糜蛋白酶抑制剂对糖尿病仓鼠肾功能保护作用的实验研究

Experiment study of renal protective function of chymotrypsin inhibitor in diabetes hamsters model

目的:观察糜蛋白酶抑制剂对糖尿病仓鼠肾脏的保护作用,进而探讨其保护肾脏损伤的机制。方法:(1)通过链脲佐菌素(streptozotocin,STZ)腹腔注射法制备仓鼠糖尿病模型。实验分为5组,每组8只。(2)通过Western blot检测各组动物糜蛋白酶抗体、血管紧张素转化酶抑制剂(angiotensin I-converting enzyme,ACE)、过氧化物酶4(NADPH oxidase-4,NOX-4)、还原型辅酶Ⅱ(NADPH)氧化酶亚单位p22phox的表达情况。(3)通过免疫荧光检测各组动物糜蛋白抑制剂对血管紧张素系统(reninangiotensin system,RAS)的成分,氧化应激的影响。(4)通过q RT-PCR及Western blot检测糜蛋白抑制剂对人血管紧张素Ⅱ(angiotensinⅡ,ANG-Ⅱ)来源的基质蛋白合成影响。结果:(1)免疫组化结果显示糖尿病组仓鼠肾小管和血管球糜蛋白酶表达明显。qRT-PCR结果显示糜蛋白酶m RNA水平较对照组普遍增高(P<0.01)。而胰岛素治疗后降至正常水平。(2)糜蛋白酶抑制剂抑制了糖尿病仓鼠肾内ANG-Ⅱ升高,同时明显降低了8-羟化脱氧鸟苷(8-hydroxy-2’-deoxyguanosine,8-OHd G)分泌水平。(3)NOX-4和p22phox染色表明糖尿病组血管球和肾小管区域氧化产物明显增高,而糜蛋白酶抑制剂抑制氧化产物的升高(P<0.01)。(4)Western blot证实了糖尿病组血管球NOX-4和p22phox蛋白的水平较对照组高,糜蛋白酶抑制剂明显降低了其表达。(5)血管球转化生长因子-β1(transforming growth factor-β,TGF-β1)表达糖尿病组高于对照组,同样糜蛋白酶抑制剂治疗后大大降低了TGF-β1水平。结论:糜蛋白酶抑制剂能保护糖尿病仓鼠的肾功能,其机制是通过降低肾内ANG-Ⅱ水平和氧化应激水平来保护糖尿病肾脏损伤。

Objective：To observe the protective effect of chymotrypsin inhibitors on diabetic hamster kidney and to explore its protection mechanism. Methods：Hamster diabetes mode was established by intraperitoneal injection of streptozotocin（STZ）and all experiment hamsters were divided into five groups. Immunohistochemistry was used to detect chymotrypsin antibody,angiotensin converting enzyme inhibitors（ACE）,NADPH oxidase-4（NOX-4）,NADPH oxidase subunit p22 phox in each group. Immunofluorescence was used to detect the animal protein inhibitor of angiotensin system Mi（renin-angiotensin system,RAS）component,oxidative stress. Realtime PCR and Western blot were used to detect the effect of protein inhibitor of matrix proteins Mi on synthesis of human angiotensinⅡ（ANG-Ⅱ）. Results：Immunohistochemistry showed that tubular and glomerular chymotrypsin in diabetic hamsters were significantly increased. Real-time PCR showed that chymotrypsin m RNA levels were generally higher in diabetic hamsters group than in control group（P〈0.01）,which returned to normal levels after insulin therapy. Chymotrypsin inhibitors inhibited the increase of angiotensin Ⅱ（ANG-Ⅱ）in kidney of diabetes hamsters,while significantly reduced the level of 8-hydroxy-2＇-deoxyguanosine secretion. NOX-4 and p22 phox staining indicated that oxidation products increased significantly in diabetic glomerular and tubular regional groups,and chymotrypsin inhibitors inhibited the increase of oxidation products（P〈0.01）. Western blot confirmed that the levels of NOX-4 and p22 phox protein were higher in glomerular diabetic group than in control group;chymotrypsin inhibitor significantly reduced its expression. Glomerular transforming growth factor-β（TGF-β1）expression was higher in diabetic group than in control groups;chymotrypsin inhibitor therapy significantly reduced the levels of TGF-β1. Conclusion：Chymotrypsin inhibitors protect diabetic hamster kidney function,the mechanism of which is through reducing renal ANG-Ⅱ levels and oxidative stress levels to protect diabetic kidney damage.