雷公藤甲素通过调控Toll-样受体(TLR)/核因子κB(NF-κB)信号通路对糖尿病肾病模型小鼠足细胞的作用研究

Effect of Triptolide on Podocytes in Mice with Diabetic Nephropathy by Regulating Toll-Like Receptor(TLR)/Nuclear Factor-Κb(NF-κB)Signal Pathway

目的观察雷公藤甲素治疗糖尿病肾病(DN)模型小鼠的疗效,并探讨其通过Toll-样受体(TLR)/核因子κB(NF-κB)信号通路对DN足细胞的作用机制。方法6只9周龄db/m和18只9周龄db/db雄性小鼠,按db/m正常对照组(等量0.9%生理盐水)、db/db DN对照组(等量0.9%生理盐水)、替米沙坦治疗组(5mg·kg^-1·d^-1)、雷公藤甲素组(50μg·kg^-1·d^-1)分组给药,治疗8周。记录各组小鼠一般情况、24h尿白蛋白定量、体质量、肾脏指数和血生化指标,光镜观察肾小球病变情况,免疫组化计算足细胞数量,Western blot检测肾脏足细胞相关蛋白Nephrin、Podocin以及肾脏组织TLR4和NF-κB蛋白定量表达。结果与DN对照组比较,雷公藤甲素组小鼠治疗后24h尿蛋白定量[(98.28±11.10)mg/24h比(239.89±44.26)mg/24h]、血清肌酐[(38.82±3.66)μmol/L比(45.32±7.63)μmol/L]、总胆固醇[(3.05±0.54)mmol/L比(4.00±0.67)mmol/L]及三酰甘油[(1.23±0.15)mmol/L比(2.09±0.48)mmol/L]均明显下降(P均<0.01),血清白蛋白水平上升[(39.21±4.33)g/L比(25.46±1.54)g/L,P<0.01],肾小球肥大和足细胞损伤减轻[(95.50±5.01)个/肾小球比(58.78±2.86)个/肾小球,P<0.05],Nephrin[(2.05±0.44)比(1.15±0.22)]、Podocin[(1.32±0.25)比(0.72±0.20)]蛋白表达量上升(P均<0.01),TLR4[(0.37±0.04)比(0.61±0.04)]和NF-κB[(0.41±0.05)比(0.76±0.06)]蛋白表达量下降(P均<0.01)。结论雷公藤甲素可能通过抑制TLR/NF-κB信号通路相关因子表达,减轻糖尿病肾病模型小鼠足细胞损伤,减低蛋白尿,保护肾功能,起到治疗糖尿病肾病的作用。

Objective To observe the therapeutic effect of triptolide on diabetic nephropathy(DN)mice and explore the mechanism of triptolide on DN podocytes via toll-like receptor(TLR)/nuclear factor-κB(NF-κB)signaling pathway.Methods Six db/m and eighteen db/db male mice of 9-week-old,were divided into db/m normal control group fed with 0.9%normal saline,db/db DN control group fed with 0.9%normal saline,telmisartan group treated with 5mg·kg^-1·d^-1 telmisartan,triptolide group treated with 50μg·kg^-1·d^-1 triptolide,for 8 weeks.Record the general situation of mice in each group,24h urinary albumin quantification,body weight,kidney index and blood biochemical indicators,observe the glomerular lesions with light microscope,calculate the number of podocytes by immunohistochemistry,and detect nephrin,podocin,TLR4 and NF-κB protein quantitative expression in kidney tissues by Western blot.Results Compare to db/db DN control group,after treatment with triptolide in db/db mice,the levels of 24h urinary protein,blood creatinine,total cholesterol and triacylglycerol decreased significantly,i.e.,24h urinary protein:[(98.28±11.10)mg/24h vs(239.89±44.26)mg/24h],blood creatinine:[(38.82±3.66)μmol/L vs(45.32±7.63)μmol/L],total cholesterol:[(3.05±0.54)mmol/L vs(4.00±0.67)mmol/L],triacylglycerol:[(1.23±0.15)mmol/L vs(2.09±0.48)mmol/L],P<0.01,respectively;serum albumin level increased significantly[(39.21±4.33)g/L vs(25.46±1.54)g/L,P<0.01].Glomerular hypertrophy and reduced podocyte damage were observed,[(95.50±5.01)/glomerulus vs(58.78±2.86)/glomerulus,P<0.05].Expression of nephrin and podocin increased significantly,i.e.,nephrin:[(2.05±0.44)vs(1.15±0.22)],podocin:[(1.32±0.25)vs(0.72±0.20)],P<0.01,respectively;while expression of TLR4 and NF-κB decreased significantly,i.e.,TLR4:[(0.37±0.04)vs(0.61±0.04)],NF-κB:[(0.41±0.05)vs(0.76±0.06)],P<0.01,respectively.Conclusion Triptolide may reduce podocyte damage,reduce proteinuria,protect renal function,and treat diabetic nephropathy in diabetic nephropathy model mice by inhibiting the expression of TLR/NF-κB signaling pathway-related factors.