雷公藤甲素联合厄贝沙坦对2型糖尿病大鼠足细胞损伤的协同保护作用

Triptolide combined with irbesartan synergistically blocks podocyte injury in a type 2 diabetes rat model

目的 观察雷公藤甲素（TP）联合厄贝沙坦对2型糖尿病（ T2DM）大鼠足细胞损伤的协同保护作用,并探讨其机制.方法 实验分为正常对照组（NC,10只）和实验组（80只）,实验组为高脂高糖喂养联合小剂量链脲佐菌素建立的T2DM大鼠模型.实验组再分为糖尿病对照组（DM组,完成11只）、TP治疗组（DT组,完成12只,TP200 μg· kg-1·d-1）、厄贝沙坦治疗组（DI组,完成12只,厄贝沙坦50 mg·kg-1·d-1）和TP联合厄贝沙坦治疗组（DTI组,完成13只,厄贝沙坦50 mg·kg-1·d-1＋ TP 200 μg·kg-1·d-1）.药物干预8周后,观察足细胞的超微结构,采用ELISA法检测尿白蛋白（UAL）；免疫组化、实时定量PCR及免疫印迹技术测定肾组织nephrin、骨形态发生蛋白-7（ BMP-7）、结缔组织生长因子（CTGF）、转化生长因子β1（TGFβ1） mRNA及蛋白的表达.结果 DM组足细胞损伤、UAL的排泄量[计算公式为UAL （mg/24h）/尿肌酐（mg/24h）]明显高于NC组（0.45 ±0.09比6.36±0.87,P＜0.01）,同时肾组织nephrin、BMP-7 mRNA及蛋白表达下调,而CTGF及TGFβ1 mRNA及蛋白的表达显著上调.TP和厄贝沙坦单独干预组UAL排泄均低于NC组（2.48±0.37比6.36±0.87,2.68±0.42比6.36±0.87,P值均＜0.01）且足细胞损伤改善,nephrin、BMP-7mRNA及蛋白表达明显上调,CTGF及TGFβ1 mRNA及蛋白的表达减少,联合治疗组上述指标变化更显著.结论 TP联合厄贝沙坦对大鼠T2DM足细胞损伤有协同保护作用.其机制可能与其上调肾小球BMP-7,并抑制CTGF、TGFβ1过度表达有关.

Objective To investigate the protective effect of combination of triptolide and irbesartan on the podocytes in a type 2 diabetic（T2DM） rat model,and evaluate its mechanism.Methods T2DM rats were induced by fed with high-sucrose-high-fat diet combined with a low dose of streptozocin.The rats were randomly divided into 5 groups：normal control group （ NC,n =10）,diabetes group （ DM,n =11）,triptolide treatment group （DT,n =12）,irbesartan treatment group （DI,n =12） and triptolide combined with irbesartan treatment group （DTI,n =13）. Ultrastructure of podocytes was observed by electronic microscopy and urinary albumin （UAL） excretion by ELISA was determined after 8 weeks.The expression of nephrin and bone morphogenetic protein-7 （BMP-7）, connective tissue growth factor （CTGF）,transforming growth factor （TGF）β1 mRNA and proteins were detected by immunohistochemistry,real-time PCR and Western blot. Results Increased UAL was significantly attenuated in all treatment groups.Compared to NC group,UAL in DM group was increased significantly （0.45 ± 0.09 vs 6.36 ± 0.87,P 〈 0.01 ）,while decreased in triptolide or irbesartan alone treatment group （2.48 ± 0.37 and 2.68 ±0.42,both P 〈 0.01 ）.Compared with those in control groups,kidney expression of nephrin,BMP-7 mRNA and proteins were downregulated while CTGF, TGFβ1 mRNA and proteins were significantly upregulated in T2DM rats. Triptolide or irbesartan each alone moderately ameliorated albuminuria and podocyte damage.However,their combined usage showed a dramatic therapeutic synergism,manifested by prevention of progressive albuminuria,restoration of the glomerular filtration barrier,reversal of the decline in slit diaphragm proteins,reduction expression of CTGF,TGFβ1,and upregulation of BMP-7.Conclusion Our findings show that triptolide can increase the efficacy of irbesartan,leading to a more effective prevention of kidney disease in T2DM rat model,which may through upregulation of BMP-7 and inhibition the overexpression of CTGF and TGFβ1.