Study On The Mechanism Of Promoting Ovarian Follicle Development Through Tonifying Kidney,Nourishing The Blood And Promoting Blood Circulation Therapy By Regulating Oxidative Stress And Autophagy

Objective:To study the substance and molecular mechanism of Xin Jia Cong Rong Tu Si Decoction（XJCRTSD）,a traditional Chinese medicine prescription of tonifying the kidney,nourishing blood and activating blood circulation（TKNBPBCT）,which regulates oxidative stress and autophagy and promotes follicular development through SIRT1/AMPK signal pathway.Methods:（1）XJCRTSD’s compounds were identified by LC-MS/MS,effective small molecules were obtained by the algorithm,new targets were predicted by enrichment analysis,and the prediction results were preliminarily verified by molecular docking.（2）400μg and 500μg triptolide（TP）was used to make models for 40 and 60days on SD rats aged 7-8 weeks respectively.The development of follicles,autophagy,apoptosis and oxidative stress of granulosa cells（GCs）were observed by estrous cycle,ovarian tissue sections,transmission electron microscope（TEM）,TUNEL-POD and spectrophotometry.Optimize the follicular development disorder model dominated by oxidative stress and autophagy.（3）The model was replicated in vivo.The rats without model were randomly divided into Control group and to-be-Model group.The rats were gavaged with normal saline as Model group,and the rest were treated with high,medium and low doses of traditional Chinese medicine,namely HD group,MD group and LD group.The follicular development,autophagy,apoptosis and oxidative stress of GCs were observed by estrous cycle,histomorphology,TEM,Annexin/PI double staining flow cytometry and spectrophotometry.The proteins of SIRT1/AMPK signal pathway and expression of 8-Ohd G was located and semi quantitatively analyzed by immunohistochemistry,and the autophagy and apoptosis related proteins were quantitatively analyzed by Western blot.（4）In vitro experiments,GCs system was taken,the Control group without intervention,TP intervention as the model group,cultured with TP combined with drug containing serum was the TCM group,TP combined with chloroquine（CQ）as the CQ group,TP combined with N-acetyl-L-cysteine（NAC）as the NAC group,the above two groups combined with drug containing serum were TCM+CQ group and TCM+NAC group respectively,coenzyme Q10（Co-Q10）and DMSO as the control,CCK-8,Annexin/PI double staining flow cytometry,spectrophotometry,ELISA,Western blot,cell activity,apoptosis rate,oxidative stress,reproductive hormone,autophagy and apoptosis related protein,and autophagosome of GCs were observed by TEM.Finally,the two pathways were interfered,and the expression of key proteins in the pathway was observed by Western blot.Results:（1）A total of 143 kinds of small molecules with good oral availability and drug like properties were identified by pharmacomics,and 15 core drug components and 52 core targets were analyzed;The results of enrichment analysis showed that traditional Chinese medicine prescription could treat follicular development disorders through AMPK,m TOR and other signal pathways,as well as biological processes such as redox reaction and PI3K signal pathway regulation;The results of molecular docking potential energy are well.（2）In the model optimization experiment,the estrous cycle of all rats was disordered after modeling;Compared with the control group,the number of atresia follicles and GCs death increased after modeling;Except for the 400-40d group,AMH and E₂ decreased significantly and FSH increased significantly in each group;The activities of SOD and GPx in follicular fluid decreased significantly,and the content of MDA increased significantly;The degree of autophagy increased under TEM,and GCS in 500-60d group showed necrosis;The apoptosis rate of GCs increased significantly in 400-40 and 500-60 group（all p<0.05 or p<0.01）.The abnormal secretion of ovarian hormone and pituitary hormone was positively correlated with apoptosis,autophagy and oxidative stress（p<0.05）.（3）In vivo experiment,compared with the Control group,the estrous cycle in the Model group was significantly disordered;Microscopically,the number of atretic follicles increased,and the thickness of GCs layer,primary follicles and secondary follicles decreased significantly;The levels of ovarian hormones AMH,E₂ and INH-B decreased significantly,and the level of pituitary hormone FSH increased significantly;The apoptosis rate and cleaved-caspase-3 increased significantly;The activities of antioxidant enzymes SOD and GPx decreased significantly,and the content of MDA protein and the expression of 8-Ohd G increased significantly;The expression of SIRT1and AMPK increased significantly,the autophagy flow node proteins Beclin-1 and LC3Ⅱ/I increased significantly,and p62 decreased significantly（all P<0.05 or P<0.01）.Compared with the Model group,the estrous cycle of HD group recovered significantly,and the number of atretic follicles and total follicles decreased significantly;The levels of AMH,E₂ and INH-B increased significantly,while the level of FSH decreased significantly;The expression of SOD and GPx increased significantly,the MDA and 8-Ohd G decreased significantly;The expression of SIRT1 and AMPK decreased significantly;Autophagy flow was significantly inhibited;The apoptosis rate of each treatment group decreased significantly（all P<0.05 or P<0.01）.Under TEM,autophagy in model group increased significantly,mainly macroautophagy,and autophagosomes in HD group decreased,showing mitochondrial selective autophagy.Autophagy in MD group was more than that in HD group,and macroautophagy and selective autophagy were both seen,suggesting that after compound intervention,damaging autophagy changed to protective autophagy.The increase of apoptosis rate,autophagy related protein and oxidative stress were positively correlated with follicular development disorder,and the increase of oxidative stress was positively correlated with the increase of apoptosis and autophagy（p<0.05）.（4）In vitro,compared with the Control group,the cell viability of the model group decreased significantly;The apoptosis rate and cleaved-caspase-3 increased significantly;MDA and 8-Ohd G were significantly increased,and the activities of antioxidant enzymes SOD and GPx protein were significantly decreased;The autophagy flow node proteins Beclin-1 and LC3Ⅱ/I increased significantly,and p62decreased significantly;Pathway proteins p-SIRT1/SIRT1,p-AMPKα-1/AMPKα-1increased significantly;The expression of p-Akt/Akt protein decreased significantly;The levels of ovarian hormones AMH,E₂ and INH-B decreased significantly（all p<0.05 or p<0.01）;Compared with Model group,the cell viability of TCM,TCM+CQ,TCM+NAC,NAC and CQ groups increased significantly;Apoptosis related indexes decreased significantly;The level of E₂ increased slightly in CQ and NAC groups,and the level of other ovarian hormones increased significantly;The activity of antioxidant enzyme protein increased significantly,the product of oxidative stress decreased significantly,and the content of 8-Ohd G decreased slightly in CQ and NAC groups;Autophagy flow was significantly inhibited in TCM,TCM+CQ and TCM+NAC groups;Pathway node proteins p-SIRT1/SIRT1,p-AMPKα-1/AMPKα-1 significantly decreased;The expression of p-Akt/Akt protein was significantly increased（all p<0.05or p<0.01）,but there was no significant difference between CQ group and NAC group（p>0.05）.After m TOR activation,the relative expression of p-AMPKα-1 increased significantly and p-Akt and p-m TOR decreased significantly,which may activate the compensatory increase upstream of PI3K signal pathway and interfere with SIRT1/AMPK signal pathway;After AMPK inhibition,the relative expression of p-AMPKα-1 increased significantly,and p-Akt and p-m TOR decreased significantly（all p<0.05 or p<0.01）.SIRT1/AMPK signal pathway had a negative effect on PI3K/Akt signal pathway.Conclusion:Continuous intragastric administration of TP with 500μg/Kg for 40days can create a follicular development disorder model dominated by oxidative stress-induced GCs damaging autophagy and mediated by apoptosis.XJCRTSD may improve the activity of antioxidant enzymes and scavenge oxidative stress products,repair DNA anti GCs oxidative stress damage,prevent caspase-3 shear anti apoptosis,restore GCs protective autophagy,promote near follicular development and improve ovarian reserve;The mechanism may involve SIRT1/AMPK and PI3K/Akt signaling pathways and their crosstalk.The regulation of autophagy is based on SIRT1/AMPK signaling pathway itself and the negative regulation of PI3K/Akt signaling pathway.The regulation of oxidative stress may be based on the appropriate activation of PI3K/Akt signaling pathway to regulate damaging autophagy,restore protective autophagy,eliminate oxidative stress sources and improve antioxidant activity.