芪蛭降糖胶囊对糖尿病肾病大鼠肾实质小动脉内膜/中膜厚度比与炎性因子的影响

Effect of Qizhi Jiangtang Capsule on the Ratio of Intima /media Thickness and Inflammatory Factors in Renal Small Artery of Diabetic Nephropathy Rats

目的:观察芪蛭降糖胶囊对糖尿病肾病(diabetic nephropathy,DN)大鼠肾实质小动脉内膜/中膜厚度比与炎性因子的影响。方法:48只清洁级雄性Wistar大鼠按体重随机抽取40只,采用切除右肾加腹腔注射链脲菌素(streptozotocin,STZ)的方法制备DN模型,另外8只大鼠行右肾假切术。造模成功后按血糖高低,两头随机抽取分为模型组、缬沙坦对照组、芪蛭降糖胶囊低剂量组、芪蛭降糖胶囊高剂量组。成模2 d起各组给予相应浓度和剂量的药物,分别于4、8、12周观察DN尿蛋白定量(TP)、尿视黄醇结合蛋白(RBP)、尿N-乙酰-β-D-氨基葡萄糖苷酶(NAG酶)和血尿素氮(BUN)、肌酐(Scr)、β2微球蛋白(β2-MG)的变化。12周后,处死所有动物,肾组织行HE染色,光镜下观察肾组织病变,测量肾实质小动脉内膜/中膜厚度比;电镜观察肾小球毛细血管超微结构;免疫组化检测肾组织中单核细胞趋化蛋白-1(MCP-1)和肾小动脉CD31的表达;原位杂交法检测MCP-1 mRNA在肾组织中的表达;免疫荧光检测肾小动脉α-平滑肌肌动蛋白(α-SMA)表达。结果:在各个时间点,芪蛭降糖胶囊能明显减少DN大鼠TP、RBP、NAG酶(P<0.01),改善肾功能。HE染色显示,芪蛭降糖胶囊能减轻肾组织及其血管病理损害,比较各组大鼠肾小动脉内膜/中膜厚度比表明,模型组显著高于假手术组(P<0.01),对照组和低剂量组明显低于模型组(P<0.05),高剂量组显著低于对照组(P<0.01)。电镜观察显示,芪蛭降糖胶囊能减轻肾小球基底膜增厚和系膜增生,保护内皮细胞之间窗孔形态,减轻足细胞足突融合。免疫组化检查显示芪蛭降糖胶囊能减少肾组织中MCP-1表达和下调肾小动脉CD31的表达(P<0.01);原位杂交法检测显示芪蛭降糖胶囊能下调MCP-1mRNA在肾组织中的表达;免疫荧光检测显示芪蛭降糖胶囊能下调肾小动脉α-SMA的表达。结论:芪蛭降糖胶囊可以改善肾组织和血管结构病理损害,而此作用可能部分是由下调MCP-1、MCP-1 mRNA在肾组织的表达阻断炎性反应而实现的。

Objective：To observe effects of qizhi jiangtang capsule on the ratio of intima /media thickness and inflammatory factors in renal small artery of DN rats.Methods：48 male Wistar rats were randomly divided into 5 groups（n=8） ：the sham operation group,the model group,Valsartan group（control group）,the low-dosage of qizhi jiangtang granules group（low dosage group） and high dosage of Qizhi Jiangtang granules group（high dosage group）.Except the rats in the sham operation group,the other rats were established DN animal model by removal of the right kidney with intraperitoneal injection of STZ.Medicine was given to each group according to the designed concentration and dosage from 2 days of the model established.Urine protein quantitative,urine retinol binding protein（RBP）,urinary N-acetyl-β-D-amino glucoside enzyme（NAG） and blood urea nitrogen（BUN）,serum creatinine（Scr）,β2-microglobulin（β2-MG） of DN rats were tested and recorded every 4 weeks.All animals were put to death after treated12 weeks.Histological change in the kidney tissues was examined by HE stain.Thickness ratio of intima/media of small artery in renal parenchyma were measured.Glomerular capillary ultrastructure was observed by electron microscopy.The expression of monocyte chemotactic protein-1（MCP-1） and renal arteriole CD31was detected by immunohistochemical.The expression of MCP-1 mRNA in renal tissue was detected in situ hybridization method,while the expression of α-smooth muscle actin（α-SMA） in renal arteriole was detected by immunofluorescence.Results：At all time points,TP、RBP、NAG of DN rats were significantly decreased by qizhi jiangtang capsule（P〈0.01）,and renal function was improved in the low and high dosage groups.HE staining showed that renal and vessel pathological damage were lessened by qizhi jiangtang capsule.Comparison of thickness ratio of intima/media of small artery in renal parenchyma of each group showed that：the ratio of the model group was significantly higher than that of the sham operation group（P〈0.01）,the ratio of the control group and the low-dosage group was significantly lower that of the model group（P0.05）,the ratio of the high dosage group was significantly lower that of the low-dosage group（P〈0.01）.Observations of electron microscopy show that thickening of glomerular basement membrane and mesangium hyperplasia were reduced by qizhi jiangtang capsule,the form of hole between the endothelial cells protected,podocyte foot process fusion reduced.Immunohistochemical tests show that the expression of MCP-1 and renal arteriole CD31were down-regulated by qizhi jiangtang capsule（P〈0.01）.The expression of MCP-1 mRNA in renal tissue were down-regulated in situ hybridization method.while the expression of renal arteriole α-SMA were down-regulated by immunofluorescence test.Conclusion：Pathological damage of renal tissues and vascular structures was improved by qizhi jiangtang capsule in DN rats.This mechanism is partly related to the expression of MCP-1 and MCP-1 mRNA in the renal tissues were down-regulated,and inflammatory reaction was blocked.