Protective Effects And Mechanism Of Oviductus Ranae-containing Serum On Oxidative Stress-induced Apoptosis In Rat Ovarian Granulosa Cells

Granulosa cells are capable of performing steroidogenesis in response to regulate physiological function,which play a critical role in follicular development.Moreover,the apoptosis of granulosa cells leads to follicular atresia,which plays an important role in age-related decline in fertility of perimenopausal women.Some modern studies indicate that oxidative stress contributes to aging,and is one of the most important induced factors of aging.The age-related decline in fertility may be modulated by oxidative stress in perimenopausal women.Therefore,the salvation of granulosa cells,especially in the presence of oxidative stress,might be of important therapeutic value in the treatment of female reproductive aging.Oviductus Ranae(Rana temporaria chensinensis David,OR),a traditional Chinese medicine,is used to be applied to cure climacteric syndrome and anti-aging.OR contains many types of biologically active substances.Modern pharmacological studies have demonstrated that OR delays ovarian aging by increasing the synthesis of extradiol(E2),up-regulating antioxidants,promoting follicular development,and improving ovarian function.In vitro studies have shown that OR has also been proven to increase proliferation of granulosa cells.However,the cellular and molecular mechanisms via which OR exerts its ovarian protective effect are not yet elucidated.In this study,oxidative stress was induced by exposing ovarian granulosacells to H2O2.The protective effect of OR against oxidative stress in granulosa cells,and the underlying molecular mechanisms were investigated.Moreover,to explore the therapeutical targets of Chinese medicine may be beneficial to provide theoretical basis for clinical application.Objective:To establish cultural method of rat ovarian granulosa cells in vitro,and investigate the function and mechanism of OR-containing serum in protecting rat ovarian granulosa cells from hydrogen peroxide(H2O2)-induced oxidative damage.Methods:1.The primary cultural system of ovarian granulosa cells in rats was established,and CCK-8 assay was used to determine the effects of OR-containing serum on proliferation,to analyze the best optimal concentration for the next experiments in vitro.2.The oxidative stress model of cultured granulosa cells was induced with H2O2,and CCK-8 assay was used to evaluate the protective effects of OR-containing serum on H2O2-induced apoptosis in granulosa cells.3.H2O2-treated granulosa cells were pretreated with OR-containing serum,and apoptotic granulosa cells were observed microscopically using4',6-diamidino-2-phenylindole(DAPI),and the apoptotic rate was quantified via Annexin V/propidium iodide(PI)staining combined with flow cytometry.The levels of reactive oxygen species(ROS)and mitochondrial membrane potential(??m)in the cells were measured using2,7-dichlorofluoresceinacetate(DCFH-DA)and rhodamine 123,respectively,and analyzed by flow cytometry.4.Mitogen-activated protein kinases(MAPKs),including ERK1/2,JNK,and p38 MAPK,and other apoptosis-related proteins(p53,Bcl-2,Bax,caspase-3,caspase-9),were detected by western blot analysis,and the related mRNA levels were detected by qRT-PCR.Results:1.Granulosa cells were identified using immunofluorescence staining for follicle stimulating hormone receptor(FSHR),and the final cell cultures contained > 95% granulosa cells.2.The results of CCK-8 assay showed that OR-containing serum significantly promoted the proliferation of granulosa cells in a dose-dependent manner.Treated with the OR-containing serum for 48 h,the volume percent of 20% serum in OR groups was significantly promoted the proliferation of granulosa cells(P < 0.05).3.Exposure of granulosa cells to different concentrations of H2O2 for 2h resulted in a concentration-dependent decrease in cell viability.At a concentration of 150 ?M,H2O2 caused a 51.18% ± 0.8% decrease in cell viability(P < 0.01).Thus,the cells were treated with this concentration of H2O2 in subsequent experiments.Granulosa cells were pretreated with OR-containing serum prior to exposure to H2O2(150 ?M).Pretreatment with the OR-containing serum for 48 h increased the cell viability.4.Chromatin condensation,nuclear fragmentation,and apoptotic bodies in the cells were visualized after treatment with 150 ?M H2O2 for 2 h.Pretreatment with the OR-containing serum for 48 h led to a significant decrease in the number of DAPI-positive apoptotic cells.A quantitative evaluation of the number of apoptotic granulosa cells was performed using Annexin V/PI staining combined with flow cytometry.Pretreatment of the granulosa cells with the OR-containing serum for 48 h reduced the apoptotic rates.5.DCFH-DA and rhodamine 123 staining results found that pretreatment with OR-containing serum contributed to a remarkable decrease in ROS accumulation,and an obvious increase in ??m,which inhibited H2O2-induced depolarization.6.After stimulation with H2O2(150 ?M)for 2 h,the expression of ERK2 and Bcl-2 mRNA increased significantly in the OR-containing serum groups incomparison with that in the H2O2 model group.The mRNA level of p53,Bcl-2,Bax,caspase-3,JNK,and p38 MAPK decreased significantly in the OR-containing serum groups compared to that in the H2O2 model group(P < 0.05).7.The results obtained from Western blotting showed that treatment with H2O2 elevated p53,Bax,and caspase-3 expression,and reduced Bcl-2 expression.Nevertheless,pretreatment with the OR-containing serum enhanced the level of Bcl-2,and suppressed the level of p53,Bax,and caspase-3.We further explore the effects of the OR-containing serum on MAPK signaling.The results showed that the OR-containing serum attenuated phosphorylation of JNK and p38 MAPK,and improved ERK1/2 phosphorylation.Conclusions:1.Optimize the separated method and obtain a lot of high purity of ovarian granulosa cells in vitro,to lay the foundation for further research on ovarian function and mechanism of OR and other traditional Chinese medicine.2.OR-containing serum protected rat ovarian granulosa cells against H2O2-induced apoptosis,by reducing ROS production and improving mitochondrial membrane potential,through down-regulation of negative regulators of proliferation,and activation of MAPK signaling pathways.