. All representative images presented were repeated in three independent experiments. Student's t-test was used to evaluate the significance in differences between two groups of observations, * P < 0.05; * * P < 0.01; * * * P < 0.001. (G) Liver tissue were prepared as rapid freezing section and subjected to ORO staining for detection of lipids accumulation. Representative images of liver ORO staining are shown (GI) (n = 3 sections/mouse/group, n = 8 mice/group). Densitometric quantification was conducted using Image J and values are expressed as the fold change compared with control and are presented as mean ± SEM (n = 8 mice/group). Student's t-test was used to evaluate the significance in differences between two groups of observations, * P < 0.05; * * P < 0.01; * * * P < 0.001. and THP-1 cells were subsequently cultured with or without p-mTOR inhibitor AZD8055 (20 nM) for 48 h, cells were harvested for western blotting, representative blotting images (FI,GI) and quantification (FII,GII) of mTOR, p-mTOR, pre-SREBP-1 and n-SREBP-1, and ESRα. Data illustrated on the bar graph are the mean ± SD. All representative images presented were repeated in three independent experiments. Student's t-test was used to evaluate the significance in differences between two groups of observations, * P < 0.05; * * P < 0.01; * * * P < 0.001. regulated SREBP-1 activity (at least in part) via the mTOR/p-mTOR pathway in hepatocytes.

E2 Enhanced Cellular Sensitivity to Statins in vivo and in vitro
Statins are competitive inhibitors of 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR). Statins are a class of lipidlowering medications that reduce illness and mortality in those who are at high risk of cardiovascular disease. We wished to assess the relationship between inhibition of HMGCR expression and E2 regulation in postmenopausal atherosclerotic disease caused by a combination of statins and E2. Hence, E2 (2 nM) or its vehicle alone, or combined with rosuvastatin (5 µM), were used to treat HS-stimulated THP-1 foam cells. Compared with the vehicle group, treatment with E2 and rosuvastatin decreased lipid accumulation in foam cells ( Figure 4A). Noticeably, the combination of E2 with rosuvastatin dramatically reduced lipid accumulation in THP-1 foam cells when compared with that in the vehicle group or monotherapy group (P < 0.01). We wished to investigate the effect on hepatic lipid disposition and formation of atherosclerotic plaques in arteries. In vivo, E2 (0.25 mg/kg/day) combined with rosuvastatin (5 mg/kg/day) or an equal volume of saline solution were used to treat normal diet-fed OVX ApoE −/− mice. After 12 weeks of treatment, supplementation with E2 alone (0.25 mg/kg/day) did not affect bodyweight or lipid profiles (Figures 4B-D) in contrast with the HFD-fed saline-treated OVX group. Noticeably, therapy with rosuvastatin or a combination of E2 and rosuvastatin resulted in reduced bodyweight as well as decreased plasma levels of TC and LDL-C in HFD-fed OVX ApoE −/− mice when compared with those in the HFDfed saline-treated OVX group (Figures 4B-D). Rosuvastatin significantly increased serum levels of aspartate transaminase and alanine aminotransferase; E2 and rosuvastatin co-treatment restored these effects, which suggested that E2 relieved statincaused hepatic damage ( Figure 4C). Administration of E2 or rosuvastatin alleviated aortic-root plaque formation and relieved lipid deposition compared with that in saline-treated OVX controls (Figures 4E-G). Remarkably, combined treatment of E2 and rosuvastatin enhanced these cardioprotective effects. In contrast to the monotherapy group (E2 or rosuvastatin), combined treatment resulted in less fatty-liver damage and a smaller plaque size in the aortic root.
Taken together, these results implied that E2 enhanced cellular sensitivity to statins in vivo and in vitro. Combination of E2 and statins might have synergistic cardioprotective effects by regulating lipid metabolism.

DISCUSSION
The metabolic effects of E2 range from weight gain to the development of metabolic syndrome, and are associated with cardiovascular and hepatic manifestations. Studies on animal models of atherosclerosis have provided compelling evidence that physiological E2 levels potently attenuate the early and advanced stages of atherosclerotic lesions in females.
Atheroprotection by E2 in atherogenic transgenic mouse models with targeted inactivation of the LDL receptor (28) and ApoE (29,30) has been demonstrated. In these models, E2 hindered atherosclerosis development. E2 deficiency induced by fulvestrant in female rats resulted in weight gain as well as disturbed metabolism of glucose and lipids (31). Consistent with those findings, we found that E2 supplementation attenuated lipid accumulation and LDL-uptake in cholesterol-loaded THP-1 macrophages (foam cells) and HepG2 cells. Moreover, E2 administration decreased fatty-liver damage and relieved plaque formation in the heart chambers of OVX ApoE −/− mice. Mennatallah et al. (31) reported that an OVX rat model was associated with dyslipidemia as demonstrated by significant increases in levels of TG, TC, and LDL-C as well as a significant decrease in the HDL-C level. In our study, the LDL-C level was negatively correlated with the serum E2 level in postmenopausal women but, in the OVX mouse model, inhibition of atherosclerotic lesions using exogenous E2 (supplementation for 12 and 20 weeks) did not influence serum lipid profiles or bodyweight. A likely possibility is that continuous therapy in a mouse model was different with circulating E2 levels in humans; physiological rhythmic release of hormones may have a more comprehensive impact upon metabolism kinetics.
In general, E2 exerts its physiological effects through ESRα and ESRβ. Functional ESRs are expressed in macrophages (32), vascular endothelial cells (33), and smooth muscle cells (34). Expression of ESRs has been reported to be downregulated in the atherosclerotic vessels of postmenopausal women (35). In a combined ApoE/ERα deletion model, ERα was reported to be a major mediator of E2 protection in advanced atherosclerotic lesions (36), and ERα deficiency abolished the atheroprotection effect of E2 in ApoE −/− mice (36). However, the relative contribution of E2 signaling through ESRα with regard to the metabolism of lipids and lipoprotein and ASCVD is not known.
SREBP-1 is a transcription factor expressed ubiquitously. It is an adipocyte-differentiation factor which directly regulates the transcription of > 200 genes involved in the de novo synthesis of fatty acids, TG and cholesterol. The phenotypes of published germline and tissue-specific knockout/transgenic mice for the SREBP pathway have revealed the potential of SREBP-1 to reduce fatty-acid synthesis, enhance insulin secretion, and alter the inflammatory response in the liver, intestine, pancreas, and immune system. Previously, we discovered that decreased SREBP-1 expression was a risk factor for coronary artery disease (CAD), and that expression of SREBP-1 mRNA in carotid plaques correlated with the corresponding value in circulating leukocytes in CAD patients undergoing carotid endarterectomy (19). In the present study, after stratification by various clinical parameters, we found SREBP-1 expression to correlate with the serum E2 level. Expression of ESRα protein strongly correlated with expression of SREBP-1 protein in the liver, blood and TCGA normal tissue based on the GEPIA database; moreover, manipulating ESRα expression simultaneously led to alteration of n-SREBP-1 expression. Pedram et al. showed that activation of membrane-localized ESRα by the E2 agonist prolyl-pyrazoletriol could induce adenosine monophosphate-activated protein kinase to phosphorylate SREBP-1 in the liver, thereby preventing its proteolytic cleavage by site-1 protease (37). Consequently, SREBP-1 was sequestered in the cytoplasm, thereby preventing expression of cholesterol synthesis-associated genes (37). In the present study, under standard culture conditions, SREBP-1 was localized to the nucleus in HePG2 cells, and hyperlipidemic or E2-hyperlipidemic co-treatment significantly increased the ratio of cytoplasm expression:nucleus expression of SREBP-1. As a critical lipid metabolism-related transcription factor, nuclear SREBP-1 regulates expression of target genes. Retention of cytoplasmic SREBP-1 might lead to repressed expression of cholesterol-and lipid synthesis-related genes that result in decreased accumulation of intracellular lipids in the liver. Interestingly, the pattern of SREBP-1 expression in THP-1 macrophages was different from that in HepG2 cells. Barely expressed under normal conditions, hypercholesterolemia stimulation dramatically increased its nuclear expression, whereas E2 and hyperlipidemic co-treatment increased its cytoplasmic retention. These data suggested that E2 might work as a "fine tuning" regulator to maintain lipid homeostasis during foam-cell formation. Therefore, we speculated that E2/ESRα might regulate lipid homeostasis directly/indirectly via SREBP-1 signaling. Subsequently, we globally identified the promoters occupied by ESRα in human hepatocyte lines (HepG2, LO2, LM3) using chromatin immunoprecipitation sequencing, but, the ESRα-binding DNA sequence was exclusive of the SREBP-1 promoter region. AKT/mTOR has been reported to be an upstream SREBP-1 regulating pathway which can modulate hepatic lipid kinetics by affecting COPII-dependent SREBP-1 processing (38), and mTOR is an essential regulator of lipogenic metabolism because it activates SREBP-1 cleavage (39). In the present study, an inhibitor of the AKT/mTOR pathway, AZD8055, partly abolished ESRα-induced SREBP-1 expression in HepG2 cells, which implied that SREBP-1 activation by ESRα could be related to the mTOR pathway. However, inhibition of p-mTOR did not affect SREBP-1 expression in THP-1 foam cells. The reciprocal regulation between ESRα and SREBP-1 is complex, and further research is needed to understand the regulatory mechanism between these two factors.
Our previous case-control study uncovered that downregulated SREBP-1 expression in peripheral leukocytes was an independent risk factor for CAD. However, those effects seem to contradict with the data that treatment with 10% HS upregulated SREBP-1 expression and increased lipid accumulation in foam cells. Overexpression of SREBP-1c protein has been reported to lead to disturbances in the metabolism of carbohydrates and lipids, and hepatic lipid content (40). In OVX rats, enhanced hepatic expression of SREBP-1c may be a cause of excessive hepatic lipid production through subsequent activation of fatty acid synthase (FASN). Whether an increase in SREBP-1 expression is a protective factor or a risk factor for CAD is influenced by two factors. On the one hand, estrogen-induced SREBP-1 expression regulates lipid metabolism with the aim of restoring lipid homeostasis. On the other hand, abnormal alterations in lipid levels can feedback and adjust SREBP-1 expression. Therefore, upregulated expression of SREBP-1 seems to related to irregular lipid profiles. The interactive mechanism between SREBP-1 expression and lipid metabolism merits further exploration.
SREBP-1 can produce two proteins, SREBP-1a and SREBP-1c, which are derived from different promoters (41). In humans, SREBP-1c primarily regulates fatty-acid metabolism, such as FASN (42,43). SREBP-1a, with 1147 amino acids, is 47% identical to SREBP-2 in humans. The transcriptional activity of SREBP-2 corresponds to SREBP-1a on account of the almost identical length of the N-terminal domain. SREBP-2 is mainly responsible for cholesterol-related genes, such as HMGCR (a ratelimiting enzyme in cholesterol synthesis) and the low-density lipoprotein receptor gene (44). SREBP-1a targets both sides of genes. However, there are overlapping functions between individual SREBPs.
Statins are the most commonly used class of drugs to treat and prevent CAD worldwide. Statins strongly induce HMGCR via a SREBP-2 pathway (45). If statins bind to HMGCR, they block access of the natural substrate HMG-CoA to the catalytic site and thereby interfere with cholesterol synthesis. On the basis that E2 regulated SREBP-1 expression, we speculated that E2 might influence the cellular sensitivity of statins by cross-regulating SREBPs. E2 and statin co-treatment significantly decreased lipid accumulation in vitro, after HFD consumption for 12 weeks, administration of E2 and a statin together significantly relieved blood-lipid disorders and hindered progression of atherosclerosis and fatty-liver damage in OVX ApoE −/− mice. This observation has an important translational implication because clinical studies exploring E2 supplementation as atheroprotection agents have been hindered by the increased risk of complications of high-dose E2 treatment in postmenopausal women. Investigation of the utility of low-dose, E2-based combination treatment against CAD is needed.
Amparo and colleagues also discovered that E2 inhibits and delays the development of early lesions via ERα-independent mechanisms because they found no significant interaction between the ERα genotype and hormone treatment or hormone status (46). The differences between our findings and their observations might be because the pleiotropic effects of hormone therapy on the vascular system and cells might differ depending not only on hormone status, but also on the atherosclerosis stage in the underlying physiological condition. We need to know to what extent and how the cessation of endogenous E2 production affect ESR expression/function in the regulatory network of lipid kinetics and thereby enhances/attenuates their pro-or anti-atherogenic effects.

CONCLUSION
The present study aids deeper understanding of the ESRα/SREBP-1-dependent mechanisms of E2 linked to cholesterol metabolism in postmenopausal women. We also provide evidence that E2 enhances statin sensitivity which might have synergistic anti-atherosclerotic effects in the early stages of cardiovascular lesions and mild hepatic injury. This knowledge is potentially important because deeper understanding of this system may lead to stronger comprehension of how to optimize hormone therapies for protection against cardiovascular disease in postmenopausal women.

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

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Medical Ethics Committee of Taihe Hospital of the Hubei University of Medicine. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Animal Care and Use Committee of Hubei University of Medicine.

AUTHOR CONTRIBUTIONS
CP: concept, funding, and critical review. FX and CP: design and writing. XLi, YX, and DC: materials. XX, XLv, and GY: data collection and/or processing. FX and CP: analysis and/or interpretation. FX: literature search. All authors contributed to the article and approved the submitted version.


s2fieldsofstudy: Biology Chemistry

text:
Protection of a Gold Catalyst by a Supramolecular Cage Improves Bioorthogonality

Gold catalysts exhibit poor compatibility with cellular components. We show that encapsulation of a gold catalyst within the cavity of a supramolecular cage improves the reactivity of the gold complex under biological conditions. The gold complex catalyzes an intramolecular hydroarylation to produce a fluorescent dye. The encapsulated gold is able to produce this dye in higher yields compared to the free gold under aqueous aerobic conditions and in the presence of biological additives. The substrate was found to be highly cytotoxic, meaning that a very low substrate concentration of 1 μM is required to carry its transformation inside living cells; however, catalysis in cell culture media carried out at micromolar range is found to be inhibited. Although this specific reaction cannot be applied inside living cells, we present a viable strategy to improve the reactivity of gold catalysts in vivo.

Introduction
The selective activation of prodrugs and medicines is highly desirable, as it provides the ability to obtain precise spatial and temporal control over their activity which may minimize offtarget reactivity. [1] This is particularly relevant for cancer treatment, where system-wide toxicity goes hand in hand with cancer cell toxicity, often resulting in severe side effects. [2] In order to achieve such selectivity, a bioorthogonal trigger is needed to activate the drug, which should selectivity occur at the desired location. In addition, this trigger should be inert in the off-target cells and should not influence other endogenous processes within the cell. One strategy to accomplish is to design prodrugs which are activated by new-to-nature transformations. Transition metal catalysis is a powerful tool to achieve this as it provides access to a wide scope of reactions which cannot be performed by native enzymes. [3] Gold catalyzed transformations are attractive for this purpose, as they are known to undergo bioorthogonal transformations. For example, gold complexes have been shown to promote CÀ S cross coupling reactions in biological systems, whereby aryl groups were conjugated to cysteine residues in bacterial proteins. [4] In addition, they provide routes to synthesize cyclic compounds, a scaffold commonly found in medicines, from alkynes. [5] Alkynes are known to have good bio-orthogonality as they are generally not found naturally in the cell environment, which decreases the chances of non-desired reactivity. [6] Gold catalyzed cyclizations have already been performed in vivo; however, the activity of the gold catalyst itself is typically very low. The first example of a gold catalyzed intramolecular hydroarylation was shown by Mascareñas and coworkers. [7] Although they showed that only 10 mol% of a small gold catalyst, [Au(PTA)Cl], could successfully convert the substrate to generate a fluorescent coumarin derivative in water with almost quantitative conversion, when the reaction was applied inside HeLa cells the authors estimated a turnover number (TON) of just 1.12. As accurate quantification of catalytic reactions in vitro is challenging, the authors emphasized that this TON should only be taken as an approximation. Yet, it is clear that the cellular environment has a considerable detrimental effect on the activity of the gold complex. The root of this problem is the high concentrations of strongly goldbinding thiol-containing biomolecules, such as glutathione (GSH), present in the cellular environment. [7] A need to protect the gold catalyst from the cellular environment prompted Tanaka et al. to dock an NHCÀ Au(I) complex inside an albumin scaffold, to generate an artificial metalloenzyme. Indeed, good conversions to generate an anticancer compound were achieved when the gold complex was docked inside the hydrophobic cavity of the protein, whereas the free complex was readily poisoned by thiols and therefore exhibited almost no reactivity. As such, the hydrophilic GSH molecules apparently cannot reach the complex when it is inside the hydrophobic cavity of the protein. In A549 cells it was shown that while high concentrations of gold complex were needed to synthesize the cytotoxic compound and induce cell death, much lower concentrations of metalloenzyme were needed to achieve the same levels of cytotoxicity, indicating that protection of the gold catalyst with the protein scaffold can prevent catalyst poisoning and improve reactivity. [8] This incompatibility of gold complexes with the cellular environment runs both ways. Cellular biomolecules have a detrimental effect on the catalytic activity, but gold complexes can also harm the cells by inducing toxicity themselves. Zou et al. therefore developed a system where an inactive and non-toxic gold complex was first applied in cells. This gold complex was activated by a palladium species to become cytotoxic and catalytically active for an intramolecular hydroarylation reaction to synthesize a fluorescent coumarin derivative. This dual activity of the gold complex was demonstrated in both A549 cells and in zebrafish. [9] Considering the poor compatibility of gold catalysts with the components in living cells, we aimed to design a simple system whereby protection could be provided for both the gold catalyst from the cell environment and the cell from the gold complex. To this end we looked for ways to encapsulate the gold complex in a supramolecular cage, eventually carrying out gold catalysis within the cavity of a cage. The molecular capsule should provide a physical barrier between the gold center and the surrounding environment, leading to improved compatibility. In previous work, gold complexes have been encapsulated with the aim to improve their catalytic properties in terms of activity and selectivity. Reported benefits of confinement on catalysis have been due to: 1) pre-organization of substrate and catalyst; [10] 2) positive effects of increased local concentration of catalysts; [11] 3) cage effects that control the selectivity; [12] and 4) stabilization of the catalyst by the cage. [13] Although there are several examples of gold catalysts being encapsulated in a range of different supramolecular cages, [14] we selected the small tetrahedral K 12 [Ga 4 L 6 ] cage developed by Raymond and coworkers for the current goal ( Figure 1). [15] Once encapsulated inside this cage, catalysts are known to participate in size-selective catalysis, where only substrates small enough to fit through the window are able to react. [12c] With this in mind, we hypothesized that large biomolecules may be stopped from reaching the gold center, while small substrate molecules may still access the active site. In addition, a gold catalyst, [Au(PMe 3 )] + , has been shown to bind inside the cavity of the cage. The encapsulated gold complex exhibited improved activity compared to the free complex, [16] and has also been shown to participate in a tandem reaction with enzymes, indicating its biocompatibility. [17] Here we explore the use of encapsulated gold complexes for catalysis applications under biological conditions in living cells.

Approach
The tetrahedral K 12 [Ga 4 L 6 ] cage (see Figure 1A) was prepared according to a literature procedure, and spectroscopic data were identical to those reported by Raymond et al. [16] Preparation of the encapsulated gold complex, [Au(PMe 3 )] + �cage, was achieved by mixing 1.2 eq of the cage with 1 eq of the commercially available gold catalyst, [Au(PMe 3 )Cl]. The catalytically active cationic gold species [Au(PMe 3 )] + resides within the cavity, as the highly negative charged capsule is able to encapsulate cationic guests due to electrostatic interactions. For reactions with free [Au(PMe 3 )Cl], no chloride-abstracting reagents were required as it is known that the chloride is able to dissociate from the gold center in water. [7] Catalyst encapsulation in the aqueous phase was established by 1 H NMR spectroscopy and 2D 1 H diffusion ordered NMR spectroscopy (DOSY). Two new overlapping doublets appeared in the 1 H NMR spectrum ( Figure S1) at À 1.85 and À 1.89 ppm which correspond to the phosphine alkyl protons of [Au(PMe 3 )Cl], and these peaks are shifted upfield with respect to those of free [Au(PMe 3 )Cl], which would appear at 1.64 ppm. Besides this, all the aromatic peaks belonging to the empty cage appear between 7.80 and 6.52 ppm, but in the presence of [Au-(PMe 3 )Cl] they are all shifted downfield to between 7.84 and 6.57 ppm. In addition, the DOSY spectrum ( Figure S3) had only one band with a logD value of À 9.62, indicating that only one species is diffusing in solution. Together these data are characteristic of encapsulation of the gold complex within the cavity of the cage. In order to monitor catalytic conversions in the highly complex environment of the cell, typically substrates are used that become fluorescent upon product formation. [18] However, many fluorophores contain large aromatic groups in their structure, which may be too large to fit through the small windows of the cage (see supplementary information). We therefore designed a small alkyne-containing substrate, 1, which upon a gold catalyzed hydroarylation reaction generates a coumarin dye, 2 a. As a by-product, a non-fluorescent regioisomer 2 b can also be formed ( Figure 1B). Substrate 1 was prepared using standard synthetic steps from commercially available building blocks (see supplementary information). To successfully demonstrate that catalysis can be performed in living cells, we need to: 1) explore the cytotoxicity of the substrate, catalyst, and product, which will set the boundary conditions for catalysis; 2) demonstrate that the caged catalyst enters the living cell in order to perform the catalytic transformation; and 3) explore the compatibility of the catalyst system with commonly abundant components in living cells. In the following sections these will be discussed.

Cytotoxicity and catalyst conditions
Before catalysis can be carried out, appropriate reaction conditions need to be found. The reaction conditions for transition metal catalysis inside cells is often vastly different to that of catalytic reactions for synthetic purposes. While catalysis reactions for synthesis typically are carried out at millimolar to molar concentrations of substrate, much lower concentrations are typically required for catalysis in cells. Ideally, the reaction components for catalysis should induce as little cytotoxicity as possible, in order to ensure that the reaction mainly takes place inside of living cells, rather than extracellularly in the presence of dead cells. Therefore, the biocompatibility of the reaction components with living cells was first investigated in order to determine the appropriate reaction conditions for intracellular catalysis. To this end, the viability of human cervical cancer cells (HeLa cells) was determined in the presence of substrate 1, product 2 a, the free [Au(PMe 3 )Cl], the empty cage, and the encapsulated [Au(PMe 3 )] + �cage, using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay ( Figure 2). After HeLa cells were incubated with increasing concentrations of the compounds, the percentage of cells which remained alive compared to before incubation with the compounds, was determined. The relative cytotoxicity of the different compounds was determined by comparing the cell viability in the presence of each of the compounds at a given concentration. Substrate 1 was found to induce very high levels of cytotoxicity: at only 10 μM, the cell viability was just 23 %. In order to minimize cytotoxicity induced by substrate 1, it should be applied in very low concentrations for catalysis, ideally at just 1 μM, as at this concentration a cell viability of 74 % was observed. The cell viability of product 2 a at concentrations above 10 μM was higher than that for substrate 1, and below these concentrations the cell viabilities of substrate 1 and product 2 are similar (67-74 % and 57-53 % respectively). This means that any product which would be formed during catalysis would not induce any significant cytotoxicity at this concentration. Both of the gold complexes were also found to induce less cytotoxicity than the substrate. Gratifyingly, the encapsulated [Au(PMe 3 )] + �cage complex exhibited reduced cytotoxicity compared to the free [Au(PMe 3 )Cl]: 79 % cell viability was observed for [Au(PMe 3 )] + �cage at 50 μM, while at the same concentration for free [Au(PMe 3 )Cl] the cell viability was only 59 %. The cage itself was essentially non-cytotoxic, even at cage concentrations up to 500 μM. Although the cytotoxicity of the gold complex was reduced by encapsulation within the cage, the lack of cytotoxicity across all concentrations of cage brought into question the cell membrane permeability of the cage. The [Au(PMe 3 )] + �cage complex has a total negative charge of À 11, and it is often challenging to transport large hydrophilic or charged cargo across the cell membrane. [19]

Uptake of cages in living cells
Confocal microscopy was used in order to visualize uptake of the cage using fluorescence. As the [Au(PMe 3 )] + �cage complex itself is not fluorescent, the cage was labelled with the fluorescent dye RhodamineB (RhoB), generating a fluorescent RhoB-cage complex. Although RhoB is very large, we hypothesized that electrostatic interactions could bind the dye to the cage, either by partial encapsulation of the alkyl groups within the cavity, or by binding to the exterior of the cage. A solution of 1 eq RhoB and 1.2 eq cage in water was stirred for 30 min at room temperature, and analyzed by 1 H NMR spectroscopy, DOSY, UV/Vis spectroscopy, fluorescence spectroscopy, and cryospray ionization high resolution mass spectrometry (CSI-HRMS). In the 1 H NMR spectrum of the RhoB and cage mixture ( Figure S4), the quartet at 3.49 ppm and the triplet at 1.16 ppm of the free RhoB were shifted upfield and appeared as two broad singlets at 3.06 ppm and 0.94 ppm respectively. In addition, the aromatic signals of the free RhoB between 8.17-6.57 ppm and the aromatic signals of the empty cage between 7.80-6.47 ppm were all shifted to give a set of overlapping peaks between 7.53-6.44 ppm. All of these signals in the RhoB and cage mixture gave one band in the DOSY spectrum with a logD value of À 9.63 ( Figure S5), indicating that there is one species in solution. In addition, the CSI-HRMS spectrum had signals which correspond to 3-, 4-, and 5-species containing one cage and one RhoB ( Figure S7). Finally, the UV/Vis spectrum of RhoB in the presence of cage also indicated binding ( Figure S6). The absorption at 554 nm decreased from 1.09 for free RhoB to 0.86 for RhoB in the presence of cage, which is typical for of a binding event. Having established that RhoB indeed binds to the cage, the fluorescence properties of the resultant complex was evaluated. Although the RhoB-cage complex indeed displays fluorescence, the intensity of the emission spectrum is lower than that of the free RhoB, indicating that fluorescence of the RhoB is partially quenched upon binding ( Figure 3B). Therefore, if fluorescence is detected after incubation with cells with the RhoB-cage complex, comparing the fluorescence intensity with that of the free RhoB gives an indication of whether or not the RhoB is still bound to the cage and therefore shows whether or not the cage has entered the cell. To this end, HeLa cells were incubated with a 1 μM solution of either RhoB or RhoB-cage for 30 min, followed by washing twice with PBS buffer. While fluorescence was observed in the cytoplasm of the cells incubated with free RhoB, only background fluorescence was seen for the RhoBcage ( Figure 3C). Therefore, the cage together with its cargo is not cell permeable, and the reason for the cage being non-toxic is due to the fact that it does not in fact enter the cell. This means that in order to carry out the reaction inside cells, the [Au(PMe 3 )] + �cage complex would need to be first brought into the cell using a cell-penetrating vehicle, such as a cellpenetrating peptide or lipid nanoparticle (LNP).

Effect of catalyst encapsulation on biocompatibility
In order to gain insights into catalytic conversion of substrate 1 under biologically relevant conditions, we first carried out the catalysis at high substrate concentrations, under the typical catalysis conditions used in the literature for [Au(PMe 3 )] + �cage. [16,17] Therefore 0.2 M of substrate 1 was stirred at 37°C for 16 h in the presence of 2.5 mol% catalyst. Almost identical yields of the fluorescent product 2 a were obtained for both free [Au(PMe 3 )Cl] and encapsulated [Au(PMe 3 )] + �cage in water, with 69 % and 70 % yields respectively ( Figure 4A). Due to solubility reasons, the reaction mixtures could not be analyzed by 1 H NMR spectroscopy, and the yield of 2 a was instead determined by fluorescence intensity: the concentration of product 2 a was calculated from a fluorescence calibration curve, and the final yield determined from the ratio between the observed concentration and theoretical maximum concentration of product 2 a. When the catalysis was carried out in CH 3 CN using [Au(PPh 3 )Cl] as catalyst, full conversion was achieved, resulting in a 7 : 3 ratio of fluorescent product 2 a and the non-fluorescent regio-isomer 2 b. Therefore, it is likely that 2 b is also being formed during the catalysis water, and that the overall conversion is higher than the yield of 2 a. Having established the successful formation of 2 a by [Au(PMe 3 )] + �cage, we next looked to investigate the effect of the in the Cl] complex and therefore reduces the amount of active species in solution, it appears that this is not the case in the presence of the cage: once encapsulated inside the cage, recombination of the gold center with the chloride is prevented. As expected, GSH behaved as a strong poison for the catalysis. Even in the presence of only 0.5 eq of GSH compared to the gold, free [Au(PMe 3 )Cl] only generated 2 a in a 2 % yield. However, upon encapsulation a 6fold increase in yield was observed, with a 13 % yield for [Au(PMe 3 )] + �cage. When the reaction mixture was analyzed by high resolution electrospray ionization mass spectrometry (Figure 4C), a peak at 852.0034 was observed, which corresponds to a species containing one GSH molecule and two [Au(PMe 3 )] + cations. No species corresponding to one GSH molecule and one [Au(PMe 3 )] + cation was observed. It appears that one GSH molecule poisons the catalysis by binding two gold centers. As GSH is found in concentrations of 2-5 mM in cells, [20] we looked to see the extent to which the cage could protect against this thiol poisoning. Catalysis was carried out in the presence of increasing equivalents of GSH with respect to the gold, and, unsurprisingly, no yield at all of 2 a was observed above 0.5 eq for free [Au(PMe 3 )Cl] ( Figure 4B). However, for the [Au(PMe 3 )] + �cage complex 2 a was still observed with up to 2 eq of GSH, albeit in very low yields (< 5 %). Catalysis was also carried out in the presence of all three biological additives combined. When both GSH and His are combined together in PBS buffer no yield at all is observed for free [Au(PMe 3 )Cl], however 2 a was still produced in a 4 % yield for the encapsulated [Au(PMe 3 )] + �cage. Finally, catalysis was also carried out in the presence of cell culture medium (DMEM + 10 % FBS), as such media contains a complex mixture of proteins and biomolecules, which is similar to what is found inside of cells. Using free [Au(PMe 3 )Cl] again resulted no yield; however, encapsulated [Au(PMe 3 )] + �cage was able to generate 2 a in a 10 % yield. It is likely that the lower yields observed when DMEM + 10 % FBS was used as solvent compared to water is again due to poisoning from thiols found in the amino acids and proteins in the cell culture medium. Although the catalytic activity of encapsulated [Au-(PMe 3 )] + �cage is quite drastically reduced in the presence of these biological additives, 2 a is still generated in consistently higher yields compared to free [Au(Me 3 P)Cl]. Therefore, while poisoning still takes place, the cage indeed is able to provide some protection against these biological additives. While it is clear that GSH is one source of catalysis inhibition under biological conditions, it should be noted that while these data give an indication of what may happen in vivo, they may not necessarily translate to the cell environment; the cell is a highly complex, heterogeneous environment, [21] and such simple test reactions under physiological conditions may not paint an accurate picture of how the system will behave inside a cell. The reactivity of gold catalysts towards a variety of transformations, including hydroarylations, has been shown to be highly dependent on the counterion of the gold complex; [22] for example, DFT studies into a propargylic amide cyclization using an LAuNTf 2 complex as catalyst show that (NTf 2 ) À counterions interact with the amide group to promote (de-)protonation during the catalytic cycle. [23] Therefore it is highly possible that the reactivity of the encapsulated and free gold complexes may be different if the chloride counterion is substituted by other anions found in the cell.
With the promising results of gold catalyst protection from typical biological metal poisons by encapsulation in the gallium-cage and established cytotoxicity of substrate 1, next the biocompatibility of the reaction itself was investigated under the required concentrations of all components. The reaction was carried out under physiological conditions, rather than in cells, in order to gain insight into how the reaction behaves in the presence of biological components, as both the set-up and analysis is more facile under physiological conditions compared to in cells. Catalysis was therefore carried out under dilute conditions in various cell culture media in order to determine if the reaction would be feasible in vivo (Table S2, SI). Solutions of the catalysts were first prepared in water or DMSO before addition of the substrate. A solution of 50 mol% of either encapsulated [Au(PMe 3 )] + �cage in water or free [Au(PMe 3 )Cl] in DMSO was therefore added to cell culture media (either DMEM + 10 % FBS, FBS, or FBS/PBS mixtures), followed by the addition of 1 eq of substrate 1 to give a final concentration of 10 μM of substrate. This concentration of substrate was selected so that even at low yields, product 2 a would still be able to be detected. The reaction mixtures were stirred at 37°C for 16 h, after which they were directly analyzed by fluorescence spectroscopy. Unfortunately, in all cases, for both encapsulated [Au(PMe 3 )] + �cage and free [Au(PMe 3 )Cl], no fluorescence was detected, meaning that no product was formed. Therefore, catalysis with either free or encapsulated gold is not possible under the conditions which would be required for in cells. While we demonstrated clearly a protective effect of the nanocage for encapsulated gold catalyst under semi-biological conditions in the presence of biological metal poisons, further substrate design and optimization is required for the application of such systems in living matter.

Conclusion
An encapsulated gold catalyst was able to catalyze an intramolecular hydroarylation reaction to produce a fluorescent coumarin dye. Under catalytic conditions in water the encapsulated [Au(PMe 3 )] + �cage complex was able to generate the product with about the same yields as the free catalyst. Importantly, under identical conditions in the presence of biological additives such GSH, Histidine, PBS buffer, and cell culture medium, the encapsulated catalyst [Au(PMe 3 )] + �cage provided much higher yields than the free [Au(PMe 3 )Cl], indicating the cage was providing protection for the gold catalyst. The [Au(PMe 3 )] + �cage complex afforded product 2 a in the presence of GSH, His, PBS buffer, and cell culture medium, whereas free [Au(PMe 3 )Cl] was completely inactive under the same conditions. Although the proof of principle that catalyst protection can be achieved by encapsulation in synthetic molecular cages, several issues need to be solved before these systems can be applied in cells. At very low substrate concentrations (10 μM) required for reactions to be carried out in cells, neither the free [Au(PMe 3 )Cl] nor encapsulated [Au(PMe 3 )] + �cage was able to generate product 2 a in cell culture media, so activity or stability under these conditions need to be improved. The cage itself was found to be not cell permeable and vehicles known to transport non-permeable cargo into cells may be required. [24] For example, encapsulation of the [Au(PMe 3 )] + �cage complex inside lipid nanoparticles (LNPs) may provide a viable route to deliver the protected catalyst into the cell. [25] Another potential route to cell permeability may be bioconjugation of the cage to a cell penetrating peptide, a strategy which has previously been applied to Pd 2 L 4 metallocages. [26] Also, the substrate should be redesigned as such so that it is less toxic and can be used at higher substrate concentrations. In addition, an even smaller substrate may further improve the protection provided by the cage, and further enhance the activity of the encapsulated gold under biological conditions.

Cell experiments
HeLa cells were incubated for 30 min with RhoB or RhoB-cage complex in DMEM + 10 % FBS to give a final concentration of 500 nM. The cells were then washed 3 times with PBS and then imaged by confocal microscopy.


s2fieldsofstudy:
Biology
Agricultural And Food Sciences

text:
Prevalence of fish-borne zoonotic trematode infection in Jilin Province, China

Fish-borne zoonotic trematodes (FZTs) are the most serious food-borne parasites in Asia and have become a burden to public health and a new challenge in food safety. In Jilin Province, China, the prevalence of FZTs in intermediate and definitive hosts has not been extensively explored. In the present study, we investigated the prevalence of FZTs in Jilin Province, China. From July to November 2020, a total of 132 freshwater snails (the first intermediate host of FZTs), 4122 wild freshwater fishes (the second intermediate host of FZTs) and 143 fecal samples from canines, ducks and swine (the definitive host of FZTs) were collected from the Yitong River basin of Jilin Province. FZT species were identified by morphological observation combined with internal transcribed spacer (ITS) sequence analysis. The prevalence of FZTs was then calculated. The results showed that the prevailing species of FZTs in Jilin Province, China, were Clonorchis sinensis, Metorchis orientalis and Echinochasmus japonicus. The total prevalence of FZTs was 29.74% (1226/4122) in fish, the total infection rates were 2.27% (3/132) in snails, 75.00% (21/28) in canines and 37.18% (29/78) in ducks. The coinfection rates of the two trematodes were 13.39% (552/4122) in fish, 35.71% (10/28) in canines and 7.69% (6/78) in ducks. The coinfection rate of the three flukes was 2.60% (107/4122) in fish. Nine of the 12 fish species examined were infected with FZT metacercariae.

Introduction
Fish-borne zoonotic trematodes (FZTs) are an important group of zoonotic parasites transmitted by fish and fish products (Hop et al., 2007). A total of 59 species of FZTs have been found to parasitize humans. FZTs' life cycle involve a first intermediate host (freshwater snails), a second intermediate host (freshwater fish) and a definitive host (human or other animals). These trematodes can be grouped into two main categories, the liver fluke group (Opisthorchiidae: 12 species) and the intestinal fluke group (Heterophyidae: 36 species, Echinostomatidae: 10 species and Nanophyetidae: 1 species) (Qiu et al., 2017). They are primarily related to liver and biliary disorders and intestinal diseases, causing severe disease and economic burden (Tang et al., 2016;Yu and Mott, 1994).
FZTs are primarily distributed in Asia, including China, South Korea, Japan, Laos, Vietnam, Thailand and the Far East of Russia (Qian et al., 2016;Stefan et al., 2013;WHO, 1995). More than 26 million people have been infected with liver flukes, including 15 million infected with C. sinensis (Qian et al., 2016;Sripa et al., 2007). In cases of intestinal FZTs, an estimated 40 to 50 million people are infected with one or several species (Hung et al., 2015;Keiser and Utzinger, 2009). The estimates of at-risk populations for FZTs are more than 1.1 billion (Santos and Howgate, 2011;Sripa et al., 2007Sripa et al., , 2010. The World Health Organization recently added FZT infections to its list of emerging infectious diseases (Clausen et al., 2012). The prevalence of C. sinensis in freshwater fish is 67.5% in Korea and 12.5% in Vietnam (Sohn et al., 2018). In China, the prevalence of C. sinensis metacercariae in fish is 35.1% (in 19 provinces/municipalities) . FZT (Choi et al., 2006;Qiu et al., 2017). Jilin Province, located in the northeast region of China, has a high infection rate of C. sinensis in humans (Li et al., 2011) because the local people have a habit of eating raw and undercooked fish (Lun et al., 2005). The prevalence of C. sinensis in the Songhua River, Fuyu County, Western Jilin Province, is 31.96% (Zhang et al., 2014. However, data on the FZT species and prevalence in Jilin Province have not been explored extensively, which is very detrimental to the prevention and control of FZTs in Jilin Province. Therefore, this study conducted an epidemiological investigation on FZTs in Jilin Province from July to November 2020.

Description of sampling sites and sampling time
The Yitong River basin runs through the central area of Jilin Province, which is located in multiple important economic locations and is an important supply source for freshwater fish in Jilin Province. It is the second largest tributary of the Songhua River, with a basin area of 8440.00 square kilometers and a total length of 342.50 km. Freshwater fish and snail samples were collected in the upstream, midstream and downstream areas of the Yitong River basin, with 13 sampling points covering reservoirs, ponds, river mainstems, river tributaries, streams and wetlands. The fish samples were continuously harvested from July to November 2020. Fecal samples were collected from the definitive hosts living in villages or wildlife living around these 13 collection sites (Fig. 1). The definitive host feces and wild freshwater snails were harvested from July to November 2020.  Regan) were randomly collected from the Yitong River basin. We collected freshwater fish using fishing nets or purchased them from fishermen, and the collected fish are commonly and popularly consumed species in the area. The sampling information for the freshwater fish is provided in Additional File Table S1.

Freshwater snails
Freshwater snails were collected during the morning for 20 min per site by fully randomly handpicking or catching them with a net. In 9 of 13 sampling collection sites, a total 132 snails from 4 species (46 Parafossarulus striatulus, 11 Bithynia. sp., 68 Viviparidae gen. sp. and 7 Radix auricularia) were obtained for trematode detection.

Fecal samples of canines, swine and ducks
A total of 143 fecal samples from canines (28 samples), ducks (78 samples) and swine (37 samples) were randomly collected. The majority of animals were domestic and feral (12 ducks) and more than 12 months old (100% canines, 100% swine, 46.15% ducks). Feces from canines, ducks and swine were directly sampled immediately after defecation. The duck breeds were Partridge duck and Anas platyrhynchos. The dogs were primarily Chinese field dogs, and the swine were Landrace and Large Yorkshire. The sampling information from the definitive hosts is provided in Additional File Table S2.

FZT metacercariae observation in freshwater fishes
FZT metacercariae were detected using a combination of compression and artificial digestion methods as previously described (WHO, 1995). Briefly, a compression process was used for small-sized fish (≤10 g): each fish was compressed using two glass slides and observed under a microscope. The fish were repeatedly compressed and observed three times. Then, FZT-positive fish were used to isolate metacercariae. Artificial digestion was used for large-sized fish or to isolate metacercariae. Each whole fish was cut into small pieces and ground using a homogenizer, which was placed in a beaker with digestion solution (10 g pepsin, 6 g NaCl and 10 mL concentrated HCl in 1 L distilled water) at a ratio of 1 g tissue per 10 mL solution. The mixture was thoroughly stirred at 37 • C for 3 h. After digestion, the large particles were removed by filtering through a 250 μm/aperture copper sieve. The filtered solution was transferred to a triangle measuring cup, diluted in tap water and kept still for 2 h. The supernatant was discarded, and the sediment was retained. This procedure was repeated until the supernatant became clear. The sediment was transferred to a cell culture dish containing 5 mL sterile PBS, and the metacercariae were morphologically identified and classified under a microscope and counted.
The prevalence, metacercariae abundance and calculated intensity/ 100 g fish of FZTs were calculated as shown (Bush et al., 1997).

Prevalence =
The number of metacercariae − infected fish The number of fish examined × 100% Calculate intensity/100 g fish = The total number of metacercariae The total weight of each fish species (g) × 100 Metacercariae abundance: The number of metacercariae in each fish.

FZT examination in snails
Small snails were crushed using two glass slides, and FZT larvae were observed under a microscope. The large hard-shelled snails were opened, and visceral tissue and hemolymph were transferred onto glass slides for microscopic observation (Hung et al., 2015). The infection rate of FZTs was calculated (Bush et al., 1997).

FZT egg detection in fecal samples
Fecal samples were examined for FZT eggs using direct smears and microscopic observation. The smear was repeated three times for each sample to improve the detection rate. The infection rate of FZTs was calculated in definitive hosts (Bush et al., 1997).

Genomic DNA extraction
All of the fecal samples and freshwater snails were stored at 4 • C until DNA extraction. Different kinds of metacercariae species from three independent experiments were stored in Alsever's solution at 4 • C prior to DNA extraction. Genomic DNA from different metacercariae species and freshwater snail samples was extracted using a genomic DNA Kit (TIANGEN BIOTECH, Beijing, China). DNA from the definitive host fecal samples was performed strictly according to the instructions of the Fecal Genomic DNA Kit (TIANGEN BIOTECH, Beijing, China). All DNA precipitates were stored at − 80 • C.

Detection of ITS sequences for FZTs
The internal transcribed spacer (ITS) sequences of FZTs from all the extracted DNA were amplified by PCR using ITS sequence primers (Additional file Table S3) as previously described (Qiu et al., 2019;Zhou et al., 2010). After amplification, 10 μL of PCR product was examined by agarose gel (1.5%) electrophoresis and ethidium bromide substitution (Applygen Technologies Inc. Beijing, China) staining. All positive products were sequenced by Life Technology Company (Comate Bioscience Co., Ltd., Jilin, China).

Phylogenetic analysis of ITS sequences
To study the phylogenetic relationships of the isolated and other FZTs, ITS sequences of liver flukes (M. orientalis, M. ussuriensis, C. sinensis, Opisthorchis sudarikovi, Opisthorchis felineus, Erschoviorchis anuiensis and Euryhelmis costaricensis) and intestinal flukes (Echinostoma trivolvis, Echinoparyphium mordvilkowi, Stephanoprora uruguayense, Stephanoprora chasanensis, Stephanoprora pseudoechinata, E. japonicus, Echinochasmus milvi, Echinochasmus coaxatus) from GenBank of the National Center of Biotechnology Information (NCBI) were used for phylogenetic analysis using Aspidogaster chongqingens as the outgroup. The phylogenetic tree for ITS genes of FZTs was reconstructed using MEGA X software. The methods of maximum likelihood (ML), maximum parsimony (MP) and neighbor joining (NJ) were used for phylogenetic construction, and the initial trees were automatically obtained by selecting the topology with a superior log likelihood value .

Data analysis
The overall prevalence of FZT metacercariae in freshwater fish and their respective prevalence with respect to different factors (fish species, sampling location, sampling water source, sampling time) were statistically analyzed using SPSS24.0 (IBM, Armonk, NY, USA), and the analysis shows the 95% confidence intervals (95% CI). Differences in data between groups and statistical graph plotting were performed using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). A p value < 0.05 was considered to indicate a significant difference.

The prevalence of FZTs in wild freshwater fish in Jilin Province
Morphological observation of metacercariae combined with ITS sequence analysis revealed that the prevailing FZTs in Jilin Province were C. sinensis, M. orientalis and E. japonicus (Figs. 2 and 3). The metacercariae of C. sinensis were oblate or elliptical, 135.65 ± 27.65 μm × 93.23 ± 12.76 μm in size and contained an active larva with two layers of thin cyst walls. The excretory sac of the larva was dark brown, and the oral sucker and ventral sucker were similar in size (Fig. 2 A and D). The metacercariae of M. orientalis were oval or round, 113.37 ± 12.52 μm × 101.89 ± 13.87 μm in size, with two layers of thick cyst walls (10.05 ± 2.50 μm thick). The metacercariae contained a movable larva with a slower activity frequency than that of C. sinensis, with a brownish yellow excretory sac with a similar size between the oral and ventral suckers ( Fig. 2 B and E). The metacercariae of E. japonicus were elliptical and very small, approximately 78.63 ± 12.18 μm × 43.66 ± 8.95 μm in size.
The metacercariae were translucent, with various round particles distributed in the excretion tube ( Fig. 2 C and F).
The ITS sequences of C. sinensis, M. orientalis and E. japonicus metacercariae from different fish species were analyzed by molecular biology and deposited in NCBI GenBank (Nos. MW828640, MW828729 and MW828605), which were 100% homologous to the reported ITS sequences of the trematodes deposited in NCBI GenBank (Nos. MF319654, MK482055 and KT873314) (Fig. 3) (Besprozvannykh et al., 2017;Na et al., 2016;Tatonova et al., 2017). The ITS sequence phylogenetic tree indicated that the sequences were classified as C. sinensis, M. orientalis and E. japonicus (marked with * in Fig. 3).

Association of the size of fish with FZT prevalence
To understand the relationship between fish size and FZT metacercariae infection, we counted FZT metacercariae in fish of different weights. The results showed that small-sized fish (0<fish≦5 g) were more vulnerable to three FZTs than middle-sized fish (5<fish≦50 g), and no FZT metacercariae infection was observed in large-sized fish (>50 g) (Fig. 4).

The FZT prevalence in different reaches and water sources
Wild freshwater fish from upstream, midstream and downstream were examined. The results showed that C. sinensis, M. orientalis and E. japonicus were present in all three reaches of the Yitong River basin.  (Table 3).
3.1.5 The effect of different months on FZT prevalence in wild freshwater fish.
We continuously examined FZT prevalence in freshwater fish from July to November. The prevalence of C. sinensis, M. orientalis and E. japonicus in wild freshwater fish exhibited similar trends, increasing from July to August or September. The highest prevalence of C. sinensis (31.96%) and E. japonicus (30.25%) was observed in August, while that of M. orientalis (19.03%) was observed in September (Fig. 5).

Infection rate of FZTs in wild freshwater snails
We determined FZT infection rates in freshwater snails using direct microscopic observation combined with ITS sequence analysis. No FZT larvae were detected by direct observation in snails. ITS sequence analysis showed that C. sinensis ITS sequences were amplified in two P. striatulus samples (4.35%, 2/46), the M. orientalis ITS sequence was amplified in one P. striatulus sample (2.17%, 1/46), and no E. japonicus ITS sequences were amplified in snail samples. The overall infection rate of FZTs in snails was 2.27% (3/132), and no coinfection was observed.

FZT infection in the definitive hosts
Morphological identification and ITS sequence analysis were combined to assess FZT infection in fecal samples from canines, swine and ducks. The infection rates of C. sinensis and E. japonicus in canines were 46.43% (13/28) and 64.29% (18/28), respectively, and the infection rates of M. orientalis and E. japonicus in ducks were 19.23% (15/78) and  The eggs of C. sinensis were bulb-shaped and yellow colored, approximately 24 ± 2.58 μm × 15.6 ± 2.33 μm in size, with egg operculum and shoulder peaks, containing a well-developed miracidium and a small spine at the bottom (Fig. 2 G). M. orientalis eggs were oval and yellow-brown colored, 25.87 ± 3.21 μm × 16.32 ± 1.65 μm in size, with egg operculum and without shoulder peaks, containing a welldeveloped miracidium and a small spine at the bottom (Fig. 2 H). The eggs of E. japonicus were oval and large, golden yellow colored, 84.25 ± 3.17 μm × 62.5 ± 2.55 μm in size, with a small egg operculum and containing yellow follicles (Fig. 2 I and J).

Discussion
In the present study, infection by FZTs in the first intermediate hosts (snails), the second intermediate hosts (freshwater fish) and the definitive hosts (canines, swine and ducks) in Jilin Province of China was investigated. Three trematodes (C. sinensis, M. orientalis and E. japonicus) were detected to be prevalent in this region. Nine species of fish could serve as the second intermediate hosts of FZTs, with P. striatulus serving as the first intermediate host and canines and ducks serving as the definitive hosts.
The different prevalences in different regions might be attributed to geographical conditions and local ecological status (climate, temperature, water quality, environment, etc.). The prevalence of C. sinensis was 37.09% (2160/5824) in Guangdong (Chen et al., 2010), 16.97% (52/307) in Guangxi (Sohn et al., 2009,b) and 19.96% (643/3221) in Heilongjiang (Zhang et al., 2014). The prevalence of M. orientalis was 10.54% (718/6815) in Heilongjiang (Qiu et al., 2017) and 6.33% (19/300) in Anhui . Although E. japonicus metacercariae was found in freshwater fish (Sohn, 2009), the prevalence data for this trematode in freshwater fish are still lacking. In this study, the prevalence rates of C. sinensis, M. orientalis, and E. japonicus in freshwater fish in Jilin Province were 22.59%, 9.92% and 18.10%, respectively. Among 4122 samples, 13.39% of fish were infected with two species of trematodes, and 2.60% of fish were coinfected with three species of trematodes. This is the first report of the coinfection rate of FZTs in freshwater fish in Jilin Province. The suggests that ingestion of small amounts of raw fish might also cause FZT coinfection in definitive hosts.
A total of nine species of fish were found to be infected with FZTs in our survey, of which eight species were infected with C. sinensis, five species with M. orientalis and six species with E. japonicus. Unlike the survey in Heilongjiang Province (Qiu et al., 2017;Zhang et al., 2014), we did not find C. sinensis and M. orientalis in M. anguillicaudatu and C. auratus. In Guangdong Province, C. sinensis was prevalent in C. idellus, H. nobilis, C. molitorella, T. mossambica, C. auratus, H. molitrix, P. pekinensis and P. parva (Chen et al., 2010). The prevalence of FZTs in different fishes in Jilin Province was not consistent with that in other regions, which might be influenced by factors such as sampling time, sampling location, sampling water sources and sample size. Of note, P. parva was the most vulnerable fish to C. sinensis and M. orientalis in Jilin Province, which is consistent with observations in other provinces (Chen et al., 2010;Qiu et al., 2017;Zhang et al., 2014). Locally, the most commonly eaten fish are P. parva, H. leuciclus, R. sinensis, C. auratus, S. dabryi, and C. carpio. P. parva, H. leuciclus, R. sinensis and S. dabryi, which are susceptible to FZTs, are often eaten by deep-frying or stewing. However, insufficient cooking can result in incomplete extinction of the metacercariae in the muscle of fish and cause infection. Additional advice should be given to strengthen local disease publicity to avoid raw and accidental ingestion of susceptible fish. This is the first study to report the prevalence of E. japonicus in R. sinensis, H. leuciclus, C. giurinus, and S. dabryi, which enriches data on the second intermediate host of E. japonicus.
Interestingly, our study found that the sampling water sources acted as constraints on the prevalence of FZTs, demonstrating that C. sinensis, E. japonicus and M. orientalis have selectivity for the environment, which has not been reported in previous investigations. A previous report demonstrated that the prevalence of C. sinensis varied in different water sources, with the prevalence of C. sinensis in rivers being much higher than that in ponds, reservoirs and streams . In Heilongjiang Province, the prevalence of C. sinensis and M. orientalis was higher in the Songhua and Nenjiang Rivers than in lakes and ponds (Qiu et al., 2017;Zhang et al., 2014). In this study, the prevalence of C. sinensis was higher in ponds than in other water sources in Jilin Province. We found that the ponds had freshwater snails living in them with the necessary conditions for the transmission of C. sinensis. Additionally, the definitive hosts around the ponds may be responsible for  The prevalence of FZTs in freshwater fish gradually increased and then decreased, with the highest prevalence of C. sinensis and E. japonicus in August and the highest prevalence of M. orientalis in September. *p < 0.05 was considered to be a significant difference, and the prevalence in November was used as a control. The significances of C. sinensis, M. orientalis and E. japonicus in different months are marked in red *, green * and blue *, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) the high prevalence of C. sinensis in the ponds. The prevalence of M. orientalis was higher in the reservoirs than in other water sources in our study due to the abundance of water, fish resources, and a large number of wild birds. M. orientalis is transmitted in a variety of wild birds , which may be responsible for the high prevalence of M. orientalis in these reservoirs in our study. The prevalence of E. japonicus was higher in the wetlands than in other water sources due to the rich biodiversity of the wetland park, which had all the hosts necessary for the prevalence of E. japonicus. Wetland waters are virtually immobile, making them more conducive to E. japonicus infection. We also found that M. orientalis was primarily prevalent upstream, while the prevalence of C. sinensis and E. japonicus was similar among the three reaches. It is recommended that surveys of FZTs should cover various water sources and basins as much as possible to avoid data bias or errors. The differences in FZT prevalence in wild fish in Jilin Province among different months reflect the important influence of temperature and precipitation on FZT prevalence. Analyzing the prevalence of C. sinensis in freshwater fish in different seasons revealed that the prevalence of C. sinensis was higher in summer, but there were no significant differences among the four seasons . However, that study did not mention sampling locations, and the influence of sampling time on the prevalence of FZTs may not be suitable for Jilin Province, China. It is known that precipitation and temperature variation affect the prevalence of C. sinensis metacercariae in fish . The prevalence of FZTs in cultured freshwater fish in Jilin Province was highest in July (Yang et al., 2019), which was earlier than our finding (the prevalence of FZTs was highest in August and September), which may be caused by the lower temperature in 2020. The earlier increases in temperature and precipitation are more favorable for the development of FZTs in freshwater snails.
Transmission of FZTs is strictly restricted by the first and second intermediate hosts and the definitive hosts . Our study identified FZT infection in the first intermediate hosts and in the definitive hosts in Jilin Province. These findings suggested that a complete life cycle of FZTs has formed in Jilin Province. The activities of definitive hosts may pose new challenges to local public safety to enact measures against FZTs, and the surveillance and treatment of FZTs in local livestock and poultry may be needed. The results of two FZT coinfections in canines and ducks further supported the inference that FZT coinfection in freshwater fish could ultimately result in FZT coinfection in definitive hosts.
This study identified three FZTs in intermediate and definitive hosts in Jilin Province. However, the low number of samples of the definitive hosts and the first intermediate hosts may affect the assessment of the prevalence of FZTs among the first intermediate hosts and definitive hosts in Jilin Province, and more work is required in future studies to confirm these findings.

Conclusion
This study is a detailed epidemiological investigation of FZTs in Jilin Province, including the first and second intermediate hosts, definitive hosts, species and prevalence of FZTs and coinfection rates.

Funding
This study was supported by National Key R&D Program of China (Nos. 2017YFD0501200, 2017YFD0501305), Science and Technology Development Program of Jilin Province (Nos. 20200402044NC). The experiments conducted in this study complied with the current laws of China.

Data availability statement
The newly generated sequences were submitted to the NCBI Gen-Bank database under the accession No. MW828640, MW828729 and MW828605.