补肾化痰方对多囊卵巢综合征雌鼠TLR4/NF-κB炎性信号通路、氧化应激和胰岛素抵抗的影响

Effect of Bushen Huayu Recipe Combined with Metformin on TLR4/NF-κB Inflammatory Signal Pathway, Oxidative Stress and Insulin Resistance in Female Rats with Polycystic Ovary Syndrome

目的:观察补肾化痰方联合二甲双胍对多囊卵巢综合征(PCOS)雌鼠TLR4/NF-κB炎性信号通路、氧化应激和胰岛素抵抗的影响。方法:将120例6周龄SD雌性大鼠随机分为空白组、模型组、二甲双胍组、二甲双胍+中药组,每组各10只;建立PCOS模型后给予药物干预,空白组和模型组给予生理盐水灌胃处理,二甲双胍组给予接受二甲双胍(270mg/kg·d)灌胃处理,二甲双胍+中药组在二甲双胍组基础上加用(34.5mg/kg·d)灌胃处理,4组均连续干预4周。记录各组大鼠卵巢排卵情况;检测干预前后的大鼠外周血TLR4蛋白、NF-κB蛋白、IL-6和TNF-α表达水平;采用免疫组化法(SP)检测各组大鼠卵巢组织TLR4蛋白、NF-κB蛋白表达水平;并检测各组大鼠干预前后的外周血氧化应激和胰岛素抵抗相关指标。结果:PCOS各组(二甲双胍+中药组、二甲双胍组、模型组)平均排卵数、排卵率显著低于空白组,囊性扩张性排卵数高于空白组(P<0.05),提示建模成功。模型组、二甲双胍组、二甲双胍+中药组平均排卵数和排卵率逐渐升高,比较差异存在统计学意义(P<0.05)。PCOS各组外周血TLR4蛋白、NF-κB蛋白、IL-6和TNF-α表达水平均高于空白组(P<0.05),二甲双胍+中药组干预后的以上指标均显著低于二甲双胍组和模型组(P<0.05),二甲双胍组低于模型组(P<0.05)。PCOS各组卵巢组织TLR4蛋白、NF-κB蛋白表达量均高于空白组(P<0.05),二甲双胍+中药组以上指标低于二甲双胍组和模型组(P<0.05),二甲双胍组低于模型组(P<0.05)。PCOS各组外周血CAT、SOD、GSH-Px活性水平低于空白组,MAD水平高于空白组(P<0.05),双胍+中药组干预后的以上指标(除MAD)高于二甲双胍组和模型组(P<0.05),二甲双胍组高于模型组(P<0.05)。PCOS各组PFG、2hPG、FINS、HOMA-IR均高于空白组(P<0.05),双胍+中药组干预后的以上指标低于二甲双胍组和模型组(P<0.05),二甲双胍组低于模型组(P<0.05)。结论:补肾化痰方联合二甲双胍能够显著改善PCOS雌鼠胰岛素抵抗和糖代谢异常状态,其机制可能与下调TLR4/NF-κB炎性信号通路,降低下游IL-6和TNF-α等炎性细胞因子表达,抑制氧化应激有关。

Objective:To observe the effect of Bushen Huayu Recipe combined with Metformin on TLR4/NF-κB inflammatory signaling pathway, oxidative stress and insulin resistance in female rats with polycystic ovary syndrome(PSOS). Methods:120 female Sprague-Dawley rats aged 6 weeks were randomly divided into blank group, model group, Metformin group, Metformin+Chinese medicine group, 10 rats in each group. After the PSOS model was established, the drug intervention was given. The blank group and the model group were given normal saline. After gavage treatment, the Metformin group was treated with Metformin(270 mg/kg·d), the Metformin+Chinese medicine group was treated with Metformin(34.5 mg/kg·d), and 4 groups were treated for 4 weeks. Ovarian ovulation was recorded in each group. The expression of TLR4 protein, NF-κB protein, IL-6 and TNF-αin peripheral blood of rats before and after intervention was detected. The ovarian tissue of each group was detected by immunohistochemistry(SP). The expression levels of TLR4 protein and NF-κB protein were detected. Peripheral blood oxidative stress and insulin resistance were measured before and after intervention in each group. Results:The average number of ovulation and ovulation rate in the PCOS group(the Metformin+Chinese medicine group, the metformin group, the model group) was significantly lower than that in the blank group. The number of cystic dilatation ovulation was higher than that in the blank group(P<0.05), suggesting successful modeling. The average number of ovulation and ovulation rate in the model group, the Metformin group, the Metformin+Chinese medicine group gradually increased, and the difference was statistically significant(P<0.05). The expression levels of TLR4 protein, NF-κB protein, IL-6 and TNF-α in peripheral blood of the PCOS group were higher than those in the blank group(P<0.05). The above indexes after the Metformin+Chinese medicine group were significantly lower than the Metformin group and the model group(P<0.05), the Metformin group was lower than the model group(P<0.05). The expressions of TLR4 protein and NF-κB protein in ovarian tissue of the PCOS group were higher than those in the blank group(P<0.05). The above indicators in the Metformin+Chinese medicine group were lower than the Metformin group and the model group(P<0.05), and the Metformin group was lower than the model group(P<0.05). The activity levels of CAT, SOD and GSH-Px in peripheral blood of the PCOS group were lower than those in the blank group, MAD level was higher than that in the blank group(P<0.05), and the above indicators(except MAD) after intervention in Metformin+Chinese medicine group were higher than the Metformin group and the model group(P<0.05) was higher in the Metformin group than in the model group(P<0.05). The PFG, 2 hPG, FINS and HOMA-IR of PCOS group were higher than those of the blank group(P<0.05). The above indexes after the intervention of the Metformin +Chinese medicine group were lower than that of the Metformin group and the model group(P<0.05), and the Metformin group was lower than the model group(P<0.05). Conclusion:Bushen Huayu Recipe combined with Metformin can significantly improve insulin resistance and abnormal glucose metabolism in female PCOS, which may down-regulate TLR4/NF-κB inflammatory signaling pathway and reduce downstream inflammatory cytokines such as IL-6 and TNF-α. Expression is associated with inhibition of oxidative stress.