Based On NEK7-NLRP3 Inflammasome Signaling Pathway To Explore The Mechanism Of Cangfu Daotan Decoction In Polycystic Ovary Syndrome Of Phlegm-dampness

Objective:1.To study the expression levels of NEK7-NLRP3 inflammasome signaling pathway related factors,including NEK7,NLRP3,Caspase-1,ASC,IL-1b and IL-18,in ovarian granulosa cells of patients with polycystic ovary syndrome,as well as the expression levels of IL-18 and IL-1b in peripheral blood.To understand their clinical significance in polycystic ovary syndrome.The NEK7-NLRP3 inflammasome signaling pathway was used as the entry point to explore the inflammatory mechanism of phlegm-dampness PCOS.2.To observe the clinical efficacy of Cangfu Daotan Decoction in the process of in vitro fertilization-embryo transfer（IVF-ET）of phlegm-dampness PCOS patients,and to detect the expression levels of NEK7-NLRP3 inflammatory vesicle signaling pathway-related factors in ovarian granulosa cells of patients with phlegm-damp PCOS after the intervention of Cangfu Daotan Decoction.To further explore the mechanism of Cangfu Dao Tan Decoction to improve the clinical symptoms of phlegm-dampness PCOS patients.Method:Part Ⅰ:33 patients with phlegm-dampness PCOS,33 patients with non-phlegm-dampness PCOS,and 33 patients with normal control group were selected.Basic information of patients in the three groups was collected,including age,body mass index（BMI）,infertility years,basal follicle stimulating hormone（FSH）,basal luteinizing hormone（LH）,and basal testosterone（T）.All three groups were treated with antagonist regimen.Real-time quantitative PCR and Western blot were used to detect NEK7-NLRP3inflammatory body signaling pathway related factors in ovarian granulosa cells,and ELISA was used to detect the expression levels of IL-18 and IL-1b in peripheral blood.The days and doses of LH,progesterone（P）,estradiol（E₂）and gonadotropin（Gn）,the number of oocytes obtained,the rate of normal fertilization,the rate of embryo formation,the rate of excellent embryo and the changes of TCM symptom score before and after treatment were compared among the three groups on trigger day.We also analyzed the correlation between factors related to NEK7-NLRP3 inflammatory vesicle signaling pathway,factors and BMI,LH,FSH,T,Gn days,Gn dose,trigger day LH,trigger day P,trigger day E₂,number of eggs gained,normal fertilization rate,embryo success rate,eugenics rate and TCM evidence score in patients with phlegmatic and wet PCOS.Part Ⅱ:60 patients with phlegm-damptype PCOS were selected and randomly divided into control group（30 cases）and experimental group（30 cases）,both groups were given antagonist scheme.In the period before ovulation,the experimental group began to take Cangfu Daotan Tang granules on the fifth day of menstruation or repulsive bleeding,and the control group began to take placebo granules on the fifth day of menstruation or repulsive bleeding,and both groups were taken until the trigger day.The age,infertility duration,basal LH,basal FSH,basal T,BMI before and after treatment,trigger day LH,trigger day P,trigger day E₂,Gn days and dosage,number of oocytes retrieved,normal fertilization rate,embryo formation rate,high-quality embryo rate and symptom scores before and after treatment were collected and recorded.At the same time,real-time quantitative PCR was used to detect the expression levels of NEK7-NLRP3 inflammasome signaling pathway related factors in ovarian granulosa cells of the two groups,and statistical analysis was conducted.Results:Part Ⅰ:1.BMI,basal LH,basal T,TCM symptom score,Gn days and Gn dosage in patients with phlegm-dampness PCOS were significantly higher than those in normal control group（P<0.05）.2.The number of oocytess obtained in the phlegm-dampness PCOS group and the non-phlegm-dampness PCOS group was significantly higher than that in the normal control group（P<0.05）,and the normal fertilization rate in the phlegm-dampness PCOS group was significantly lower than that in the non-phlegm-dampness PCOS group and the normal control group（P<0.05）.The embryo rate of phlegm-dampness PCOS group and non-phlegm-dampness PCOS group was significantly lower than that of normal control group（P<0.05）,and the optimal embryo rate was basically the same among the three groups（P>0.05）.3.m RNA expression levels of NEK7-NLRP3 inflammasome signaling pathway related factors（NEK7,NLRP3,ASC,Csapase-1,IL-18 and IL-1b）were significantly different among the three groups（P<0.05）.The protein expression of above factors in phlegm-dampness PCOS group was significantly higher than that in normal control group（P<0.05）.4.There were significant differences in the expression levels of IL-18 and IL-1b in peripheral blood among the three groups（P<0.001）.5.The m RNA levels of NEK7 and ASC,Caspase-1 and IL-1b,and ASC and Caspase-1 were significantly positively correlated in patients with phlegm-damp PCOS（P<0.05）;IL-1b m RNA levels were significantly negatively correlated with the TCM evidence score,IL-18 m RNA levels with the number of normal embryos,and IL-18m RNA levels with the number of available embryos（P<0.05）.Part Ⅱ:1.Comparison of patients’baseline conditions:years of infertility,BMI,age and basal sex hormone levels were basically the same between the two groups（P>0.05）.2.The number of Gn days and dosage in the experimental group were significantly lower than those in the control group（P<0.05）.3.After treatment,the BMI level in the experimental group was significantly lower than that in the control group（P<0.05）.4.The fertilization rate and embryo rate in the experimental group were significantly higher than those in the control group（P<0.05）and the rate of superior embryos was improved,but the difference was not statistically significant（P>0.05）.5.The TCM evidence score in the experimental group decreased significantly after treatment,which was lower than that in the control group（P<0.001）.6.The NEK7-NLRP3 inflammatory vesicle-related factors （NEK7,NLRP3,ASC,Csapase-1,IL-18 and IL-1b）in the experimental group m RNA expression was significantly lower than that of the control group（P<0.05）.Conclusion:1.Abnormal activation of NEK7-NLRP3 inflammatories signaling pathway in ovarian granulosa cells of patients with phlegm-dampness PCOS is an important reason for the inflammatory response and chronic low-grade inflammation of phlegm-dampness PCOS,and the NEK7-NLRP3 inflammatories signaling pathway is a potential effective therapeutic target for PCOS.2.Cangfu Tongtan Decoction can significantly reduce the phlegm-dampness performance of PCOS patients,improve the ovarian accumulation and loss of fluid in PCOS patients,reduce the number of days of Gn use,the amount of Gn,and BMI of phlegm-dampness PCOS patients,and improve the rate of fertilization,embryo formation and embryo optimization.3.Cangfu Tongtan Decoction may relieve the inflammatory state of patients with phlegm-dampness PCOS by inhibiting the activation of NEK7-NLRP3 inflammatory body signaling pathway,improve endocrine disorders,obesity and other pathological changes,and thus improve the developmental potential of follicles and embryo quality,laying a foundation for good pregnancy outcomes in infertile patients with phlegm-dampness PCOS.