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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 inhibitor on the diabetic hamster kidney and to explore its mechanism of protecting kidney injury. Methods: (1) Hamster diabetic model was established by intraperitoneal injection of streptozotocin (STZ). The experiment was divided into 5 groups, 8 in each group. (2) Western blot was used to detect the expression of chymotrypsin antibody, angiotensin I-converting enzyme (ACE), NADPH oxidase-4 (NOX-4) Ⅱ (NADPH) oxidase subunit p22phox expression. (3) Immunofluorescence was used to detect the effects of chymase inhibitor on the composition and oxidative stress of reninangiotensin system (RAS). (4) The effects of chymase inhibitor on matrix protein synthesis of human angiotensin II (ANG-Ⅱ) were detected by q RT-PCR and Western blot. Results: (1) The results of immunohistochemistry showed that the expression of renal tubular and chymase in hamsters in diabetic group was significant. The result of qRT-PCR showed that the m RNA level of chymotrypsin was generally higher than that of the control group (P <0.01). The insulin treatment dropped to normal levels. (2) Chymotrypsin inhibitor could inhibit the increase of intrarenal ANG-II in diabetic hamsters and at the same time significantly reduce the level of 8-hydroxy-2’-deoxyguanosine (8-OHd G) secretion. (3) NOX-4 and p22phox staining showed that the oxidative products of glomeruli and tubules in diabetic group were significantly increased, while the increase of oxidized products was inhibited by chymotrypsin inhibitor (P <0.01). (4) Western blot confirmed that the level of NOX-4 and p22phox in the glomerulus of diabetic group was higher than that of the control group, and chymotrypsin inhibitor significantly reduced the expression of NOX-4 and p22phox. (5) The expression of transforming growth factor-β1 (TGF-β1) in diabetic group was significantly higher than that in the control group. Similarly, the treatment with chymotrypsin inhibitor significantly reduced the level of TGF-β1. Conclusion: Chymotrypsin inhibitor can protect renal function of diabetic hamsters by decreasing ANG-Ⅱ level in renal and oxidative stress to protect diabetic nephropathy.