基于miR-17-5p/ATG7介导自噬探讨苍附导痰汤对痰湿型多囊卵巢综合征不孕症的影响

Effects of Cangfu Daotan Decoction on phlegm-dampness type of polycystic ovary sydrome based on miR-17-5p/ATG7 mediated autophagy

目的:基于miR-17-5p/ATG7介导自噬探讨苍附导痰汤对痰湿型多囊卵巢综合征(PCOS)不孕症患者的影响。方法:选取符合痰湿型PCOS诊断标准的不孕患者76例,随机分为试验组(苍附导痰汤+IVF)、观察组(安慰剂+IVF),各38例。另选取因男方因素行辅助生殖的患者38例为对照组(IVF)。3组患者皆采用拮抗剂方案进行控制性卵巢刺激,统计分析试验组和观察组患者治疗前后中医痰湿证候积分、体质量指数(BMI)变化,比较3组患者促性腺激素(Gn)使用天数、Gn用量、性激素水平、获卵数、2PN受精率、优质胚胎率等临床指标,采用qRT-PCR检测颗粒细胞中miR-17-5p及ATG7 mRNA表达水平,Western Blot检测ATG7蛋白水平。结果:试验组治疗后痰湿证候积分、BMI显著降低(P<0.05),治疗前后痰湿证候积分差值、BMI差值显著高于观察组(P<0.05);Gn用药天数试验组显著低于观察组(P<0.05),试验组和观察组均显著高于对照组(P<0.05);Gn用量试验组和观察组高于对照组(P<0.05);扳机日E2水平试验组高于观察组和对照组(P<0.05);获卵数对照组显著低于试验组和观察组(P<0.05);优质胚胎率试验组和对照组显著高于观察组(P<0.05);miR-17-5p相对表达量试验组和观察组均显著低于对照组(P<0.05),观察组显著低于试验组(P<0.05);ATG7 mRNA相对表达量试验组和观察组显著高于对照组(P<0.05);ATG7蛋白表达水平观察组显著高于试验组和对照组(P<0.05),试验组显著高于对照组(P<0.05)。结论:与单纯男子因素不孕女性相比,痰湿型PCOS患者颗粒细胞中miR-17-5p/ATG7表达存在差异,低表达的miR-17-5p可能通过上调ATG7的表达使颗粒细胞过度自噬,从而参与痰湿型PCOS的发生;苍附导痰汤可使miR-17-5p表达上调,ATG7表达下调,显著改善痰湿型PCOS不孕患者的临床症状,提高2PN受精率和优质胚胎率。

Objective:To explore the effects of Cangfu Daotan Decoction on infertility patients with phlegm-dampness type of polycystic ovary sydrome(PCOS),based on miR-17-5p/ATG7 mediated autophagy.Methods:A total of 76 patients who met the diagnostic criteria of phlegm-dampness type of polycystic ovary sydrome were randomly divided into experimental group(EG,Cangfu Daotan Decoction+IVF)and observation group(OG,placebo+IVF).Another 38 patients who underwent assisted reproduction due to male factors were selected as the control group(CG,IVF).The three groups of patients were treated with antagonist regimen for controlled ovarian stimulation.The changes of TCM phlegm-dampness syndrome score and body mass index(BMI)in the EG and the OG before and after treatment were analyzed.Clinical indicators such as the use days of gonadotropin(Gn),the dosage of Gn,the level of sex hormone level,the number of oocytes retrieved,2PN fertilization rate,high-quality embryo rate and other indicators of the three groups were analyzed.qPCR and Western Blot were used to detect the expression levels of autophagy-related gene ATG7 and its associated microRNA in granulosa cells of the two groups of patients.Results:The score of phlegm-dampness syndrome and BMI decreased in the EG after treatment(P<0.05);The integral difference of phlegm-dampness syndrome and BMI before and after treatment in the EG was higher than that in the OG(P<0.05).Gn days in the EG was lower than that in the OG(P<0.05),and both the EG and the OG were higher than that in the CG(P<0.05);The dosage of Gn in the EG and the OG was higher than that in the CG(P<0.05).The E,levels on the day of ovulation triggering in the EG were higher than those in the OG and the CG(P<0.05).The number of eggs retrieved in the CG was lower than that in the EG and the OG(P<0.05).The rate of high-quality embryos in the EG and the CG was higher than that in the OG(P<0.05).The relative expression of miR-17-5p in the EG and the OG was significantly lower than that in the CG(P<0.05),and that in the OG was lower than that in the EG(P<0.05);The relative expression of ATG7 in the EG and the OG was higher than that in the CG(P<0.05).The ATG7 protein expression level in the OG was higher than that in the EG and the CG(P<0.05),and that in the EG was higher than that in the CG(P<0.05).Conclusion:Compared to women with infertility caused solely by male factors,the expression of miR-17-5p/ATG7 in granulosa cells of patients with phlegm-dampness type of PCOS was different,low expression of miR-17-5p may lead to excessive autophagy of granulosa cells by up regulating the expression of ATG7,thus participating in the occurrence of PCOS.Cangfu Daotan Decoction can up-regulate the expression of miR-17-5p and down-regulate the expression of ATG7,and significantly improve the clinical symptoms of patients with phlegm-dampness PCOS,improve the 2PN fertilization rate and highquality embryo rate.