The Molecular Mechanism Of Tonifying Kidney,Nourishing And Activating Blood Therapy In Regulating Apoptosis And Autophagy Flux Through PI3K/AKT/mTOR Signaling Pathway

Experiment Ⅰ:The study on promoting follicular development and repairing ovarian damage through PI3K/AKT/mTOR signaling pathway by tonifying kidney,nourishing and activating bloodObjective:To observe the effect of traditional Chinese medicine compound prescription XJCRTSZW with the therapy of tonifying kidney,nourishing and activating blood to promote follicular development and repair ovarian damage through PI3K/AKT/mTOR signaling pathway.Methods:1.56 female SD rats with normal estrous cycles were selected.8 were randomly selected from the normal control group and given distilled water for gavage,and the remaining 48 were built follicular dysplasia models by continuous gavage of 400μg/kg.d triptolide（TP）for 60 days in accordance with the preliminary experimental results.2.After modeling,the rats were randomly divided into 6 groups,and 8 rats in each group.They were triptolide model group,western drug（EV+DMPA）group,XJCRTSZW low dose group,XJCRTSZW high dose group,NVP-BEZ235 inhibitor group,XJCRTSZW high dose+inhibitor（NVP-BEZ235）group.Western medicine control group was given sequential therapy of estradiol valerate and medroxyprogesterone acetate.The low and high dose groups of traditional Chinese medicine were treated with low and high dose extractum of XJCRTSZW.The inhibitor group was given NVP-BEZ235 by gavage.The high dose XJCRTSZW+inhibitor（NVP-BEZ235）group was given Chinese traditional medicine and NVP-BEZ235 by gavage.The normal control group and TP model group were given the same amount of distilled water by gavage.All animals were given intragastric administration for 2weeks,during which vaginal smears were performed to observe the changes of estrous cycle in rats.3.After gavage,serum E₂,P,FSH,LH,AMH and ET were detected by ELISA.NO was detected by nitrate reductase method and ratio of ET/NO was calculated.After blood collection,rats were sacrificed and bilateral ovarian tissue was extracted.The wet weight of ovary was weighed and the ovarian index was calculated.Ovarian sections were stained with HE.The numbers of primary and secondary follicles,antral follicles,atresia follicles and corpus luteum were counted under optical microscope and the granular cell layer thickness of antral follicles was measured.WB method was used to detect the protein content of p-PI3K、p-AKT、p-mTOR in ovarian tissue.Results:1.Effects of XJCRTSZW on serum sex hormones in rats:Compared with the normal control group,the contents of E₂,P and AMH in the serum of the TP model group decreased significantly,while the contents of FSH and LH increased significantly（p<0.01）.Compared with the model group,XJCRTSZW could increase the content of E₂,P,AMH and reduce the content of FSH and LH（p<0.01）,and the XJCRTSZW high-dose group had the same effect as the western medicine group（p>0.05）.After using the pathway inhibitor NVP-BEZ235,the ability to increase E₂,P,AMH and decrease FSH,LH disappeared in the XJCRTSZW high-dose group（p<0.01）.2.Effects of XJCRTSZW on serum ET,NO in rat and the ratio of ET/NO:Compared with the normal control group,the serum ET content in the TP model group increased,NO content decreased,and the ET/NO ratio increased significantly（p<0.01）.Compared with the TP model group,XJCRTSZW could increase the content of NO,reduce the content of ET and the ratio of ET/NO（p<0.01）,and the XJCRTSZW high-dose group had the same effect as the western medicine group（p>0.05）.After the use of PI3K/AKT/mTOR pathway inhibitor NVP-BEZ235,the NO content in the ovaries of rats decreased,and the ET content,ET/NO ratio increased,but there was no statistical significance（p>0.05）.The protective effect of XJCRTSZW high-dose group on the ovaries disappeared to a certain extent.3.Effects of XJCRTSZW on wet weight and ovarian index of rats:Compared with the normal control group,the ovarian wet weight and ovarian index of the TP model group decreased significantly（p<0.01）.Compared with the TP model group,XJCRTSZW could increase the ovarian wet weight and ovarian index（p<0.01）to some extent,and the XJCRTSZW high-dose group had the same effect as the western medicine group（p>0.05）.After the use of PI3K/AKT/mTOR pathway inhibitor NVP-BEZ235,the ability to improve ovarian wet weight and ovarian index disappeared in the XJCRTSZW high-dose group（p<0.01）.4.Effects of XJCRTSZW on follicular development,corpus luteum number and granular cell layer thickness of antral follicles in rats:Compared with the normal control group,the number of secondary follicles,antral follicles,corpus luteum and the thickness of granular cell layer in the TP model group decreased（p<0.01）,and the number of atretic follicles increased（p<0.01）,while TP had no significant effect on the number of primary follicles（p>0.05）.Compared with the TP model group,XJCRTSZW could increase the number of secondary follicles,antral follicles,corpus luteum and the granulosa cell layer thickness of antral follicles in rats（p<0.01）,and reduce the number of atretic follicles（p<0.01）,and the XJCRTSZW high-dose group had the same effect as the western medicine group（p>0.05）.After the use of PI3K/AKT/mTOR pathway inhibitor NVP-BEZ235,the protective effect of XJCRTSZW group on follicular development disappeared（p<0.01）.5.Effects of XJCRTSZW on p-PI3K,p-AKT,p-mTOR in ovarian tissue of rats:Compared with the normal control group,the protein levels of p-PI3K,p-AKT and p-mTOR in the ovarian tissue of the TP model group were significantly decreased（p<0.05 or p<0.01）.The XJCRTSZW high-dose group could improve the effects of TP on the above molecules to varying degrees（p<0.05 or p<0.01）.While the ability of the XJCRTSZW high-dose group to increase the above molecules disappeared after the use of pathway inhibitor NVP-BEZ235（p<0.05 or p<0.01）.Conclusions:Using triptolide 400μg/kg.d continuous gavage for 60 days,the in vivo animal model of follicular development disorder in rats can be successfully constructed,and the molecular mechanism may be that TP inhibits the PI3K/AKT/mTOR signaling pathway by down-regulating the key proteins p-PI3K,p-AKT and p-mTOR.The traditional Chinese medicine compound prescription XJCRTSZW with the therapy of tonifying kidney,nourishing and activating blood can improve the sex hormone level of rats by activating the PI3K/AKT/mTOR signaling pathway,and increase the wet weight and ovarian index of the rats.XJCRTSZW can restore the number of follicles at all levels,luteal number of the rats and the thickness of granulosa cell layer of the growing follicles,improve the state of"blood stasis"of the rats,protect the follicular development of the rats,and repair the ovarian damage caused by TP.Experiment Ⅱ:The molecular mechanism of tonifying kidney,nourishing and activating blood therapy in regulating apoptosis and autophagy flux of granulosa cells through PI3K/AKT/mTOR signaling pathwayObjective:To investigate the molecular mechanism of tonifying kidney,nourishing and activating blood therapy in regulating apoptosis and autophagy flux of ovarian granulosa cells through PI3K/AKT/mTOR signaling pathway.Methods:1.Primary rat ovarian GCs cultured in vitro were divided into 9 groups,they are:normal control group,TP model group,western medicine FSH group,XJCRTSZW high-dose group,autophagy inducer group,XJCRTSZW high-dose+autophagy inducer group,apoptosis inducer group,XJCRTSZW high-dose+apoptosis inducer group.The normal control group was given distilled water by gavage.After the other 8groups were induced with TP for 12h,the rats serum in the model group was given by gavage with distilled water.The FSH group was treated with FSH-containing rat serum.Low and high dose XJCRTSZW groups were treated with corresponding doses of rat serum containing drugs.In the autophagy and apoptosis inducer groups,rat serum given distilled water and autophagy or apoptosis inducer were added.XJCRTSZW high-dose+autophagy inducer group was added with XJCRTSZW high-dose serum and autophagy inducer RAPA.XJCRTSZW high-dose+apoptosis inducer group was added with XJCRTSZW high-dose serum and apoptosis inducer CPT.2.The above drugs and corresponding rat serum were incubated for 48 hours and then tested as follows.Cell viability was detected by CCK8 assay.Flow cytometry was used to detect the apoptosis rate of GCs in each group.The levels of E2,P and AMH in the supernatant of cell culture were detected by ELISA.After mRFP-GFP-LC3adenovirus transfection,autophagic flow was observed and the number of autophagosomes and autophagolysosomes was counted under laser confocal microscope.The formation of autophagosomes and autophagolysosomes in GCs of each group was observed under transmission electron microscope.WB and RT-qPCR were used to detect protein expressions and mRNA levels of caspase-3,Cleaved caspase-3,LC3-II、LC3-I,beclin-1,p62,LAMP2,cathepsin-d and PI3K/AKT/mTOR axis-related molecules.The ratio of LC3-II/LC3-I was then calculated.Results:1.Effects of XJCRTSZW on the proliferation and apoptosis of GCs:（1）Cell viability test:The cell viability of ovarian GCs was significantly decreased after TP modeling（p<0.01）,indicating that TP significantly inhibited the proliferation of GCs.The activity of GCs was significantly increased in the XJCRTSZW high-dose serum group（p<0.05）.However,the ability of XJCRTSZW high-dose serum group to protect cell viability was somewhat impaired after the addition of CPT（p<0.05 or p<0.01）.This indicated that XJCRTSZW promoted cell viability and cell proliferation by inhibiting the apoptosis of GCs.（2）WB:After TP modeling,the expression of Cleaved caspase-3（CC3）protein was significantly increased（p<0.01）,but there was no significant effect on caspase-3.XJCRTSZW high-dose group significantly improved the effect of TP on CC3 after modeling（p<0.01）.However,after the use of CPT,the ability to down-regulate the expression of CC3 protein in the XJCRTSZW high-dose group disappeared,indicating that the traditional Chinese medicine protected the proliferation of GCs by down-regulating the apoptotic factor CC3.（3）Flow cytometry:The apoptosis rate of GCs after TP modeling was as high as39.54%（second only to CPT group with apoptosis inducer）,and the apoptosis rate increased significantly compared with the normal control group（p<0.01）.After intervention in the XJCRTSZW high and low dose groups,the apoptosis rate of GCs in rats decreased significantly（p<0.01）.The XJCRTSZW high-dose group showed the most significant decline（apoptosis rate 12.67%）,with no difference from the western medicine FSH group（p>0.05）.After the addition of the apoptotic inducer CPT,the ability of XJCRTSZW high-dose serum group to reduce apoptosis rate and inhibit apoptosis disappeared（p<0.01）.2.Effect of XJCRTSZW on the autophagy flux of GCs:（1）WB method and RT-qPCR method:After TP modeling,protein expression and mRNA level of autophagy flux related molecular LC3Ⅱ,Beclin1,LAMP2,Cathepsin-D increased significantly,and the ratio of LC3-II/LC3-I increased（p<0.05or p<0.01）.Protein expression and mRNA level of p62 were significantly down-regulated（p<0.01）.XJCRTSZW high dose group can significantly cut protein expression and mRNA level of LC3Ⅱ,Beclin1,LAMP2,Cathepsin-D（p<0.05 or p<0.01）,and up-regulate the protein expression and mRNA level of p62（p<0.01）.However,the ability of XJCRTSZW high-dose group to protect GCs disappeared after the use of autophagy inducer RAPA.（2）TEM:Compared with the normal control group,the number of autophagosomes and autophagolysosomes in the ovarian GCs of rats in the TP model group increased.Compared with the TP model group,the number of autophagosomes and autophagolysosomes in the ovarian granulosa cells of rats in the XJCRTSZW serum group decreased.After RAPA intervention,the ability of XJCRTSZW high-dose group to inhibit the production of autophagosomes and autophagosomes decreased.（3）mRFP-GFP-LC3 adenovirus transfection:The number of autophagosomes（yellow puncta）and autophagic lysosomes（red puncta）in the ovarian GCs of rats was significantly increased after TP modeling（p<0.01）.Compared with the TP model group,the number of autophagosomes and autophagolysosomes in the ovarian granulosa cells of rats in the XJCRTSZW high-dose and low-dose group was significantly reduced（p<0.01）,and there was no difference between the XJCRTSZW high-dose group and the western medicine FSH group（p>0.05）.When cells were treated with autophagy inducer RAPA,the ability of XJCRTSZW high-dose group to inhibit the production of autophagosomes and autophagosomes was significantly decreased（p<0.01）.3.Effect of XJCRTSZW on the secretion of E₂,P and AMH in the supernatant of rat ovarian GCs:Compared with the normal control group,the contents of E_2,P and AMH in the supernatant of rat ovarian granulosa cells in the TP model group decreased significantly（p<0.01）.Compared with the model group,the contents of E₂,P and AMH in the superscript of GCs in the ovary of rats in the XJCRTSZW high-dose group were significantly increased（p<0.01 or p<0.05）,and the effect of the XJCRTSZW high-dose group was similar to that of the FSH group（p>0.05）.After the intervention of RAPA and CPT,the upregulation of E₂,P and AMH in the XJCRTSZW high-dose group disappeared to a certain extent,but there was no statistical significance（p>0.05）.4.Effect of XJCRTSZW on PI3K/AKT/mTOR pathway axis-related molecules in rat ovarian GCs:Through WB detection,we found that the PI3K/AKT/mTOR pathway axis-protein phosphorylation levels of PI3K,AKT and mTOR decreased significantly after TP modeling（p<0.05 or p<0.01）.XJCRTSZW high-dose group can significantly improve the influence of TP on the above molecules after molding（p<0.05 or p<0.01）.After using RAPA and CPT,the therapeutic ability of XJCRTSZW high-dose group disappeared（p<0.05 or p<0.01）.Conclusions:TP,may through the inhibition of PI3K/AKT/mTOR signaling pathway on the one hand,promote the GCs apoptosis by increasing the rate of GCs apoptosis and up-regulating apoptosis related molecular Cleaved Caspase 3,on the other hand activate the GCs autophagy flux by increasing the number of GCs autophagosome and autophagy-lysosome,raising autophagy related factor LC3Ⅱ,Beclin1,LAMP2,Cathepsin D and cutting p62,which damages the rat ovarian GCs and suppresses the follicle development.Chinese medicine compound prescription XJCRTSZW with the therapy of tonifying kidney,nourishing and activating blood,may through the activation of PI3K/AKT/mTOR signaling pathway on the one hand,depress GCs apoptosis by decreasing the rate of GCs apoptosis and down-regulating apoptosis related molecular Cleaved Caspase 3,on the other hand depress the excessive activation of GCs autophagy flux by decreasing the number of GCs autophagosome and autophagy-lysosome,cutting autophagy related factor LC3Ⅱ,Beclin1,LAMP2,Cathepsin D and raising p62,so as to fix the ovarian GCs damage caused by TP and protect the development of rat follicles.This study provides a new target for prevention and treatment of follicular development disorders and a new possible repair scheme for ovarian damage caused by tripterygium wilfordii.