Study On The Mechanism Of Modified Banxia Xiexin Decoction In Regulating PCOS By Improving IR Based On PI3K/AKT/GLUT4 Signaling Pathway

Objective To observe the effects of Modified Banxia Xiexin Decoction on reproductive endocrinology,glucolipid metabolism and phosphatidylinositol 3-kinase/protein kinase B/glucose transporter 4(PI3K/AKT/GLUT4)signaling pathway in rats with polycystic ovary syndrome(PCOS)combined with insulin resistance(IR).The mechanism of alleviating PCOS by Modified Banxia Xiexin Decoction was discussed.Methods A total of 42 female,21-day-old SPF SD rats were selected,and 9 rats were randomly selected as control group(Group Z),and the remaining 33 rats were assigned as Group M0.The PCOS IR model was constructed using high-fat diet combined with letrozole.Group M0 was randomly divided into model group(Group M),metformin group(Group X),Chinese medicine high-dose group(Group A),Chinese medicine medium-dose group(Group B)and Chinese medicine low-dose group(Group C).Blood Glucose(Glu)was measured in rats with Glucose analyzer.Histopathological changes of ovary were observed after HE staining.The serum levels of testosterone(T),luteinizing hormone(LH),estradiol(E2),follicle stimulating hormone(FSH),total cholesterol(TC),triglyceride(TG)and fasting insulin(FINS)were determined by enzymic linked immunosorbent assay(ELISA).LH/FSH and Homeostasis Model Assessment of insulin Resistance(HOMA-IR)were calculated.The gene and protein expressions of PI3K/AKT/GLUT4 signaling pathway related factors in ovary,liver and adipocytes were detected by Realtime PCR(RT-PCR),immunohistochemical(IHC)staining and western blot(WB).Results 1.Effect on body weight:Compared with group Z,body weight of rats in group M was significantly increased(P<0.05);Compared with M group,body weight of rats in groups X,A and B was significantly decreased(P<0.05).2.Influence on ovarian histopathology:Under microscope,mature follicles were observed in group Z,and granulosa cells were distributed in multilayer and orderly arrangement.There were no mature follicles in group M,and the follicles dilated like empty sacs,and the number of granular cell layers decreased.Compared with M group,granulosa cell layer was significantly thickened in all treatment groups,and mature follicles were observed under microscope.3.Effects on serum reproductive endocrine indexes:Compared with group Z,the level of T,LH and LH/FSH in group M was significantly increased(P<0.05),while the level of E2 was significantly decreased(P<0.05).Compared with group M,the level of LH/FSH in group X,group A,group B,group C was significantly decreased(P<0.05);the level of T in group X,group A was significantly decreased(P<0.05);the level of E2 in group C was significantly increased(P<0.05).4.Effects on glucose and lipid metabolism indexes:Compared with group Z,the level of HOMA-IR,TC and TG in group M was significantly increased(P<0.05);the level of Glu,FINS level had an increasing trend(P>0.05).Compared with group M,the level of FINS in group A was significantly decreased(P<0.05);the level of HOMA-IR in group X,group A,group B was significantly decreased(P<0.05);the level of TC,TG in group A was significantly decreased(P<0.05).5.Effects on gene expression of PI3K/AKT/GLUT4 signaling pathway related factors in ovary,liver and adipose tissue:(1)Ovarian tissue:Compared with group Z,the mRNA level of AKT1,PI3K in group M was significantly decreased(P<0.05),while the mRNA level of IRS 1,IRS2 and GLUT4 showed a downward trend(P>0.05).Compared with group M,the mRNA level of IRS 1,IRS2,PI3K,AKT1 and GLUT4 in group X,group A and group B showed an upward trend(P>0.05).(2)Liver tissue:Compared with group Z,the mRNA level of PI3K and AKT1 in group M was significantly decreased(P<0.05);the mRNA level of IRS 1,IRS2 and GLUT4 showed a downward trend(P>0.05).Compared with group M,the mRNA level of IRS 1 in group X,group A and group B showed an increasing trend(P>0.05);the mRNA level of IRS2 in group B and group C showed an increasing trend(P>0.05);the mRNA level of PI3K in group C was significantly increasd(P<0.05),the mRNA level of PI3K in group X,group A,group B showed an increasing trend(P>0.05);the mRNA level of AKT1 in group X,group A and group B showed an increasing trend(P>0.05);the mRNA level of GLUT4 in group X,group A,group B and group C showed an upward trend(P>0.05).(3)Adipose tissue:Compared with group Z,the mRNA level of IRS1,IRS2 in group M was significantly decreased(P<0.05);the mRNA level of AKT1 and GLUT4 showed a downward trend(P>0.05).Compared with group M,the mRNA level of IRS 1 in group A was significantly increased(P<0.05),and the mRNA level of IRS1 in group X,group B and groups C had an upward trend(P>0.05);the mRNA level of IRS2,AKT1 and GLUT4 in group X,group A,Group B and group C showed an upward trend(P>0.05).6.Effects of IHC staining on expression of PI3K/AKT/GLUT4 signaling pathway related factor proteins in ovary,liver and adipose tissue:(1)Ovarian tissue:Compared with group Z,the expression of IRS 1,IRS2,PI3K,p-PI3K,AKT1,p-AKT1 and GLUT4 in group M wsa significantly decreased(P<0.05).Compared with M group,the expression of IRS2,p-PI3K,GLUT4 in group X,group A,group B and group C was significantly increased(P<0.05);the expression of IRS1,PI3K,AKT1 and p-AKT1 in group X,group A,group B and group C showed an upward trend(P>0.05).(2)Liver tissue:Compared with group Z,the expression of p-PI3K,AKT1,and GLUT4 in group M was significantly decreased(P<0.05);the expression of IRS1,IRS2,PI3K and p-AKT1 showed a downward trend(P>0.05).Compared with M group,the expression of IRS1 and PI3K in group X,group A,group B and group C showed an upward trend(P>0.05).The expression of IRS2 in group X,group A and group B showed an upward trend(P>0.05).The expression of p-PI3K in group X,group A,group B and group C was significantly increased(P<0.05);the expression of GLUT4 in group X,group A and group B was significantly increased(P<0.05);the expression of AKT1 in groups B and C was significantly increased(P<0.05),and the expression of AKT1 in groups X and A had an upward trend(P>0.05).The expression of p-AKT1 in group A was significantly increased(P<0.05),and the expression of P-AKT1 in group X,group B and group C had an upward trend(P>0.05).(3)Adipose tissue:Compared with group Z,the expression of IRS1,IRS2,p-PI3K,AKT1 and GLUT4 in group M was significantly decreased(P<0.05),while the expression of PI3K and p-AKT1 had a downward trend(P>0.05).Compared with group M,the expression of IRS1 in group C was significantly increased(P<0.05),and the expression of IRS1 in groups X and B had an increasing trend(P>0.05).The expressions of IRS2,PI3K and p-AKT1 in group X,group A,group B and group C showed an increasing trend(P>0.05).The p-PI3K expression in group X,group A,group B and group C was significantly increased(P<0.05).The expression of AKT1 and GLUT4 in group X,group A,group B and group C showed an upward trend(P>0.05).7.Western Blot was used to detect the influence of PI3K/AKT/GLUT4 signaling pathway related factor protein expression in ovary,liver and adipose tissue:(1)Ovarian tissue:Compared with group Z,the protein expression of IRS1,IRS2,PI3K and p-AKT1 in group M showed a downward trend(P>0.05).Compared with group M,the protein expression of IRS 1 in group X,group A,group B and group C showed an upward trend(P>0.05).The expression of IRS2,p-AKT1 protein in group A,group B and group C was increased(P>0.05);the expression of PI3K in group X,group A and group C was increased(P>0.05),the expression of GLUT4 protein in group X,group A and group B was increased(P>0.05).(2)Liver tissue:Compared with group Z,the protein expression of IRS2 in group M was significantly decreased(P<0.05);the protein expression of IRS1,PI3K,p-AKT1 and GLUT4 in group M showed a downward trend(P>0.05).Compared with group M,the protein expression of IRS 1,IRS2,PI3K,p-AKT1 and GLUT4 in group X,group A,group B and group C showed an upward trend(P>0.05).(3)Adipose-tissue:Compared with group Z,the protein expression of IRS1,IRS2,PI3K,p-AKT1 and GLUT4 in group M showed a decreasing trend(P>0.05).Compared with group M,the protein expression of IRS1,PI3K in group X and group A had an upward trend(P>0.05);the protein expression of IRS2,p-AKT1 and GLUT4 in group X,group A,group B and group C had an upward trend(P>0.05).Conclusion PCOS IR rats had abnormal reproductive hormones,glucose and lipid metabolism,and abnormal expression of PI3K/AKT/GLUT4 pathway related factors.Modified Banxia Xiexin Decoction can improve follicular development and disorder of reproductive endocrine hormone and glucolipid metabolism in PCOS IR rats,and the mechanism is related to regulating signal transduction of PI3K/AKT/GLUT4 pathway.