| Objective:This project was supported by the National Natural Science Foundation of China: "Based on the theory of ’jealous infertility’ in Chinese medicine,the study on the influence of uncontrollable inflammation caused by psychological stress on SIRT1-mediated autophagy-apoptosis homeostasis of follicles"(No.: 8177150454).In this study,SIRT1 gene silence mice were used as the research object,and the effect of Kaiyu Zhongyu Decoction on reproductive hormones,serum inflammatory cytokines and autophagy-apoptosis related proteins of mice with chronic psychological stress was observed,so as to explore the mechanism of Kaiyu Zhongyu Decoction improving ovarian reserve of mice with psychological stress through SIRT1 pathway.Methods:Experiment 1: The effect of Kaiyu Zhongyu Decoction on inflammatory factors in mice under psychological stressA total of 100 healthy non-pregnant female SPF ICR mice were randomly divided into 5groups.(1)Blank Control group(Control)(2)psychological Stress group(Stress)(3)psychological Stress + Kaiyu Zhongyu Decoction group(KYZY)(4)SIRT1 gene silence +psychological Stress group(SIRT1si)(5)SIRT1 gene silence + psychological Stress + Kaiyu Zhongyu Decoction group(SIRT1si+ KYZY).(1)The behavioral changes of mice were evaluated by body weight change,open field test and sugar water preference test.(2)The contents of CRH,ACTH and CORT in serum were detected by ELISA.(3)The contents of IL-1β,IL-6,IL-8,TNF-α and INF-γ in serum were determined by ELISA.Experiment 2: The effect of Kaiyu Zhongyu Decoction on ovarian reserve in mice under psychological stress(1)The contents of FSH,LH,E2 and AMH in serum were determined by ELISA.(2)The ovarian tissue of mice was stained with HE,and the morphology of follicles at all levels was observed under light microscope.(3)The apoptotic degree of oocytes and granulosa cells in mouse ovary tissue was detected by TUNEL assay.Experiment 3:Effect of Kaiyu Zhongyu Decoction on "autophagy-apoptosis" protein in mice under psychological stress(1)The mRNA expression levels of SIRT1,Fox O1,AMPK,Caspase-3,Bcl-2 and Bax in ovarian tissues of mice in each group were detected by qPCR.(2)The protein expression levels of Atg12-Atg5,AMPK,SIRT1,Fox O1,Caspase-3 and Beclin1 in mouse ovarian tissues were detected by immunohistochemistry.(3)The protein expression levels of Bcl-2 and Bax in mouse ovarian tissues were detected by Western Blot.Results:1 Comparison of serum CRH,CORT,ACTH contents and behavioral changes of mice in each groupCompared with the control group,mice in the Stress group and SIRT1 si group gained more slowly(P < 0.05);The sugar water consumption decreased and the sugar water preference coefficient decreased significantly(P < 0.05);The total distance and time of movement of mice in the stress group and SIRT1 si group were significantly decreased(P<0.05),the static time was prolonged(P<0.05),the frequency of entering the central region was significantly decreased(P<0.05).Compared with the control group,serum CRH,CORT and ACTH contents in the stress group were significantly increased(P<0.05);The contents of CORT and ACTH in KYZY group were significantly lower than those in Stress group(P<0.05);CRH and CORT contents in SIRT1 si group were significantly higher than those in control group(P<0.05);Compared with the stress group,the contents of CRH,CORT and ACTH in SIRT1 si group were significantly decreased(P < 0.05);The contents of CORT,CRH and ACTH in SIRT1 si +KYZY group were significantly lower than those in Stress group(P<0.05).2 Comparison of the contents of IL-1β,IL-6,IL-8,TNF-α and INF-γ in serum of mice in each groupCompared with the control group,IL-1β,IL-6,IL-8 and TNF-α contents in the stress group were significantly increased(P<0.05);The contents of IL-1β,IL-6,IL-8 and TNF-α in serum of KYZY group and SIRT1 si group were significantly lower than those of stress group(P<0.05),there was no significant difference in INF-γ content(P > 0.05);Compared with SIRT1 si group,the contents of IL-1β and TNF-α in SIRT1 si + KYZY group were significantly decreased(P<0.05).3 Comparison of ovarian function indexes in each group3.1 Comparison of serum FSH,LH,E2 and AMH contents in each groupCompared with the control group,the concentrations of FSH,LH,E2 and AMH in the stress group were significantly decreased(P<0.05);The concentrations of FSH,LH,E2 and AMH in SIRT1 si group and KYZY group were significantly higher than those in stress group(P<0.05);Compared with SIRT1 si group,the concentrations of FSH,LH and E2 in SIRT1 si +KYZY group were significantly increased(P<0.05).3.2 Comparison of follicular development in each groupIn the control group,the follicle development morphology was better,the granulosa cells of follicles were orderly,and the number of primary follicles was more.The number of follicles in the stress group decreased and atresia follicles were visible.The number of follicles and corpus luteum increased in KYZY group.Primary follicles were observed in SIRT1 si group.In SIRT1 si + KYZY group,the number of ovarian follicles increased,but the morphology was not good,and the number of corpus luteum increased.3.3 Comparison of apoptotic degree of ovary oocytes and granulosa cells in each groupCompared with the control group,the mean fluorescence intensity of oocytes and granulocytes in the stress group was significantly increased(P<0.05);The mean fluorescence intensity of oocytes and granuloids in KYZY group was significantly lower than that in stress group(P < 0.05);The mean fluorescence intensity of oocytes and granulocytes in SIRT1 si group was significantly lower than that in stress group(P<0.05).4 Changes in apoptosis and mRNA levels of SIRT1 pathway in each groupCompared with the control group,Caspase-3 mRNA expression was increased in the stress group(P<0.05),there was no significant difference in the expression levels of Bcl-2and BaxmRNA(P>0.05);The mRNA levels of Caspase-3 and Bcl-2 in KYZY group were significantly lower than those in stress group(P < 0.05);The mRNA expression levels of caspase-3 and bcl-2 in SIRT1 si group were significantly lower than those in stress group(P<0.05).Compared with the control group,Fox O1 mRNA expression was significantly increased in the stress group(P<0.05);The expression level of SIRT1 mRNA in SIRT1 si group was significantly lower than that in stress group(P<0.05).5 Changes of autophagy-apoptosis protein levels in each groupCompared with the control group,protein expression levels of Atg12-Atg5,Caspase-3and Bax were increased in the stress group(P<0.05),the protein expression levels of Bcl-2and Beclin-1 were decreased(P<0.05);The protein expression levels of Beclin-1 and Bcl-2in KYZY group were significantly higher than those in stress group(P<0.05);The expression level of Bcl-2 protein in SIRT1 si group was significantly higher than that in stress group(P<0.05),the protein expression levels of Atg12-Atg5,Caspase-3 and Bax were significantly decreased(P<0.05).Compared with the control group,the expression levels of AMPK and SIRT1 in the stress group were significantly decreased(P < 0.05);The expression level of SIRT1 in KYZY group was significantly higher than that in stress group(P<0.05);The expression levels of AMPK and SIRT1 in SIRT1 si group were significantly lower than those in stress group(P<0.05).Conclusion:1.Chronic psychological stress significantly increased the content of serum inflammatory factors,increased the expression of apoptotic proteins Bax and Caspase-3,decreased the expression of autophagy protein Beclin-1,increased the degree of apoptosis of ovarian tissue cells,and decreased the secretion of reproductive hormones,leading to decreased ovarian reserve;2.Chronic psychological stress resulted in the decrease of SIRT1 and AMPK protein expression in mouse ovarian tissue;3.SIRT1 gene silencing can inhibit the increase of inflammatory factors mediated by psychological stress,the increase of apoptotic proteins and the decrease of reproductive hormones;4.Kaiyu Zhongyu Decoction can reduce the content of inflammatory factors,inhibit apoptosis,promote autophagy,increase the secretion of reproductive hormones,and improve ovarian reserve in mice under psychological stress;5.The mechanism of Kaiyu Zhongyu Decoction improving ovarian reserve in mice under psychological stress may be related to SIRT1 pathway. |
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