黄芪皂苷Ⅰ通过抑制足细胞焦亡干预糖尿病肾脏疾病的机制研究

Study on the mechanism of astragalosideⅠinhibiting podocyte pyroptosis in diabetic kidney disease

目的探讨黄芪活性成分黄芪皂苷Ⅰ抑制足细胞损伤、改善糖尿病肾脏疾病的作用机制。方法60只雄性db/db小鼠按体质量随机分为模型组、黄芪皂苷Ⅰ低剂量组(10 mg/kg)、黄芪皂苷Ⅰ中剂量组(20 mg/kg)、黄芪皂苷Ⅰ高剂量组(40 mg/kg)及缬沙坦组(10 mg/kg),每组12只;12只db/db同窝对照db/m小鼠作为对照组。灌胃给药8周。透射电子显微镜观察肾脏超微结构;免疫组织化学法、蛋白质印迹法检测肾脏足细胞标志物肾病蛋白(nephrin)的表达情况;酶联免疫吸附测定法检测小鼠血清白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)含量;蛋白质印迹法检测肾脏组织NOD样受体热蛋白结构域相关蛋白3(NLRP3)、胱天蛋白酶-1(Caspase-1)、消皮素D(GSDMD)蛋白表达。结果与对照组比较,模型组小鼠肾小球出现明显的足细胞丢失、足突融合等现象;肾脏nephrin蛋白表达下降(P<0.05);血清IL-1β、IL-18含量升高(均P<0.05);NLRP3、裂解型Caspase-1(Cleaved-Caspase-1)、GSDMD-N蛋白表达升高(均P<0.05)。与模型组比较,黄芪皂苷Ⅰ给药组肾脏病理损伤得到缓解;Nephein蛋白表达升高(P<0.05);血清IL-1β、IL-18含量下降(均P<0.05);NLRP3、Cleaved-Caspase-1、GSDMD-N蛋白表达下降(均P<0.05)。结论黄芪皂苷I可能通过抑制细胞焦亡、改善足细胞损伤发挥干预糖尿病肾脏疾病的作用。

Objective To investigate the mechanism of astragaloside I,the active constituent of milkvetch root,in inhibiting podocyte injury and improving diabetic kidney disease.Methods According to the body weight,60 male db/db mice were randomly divided into the model group,astragalosideⅠlow-dose group(10 mg/kg),astragaloside I medium-dose group(20 mg/kg),astragaloside I high-dose group(40 mg/kg),and valsartan group(10 mg/kg),with 12 mice per group.Twelve db/db littermate control db/m mice were used as the control group.The drug was administered by gavage for 8 weeks.Transmission electron microscope was used to observe the ultrastructure of the kidney;immunohistochemistry and Western blotting were used to detect the expression of nephrotic protein(nephrin),a marker of renal podocytes;enzyme-linked immunosorbent assay was used to detect the contents of interleukin-1β(IL-1β)and interleukin-18(IL-18)in the serum of mice;Western blotting was used to detect the protein expressions of NOD-like receptor thermoprotein domain-related protein 3(NLRP3),cysteinyl aspartate specific proteinase 1(Caspase-1),and Gasdermin D(GSDMD)in kidney tissue.Results Compared with the control group,the glomeruli of the model group showed obvious podocyte loss and foot process fusion;the protein expression of nephrin was decreased(P<0.05);the contents of IL-1βand IL-18 in serum were increased(P<0.05);the protein expressions of NLRP3,Cleaved-Caspase-1,and GSDMD-N were increased(P<0.05).Compared with the model group,the renal pathological damage in the astragalosideⅠadministration groups were alleviated;the protein expression of nephrin was increased(P<0.05);the contents of IL-1βand IL-18 in serum were decreased(P<0.05);the protein expressions of NLRP3,Cleaved-Caspase-1,and GSDMD-N were decreased(P<0.05).Conclusion Astragaloside I may play a role in intervening diabetic kidney disease by inhibiting pyroptosis and improving podocyte injury.