Gallic acid regulates autophagy to alleviate hydrogen peroxide-induced oxidative damage in granulosa cells

Gallic acid regulates autophagy to alleviate hydrogen peroxide-induced oxidative damage in granulosa cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gallic acid regulates autophagy to alleviate hydrogen peroxide-induced oxidative damage in granulosa cells Minghao Yu, Ruiyang Fan, Sheng-Mei Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8528931/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Autophagy is closely related to the development of the ovaries. Gallic acid (GA) exhibits various biological activities, including antioxidant, anti-inflammatory, and potential regulatory effects, on autophagy. However, the relationship of GA with autophagy and oxidative damage in granulosa cells (GCs) has not been elucidated. This study aimed to investigate the role of GA in alleviating hydrogen peroxide (H 2 O 2 )-induced oxidative damage in GCs via autophagy regulation.Herein, cell viability, oxidative stress (OS), inflammatory markers and reproductive-related hormones were measured to evaluate the effects of GA. Notably, GA alleviated H 2 O 2 -induced decrease in GC viability. Reactive oxygen species, malondialdehyde, tumor necrosis factor-α, interleukin (IL)-6, and IL-1β were all markedly increased in GCs after H 2 O 2 treatment. Moreover, this treatment reduced the levels of superoxide dismutase, catalase, total antioxidant capacity and reproductive-related hormone. After the H 2 O 2 treatment, terminal deoxynucleotidyl transferase dUTP nick end labeling staining demonstrated a significant rise in cell death. However, GA treatment inhibited H 2 O 2 -induced oxidative damage, inflammation, and apoptosis. Western blotting analysis for evaluating the effect of GA on autophagy by detecting autophagy-related protein levels and related pathways revealed that GA reduced the H 2 O 2 -induced increase in autophagy. Altogether, the results of this study show that GA alleviates OS and cell death in GCs by regulating autophagy, Therefore, from the standpoint of autophagy control, GA might contribute to ovarian damage prevention. gallic acid ovarian granulosa cells oxidative stress autophagy hydrogen peroxide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1.Introduction The ovary is an important reproductive organ in females and is involved in multiple functions such as oocyte production, sex hormone secretion, and estrous cycle maintenance (Yang et al., 2021 ). Ovarian granulosa cells (GCs), the ovarian somatic cells surrounding oocytes, play a vital role in normal follicular growth and development (Liu et al., 2023 ) by promoting follicle development and supporting normal oocyte growth through nutrient supply and signal transmission (Alam and Miyano, 2020 ). Ovarian GCs have been widely employed to study ovarian endocrine and reproductive functions in vitro, with many studies reporting oxidative stress (OS)-induced pathological damage in the ovarian microenvironment, which can further lead to GC apoptosis in follicles and follicular atresia, thereby damaging oocytes (Gao et al., 2023 ). OS is caused by excessive reactive oxygen species (ROS) production in cells, which then damages cellular proteins, lipids, and DNA, ultimately leading to cell necrosis and apoptosis (Xie et al., 2023 ). Cellular ROS accumulation can damage many key physiological functions in females, such as oocyte maturation, ovulation, luteolysis, and luteal maintenance during pregnancy (An et al., 2021 ). Hydrogen peroxide (H 2 O 2 ) is a common OS inducer, and H 2 O 2 -treated cells have been shown to produce large amounts of ROS in a short time, which, in turn, induce apoptosis (An et al., 2021 ). Conversely, antioxidants may reduce the negative impact of OS on GCs via reduced ROS production and activity by increasing antioxidant gene expression and restoring mitochondrial function (An et al., 2021 ). Therefore, identifying suitable antioxidants to repair OS damage and improve OS resistance in GCs is crucial. Autophagy is an important intracellular mechanism involved in maintaining cellular homeostasis and responding to destructive events, such as clearance of long-lived, aggregated, and misfolded proteins and damaged organelles (Kumariya et al., 2021 ). Various classical pathways, including phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) can induce autophagy (Bhardwaj et al., 2022 ). Autophagy in ovarian GCs and oocytes is critically implicated in follicular development, primordial follicle pool formation, follicular recruitment, and follicular atresia (Tong et al., 2022 ), and regulation of GC differentiation, with insufficient autophagy being associated with ovarian dysfunction (Xu et al., 2022 ). For example, in the GCs of patients with polycystic ovary syndrome (PCOS), Beclin 1 (BECN1) and autophagy-related gene 7 expression was markedly increased, suggesting the involvement of abnormal activation of GC autophagy in abnormal ovary development (Liu et al., 2021 ). Furthermore, GC autophagy inhibition through the PI3K/AKT/mTOR pathway has been shown to normalize GC proliferation, improve abnormal follicular development, and alleviate ovulation disorders in rats with PCOS (Kobayashi et al., 2020 ). Follicle atresia could result from GC apoptosis, induced by increased autophagy and imbalanced internal homeostasis. which indicates that regulation of GC autophagy may provide a new strategy to improve follicular development. Brandt's vole ( Lasiopodomys brandtii ), belonging to the Cricetidae family of Rodentia, mainly inhabits Inner Mongolia, Mongolia, and other degraded areas of pasture (Yu et al., 2024a ). They play an important role in the grassland ecosystems and feed on tannin-containing plants, including tannic acid (TA) (Yu et al., 2024b ). Reportedly, TA can affect the reproductive performance of Brandt's voles; however, its mechanism of action remains unknown (Yu et al., 2021 ). TA decomposes into gallic acid (GA) and glucose in the digestive tract of mammals (Baldwin and Booth, 2022 ). GA is a phenolic compound and a plant secondary metabolite that is widely present in nature and has a strong antioxidant capacity (Baldwin and Booth, 2022 ). Notably, phenolic compounds can affect cellular autophagy, apoptosis, and the reproductive capacity of animals (Zahedi et al., 2025 ). In this study, we aimed to create an OS model of GCs by examining how GA affect OS in the ovarian GCs of Brandt's voles that received H 2 O 2 treatment. GCs with oxidative damage displayed the effects of GA on ROS, cell survival, antioxidant function, apoptosis, and autophagy. Additionally, the underlying mechanisms of GA in alleviating H 2 O 2 -induced oxidative damage in GCs were explored at the cellular level. 2.Materials and methods 2.1.Animals Herein, 3-week-old female Brandt's voles were obtained from a grassland in Inner Mongolia and bred in pairs in the animal breeding room of the Ecology Laboratory of Yangzhou University under a 12L:12D photoperiod and 22 ± 1°C. The voles were subcutaneously injected with 8 IU of pregnant mare serum gonadotropin (Ningbo No. 2 Hormone Factory). All of the voles were sacrificed by cervical dislocation after 48 h, and their ovaries were separated for further in vitro testing. 2.2.Isolation and culture of ovarian GCs GCs were isolated from the mature follicles on the ovaries of Brandt's voles by puncturing them with a skin test needle, and the follicular fluid that spilled over was collected. A GC solution was prepared, filtered through a 200-mesh cell sieve, and centrifuged once more for five min at 1000 rpm to extract GCs. These were then resuspended in DMEM/F12. Prior to cell treatments, the cells were cultured for two days at 37°C with 5% CO2 in DMEM/F12 supplemented with 10% fetal bovine serum (FBS; HyClone, USA) and 1% penicillin/streptomycin. GCs were used to establish a damage model by treatment with H 2 O 2 (China Hushi) for 24 h. GA was purchased from Aladdin, China, and GCs were pretreated with GA for 24 h prior to subsequent experiments to study its effects on cell damage and autophagy. 2.3.Identification of GCs GCs cultured for 48 h were digested with 0.25% trypsin; the digestion was terminated by adding DMEM/F12. Then they were seeded into 24-well plates with small coverslips pre-placed in the wells.. After achieving 70% confluence, the slides were removed, and the cells were stained for immunofluorescence. The cells were treated with the main antibody, follicle-stimulating hormone receptor (FSHR) rabbit polyclonal antibody (1:200), Thereafter, they were incubated overnight at 4°C. The secondary IgG antibody was added and the samples were incubated for 60 min at 37 C in the dark following three PBS washes (5 min each). Three PBS washes (5 min each) were performed on the cells. Red fluorescence was excited with a halogen lamp under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) to observe fluorescence. 2.4.Cell viability assay The Cell Viability Kit-8 (CCK-8) (Vazyme, China) was used to measure cell viability. Briefly, GCs were seeded into 96-well plates and treated with the medicines listed above. After adding 10 µL of the CCK-8 reagent to each well, the cells were cultured in the cell culture box for 2 h at 37°C with 5% CO2. The optical density (OD) of each well was measured at 450 nm, and the average OD for each treatment was used to assess cell viability. 2.5.Analysis of antioxidant defense mechanism of GCs Using matched detection kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China), the activities of total antioxidant capacity (T-AOC), catalase (CAT), and superoxide dismutase (SOD) as well as the malondialdehyde (MDA) levels were assessed in accordance with the manufacturer's instructions. 2.6.Determination of cytokine levels As instructed by the manufacturer, ELISA kits (Jianglai Biological Technology Ltd., Shanghai, China) were used to quantify the cytokine levels of IL-6, TNF-α, IL-1β, E2, AMH and progesterone. In summary, an ELISA plate was coated with a standard or sample, then with the horseradish peroxidase-tagged detection antibody. The plate was then incubated for 60 min at 37 C. Next, substrates A and B were added, and the mixture was incubated for 15 min at 37°C in the dark. The termination solution was used to halt the reaction, and a microplate reader was used to measure the OD at 450 nm. The intra- and inter-assay coefficients of variance for all three hormones and cytokines were below 9% and 11%, respectively. 2.7.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay GC apoptosis was measured by the TUNEL Cell Apoptosis Detection Kit (Beyotime Biotechnology, China). In short, the cells were permeabilized with 0.5% Triton X-100 for 5 min after being fixed with 4% paraformaldehyde at room temperature. The cells were rinsed with PBS, then rehydrated and exposed to the TUNEL reaction mixture, consisted of an enzyme-to-labeling solution ratio of 1:9, for 1h at 37°C in the dark. To identify nuclei, GCs were subsequently stained with 4′,6-diamidino-2-phenylindole for five minutes in the dark. TUNEL-positive GCs were photographed after washing using an Olympus fluorescent microscope. 2.8.Western blotting For western blotting, cell pellets were lysed and centrifuged to collect total protein from the supernatant. The bicinchoninic acid test was used to determine the protein content. The materials were denatured and electrophoresed after adding the loading buffer before being deposited to the membranes. Tris-buffered saline with Tween 20 (TBST) was used to wash the membranes thrice after transfer. The blocking solution (1×5000) was used to dilute the secondary antibody. After agitating for 2 h at room temperature and rinsing thrice with TBST, the polyvinylidene fluoride membrane was placed in the working solution. For chromogenic reactions, the enhanced chemiluminescence chromogenic solution was used, and ImageJ 6.0 was used for band analysis. 2.9.ROS detection To detect ROS, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was used. Following the removal of the cell culture medium and three PBS washes, DCFH-DA (1:1000) had been diluted to a final dosage of 10 µM and added to the cells. After 20 min of incubation at 37°C, the cells were examined and imaged using a fluorescent microscope. 2.10.Statistical analyses In this study, group differences were compared using Tukey's post-hoc test after one-way analysis of variance. GraphPad Prism (version 8.0) was used to conduct the graphic analysis. A significance level of P < 0.05 was established. SPSS was used for all statistical analyses (version 26.0; SPSS Inc., Chicago, Illinois, USA). 3.Results 3.1.Identification of Brandt's vole ovarian GCs Hematoxylin–eosin (HE) staining showed that ovarian GCs were polygonal or fusiform (Fig. 1 A), and they expressed FSHR. The results of FSHR immunocytochemical fluorescence staining revealed considerable red fluorescence under fluorescence excitation by the halogen light source, indicating FSHR-positive staining in the GCs (Fig. 1 B). The average positive expression rate of FSHR in the GCs reached up to 90%, which met the requirements for subsequent experiments. 3.2.Effect of GA on the viability of GCs with oxidative damage 3.2.1.Establishment of OS injury model of GCs To assess the H 2 O 2 -induced oxidative damage in GCs, ovarian GCs were treated with H 2 O 2 at concentrations of 10, 50, 100, 150, 200, and 250 µM for 24 h, and the CCK-8 test was used to evaluate cell viability. Furthermore, 200 µM H 2 O 2 dramatically reduced cell viability compared to the control (P < 0.01). (Fig. 2 ). Therefore, 200 µM H 2 O 2 was selected to establish the cell oxidative damage model. 3.2.2.Effect of GA on cell viability The effect of GA on the activity of ovarian GCs was assessed by treating GCs with GA at concentrations of 6, 12, 25, 50, 100, 200, and 400 µM for 24 h, and the cell viability was detected through the CCK-8 assay. Overall, no discernible alteration was observed between the cell viability of control and that of the 6–100 µM GA treatment groups, and the cell viability decreased significantly when the concentration increased to 200 µM (Fig. 3 ). Therefore, the concentration range of 6–100 µM was selected for subsequent concentration screening. 3.2.3.Protective effect of GA on oxidative damage in cells The protective effects and the optimal concentration of GA for alleviating oxidative damage in GCs were determined by treating the cells with different concentrations of GA (6, 12, 25, 50, and 100 µM) for 24 h, and then subjecting the pretreated cells to 200µM H 2 O 2 for 24 h to induce damage. The CCK-8 assay was employed to assess cell viability. Notably, compared with the damage model group, different concentrations of GA improved cell viability to different degrees, thereby confirming that GA could protect GCs from oxidative damage. The highest cell viability was detected with 50 µM GA (Fig. 4 ); therefore, this concentration was selected for subsequent experiments. 3.3.The effect of GA on apoptosis in cells with oxidative damage Apoptosis was detected using the TUNEL test. In this instance, the GC apoptosis rate of the H 2 O 2 model group was noticeably higher than that of the control group (P < 0.01). However, GA pretreatment markedly reduced the apoptosis of GCs with oxidative damage (P < 0.01) (Fig. 5 ). 3.4.Effects of GA on T-AOC, CAT, SOD, MDA, and ROS in GCs with oxidative damage The effects of H 2 O 2 on the levels of T-AOC, CAT, SOD, and MDA were detected in GCs. Notably, the activities of T-AOC, CAT, and SOD in the H 2 O 2 model group substantially decreased, whereas MDA levels increased. However, GA pretreatment significantly reversed these phenomena by increasing the activities of T-AOC, SOD, and CAT, and decreasing MDA levels (P < 0.01) (Fig. 6 A). The DCFH-DA fluorescent probe was used to detect cellular ROS levels, which were significantly increased in the H 2 O 2 model group, whereas GA pretreatment significantly decreased ROS levels (Fig. 6 B). 3.5.The effect of GA on TNF-α, IL-6, and IL-1β in cells with oxidative damage Compared to the control, the TNF-α, IL-6, and IL-1β activities in the H 2 O 2 model group were considerably higher. However, after GA pretreatment, this tendency was considerably reversed, resulting in lower TNF-α, IL-6, and IL-1β levels compared to those in the model group (P < 0.05) (Fig. 7 ). 3.6.The effect of GA on E2,AMH and P4 in cells with oxidative damage Compared with the control group, the activities of E2, AMH and P4 in the H 2 O 2 model group were significantly decreased (P < 0.05). However, after GA pretreatment, this trend was significantly reversed, resulting in its E2, AMH and P4 levels being higher than those of the model group (P < 0.01) (Fig. 8 ). 3.7.Effect of GA on the expression of autophagy-related proteins Western blotting revealed the expression profile of the autophagy-related proteins BECN1, light chain 3 (LC3)AB, and sequestosome 1 (P62/SQSTM1), along with that of the autophagy pathway-related proteins AKT, phosphorylated AKT (P-AKT), mTOR, and phosphorylated mTOR (P-mTOR). In the injury model group, P-AKT and P-mTOR levels were significantly decreased. GA pretreatment significantly increased P-AKT and P-mTOR levels (P < 0.01). Compared with the control group, the expression of BECN1 and LC3BⅡ/Ⅰ in the model group were was markedly upregulated, whereas that of P62 was decreased. After pretreatment with GA, the expression of BECN1 and LC3Ⅱ/Ⅰ significantly decreased, and that of P62 significantly increased (P < 0.01) (Fig. 9 ). 3.8.Effect of GA on cell lysosomes Herein, a LysoTracker Red fluorescent probe was used to detect intracellular autophagolysosomes. The results showed that H 2 O 2 -induced OS markedly increased the number of autolysosomes and the level of autophagy compared with those in the control group. However, GA pretreatment significantly reversed H 2 O 2 -induced damage and reduced the number of autolysosomes (Fig. 10 A). Monodansylcadaverine (MDC) staining was used to observe autophagosomes, which emitted green fluorescence. Notably, the number of autophagosomes in the cells increased after H 2 O 2 treatment, whereas GA pretreatment decreased their number (Fig. 10 B).The schematic diagram illustrating the mechanism by which gallic acid improves oxidative damage in ovarian granulosa cells is shown in Fig. 11 . Gallic acid regulates autophagy through the AKT-mTOR pathway to improve oxidative damage. 4.Discussion GCs play a critical role in maintaining follicular development and female physiology by providing physical support and creating a favorable microenvironment by secreting various growth factors and hormones (Zeng et al., 2023 ). Consequently, GC dysfunction can lead to abnormal follicular development and ovulation, thereby affecting female fertility (Zhang et al., 2023 ). Herein, in vitro primary culture of ovarian GCs of Brandt's voles were employed to observe cell morphology by HE staining, and FSHR expression was detected in the cells by immunofluorescence. Notably, the purity of the isolated GCs was > 90%, making them suitable for subsequent experiments. Reportedly, excessive ROS can oxidatively damage intracellular macromolecules (such as proteins and lipids) (Yan et al., 2022 ). Therefore, cells were treated with different concentrations of H 2 O 2 in this study, and their cell viability was determined through the CCK-8 assay. Cell viability gradually decreased with increasing H 2 O 2 concentration, with the most significant decrease observed at 200 µM H 2 O 2 . Therefore, 200 µM H 2 O 2 was selected to induce OS for 24 h to successfully establish the OS model of GCs. Additionally, GA was found to exhibit notable safety and efficacy in most cells at low concentrations, albeit it was toxic at relatively high concentrations. These results indicated the low toxicity profile of GA in vivo. Herein, GA-treated cells (24 h) did not exhibit any significant change in cell viability at concentrations < 100 µM, but at 200 µM, the cell activity markedly decreased. Therefore, 6–100 µM GA was considered a safe concentration range for GCs. Consequently, the protective effects of different concentrations of GA against oxidative damage were screened, and compared with the oxidative damage group, GA pretreatment at 50 µM for 24 h was shown to significantly increase the cell viability after 200 µM H 2 O 2 treatment for 24 h. Next, cell apoptosis was evaluated through TUNEL assay. Our results demonstrated that the H 2 O 2 treatment markedly increased apoptosis, whereas the rate of apoptosis decreased significantly after GA pretreatment. GA can be easily obtained by hydrolyzing tannins under alkaline or acidic conditions, which are present in many plants (Xu et al., 2021 ). Reportedly, The phenolic hydroxyl groups of GA display the ability to scavenge already existing ROS and prevent further production (Sohrabi et al., 2021 ), thereby inhibiting the oxidation of associated lipids, DNA, proteins, and enzymes (Shabani et al., 2020 ). In the present study, GA pretreatment markedly inhibited H 2 O 2 -induced ROS over-accumulation, which is consistent with the findings of previous studies. The enzymatic antioxidant system is an important free radical scavenging system in the body (Esmaeilzadeh et al., 2020 ). Consistently, these results showed that H 2 O 2 -induced oxidative damage subsequently decreased T-AOC, CAT, and SOD activities, along with increasing MDA levels, which were reversed by GA pretreatment. Taken together, our results imply that GA shields GCs from oxidative damage. TNF-α is an inflammatory factor that stimulates the proliferation of theca cells, thereby playing a vital role in regulating normal ovarian activity and follicular development (Tylutka et al., 2024 ). Additionally, IL-6 and IL-1β, as inflammatory cytokines, can produce ROS by up-regulating nicotinamide adenine dinucleotide phosphate oxidase (Akhtar et al., 2020 ), along with reducing the activity of aromatase in follicles, thereby resulting in a decrease in estradiol concentration, fertility, and fertilization ability in follicles (Alkhuriji et al., 2020 ). The results of this study demonstrated that GA pretreatment reduced the rise in TNF-α, IL-6, and IL-1β levels in the H 2 O 2 -induced GC damage model, which were considerably higher. This indicated that GA can alleviate the OS-induced increase in inflammatory factor levels in GCs. Autophagy is a highly conserved catabolic process regulated by multiple autophagy-related genes that can respond to different environments and orderly degrade and utilize cell components (Ding et al., 2024 ). With increasing basal autophagy levels, cellular ROS clearance is promoted to protect cells, albeit an excessive increase in autophagy levels can cause autophagy disorder and programmed cell death (Duan et al., 2024 ). Herein, BECN1 and LC3 expression in GCs was upregulated after H 2 O 2 treatment, whereas that of P62 protein decreased. However, treatment with GA reduced the expression of BECN1 and LC3 and increased that of P62. Lysosomes are the final effector organelles responsible for autophagic degradation, and their number and quality can represent autophagy activity (Ommati et al., 2025 ). Herein, LysoTracker Red, a red fluorescent probe, was used for specific fluorescent staining of lysosomes in living cells. Lysosomes were observed under a fluorescence microscope, where red fluorescence accumulated, with the relative fluorescence intensity and expression indirectly reflecting the quality and quantity of lysosomes. Furthermore, MDC, an acidophilic dye, was used to label acidic endosomes, lysosomes, and late autophagosomes. The qualitative assessment of these autophagosomes indicated their significant accumulation in GCs after H 2 O 2 treatment. However, by altering autophagy-related protein expression, GA pretreatment might lessen the H 2 O 2 -induced autophagy increase. Numerous studies have examined ovarian damage-related autophagy and apoptosis, and ovarian cell apoptosis has been shown to cause extensive follicular atresia or regression (Dou et al., 2024 ). Reportedly, both autophagy and apoptosis regulate ovarian follicular development and atresia (Jin et al., 2024 ). Notably, H 2 O 2 can induce ROS production and activate the mitochondrial apoptosis pathway (Wang et al., 2025 ). Activation of the PI3K/AKT pathway has been closely related to cell proliferation, differentiation, apoptosis, and autophagy, and is a well-recognized survival signal transduction pathway (Liu et al., 2024 ). The PI3K/AKT/mTOR pathway has been extensively studied in association with GC autophagy (Zhou et al., 2024 ). Although the study of autophagy has considerably advanced in recent years, studies on autophagy and the signal transduction regulation mechanism of ovarian GCs remain limited and are worth exploring. The results of this study show that the oxidative damage model established by H 2 O 2 induced autophagy and apoptosis in granulosa cells. H 2 O 2 regulates autophagy by activating the AKT/mTOR signaling pathway. Pretreatment with GA can reduce the levels of autophagy and apoptosis in granulosa cells in ovarian injury. 5.Conclusion Our results showed that gallic acid alleviated H2O2-induced granulosa cell injury by increasing anti-oxidation, anti-inflammation, and decreasing excessive autophagy and apoptosis levels, and provided new insights into the prevention of ovarian oxidative damage from a molecular perspective. Declarations Funding This work was supported by the National Natural Science Foundation of China [grant number 31971418] . Author contributions Ming-Hao Yu: Formal analysis, investigation, and conceptualization. Rui-Yang Fan: Composing, editing, and reviewing. Sheng-Mei Yang: Composing, editing, and reviewing. Each author has read and approved the study. Declaration of competing interests The authors declare no competing interests. IRB approval All procedures performed in studies involving animals were approved by the Animal Care and Use Committee of Yangzhou University (No. SJXY-2).This study was performed in line with the principles of the Declaration of Helsinki. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments. Data statement The data will be made available upon request. References Akhtar M, Guo S, Guo Y-f, Zahoor A, Shaukat A, Chen Y, Umar T, Deng G, Guo M (2020) Upregulated-gene expression of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) via TLRs following NF-κB and MAPKs in bovine mastitis. Acta tropica 207: 105458, DOI: 10.1016/j.actatropica.2020.105458 Alam MH, Miyano T (2020) Interaction between growing oocytes and granulosa cells in vitro. Reproductive medicine and biology 19: 13-23, DOI: 10.1002/rmb2.12292 Alkhuriji AF, Al Omar SY, Babay ZA, El-Khadragy MF, Mansour LA, Alharbi WG, Khalil MI (2020) Association of IL-1β, IL-6, TNF-α, and TGFβ1 gene polymorphisms with recurrent spontaneous abortion in polycystic ovary syndrome. Disease Markers 2020: 6076274, DOI: 10.1155/2020/6076274 An R, Wang X, Yang L, Zhang J, Wang N, Xu F, Hou Y, Zhang H, Zhang L (2021) Polystyrene microplastics cause granulosa cells apoptosis and fibrosis in ovary through oxidative stress in rats. Toxicology 449: 152665, DOI: 10.1016/j.tox.2020.152665 Baldwin A, Booth BW (2022) Biomedical applications of tannic acid. J Biomater Appl 36: 1503-1523, DOI: 10.1177/08853282211058099. Bhardwaj JK, Paliwal A, Saraf P, Sachdeva SN (2022) Role of autophagy in follicular development and maintenance of primordial follicular pool in the ovary. Journal of cellular physiology 237: 1157-1170, DOI: 10.1002/jcp.30613 Ding Y, Huang X, Ji T, Qi C, Gao X, Wei R (2024) The emerging roles of miRNA-mediated autophagy in ovarian cancer. Cell Death Dis 15: 314, DOI: 10.1038/s41419-024-06677-8. Dou J, Wu Y, Hu R, Liu J, Zhang Y, Zhen X, Wu T, Zhang C, Liu Y, Zheng R (2024) Quinoa ameliorates polycystic ovary syndrome via regulating gut microbiota through PI3K/AKT/mTOR pathway and autophagy. Nutrition & Metabolism 21: 80, DOI: 10.1186/s12986-024-00855-3 Duan H, Yang S, Yang S, Zeng J, Yan Z, Zhang L, Ma X, Dong W, Zhang Y, Zhao X, Hu J, Xiao L (2024) The mechanism of curcumin to protect mouse ovaries from oxidative damage by regulating AMPK/mTOR mediated autophagy. Phytomedicine 128: 155468, DOI: 10.1016/j.phymed.2024.155468. Esmaeilzadeh M, Heidarian E, Shaghaghi M, Roshanmehr H, Najafi M, Moradi A, Nouri A (2020) Gallic acid mitigates diclofenac-induced liver toxicity by modulating oxidative stress and suppressing IL-1β gene expression in male rats. Pharmaceutical biology 58: 590-596, DOI: 10.1080/13880209.2020.1777169 Gao Y, Zou Y, Wu G, Zheng L (2023) Oxidative stress and mitochondrial dysfunction of granulosa cells in polycystic ovarian syndrome. Frontiers in Medicine 10: 1193749, DOI: 10.3389/fmed.2023.1193749 Jin R, Chen A, Ye Y, Ren Y, Lu J, Xuan F, Zhou W (2024) Effect of berberine combined with metformin on autophagy in polycystic ovary syndrome by regulating AMPK/AKT/mTOR pathway. Mol Reprod Dev 91: e23768, DOI: 10.1002/mrd.23768. Kobayashi M, Yoshino O, Nakashima A, Ito M, Nishio K, Ono Y, Kusabiraki T, Kunitomi C, Takahashi N, Harada M (2020) Inhibition of autophagy in theca cells induces CYP17A1 and PAI-1 expression via ROS/p38 and JNK signalling during the development of polycystic ovary syndrome. Molecular and Cellular Endocrinology 508: 110792, DOI: 10.1016/j.mce.2020.110792 Kumariya S, Ubba V, Jha RK, Gayen JR (2021) Autophagy in ovary and polycystic ovary syndrome: role, dispute and future perspective. Autophagy 17: 2706-2733, DOI: 10.1080/15548627.2021.1938914 Liu M, Zhu H, Zhu Y, Hu X (2021) Guizhi Fuling Wan reduces autophagy of granulosa cell in rats with polycystic ovary syndrome via restoring the PI3K/AKT/mTOR signaling pathway. Journal of Ethnopharmacology 270: 113821, DOI: 10.1016/j.jep.2021.113821 Liu S, Jia Y, Meng S, Luo Y, Yang Q, Pan Z (2023) Mechanisms of and potential medications for oxidative stress in ovarian granulosa cells: a review. International Journal of Molecular Sciences 24: 9205, DOI: 10.3390/ijms24119205 Liu W, Liu H, Zhang S, Hao H, Meng F, Ma W, Guo Z, Jiang S, Shang X (2024) Silica nanoparticles cause ovarian dysfunction and fertility decrease in mice via oxidative stress-activated autophagy and apoptosis. Ecotoxicol Environ Saf 285: 117049, DOI: 10.1016/j.ecoenv.2024.117049. Ommati MM, Zuo Q, Sabouri S, Retana-Marquez S, Nategh Ahmadi H, Gholami A, Eftekhari A, Shojaei S, Lijuan L, Heidari R (2025) Fluoride-induced autophagy and apoptosis in the mouse ovary: genomic insights into IL-17 signaling and gut microbiota dysbiosis. Journal of Agricultural and Food Chemistry 73: 2138-2155, DOI: 10.1021/acs.jafc.4c10165 Shabani S, Rabiei Z, Amini-Khoei H (2020) Exploring the multifaceted neuroprotective actions of gallic acid: A review. International Journal of Food Properties 23: 736-752, DOI: 10.1007/s11011-019-00400-9 Sohrabi F, Dianat M, Badavi M, Radan M, Mard SA (2021) Gallic acid suppresses inflammation and oxidative stress through modulating Nrf2-HO-1-NF-κB signaling pathways in elastase-induced emphysema in rats. Environmental Science and Pollution Research 28: 56822-56834, DOI: 10.1007/s11356-021-14513-1 Tong C, Wu Y, Zhang L, Yu Y (2022) Insulin resistance, autophagy and apoptosis in patients with polycystic ovary syndrome: Association with PI3K signaling pathway. Frontiers in endocrinology 13: 1091147, DOI: 10.3389/fendo.2022.1091147 Tylutka A, Walas Ł, Zembron-Lacny A (2024) Level of IL-6, TNF, and IL-1β and age-related diseases: A systematic review and meta-analysis. Frontiers in immunology 15: 1330386, DOI: 10.3389/fimmu.2024.1330386 Wang S, Ou Y, Cao S, Sun X, Qin N, Liswaniso S, Xu R (2025) Effects of Melatonin on H2O2-Induced Oxidative Damage of the Granulosa Cells in Hen Ovarian Follicles. Genes 16: 362, DOI: 10.3390/genes16040362 Xie Q, Hong W, Li Y, Ling S, Zhou Z, Dai Y, Wu W, Weng R, Zhong Z, Tan J (2023) Chitosan oligosaccharide improves ovarian granulosa cells inflammation and oxidative stress in patients with polycystic ovary syndrome. Frontiers in Immunology 14: 1086232, DOI: 10.3389/fimmu.2023.1086232 Xu B, Dai W, Liu L, Han H, Zhang J, Du X, Pei X, Fu X (2022) Metformin ameliorates polycystic ovary syndrome in a rat model by decreasing excessive autophagy in ovarian granulosa cells via the PI3K/AKT/mTOR pathway. Endocrine Journal 69: 863-875, DOI: 10.1507/endocrj.EJ21-0480 Xu Y, Tang G, Zhang C, Wang N, Feng Y (2021) Gallic Acid and Diabetes Mellitus: Its Association with Oxidative Stress. Molecules 26: 7115, DOI: 10.3390/molecules26237115. Yan F, Zhao Q, Li Y, Zheng Z, Kong X, Shu C, Liu Y, Shi Y (2022) The role of oxidative stress in ovarian aging: a review. J Ovarian Res 15: 100, DOI: 10.1186/s13048-022-01032-x. Yang L, Chen Y, Liu Y, Xing Y, Miao C, Zhao Y, Chang X, Zhang Q (2021) The role of oxidative stress and natural antioxidants in ovarian aging. Frontiers in Pharmacology 11: 617843, DOI: 10.1186/s13048-022-01032-x Yu M, Fan R, Wang D, Han Y, Dai X, Yang S-M (2024a) Tannic acid alleviates 3-nitropropionic acid-induced ovarian damage in Brandt’s vole (Lasiopodomys brandtii). Reproductive Sciences 31: 2261-2272, DOI: 10.1007/s43032-024-01543-6 Yu M, Fan R, Yang S-M (2024b) Effect of tannic acid on adiponectin and gonads in male Brandt’s voles (Lasiopodomys brandtii). General and Comparative Endocrinology 357: 114592, DOI: 10.1016/j.ygcen.2024.114592 Yu M, Sun X, Dai X, Gu C, Gu M, Wang A, Wei W, Yang S (2021) Effects of tannic acid on antioxidant activity and ovarian development in adolescent and adult female Brandt’s voles. Reproductive Sciences 28: 2839-2846, DOI: 10.1007/s43032-021-00578-3 Zahedi F, Kowsar R, Khodabandeh Z, Dara M, Alaee S (2025) Protective Effects of Gallic Acid Against Lead Acetate‐Induced Toxicity in Mice Ovary: Focus on Apoptosis, Inflammation, and Folliculogenesis. Food Science & Nutrition 13: e70638, DOI: 10.1002/fsn3.70638 Zeng L, Zhou C, Xu W, Huang Y, Wang W, Ma Z, Huang J, Li J, Hu L, Xue Y, Luo T, Zheng L (2023) The ovarian-related effects of polystyrene nanoplastics on human ovarian granulosa cells and female mice. Ecotoxicol Environ Saf 257: 114941, DOI: 10.1016/j.ecoenv.2023.114941. Zhang CH, Liu XY, Wang J (2023) Essential Role of Granulosa Cell Glucose and Lipid Metabolism on Oocytes and the Potential Metabolic Imbalance in Polycystic Ovary Syndrome. Int J Mol Sci 24: 16247, DOI: 10.3390/ijms242216247. Zhou XY, Lai YH, Zhang J, Li Y, Wu XM, Yang YZ, Zhang XF, Ma LZ, Zheng KM, Liu YD, Wang Z, Chen SL (2024) Advanced oxidation protein products attenuate the autophagy-lysosome pathway in ovarian granulosa cells by modulating the ROS-dependent mTOR-TFEB pathway. Cell Death Dis 15: 161, DOI: 10.1038/s41419-024-06540-w. Additional Declarations No competing interests reported. Supplementary Files AKT.jpg gapdh12.21.jpg 10.10pmtor11.jpg gapdh4.jpg mtor2.jpg 7.png 1.png 1.tif 2.png 2.tif 3.png 4.png 5.png 6.png 7.png 8.png 9.png 10.png 12.png 13.png 14.png 15.png 16.png 12.21PAKT.jpg 12.21BECLIN1.tif 12.26LC3.tif 11.27p624.jpg 10.10pmtor11.jpg 12.26gapdh.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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(B) Identification of ovarian GCs; red fluorescence, follicle-stimulating hormone receptor (FSHR)-positive cells; blue fluorescence, 4′,6-diamidino-2-phenylindole-stained nuclei. FSHR expression can be observed on the cell membrane.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/49e16f0ebbf2fa99024a9d16.png"},{"id":100151632,"identity":"cedfc6f4-ef31-48de-9edf-0819587dcfc8","added_by":"auto","created_at":"2026-01-13 13:30:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39482,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of hydrogen peroxide (H2O2) on the viability of granulosa cells\u003c/p\u003e\n\u003cp\u003eCells were treated with different concentrations of H2O2 (10–250 μM) for 24 h. Cell viability was measured using the Cell-Counting Kit 8 assay.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± standard error of the mean.** P \u0026lt; 0.01; *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/f96fa9df0f79e7f8a11ff527.png"},{"id":100367353,"identity":"c8cf425d-9ced-4ddb-ac17-f3483fe2b509","added_by":"auto","created_at":"2026-01-16 07:57:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43235,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of gallic acid (GA) on cell viability\u003c/p\u003e\n\u003cp\u003eCells were treated with different concentrations of GA (6–400 μM) for 24 h. Cell viability was measured using the Cell-Counting Kit 8 assay. Data are presented as mean ± standard error of the mean. ** P \u0026lt; 0.01; *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/21ed6a52b4630d0be0485376.png"},{"id":100151623,"identity":"6c58dfbf-f948-44b0-b5e5-ac593af2d6d2","added_by":"auto","created_at":"2026-01-13 13:30:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43828,"visible":true,"origin":"","legend":"\u003cp\u003eProtective effect of gallic acid (GA) against oxidative damage in cells\u003c/p\u003e\n\u003cp\u003eCells were pretreated with different concentrations of GA (6–100 μM) for 24 h, followed by exposure to 200 μΜ H2O2 for 24 h. Cell viability was measured using the Cell-Counting Kit 8 assay. Data are presented as mean ± standard error of the mean. ** P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/9d914c526b9e4f77a39cd530.png"},{"id":100367485,"identity":"9754df86-454c-40ca-83dd-8c638a1900f7","added_by":"auto","created_at":"2026-01-16 07:57:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3336329,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of gallic acid on cell apoptosis assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay\u003c/p\u003e\n\u003cp\u003e(A) Fluorescence between groups was observed under confocal microscope. (B) Fluorescence intensity ratio between groups. ** P \u0026lt; 0.01 versus Control; ## P \u0026lt; 0.01 versus H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/076a4f87de3aecc83fb53ae1.png"},{"id":100368308,"identity":"02e9321e-2d9b-4c75-85d8-6770f079189c","added_by":"auto","created_at":"2026-01-16 07:57:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2972568,"visible":true,"origin":"","legend":"\u003cp\u003eGallic acid (GA) can alleviate hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)-induced oxidative damage of granulosa cells\u003c/p\u003e\n\u003cp\u003e(A) Effect of GA on total antioxidant capacity, catalase, superoxide dismutase, and malondialdehyde levels in oxidative stress-injured cells. (B) Cell staining after treatment with GA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for detecting reactive oxygen species (ROS); the intracellular ROS levels of each group were observed under a confocal microscope. The data are mean ± standard error of the mean. ## P \u0026lt; 0.01 versus Control, ** P \u0026lt; 0.01 versus H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/3c19d6380c937a7d25608624.png"},{"id":100151638,"identity":"94be01f4-b00b-4928-ac8c-17955ea79b06","added_by":"auto","created_at":"2026-01-13 13:30:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":100227,"visible":true,"origin":"","legend":"\u003cp\u003eGallic acid alleviates hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)-induced inflammatory damage in granulosa cells\u003c/p\u003e\n\u003cp\u003eThe Inflammatory cytokines levels of (A) tumor necrosis factor-α; (B) interleukin (IL)-6; and (C) IL-1β. Data are presented as mean ± standard error of the mean. # P \u0026lt; 0.05 versus Control; ## P \u0026lt; 0.01 versus Control; * P \u0026lt; 0.01 versus H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/65f2131790a3715f519a2cf7.png"},{"id":100151671,"identity":"13819ffb-0efd-4fc5-bf93-330bf3da2648","added_by":"auto","created_at":"2026-01-13 13:30:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":101649,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of gallic acid and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on reproductive hormone levels of granulosa cells\u003c/p\u003e\n\u003cp\u003eThe hormones levels of (A)estradiol; (B)Anti-Müllerian hormone; and (C)progesterone. Data are presented as mean ± standard error of the mean. ## P \u0026lt; 0.01 versus Control; * P \u0026lt; 0.05 versus H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e; ** P \u0026lt; 0.01 versus H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/a6830512b8de978ca8de7575.png"},{"id":100368531,"identity":"7dabab56-d0f4-4ad1-adc4-65d2725486aa","added_by":"auto","created_at":"2026-01-16 07:58:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1279163,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of gallic acid on autophagy and related pathway protein expression\u003c/p\u003e\n\u003cp\u003eWestern blotting and semi-quantitative analysis of (A and B) Beclin 1, light chain 3, and sequestosome 1 levels and (C and D) , phosphorylated protein kinase B and phosphorylated mechanistic target of rapamycin levels in granulosa cells. Data are presented as mean ± standard error of the mean. # P \u0026lt; 0.05 versus Control; ## P \u0026lt; 0.01 versus Control, * P \u0026lt; 0.05 versus H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e; ** P \u0026lt; 0.01 versus H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/27cdbe45fb1f5ef1fe061c56.png"},{"id":100368330,"identity":"22add976-572c-417e-b83e-a6562eae3bce","added_by":"auto","created_at":"2026-01-16 07:57:51","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":3686200,"visible":true,"origin":"","legend":"\u003cp\u003eAutophagolysosome staining in granulosa cells treated with gallic acid and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eAutophagic lysosomes (red fluorescence) were observed by confocal fluorescence microscopy. 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07:58:26","extension":"tif","order_by":25,"title":"","display":"","copyAsset":false,"role":"supplement","size":1519536,"visible":true,"origin":"","legend":"","description":"","filename":"12.26LC3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/78758f252fd7ad9de7589e62.tif"},{"id":100151657,"identity":"b02cffa4-3238-4166-a7e0-da31f26372ab","added_by":"auto","created_at":"2026-01-13 13:30:10","extension":"jpg","order_by":26,"title":"","display":"","copyAsset":false,"role":"supplement","size":3525,"visible":true,"origin":"","legend":"","description":"","filename":"11.27p624.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/8b417dc42877955df02c9a98.jpg"},{"id":100368533,"identity":"d8e9937f-9754-4506-bc47-699c14b52c3a","added_by":"auto","created_at":"2026-01-16 07:58:03","extension":"jpg","order_by":27,"title":"","display":"","copyAsset":false,"role":"supplement","size":3721,"visible":true,"origin":"","legend":"","description":"","filename":"10.10pmtor11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/9d28220d670344624effded3.jpg"},{"id":100151679,"identity":"79290f72-40f6-4cbf-91eb-7c9ecb189685","added_by":"auto","created_at":"2026-01-13 13:30:10","extension":"tif","order_by":28,"title":"","display":"","copyAsset":false,"role":"supplement","size":1519536,"visible":true,"origin":"","legend":"","description":"","filename":"12.26gapdh.tif","url":"https://assets-eu.researchsquare.com/files/rs-8528931/v1/8e323de46a6dadab353fb9b0.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gallic acid regulates autophagy to alleviate hydrogen peroxide-induced oxidative damage in granulosa cells","fulltext":[{"header":"1.Introduction","content":"\u003cp\u003eThe ovary is an important reproductive organ in females and is involved in multiple functions such as oocyte production, sex hormone secretion, and estrous cycle maintenance (Yang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ovarian granulosa cells (GCs), the ovarian somatic cells surrounding oocytes, play a vital role in normal follicular growth and development (Liu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) by promoting follicle development and supporting normal oocyte growth through nutrient supply and signal transmission (Alam and Miyano, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Ovarian GCs have been widely employed to study ovarian endocrine and reproductive functions in vitro, with many studies reporting oxidative stress (OS)-induced pathological damage in the ovarian microenvironment, which can further lead to GC apoptosis in follicles and follicular atresia, thereby damaging oocytes (Gao et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOS is caused by excessive reactive oxygen species (ROS) production in cells, which then damages cellular proteins, lipids, and DNA, ultimately leading to cell necrosis and apoptosis (Xie et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Cellular ROS accumulation can damage many key physiological functions in females, such as oocyte maturation, ovulation, luteolysis, and luteal maintenance during pregnancy (An et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) is a common OS inducer, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated cells have been shown to produce large amounts of ROS in a short time, which, in turn, induce apoptosis (An et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Conversely, antioxidants may reduce the negative impact of OS on GCs via reduced ROS production and activity by increasing antioxidant gene expression and restoring mitochondrial function (An et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, identifying suitable antioxidants to repair OS damage and improve OS resistance in GCs is crucial.\u003c/p\u003e \u003cp\u003eAutophagy is an important intracellular mechanism involved in maintaining cellular homeostasis and responding to destructive events, such as clearance of long-lived, aggregated, and misfolded proteins and damaged organelles (Kumariya et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Various classical pathways, including phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) can induce autophagy (Bhardwaj et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Autophagy in ovarian GCs and oocytes is critically implicated in follicular development, primordial follicle pool formation, follicular recruitment, and follicular atresia (Tong et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and regulation of GC differentiation, with insufficient autophagy being associated with ovarian dysfunction (Xu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For example, in the GCs of patients with polycystic ovary syndrome (PCOS), Beclin 1 (BECN1) and autophagy-related gene 7 expression was markedly increased, suggesting the involvement of abnormal activation of GC autophagy in abnormal ovary development (Liu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, GC autophagy inhibition through the PI3K/AKT/mTOR pathway has been shown to normalize GC proliferation, improve abnormal follicular development, and alleviate ovulation disorders in rats with PCOS (Kobayashi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Follicle atresia could result from GC apoptosis, induced by increased autophagy and imbalanced internal homeostasis. which indicates that regulation of GC autophagy may provide a new strategy to improve follicular development.\u003c/p\u003e \u003cp\u003eBrandt's vole (\u003cem\u003eLasiopodomys brandtii\u003c/em\u003e), belonging to the Cricetidae family of Rodentia, mainly inhabits Inner Mongolia, Mongolia, and other degraded areas of pasture (Yu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). They play an important role in the grassland ecosystems and feed on tannin-containing plants, including tannic acid (TA) (Yu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Reportedly, TA can affect the reproductive performance of Brandt's voles; however, its mechanism of action remains unknown (Yu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). TA decomposes into gallic acid (GA) and glucose in the digestive tract of mammals (Baldwin and Booth, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). GA is a phenolic compound and a plant secondary metabolite that is widely present in nature and has a strong antioxidant capacity (Baldwin and Booth, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Notably, phenolic compounds can affect cellular autophagy, apoptosis, and the reproductive capacity of animals (Zahedi et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we aimed to create an OS model of GCs by examining how GA affect OS in the ovarian GCs of Brandt's voles that received H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment. GCs with oxidative damage displayed the effects of GA on ROS, cell survival, antioxidant function, apoptosis, and autophagy. Additionally, the underlying mechanisms of GA in alleviating H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage in GCs were explored at the cellular level.\u003c/p\u003e"},{"header":"2.Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.Animals\u003c/h2\u003e \u003cp\u003eHerein, 3-week-old female Brandt's voles were obtained from a grassland in Inner Mongolia and bred in pairs in the animal breeding room of the Ecology Laboratory of Yangzhou University under a 12L:12D photoperiod and 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The voles were subcutaneously injected with 8 IU of pregnant mare serum gonadotropin (Ningbo No. 2 Hormone Factory). All of the voles were sacrificed by cervical dislocation after 48 h, and their ovaries were separated for further \u003cem\u003ein vitro\u003c/em\u003e testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2.Isolation and culture of ovarian GCs\u003c/h2\u003e \u003cp\u003eGCs were isolated from the mature follicles on the ovaries of Brandt's voles by puncturing them with a skin test needle, and the follicular fluid that spilled over was collected. A GC solution was prepared, filtered through a 200-mesh cell sieve, and centrifuged once more for five min at 1000 rpm to extract GCs. These were then resuspended in DMEM/F12. Prior to cell treatments, the cells were cultured for two days at 37\u0026deg;C with 5% CO2 in DMEM/F12 supplemented with 10% fetal bovine serum (FBS; HyClone, USA) and 1% penicillin/streptomycin. GCs were used to establish a damage model by treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (China Hushi) for 24 h. GA was purchased from Aladdin, China, and GCs were pretreated with GA for 24 h prior to subsequent experiments to study its effects on cell damage and autophagy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3.Identification of GCs\u003c/h2\u003e \u003cp\u003eGCs cultured for 48 h were digested with 0.25% trypsin; the digestion was terminated by adding DMEM/F12. Then they were seeded into 24-well plates with small coverslips pre-placed in the wells.. After achieving 70% confluence, the slides were removed, and the cells were stained for immunofluorescence. The cells were treated with the main antibody, follicle-stimulating hormone receptor (FSHR) rabbit polyclonal antibody (1:200), Thereafter, they were incubated overnight at 4\u0026deg;C. The secondary IgG antibody was added and the samples were incubated for 60 min at 37 C in the dark following three PBS washes (5 min each). Three PBS washes (5 min each) were performed on the cells. Red fluorescence was excited with a halogen lamp under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) to observe fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4.Cell viability assay\u003c/h2\u003e \u003cp\u003eThe Cell Viability Kit-8 (CCK-8) (Vazyme, China) was used to measure cell viability. Briefly, GCs were seeded into 96-well plates and treated with the medicines listed above. After adding 10 \u0026micro;L of the CCK-8 reagent to each well, the cells were cultured in the cell culture box for 2 h at 37\u0026deg;C with 5% CO2. The optical density (OD) of each well was measured at 450 nm, and the average OD for each treatment was used to assess cell viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5.Analysis of antioxidant defense mechanism of GCs\u003c/h2\u003e \u003cp\u003e Using matched detection kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China), the activities of total antioxidant capacity (T-AOC), catalase (CAT), and superoxide dismutase (SOD) as well as the malondialdehyde (MDA) levels were assessed in accordance with the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6.Determination of cytokine levels\u003c/h2\u003e \u003cp\u003eAs instructed by the manufacturer, ELISA kits (Jianglai Biological Technology Ltd., Shanghai, China) were used to quantify the cytokine levels of IL-6, TNF-α, IL-1β, E2, AMH and progesterone. In summary, an ELISA plate was coated with a standard or sample, then with the horseradish peroxidase-tagged detection antibody. The plate was then incubated for 60 min at 37 C. Next, substrates A and B were added, and the mixture was incubated for 15 min at 37\u0026deg;C in the dark. The termination solution was used to halt the reaction, and a microplate reader was used to measure the OD at 450 nm. The intra- and inter-assay coefficients of variance for all three hormones and cytokines were below 9% and 11%, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay\u003c/h2\u003e \u003cp\u003eGC apoptosis was measured by the TUNEL Cell Apoptosis Detection Kit (Beyotime Biotechnology, China). In short, the cells were permeabilized with 0.5% Triton X-100 for 5 min after being fixed with 4% paraformaldehyde at room temperature. The cells were rinsed with PBS, then rehydrated and exposed to the TUNEL reaction mixture, consisted of an enzyme-to-labeling solution ratio of 1:9, for 1h at 37\u0026deg;C in the dark. To identify nuclei, GCs were subsequently stained with 4\u0026prime;,6-diamidino-2-phenylindole for five minutes in the dark. TUNEL-positive GCs were photographed after washing using an Olympus fluorescent microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8.Western blotting\u003c/h2\u003e \u003cp\u003eFor western blotting, cell pellets were lysed and centrifuged to collect total protein from the supernatant. The bicinchoninic acid test was used to determine the protein content. The materials were denatured and electrophoresed after adding the loading buffer before being deposited to the membranes. Tris-buffered saline with Tween 20 (TBST) was used to wash the membranes thrice after transfer. The blocking solution (1\u0026times;5000) was used to dilute the secondary antibody. After agitating for 2 h at room temperature and rinsing thrice with TBST, the polyvinylidene fluoride membrane was placed in the working solution. For chromogenic reactions, the enhanced chemiluminescence chromogenic solution was used, and ImageJ 6.0 was used for band analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9.ROS detection\u003c/h2\u003e \u003cp\u003eTo detect ROS, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was used. Following the removal of the cell culture medium and three PBS washes, DCFH-DA (1:1000) had been diluted to a final dosage of 10 \u0026micro;M and added to the cells. After 20 min of incubation at 37\u0026deg;C, the cells were examined and imaged using a fluorescent microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10.Statistical analyses\u003c/h2\u003e \u003cp\u003eIn this study, group differences were compared using Tukey's post-hoc test after one-way analysis of variance. GraphPad Prism (version 8.0) was used to conduct the graphic analysis. A significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was established. SPSS was used for all statistical analyses (version 26.0; SPSS Inc., Chicago, Illinois, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3.Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1.Identification of Brandt's vole ovarian GCs\u003c/h2\u003e \u003cp\u003eHematoxylin\u0026ndash;eosin (HE) staining showed that ovarian GCs were polygonal or fusiform (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and they expressed FSHR. The results of FSHR immunocytochemical fluorescence staining revealed considerable red fluorescence under fluorescence excitation by the halogen light source, indicating FSHR-positive staining in the GCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The average positive expression rate of FSHR in the GCs reached up to 90%, which met the requirements for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2.Effect of GA on the viability of GCs with oxidative damage\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1.Establishment of OS injury model of GCs\u003c/h2\u003e \u003cp\u003eTo assess the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage in GCs, ovarian GCs were treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at concentrations of 10, 50, 100, 150, 200, and 250 \u0026micro;M for 24 h, and the CCK-8 test was used to evaluate cell viability. Furthermore, 200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e dramatically reduced cell viability compared to the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, 200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was selected to establish the cell oxidative damage model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2.Effect of GA on cell viability\u003c/h2\u003e \u003cp\u003eThe effect of GA on the activity of ovarian GCs was assessed by treating GCs with GA at concentrations of 6, 12, 25, 50, 100, 200, and 400 \u0026micro;M for 24 h, and the cell viability was detected through the CCK-8 assay. Overall, no discernible alteration was observed between the cell viability of control and that of the 6\u0026ndash;100 \u0026micro;M GA treatment groups, and the cell viability decreased significantly when the concentration increased to 200 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, the concentration range of 6\u0026ndash;100 \u0026micro;M was selected for subsequent concentration screening.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3.Protective effect of GA on oxidative damage in cells\u003c/h2\u003e \u003cp\u003eThe protective effects and the optimal concentration of GA for alleviating oxidative damage in GCs were determined by treating the cells with different concentrations of GA (6, 12, 25, 50, and 100 \u0026micro;M) for 24 h, and then subjecting the pretreated cells to 200\u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 h to induce damage. The CCK-8 assay was employed to assess cell viability. Notably, compared with the damage model group, different concentrations of GA improved cell viability to different degrees, thereby confirming that GA could protect GCs from oxidative damage. The highest cell viability was detected with 50 \u0026micro;M GA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e); therefore, this concentration was selected for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3.The effect of GA on apoptosis in cells with oxidative damage\u003c/h2\u003e \u003cp\u003eApoptosis was detected using the TUNEL test. In this instance, the GC apoptosis rate of the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e model group was noticeably higher than that of the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, GA pretreatment markedly reduced the apoptosis of GCs with oxidative damage (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4.Effects of GA on T-AOC, CAT, SOD, MDA, and ROS in GCs with oxidative damage\u003c/h2\u003e \u003cp\u003eThe effects of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on the levels of T-AOC, CAT, SOD, and MDA were detected in GCs. Notably, the activities of T-AOC, CAT, and SOD in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e model group substantially decreased, whereas MDA levels increased. However, GA pretreatment significantly reversed these phenomena by increasing the activities of T-AOC, SOD, and CAT, and decreasing MDA levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The DCFH-DA fluorescent probe was used to detect cellular ROS levels, which were significantly increased in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e model group, whereas GA pretreatment significantly decreased ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5.The effect of GA on TNF-α, IL-6, and IL-1β in cells with oxidative damage\u003c/h2\u003e \u003cp\u003eCompared to the control, the TNF-α, IL-6, and IL-1β activities in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e model group were considerably higher. However, after GA pretreatment, this tendency was considerably reversed, resulting in lower TNF-α, IL-6, and IL-1β levels compared to those in the model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.6.The effect of GA on E2,AMH and P4 in cells with oxidative damage\u003c/h2\u003e \u003cp\u003eCompared with the control group, the activities of E2, AMH and P4 in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e model group were significantly decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, after GA pretreatment, this trend was significantly reversed, resulting in its E2, AMH and P4 levels being higher than those of the model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.7.Effect of GA on the expression of autophagy-related proteins\u003c/h2\u003e \u003cp\u003eWestern blotting revealed the expression profile of the autophagy-related proteins BECN1, light chain 3 (LC3)AB, and sequestosome 1 (P62/SQSTM1), along with that of the autophagy pathway-related proteins AKT, phosphorylated AKT (P-AKT), mTOR, and phosphorylated mTOR (P-mTOR). In the injury model group, P-AKT and P-mTOR levels were significantly decreased. GA pretreatment significantly increased P-AKT and P-mTOR levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the control group, the expression of BECN1 and LC3BⅡ/Ⅰ in the model group were was markedly upregulated, whereas that of P62 was decreased. After pretreatment with GA, the expression of BECN1 and LC3Ⅱ/Ⅰ significantly decreased, and that of P62 significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.8.Effect of GA on cell lysosomes\u003c/h2\u003e \u003cp\u003eHerein, a LysoTracker Red fluorescent probe was used to detect intracellular autophagolysosomes. The results showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced OS markedly increased the number of autolysosomes and the level of autophagy compared with those in the control group. However, GA pretreatment significantly reversed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced damage and reduced the number of autolysosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). Monodansylcadaverine (MDC) staining was used to observe autophagosomes, which emitted green fluorescence. Notably, the number of autophagosomes in the cells increased after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, whereas GA pretreatment decreased their number (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB).The schematic diagram illustrating the mechanism by which gallic acid improves oxidative damage in ovarian granulosa cells is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. Gallic acid regulates autophagy through the AKT-mTOR pathway to improve oxidative damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4.Discussion","content":"\u003cp\u003eGCs play a critical role in maintaining follicular development and female physiology by providing physical support and creating a favorable microenvironment by secreting various growth factors and hormones (Zeng et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Consequently, GC dysfunction can lead to abnormal follicular development and ovulation, thereby affecting female fertility (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Herein, in vitro primary culture of ovarian GCs of Brandt's voles were employed to observe cell morphology by HE staining, and FSHR expression was detected in the cells by immunofluorescence. Notably, the purity of the isolated GCs was \u0026gt;\u0026thinsp;90%, making them suitable for subsequent experiments.\u003c/p\u003e \u003cp\u003eReportedly, excessive ROS can oxidatively damage intracellular macromolecules (such as proteins and lipids) (Yan et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, cells were treated with different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in this study, and their cell viability was determined through the CCK-8 assay. Cell viability gradually decreased with increasing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration, with the most significant decrease observed at 200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Therefore, 200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was selected to induce OS for 24 h to successfully establish the OS model of GCs. Additionally, GA was found to exhibit notable safety and efficacy in most cells at low concentrations, albeit it was toxic at relatively high concentrations. These results indicated the low toxicity profile of GA in vivo. Herein, GA-treated cells (24 h) did not exhibit any significant change in cell viability at concentrations\u0026thinsp;\u0026lt;\u0026thinsp;100 \u0026micro;M, but at 200 \u0026micro;M, the cell activity markedly decreased. Therefore, 6\u0026ndash;100 \u0026micro;M GA was considered a safe concentration range for GCs. Consequently, the protective effects of different concentrations of GA against oxidative damage were screened, and compared with the oxidative damage group, GA pretreatment at 50 \u0026micro;M for 24 h was shown to significantly increase the cell viability after 200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment for 24 h. Next, cell apoptosis was evaluated through TUNEL assay. Our results demonstrated that the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment markedly increased apoptosis, whereas the rate of apoptosis decreased significantly after GA pretreatment. GA can be easily obtained by hydrolyzing tannins under alkaline or acidic conditions, which are present in many plants (Xu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Reportedly, The phenolic hydroxyl groups of GA display the ability to scavenge already existing ROS and prevent further production (Sohrabi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), thereby inhibiting the oxidation of associated lipids, DNA, proteins, and enzymes (Shabani et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the present study, GA pretreatment markedly inhibited H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced ROS over-accumulation, which is consistent with the findings of previous studies. The enzymatic antioxidant system is an important free radical scavenging system in the body (Esmaeilzadeh et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Consistently, these results showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage subsequently decreased T-AOC, CAT, and SOD activities, along with increasing MDA levels, which were reversed by GA pretreatment. Taken together, our results imply that GA shields GCs from oxidative damage.\u003c/p\u003e \u003cp\u003eTNF-α is an inflammatory factor that stimulates the proliferation of theca cells, thereby playing a vital role in regulating normal ovarian activity and follicular development (Tylutka et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, IL-6 and IL-1β, as inflammatory cytokines, can produce ROS by up-regulating nicotinamide adenine dinucleotide phosphate oxidase (Akhtar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), along with reducing the activity of aromatase in follicles, thereby resulting in a decrease in estradiol concentration, fertility, and fertilization ability in follicles (Alkhuriji et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The results of this study demonstrated that GA pretreatment reduced the rise in TNF-α, IL-6, and IL-1β levels in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced GC damage model, which were considerably higher. This indicated that GA can alleviate the OS-induced increase in inflammatory factor levels in GCs.\u003c/p\u003e \u003cp\u003eAutophagy is a highly conserved catabolic process regulated by multiple autophagy-related genes that can respond to different environments and orderly degrade and utilize cell components (Ding et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). With increasing basal autophagy levels, cellular ROS clearance is promoted to protect cells, albeit an excessive increase in autophagy levels can cause autophagy disorder and programmed cell death (Duan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Herein, BECN1 and LC3 expression in GCs was upregulated after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, whereas that of P62 protein decreased. However, treatment with GA reduced the expression of BECN1 and LC3 and increased that of P62. Lysosomes are the final effector organelles responsible for autophagic degradation, and their number and quality can represent autophagy activity (Ommati et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Herein, LysoTracker Red, a red fluorescent probe, was used for specific fluorescent staining of lysosomes in living cells. Lysosomes were observed under a fluorescence microscope, where red fluorescence accumulated, with the relative fluorescence intensity and expression indirectly reflecting the quality and quantity of lysosomes. Furthermore, MDC, an acidophilic dye, was used to label acidic endosomes, lysosomes, and late autophagosomes. The qualitative assessment of these autophagosomes indicated their significant accumulation in GCs after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment. However, by altering autophagy-related protein expression, GA pretreatment might lessen the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced autophagy increase.\u003c/p\u003e \u003cp\u003eNumerous studies have examined ovarian damage-related autophagy and apoptosis, and ovarian cell apoptosis has been shown to cause extensive follicular atresia or regression (Dou et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Reportedly, both autophagy and apoptosis regulate ovarian follicular development and atresia (Jin et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Notably, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can induce ROS production and activate the mitochondrial apoptosis pathway (Wang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Activation of the PI3K/AKT pathway has been closely related to cell proliferation, differentiation, apoptosis, and autophagy, and is a well-recognized survival signal transduction pathway (Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The PI3K/AKT/mTOR pathway has been extensively studied in association with GC autophagy (Zhou et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although the study of autophagy has considerably advanced in recent years, studies on autophagy and the signal transduction regulation mechanism of ovarian GCs remain limited and are worth exploring. The results of this study show that the oxidative damage model established by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e induced autophagy and apoptosis in granulosa cells. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e regulates autophagy by activating the AKT/mTOR signaling pathway. Pretreatment with GA can reduce the levels of autophagy and apoptosis in granulosa cells in ovarian injury.\u003c/p\u003e"},{"header":"5.Conclusion","content":"\u003cp\u003eOur results showed that gallic acid alleviated H2O2-induced granulosa cell injury by increasing anti-oxidation, anti-inflammation, and decreasing excessive autophagy and apoptosis levels, and provided new insights into the prevention of ovarian oxidative damage from a molecular perspective.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [grant number 31971418] .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMing-Hao Yu: Formal analysis, investigation, and conceptualization. Rui-Yang Fan: Composing, editing, and reviewing. Sheng-Mei Yang: Composing, editing, and reviewing. Each author has read and approved the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIRB approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving animals were approved by the Animal Care and Use Committee of Yangzhou University (No. SJXY-2).This study was performed in line with the principles of the Declaration of Helsinki. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data will be made available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAkhtar M, Guo S, Guo Y-f, Zahoor A, Shaukat A, Chen Y, Umar T, Deng G, Guo M (2020) Upregulated-gene expression of pro-inflammatory cytokines (TNF-\u0026alpha;, IL-1\u0026beta; and IL-6) via TLRs following NF-\u0026kappa;B and MAPKs in bovine mastitis. Acta tropica 207: 105458, DOI: 10.1016/j.actatropica.2020.105458\u003c/li\u003e\n \u003cli\u003eAlam MH, Miyano T (2020) Interaction between growing oocytes and granulosa cells in vitro. Reproductive medicine and biology 19: 13-23, DOI: 10.1002/rmb2.12292\u003c/li\u003e\n \u003cli\u003eAlkhuriji AF, Al Omar SY, Babay ZA, El-Khadragy MF, Mansour LA, Alharbi WG, Khalil MI (2020) Association of IL-1\u0026beta;, IL-6, TNF-\u0026alpha;, and TGF\u0026beta;1 gene polymorphisms with recurrent spontaneous abortion in polycystic ovary syndrome. Disease Markers 2020: 6076274, DOI: 10.1155/2020/6076274\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAn R, Wang X, Yang L, Zhang J, Wang N, Xu F, Hou Y, Zhang H, Zhang L (2021) Polystyrene microplastics cause granulosa cells apoptosis and fibrosis in ovary through oxidative stress in rats. Toxicology 449: 152665, DOI: 10.1016/j.tox.2020.152665\u003c/li\u003e\n \u003cli\u003eBaldwin A, Booth BW (2022) Biomedical applications of tannic acid. J Biomater Appl 36: 1503-1523, DOI: 10.1177/08853282211058099.\u003c/li\u003e\n \u003cli\u003eBhardwaj JK, Paliwal A, Saraf P, Sachdeva SN (2022) Role of autophagy in follicular development and maintenance of primordial follicular pool in the ovary. Journal of cellular physiology 237: 1157-1170, DOI: 10.1002/jcp.30613\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDing Y, Huang X, Ji T, Qi C, Gao X, Wei R (2024) The emerging roles of miRNA-mediated autophagy in ovarian cancer. Cell Death Dis 15: 314, DOI: 10.1038/s41419-024-06677-8.\u003c/li\u003e\n \u003cli\u003eDou J, Wu Y, Hu R, Liu J, Zhang Y, Zhen X, Wu T, Zhang C, Liu Y, Zheng R (2024) Quinoa ameliorates polycystic ovary syndrome via regulating gut microbiota through PI3K/AKT/mTOR pathway and autophagy. Nutrition \u0026amp; Metabolism 21: 80, DOI: 10.1186/s12986-024-00855-3\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDuan H, Yang S, Yang S, Zeng J, Yan Z, Zhang L, Ma X, Dong W, Zhang Y, Zhao X, Hu J, Xiao L (2024) The mechanism of curcumin to protect mouse ovaries from oxidative damage by regulating AMPK/mTOR mediated autophagy. Phytomedicine 128: 155468, DOI: 10.1016/j.phymed.2024.155468.\u003c/li\u003e\n \u003cli\u003eEsmaeilzadeh M, Heidarian E, Shaghaghi M, Roshanmehr H, Najafi M, Moradi A, Nouri A (2020) Gallic acid mitigates diclofenac-induced liver toxicity by modulating oxidative stress and suppressing IL-1\u0026beta; gene expression in male rats. Pharmaceutical biology 58: 590-596, DOI: 10.1080/13880209.2020.1777169\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGao Y, Zou Y, Wu G, Zheng L (2023) Oxidative stress and mitochondrial dysfunction of granulosa cells in polycystic ovarian syndrome. Frontiers in Medicine 10: 1193749, DOI: 10.3389/fmed.2023.1193749\u003c/li\u003e\n \u003cli\u003eJin R, Chen A, Ye Y, Ren Y, Lu J, Xuan F, Zhou W (2024) Effect of berberine combined with metformin on autophagy in polycystic ovary syndrome by regulating AMPK/AKT/mTOR pathway. Mol Reprod Dev 91: e23768, DOI: 10.1002/mrd.23768.\u003c/li\u003e\n \u003cli\u003eKobayashi M, Yoshino O, Nakashima A, Ito M, Nishio K, Ono Y, Kusabiraki T, Kunitomi C, Takahashi N, Harada M (2020) Inhibition of autophagy in theca cells induces CYP17A1 and PAI-1 expression via ROS/p38 and JNK signalling during the development of polycystic ovary syndrome. Molecular and Cellular Endocrinology 508: 110792, DOI: 10.1016/j.mce.2020.110792\u003c/li\u003e\n \u003cli\u003eKumariya S, Ubba V, Jha RK, Gayen JR (2021) Autophagy in ovary and polycystic ovary syndrome: role, dispute and future perspective. Autophagy 17: 2706-2733, DOI: 10.1080/15548627.2021.1938914\u003c/li\u003e\n \u003cli\u003eLiu M, Zhu H, Zhu Y, Hu X (2021) Guizhi Fuling Wan reduces autophagy of granulosa cell in rats with polycystic ovary syndrome via restoring the PI3K/AKT/mTOR signaling pathway. Journal of Ethnopharmacology 270: 113821, DOI: 10.1016/j.jep.2021.113821\u003c/li\u003e\n \u003cli\u003eLiu S, Jia Y, Meng S, Luo Y, Yang Q, Pan Z (2023) Mechanisms of and potential medications for oxidative stress in ovarian granulosa cells: a review. International Journal of Molecular Sciences 24: 9205, DOI: 10.3390/ijms24119205\u003c/li\u003e\n \u003cli\u003eLiu W, Liu H, Zhang S, Hao H, Meng F, Ma W, Guo Z, Jiang S, Shang X (2024) Silica nanoparticles cause ovarian dysfunction and fertility decrease in mice via oxidative stress-activated autophagy and apoptosis. Ecotoxicol Environ Saf 285: 117049, DOI: 10.1016/j.ecoenv.2024.117049.\u003c/li\u003e\n \u003cli\u003eOmmati MM, Zuo Q, Sabouri S, Retana-Marquez S, Nategh Ahmadi H, Gholami A, Eftekhari A, Shojaei S, Lijuan L, Heidari R (2025) Fluoride-induced autophagy and apoptosis in the mouse ovary: genomic insights into IL-17 signaling and gut microbiota dysbiosis. Journal of Agricultural and Food Chemistry 73: 2138-2155, DOI: 10.1021/acs.jafc.4c10165\u003c/li\u003e\n \u003cli\u003eShabani S, Rabiei Z, Amini-Khoei H (2020) Exploring the multifaceted neuroprotective actions of gallic acid: A review. International Journal of Food Properties 23: 736-752, DOI: 10.1007/s11011-019-00400-9\u003c/li\u003e\n \u003cli\u003eSohrabi F, Dianat M, Badavi M, Radan M, Mard SA (2021) Gallic acid suppresses inflammation and oxidative stress through modulating Nrf2-HO-1-NF-\u0026kappa;B signaling pathways in elastase-induced emphysema in rats. Environmental Science and Pollution Research 28: 56822-56834, DOI: 10.1007/s11356-021-14513-1\u003c/li\u003e\n \u003cli\u003eTong C, Wu Y, Zhang L, Yu Y (2022) Insulin resistance, autophagy and apoptosis in patients with polycystic ovary syndrome: Association with PI3K signaling pathway. Frontiers in endocrinology 13: 1091147, DOI: 10.3389/fendo.2022.1091147\u003c/li\u003e\n \u003cli\u003eTylutka A, Walas Ł, Zembron-Lacny A (2024) Level of IL-6, TNF, and IL-1\u0026beta; and age-related diseases: A systematic review and meta-analysis. Frontiers in immunology 15: 1330386, DOI: 10.3389/fimmu.2024.1330386\u003c/li\u003e\n \u003cli\u003eWang S, Ou Y, Cao S, Sun X, Qin N, Liswaniso S, Xu R (2025) Effects of Melatonin on H2O2-Induced Oxidative Damage of the Granulosa Cells in Hen Ovarian Follicles. Genes 16: 362, DOI: 10.3390/genes16040362\u003c/li\u003e\n \u003cli\u003eXie Q, Hong W, Li Y, Ling S, Zhou Z, Dai Y, Wu W, Weng R, Zhong Z, Tan J (2023) Chitosan oligosaccharide improves ovarian granulosa cells inflammation and oxidative stress in patients with polycystic ovary syndrome. Frontiers in Immunology 14: 1086232, DOI: 10.3389/fimmu.2023.1086232\u003c/li\u003e\n \u003cli\u003eXu B, Dai W, Liu L, Han H, Zhang J, Du X, Pei X, Fu X (2022) Metformin ameliorates polycystic ovary syndrome in a rat model by decreasing excessive autophagy in ovarian granulosa cells via the PI3K/AKT/mTOR pathway. Endocrine Journal 69: 863-875, DOI: 10.1507/endocrj.EJ21-0480\u003c/li\u003e\n \u003cli\u003eXu Y, Tang G, Zhang C, Wang N, Feng Y (2021) Gallic Acid and Diabetes Mellitus: Its Association with Oxidative Stress. Molecules 26: 7115, DOI: 10.3390/molecules26237115.\u003c/li\u003e\n \u003cli\u003eYan F, Zhao Q, Li Y, Zheng Z, Kong X, Shu C, Liu Y, Shi Y (2022) The role of oxidative stress in ovarian aging: a review. J Ovarian Res 15: 100, DOI: 10.1186/s13048-022-01032-x.\u003c/li\u003e\n \u003cli\u003eYang L, Chen Y, Liu Y, Xing Y, Miao C, Zhao Y, Chang X, Zhang Q (2021) The role of oxidative stress and natural antioxidants in ovarian aging. Frontiers in Pharmacology 11: 617843, DOI: 10.1186/s13048-022-01032-x\u003c/li\u003e\n \u003cli\u003eYu M, Fan R, Wang D, Han Y, Dai X, Yang S-M (2024a) Tannic acid alleviates 3-nitropropionic acid-induced ovarian damage in Brandt\u0026rsquo;s vole (Lasiopodomys brandtii). Reproductive Sciences 31: 2261-2272, DOI: 10.1007/s43032-024-01543-6\u003c/li\u003e\n \u003cli\u003eYu M, Fan R, Yang S-M (2024b) Effect of tannic acid on adiponectin and gonads in male Brandt\u0026rsquo;s voles (Lasiopodomys brandtii). General and Comparative Endocrinology 357: 114592, DOI: 10.1016/j.ygcen.2024.114592\u003c/li\u003e\n \u003cli\u003eYu M, Sun X, Dai X, Gu C, Gu M, Wang A, Wei W, Yang S (2021) Effects of tannic acid on antioxidant activity and ovarian development in adolescent and adult female Brandt\u0026rsquo;s voles. Reproductive Sciences 28: 2839-2846, DOI: 10.1007/s43032-021-00578-3\u003c/li\u003e\n \u003cli\u003eZahedi F, Kowsar R, Khodabandeh Z, Dara M, Alaee S (2025) Protective Effects of Gallic Acid Against Lead Acetate‐Induced Toxicity in Mice Ovary: Focus on Apoptosis, Inflammation, and Folliculogenesis. Food Science \u0026amp; Nutrition 13: e70638, DOI: 10.1002/fsn3.70638\u003c/li\u003e\n \u003cli\u003eZeng L, Zhou C, Xu W, Huang Y, Wang W, Ma Z, Huang J, Li J, Hu L, Xue Y, Luo T, Zheng L (2023) The ovarian-related effects of polystyrene nanoplastics on human ovarian granulosa cells and female mice. Ecotoxicol Environ Saf 257: 114941, DOI: 10.1016/j.ecoenv.2023.114941.\u003c/li\u003e\n \u003cli\u003eZhang CH, Liu XY, Wang J (2023) Essential Role of Granulosa Cell Glucose and Lipid Metabolism on Oocytes and the Potential Metabolic Imbalance in Polycystic Ovary Syndrome. Int J Mol Sci 24: 16247, DOI: 10.3390/ijms242216247.\u003c/li\u003e\n \u003cli\u003eZhou XY, Lai YH, Zhang J, Li Y, Wu XM, Yang YZ, Zhang XF, Ma LZ, Zheng KM, Liu YD, Wang Z, Chen SL (2024) Advanced oxidation protein products attenuate the autophagy-lysosome pathway in ovarian granulosa cells by modulating the ROS-dependent mTOR-TFEB pathway. Cell Death Dis 15: 161, DOI: 10.1038/s41419-024-06540-w.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"gallic acid, ovarian granulosa cells, oxidative stress, autophagy, hydrogen peroxide","lastPublishedDoi":"10.21203/rs.3.rs-8528931/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8528931/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAutophagy is closely related to the development of the ovaries. Gallic acid (GA) exhibits various biological activities, including antioxidant, anti-inflammatory, and potential regulatory effects, on autophagy. However, the relationship of GA with autophagy and oxidative damage in granulosa cells (GCs) has not been elucidated. This study aimed to investigate the role of GA in alleviating hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)-induced oxidative damage in GCs via autophagy regulation.Herein, cell viability, oxidative stress (OS), inflammatory markers and reproductive-related hormones were measured to evaluate the effects of GA. Notably, GA alleviated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced decrease in GC viability. Reactive oxygen species, malondialdehyde, tumor necrosis factor-α, interleukin (IL)-6, and IL-1β were all markedly increased in GCs after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment. Moreover, this treatment reduced the levels of superoxide dismutase, catalase, total antioxidant capacity and reproductive-related hormone. After the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, terminal deoxynucleotidyl transferase dUTP nick end labeling staining demonstrated a significant rise in cell death. However, GA treatment inhibited H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage, inflammation, and apoptosis. Western blotting analysis for evaluating the effect of GA on autophagy by detecting autophagy-related protein levels and related pathways revealed that GA reduced the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced increase in autophagy. Altogether, the results of this study show that GA alleviates OS and cell death in GCs by regulating autophagy, Therefore, from the standpoint of autophagy control, GA might contribute to ovarian damage prevention.\u003c/p\u003e","manuscriptTitle":"Gallic acid regulates autophagy to alleviate hydrogen peroxide-induced oxidative damage in granulosa cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 13:30:02","doi":"10.21203/rs.3.rs-8528931/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ca64cf9d-1f4a-4bd3-8984-cda07e336a5d","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-04T14:27:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 13:30:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8528931","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8528931","identity":"rs-8528931","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}