通络方剂对1型糖尿病大鼠肾脏Ang Ⅱ、VEGF、ERK1/2表达的影响

Effects of Tongluo recipe on renal expression of Ang Ⅱ,VEGF,and ERK1/2 in rat models of type 1 diabetes mellitus

目的研究通络方剂对链脲佐菌素(STZ)诱导的1型糖尿病大鼠肾脏组织ERK1/2信号转导通路的影响及对肾脏的保护机制。方法健康SD大鼠随机分为正常组(10只)和造模组。造模组给予单次腹腔注射STZ 60mg/kg,造模成功后按体质量随机分为5组:糖尿病非治疗组,通络方剂低剂量组[0.5g/(kg.d)]、中剂量组[1.0g/(kg.d)]、高剂量组[2.0g/(kg.d)]和氯沙坦组[30mg/(kg.d)],每组10只。适应性饲养1周后,按分组分别给予不同处理,持续给药12周后处死,称体质量,取血检测血糖、血脂、血肌酐、尿素氮,留24h尿检测尿肌酐、尿蛋白、尿微量白蛋白,ELISA法检测血清及肾脏组织中血管紧张素Ⅱ(AngⅡ)的含量,取左肾称质量,H-E、PAS染色观察肾脏病理改变,免疫组织化学法检测p-ERK1/2蛋白的表达,蛋白质印迹法检测VEGF蛋白的表达。结果与糖尿病非治疗组比较,糖尿病大鼠系膜细胞增殖受到抑制,蛋白尿明显降低,体质量增加,肾肥大指数降低,肌酐清除率、血尿素氮降低,肾功能改善,血清及肾组织AngⅡ含量降低,VEGF及p-ERK1/2蛋白的表达降低(P<0.05)。中剂量通络方剂与氯沙坦在改善糖尿病大鼠肾功能及抑制AngⅡ、VEGF、p-ERK1/2表达方面差异无统计学意义。结论通络方剂对糖尿病大鼠肾脏的保护作用可能与抑制AngⅡ、VEGF表达,阻断ERK1/2信号转导通路有关。

Objective To observe the effects of Tongluo recipe on extracellular signal-regulated protein kinase（ERK1/2） pathway in the kidneys of streptozotocin-induced rat models of type 1 diabetes mellitus,and to explore the renoprotective mechanism of Tongluo recipe.Methods Adult male Sprague-Dawley rats were randomly divided into 2 groups： control group（A,n=10） and experimental group.Rats in the experimental group were induced by intraperitoneal injection of streptozotocin（60 mg/kg body mass）,and were randomly subdivided into group B（diabetic control,n=10）,group C（diabetic rats treated with low dose of Tongluo recipe,0.5 g/[kg·d],n=10）,group D（diabetic rats treated with medium dose of Tongluo recipe,1.0 g/[kg·d],n=10）,group E（diabetic rats treated with high dose of Tongluo recipe,2.0 g/[kg·d],n=10） and group F（diabetic rats treated with losartan,30 mg/[kg·d],n=10）.At the end of the 12th week rats were sacrificed,and the serum glucose,triacylglycerol（TG）,total cholesterol（Chol）,serum creatinine（Scr）,urea nitrogen（BUN） in blood,24-hour urine protein（Upro24）,microalbuminuria（MAU）,and creatinine in urine（UCR） were examined.The kidney mass/body mass（KM/BM） and creatinine clearance rate（Ccr） were calculated.The left kidneys were collected,weighed and subjected to H-E staining and PAS staining to observe the pathological changes;serum and renal tissue angiotensin Ⅱ（Ang Ⅱ） levels were determined by ELISA;the expression of phosphorylated ERK1/2（p-ERK1/2） was detected by immunohistochemical staining and vascular endothelial growth factor（VEGF） protein expression was determined by Western blotting analysis.Results Compared with group B,group D had a slighter mesangial proliferation under light microscope.Besides,rats in group D had significantly decreased Ccr,BUN,MAU,and Upro24 in the serum,and decreased Ang Ⅱ in the serum and renal tissue,and decreased KM/BM,VEGF and p-ERK1/2 levels in renal tissue compared with rats in group B（P0.05）.The medium dose of Tongluo recipe and losartan showed similar effects in improving the renal function and inhibiting Ang Ⅱ,VEGF,and p-ERK1/2 expression of diabetic rats.Conclusion The renoprotective effect of Tongluo recipe may be related to inhibition of ERK1/2 cascade pathway by decreasing the expression of Ang Ⅱ and VEGF.