{"paper_id":"9855379b-cf28-4f06-8438-a38bc9b7c233","body_text":"1 Department of Clinical Medicine, School of Queen Mary, Nanchang University, 330006 Nanchang, Jiangxi, China\n2 Department of Gynecology and Obstetrics, West China Second University Hospital of Sichuan University, 610000 Chengdu, Sichuan, China\n3 Metabolic Control and Aging-Jiangxi Key Laboratory of Aging and Diseases, Human Aging Research Institute (HARI), School of Life Science, Nanchang University, 330031 Nanchang, Jiangxi, China\nAbstract\nAvailable treatments for endometriosis remain unsatisfactory; therefore, there is an urgent demand for novel and effective therapeutic strategies. This study focused on the therapeutic effect of cepharanthine, a monomer derived from a Chinese herb, on endometriosis in vitro, in patient-derived eutopic endometrial organoids, and in vivo.\nPatient-derived ectopic endometrial stromal cells were isolated from ovarian endometriomas. Organoids were generated from the eutopic endometrium of patients with endometriosis. Ectopic endometrial stromal cells, eutopic endometrial organoids, and immortalized endometrial stromal cells were used to evaluate the effects of cepharanthine on cell viability, growth, and apoptosis. Female BALB/c mice were used to develop a peritoneal endometriosis model.\nCepharanthine treatment decreased the viability of immortalized endometrial stromal cells, patient-derived endometriotic stromal cells, as well as eutopic endometrial organoids. It induced DNA damage, downregulated cyclin D1, and caused cell-cycle arrest at the G0/G1 phase. It also promoted apoptosis by enhancing cytochrome C release, activating caspase-9 and caspase-3, increasing the expression of proapoptotic factor Bax, and decreasing the expression of antiapoptotic factor B-cell lymphoma 2 (Bcl-2). Intraperitoneal administration of cepharanthine significantly inhibited the growth of murine peritoneal endometriosis model. The treatment significantly downregulated the protein expression of cyclin D1 and DNA repair protein RAD51 (RAD51), and increased phosphorylated histone H2AX (γ-H2AX) expression in endometriosis lesions, indicating that it induced DNA damage and impaired DNA repair. Additionally, Ki-67 expression was significantly decreased, and apoptosis was markedly increased in the lesions.\nOur results indicate that cepharanthin may represent a promising treatment option for endometriosis.\nKeywords\n- apoptosis\n- cepharanthine\n- DNA damage\n- endometriosis\n- organoids\nEndometriosis is a prevalent chronic gynecological disorder characterized by the presence of endometrial glands and stroma outside the uterine cavity. It mainly affects about 10% of women of reproductive age [1]. The disease is often accompanied by severe pelvic pain, dysmenorrhea, deep dyspareunia, and infertility, and is also a leading cause of miscarriage and implantation failure. It is a chronic condition without a definitive medical or surgical cure. Treatment of endometriosis consists of surgical removal of lesions and hormone treatment (combined oral contraceptives, progestins, or gonadotropin-releasing hormone agonists), often with side effects and variable efficacy. The recurrence rate is up to 50% within 5 years after surgery [2]. Studies have begun to focus on plant-derived agents as natural treatments, aiming for high efficacy, fewer side effects, and preserved fertility [3].\nStephania cepharantha Hayata has been used by Traditional Chinese Medicine practitioners to treat endometriosis-related symptoms for many years. Cepharanthine has been approved for the treatment of leukopenia in Japan and in China [4,5]. It has anti-cancer, anti-inflammation, anti-virus, and other pharmacological activities without major side effects [6,7]. Cepharanthine can inhibit the proliferation of multiple cell types in vitro, including peripheral mononuclear cells and T-cells [7]. It can also induce apoptosis of many cancer cells, including ovarian [8] and breast cancer cells [9].\nThe survival of ectopic endometriotic lesions relies critically on key biological processes, such as proliferation, apoptosis, inflammation, and angiogenesis [10]. Newly developed endometriotic lesions exhibit more cell proliferation than does eutopic endometrium, and endometrial tissue from affected women is more apoptosis-resistant [3]. This imbalance between cell proliferation and cell death promotes pathological tissue overgrowth. In this regard, we hypothesized that cepharanthine, which increases apoptosis in some cancer cells, has therapeutic value in treating endometriosis.\nIn 2019, Boretto et al. [11] established organoid models for endometrial diseases (endometriosis, hyperplasia, endometrial cancer) that exhibited long-term expandability, genomic/transcriptomic stability, replicated disease diversity, and served as promising preclinical models and tools for drug screening/discovery. Other studies have similarly noted that, as an ideal research model, endometrial organoids hold potential for pathophysiological studies, novel therapeutic drug screening, and personalized medicine drug testing [12,13]. Our team has previously reported the successful establishment and culture of endometrial organoids [14]. We tested cepharanthine in vitro, in patient-derived endometrial organoids, and in vivo.\nThe Ethics Committee of West China Second University Hospital of Sichuan University approved the study on March 7, 2018 (ethical approval number: Medical Research 2018 No. 48). Informed consent was obtained from each participant. Samples of ovarian endometrioma and eutopic endometrium were collected from 3 patients of reproductive age who had not received any medication before surgery. Tissue samples were placed in an iced DMEM/F12 culture medium (Gibco, 11330-032, Melbourne, VIC, Australia) containing 1% penicillin-streptomycin (Gibco, 15140-122) and then were immediately transferred to the laboratory on ice.\nThis was done as previously described [15]. After rinsing 3 times with iced sterile PBS, the ovarian endometrioma was cut into 1-mm3 pieces and digested with 0.1% collagenase IV solution (Gibco, Catalog No. 17104-019, Waltham, MA, USA) for 1 h. The mixture was then filtered through a 70 μm filter (Falcon, 352350, Franklin Lakes, NJ, USA) and a 40 μm filter (Falcon, 352340). After centrifugation at 1000 g for 10 min, stromal cells were collected from the bottom of the tube. The cells were resuspended in DMEM/F-12 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B (Sigma-Aldrich, Catalog No. A2942, St. Louis, MO, USA), and then incubated in 5% CO2 at 37 °C overnight. Cells from two passages were used for subsequent experiments. Isolated ectopic endometrial stromal cells were validated for purity via immunofluorescence staining targeting vimentin, a specific marker of stromal cells.\nThis was performed as previously described [14,16]. Endometrial samples were placed in cooled normal saline with 1% antibiotic-antimycotic, transported to the laboratory within 1 h, rinsed with 0.01% benzalkonium bromide, then 3 times with phenol red-free DMEM/F12 plus 1% antibiotic-antimycotic. Tissue was transferred to 100-mm dishes with 10 mL digestion solution (cooled PBS with 0.1% collagenase IV), minced into 1-mm3 fragments, and incubated at 37 °C under 5% CO2 for 40 min, before neutralizing medium was added. The suspension was filtered through a 70-μm strainer (Falcon, 431751), then a 40-μm strainer (Falcon, 431750). The 40-μm strainer was inverted and rinsed with 6 mL medium to collect epithelial cells. After centrifugation at 1500 rpm for 10 min, the pellet was resuspended in 1 mL advanced DMEM/F12 (Gibco, 12634010, USA), mixed with Matrigel (Corning, 356231, Corning, NY, USA), and kept on ice. Then, 30-μL droplets were seeded into 24-well plates (1/well), incubated at 37 °C for 15 min, and then overlaid with organoid medium. Organoids formed in 4–7 days. They were passaged when >400 μm in diameter; those from one passage were used for experiments.\nThe immortalized endometrial stromal cells (iCell Bioscience Inc., iCell-0187a, Shanghai, China) were confirmed by short tandem repeat (STR) validation. All cell lines tested negative for mycoplasma. Cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C in DMEM/F12 supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Cepharanthine was purchased from APExBIO, N2771, Houston, TX, USA.\nThis was carried out as previously reported [17,18]. Cells were cultured with different concentrations of cepharanthine for 48 h. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Sigma). Cell survival was calculated by normalizing the absorbance to that of untreated controls.\nCells were treated with 14.16 μM cepharanthine for 48 h. Quantitative evaluation of cellular apoptosis was carried out by flow cytometric analysis using the Annexin V/propidium iodide (PI) staining method. Briefly, cells were digested with 0.25% trypsin-free EDTA, then washed with PBS once, and then incubated with Annexin V-Alexa Fluor 747/PI Apoptosis Detection Kit (Yeasen, 40304-ES20, Shanghai, China). Cells were then resuspended using 100 μL 1× Binding Buffer, and then 5 μL Annexin V plus 10 μL staining solution were added in the dark for 15 min. The ratio of apoptotic cells was determined by flow cytometry (Beckman Coulter, Model CytoFLEX S, Brea, CA, USA), and data were analyzed by software (Version 2.4, Beckman Coulter, Brea, CA, USA). Cell cycle analysis was performed according to a previously published protocol [19,20].\nCells were incubated with 14.16 μM cepharanthine for 48 h. The medium was then changed with fresh DMEM/F12, and the cells were cultured for another 2 weeks. Cells were then fixed in 4% paraformaldehyde (PFA) for 10 min and stained with crystal violet using ImageJ software (Version 1.54f, National Institutes of Health, Bethesda, MD, USA) for 10 min to visualize and quantify the colonies. Colony numbers were determined both manually and using ImageJ in triplicate.\nThe experiment was conducted as described previously [18,21]. Primary antibodies including cyclin D1 (Abcam, ab134175, Cambridge, UK, 1:1000), γH2AX (Abcam, ab26350, 1:1000), caspase-9 (Proteintech, 10380-1-AP, Rosemont, IL, USA, 1:1000), caspase-3 (Proteintech, 66470-2-Ig, 1:1000), B-cell lymphoma 2 (Bcl-2) (Proteintech, 12789-1-AP, 1:1000), Bax (Proteintech, 60267-1-Ig, 1:1000), cytochrome C (Proteintech, 66264-1-IG, 1:1000) and β-actin (Atagenix Laboratory, ATPA00014Rb, Wuhan, Hubei, China, 1:1000) were used. Briefly, 20 μg of whole-cell lysate per sample were loaded onto a 12% polyacrylamide gel for electrophoresis and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk in 1× Tris-buffered saline (pH 7.6) containing 0.05% Tween-20, followed by incubation with primary antibodies. Secondary antibodies were used at 1:5000. Proteins were visualized using the Western Bright enhanced chemiluminescence (ECL) detection kit (Junengbio, K-12043-D10, Guangzhou, Guangdong, China) and imaged with a Bio-Rad ChemiDocXRS+ chemiluminescence system (Bio-Rad, 1708265, Hercules, CA, USA). Each band was obtained after 3 independent repeated experiments. Band densities were quantified via ImageJ software.\nThe animal model was established using the method of a previously published study [22]. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of West China Second University Hospital of Sichuan University (Approval No. 2020028) and performed in compliance with the university’s Guide for the Care and Use of Laboratory Animals. Female BALB/c mice (6–8 weeks of age) were housed in a specific pathogen-free (SPF) facility and used to establish a mouse model of peritoneal endometriosis. After adaptive feeding for one week, donor mice were randomly selected and euthanized to collect the uteri, all surgery was performed under anesthesia (pentobarbital sodium, i.p., 50 mg/kg). All uterine horns were identically managed, including a longitudinal split to isolate endometrial tissue and careful dissection into consistent pieces smaller than 1 mm. The fragments were then suspended in sterile PBS. The peritoneal endometriosis model was established by intraperitoneal injection of endometrial tissue pieces. Fragments from one donor mouse were injected into the peritoneal cavity of two recipient mice. Fourteen days after injection, 5 mice were euthanized to check the formation of endometriosis lesions. After hematoxylin and eosin (HE) stain verification of the endometriotic lesions (all of the 5 mice had endometriosis lesions), the remaining 12 mice were randomly divided into two groups. In the literature, the doses of i.p. injections of cepharanthine have varied from 10 mg/kg/day to 20 mg/kg/day [23,24,25]; we chose to use an i.p. injection dose of 10 mg/kg/day, and the control group received i.p. injections of saline. Injections began on the 15th day after the operation and lasted for 4 weeks. The mice were then euthanized, and the endometriotic lesions were collected. Mice were euthanized with 100% CO2 at a flow rate of 30-70% chamber volume per minute until respiratory arrest. Death was verified by the lack of respiration and reflex response.\nImmunohistochemical staining was performed as previously described [18]. Sections were dewaxed with xylene and dehydrated using a graded ethanol series. The primary antibodies, anti-cyclin D1 (CCND1) (ab134175, Abcam), anti-Ki67 (27309-1-AP, Proteintech), anti-DNA repair protein RAD51 (RAD51) (ab133534, Abcam), and anti-γ-H2AX (ab26350, Abcam) were added to the slides and left overnight at 4 °C. After washing with PBS, the secondary antibody (KIHC-5, Proteintech) was added to the slides. One hour later, the signals were magnified by 3,3′-diaminobenzidine (DAB), and the nuclei were counterstained with haematoxylin. At least fifteen non-overlapping, independent fields of view were randomly selected and analyzed under a microscope.\nAnalysis of apoptotic cells within endometriotic lesions was done by using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Promega, Madison, WI, USA), following the manufacturer’s protocol. The photographs were captured by Olympus FV-3000 confocal microscopy (Olympus Corporation, Tokyo, Japan) and saved in TIFF format. The number of TUNEL-positive nuclei was counted by ImageJ (1.53 K). Six fields of view were randomly selected from each sample, and each group included six independent replicates.\nData were presented as means ± standard deviation of at least 3 independent experiments. The two-tailed Student’s t-test was used to compare two independent groups. Statistical significance was set at p ≤ 0.05. The statistical analyses were done by using SPSS 20.0 (IBM Corp., 20100901, Armonk, NY, USA) and R 2.10.0 (R Foundation for Statistical Computing, Vienna, Austria).\nWe first tested the effect of cepharanthine on an immortalized endometrial stromal cell line. Our results revealed that cepharanthine treatment reduced cell viability in a dose‑dependent manner. The half-maximal inhibitory concentration (IC50) was 14.16 μM (Fig. 1A). We then tested the effect of cepharanthine on patient-derived ectopic stromal cells. To verify the primary ectopic stromal cells, we stained the cells with an antibody to vimentin, which is a marker of stromal cells (Fig. 1B). Data showed that cell viability was decreased in a dose-dependent manner by cepharanthine treatment. The IC50 was 2.816 μM (Fig. 1C).\nWe previously reported the successful establishment and culture of endometrial organoids [14]. This time, we generated organoids from the eutopic endometrium of patients with endometriosis and investigated the impact of cepharanthine on the viability of these organoids. Fig. 2A shows the growth status of endometrial organoids treated with increasing concentrations of cepharanthine. Cepharanthine treatment resulted in a dose-dependent reduction in the viability of patient-derived eutopic endometrial organoids. The IC50 was 9.058 μM. Fig. 2B shows the morphology of the organoids. Hematoxylin-eosin staining confirmed the presence of well-defined epithelial cells around the lumen in the eutopic endometrial organoid.\nThe colony-formation assay indicated that the growth ability of the endometrial stromal cells was markedly inhibited by cepharanthine, as indicated by the decreased colony numbers (Fig. 3A). To clarify the association between cepharanthine’s antiproliferative activity and apoptosis induction, Annexin V/PI-based flow cytometric analysis was utilized. As shown in Fig. 3B, the number of apoptotic cells was significantly greater in the cepharanthine-treated group than in the control group. Cell-cycle analyses were also conducted, and results showed that cepharanthine-treated cells were blocked at the G0/G1 phase; concomitantly, the percentages of S-phase and G2/M-phase cells were reduced (Fig. 3C).\nAfter HE stain verification of the endometriotic lesions (Fig. 4A), 12 mice were randomized into two groups. The reported doses of intraperitoneal injections of cepharanthine varied from 10 mg/kg/day to 20 mg/kg/day [23,24,25]. The study group chose to use an intraperitoneal injection dose of 10 mg/kg/day, and the control group received an intraperitoneal injection of saline. Compared to the control group (mean lesion weight = 0.445 [0.085] g), treatment with cepharanthine resulted in a statistically significant reduction in lesion weight (mean lesion weight = 0.183 [0.035] g); p < 0.001 (Fig. 4B, Supplementary Fig. 1). Immunostaining revealed that Ki-67 expression (a proliferation marker) was significantly reduced in the cepharanthine-treated group, p < 0.01 (Fig. 4C). The TUNEL assay was used to detect cellular apoptosis within the endometriotic lesion. Fig. 4D shows significantly more TUNEL-positive nuclei in the cepharanthine-treated group than in the control group (p < 0.05).\nThe Western Blot analysis showed that cepharanthine upregulated cytochrome C in the cytoplasm, enhanced the expression of cleaved-caspase-9, cleaved-caspase-3, and Bax, and downregulated the expression of Bcl-2 in endometrial stromal cells. These results suggested that cepharanthine triggered apoptosis via the release of cytochrome C from mitochondria to the cytoplasm and subsequent activation of caspase-9 and caspase-3 [26] (Fig. 5A,B).\nCepharanthine has been reported to induce DNA damage in lung cancer cells [27]. Given that histone H2AX phosphorylation is a well-established marker of DNA double-strand breaks [28], we examined the levels of phosphorylated H2AX (γ-H2AX) in immortalized endometrial stromal cells after cepharanthine treatment. Our results demonstrated that cepharanthine treatment produced significantly more H2AX phosphorylation than in the control group (Fig. 5A,B), indicating its potential to induce DNA damage in immortalized endometrial stromal cells.\nGiven that cepharanthine treatment induced G0/G1 phase cell-cycle arrest accompanied by decreased proportions of cells in S and G2/M phases, we examined CCND1 expression in the treated cells. The results demonstrated that cepharanthine significantly downregulated cyclin D1 expression (Fig. 5A,B).\nPrevious studies have shown that downregulation of cyclin D1 can impair cellular DNA repair [18,21,29]. Since the in vitro result showed that cepharanthine treatment could downregulate cyclin D1, we examined cyclin D1 expression in endometriotic lesions from cepharanthine-treated mice. Consistent with the in vitro test, the cepharanthine treatment downregulated cyclin D1, concurrently increased the expression of γ-H2AX, and decreased the expression of RAD51 (a protein critical in DNA repair) (Fig. 5C,D). These results were consistent with those of previous studies.\nEndometriosis is a chronic disease that presents with recurring symptoms and is difficult to cure [30]. Available treatments for endometriosis are generally considered to be unsatisfactory [31]. Common medical treatments, such as gonadotropin-releasing hormone agonists, progestins, aromatase inhibitors, and androgens, are associated with various side effects and are not fully effective; the disease frequently returns [32]. Recently, a growing number of studies have identified plant-derived agents as a treatment option for endometriosis to achieve high efficacy and minimize adverse effects [33]. This study focused on the therapeutic effect of cepharanthine, an approved Traditional Chinese Medicine monomer, in treating endometriosis. Cepharanthine was purified by Kondo from Stephania cepharantha Hayata in 1934, then tested in humans, based on the traditional use of its original plant [7]. It has been widely used for the past seventy years to treat a variety of acute and chronic diseases, including radiation-induced leukopenia, venomous snakebites, HIV, and the novel coronavirus [4,34], with few known side effects. Studies have already reported various pharmacological effects of cepharanthine, such as antitumor [6,8,9], antimalarial [35], antioxidant [36], anti-allergic [35], and anti-inflammatory [37] effects. However, this drug was not previously tested for endometriosis.\nIn the present study, cepharanthine inhibited the viability of immortalized endometrial and patient-derived ectopic stromal cells in vitro. Recent studies have reported that newly developed endometriosis organoids function as patient-specific avatars by providing expandable biological materials suitable for drug screening [38]. Given that the eutopic endometrium of endometriosis patients exhibits marked changes in proliferation, adhesion, and angiogenesis compared with healthy endometrium [39], we generated patient-derived eutopic endometrial organoids to test the effects of cepharanthine treatment in vitro. Results showed that cepharanthine decreased the viability of endometrial organoids with an IC50 of 9.058 μM. We also established a mouse peritoneal endometriosis model and found that cepharanthine treatment prevented the formation of endometriosis in vivo. The formation and survival of endometriotic lesions at ectopic sites have been reported to be mainly dependent on biological processes such as proliferation and apoptosis [40,41,42], and the imbalance between cellular proliferation and cell death contributes to the tumor-like, uncontrolled growth of ectopic tissues. We therefore conducted a series of experiments to illustrate the potential mechanism of cepharanthine against endometriosis. The colony-formation assay indicated that the growth ability of the immortalized endometrial stromal cells was markedly inhibited by cepharanthine treatment. The anti-proliferation and apoptosis-inducing ability of cepharanthine was further confirmed by immunohistochemistry in vivo, as indicated by decreased Ki67 expression and increased TUNEL-positive nuclei inside the endometriotic lesions of cepharanthine-treated mice. Cellular apoptosis has been reported to be induced by the downregulation of anti-apoptotic proteins (e.g., Bcl-2) or the upregulation of pro-apoptotic proteins (e.g., Bax) [43]. Our Western Blot results indicated that cepharanthine upregulated cytochrome C in the cytoplasm, enhanced the expression of cleaved-caspase-9, cleaved-caspase-3, and Bax, and downregulated the expression of Bcl-2 in immortalized endometrial stromal cells, consistent with the previous study [44]. Furthermore, cepharanthine caused DNA damage, which was further confirmed by the elevated expression of γ-H2AX (a well‑established biomarker for DNA double-strand breaks [45] in cepharanthine-treated immortalized endometrial stromal cells and inside the endometriotic lesions of cepharanthine-treated mice). Previous bioinformatic analysis revealed that the elevated level of cyclin D1 is closely related to the pathogenesis of endometriosis [46]. Studies have also shown that the downregulation of cyclin D1 impairs cellular DNA repair [18,21,29,47]. Our in vitro test demonstrated that cepharanthine treatment downregulatedcyclin D1expression, thereby inducing G0/G1 phase cell-cycle arrest. We then checked cyclin D1 expression inside the endometriotic lesions of cepharanthine-treated mice. Consistent with the in vitro test, cepharanthine treatment downregulated cyclin D1, concurrently increased the expression of γ-H2AX, and decreased the expression of RAD51 (a protein critical in DNA repair), which was consistent with previous studies [18,21].\nTaken together, the mechanisms by which cepharanthine operates against endometriosis involve triggering apoptosis through the caspase-9/caspase-3 pathway, inducing cell cycle arrest, causing cellular DNA damage, and impairing DNA repair by downregulating cyclin D1 (Fig. 6).\nHowever, this study has limitations that must be acknowledged. First, although the mouse model is standard, it does not fully replicate the complex hormonal and immune microenvironment of human endometriosis. Second, our focus was primarily on stromal cells; the effect on epithelial cells within lesions warrants further investigation. Third, the precise upstream signaling responsible for cyclin D1 downregulation by cepharanthine remains to be elucidated. Investigations using more advanced models, such as patient-derived xenografts or humanized systems, would better predict clinical efficacy. Exploring potential synergies between cepharanthine and existing hormonal therapies could reveal combination strategies to prevent recurrence. Moreover, given the known anti-inflammatory properties of cepharanthine [36], its impact on the pelvic inflammatory milieu of endometriosis is a compelling avenue for study.\nIn our research, we verified the cepharanthine therapeutic potential for endometriosis in cell model, animal model and organoid derived from patients, but the clinical trial of this drug monomer on patients has not yet been conducted, and there is a lack of clinical data. The exploration of the pharmacological and mechanistic aspects of drug action will be carried out in future research.\nOur findings suggested that cepharanthine may serve as a promising cytotoxic agent for the treatment of endometriosis. As cepharanthine tablets are commercially available in Chinese pharmacies, this agent could be rapidly advanced into clinical trials, providing potential therapeutic benefits for patients with endometriosis.\nAll original data generated in this study are available within the manuscript. Further relevant information can be obtained from the corresponding author upon reasonable request.\nWXC researched the literature, conducted the experiments, and drafted the manuscript. HTL conducted literature searches and performed animal experiments. HXY supervised the experiments and conducted part of them. YingX did the surgeries and provided the samples. YangX conceptualized the study and edited the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.\nThe study was carried out in accordance with the guidelines of the Declaration of Helsinki. The use of human resected tissues for this experiment was approved by the Ethics Committee of West China Second University Hospital of Sichuan University on March 7th, 2018 (Ethical Approval No.: Medical Research 2018 No.48). Written informed consent was acquired from each participant before the sample was obtained. All animal procedures were approved by the Institutional Animal Care and Use Committee of West China Second University Hospital of Sichuan University (Approval No.2020028) and conducted in accordance with ethical standards and guidelines. All animal experimental procedures were conducted in accordance with the principles of the 3Rs (Replacement, Reduction, and Refinement).\nNot applicable.\nThis work was supported by a research grant from Nanchang University for Wenxi Chen.\nThe authors declare no conflicts of interest.\nSupplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/CEOG48879.\nReferences\nPublisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.","source_license":"CC0","license_restricted":false}