Study On The Molecular Mechanism Of Nourishing Kidney And Activating Blood Therapy In Regulating Granulosa Cells Autophagy Of Rat Ovary Based On PI3K/AKT/mTOR Signaling Pathway

Experiment ?:Study on autophagy induced by triptolide in primary cultured rat ovarian granulosa cells in vitroAim:To observe the concentration,time and mechanism of autophagy induced by triptolide?TP?in granulosa cells?GCs?.Methods:CCK-8 method was used to compare the inhibitory effects of TP at different concentrations on primary cultured rat ovary GCs and IC50 was calculated.The effects of TP at different concentrations and time points on the expression of GCs autophagy factor protein and the cascade of PI3K/AKT/mTOR pathway were detected by WB method.The effects of TP,autophagy inducer?BrefeldinA?and PI3K/mTOR inhibitor?NVP-BEZ235?on the expression of PI3K/AKT/mTOR cascade and autophagy related factor protein were detected by WB method.Results:1.The IC50 of different concentrations of TP on GCs of rat ovary was 14.65?M,and the minimum inhibitory concentration of TP was 0.1?M?100nM?;2.Compared with the control group,the expression levels of Beclin1 and LC3II in each group were significantly higher than those in the control group?p<0.05 or p<0.01?;3.After the GCs12h of rats was treated with TP,BrefeldinA and NVP-BEZ235,compared with the control group,TP could significantly promote the expression level of downstream autophagy effector molecule Beclin1?LC3II and inhibit the expression level of LC3I?p62 protein?p<0.05 or p<0.01?.Moreover,the expression of Beclin 1and LC3II/LC3I in TP group was higher than that in Brefeldin A group?p<0.05 or p<0.01?,and the expression of Beclin1 and LC3II/LC3I in TP group was higher than that in Brefeldin A group?p<0.05 or p<0.01?.At the same time,TP could significantly inhibit the expression of p-PI3K,p-AKT,p-mTOR protein,and the inhibitory effect of TP was better than that of NVP-BEZ235 group.Conclusion:TP?100nM?can induce GCs autophagy successfully in cultured rat ovary for 12h;TP may induce GCs autophagy by inhibiting PI3k/Akt/mTOR signaling pathway.Experiment ?.Nourishing Kidney and Activating Blood Therapy Regulat granulosa cell autophagy in rats based on PI3K/AKT/mTOR signaling pathwayAim:To explore the molecular mechanism of regulating ovarian GCs autophagy in vitro by the Nourishing Kidney and Activating Blood Therapy based on PI3K/AKT/mTOR pathway as the entry point.Methods:1.Pharmacological experiment:The primary cultured rat ovary GCs was divided into 6 groups:blank control group,model group,low-dose Chinese medicine group,middle-dose Chinese medicine group,high-dose Chinese medicine group and western medicine?FSH?group.The rats in the blank control group were given the serum of normal saline,the other five groups were treated with TP for 12 hours,and the model group was given the serum of normal saline for 12 hours.Serum of rats with FSH was added to each dose of Chinese medicine group and FSH group respectively.After 48hours of serum incubation,the secretion of E₂ and P in GCs of each group was detected by Elisa method;The mRNA expression of PI3K,AKT,mTOR,Beclin1,LC3,p62 and the protein expression of Beclin1,LC3,p62,p-PI3K,p-AKT,p-mTOR in GCs were detected by RT-qPCR,WB and immunofluorescence respectively.2.Mechanism experiment:Primary cultured rat ovary GCs was divided into 5groups:blank control group,model group,high-dose Chinese medicine group,inhibitor group,high-dose Chinese medicine+inhibitor group.The rats in the blank control group were given normal saline intragastric serum,the other four groups were first induced by TP for 12 hours,the model group was given serum of rat gavaged with normal saline,high-dose Chinese medicine group was given high-dose Chinese medicine rat serum containing drug,the inhibitor group was given normal saline gastric perfusion rat serum containing drug and inhibitor,and the high-dose Chinese medicine+inhibitor group were given serum containing drug and inhibitor of rat gavaged with high-dose Chinese medicine.After 48 hours,the number of autophagy bodies in GCs of each group was observed by transmission electron microscope,and the other indexes and methods were the same as the drug effect experiment.Results:1.Pharmacological experiment:1)Elisa method:Compared with the blank control group,the E₂ content in the model group was significantly lower than that in the control group.Compared with the model group,the E₂ content in each dose group of Chinese medicine was all increased?p<0.05?,and the E₂ content in the high-dose group and FSH group was higher than that in the control group?p<0.05?.There was no significant difference between the two groups?p>0.05?.Compared with the blank control group,each group had no effect on the content?p>0.05?.2)RT-qPCR method:Compared with the control group,there was no significant change in the mRNA level of PI3KPK AK TOR in the GCs of each group?p>0.05?,while in the model group,the mRNA levels of the Beclin 1,LC3I,LC3II and p62were significantly higher than that of the control group?p<0.01?;The mRNA levels of Beclin 1,LC3I,LC3IIand p62 in each dose group and FSH group were significantly decreased?all p<0.05?;The effect of high-dose Chinese medicine group was better than that of western medicine FSH group?all p<0.05?.3)WB method:Compared with the control group,the expression of p-PI3K,p-AKT,p-mTOR and p62 in the model group was significantly lower than that in the control group?p<0.01?;The levels of p-PI3K,p-AKT,p-mTOR and the expression of p62 in each dose group and FSH group were up-regulated and the expression of Beclin 1 and LC3II/I were down-regulated,and the effect of high-dose group was better than that of FSH group?all p<0.05?.4)Immunofluorescence method:The expression of p-PI3K,p-AKT,p-mTOR and p62 decreased in model group,the expression of p-PI3K,p-AKT,p-mTOR and p62were up-regulated and down-regulated in each dosage group and FSH group.The effect of high-dose group was better than that of FSH group?all p<0.05?.2.Mechanism experiment:1)Elisa method:Compared with the blank control group,the E₂ content in the model group was significantly lower than that in the control group.Compared with the model group,the E₂ content in the high-dose group of TCM was significantly higher than that in the high-dose group of TCM+inhibitor group?p<0.01?,and that the E2 content in the high-dose group of TCM was significantly higher than that in the high-dose group of TCM+inhibitor group?p<0.01?;But compared with the blank control group,the content of P in each group had no effect?p>0.05?.2)Transmission electron microscope results:The number of autophagy bodies in the model group was more than that in the blank control group,the number of autophagy bodies in the high dose group was less than that in the model group,and the number of autophagy bodies in the high-dose group was less than that in the Chinese medicine+inhibitor group.3)RT-qPCR method:Compared with the control group,there was no significant change in the mRNA levels of PI3KAK,AKT and mTOR in each group?p>0.05?,but the mRNA levels of LC3I,LC3II and p62 in the control group were significantly higher?p<0.01?;The mRNA levels of Beclin1,LC3IzLC3II and p62 in the high-dose group were significantly lower than those in the Chinese medicine+inhibitor group?p<0.01?,and the level of mRNA in the high dose group was significantly lower than that in the Chinese medicine+inhibitor group?p<0.01?.4)WB method:Compared with the control group,the expression of p-PI3K,p-AKT,p-mTOR and p62 in the model group was significantly lower than that in the control group;The levels of p-PI3K,p-AKT,p-mTOR and the expression of p62 in high-dose group were up-regulated,and the expression of Beclin 1 and LC3II/I were down-regulated.The expression of p-PI3K,p-AKT,p-mTOR and p62 in high-dose group was significantly higher than that in high-dose+inhibitor group?p<0.01?.5)Immunofluorescence method:The expression of p-PI3K,p-AKT,p-mTOR and p62 decreased in the model group,the expression of p-PI3K,p-AKT,p-mTOR and p62 in the high dose group of traditional Chinese medicine increased,and the expression of p-PI3K,p-AKT,p-mTOR and p62 up-regulated the expression of Beclin 1 and LC3II.The expression of p-PI3K,p-AKT,p-mTOR and p62 in the high-dose group was significantly higher than that in the high-dose group,and the expression of Beclin 1 and LC3II in the high-dose group was significantly lower than that in the traditional Chinese medicine+inhibitor group.Conclusion:The effect of TP100nM on rat ovary GCs12h can significantly inhibit the level of E₂ secreted by GCs,and the high-dose group of Chinese herbal medicine can significantly improve the inhibition of TP on the secretion of E₂ by GCs in vitro;The Bushen Huoxue recipe Guishen pills may repair TP induced ovarian GCs autophagy injury in rats by activating the PI3K/AKT/mTOR pathway,down-regulating the expression of downstream autophagy molecules Beclin1 and LC3 and reducing the consumption of p62.