Related Mechanism Study Of Jianpi Yishen Huazhuo Formula Improves Polycystic Ovary Syndrome Through PI3K/AKT/mTOR Pathway

Objective To study the effect of Jianpi Yishen Huazhuo Formula（JPYSHZF）on ovarian inflammation and apoptosis in rats with polycystic ovary syndrome（PCOS）through PI3K/AKT/mTOR signal pathway,and to explore the mechanism of JPYSHZF in improving the ovarian function of PCOS rats,so as to provide scientific basis for the clinical treatment of PCOS by JPYSHZF.Methods Seventy female SD rats without specific pathogens were randomly divided into two groups:the blank group（n=15）was given physiological saline by gavage daily,and the model group（n=55）was given letrozole 1mg/kg by gavage daily in combination with high-fat diet.After 21 days,five rats were randomly selected from each of the above two groups to observe the estrous cycle,pathological section of ovarian tissue（HE）staining and sex hormone changes to verify the model.The rats（n=50）with successful modeling were randomly divided into model group,metformin group,low,medium and high dose groups of Jianpi Yishen Huazhuo Formula,with 10 rats in each group.The blank group and model group were given equal volume of normal saline by gavage;The metformin group was given metformin hydrochloride tablets 100 mg/kg by gavage;The low,middle and high dose groups of Jianpi Yishen Huazhuo Formula were given 1.275,2.55 and 5.10 g/kg of Jianpi Yishen Huazhuo Formula drug suspension respectively.After 30 days,vaginal secretion smear,ovarian HE staining and ELISA were used to evaluate the therapeutic effect of Jianpi Yishen Huazhuo Formula on PCOS model rats;IHC and Western Blot methods were used to detect the expression of NALP3,ASC,BAX,Caspase-3,PI3K,AKT,mTOR and other proteins in ovarian tissue,so as to explore the effect of Jianpi Yishen Huazhuo Formula on inflammatory and apoptosis-related factors in ovarian tissue of PCOS model rats and its possible mechanism.Results1.Evaluation of PCOS modelCompared with the blank group,the estrous cycle of the model group was disordered,and the performance of duration interval appeared;body mass and fasting blood glucose increased significantly（P<0.01）;HE staining showed that the ovary was mainly cystic empty vacuoles,follicular fluid decreased,granular cell layer became thin,interstitial hyperplasia was obvious,no normal follicles at different stages were found,and the number of corpus luteum decreased;serumT,LH,AMH increased significantly（P<0.05）,E₂and FSH decreased significantly（P<0.05,P<0.01）.2.Effect of Jianpi Yishen Huazhuo Formula on estrus cycle,ovarian morphology and endocrine of PCOS ratsCompared with the model group,after the treatment of each dose group of Jianpi Yishen Huazhuo Formula and metformin,the estrus cycle of rats gradually recovered;the appearance of ovarian tissue showed that the cystic processes in each treatment group were improved;body weight growth rate and ovarian index decreased significantly（P<0.05,P<0.01）;HE staining showed that polycystic changes in ovarian tissue were significantly improved;follicle counting results showed that the number of follicles and corpus luteum at different stages increased significantly（P<0.05,P<0.01）and the number of cystic follicles decreased significantly（P<0.05,P<0.01）;serum hormone T,LH and AMH were significantly decreased（P<0.05,P<0.01）,E₂and FSH were significantly increased（P<0.05）.3.Mechanism of Jianpi Yishen Huazhuo Formula in treating PCOS rats（1）Effect of Jianpi Yishen Huazhuo Formula on the expression of inflammatory and apoptosis-related proteins in ovarian tissue of PCOS ratsIHC results showed that:compared with the blank group,the positive expression of NALP3,ASC,Caspase-1,NF-κB,IL-1β,IL-18,IL-6,BAX,Caspase-3,Caspase-9 protein increased significantly in ovarian tissue of model group rats（P<0.05,P<0.01）,however,after treatment with various doses of Jianpi Yishen Huazhuo Formula and metformin,the positive expression of the above proteins decreased significantly（P<0.05,P<0.01）;Western Blot results showed that:compared with the blank group,the relative expression of NALP3,ASC,Caspase-1,p-NF-κB,NF-κB,IL-1β,IL-18,IL-6,BAX/Bcl-2,Caspase-3,Caspase-9 and Cytochrome protein increased significantly in ovarian tissue of model group rats（P<0.05,P<0.01）,but decreased significantly after the treatment with different doses of Jianpi Yishen Huazhuo Formula and metformin（P<0.05,P<0.01）.（2）Effect of Jianpi Yishen Huazhuo Formula on PI3K/AKT/mTOR pathway protein in ovarian tissue of PCOS ratsThe results of IHC showed that:compared with the blank group,the positive expression of PI3K,AKT and mTOR protein in the model group decreased（P<0.05）;compared with the model group,different doses of Jianpi Yishen Huazhuo Formula and metformin can significantly increase the positive expression of PI3K,AKT and mTOR protein（P<0.05,P<0.01）;Western Blot results showed that:compared with the blank group,the relative expression of PI3K and the protein phosphorylation levels of PI3K,AKT,mTOR were significantly reduced in the model group（P<0.05,P<0.01）,however,it increased significantly after treatment with different doses of Jianpi Yishen Huazhuo Formula and metformin（P<0.05,P<0.01）.Conclusion The Jianpi Yishen Huazhuo Formula not only effectively improves polycystic ovary changes,sexual hormone disorders,and ovarian function in PCOS rats,but also has a significant inhibitory effect on inflammation and apoptosis related proteins in ovarian tissue of PCOS rats.Its mechanism may be related to the activation of the PI3K/AKT/mTOR pathway.