解郁化痰法对多囊卵巢综合征大鼠肝脏组织IRS-1及IRS-2表达的影响

目的:观察二陈汤、柴胡疏肝散、二陈汤+柴胡疏肝散及二甲双胍对多囊卵巢综合征(PCOS)模型大鼠的肝脏组织胰岛素受体底物-1(IRS-1),胰岛素受体底物-2(IRS-2) mRNA和蛋白表达的影响,探讨解郁化痰法改善多囊卵巢综合征胰岛素抵抗的生物学机制。方法:通过颈背部皮下注射脱氢表雄酮(DHEA)复制多囊卵巢综合征模型大鼠。将造模成功大鼠分为模型组、二陈汤组(9.135 g·kg^-1),柴胡疏肝散组(6.615 g·kg^-1),二陈汤+柴胡疏肝散组(合方组,9.135 g·kg^-1+6.615 g·kg^-1),二甲双胍组(0.158 mg·kg^-1),并以正常组作为对照。给予相应药物灌胃,每日1次,连续4周。于末次给药后,麻醉处死,分离大鼠卵巢、肝脏组织,采用酶联免疫吸附测定(ELISA)检测各组大鼠血清睾酮(T),雌二醇(E2)激素水平,苏木素-伊红(HE)染色检测各组大鼠卵巢组织病理学变化,采用实时荧光定量PCR(Real-time PCR)和蛋白免疫印迹法(Western blot)检测各组大鼠肝脏组织的IRS-1,IRS-2 mRNA及蛋白表达水平。结果:与正常组比较,模型组大鼠血清T水平显著上升,E2水平显著下降(P<0.01),卵巢组织病理可见大量囊状扩张卵泡,颗粒细胞层较薄,黄体少见,肝脏组织IRS-1,IRS-2 mRNA及蛋白水平显著下降(P<0.01);与模型组比较,各给药组血清T水平显著下降,E2水平显著上升(P<0.01),卵巢组织病理可见囊状扩张卵泡数目减少,颗粒细胞层增厚,可见各级卵泡,二陈汤组IRS-1,IRS-2 mRNA及蛋白表达明显上升(P<0.05,P<0.01);柴胡疏肝散组IRS-1 mRNA及蛋白表达显著上升(P<0.01),IRS-2 mRNA及蛋白表达有上升趋势;合方组IRS-1 mRNA及蛋白表达和IRS-2 mRNA表达明显上升(P<0.05),IRS-2 mRNA有上升趋势;二甲双胍组IRS-1蛋白和IRS-2 mRNA和蛋白表达显著上调(P<0.01),IRS-1 mRNA有上升趋势。结论:二陈汤改善PCOS模型大鼠的胰岛素抵抗效果较柴胡疏肝散更为显著,其机制可能与调控胰岛素信号转导通路的关键信号分子IRS-1,IRS-2 mRNA和蛋白表达水平有关。

Effect of Method of Relieving Depression and Resolving Phlegm on Expression of IRS-1 and IRS-2 in Liver Tissues of Rats with Polycystic Ovary Syndrome

Objective: To observe the effect of Erchentang, Chaihu Shugansan, Erchentang+Chaihu Shugansan and metformin on insulin receptor substrate-1(IRS-1) and insulin receptor substrate-2(IRS-2) mRNA and protein expression in liver tissues of model rats with polycystic ovary syndrome(PCOS),and to explore the biological mechanism of relieving depression and resolving phlegm to improve insulin resistance of polycystic ovary syndrome. Method: A model of polycystic ovary syndrome was replicated in rats by subcutaneous injection of dehydroepiandrosterone(DHEA) under the neck and back.The control group was the normal group,The model rats were divided into model group, Erchentang group(9.135 g·kg^-1),Chaihu Shugansan group(6.615 g·kg^-1),Erchentang+Chaihu Shugansan group (Hefang group,9.135 g·kg^-1+6.615 g·kg^-1), and metformin group(0.158 mg·kg^-1).The corresponding drugs were given by intragastric administration once a day for 4 weeks. The ovarian and liver tissues of rats were isolated, and serum testosterone(T) and estradiol(E₂) hormone levels in each group were detected by enzyme-linked immunosorbent assay(ELISA), the histopathological changes of ovary in each group were detected by hematoxylin-eosin (HE) staining,IRS-1 and IRS-2 mRNA and protein expression levels in rat liver tissues were detected by quantitative real-time fluorescence PCR(Real-time PCR) and Western blot. Result: Compared with normal group, the serum T and E₂ level of model group were significantly decreased (P<0.01),the histology of ovary showed a large number of saccate dilated follicles, thin granular cell layer and rare corpus luteum,IRS-1, IRS-2 mRNA and protein levels in liver tissues decreased significantly (P<0.01).Compared with model group, the serum T and E₂ level of each group significantly decreased (P<0.01),histopathology of ovarian tissue showed that the number of saccular dilated follicles decreased, and the granulosa cell layer thickened, showing follicles of all levels,IRS-1, IRS-2 mRNA and protein expression were significantly increased in Erchentang group(P<0.05,P<0.01),IRS-1 mRNA and protein expression were significantly increased in Chaihu Shugansan group(P<0.01), IRS-2 mRNA and protein expression were on the rise,IRS-1 mRNA and protein expression and IRS-2 mRNA expression were significantly increased in the Hefang group (P<0.05), while IRS-2 mRNA was on the rise,IRS-1 protein and IRS-2 mRNA and protein expression were significantly increased in metformin group (P<0.01), while IRS-1 mRNA showed an upward trend. Conclusion: Erchentang improved the insulin resistance of PCOS model rats more significantly than Chaihu Shugansan, and its mechanism may be related to IRS-1, IRS-2 mRNA and protein expression levels of key signaling molecules regulating insulin signal transduction pathway.