Effect Of Guizhi Fuling Wan On Apoptosis And Autophagy Of Granulosa Cells On Ovulation Disorder Of PCOS-IR Rats

Objective:To investigate the mechanism of Guizhi Fuling Wan treating ovulation disorder in rats with polycystic ovary syndrome with insulin resistance(PCOS-IR)by observing the regulation of endocrine level,ovarian morphology,granulosa cell apoptosis and autophagy related proteins in the rats.Methods:72 SD female rats were divided into blank group,model group,metformin group and Guizhi Fuling Wan low-dose,medium-dose and high-dose groups according to random number table method.Except blank group,rats in other groups were given letrozole sodium carboxymethyl cellulose solution 1mg·kg-1 combined with high-fat emulsion 15 m L ·kg-1 for continuous gavage for 30 days to establish PCOS-IR rat model.At the same time,the drug treatment group was given drug intervention.Vaginal exhumed cell smear was performed on the 21 st day of intragastric administration to observe the estrous cycle of rats.Serum sex hormone levels were detected by enzymelinked immunoassay(ELISA).The ratio of LH to FSH and HOMA-IR were calculated.The wet weight of the ovaries was weighed and the long diameter and short diameter were measured,and the ovarian index and ovarian volume were calculated.TUNEL was used to inspect the number of apoptotic positive cells.Morphological changes of ovarian tissue were observed by HE staining.The protein expression of cleaved PARP,cleaved caspase3,cleaved caspase9,p62,Beclin1 and LC3 II in rat ovarian granulosa cells were observed by Western Blot.Results:1.Effect of Guizhi Fuling Wan on estrus cycle of PCOS-IR ratsThe estrus cycle of control group showed regular changes,which could be seen in all four periods;The estrous cycle of the female rats in the model group was not regular and stayed in the estrous interphase but little estrous phase.However,the estrus cycle of rats in Guizhi Fuling Wan low,medium and high groups and metformin groups basically recovered regularly,showing periodic changes.2.Effect of Guizhi Fuling Wan on ovarian index and ovarian volume of PCOS-IR ratsCompared to the control group,ovarian coefficient was significantly decreased,ovarian volume was significantly increased in model group(P <0.01),Compared to the model group in Guizhi Fuling Wan medium-dose and high-dose groups and metformin groups was significantly increased,ovarian volume was decreased(P <0.01).3.Effect of Guizhi Fuling Wan on histomorphological changes of ovary in PCOSIR ratsPrimary follicles,primary follicles and secondary follicles were observed in ovary of control group,which were more numerous and well developed;There are also mature follicles with large volume and more follicular fluid.There are more granulosa cells in follicle.The corpus luteum is also well developed and numerous.The number of cystic follicles and atretic follicles were increased in the model group and the thickness of the granulosa cell layer of the ovary was significantly decreased.The number of cystic follicles and atretic follicles were decreased in the Guizhi Fuling Wan low-dose,medium-dose and high-dose groups and the metformin groups,and there were mature follicles with larger volume and more follicular fluid.4.Effect of Guizhi Fuling Wan on sex hormone levels in PCOS-IR ratsCompared to the control group,serum T,LH levels and LH/FSH in model group were increased(P<0.01),and the difference in FSH was not statistically significant.LH and LH/FSH decreased in Guizhi Fuling Wan low-dose,medium-dose and high-dose groups(P<0.05,P<0.01),while serum T level in Guizhi Fuling Wan low-dose,mediumdose and high-dose groups and metformin groups were significantly decreased compared to the model group(P<0.05,P<0.01).5.Effect of Guizhi Fuling Wan on insulin resistance of PCOS-IR ratsCompared to the control group,serum FINS level and HOMA-IR of model group were increased(P<0.01),Compared to the model group,serum FINS level and HOMAIR of Guizhi Fuling Wan low-dose,medium-dose and high-dose groups and metformin groups were significantly decreased(P<0.01).6.Effect of Guizhi Fuling Wan on TUNEL apoptotic positive cells in ovarian granulosa cells of PCOS-IR ratsCompared to the control group,the number of apoptotic positive cells in ovarian granulosa cells of model group was significantly increased(P<0.05);Compared to the model group,the number of apoptotic positive cells in Guizhi Fuling Wan low-dose,medium-dose and high-dose groups and metformin groups was significantly decreased,especially in Guizhi Fuling Wan high-dose group(P<0.05).7.Effects of Guizhi Fuling Wan on cleaved-PARP,cleaved caspase-3,cleaved caspase-9 protein in ovarian granulosa cells of PCOS-IR ratsWestern blot results showed that cleaved-PARP,cleaved-Caspase3,cleavedCaspase9 protein expression was significantly increased in model group compared to the control group(P<0.01,P<0.05),Compared to the model group,cleaved-PARP,cleaved-Caspase3,cleaved-Caspase9 protein expression in ovarian tissue of rats in Guizhi Fuling Wan low-dose,medium-dose and high-dose groups and metformin groups was significantly decreased(P<0.01,P<0.05).8.Effects of Guizhi Fuling Wan on P62,Beclin1,LC3 II proteins in ovarian granulocyte cells of PCOS-IR ratsWestern blot results showed that the expression of autophagy-related protein P62 in ovarian tissue of rats showed a decreasing trend,but the difference was not statistically significant in model group compared to the control group.Compared to the model group,the expression of P62 protein in Guizhi Fuling Wan high-dose group showed an increasing trend,but the difference was not statistical difference.Compared to the model group,the expression of autophagy related protein Beclin1,LC3 II in ovarian tissue of rats was significantly increased in model group compared to the control group(P<0.05),compared with model group,the protein expressions of Beclin1 and LC3II in Guizhi Fuling Wan medium and high dose groups were significantly decreased(P<0.05).Conclusion:1.Guizhi Fuling Wan restores the normal morphology of ovary in PCOS-IR rats to a certain extent,improves the ovarian index and reduces ovarian volume,and improves the ovarian function of rats.2.Guizhi Fuling Wan corrects sex hormone and islet dysfunction in PCOS-IR rats,improves islet resistance,and promotes follicle maturation and ovulation.3.Guizhi Fuling Wan significantly reduces the PCOS-IR rat granulosa cell apoptosis positive cells number,cuts the expression of cleaved-PARP,cleaved-Caspase3,cleaved-Caspase9,at the same time,cuts the expression of Beclin1 and LC3 II protein,inhibits the excessive apoptosis of granulosa cells and autophagy,to improve ovarian microenvironment and function of rats and PCOS-IR ovulation.