Exploring The Mechanism Of Cangfu Daotan Decoction In The Treatment Of Phlegm Dampness Polycystic Ovary Syndrome Based On INSR Gene Methylation

Objective:Through the study of the methylation level of insulin receptor（INSR）gene promoter region,the expression level of INSR/PI3K/GLUT4signaling pathway and the intervention of Cangfu Daotan Decoction in ovarian granulosa cells of patients with phlegm-dampness polycystic ovary syndrome（PCOS）.To explore the possible pathogenesis of PCOS of phlegm-dampness type in epigenetic point of view and the therapeutic mechanism of Cangfu Daotan Decoction,and to enrich the understanding of PCOS disease at the theoretical and molecular levels.Methods:Sixty-six phlegm-dampness PCOS patients who were scheduled to receive IVF-ET were randomly divided into experimental group（EG）（Cangfu Daotan Decoction）（n=33）and control group（CG）（placebo）（n=33）.In addition,33 infertile patients with male factors who received IVF-ET assisted pregnancy were selected as the blank group（BG）（placebo）.The patients in the EG and CG were pretreated with Diane-35,and the patients in the three groups were treated with fixed antagonist protocol for ovarian hyperstimulation.The changes of phlegm dampness syndrome score,insulin resistance index（HOMA-IR）and body mass index（BMI）were statistically compared between the EG and the CG before and after treatment.The levels of blood estradiol（E₂）,luteinizing hormone（LH）and progesterone（P）on the day of HCG injection,the dosage of gonadotropin（Gn）,Gn days,the number of eggs retrieved,fertilization rate,high-quality embryo rate and cumulative pregnancy rate were compared among the three groups.Bisulfite amplicon sequencing（BSAS）was used to determine the changes in the methylation levels of the INSR gene promoter region in ovarian granulosa cells of the three groups of patients;the contents of INSR/PI3K/GLUT4 m RNA and protein were determined by real-time nucleic acid amplification assay system（q PCR）and Western blot.Results:1.The score of phlegm dampness syndrome and HOMA-IR score in the EG decreased after treatment,and the difference between the EG and the CG before and after treatment was statistically significant（P<0.05）.2.After treatment,the BMI of the EG was lower than that before treatment,and the difference was statistically significant（P<0.05）;There was no significant change in BMI in the CG before and after treatment（P>0.05）.3.There was no significant difference in Gn treatment days among the three groups（P>0.05）;The dosage of Gn in the EG and the BG was significantly lower than that in the CG（P<0.05）.4.There was no significant difference in the levels of E₂,LH and P on HCG day among the three groups（P>0.05）.However,the daily E₂ level of HCG in the EG was higher than that in the CG.5.There was no significant difference in the number of eggs obtained between the EG and the CG（P>0.05）,and the number of eggs obtained in the EG was significantly higher than that in the BG（P<0.05）;The fertilization rate and high-quality embryo rate in the EG and the BG were significantly higher than those in the CG（P<0.05）;There was no significant difference in the cumulative pregnancy rate among the three groups（P>0.05）,but the cumulative pregnancy rate in the EG and the BG was higher than that in the CG.6.Comparison of methylation level of INSR gene promoter region in granulosa cells of three groups:the average methylation level of INSR pair1 fragment 38,47,56,94 and 143 in the EG and the BG was significantly lower than that in the CG（P<0.05）;The average methylation levels of 47,56,94 and 143 sites in the EG were significantly lower than those in the CG（P<0.05）;The average methylation levels of 38,47,56,94 and 143 sites in the BG were significantly lower than those in the CG（P<0.05）;The average methylation levels of 47,94 and 143 sites in the BG were significantly lower than those in the EG（P<0.05）.7.The m RNA expressions of INSR,PI3K and GLUT4 genes in granulosa cells of the ex-perimental group and the blank group were significantly higher than those in the control group（P<0.05）;The m RNA expressions of INSR and PI3K in the blank group were significantly higher than those in the experimental group（P<0.05）;There was no significant difference in the expression of GLUT4 m RNA between the experimental group and the blank group（P>0.05）.8.The ex-pressions of INSR,PI3K and GLUT4 genes and proteins in granulosa cells in the experimental group and the blank group were higher than those in the control group,and the difference was statistically significant（P<0.05）;The protein expressions of INSR and GLUT4 genes in the blank group were significantly higher than those in the experimental group（P<0.05）;There was no significant difference in PI3K protein expression between the ex-perimental group and the blank group（P>0.05）.9.The relative expression of INSR gene m RNA in granulosa cells of the three groups was negatively correlated with the methylation level of INSR gene（P<0.05）.Conclusion:1.Cangfu Daotan decoction has a positive regulatory effect on improving the clinical symptoms of patients with phlegm dampness PCOS.2.The pathogenesis of phlegm dampness PCOS may be related to the increase of methylation level of INSR gene promoter and the down-regulation of INSR/PI3K/GLUT4 signal pathway expression in granulosa cells.3.The down-regulation of INSR/PI3K/GLUT4 signal pathway expression in ovarian granulosa cells of patients with phlegm dampness PCOS may be related to the increase of methylation level in the promoter region of INSR gene.4.The method of invigorating the spleen,drying dampness and resolving phlegm may improve phlegm dampness and insulin resistance by reducing the methylation level of INSR gene promoter and up regulating the m RNA and protein expression of INSR/PI3K/GLUT4 pathway,increase the energy supply of follicular de-velopment,improve the quality of eggs,and obtain better sperm receiving rate and high-quality embryo rate,so as to improve the pregnancy rate.Further enrich the connotation of“ovarian retention and drainage”theory and“invigorating the spleen,drying dampness,resolving phlegm and promoting reproduction”.