加味芍药甘草汤基于Toll样受体4/核因子κB信号通路对多囊卵巢综合征大鼠妊娠结局及免疫机制的影响

Effects of Modified ShaoyaoGancao Decoction on pregnancy outcome and immune mechanism in rats with polycystic ovary syndrome by regulating the TLR4/NF-κB signaling pathway

目的观察加味芍药甘草汤基于Toll样受体4/核因子κB(TLR4/NF-κB)信号通路对多囊卵巢综合征(PCOS)大鼠妊娠结局及免疫机制的影响。方法将80只大鼠按照随机数字表法分为2组,空白组10只和造模组70只。造模组制备PCOS大鼠模型,将造模成功的70只大鼠按照随机数字表法分为7组,模型组10只予等容积0.9%氯化钠注射液灌胃,加味芍药甘草汤低、中、高剂量组各10只,分别予加味芍药甘草汤12.5、25.0、50.0 g/kg灌胃,炔雌醇环丙孕酮片组10只予炔雌醇环丙孕酮片0.2035 g/kg灌胃,抑制剂组10只经尾静脉注射TAK-24210 mg/kg,联合组10只予加味芍药甘草汤50 g/kg灌胃同时尾静脉注射TAK-24210 mg/kg。空白组大鼠予等容积0.9%氯化钠注射液灌胃。检测各组大鼠体质量和卵巢质量;检测各组大鼠T淋巴细胞亚群CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+);观察各组大鼠卵巢组织中TLR4和磷酸化NF-κB p65(p-NF-κB p65)蛋白表达;比较各组大鼠妊娠率和各组孕鼠胚泡着床数。结果与空白组比较,模型组体质量和卵巢质量均升高(P<0.05),CD4^(+)、CD4^(+)/CD8^(+)降低(P<0.05),CD8^(+)升高(P<0.05),TLR4和p-NF-κB p65蛋白表达均升高(P<0.05),妊娠率和胚泡着床数均降低(P<0.05);与模型组比较,各给药组大鼠体质量和卵巢质量均降低(P<0.05),各给药组大鼠CD4^(+)、CD4^(+)/CD8^(+)均升高,CD8^(+)均降低(P<0.05),卵巢组织中TLR4和p-NF-κB p65蛋白表达均降低(P<0.05),妊娠率和胚泡着床数均升高(P<0.05);加味芍药甘草汤低、中、高剂量组体质量和卵巢质量、CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+)、TLR4和p-NF-κB p65蛋白表达、妊娠率和胚泡着床数组间两两比较差异均有统计学意义(P<0.05);加味芍药甘草汤高剂量组、炔雌醇环丙孕酮片组和抑制剂组大鼠体质量和卵巢质量、CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+)、TLR4和p-NF-κB p65蛋白表达、妊娠率和胚泡着床数比较差异均无统计学意义(P>0.05);与加味芍药甘草汤高剂量组比较,联合组大鼠体质量和卵巢质量均降低(P<0.05),CD4^(+)、CD4^(+)/CD8^(+)均升高(P<0.05),CD8^(+)降低(P<0.05),TLR4和p-NF-κB p65蛋白表达均升高(P<0.05),妊娠率和胚泡着床数升高(P<0.05)。结论加味芍药甘草汤可调节PCOS大鼠免疫功能,增加PCOS大鼠妊娠率和胚泡着床率,抑制卵巢组织中TLR4和p-NF-κB p65蛋白表达。

Objective To explore the Modified ShaoyaoGancaoDecoction on pregnancy outcomes and immune mechanisms in rats with polycystic ovary syndrome(PCOS)by regulating the Toll-like receptor 4/nuclear factor kappa B(TLR4/NF-κB)signaling pathway.Methods A total of 80 rats were used in this experiment,and 10 randomly selected rats were included in the control group(gavage of the isodose 0.9%NaCl solution).The PCOS model was created in the remaining 70 rats,and they were randomly divided into model group(gavage of the isodose 0.9%NaCl solution),low-dose group(gavage of 12.5 g/kg Modified ShaoyaoGancaoDecoction),medium-dose group(gavage of 25.0 g/kg Modified ShaoyaoGancaoDecoction),high-dose group(gavage of 50.0 g/kg Modified ShaoyaoGancaoDecoction),ethinylestradiol/cyproterone acetate group(gavage of 0.2035 g/kg ethinylestradiol/cyproterone acetate),inhibitor group(tail vein administration of 10 mg/kg TAK-242)and combination group(gavage of 50.0 g/kg Modified ShaoyaoGancao Decoction plus tail vein administration of 10 mg/kg TAK-242),with 10 rats per group.The body mass and ovarian mass of rats in each group were measured.T lymphocyte subpopulations CD4^(^(+)),CD8^(^(+)),and CD4^(^(+))/CD8^(^(+))were detected by flow cytometry.Protein expressions of TLR4 and p-NF-κB p65 in rat ovary were detected by Western blot.The pregnancy rate and the number of embryo implantation were recorded.Results Compared with those of the control group,rats in the model group presented significantly greater body mass and ovarian mass,higher CD8^(^(+))and protein expressions of TLR4 and p-NF-κB p65 in rat ovary,and lower CD4^(^(+)),CD4^(^(+))/CD8^(^(+)),pregnancy rate and number of embryo implantation(all P<0.05).Compared with those of the model group,rats administrated with the Modified ShaoyaoGancaoDecoction presented significantly less body mass and ovarian mass,lower CD8^(^(+))and protein expressions of TLR4 and p-NF-κB p65 in rat ovary,and higher CD4^(^(+)),CD4^(^(+))/CD8^(^(+)),pregnancy rate and number of embryo implantation(all P<0.05).There were significant differences in the body mass,ovarian mass,CD4^(^(+)),CD8^(^(+)),CD4^(^(+))/CD8^(^(+)),protein expressions of TLR4 and p-NF-κB p65 in rat ovary,pregnancy rate and number of embryo implantation in the pairwise comparison of the low-dose,medium-dose and high-dose group(P<0.05).No significant differences were detected in the body mass,ovarian mass,CD4^(^(+)),CD8^(^(+)),CD4^(^(+))/CD8^(^(+)),protein expressions of TLR4 and p-NF-κB p65 in rat ovary,pregnancy rate and number of embryo implantation among the high-dose group,ethinylestradiol/cyproterone acetate group and inhibitor group(P>0.05).Compared with those of the high-dose group,rats in the combination group presented significantly greater body mass and ovarian mass,higher CD8^(^(+))and protein expressions of TLR4 and p-NF-κB p65 in rat ovary,and lower CD4^(^(+)),CD4^(^(+))/CD8^(^(+)),pregnancy rate and number of embryo implantation(all P<0.05).Conclusion Modified ShaoyaoGancaoDecoction J can regulate the immune function,increase the pregnancy rate and embryo implantation rate of PCOS rats by downregulating TLR4 and p-NF-κB p65 in rat ovary.