目的:观察养血补肾片对多囊卵巢综合征(PCOS)胰岛素抵抗模型大鼠卵巢组织病理学、糖脂代谢水平、胰岛素水平、激素水平、炎症因子水平及卵巢组织蛋白表达水平的影响,探讨养血补肾片治疗多囊卵巢综合征的作用机制。方法:将60只SPF级雌性SD大鼠随机分为正常组、模型组、西药组、中药小剂量组、中药大剂量组,每组12只。除正常组外,其他各组大鼠均通过高脂饲料喂养联合来曲唑溶液灌胃建成PCOS胰岛素抵抗模型。建模后第31天起,西药组大鼠每日按0.18 g/kg的剂量给予二甲双胍灌胃,中药小剂量组和中药大剂量组大鼠每日分别按生药1.62 g/kg、3.24 g/kg的剂量给予养血补肾片灌胃。上述3组灌胃容积均为2 mL/kg,每日灌胃1次,连续灌胃4周。给药结束后第2天,取大鼠腹主动脉血和卵巢用于指标观察。HE染色法观察各组大鼠卵巢组织形态;已糖激酶法检测各组大鼠空腹血糖(FBG)水平;酶比色法检测各组大鼠血清脂质代谢指标水平;酶联免疫吸附测定(ELISA)法检测各组大鼠血清激素、炎症因子及空腹胰岛素(FINS)水平;Western Blotting法检测各组大鼠卵巢组织蛋白表达。结果:与正常组比较,模型组大鼠各级卵泡数量减少,颗粒细胞层次减少,卵泡结构不规则,闭锁卵泡和囊性扩张卵泡增多;与模型组比较,各给药组大鼠上述卵巢组织形态均有不同程度改善,其中中药大剂量组改善效果最佳。给药后,各给药组大鼠血清FBG、脂质代谢指标、激素、炎症因子及FINS水平较模型组均有不同程度的改善。其中,中药大剂量组大鼠的血清FBG、总胆固醇(TC)、白细胞介素-6(IL-6)、游离睾酮(FT)、黄体生成素(LH)、促卵泡激素(FSH)水平较模型组均显著降低,差异有统计学意义(P<0.05);血清FINS、瘦素(LEP)、白细胞介素-10(IL-10)水平较模型组显著升高,差异有统计学意义(P<0.05)。与模型组比较,各给药组大鼠卵巢组织磷酸化核因子-κBp65(P-NF-κBp65)蛋白表达水平均显著降低,磷酸化胰岛素受体底物-1(P-IRS-1)蛋白表达水平均显著升高,差异有统计学意义(P<0.05)。西药组大鼠上述各指标水平与中药各剂量组比较,除西药组大鼠血清FT水平高于中药各剂量组(P<0.05),其他指标水平差异均无统计学意义(P> 0.05)。结论:养血补肾片对PCOS胰岛素抵抗模型大鼠具有改善糖脂代谢、降低炎症因子水平和促进LEP分泌的作用,其机制与抑制NF-κB通路激活、提高P-IRS-1蛋白活性、改善胰岛素抵抗有关。

Objective:To explore the mechanism of Yangxue Bushen Tablet ( 养血补肾片 ) in treating polycystic ovary syndrome(PCOS) by observing its effects on ovarian morphology,glycolipids metabolism,insulin,hormone,inflammatory factors and ovarian tissue protein expression levels of model rats with PCOS and insulin resistance. Methods:A total of 60 SPF grade female SD rats were randomly divided into normal group,model group,western medicine group,small dose of Chinese medicinal group and large dose of Chinese medicinal group,with 12 rats in each group. Except the rats in the normal group,others were fed with high-fat diet and given letrozole solution by gavage to establish the model of PCOS with insulin resistance.Since the 31 th day after the establishment of the model,the rats in the western medicine group were given metformin by gavage at a dose of 0.18 g/kg. The rats in the small dose of Chinese medicinal group and the large dose of Chinese medicinal group were given Yangxue Bushen Tablet by gavage at a dose of 1.62 g/kg and 3.24 g/kg respectively. The volume of the medicinal solution given to the rats in the above three groups were 2 m L/kg,once a day for 4 weeks. On the second day after the end of drug administration,the abdominal aorta blood and ovaries of the rats were taken out for indicator test. The HE stain was performed for observing the ovarian morphology of the rats in each group. The hexokinase method was used to test the fasting blood glucose(FBG) level of the rats in each group. The serum lipid metabolism level of the rats in each group was measured by enzymatic colorimetry. The enzyme-linked immuno sorbent assay(ELISA)method was used to examine the levels of serum hormone,inflammatory factors and fasting insulin(FINS) of the rats in each group. The ovarian tissue protein expression level of the rats in each group was tested by Western Blotting method. Results:Compared with the rats in the normal group,the rats in the model group had less follicles and granular cell layers,more atresic follicles and follicular cystic dilatation,and their follicular structure was irregular. Compared with the rats in the model group,other rats that were given medicine got improvements in their ovarian tissue morphology of different levels,among which the large dose of Chinese medicinal group displayed the best results. After medication,the levels of serum FBG,lipid metabolism,hormone,inflammatory factors and FINS of rats in all the medication groups were improved. The serum FBG,total cholesterol(TC),interleukin-6(IL-6),free testosterone(FT),luteinizing hormone(LH) and follicle stimulating hormone(FSH) levels of rats in the large dose of Chinese medicinal group were lower than those in the model group(P<0.05). The serum FINS,leptin(LEP) and interleukin-10(IL-10) levels of rats in the large dose of Chinese medicinal group were higher than those in the model group(P<0.05). Compared with the model group,the level of P-NF-κBp65 protein expression in the ovarian tissue of rats in all the medication groups decreased(P<0.05),and the level of P-IRS-1 protein expression in the ovarian tissue increased(P<0.05). For the comparison of all the above indicators between the western medicine group and both the two Chinese medicinal groups,except the higher serum FT level in the western medicine group which was statistically different from the Chinese medicinal groups(P<0.05),the differences of other indicators were not statistically significant(P> 0.05). Conclusions:Yangxue Bushen Tablet has a positive effect on model rats with PCOS and insulin resistance in improving glycolipid metabolism,decreasing levels of inflammatory factors,and promoting the LEP secretion. Its mechanism is related to inhibiting the activation of NF-κB pathway,increasing the activity of P-IRS-1 protein and ameliorating the state of insulin resistance.