益气散聚方对肥胖相关肾小球病模型小鼠AMPK信号通路的影响及机制研究

Effect of Yiqi Sanju Recipe on AMPK signaling pathway of obesity-related glomerulopathy model mice and its mechanism

目的探讨益气散聚方对肥胖相关性肾小球病模型小鼠体内AMPK与炎症诱发的关系以及对肾损害的影响,阐明益气散聚方防治肥胖相关性肾小球的作用机制。方法随机将6周龄的60只雄性C57BL/6试验小鼠分为正常组(6只)、西药组(12只)、中药组(18只)、中西医结合组(12只)、模型组(12只),正常组小鼠用生理盐水与普通饲料灌胃、西药组小鼠用二甲双胍与高脂饲料灌胃、中药组小鼠用益气散聚方与高脂饲料灌胃、中西医结合组小鼠用二甲双胍、益气散聚方与高脂饲料灌胃、模型组小鼠用生理盐水与高脂饲料灌胃。在药物干预的第4周末、第8周末观察各组小鼠的肥胖体质量,并检测24 h尿蛋白定量、尿RBP、尿NAG的变化情况,利用HE染色、免疫组化于镜下观察肾脏组织的病理变化,同时对肾脏组织内转化生长因子β_(1)蛋白(TGF-β_(1))、单核细胞趋化蛋白-1(MCP-1)、磷酸化腺苷酸活化蛋白激酶α(P-AMPKα)mRNA含量进行检测。结果在用药的第4周末和第8周末,正常组、中药组、模型组、西药组的小鼠体质量较前一次测量均增大,但差异无统计学意义(P>0.05);中西医结合组小鼠与其他各组相比体质量下降明显,其差异均具有统计学意义(P<0.05);在用药的第4周末和第8周末,中药组、中西医结合组、西药组与模型组相比,小鼠的24 h尿蛋白定量、尿RBP、尿NAG含量均降低,差异均具有显著统计学意义(P<0.01),且五组进行组间比较发现,中西医结合组与中药组的治疗效果较好,差异具有统计学意义(P<0.05);模型组、中药组、西药组、中西医结合组小鼠治疗的第4周末肾小球体积增大,肾小球囊腔变小,固有细胞无增生,系膜区未见明显增生,肾小管间质正常,未见细胞浸润;治疗第8周末检查发现与西药组、模型组进行比较,中西医结合组与中药组小鼠的肾小球直径均减小,但差异无统计学意义(P>0.05);药物干预第8周末对五组小鼠免疫组化的结果进行比较发现,中西医结合组、西药组、中药组比模型组的PAMPK-α含量升高明显,差异具有显著统计学意义(P<0.01);实时定量PCR检测结果显示:中药组、中西医结合组小鼠治疗的第4周末、第8周末MCP-1 mRNA、TGF-β_(1) mRNA表达下降,两组较模型组明显降低,差异均具有显著统计学意义(P<0.01);中药组、西药组、中西医结合组小鼠用药的第8周末PAMPK-αmRNA表达升高,三组较模型组显著升高,差异均具有统计学意义(P<0.05)。结论益气散聚方可通过提高肥胖相关肾小球病模型小鼠AMPK表达,抑制MCP-1、TGF-β_(1)等炎症因子表达,从而改善肥胖引起的肾脏损伤。

Objective To investigate the effect of Yiqi Sanju Recipe on AMPK signaling pathway,inflammation,renal damage in obesity-related glomerulopathy model mice,and to clarify the mechanism of Yiqi Sanju Recipe in the prevention and treatment of obesity-related glomerulopathy.Methods Sixty male C57BL/6 test mice at 6 weeks of age were randomly divided into normal group(n=6);western medicine group(n=12);traditional Chinese medicine group(n=18);integrated traditional Chinese and western medicine group(n=12);model group(n=12);which were gavaged with normal saline and common feed;metformin and high-fat feed;Yiqi Sanju Recipe and high-fat feed;metformin,Yiqi Sanju Recipe,and high-fat feed;normal saline and high-fat feed;respectively.At the end of the 4 th and 8 th week of treatment,the obesity body mass of mice in each group was observed,and the changes of 24 h urine protein,urine retinol-binding protein(RBP),and urine NAG were detected.The pathological changes of renal tissues were observed by HE staining and immunohistochemistry under microscope.Meanwhile,the mRNA levels of transforming growth factorβ_(1)(TGF-β_(1)),monocyte chemotaxis protein-1(McP-1),and phosphorylated adenylate activated protein kinaseα(P-AMPKα)in renal tissues were detected.Results At the end of the 4 th and 8 th week of treatment,the body mass of mice in normal group,traditional Chinese medicine group,model group,and western medicine group increased compared with the previous measurement,but the difference was not statistically significant(P>0.05);compared with other groups,the body mass of mice in the integrated traditional Chinese and western medicine group decreased significantly,and the difference was statistically significant(P<0.05).At the end of the 4 th and 8 th week of treatment,compared with the model group,the quantitative 24-h urinary protein,urinary RBP,and urinary NAG of mice in the traditional Chinese medicine group,integrated traditional Chinese and western medicine group,and western medicine group decreased,and the differences were statistically significant(P<0.01);comparison among the five groups showed that the treatment efficacy of integrated traditional Chinese and western medicine group and traditional Chinese medicine group was better,and the difference was statistically significant(P<0.05).At the end of the 4 th week of treatment,in model group,traditional Chinese medicine group,western medicine group,and integrated traditional Chinese and western medicine group,the glomerular volume of mice increased,and the glomerular capsule became smaller,with no obvious proliferation in inherent cells and in mesangial area;the renal tubulointerstitial was normal,and there was no cell infiltration.At the end of the 8 th week of treatment,compared with western medicine group and model group,the glomerular diameter of mice in integrated traditional Chinese and western medicine group and traditional Chinese medicine group decreased,but the difference was not statistically significant(P>0.05).At the end of the 8 th week of treatment,the immunohistochemical results of the five groups were compared,which showed that that PAMPK-αlevel of integrated traditional Chinese and western medicine group,western medicine group,and traditional Chinese medicine group was higher than that of the model group,and the differences were statistically significant(P<0.01).The results of real-time quantitative PCR showed that MCP-1 mRNA and TGF-β_(1) mRNA in traditional Chinese medicine group,integrated traditional Chinese,and western medicine group at the end of the 4 th and 8 th week of treatment reduced,which were significantly lower than those in the model group(P<0.01).PAMPK-αmRNA level at the end of 8 th week of treatment in traditional Chinese medicine group,western medicine group,and integrated traditional Chinese and western medicine group increased,which were significantly higher than that of the model group(P<0.05).Conclusion Yiqi Sanju Recipe can improve the expression of AMPK in obesity-related glomerulopathy model mice,and inhibit the expression of inflammatory factors such as MCP-1 and TGF-β_(1),thereby improving the kidney damage caused by obesity.