Morroniside protect human granulosa cells against H2O2-induced oxidative damage via regulating Nrf2 and MAPK signaling pathway

Morroniside protect human granulosa cells against H2O2-induced oxidative damage via regulating Nrf2 and MAPK signaling pathway | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Morroniside protect human granulosa cells against H2O2-induced oxidative damage via regulating Nrf2 and MAPK signaling pathway Yucong Ma, Guimin Hao, Xiaohua Lin, Zhiming Zhao, Aimin Yang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-520225/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 Background Morroniside is the main ingredient of Cornus officinalis, which has an antioxidant effect. Ovarian granulosa cells (GCs) are responsible for regulating the development and atresia of follicles, which are susceptible to oxidative stress. In this study, we investigated whether morroniside could inhibit oxidative stress of GCs induced by hydrogen peroxide (H 2 O 2 ), thus leading to improve oocyte quality. Methods The study was divided into 5 groups: control group, H 2 O 2 group, morroniside (5 µM) + H 2 O 2 , morroniside (10 µM) + H 2 O 2 , Morroniside (20 µM) + H 2 O 2 . Cell survival rate was determined by CCK-8, ROS fluorescence level was determined by DCFH-DA probe, MDA, 8-OHdG, T-AOC, SOD, NQO1 and caspase-3 were determined by ELISA, SOD, NQO1, Bax, Bcl-2, caspase-3, caspase-9, Nrf2 and MAPKs protein expression were determined by Western blot, and Nrf2 nuclear translocation level was determined by immunofluorescence method. SPSS21.0 was used for statistical data analysis. Results After pretreatment with morroniside, the levels of ROS, MDA and 8-OHdG in ovarian GCs were significantly decreased. Morroniside significantly upregulated the level of p-Nrf2 and promoted the nuclear translocation level of Nrf2, which transcriptionally activated antioxidase SOD and NQO1. In addition, the levels of apoptosis-related proteins Bax, Bcl-2, caspase-3 and caspase-9 were significantly regulated via p38 and JNK pathway by morroniside. Conclusions These results suggested that morroniside could reduce oxidative damage and apoptosis of ovarian GCs induced by H 2 O 2 in multiple ways, which provided a new idea for clinical improvement of oxidative stress in female reproductive system. Endocrinology & Metabolism Morroniside Human ovarian granulosa cells Oxidative stress Apoptosis Nuclear factor erythroid 2 related factor 2 (Nrf2) Mitogen-activated protein kinases (MAPKs) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ovarian granulosa cells (GCs) are located in the follicles and around the oocytes. They produce steroids, which are responsible for regulating the development and atresia of follicles. GCs are very important for oocyte maturation, oocyte quality and embryo development [ 1 , 2 ]. Adenosine triphosphate in ovarian GCs can be directly transferred to oocytes through the gap of cumulus GCs [ 3 ]. Furthermore, the GCs can also convert glucose into pyruvate which is the energy substrate of oocytes, and transfer it to oocytes [ 4 ]. Evidence shows that the dysfunction of GCs is related to ovarian senescence, the fewer oocytes retrieved, poor oocyte and embryo quality, and low pregnancy rate of in vitro fertilization-embryo transfer (IVF-ET) [ 5 , 6 ]. In addition, women with polycystic ovary syndrome and endometriosis have a higher rate of GCs apoptosis, and thus reduced fertility and pregnancy rates [ 5 , 7 ]. Therefore, normal ovarian GCs are necessary to maintain reproductive function. Physiological levels of reactive oxygen species (ROS) are necessary for follicular growth, oocyte maturation, normal ovulation and ovarian hormone synthesis [ 8 ]. Oxidative stress occurs with the generation of excessive ROS or when antioxidant defense mechanisms are weakened [ 9 ]. Oxidative stress is the basic pathogenesis of a variety of reproductive system diseases, which can damage fertility, decrease pregnancy and delivery rate, and result in recurrent abortion [ 10 ]. Eight-hydroxy-2'-deoxyguanosine (8-OHdG) is a sensitive indicator of DNA damage as the result of oxidative stress. The increase of 8-OHdG content in ovarian GCs is related to the low fertilization rate of oocytes and poor embryo quality during IVF-ET [ 11 ]. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a key antioxidant transcription factors in response to ROS, and it can bind to antioxidant response element (ARE) and induces the expression of numerous antioxidant enzyme including superoxide dismutase (SOD) [ 12 , 13 ], high level of SOD is positively correlated with IVF pregnancy rate [ 14 ]. In addition, excessive ROS generation can trigger GCs apoptosis through MAPK, AKT/FoxO3a and mTOR pathways and increased the apoptosis-related gene expressions, including caspase 9 and caspase 3, causing the GCs cycle arrest and reduces its supporting effect on oocytes, thus affecting oocyte development, ovarian reserve and reproductive potential [ 15 , 16 ]. Therefore, in order to improve female reproductive function, the discovery of drugs that can improve GCs oxidative stress is an urgent problem to be solved. Morroniside is an important active ingredient in Cornus officinalis (Fig. 1 A). It has a variety of biological activities, such as antioxidant, anti-apoptotic, anti-inflammatory [ 17 – 19 ], which can relieve nerve pain, improve cardiovascular and liver functions [ 20 , 21 ]. Deng et al. have confirmed that morroniside inhibitd autophagy in rats ovarian GCs by regulating the PI3K/AKT/mTOR pathway [ 22 ]. However, the effect of morroniside on oxidative stress of GCs induced by H 2 O 2 is largely unclear. Therefore, the aim of this study was to investigate the effect of morroniside on GCs of oxidative stress induced by H 2 O 2 and elucidated the molecular mechanisms by which morroniside protect human granulosa cells against H 2 O 2 -induced oxidative damage which could improve oocyte development. Materials And Methods Cell Culture and Treatment Ovarian GCs were obtained from patients undergoing IVF-ET due to fallopian tube factors in the Reproductive Department of the Second Hospital of Hebei Medical University, and were approved by the ethics committee (Shijiazhuang, China) and informed consent of the patients. The follicular fluid containing GCs were centrifuged at 4˚C (2000rpm, 10min). Remove the upper follicular fluid, and 5ml PBS was added into the below sediment and mixed them. 5ml human lymphocyte separation fluid (Lympholyte-H, Cedarlane Laboratories, Canada) was added to another 10ml centrifuge tube and inclined angle of 45°. PBS suspension was slowly added to the surface of the human lymphocyte separation solution, and then centrifuged at 4˚C (2500rmp, 10min). The white floc in the middle layer is GCs. The GCs were cultured in DMEM/F12 medium (Gibco, Thermo, USA) at 37˚C in a 5% CO 2 incubator. After the cells adhered to the wall, the cells were incubated with H 2 O 2 for 24 h after the different concentrations morroniside ranging from 5 to 20μM pretreated for 24 h. CCK-8 assay GCs were pretreated with different concentrations of morroniside and H 2 O 2 which determined by CCK-8 assay kits (MCE, China). 96-well plate cells were added with 10μl CCK-8 reagent per well and incubated at 37℃ for 2 h. The OD value of each hole was read at 450nm with a microplate reader (Vesar Max, Santak, USA). Intracellular ROS Detection Intracellular ROS levels in each group were detected with ROS assay kit (Beyotime, China). The culture medium containing serum was removed, the cells were incubated with diluted DCFH-DA (1:1000) for 20 min at 37℃ in 5% CO 2 incubator and then washing three times with serum-free medium. ROS content was detected by fluorescence microscope (EVOS® FL, Thermo, USA) and fluorescence intensity was analyzed by Image J software. ELISA assay The ovarian GCs sample lysis fluid was diluted to the optimal concentration. The biomarkers related to oxidative stress and apoptosis, including ROS, malondialdehyde (MDA), 8-OHdG, total antioxidant capacity (T-AOC), SOD, NAD(P)H quinone oxidoreductase 1 (NQO1), and caspase-3, were detected by ELISA assay kit (Jianglai Biological Co., Ltd, Shanghai, China; Jiancheng Bioengineering Institute, Nanjing, China; Abcam, USA; Tongwei Industrial Co., Ltd, Shanghai, China) following the manufacturer’s instructions. The absorbance values were measured by the microplate reader (VersaMax, Molecular Devices, USA). Western blot analysis The collected GCs were lysed to extract total protein and the protein concentration was determined using BCA protein assay kit (SolarBio, Beijing, China). The same amount of total protein in each well (10-15 μg) was separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore, Merck KGAA, Darmstadt, Germany) at the end of electrophoresis. The PVDF membrane was sealed in 5% skimmed milk powder for 2 h and incubated with different primary antibodies overnight at 4℃. The primary antibodies used in this study mainly included 1:1000 SOD (ab68155, Abcam, USA), 1:1000 NQO1(ab80588, Abcam, USA), 1:1000 Bax (ab32503, Abcam, USA), 1:1000 Bcl-2 (26593-1-AP, Proteintech, USA), 1:1000 caspase-3 (YT6113, Immunoway, USA), 1:1000 caspase-9 (ab202068, Abcam, USA), 1:500 p-Nrf2 (ab76026, Abcam, USA), 1:500 Nrf2 (ab62352, Abcam, USA), 1:500 p-ERK (YP0101, Immunoway, USA), 1:500 ERK (16443-1-AP, Proteintech, USA), 1:500 JNK (66210-1-lg, Proteintech, USA), 1:500 p-JNK (YP0156, Immunoway, USA), 1:500 p38 (ab181602, Abcam, USA) and 1:500 p-p38 (ab4822, Abcam, USA). The PVDF membrane was washed with TBST for 3 times and incubated with the secondary antibody (SA00001-2, Proteintech, USA) and incubated for room temperature for 1 h. After the PVDF membrane was washed three times with TBST, antibody-antigen complexes were visualized using a Chemiluminescence Plus Western immunoblot analysis kit (Millipore, USA). Immunofluorescence staining The treated GCs were fixed with 4% paraformaldehyde for 20min, drilled with 1% Triton and sealed with 10% goat serum for 30min, and then incubated overnight with Nrf2 antibody (1:200) at 4℃. After the cells were incubated with fluorescence-labeled secondary antibodies at room temperature for 2 h, the cells were stained with DAPI for 10min. Finally, it was observed under a laser scanning confocal microscope (Leica, Germany). Statistical analysis All data were expressed as mean ± standard deviation and statistical analysis was performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Multiple groups comparison was performed by ANOVA and post-hot analysis. Values of P < 0.05 were considered statistically significant. Results Morroniside increased GCs viability CCK-8 assay were used to detect the survival rate of cells with different concentrations of morroniside and H 2 O 2 . Compared with the control group, there was no significant change in the survival rate of GCs after pretreatment with 1, 5, 10, 20 μM morroniside except for the 50 μM group (Fig. 1B). With the increase of H 2 O 2 concentration, the survival rate of GCs treated with H 2 O 2 decreased gradually in a concentration-dependent manner. 600 μM H 2 O 2 could significantly reduce the cell survival rate without causing more serious cell damage, so this concentration was used in our subsequent experiments (Fig. 1C). We tested the effect of different concentrations of morroniside on the cell survival rate induced by H 2 O 2 , and the results showed that after pre-incubated with morroniside, the survival rate of GCs induced by H 2 O 2 increased significantly compared with H 2 O 2 group (Fig. 1D). Morroniside inhibited GCs oxidative stress To test the effect of morroniside on GCs ROS levels induced by H 2 O 2 , we performed with ROS assay kit and ELISA. As shown in Fig. 2A and B, compared with the control group, ROS level of GCs which were treated with 600 μM H 2 O 2 for 24 h increased significantly ( P < 0.05). Compared with the H 2 O 2 group, ROS levels of GCs which were pretreated with morroniside significantly decreased followed a concentration (5, 10, 20 μM)-dependent manner, expression peaked at 20 μM ( P < 0.05). MDA, 8-OHdG and T-AOC content levels were detected by ELISA to evaluate the degree of oxidative stress. The oxidative damage products content of MDA and 8-OHdG in morroniside group were significantly reduced compared with H 2 O 2 group ( P < 0.05), which is consistent with the result of ROS level of GCs (Fig. 2C and D). In addition, the activity of T-AOC was detected to evaluate the antioxidant level of morroniside. The levels of T-AOC in GCs were significantly reduced after H 2 O 2 treatment, while morroniside could significantly increase the activities of T-AOC ( P < 0.05) (Fig. 2E). These results demonstrated that morroniside protects GCs by reducing oxidative damage induced by H 2 O 2 . Morroniside increased the expression of SOD and NQO1 in GCs inhibited by H 2 O 2 NQO1 is believed to be partly the reduction of free radical load in cells and the detoxification of xenobiotics. SOD is one of the most important antioxidant enzymes, enabling organisms to survive in an oxygen containing atmosphere [23]. The activities of SOD and NQO1 in ovarian GCs were detected and found that H 2 O 2 significantly reduced their activities, while morroniside could significantly increase the activities of them ( P < 0.05) (Fig. 3A and B). The protein expression of SOD and NQO1 were also performed by Western blot, the results showed that H 2 O 2 reduced the protein levels of SOD and NQO1, while different concentrations of morroniside significantly increased the protein levels in a dose dependent manner (Fig. 3C-E). These results demonstrated that morroniside upregulated the protein levels and activities of antioxidant enzymes to protect GCs against oxidative damage induced by H 2 O 2 . Morroniside inhibited GCs apoptosis induced by oxidative stress High concentration of ROS can damage the structure of mitochondria, make the polar pores in the inner mitochondrial membrane in an expanded state which led to the outflow of calcium ions and cytochrome C, finally membrane potential disappeared and cells initiates apoptosis [24]. The expression of apoptosis-related proteins were detected by Western blot analysis. The results showed that the protein expressions of Bax, caspase-9 and caspase-3 in the H 2 O 2 group were significantly increased, while the Bcl-2 expression was significantly decreased ( P < 0.05). However, compared with the H 2 O 2 group, the protein expression levels of Bax, caspase-9 and caspase-3 were significantly decreased, while Bcl-2 levels were significantly increased after pretreated with morroniside (Fig. 4A-E) ( P < 0.05). Activity of casepase-3 were detected and showed that H 2 O 2 increased its activity, while morroniside at different concentrations decreased its activity (Fig. 4F) ( P < 0.05). These results suggested that morroniside can attenuate oxidative stress induced apoptosis of GCs. Morroniside activates Nrf2, p38 and JNK signaling pathway in GCs When the cell is stimulated by ROS, Nrf2 is activated, transported into the nucleus, and then combined with the antioxidant response element (ARE) to activate the express of anti-oxidase genes, thus play an important anti-oxidative damage effect [25]. In order to further study the mechanism of the anti-oxidation of morroniside, we observed the effect of morroniside on the nuclear translocation of Nrf2 in GCs cultured in vitro. In the control and H 2 O 2 group, Nrf2 was almost located in the cytoplasm. When GCs were pretreated with different concentrations morroniside, Nrf2 transported into the nucleus, and 20μM morroniside group almost located in the nucleus (Fig. 5A). Western blot results also showed that the level of Nrf2 in the nucleus in the morroniside group was significantly higher than that in the H 2 O 2 group ( P < 0.05) (Fig. 5B), which is consistent with Fig. 5A. Furthermore, phosphorylation level of Nrf2 was detected by Western blot analysis. Compared with the H 2 O 2 group, phosphorylated Nrf2 (p-Nrf2) levels were increased in different morroniside groups ( P < 0.05) (Fig. 5C). The results demonstrated that morroniside promoted p-Nrf2 expression, and the activated Nrf2 was transfer into the nucleus to regulate the expression of antioxidant enzymes and thus exert antioxidant effects. Mitogen-activated protein kinases (MAPKs) are activated in response to oxidative stress. Several studies have demonstrated that ROS production and activation of MAPKs play a vital role in Aβ-induced apoptosis [26]. To further elucidate the signaling pathway that involved in morroniside preventing H 2 O 2 -induced apoptosis, we examined the effect of morroniside on activation of MAPKs in GCs. Our data showed that H 2 O 2 upregulated phospho-JNK and phosphor-p38MAPK expression, but not ERK1/2 phosphorylation (Fig. 5D). Furthermore, morroniside significantly suppressed H 2 O 2 -induced upregulation of p-JNK and p-p38 MAPK in GCs. These results suggested that morroniside could inhibite JNK and p38 signaling pathway which induced cell apoptosis. Discussion In this study, we firstly investigated the protective effect of morroniside on ovarian GCs from the perspective of oxidative stress. We showed that morroniside increased the expression level of p-Nrf2, promoted the nuclear translocation of Nrf2, and then up-regulated the expression of antioxidant enzymes such as SOD and NQO1, and reduced the oxidative damage induced by H 2 O 2 . In addition, morroniside ameliorated p38 and JNK pathway-induced apoptosis by reducing ROS levels. Morroniside can be used as a potential drug to improve the quality of follicles by protecting GCs. Oxidative stress is closely related to the injury of female reproductive function. With the increase of age, the antioxidant capacity of the ovary decreased, and the imbalance between oxidation and antioxidant in the ovary led to the apoptosis of oocytes and ovarian GCs [ 27 ]. The level of ROS and the expression of apoptotic proteins induced by ROS in ovarian GCs of patients with polycystic ovary syndrome (PCOS) were significantly higher than those of non-PCOS women [ 28 ]. Prieto L et al confirmed that the levels of ROS in the follicular fluid of infertility patients with endometriosis are increased, while the levels of T-AOC and SOD are generally decreased [ 29 ]. Morroniside could suppressed autophagy and apoptosis in rat ovarian GCs through the PI3K/AKT/mTOR pathway [ 22 ]. Our study showed that morroniside could inhibited the levels of ROS and 8-OHdG and MDA content and increased the expression of SOD and NQO1in human ovarian GCs (Figs. 2 and 3 ). The oxidative damage of GCs caused by various reasons such as aging directly affects female reproduction, the results suggested that morroniside protected GCs from oxidative damage. Nrf2 is important for antioxidant stress. When cells explored to a variety of stimuli including antioxidants and xenobiotics, Nrf2 was activated and transferred into the nucleus, formed a complex with the maf protein and bond to ARE, and regulate ARE-mediated antioxidant enzyme gene expression such as SOD and NQO1 [ 12 , 13 ]. The expression of Nrf2 detected in cumulus cells might be related to oocyte quality [ 30 ], while upregulation of Nrf2 in oocytes and cumulus cells might affect the GSH level in matured COCs [ 31 ]. Under the stimulation of harmful conditions (such as heat stress and heavy metals), the activation of Nrf2 pathway can affect the activity and proliferation of ovarian GCs [ 32 , 33 ]. However, Nrf2 activators such as quercetin and dimethyl fumarate [ 34 ] can up-regulate the expression of Nrf2 and its downstream SOD and CAT in ovarian GCs to reduce the level of ROS, thus playing an antioxidant role [ 35 ]. Consistent with these studies, our results show that morroniside promoted the nuclear translocation of Nrf2, thereby regulating the expression of downstream antioxidant genes and playing a protective role on GCs. Thus, the morroniside through regulating the Nrf2 attenuating the ROS level (Fig. 5 A and B). Endogenic ROS as a second messenger is involved in multiple signaling pathways of cascading effect [ 36 ] and is an upstream activator of p38 and JNK which are the member of the MAPK family and are involved in the activation of apoptotic factor such as caspase 3 [ 28 , 37 , 38 ]. The release of cytochrome c (Cyt c) is the key to mitochondria-mediated activation of apoptosis protein, which activates caspase-3 together with its cofactor caspase-9 [ 39 ], and then activates the apoptotic signaling pathway. Bcl-2 and Bax are involved in this process. Bcl-2 inhibits the release of Cyt c while Bax promotes its release [ 40 ]. We got the same results that the morroniside reduced the phosphorylation level of p38 and JNK, decreased the expression of Bax, caspase-3 and caspase-9, and increased the level of Bcl-2 in ovarian GCs. ERK also belongs to the MAPK family and is widely expressed in the GCs. Different from the apoptotic effects of p38 and JNK, ERK is very necessary for oocyte maturation and embryo development [ 41 ]. Ma et al. found that the p-ERK level in GCs of women with low ovarian function was lower than that of women with normal ovarian function and confirmed that activation of ERK pathway could inhibit apoptosis and reduce the expression level of cleavaged caspase-3 [ 42 ]. Our results showed that morroniside reduce H 2 O 2 -induced GCs apoptosis by regulating p38 and JNK pathway but not by ERK pathway (Figs. 4 and 5 D). In summary, our study suggested that the morroniside had a protective effect on GCs stimulated by H 2 O 2 . Morroniside increased the expression level of p-Nrf2, promoted the nuclear translocation of Nrf2, and then up-regulated the expression of antioxidant enzymes such as SOD and NQO1 which reduced the oxidative damage induced by H 2 O 2 . In addition, morroniside ameliorated p38 and JNK pathway-induced apoptosis by reducing ROS levels. This study also provides a new idea for the clinical treatment of reproductive diseases caused by oxidative stress. Abbreviations ARE: antioxidant response element; Cyt c: cytochrome c; GCs: Granulosa cells; H 2 O 2 : Hydrogen peroxide; IVF-ET: In vitro fertilization-embryo transfer; MDA: Malondialdehyde; MAPKs: Mitogen-activated protein kinases; NQO1: NAD(P)H quinone oxidoreductase 1; Nrf2: Nuclear factor erythroid 2 related factor 2; ROS: Reactive oxygen species; SOD: Superoxide dismutase; T-AOC: total antioxidant capacity; 8-OHdG: 8-hydroxy-2-deoxyguanosine Declarations Acknowledgements Thanks to all those who helped with this research. Authors’ contributions YCM and GMH contributed equally to this work. YCM, GMH, MH and HLD jointly participated in the design of this study. XHL, ZMZ and AMY were responsible for collecting cell samples, YCM, XHL, AMY, YCC, SCZ, LJF, JRG, YZ and JWC performed the experiments, YCM, GMH and ZMZ were responsible for data statistics and wrote the paper. All authors have seen and approved the manuscript. Funding This study was supported by the National Natural Science Foundation of China (No. 81774359, 82074247 and 81803924). Availability of data and materials The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate This study was approved by the Ethics Committee of Hebei University of Chinese Medicine (Shijiazhuang, China). Cell samples were collected with the patient's informed consent. Consent for publication Not applicable. Competing interests The authors declared that they have no conflicts of interest. References Coticchio G, Dal Canto M, Mignini Renzini M, Guglielmo MC, Brambillasca F, Turchi D, Novara PV, Fadini R. Oocyte maturation: gamete-somatic cells interactions, meiotic resumption, cytoskeletal dynamics and cytoplasmic reorganization. Hum Reprod Update. 2015; 21(4): 427-54. Dumesic DA, Meldrum DR, Katz-Jaffe MG, Krisher RL, Schoolcraft WB. Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health. Fertil Steril. 2015; 103(2): 303-16. Downs SM. The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocyte. Dev Biol. 1995; 167(2): 502-12. Pacella-Ince L, Zander-Fox DL, Lane M. Mitochondrial SIRT3 and its target glutamate dehydrogenase are altered in follicular cells of women with reduced ovarian reserve or advanced maternal age. Hum Reprod. 2014; 29(7): 1490-9. Tatone C, Amicarelli F, Carbone MC, Monteleone P, Caserta D, Marci R, Artini PG, Piomboni P, Focarelli R. Cellular and molecular aspects of ovarian follicle ageing. Hum Reprod Update; 2008; 14: 131-42. Nakahara K, Saito H, Saito T, Ito M, Ohta N, Sakai N, Tezuka N, Hiroi M, Watanabe H. Incidence of apoptotic bodies in membrana granulosa of the patients participating in an in vitro fertilization program. Fertil Steril. 1997; 67(2): 302-8. Das M, Djahanbakhch O, Hacihanefioglu B, Saridogan E, Ikram M, Ghali L, Raveendran M, Storey A. Granulosa cell survival and proliferation are altered in polycystic ovary syndrome. J Clin Endocrinol Metab. 2008; 93(3): 881-7. Shkolnik K, Tadmor A, Ben-Dor S, Nevo N, Galiani D, Dekel N. Reactive oxygen species are indispensable in ovulation. Proc Natl Acad Sci U S A. 2011; 108(4): 1462-7. Lu J, Wang Z, Cao J, Chen Y, Dong Y. A novel and compact review on the role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2018; 16(1): 80. Ishii T, Miyazawa M, Takanashi Y, Tanigawa M, Yasuda K, Onouchi H, Kawabe N, Mitsushita J, Hartman PS, Ishii N. Genetically induced oxidative stress in mice causes thrombocytosis, splenomegaly and placental angiodysplasia that leads to recurrent abortion. Redox Biol. 2014; 2: 679-85. Seino T, Saito H, Kaneko T, Takahashi T, Kawachiya S, Kurachi H. Eight-hydroxy-2'-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program. Fertil Steril. 2002; 77(6): 1184-90. Jaiswal AK. Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic Biol Med. 2004; 36(10): 1199-207. Wang Z, Hao Y, Wang Y, Liu J, Yuan X, Sun B, Wang J. Wheat alkylresorcinols protect human retinal pigment epithelial cells against H 2 O 2 -induced oxidative damage through Akt-dependent Nrf2/HO-1 signaling. Food Funct. 2019; 10(5): 2797-804. Matos L, Stevenson D, Gomes F, Silva-Carvalho JL, Almeida H. Superoxide dismutase expression in human cumulus oophorus cells. Mol Hum Reprod. 2009; 15(7): 411-9. Zhu M, Miao S, Zhou W, Elnesr SS, Dong X, Zou X. MAPK, AKT/FoxO3a and mTOR pathways are involved in cadmium regulating the cell cycle, proliferation and apoptosis of chicken follicular granulosa cells. Ecotoxicol Environ Saf. 2021; 214: 112091. Hou J, Lei Z, Cui L, Hou Y, Yang L, An R, Wang Q, Li S, Zhang H, Zhang L. Polystyrene microplastics lead to pyroptosis and apoptosis of ovarian granulosa cells via NLRP3/Caspase-1 signaling pathway in rats. Ecotoxicol Environ Saf. 2021; 212: 112012. Zhang JX, Wang R, Xi J, Shen L, Zhu AY, Qi Q, Wang QY, Zhang LJ, Wang FC, Lü HZ et al. Morroniside protects SK-N-SH human neuroblastoma cells against H2O2-induced damage. Int J Mol Med. 2017; 39(3): 603-12. Gao X, Liu Y, Wang L, Sai N, Liu Y, Ni J. Morroniside inhibits H 2 O 2 -induced podocyte apoptosis by down-regulating NOX4 expression controlled by autophagy in vitro. Front Pharmacol. 2020; 11: 533809. Park CH, Yamabe N, Noh JS, Kang KS, Tanaka T, Yokozawa T. The beneficial effects of morroniside on the inflammatory response and lipid metabolism in the liver of db/db mice. Biol Pharm Bull. 2009; 32(10): 1734-40. Tang X, Wu H, Mao X, Li X, Wang Y. The GLP-1 receptor herbal agonist morroniside attenuates neuropathic pain via spinal microglial expression of IL-10 and β-endorphin. Biochem Biophys Res Commun. 2020; 530(3): 494-9. Liu T, Sun F, Cui J, Zheng S, Li Z, Guo D, Tian X, Zhu Z, Zheng W, Wang Y et al. Morroniside enhances angiogenesis and improves cardiac function following acute myocardial infarction in rats. Eur J Pharmacol. 2020; 872: 172954. Deng D, Yan J, Wu Y, Wu K, Li W. Morroniside suppresses hydrogen peroxide-stimulated autophagy and apoptosis in rat ovarian granulosa cells through the PI3K/AKT/mTOR pathway. Hum Exp Toxicol. 2021; 40(4): 577-86. Woith E, Stintzing F, Melzig MF. SOD activity and extremophilicity: a screening of various plant species. Pharmazie. 2017; 72(8): 490-6. Neuzil J, Wang XF, Dong LF, Low P, Ralph SJ. Molecular mechanism of ‘mitocan’-induced apoptosis in cancer cells epitomizes the multiple roles of reactive oxygen species and Bcl-2 family proteins. FEBS Lett. 2006; 580(22): 5125-9. de Haan JB. Nrf2 activators as attractive therapeutics for diabetic nephropathy. Diabetes. 2011; 60(11): 2683-4. Ebenezer PJ, Weidner AM, LeVine H, Markesbery WR, Murphy MP, Zhang L, Dasuri K, Fernandez-Kim SO, Bruce-Keller AJ, Gavilán E et al. Neuron specific toxicity of oligomeric amyloid-β: role for JUN-kinase and oxidative stress. J Alzheimers Dis. 2010; 22(3): 839-48. Tatone C, Amicarelli F. The aging ovary-the poor granulosa cells. Fertil Steril. 2013; 99(1): 12-7. Liu Y, Zhai J, Chen J, Wang X, Wen T. PGC-1α protects against oxidized low-density lipoprotein and luteinizing hormone-induced granulosa cells injury through ROS‑p38 pathway. Hum Cell. 2019; 32(3): 285-96. Prieto L, Quesada JF, Cambero O, Pacheco A, Pellicer A, Codoceo R, Garcia-Velasco JA. Analysis of follicular fluid and serum markers of oxidative stress in women with infertility related to endometriosis. Fertil Steril. 2012; 98(1): 126-30. Kwak SS, Yoon JD, Cheong SA, Jeon Y, Lee E, Hyun SH. The new system of shorter porcine oocyte in vitro maturation (18 hours) using ≥8 mm follicles derived from cumulus-oocyte complexes. Theriogenology. 2014; 81(2): 291-301. Yoon JD, Jeon Y, Cai L, Hwang SU, Kim E, Lee E, Kim DY, Hyun SH. Effects of coculture with cumulus-derived somatic cells on in vitro maturation of porcine oocytes. Theriogenology. 2015; 83(2): 294-305. Wang Y, Yang C, Elsheikh NAH, Li C, Yang F, Wang G, Li L. HO-1 reduces heat stress-induced apoptosis in bovine granulosa cells by suppressing oxidative stress. Aging (Albany NY). 2019; 11(15): 5535-47. Aglan HS, Gebremedhn S, Salilew-Wondim D, Neuhof C, Tholen E, Holker M, Schellander K, Tesfaye D. Regulation of Nrf2 and NF-κB during lead toxicity in bovine granulosa cells. Cell Tissue Res. 2020; 380(3): 643-55. Akino N, Wada-Hiraike O, Terao H, Honjoh H, Isono W, Fu H, Hirano M, Miyamoto Y, Tanikawa M, Harada M et al. Activation of Nrf2 might reduce oxidative stress in human granulosa cells. Mol Cell Endocrinol. 2018; 470: 96-104. Khadrawy O, Gebremedhn S, Saliew-Wondim D, Taqi MO, Neuhoff C, Tholen E, Hoelker M, Schellander K, Tesfaye D. Endogenous and exogenous modulation of Nrf2 mediated oxidative stress response in bovine granulosa cells: potential implication for ovarian function. Int J Mol Sci. 2019; 20(7). Forman HJ, Torres M, Fukuto J. Redox signaling. Mol Cell Biochem. 2002; 234-235(1-2): 49-62. Zou W, Zeng J, Zhuo M, Xu W, Sun L, Wang J, Liu X. Involvement of caspase-3 and p38 mitogen-activated protein kinase in cobalt chloride-induced apoptosis in PC12 cells. J Neurosci Res. 2002; 67(6): 837-43. Yang H, Xie Y, Yang D, Ren D. Oxidative stress-induced apoptosis in granulosa cells involves JNK, p53 and Puma. Oncotarget. 2017; 8(15): 25310-22. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997; 91(4): 479-89. Rosse T, Olivier R, Monney L, Rager M, Conus S, Fellay I, Jansen B, Borner C. Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature. 1998; 391(6666): 496-9. González R, Ruiz-León Y, Gomendio M, Roldan ER. The effect of glucocorticoids on ERK-1/2 phosphorylation during maturation of lamb oocytes and their subsequent fertilization and cleavage ability in vitro. Reprod Toxicol. 2010; 29(2): 198-205. Han Y, Luo H, Wang H, Cai J, Zhang Y. SIRT1 induces resistance to apoptosis in human granulosa cells by activating the ERK pathway and inhibiting NF-κB signaling with anti-inflammatory functions. Apoptosis. 2017; 22(10): 1260-72. 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. Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-520225","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":26881823,"identity":"798f55f0-b2fe-4119-8424-8902332c7029","order_by":0,"name":"Yucong Ma","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yucong","middleName":"","lastName":"Ma","suffix":""},{"id":26881824,"identity":"187645d1-3928-4766-b24e-296e16b8116e","order_by":1,"name":"Guimin Hao","email":"","orcid":"","institution":"Second Hospital of Hebei Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guimin","middleName":"","lastName":"Hao","suffix":""},{"id":26881825,"identity":"b4af2951-06a9-4da0-a1fd-ec76051d07d0","order_by":2,"name":"Xiaohua Lin","email":"","orcid":"","institution":"hebei hospital of triditional chinese medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaohua","middleName":"","lastName":"Lin","suffix":""},{"id":26881826,"identity":"3f8955d1-909e-4bca-9acc-797ff3baa770","order_by":3,"name":"Zhiming Zhao","email":"","orcid":"","institution":"Second Hospital of Hebei Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhiming","middleName":"","lastName":"Zhao","suffix":""},{"id":26881827,"identity":"02fb1a3a-2dcf-496d-a42b-9327134127d1","order_by":4,"name":"Aimin Yang","email":"","orcid":"","institution":"Second Hospital of Hebei Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aimin","middleName":"","lastName":"Yang","suffix":""},{"id":26881828,"identity":"e50135b7-2adb-4b4c-8b45-9ef3ba873977","order_by":5,"name":"Yucong Cao","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yucong","middleName":"","lastName":"Cao","suffix":""},{"id":26881829,"identity":"15ffc466-bc2b-4032-b17f-65659e23a6b9","order_by":6,"name":"Shuancheng Zhang","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuancheng","middleName":"","lastName":"Zhang","suffix":""},{"id":26881830,"identity":"1cab9d8f-1fb5-4243-89d5-b321c496b08b","order_by":7,"name":"Jingran Geng","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingran","middleName":"","lastName":"Geng","suffix":""},{"id":26881831,"identity":"0bc27b72-4bce-4202-be24-db0b70922eb1","order_by":8,"name":"Lijie Fan","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lijie","middleName":"","lastName":"Fan","suffix":""},{"id":26881832,"identity":"0ea85b2a-78cd-4b68-ac61-5cbd85d4bc22","order_by":9,"name":"Yu Zhang","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhang","suffix":""},{"id":26881833,"identity":"79f1c166-0223-4270-84c6-8d28fbf7a13c","order_by":10,"name":"Jingwei Chen","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingwei","middleName":"","lastName":"Chen","suffix":""},{"id":26881834,"identity":"67f9ed32-d6ad-4a1a-aef5-cf37163664c1","order_by":11,"name":"Ming He","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"He","suffix":""},{"id":26881835,"identity":"ddbd1e44-3ec5-444b-8f2f-e6448e074542","order_by":12,"name":"Huilan Du","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYDCCAzwMDAkFDAz8EgxsUBGitBgwMEjOIEkLA1CLwQ1itfDdyD0m8cDALs/4dvOxBz9zGOT4biQwfi7Ao0XyRl6aRIJBcrHZnWPphr3bGIwlbyQwS8/Ao8XgRo4ZUAtz4jYgQ5pxG0PihhsJbMw8hLXUJ26ekf8NpKWeWC2HEzdI5LCBtCQYENIieeaNsUWCwfHEGTfSzIF+kTCceeZhszQ+LXzHcwxv/qioTuyfkfzswc9tNvJ8x5MPfsanBR1IADFjAwkaRsEoGAWjYBRgAwD1ck19coPWWgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7289-9540","institution":"Hebei University of Chinese Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Huilan","middleName":"","lastName":"Du","suffix":""}],"badges":[],"createdAt":"2021-05-12 20:11:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-520225/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-520225/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":9272526,"identity":"330b39f1-2775-4daa-962b-4d6b5e5d8192","added_by":"auto","created_at":"2021-05-17 19:30:27","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":254764,"visible":true,"origin":"","legend":"The effect of morroniside on GCs viability decreased by H2O2. \n(A) Structure of morroniside. (B) Ovarian GCs were treated with morroniside at different concentrations (including 1, 5, 10, 20, 50 μM) for 24 h. (C) GCs were treated with H2O2 at different concentrations (including 200, 400, 600, 800, 1000, 1500 μM) for 24 h. (D) GCs were pretreated with morroniside at different concentrations (including 5, 10, and 20 μM) for 24 h and then treated with 600 μM H2O2 for 24 h. The survival rates of GCs were determined by CCK-8 assay kit. #P \u003c 0.01 versus the control group, ##P \u003c 0.001 versus the control group, *P \u003c 0.01 versus the H2O2 group.\n","description":"","filename":"Fig1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-520225/v1/5ed22053030509394ee1a853.jpeg"},{"id":9272525,"identity":"4ce4c0fb-507d-4e93-bef7-335d25c2c14f","added_by":"auto","created_at":"2021-05-17 19:30:27","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":378351,"visible":true,"origin":"","legend":"Morroniside inhibited GCs oxidative stress induced by H2O2. \n(A) Intracellular ROS level and quantitative analysis of ROS. The oxidative stress biomarkers of (B) ROS, (C) MDA, (D) 8-OHdG and (E) T-AOC content levels were test with ELISA. #P \u003c 0.05 versus the control group, *P \u003c 0.05 versus the H2O2 group. Scale bar was 200μm.\n","description":"","filename":"Fig2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-520225/v1/50bdc37764a69c8bc5515e20.jpeg"},{"id":9272265,"identity":"c8c26aa2-9a27-4621-a2a4-5e46f9b3fe8e","added_by":"auto","created_at":"2021-05-17 19:24:27","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":264905,"visible":true,"origin":"","legend":"Effect of morroniside on the activity and protein expression of SOD and NQO1 in GCs inhibited by H2O2. \nThe activity levels of antioxidant enzymes (A) SOD and (B) NQO1 were test with ELISA. (C) The protein expression levels of SOD and NQO1 were detected by western blot analysis. (D and E) The protein expression levels of SOD and NQO1 were quantitatively analyzed. #P \u003c 0.05 versus the control group, *P \u003c 0.05 versus the H2O2 group.\n","description":"","filename":"Fig3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-520225/v1/86aa34b90508a5269672a3a5.jpeg"},{"id":9272405,"identity":"864bcce3-049f-4f41-a7b8-4cbf8f73024e","added_by":"auto","created_at":"2021-05-17 19:27:27","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":357984,"visible":true,"origin":"","legend":"Effect of morroniside on GCs apoptosis induced by oxidative stress. \n(A) The protein expression levels of Bax, Bcl-2, caspase-3 and caspase-9, which were related to apoptosis, were detected by Western blot analysis. (B-E) The protein expression levels of Bax, Bcl-2, caspase-3 and caspase-9 were quantitatively analyzed. (F) The casepase-3 activity level. #P \u003c 0.05 versus the control group, *P \u003c 0.05 versus the H2O2 group.\n","description":"","filename":"Fig4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-520225/v1/c24b7974971737d0e9b5d4aa.jpeg"},{"id":9272404,"identity":"ff070b4f-e8c7-4099-b2fb-d4d0fbf8e903","added_by":"auto","created_at":"2021-05-17 19:27:27","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":417104,"visible":true,"origin":"","legend":"Effect of morroniside on the protein expression and nuclear translocation of Nrf2. \n(A) The green fluorescence represents Nrf2, and the nucleus is blue DAPI. The white arrows represent Nrf2 in the cytoplasm and the red arrows represent Nrf2 in the nucleus. Scale bars are 50μm. (B) The protein expression levels of nuclear Nrf2, (C) p-Nrf2, Nrf2, (D) p-p38, p38, JNK, p-JNK, ERK and p-ERK were detected by western blot analysis, and the protein expression levels of p-Nrf2 in nucleus, p-Nrf2/Nrf2, p-p38/p38, p-JNK/JNK and p-ERK/ERK were quantitatively analyzed. #P \u003c 0.05 versus the control group, *P \u003c 0.05 versus the H2O2 group.\n","description":"","filename":"Fig5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-520225/v1/7757a3ec3a16c25854d33cff.jpeg"},{"id":9272406,"identity":"286f2f70-429b-46a9-b434-acecc8a7d7a5","added_by":"auto","created_at":"2021-05-17 19:27:27","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":181384,"visible":true,"origin":"","legend":"Schematic model of morroniside Schematic model of morroniside protecting human granulosa cells against H2O2-induced oxidative damage.\nMorroniside increased the expression level of p-Nrf2, promoted the nuclear translocation of Nrf2, and then up-regulated the expression of antioxidant enzymes such as SOD and NQO1 which reduced the oxidative damage induced by H2O2. In addition, morroniside ameliorated p38 and JNK pathway-induced apoptosis by reducing ROS levels.\n","description":"","filename":"Fig6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-520225/v1/bfb86a145e6297437078191f.jpeg"},{"id":13693449,"identity":"b502ae65-15d6-48f1-843c-d4b89131e123","added_by":"auto","created_at":"2021-09-17 12:47:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1078136,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-520225/v1/2c351415-36f1-48c5-a404-121a11d93634.pdf"}],"financialInterests":"","formattedTitle":"Morroniside protect human granulosa cells against H2O2-induced oxidative damage via regulating Nrf2 and MAPK signaling pathway","fulltext":[{"header":"Introduction","content":" \u003cp\u003eOvarian granulosa cells (GCs) are located in the follicles and around the oocytes. They produce steroids, which are responsible for regulating the development and atresia of follicles. GCs are very important for oocyte maturation, oocyte quality and embryo development [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Adenosine triphosphate in ovarian GCs can be directly transferred to oocytes through the gap of cumulus GCs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Furthermore, the GCs can also convert glucose into pyruvate which is the energy substrate of oocytes, and transfer it to oocytes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Evidence shows that the dysfunction of GCs is related to ovarian senescence, the fewer oocytes retrieved, poor oocyte and embryo quality, and low pregnancy rate of in vitro fertilization-embryo transfer (IVF-ET) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition, women with polycystic ovary syndrome and endometriosis have a higher rate of GCs apoptosis, and thus reduced fertility and pregnancy rates [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, normal ovarian GCs are necessary to maintain reproductive function.\u003c/p\u003e \u003cp\u003ePhysiological levels of reactive oxygen species (ROS) are necessary for follicular growth, oocyte maturation, normal ovulation and ovarian hormone synthesis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Oxidative stress occurs with the generation of excessive ROS or when antioxidant defense mechanisms are weakened [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Oxidative stress is the basic pathogenesis of a variety of reproductive system diseases, which can damage fertility, decrease pregnancy and delivery rate, and result in recurrent abortion [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Eight-hydroxy-2'-deoxyguanosine (8-OHdG) is a sensitive indicator of DNA damage as the result of oxidative stress. The increase of 8-OHdG content in ovarian GCs is related to the low fertilization rate of oocytes and poor embryo quality during IVF-ET [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a key antioxidant transcription factors in response to ROS, and it can bind to antioxidant response element (ARE) and induces the expression of numerous antioxidant enzyme including superoxide dismutase (SOD) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], high level of SOD is positively correlated with IVF pregnancy rate [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, excessive ROS generation can trigger GCs apoptosis through MAPK, AKT/FoxO3a and mTOR pathways and increased the apoptosis-related gene expressions, including caspase 9 and caspase 3, causing the GCs cycle arrest and reduces its supporting effect on oocytes, thus affecting oocyte development, ovarian reserve and reproductive potential [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, in order to improve female reproductive function, the discovery of drugs that can improve GCs oxidative stress is an urgent problem to be solved.\u003c/p\u003e \u003cp\u003eMorroniside is an important active ingredient in Cornus officinalis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). It has a variety of biological activities, such as antioxidant, anti-apoptotic, anti-inflammatory [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], which can relieve nerve pain, improve cardiovascular and liver functions [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Deng et al. have confirmed that morroniside inhibitd autophagy in rats ovarian GCs by regulating the PI3K/AKT/mTOR pathway [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the effect of morroniside on oxidative stress of GCs induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is largely unclear. Therefore, the aim of this study was to investigate the effect of morroniside on GCs of oxidative stress induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and elucidated the molecular mechanisms by which morroniside protect human granulosa cells against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage which could improve oocyte development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell Culture and Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvarian GCs were obtained from patients undergoing IVF-ET due to fallopian tube factors in the Reproductive Department of the Second Hospital of Hebei Medical University, and were approved by the ethics committee (Shijiazhuang, China) and informed consent of the patients. The follicular fluid containing GCs were centrifuged at 4˚C (2000rpm, 10min). Remove the upper follicular fluid, and 5ml PBS was added into the below sediment and mixed them. 5ml human lymphocyte separation fluid (Lympholyte-H, Cedarlane Laboratories, Canada) was added to another 10ml centrifuge tube and inclined angle of 45\u0026deg;. PBS suspension was slowly added to the surface of the human lymphocyte separation solution, and then centrifuged at 4˚C (2500rmp, 10min). The white floc in the middle layer is GCs.\u003c/p\u003e\n\u003cp\u003eThe GCs were cultured in DMEM/F12 medium (Gibco, Thermo, USA) at 37˚C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. After the cells adhered to the wall, the cells were incubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 h after the different concentrations morroniside ranging from 5 to 20\u0026mu;M pretreated for 24 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCK-8 assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGCs were pretreated with different concentrations of morroniside and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e which determined by CCK-8 assay kits (MCE, China). 96-well plate cells were added with 10\u0026mu;l CCK-8 reagent per well and incubated at 37℃ for 2 h. The OD value of each hole was read at 450nm with a microplate reader (Vesar Max, Santak, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntracellular ROS Detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntracellular ROS levels in each group were detected with ROS assay kit (Beyotime, China). The culture medium containing serum was removed, the cells were incubated with diluted DCFH-DA (1:1000) for 20 min at 37℃ in 5% CO\u003csub\u003e2 \u003c/sub\u003eincubator and then washing three times with serum-free medium. ROS content was detected by fluorescence microscope (EVOS\u0026reg; FL, Thermo, USA) and fluorescence intensity was analyzed by Image J software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ovarian GCs sample lysis fluid was diluted to the optimal concentration. The biomarkers related to oxidative stress and apoptosis, including ROS, malondialdehyde (MDA), 8-OHdG, total antioxidant capacity (T-AOC), SOD, NAD(P)H quinone oxidoreductase 1 (NQO1), and caspase-3, were detected by ELISA assay kit (Jianglai Biological Co., Ltd, Shanghai, China; Jiancheng Bioengineering Institute, Nanjing, China; Abcam, USA; Tongwei Industrial Co., Ltd, Shanghai, China) following the manufacturer\u0026rsquo;s instructions. The absorbance values were measured by the microplate reader (VersaMax, Molecular Devices, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected GCs were lysed to extract total protein and the protein concentration was determined using BCA protein assay kit (SolarBio, Beijing, China). The same amount of total protein in each well (10-15 \u0026mu;g) was separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore, Merck KGAA, Darmstadt, Germany) at the end of electrophoresis. The PVDF membrane was sealed in 5% skimmed milk powder for 2 h and incubated with different primary antibodies overnight at 4℃. The primary antibodies used in this study mainly included 1:1000 SOD (ab68155, Abcam, USA), 1:1000 NQO1(ab80588, Abcam, USA), 1:1000 Bax (ab32503, Abcam, USA), 1:1000 Bcl-2 (26593-1-AP, Proteintech, USA), 1:1000 caspase-3 (YT6113, Immunoway, USA), 1:1000 caspase-9 (ab202068, Abcam, USA), 1:500 p-Nrf2 (ab76026, Abcam, USA), 1:500 Nrf2 (ab62352, Abcam, USA), 1:500 p-ERK (YP0101, Immunoway, USA), 1:500 ERK (16443-1-AP, Proteintech, USA), 1:500 JNK (66210-1-lg, Proteintech, USA), 1:500 p-JNK (YP0156, Immunoway, USA), 1:500 p38 (ab181602, Abcam, USA) and 1:500 p-p38 (ab4822, Abcam, USA). The PVDF membrane was washed with TBST for 3 times and incubated with the secondary antibody (SA00001-2, Proteintech, USA) and incubated for room temperature for 1 h. After the PVDF membrane was washed three times with TBST, antibody-antigen complexes were visualized using a Chemiluminescence Plus Western immunoblot analysis kit (Millipore, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe treated GCs were fixed with 4% paraformaldehyde for 20min, drilled with 1% Triton and sealed with 10% goat serum for 30min, and then incubated overnight with Nrf2 antibody (1:200) at 4℃. After the cells were incubated with fluorescence-labeled secondary antibodies at room temperature for 2 h, the cells were stained with DAPI for 10min. Finally, it was observed under a laser scanning confocal microscope (Leica, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were expressed as mean \u0026plusmn; standard deviation and statistical analysis was performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Multiple groups comparison was performed by ANOVA and post-hot analysis. Values of P \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMorroniside increased GCs viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCCK-8 assay were used to detect the survival rate of cells with different concentrations of morroniside and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Compared with the control group, there was no significant change in the survival rate of GCs after pretreatment with 1, 5, 10, 20 \u0026mu;M morroniside except for the 50 \u0026mu;M group (Fig. 1B). With the increase of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration, the survival rate of GCs treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003edecreased gradually in a concentration-dependent manner. 600 \u0026mu;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e could significantly reduce the cell survival rate without causing more serious cell damage, so this concentration was used in our subsequent experiments (Fig. 1C). We tested the effect of different concentrations of morroniside on the cell survival rate induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and the results showed that after pre-incubated with morroniside, the survival rate of GCs induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e increased significantly compared with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (Fig. 1D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorroniside inhibited GCs oxidative stress \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the effect of morroniside on GCs ROS levels induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, we performed with ROS assay kit and ELISA. As shown in Fig. 2A and B, compared with the control group, ROS level of GCs which were treated with 600 \u0026mu;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 h increased significantly (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, ROS levels of GCs which were pretreated with morroniside significantly decreased followed a concentration (5, 10, 20 \u0026mu;M)-dependent manner, expression peaked at 20 \u0026mu;M (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). MDA, 8-OHdG and T-AOC content levels were detected by ELISA to evaluate the degree of oxidative stress. The oxidative damage products content of MDA and 8-OHdG in morroniside group were significantly reduced compared with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), which is consistent with the result of ROS level of GCs (Fig. 2C and D). In addition, the activity of T-AOC was detected to evaluate the antioxidant level of morroniside. The levels of T-AOC in GCs were significantly reduced after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003etreatment, while morroniside could significantly increase the activities of T-AOC (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 2E). These results demonstrated that morroniside protects GCs by reducing oxidative damage induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorroniside increased the expression of SOD and NQO1 in GCs inhibited by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNQO1 is believed to be partly the reduction of free radical load in cells and the detoxification of xenobiotics. SOD is one of the most important antioxidant enzymes, enabling organisms to survive in an oxygen containing atmosphere [23]. The activities of SOD and NQO1 in ovarian GCs were detected and found that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003esignificantly reduced their activities, while morroniside could significantly increase the activities of them (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 3A and B). The protein expression of SOD and NQO1 were also performed by Western blot, the results showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reduced the protein levels of SOD and NQO1, while different concentrations of morroniside significantly increased the protein levels in a dose dependent manner (Fig. 3C-E). These results demonstrated that morroniside upregulated the protein levels and activities of antioxidant enzymes to protect GCs against oxidative damage induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorroniside inhibited GCs apoptosis induced by oxidative stress \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh concentration of ROS can damage the structure of mitochondria, make the polar pores in the inner mitochondrial membrane in an expanded state which led to the outflow of calcium ions and cytochrome C, finally membrane potential disappeared and cells initiates apoptosis [24]. The expression of apoptosis-related proteins were detected by Western blot analysis. The results showed that the protein expressions of Bax, caspase-9 and caspase-3 in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group were significantly increased, while the Bcl-2 expression was significantly decreased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). However, compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, the protein expression levels of Bax, caspase-9 and caspase-3 were significantly decreased, while Bcl-2 levels were significantly increased after pretreated with morroniside (Fig. 4A-E) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Activity of casepase-3 were detected and showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e increased its activity, while morroniside at different concentrations decreased its activity (Fig. 4F) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). These results suggested that morroniside can attenuate oxidative stress induced apoptosis of GCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorroniside activates Nrf2, p38 and JNK signaling pathway in GCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the cell is stimulated by ROS, Nrf2 is activated, transported into the nucleus, and then combined with the antioxidant response element (ARE) to activate the express of anti-oxidase genes, thus play an important anti-oxidative damage effect [25]. In order to further study the mechanism of the anti-oxidation of morroniside, we observed the effect of morroniside on the nuclear translocation of Nrf2 in GCs cultured in vitro. In the control and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003egroup, Nrf2 was almost located in the cytoplasm. When GCs were pretreated with different concentrations morroniside, Nrf2 transported into the nucleus, and 20\u0026mu;M morroniside group almost located in the nucleus (Fig. 5A). Western blot results also showed that the level of Nrf2 in the nucleus in the morroniside group was significantly higher than that in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 5B), which is consistent with Fig. 5A. Furthermore, phosphorylation level of Nrf2 was detected by Western blot analysis. Compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, phosphorylated Nrf2 (p-Nrf2) levels were increased in different morroniside groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 5C). The results demonstrated that morroniside promoted p-Nrf2 expression, and the activated Nrf2 was transfer into the nucleus to regulate the expression of antioxidant enzymes and thus exert antioxidant effects.\u003c/p\u003e\n\u003cp\u003eMitogen-activated protein kinases (MAPKs) are activated in response to oxidative stress. Several studies have demonstrated that ROS production and activation of MAPKs play a vital role in A\u0026beta;-induced apoptosis [26]. To further elucidate the signaling pathway that involved in morroniside preventing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced apoptosis, we examined the effect of morroniside on activation of MAPKs in GCs. Our data showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e upregulated phospho-JNK and phosphor-p38MAPK expression, but not ERK1/2 phosphorylation (Fig. 5D). Furthermore, morroniside significantly suppressed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced upregulation of p-JNK and p-p38 MAPK in GCs. These results suggested that morroniside could inhibite JNK and p38 signaling pathway which induced cell apoptosis.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eIn this study, we firstly investigated the protective effect of morroniside on ovarian GCs from the perspective of oxidative stress. We showed that morroniside increased the expression level of p-Nrf2, promoted the nuclear translocation of Nrf2, and then up-regulated the expression of antioxidant enzymes such as SOD and NQO1, and reduced the oxidative damage induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. In addition, morroniside ameliorated p38 and JNK pathway-induced apoptosis by reducing ROS levels. Morroniside can be used as a potential drug to improve the quality of follicles by protecting GCs.\u003c/p\u003e \u003cp\u003eOxidative stress is closely related to the injury of female reproductive function. With the increase of age, the antioxidant capacity of the ovary decreased, and the imbalance between oxidation and antioxidant in the ovary led to the apoptosis of oocytes and ovarian GCs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The level of ROS and the expression of apoptotic proteins induced by ROS in ovarian GCs of patients with polycystic ovary syndrome (PCOS) were significantly higher than those of non-PCOS women [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Prieto L et al confirmed that the levels of ROS in the follicular fluid of infertility patients with endometriosis are increased, while the levels of T-AOC and SOD are generally decreased [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Morroniside could suppressed autophagy and apoptosis in rat ovarian GCs through the PI3K/AKT/mTOR pathway [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our study showed that morroniside could inhibited the levels of ROS and 8-OHdG and MDA content and increased the expression of SOD and NQO1in human ovarian GCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The oxidative damage of GCs caused by various reasons such as aging directly affects female reproduction, the results suggested that morroniside protected GCs from oxidative damage.\u003c/p\u003e \u003cp\u003eNrf2 is important for antioxidant stress. When cells explored to a variety of stimuli including antioxidants and xenobiotics, Nrf2 was activated and transferred into the nucleus, formed a complex with the maf protein and bond to ARE, and regulate ARE-mediated antioxidant enzyme gene expression such as SOD and NQO1 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The expression of Nrf2 detected in cumulus cells might be related to oocyte quality [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], while upregulation of Nrf2 in oocytes and cumulus cells might affect the GSH level in matured COCs [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Under the stimulation of harmful conditions (such as heat stress and heavy metals), the activation of Nrf2 pathway can affect the activity and proliferation of ovarian GCs [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, Nrf2 activators such as quercetin and dimethyl fumarate [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] can up-regulate the expression of Nrf2 and its downstream SOD and CAT in ovarian GCs to reduce the level of ROS, thus playing an antioxidant role [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Consistent with these studies, our results show that morroniside promoted the nuclear translocation of Nrf2, thereby regulating the expression of downstream antioxidant genes and playing a protective role on GCs. Thus, the morroniside through regulating the Nrf2 attenuating the ROS level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003eEndogenic ROS as a second messenger is involved in multiple signaling pathways of cascading effect [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and is an upstream activator of p38 and JNK which are the member of the MAPK family and are involved in the activation of apoptotic factor such as caspase 3 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The release of cytochrome c (Cyt c) is the key to mitochondria-mediated activation of apoptosis protein, which activates caspase-3 together with its cofactor caspase-9 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and then activates the apoptotic signaling pathway. Bcl-2 and Bax are involved in this process. Bcl-2 inhibits the release of Cyt c while Bax promotes its release [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. We got the same results that the morroniside reduced the phosphorylation level of p38 and JNK, decreased the expression of Bax, caspase-3 and caspase-9, and increased the level of Bcl-2 in ovarian GCs. ERK also belongs to the MAPK family and is widely expressed in the GCs. Different from the apoptotic effects of p38 and JNK, ERK is very necessary for oocyte maturation and embryo development [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Ma et al. found that the p-ERK level in GCs of women with low ovarian function was lower than that of women with normal ovarian function and confirmed that activation of ERK pathway could inhibit apoptosis and reduce the expression level of cleavaged caspase-3 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our results showed that morroniside reduce H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced GCs apoptosis by regulating p38 and JNK pathway but not by ERK pathway (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eIn summary, our study suggested that the morroniside had a protective effect on GCs stimulated by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Morroniside increased the expression level of p-Nrf2, promoted the nuclear translocation of Nrf2, and then up-regulated the expression of antioxidant enzymes such as SOD and NQO1 which reduced the oxidative damage induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. In addition, morroniside ameliorated p38 and JNK pathway-induced apoptosis by reducing ROS levels. This study also provides a new idea for the clinical treatment of reproductive diseases caused by oxidative stress.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eARE: antioxidant response element; Cyt c: cytochrome c; GCs: Granulosa cells; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: Hydrogen peroxide; IVF-ET: In vitro fertilization-embryo transfer; MDA: Malondialdehyde; MAPKs: Mitogen-activated protein kinases; NQO1: NAD(P)H quinone oxidoreductase 1; Nrf2: Nuclear factor erythroid 2 related factor 2; ROS: Reactive oxygen species; SOD: Superoxide dismutase; T-AOC: total antioxidant capacity; 8-OHdG: 8-hydroxy-2-deoxyguanosine\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to all those who helped with this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYCM and GMH contributed equally to this work. YCM, GMH, MH and HLD jointly participated in the design of this study. XHL, ZMZ and AMY were responsible for collecting cell samples, YCM, XHL, AMY, YCC, SCZ, LJF, JRG, YZ and JWC performed the experiments, YCM, GMH and ZMZ were responsible for data statistics and wrote the paper. All authors have seen and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (No. 81774359, 82074247 and 81803924).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Hebei University of Chinese Medicine (Shijiazhuang, China). Cell samples were collected with the patient's informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCoticchio G, Dal Canto M, Mignini Renzini M, Guglielmo MC, Brambillasca F, Turchi D, Novara PV, Fadini R. Oocyte maturation: gamete-somatic cells interactions, meiotic resumption, cytoskeletal dynamics and cytoplasmic reorganization. Hum Reprod Update. 2015; 21(4): 427-54.\u003c/li\u003e\n\u003cli\u003eDumesic DA, Meldrum DR, Katz-Jaffe MG, Krisher RL, Schoolcraft WB. Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health. Fertil Steril. 2015; 103(2): 303-16.\u003c/li\u003e\n\u003cli\u003eDowns SM. The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocyte. Dev Biol. 1995; 167(2): 502-12.\u003c/li\u003e\n\u003cli\u003ePacella-Ince L, Zander-Fox DL, Lane M. Mitochondrial SIRT3 and its target glutamate dehydrogenase are altered in follicular cells of women with reduced ovarian reserve or advanced maternal age. Hum Reprod. 2014; 29(7): 1490-9.\u003c/li\u003e\n\u003cli\u003eTatone C, Amicarelli F, Carbone MC, Monteleone P, Caserta D, Marci R, Artini PG, Piomboni P, Focarelli R. Cellular and molecular aspects of ovarian follicle ageing. Hum Reprod Update; 2008; 14: 131-42.\u003c/li\u003e\n\u003cli\u003eNakahara K, Saito H, Saito T, Ito M, Ohta N, Sakai N, Tezuka N, Hiroi M, Watanabe H. Incidence of apoptotic bodies in membrana granulosa of the patients participating in an in vitro fertilization program. Fertil Steril. 1997; 67(2): 302-8.\u003c/li\u003e\n\u003cli\u003eDas M, Djahanbakhch O, Hacihanefioglu B, Saridogan E, Ikram M, Ghali L, Raveendran M, Storey A. Granulosa cell survival and proliferation are altered in polycystic ovary syndrome. J Clin Endocrinol Metab. 2008; 93(3): 881-7.\u003c/li\u003e\n\u003cli\u003eShkolnik K, Tadmor A, Ben-Dor S, Nevo N, Galiani D, Dekel N. Reactive oxygen species are indispensable in ovulation. Proc Natl Acad Sci U S A. 2011; 108(4): 1462-7.\u003c/li\u003e\n\u003cli\u003eLu J, Wang Z, Cao J, Chen Y, Dong Y. A novel and compact review on the role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2018; 16(1):\u0026nbsp;80.\u003c/li\u003e\n\u003cli\u003eIshii T, Miyazawa M, Takanashi Y, Tanigawa M, Yasuda K, Onouchi H, Kawabe N, Mitsushita J, Hartman PS, Ishii N. Genetically induced oxidative stress in mice causes thrombocytosis, splenomegaly and placental angiodysplasia that leads to recurrent abortion. Redox Biol. 2014; 2: 679-85.\u003c/li\u003e\n\u003cli\u003eSeino T, Saito H, Kaneko T, Takahashi T, Kawachiya S, Kurachi H. Eight-hydroxy-2'-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program. Fertil Steril. 2002; 77(6): 1184-90.\u003c/li\u003e\n\u003cli\u003eJaiswal AK. Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic Biol Med. 2004; 36(10): 1199-207.\u003c/li\u003e\n\u003cli\u003eWang Z, Hao Y, Wang Y, Liu J, Yuan X, Sun B, Wang J. Wheat alkylresorcinols protect human retinal pigment epithelial cells against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage through Akt-dependent Nrf2/HO-1 signaling. Food Funct. 2019; 10(5): 2797-804.\u003c/li\u003e\n\u003cli\u003eMatos L, Stevenson D, Gomes F, Silva-Carvalho JL, Almeida H. Superoxide dismutase expression in human cumulus oophorus cells. Mol Hum Reprod. 2009; 15(7): 411-9.\u003c/li\u003e\n\u003cli\u003eZhu M, Miao S, Zhou W, Elnesr SS, Dong X, Zou X. MAPK, AKT/FoxO3a and mTOR pathways are involved in cadmium regulating the cell cycle, proliferation and apoptosis of chicken follicular granulosa cells. Ecotoxicol Environ Saf. 2021; 214: 112091.\u003c/li\u003e\n\u003cli\u003eHou J, Lei Z, Cui L, Hou Y, Yang L, An R, Wang Q, Li S, Zhang H, Zhang L. Polystyrene microplastics lead to pyroptosis and apoptosis of ovarian granulosa cells via NLRP3/Caspase-1 signaling pathway in rats. Ecotoxicol Environ Saf. 2021; 212: 112012.\u003c/li\u003e\n\u003cli\u003eZhang JX, Wang R, Xi J, Shen L, Zhu AY, Qi Q, Wang QY, Zhang LJ, Wang FC, L\u0026uuml; HZ et al. Morroniside protects SK-N-SH human neuroblastoma cells against H2O2-induced damage. Int J Mol Med. 2017; 39(3): 603-12.\u003c/li\u003e\n\u003cli\u003eGao X, Liu Y, Wang L, Sai N, Liu Y, Ni J. Morroniside inhibits H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced podocyte apoptosis by down-regulating NOX4 expression controlled by autophagy in vitro. Front Pharmacol. 2020; 11: 533809.\u003c/li\u003e\n\u003cli\u003ePark CH, Yamabe N, Noh JS, Kang KS, Tanaka T, Yokozawa T. The beneficial effects of morroniside on the inflammatory response and lipid metabolism in the liver of db/db mice. Biol Pharm Bull. 2009; 32(10): 1734-40.\u003c/li\u003e\n\u003cli\u003eTang X, Wu H, Mao X, Li X, Wang Y. The GLP-1 receptor herbal agonist morroniside attenuates neuropathic pain via spinal microglial expression of IL-10 and \u0026beta;-endorphin. Biochem Biophys Res Commun. 2020; 530(3): 494-9.\u003c/li\u003e\n\u003cli\u003eLiu T, Sun F, Cui J, Zheng S, Li Z, Guo D, Tian X, Zhu Z, Zheng W, Wang Y et al. Morroniside enhances angiogenesis and improves cardiac function following acute myocardial infarction in rats. Eur J Pharmacol. 2020; 872: 172954.\u003c/li\u003e\n\u003cli\u003eDeng D, Yan J, Wu Y, Wu K, Li W. Morroniside suppresses hydrogen peroxide-stimulated autophagy and apoptosis in rat ovarian granulosa cells through the PI3K/AKT/mTOR pathway. Hum Exp Toxicol. 2021; 40(4): 577-86.\u003c/li\u003e\n\u003cli\u003eWoith E, Stintzing F, Melzig MF. SOD activity and extremophilicity: a screening of various plant species. Pharmazie. 2017; 72(8): 490-6.\u003c/li\u003e\n\u003cli\u003eNeuzil J, Wang XF, Dong LF, Low P, Ralph SJ. Molecular mechanism of \u0026lsquo;mitocan\u0026rsquo;-induced apoptosis in cancer cells epitomizes the multiple roles of reactive oxygen species and Bcl-2 family proteins. FEBS Lett. 2006; 580(22): 5125-9.\u003c/li\u003e\n\u003cli\u003ede Haan JB. Nrf2 activators as attractive therapeutics for diabetic nephropathy. Diabetes. 2011; 60(11): 2683-4.\u003c/li\u003e\n\u003cli\u003eEbenezer PJ, Weidner AM, LeVine H, Markesbery WR, Murphy MP, Zhang L, Dasuri K, Fernandez-Kim SO, Bruce-Keller AJ, Gavil\u0026aacute;n E et al. Neuron specific toxicity of oligomeric amyloid-\u0026beta;: role for JUN-kinase and oxidative stress. J Alzheimers Dis. 2010; 22(3): 839-48.\u003c/li\u003e\n\u003cli\u003eTatone C, Amicarelli F. The aging ovary-the poor granulosa cells. Fertil Steril. 2013; 99(1): 12-7.\u003c/li\u003e\n\u003cli\u003eLiu Y, Zhai J, Chen J, Wang X, Wen T. PGC-1\u0026alpha; protects against\u0026nbsp;oxidized low-density lipoprotein and\u0026nbsp;luteinizing hormone-induced granulosa cells injury through\u0026nbsp;ROS‑p38 pathway. Hum Cell. 2019; 32(3): 285-96.\u003c/li\u003e\n\u003cli\u003ePrieto L, Quesada JF, Cambero O, Pacheco A, Pellicer A, Codoceo R, Garcia-Velasco JA. Analysis of follicular fluid and serum markers of oxidative stress in women with infertility related to endometriosis. Fertil Steril. 2012; 98(1): 126-30.\u003c/li\u003e\n\u003cli\u003eKwak SS, Yoon JD, Cheong SA, Jeon Y, Lee E, Hyun SH. The new system of shorter porcine oocyte in vitro maturation (18 hours) using \u0026ge;8 mm follicles derived from cumulus-oocyte complexes. Theriogenology. 2014; 81(2): 291-301.\u003c/li\u003e\n\u003cli\u003eYoon JD, Jeon Y, Cai L, Hwang SU, Kim E, Lee E, Kim DY, Hyun SH. Effects of coculture with cumulus-derived somatic cells on in vitro maturation of porcine oocytes. Theriogenology. 2015; 83(2): 294-305.\u003c/li\u003e\n\u003cli\u003eWang Y, Yang C, Elsheikh NAH, Li C, Yang F, Wang G, Li L. HO-1 reduces heat stress-induced apoptosis in bovine granulosa cells by suppressing oxidative stress. Aging (Albany NY). 2019; 11(15): 5535-47.\u003c/li\u003e\n\u003cli\u003eAglan HS, Gebremedhn S, Salilew-Wondim D, Neuhof C, Tholen E, Holker M, Schellander K, Tesfaye D. Regulation of Nrf2 and NF-\u0026kappa;B during lead toxicity in bovine granulosa cells. Cell Tissue Res. 2020; 380(3): 643-55.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eAkino N, Wada-Hiraike O, Terao H, Honjoh H, Isono W, Fu H, Hirano M, Miyamoto Y, Tanikawa M, Harada M et al. Activation of Nrf2 might reduce oxidative stress in human granulosa cells. Mol Cell Endocrinol. 2018; 470: 96-104.\u003c/li\u003e\n\u003cli\u003eKhadrawy O, Gebremedhn S, Saliew-Wondim D, Taqi MO, Neuhoff C, Tholen E, Hoelker M, Schellander K, Tesfaye D. Endogenous and exogenous modulation of Nrf2 mediated oxidative stress response in bovine granulosa cells: potential implication for ovarian function. Int J Mol Sci. 2019; 20(7).\u003c/li\u003e\n\u003cli\u003eForman HJ, Torres M, Fukuto J. Redox signaling. Mol Cell Biochem. 2002; 234-235(1-2): 49-62.\u003c/li\u003e\n\u003cli\u003eZou W, Zeng J, Zhuo M, Xu W, Sun L, Wang J, Liu X. Involvement of caspase-3 and p38 mitogen-activated protein kinase in cobalt chloride-induced apoptosis in PC12 cells. J Neurosci Res. 2002; 67(6): 837-43.\u003c/li\u003e\n\u003cli\u003eYang H, Xie Y, Yang D, Ren D. Oxidative stress-induced apoptosis in granulosa cells involves JNK, p53 and Puma. Oncotarget. 2017; 8(15): 25310-22.\u003c/li\u003e\n\u003cli\u003eLi P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997; 91(4): 479-89.\u003c/li\u003e\n\u003cli\u003eRosse T, Olivier R, Monney L, Rager M, Conus S, Fellay I, Jansen B, Borner C. Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature. 1998; 391(6666): 496-9.\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez R, Ruiz-Le\u0026oacute;n Y, Gomendio M, Roldan ER. The effect of glucocorticoids on ERK-1/2 phosphorylation during maturation of lamb oocytes and their subsequent fertilization and cleavage ability in vitro. Reprod Toxicol.\u0026nbsp;2010; 29(2): 198-205.\u003c/li\u003e\n\u003cli\u003eHan Y, Luo H, Wang H, Cai J, Zhang Y. SIRT1 induces resistance to\u0026nbsp;apoptosis in\u0026nbsp;human granulosa cells by\u0026nbsp;activating the\u0026nbsp;ERK pathway and\u0026nbsp;inhibiting NF-\u0026kappa;B signaling with\u0026nbsp;anti-inflammatory functions. Apoptosis. 2017; 22(10): 1260-72.\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":"Morroniside, Human ovarian granulosa cells, Oxidative stress, Apoptosis, Nuclear factor erythroid 2 related factor 2 (Nrf2), Mitogen-activated protein kinases (MAPKs)","lastPublishedDoi":"10.21203/rs.3.rs-520225/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-520225/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMorroniside is the main ingredient of Cornus officinalis, which has an antioxidant effect. Ovarian granulosa cells (GCs) are responsible for regulating the development and atresia of follicles, which are susceptible to oxidative stress. In this study, we investigated whether morroniside could inhibit oxidative stress of GCs induced by hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), thus leading to improve oocyte quality.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe study was divided into 5 groups: control group, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, morroniside (5 \u0026micro;M)\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, morroniside (10 \u0026micro;M)\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, Morroniside (20 \u0026micro;M)\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Cell survival rate was determined by CCK-8, ROS fluorescence level was determined by DCFH-DA probe, MDA, 8-OHdG, T-AOC, SOD, NQO1 and caspase-3 were determined by ELISA, SOD, NQO1, Bax, Bcl-2, caspase-3, caspase-9, Nrf2 and MAPKs protein expression were determined by Western blot, and Nrf2 nuclear translocation level was determined by immunofluorescence method. SPSS21.0 was used for statistical data analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAfter pretreatment with morroniside, the levels of ROS, MDA and 8-OHdG in ovarian GCs were significantly decreased. Morroniside significantly upregulated the level of p-Nrf2 and promoted the nuclear translocation level of Nrf2, which transcriptionally activated antioxidase SOD and NQO1. In addition, the levels of apoptosis-related proteins Bax, Bcl-2, caspase-3 and caspase-9 were significantly regulated via p38 and JNK pathway by morroniside.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese results suggested that morroniside could reduce oxidative damage and apoptosis of ovarian GCs induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in multiple ways, which provided a new idea for clinical improvement of oxidative stress in female reproductive system.\u003c/p\u003e","manuscriptTitle":"Morroniside protect human granulosa cells against H2O2-induced oxidative damage via regulating Nrf2 and MAPK signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-17 19:24:25","doi":"10.21203/rs.3.rs-520225/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":"27a598b0-1c63-4e05-a589-f8a86f785e2b","owner":[],"postedDate":"May 17th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":4337419,"name":"Endocrinology \u0026 Metabolism"}],"tags":[],"updatedAt":"2021-05-17T19:24:27+00:00","versionOfRecord":[],"versionCreatedAt":"2021-05-17 19:24:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-520225","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-520225","identity":"rs-520225","version":["v1"]},"buildId":"re_ckhLnmML6MCF96OHNJ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}