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目的:研究通络方药(Tongluorecipe,TLR)对糖尿病(DM)大鼠肾小球内皮细胞增殖的作用。方法:大鼠随机分为正常对照(NC)组、糖尿病对照(DMC)组、糖尿病TLR干预(DM+TLR)组(n=7)。DMC和DM+TLR组大鼠采用链脲佐菌素(STZ,65mg/kg)腹腔注射诱导糖尿病,1周后血糖≥16.7mmol/L者为糖尿病模型制备成功;DM+TLR组按每天1g/kgTLR灌胃,NC和DMC组给予等量的生理盐水。干预12周后,测肾功能和24h尿白蛋白(UAE),比色法测血浆丙二醛(MDA)和超氧阴离子水平(O2-),ELISA法测糖基化终末产物(AGE),H-E染色及CD31免疫组化染色观察肾组织病理改变,蛋白质印迹法检测肾组织AGE受体(RAGE)、血管内皮生长因子(VEGF)、VEGF受体(Flk-1)、血管生成素(Ang)-1、Ang-2和Ang-2受体(Tie-2)蛋白表达。结果:与NC组相比,DMC组肾质量/体质量(KW/BW)、24h尿白蛋白(UAE)、MDA、O2-、AGE、RAGE、VEGF、Flk-1、Ang-2、CD31水平和肾小球系膜面积增加,肌酐清除率(Ccr)、Tie-2水平降低(P<0.01或0.05)。与DMC组相比,DM+TLR组大鼠KW/BW、UAE、MDA、O2-、AGE、RAGE、VEGF、Flk-1、Ang-2、CD31水平和肾小球系膜面积降低,Ccr、Tie-2水平升高(P<0.01或0.05)。结论:TLR可能通过降低糖尿病大鼠肾小球氧化应激水平,抑制AGE-RAGE、VEGF表达,进一步抑制肾小球内皮细胞增殖,从而减少糖尿病时肾小球新生血管形成,减轻肾损伤。
Objective: To study the effect of Tongluo prescription (TLR) on glomerular endothelial cell proliferation in diabetic rats. METHODS: Rats were randomly divided into normal control (NC) group, diabetic control (DMC) group, and diabetic TLR intervention (DM+TLR) group (n=7). Diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg) in DMC and DM+TLR rats. One week later, the diabetic model was successfully prepared when the blood glucose was ≥16.7 mmol/L; 1 g/day was administered in the DM+TLR group. kgTLR was given to the stomach, and the same dose of normal saline was given to the NC and DMC groups. Twenty-four weeks after intervention, renal function and 24-hour urinary albumin (UAE) were measured. Malondialdehyde (MDA) and superoxide anion levels (O2-) were measured by colorimetric method. Glycosylation end products (AGE) were measured by ELISA. HE staining and CD31 immunohistochemical staining were used to observe the renal pathological changes. Renal AGE receptor (RAGE), vascular endothelial growth factor (VEGF), VEGF receptor (Flk-1) and angiopoietin (Ang) were detected by Western blotting. )-1, Ang-2 and Ang-2 receptor (Tie-2) protein expression. Results: Compared with the NC group, the DMC group had kidney/body mass (KW/BW), 24-hour urinary albumin (UAE), MDA, O2-, AGE, RAGE, VEGF, Flk-1, Ang-2, and CD31 levels. And the glomerular mesangial area increased, creatinine clearance (Ccr), Tie-2 levels decreased (P <0.01 or 0.05). Compared with DMC group, KW/BW, UAE, MDA, O2-, AGE, RAGE, VEGF, Flk-1, Ang-2, CD31 levels and glomerular mesangial area were decreased in rats in DM+TLR group, Ccr, Tie-2 levels increased (P<0.01 or 0.05). Conclusion: TLR may reduce glomerular oxidative stress in diabetic rats, inhibit the expression of AGE-RAGE and VEGF, and further inhibit the proliferation of glomerular endothelial cells, thereby reducing glomerular neovascularization and reducing renal injury in diabetic rats.