Protective Mechanism Of Metformin Pretreatment On Cisplatin-induced Injury Of Human Ovarian Granulosa Cells

Objective:to investigate the anti-oxidative stress mechanism of metformin on human ovarian granulosa cell injury induced by cisplatin and to provide a new method for clinical treatment of ovarian function injury.Methods:1. Granulosa cell culture and identification:patients with a normal ovarian function and male of factors or fallopian tube factors were selected.Follicular fluid was taken when oocytes were taken and granulosa cells were isolated by centrifugation.The granulosa cells were cultured in DMEM/F12medium containing 15%fetal bovine serum.The expression of FSHR in human ovarian granulosa cells was identified by immunocytochemistry.2.Different concentrations of cisplatin（2.5μg/m L,5μg/m L,10μg/m L,20μg/m L）were added in granulosa cells for 24 hours.The propriety damage concentration was selected by CCK8 detection.Granulosa cells were pretreated with different concentrations of metformin（1m M,2m M,5m M,10m M,20m M）for 2 hours,and treated with the concentration of cisplatin for24 hours.The propriety concentration of metformin was selected by CCK8detection.3.The experiment was divided into three groups:the control group:the normal medium was added in cells for 24 hours.Experimental group:the medium containing 1m M metformin was added in cells for 2 hours,and the final concentration of 10μg/m L cisplatin was added for 24 hours.Model group:the culture medium containing 10ug/m L cisplatin was added in cells for 24hours.4.In three groups:the level of estradiol was measured by the chemiluminescence method,the activity of superoxide dismutase（SOD）was measured by the biochemical method,the activity of reactive oxygen species（ROS）and mitochondrial membrane potential（MMP）were measured by flow cytometry.The expression of apoptotic molecules Bcl-2,Bax and m RNA were detected by real-time fluorescence quantitative PCR（FQ-PCR）and Western Blot.Results:The granulosa cells attached to the culture plate completely after 24h of incubation.At 48-72h,the cells were logarithmic growth phase and outgrowing granulosa cells differentiated into fibroblast‐like cells and were connected to each other by long,thin cytoplasmic projections resembling filopodia.HE staining showed that cells had a morphology of rhomboid shape and deep-dyed pink cytoplasmic full of granules.FSHR immunocytochemistry staining demonstrated that over 95%of cells were FSHR positive,characterized by the brown-stained cytoplasm and blue-dyed nucleus.1. The data show that the inhibition of cell proliferation increased when incubated with an increasing concentration of cp.The percentage of inhibition for granulosa cells treated with various concentrations（2.5μg/m L,5μg/m L,10μg/m L,20μg/m L）was（16.30±1.47）%,（25.50±11.53）%,（36.89±1.68）%,（52.92±1.22）%,respectively（P<0.05）.CCK8 assays indicated that cp increased growth inhibition in a dose-dependent for 24.Growth inhibition was clearly observed when cp reached 10μg/m L.the growth inhibition was most marked when cells were incubated with 20μg/m L of cp than 10μg/m L and are greater than50%.（52.92±1.22%）.To minimize the damage of cells and the amount of drugs we selected 10μg/m L cp as the final concentration for the following experiments.2. To investigate the effect of metformin,the cells were pretreated with different concentrations of metformin（1m M、2m M、5m M、10m M、20m M）for 2 h and the effect on cell survival was measured by CCK8.Our data show that the percentage of cell viability for granulosa cells pretreated with various concentrations metformin（1m M、2m M、5m M、10m M、20m M）for 2h was（85.39±1.59）%,（72.86±4.39）%,（42.54±0.83）%,（34.77±2.69）%,（28.80±2.25）%,respectively（P<0.05）.The results showed that the effect of various concentrations of metformin on cell viability.At 1m M and 2m M concentration of cp,he cell viability significantly increased and at 1m M than2 m M（P<0.05）In contrast,when the cells were incubated with higher concentrations of metformin（5m M、10m M、20m M）,there was no initial rise in cell viability.The cell viability significantly declined when incubated with an increasing concentration of metformin and was in a dose-dependent manner and was most high at metformin concentration of 1m M.Thus,we selected1m M metformin as the final concentration for the following experiments.3. The data show the effect of three groups on the production of estradiol by granulosa cells.The E₂ level was decreased significantly in the model group when compared with the control group（P<0.05）.In the experiment group,the E₂ level was higher than the model group but had not reached the levels in the control group（P<0.05）.4. Effect of metformin pretreatment on oxidative stress of granulosa cells induced by cisplatin:The results show that the activities of sod were a significant decrease in SOD in the model group when compared with the control group（P<0.05）.There was a significantly higher SOD level than the model group,but had not reached the levels in the control group（P<0.05）.In the model group,cisplatin increase levels of ROS and reduced MMP compared control group（P<0.05）.In the experiment group,metformin reduced levels of ROS and increased MMP significantly compared with the model group but had not reached the levels in the normal（P<0.05）.5. Effects of metformin pretreatment on apoptosis-related molecules of granulosa cells induced by cisplatin:Western blot and PCR tests are consistent.The results show that the protein levels of anti-apoptotic protein Bcl-2significantly decreased and apoptotic-related protein Bax levels increased significantly decreased in the model group when compared with the control group（P<0.05）.Meanwhile,in the experiment group,the Bcl-2 protein levels increased,Bax levels significantly decreased compared with model group but the levels of these proteins had not reached the level in normal（P<0.05）.Compared with the control group,m RNA expression levels of Bax,significantly increased,Bcl-2 significantly decreased in model group cells（P<0.05）.In experiment,group,the Bcl-2 m RNA levels increased,Bax levels significantly decreased compared with the model group but the levels of these m RNA had not reached the level in normal（P<0.05）.Conclusions:1.The POF model of human ovarian granulosa cells induced by chemotherapeutic drugs in vitro was established.2.Cisplatin causes granulosa cell damage by producing reactive oxygen species,destroying mitochondrial function,inducing oxidative stress and apoptosis.3.Metformin pretreatment improved cisplatin-induced granulosa cell injury,which may be related to metformin reducing reactive oxygen species,improving mitochondrial function,inhibiting oxidative stress and activating Bcl-2 anti-apoptotic pathway.