Biological Basis Study Of Sputum Syndrome Of Polycystic Ovary Syndrome Based On PI3k/Akt Signaling Pathway

Objective:The dehydroepiandrosterone（DHEA）was used to copy polycystic ovary syndrome（PCOS）rats model in this research.The purpose of this research was to observe the effects of erchen Decoction on PI3K/AKT signaling pathway and insulin resistance in PCOS rats.Changes in serum sex hormone levels,ovarian morphology,insulin resistance and the PI3K/AKT signaling pathway were observed in the presence of PI3K/AKT signaling pathway blockers.The biological basis of this research was to evaluate PCOS phlegm syndrome and preliminary clear Erchen Decoction is one of the targets for the treatment of PCOS.Methods:Fifty-two 21-day-old female SPF-grade SD rats were randomly divided into normal group（10）and modeled group（50）.The model group was injected with DHEA subcutaneously on the neck and back daily while the normal group was injected with the same amount of soybean oil for 20 days.The model formation of the rats was observed according to the exfoliated cells of the vagina,ovarian histomorphology,serum sex hormone levels and insulin resistance.After successful modeling,the model rats were randomly divided into model group,Erchen Decoction(4.35g·kg^-1·d^-1),metformin group(0.3g·kg^-1·d^-1),LY294002group（LY group）,and Erchen Decoction+LY294002 group（Erchen Decoction+LY group）.The blank group was used as the control,and the intervention was conducted for 4 weeks.Serum levels of follicle-stimulating hormone（FSH）,luteinizing hormone（LH）,testosterone（T）,estradiol（E2）and insulin（INS）were determined by enzyme-linked immunosorbent assay（ELISA）.Rat homa-insulin resistance index（homa-ir）was calculated.Hematoxylin-eosin（HE）staining and ovarian histomorphology were observed.mRNA and protein expression levels of PI3k,AKT and GSK-3βin rat ovarian tissues were detected by real-time qPCR and Western Blot.Results:1.Model evaluation results:After the completion of modeling,compared with the blank group,the weight of the rats in the modeling group increased significantly（P<0.01）,Serum T,E₂ and LH levels were significantly increased in the model group（P<0.01）,and there was no significant difference between the two groups of FSH（P>0.05）,while INS and HOMA-IR levels were significantly increased（P<0.01）.The pale color of the ovary can be seen under stereoscope in the model group.Under the light microscope,the follicles in the modeling group showed cystic dilatation without oocytes,and the granular layer decreased to 1³,and the arrangement was sparse.Follicles and corpus luteum at different stages of development were rarely seen.It was proved that the rat model of PCOS with IR（PCOR-IR）was established successfully.2.Comparison of various indicators after phlegm elimination:（1）Comparison of serum sex hormone levels:Compared with the blank group,T and LH levels significantly increased in the model group（P<0.01）,and serum E₂ and FSH levels significantly decreased in the model group（P<0.01）.Compared with the model group,T levels in Erchen Decoction group and Erchen Decoction+LY group decreased significantly（P<0.01 or P<0.05）,while E₂ and FSH levels increased significantly（P<0.01）.There was no significant difference between T and E₂ levels in metformin group（P>0.05）,significantly increased FSH level（P<0.01）,and significantly decreased LH level（P<0.01）.T,E₂ and LH levels in the LY group showed no significant difference（P>0.05）,and FSH levels were significantly increased（P<0.01）.Compared with Erchen Decoction group,T and FSH levels in metformin group increased significantly（P<0.01 or P<0.05）,E₂ level decreased significantly（P<0.01）,and LH level showed no significant difference（P>0.05）.T and LH levels increased significantly in the LY group,while E₂ levels decreased significantly（P<0.01）.There was no significant difference in FSH levels（P>0.05）.（2）Morphological comparison of ovary:Multiple corpus luteum and follicles at different development stages were observed in the ovarian tissues of the blank group of rats.The granulosa cells were arranged in multiple layers,most of which were 8⁹ layers.In the model group,the corpus luteum and all levels of follicles in the ovarian tissues of the rats were rare,and the number of granulosa cell layers was reduced,most of which were 1³ layers with loose arrangement.A large number of cystic dilated follicles could be seen,the oocytes in the cystic follicles or the radiative crown disappeared,and the ovarian appearance was pale.There was improvement in Erchen Decoction group and metformin group and Erchen Decoction+LY group,and there was no significant difference between LY group and model group.（3）Comparison of insulin resistance:There was no significant difference in FPG level in each group（P>0.05）.Compared with the blank group,the serum levels of INS and HOMA-IR in the model group were significantly increased（P<0.01）.Compared with the model group,the levels of INS and HOMA-IR in Erchen Decoction group and metformin group decreased significantly（P<0.01）.There was no significant difference in the levels of INS and HOMA-IR in the LY group,Erchen Decoction+LY group（P>0.05）.Compared with Erchen Decoction group,the levels of INS and HOMA-IR in the LY group and Erchen Decoction+LY group increased significantly（P<0.01）.（4）Relative expression levels of PI3K,AKT and GSK-3βmRNA:Compared with the blank group,mRNA expression levels of PI3K,AKT and GSK-3βin the model group were significantly decreased（P<0.01）.Compared with the model group,mRNA levels of PI3K,AKT and GSK-3βin rat ovarian tissues in Erchen Decoction group and metformin group were significantly increased（P<0.01）.mRNA levels of PI3K,AKT and GSK-3βin ovarian tissues of rats in the LY group and Erchen Decoction+LY group showed no significant difference（P>0.05）.Compared with Erchen Decoction group,mRNA levels of PI3K,AKT and GSK-3βin ovarian tissues of the LY group and Erchen Decoction+LY group decreased significantly（P<0.01）.（5）Relative expression levels of PI3K,AKT and GSK-3βproteins:Compared with the blank group,the expression levels of PI3K and AKT in the model group significantly decreased（P<0.01）,while the expression levels of GSK-3βsignificantly increased（P<0.05）.Compared with the model group,the expression levels of PI3K and AKT in ovarian tissues of rats in Erchen Decoction group and metformin group were significantly increased（P<0.01）,the expression levels of GSK-3βin metformin group were significantly decreased（P<0.01）,and the expression levels of GSK-3βin Erchen Decoction were decreased without statistical significance（P>0.05）.There was no significant difference in the expression levels of PI3K,AKT and GSK-3βin rat ovarian tissues between the LY group and Erchen Decoction+LY group and the model group.Compared with the Erchen Decoction group,the levels of PI3K and AKT in ovarian tissues of the LY group and Erchen Decoction+LY group decreased significantly（P<0.01 or P<0.05）,the expression level of GSK-3βin the Erchen Decoction+LY group increased significantly（P<0.01）,and the expression level of GSK-3βin the LY group increased significantly（P<0.01）,with no statistical significance（P>0.05）.Conclusions:1.Phlegm method can improve the sex hormone level and ovary morphology of PCOS rats.2.Phlegm method improves the IR of PCOS by regulating the expression of PI3K,AKT,GSK-3βmRNA and protein.3.Phlegm syndrome affects the occurrence of IR in PCOS rats through PI3K/AKT signaling pathway.