基于内质网应激探讨糖肾平对糖尿病肾病db/db小鼠肾脏保护作用及MCP-1/CCR2信号通路的影响

Effects of Tangshenping on renal protection and MCP-1/CCR2 signaling pathway in db/db mice with diabetic kidney disease based on endoplasmic reticulum stress

目的:观察糖肾平对db/db小鼠肾脏内质网应激及其对MCP-1/CCR2信号通路的影响,探究糖肾平减轻足细胞损伤,延缓糖尿病肾病进展的作用机制。方法:24只db/db小鼠随机分为模型组、达格列净组和糖肾平组,以10只db/m作为对照组。观察小鼠一般状况,称取体质量、检测24 h尿蛋白定量;给药第0周和第12周检测各组小鼠空腹血糖和24 h尿蛋白定量;干预12周后取材,收集血清检测小鼠肾功能相关指标:血肌酐(Scr)和血尿素(UREA);检测血脂相关指标:总胆固醇(TC)、甘油三酯(TG)和低密度胆固醇(LDL)水平;采用HE染色、PAS染色、Mallory染色和透射电镜观察肾组织病理形态;免疫组化检测小鼠肾组织GRP78、pAKT、AKT、MCP-1、CCR2和Nephrin蛋白表达;Western Blot检测GRP78、MCP-1、CCR2和Nephrin蛋白表达;Real-time PCR检测小鼠肾组织GRP78和Nephrin mRNA的表达。结果:与模型组比较,糖肾平组小鼠一般状态明显改善,体质量和24 h尿蛋白定量显著减少,Scr、UREA、TC、TG和LDL显著降低,病理染色显示肾组织损伤减轻,电镜观察发现足突融合和基底膜增厚减轻,肾组织GRP78、MCP-1、CCR2蛋白和mRNA下调,Nephrin蛋白和mRNA表达上调。结论:糖肾平通过调节内质网应激水平,抑制MCP-1/CCR2激活,维持足细胞形态稳定,进而保证肾小球滤过屏障完整,减少蛋白尿的发生,从而延缓DKD进展。

Objective:To observe the effect of Tangshenping on renal endoplasmic reticulum stress(ERS)and its effect on MCP-1/CCR2 in db/db mice,and to explore the mechanism of Tangshenping in alleviating podocyte injury and delaying the progression of diabetic kidney disease(DKD).Methods:Twenty-four db/db mice were randomly divided into model group,dapagliflozin group,and Tangshenping group,with 10 db/m as control.Observe the general condition of the mice,measure body weight and 24-h proteinuria;measure fasting blood glucose and 24-h proteinuria in each group of mice at 0 and 12 weeks of administration.After 12 weeks of treatment,serum was collected to measure renal function-related parameters:serum creatinine(Scr)and urea(UREA);lipid-related parameters:cholesterol(TC),triglycerides(TG),and low-density lipoprotein(LDL).Renal histomorphology was examined by H&E staining,PAS staining,Mallory staining,and transmission electron microscopy(TEM);immunohistochemistry was performed to detect the expression of GRP78,pAKT,AKT,MCP-1,CCR2,and Nephrin proteins in mouse kidney tissues;Western Blot was performed to detect the expression of GRP78,MCP-1,CCR2,and Nephrin proteins;Real time PCR was performed to detect the expression of GRP78 and Nephrin mRNA in mouse kidney tissues.Results:Compared with the model group,the Tangshenping group showed a significant improvement in general status,a significant reduction in body mass and 24-h urinary protein,a significant reduction in Scr,Urea,TC,TG,and LDL,a reduction in pathological staining of renal tissue showing injury,a reduction in podocyte fusion and basement membrane thickening by TEM,downregulation of GRP78,MCP-1,CCR2 protein and mRNA,and upregulation of nephrin protein and mRNA expression in renal tissue.Conclusion:Tangshenping maintains podocyte morphogenetic stability by modulating ERS levels,inhibiting MCP-1/CCR2 activation,thereby ensuring integrity of the glomerular filtration barrier and reducing the occurrence of proteinuria,thereby delaying DKD progression.