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目的观察通络方剂对糖尿病大鼠肾小球足细胞的保护作用,探讨糖尿病肾病足细胞损伤的可能机制。方法通过腹腔注射链脲佐菌素(60mg/kg)制作糖尿病大鼠模型,将成模大鼠随机分为糖尿病组(DM组,12只)、糖尿病TLR干预组(DM+TLR组,12只),另设正常对照组(CON组,8只)。DM+TLR组大鼠予以TLR[0.4g/(kg.d)]灌胃,DM组和CON组每日均在同一时间予同体积蒸馏水一次性灌胃。干预12周后测定各组大鼠24h尿蛋白定量;取大鼠肾皮质,采用电子显微镜观察肾小球足细胞超微结构;采用体视学方法计数足细胞、计算足细胞相对密度;采用蛋白质印迹分析法检测足细胞nephrin蛋白的表达;测定血浆和肾皮质中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性和脂质过氧化产物丙二醛(MDA)含量。结果与CON组比较,DM组和DM+TLR组大鼠肾质量/体质量比值、血糖和24h尿蛋白升高(P均<0.01);12周时DM组大鼠肾小球基底膜增厚、足细胞足突融合、微绒毛化,足细胞计数减少(P<0.01)、足细胞相对密度降低(P<0.01)且nephrin表达减少(P<0.01)。DM+TLR组足突形态接近正常,基底膜增厚,但较DM组减轻,足细胞计数、足细胞相对密度和nephrin表达量均高于DM组(P均<0.01)。与CON组比较,DM组和DM+TLR组大鼠在观察终点血浆和肾皮质SOD、GSH-Px活性均降低(P<0.01,P<0.05),MDA含量则升高(P<0.01,P<0.05);与DM组相比较,DM+TLR组血浆和肾皮质SOD、GSH-Px活性增加(P均<0.01),MDA含量降低(P<0.01,P<0.05)。结论 TLR可以改善糖尿病大鼠肾小球的足细胞损伤,可能与其抗氧化应激作用有关。
Objective To observe the protective effect of Tongluo Prescription on glomerular podocyte in diabetic rats and to explore the possible mechanism of podocyte injury in diabetic nephropathy. Methods Diabetic rats were induced by intraperitoneal injection of streptozotocin (60mg / kg). The rats were randomly divided into four groups: diabetic group (DM group, 12 rats), diabetic TLR intervention group (DM + TLR group, , Another normal control group (CON group, 8). The rats in DM + TLR group were given gavage with TLR [0.4g / (kg · d)], and the DM group and the CON group were fed with the same volume of distilled water once a day at the same time. The urinary protein excretion of rats in each group was measured after intervention for 12 weeks. The ultrastructure of glomerular podocytes was observed by electron microscopy. The podocytes were counted by stereological method and the relative density of podocytes was calculated. The expression of nephrin protein in podocytes was detected by Western blotting. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in plasma and renal cortex were determined. )content. Results Compared with CON group, the ratio of kidney mass to body weight, blood glucose and 24h urinary protein in DM group and DM + TLR group were increased (all P <0.01); at 12 weeks, the glomerular basement membrane thickening (P <0.01). The relative density of podocytes decreased (P <0.01) and nephrin expression decreased (P <0.01). Compared with DM group, the morphology of foot process in DM + TLR group was close to normal, while the basement membrane was thicker than that in DM group. The number of podocyte, the relative density of podocyte and the expression of nephrin in DM + TLR group were higher than those in DM group (all P <0.01). Compared with CON group, the activities of SOD and GSH-Px in plasma and renal cortex of DM group and DM + TLR group decreased (P <0.01, P <0.05) and MDA content increased (P <0.01, P <0.05). Compared with DM group, the activity of SOD and GSH-Px in plasma and renal cortex of DM + TLR group increased (P <0.01) and MDA content decreased (P <0.01, P <0.05). Conclusion TLR can improve glomerular podocyte injury in diabetic rats, which may be related to its anti-oxidative stress effects.