他克莫司对2型糖尿病大鼠足细胞氧化应激的影响

发布时间：2018-07-15 20:14

【摘要】：目的:观察他克莫司(FK506)对2型糖尿病(T2DM)大鼠足细胞氧化应激作用的影响。方法:将40只正常雄性wistar大鼠随机分为正常对照组(NC组)10只,其余30只采用高脂高糖饲养(10.0%猪油,20.0%蔗糖,2.5%胆固醇,1.0%胆酸盐,66.5%常规饲料)。8周后,联合小剂量链脲佐菌素STZ(30mg/kg)一次性腹腔注射,建立2型糖尿病大鼠模型。造模成功的21只随机分为糖尿病组(DM组)10只,FK506治疗组(FK组)11只,FK组给予FK506 0.5mg/(kg·d)灌胃,NC组与DM组给予等量枸橼酸缓冲液灌胃。各组大鼠分别于药物干预前1天及干预8周后测定空腹血糖(FBG)、内生肌酐清除率(Ccr)、总胆固醇(TG)、甘油三酯(TC)、尿肌酐(UCr)、肾质量/体质量(KW/BW)、血压(SBP)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、血白细胞(WBC)。通过酶联免疫吸附测定尿白蛋白(UAL),尿白蛋白排泄率(UAER)以UAL(mg/24h)/UCr(mg/24h)比率表示,根据Ucr、Scr计算内生肌酐清除率(Ccr)。在光学显微镜和电子显微镜下观察肾组织病理学改变,通过Western-blot免疫印迹检测肾组织中NOX4蛋白的表达。结果:30只wistar大鼠造模,成功21只,造模成功率为70%。(1)与NC组比较,DM大鼠空腹血糖(FBG)、鼠尾收缩压血压(SBP)、内生肌酐清除率(Ccr)、总胆固醇(TG)、甘油三酯(TC)、24h尿白蛋白排泄率(UAER)、肾肥大脂数(KW/BW)均明显升高(P0.05);与DM组比较,FK组UAER、KW/BW明显降低(P0.05),各组血清肌酐(SCr)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)及血白细胞(WBC)间差异无统计学意义(P0.05);(2)光镜下,NC组肾小球无明显异常,DM组肾小球体积明显增大,系膜区增宽,基底膜增厚;FK组病理改变较DM组明显减轻;(3)电镜下,DM组与NC组相比,肾小球基底膜增厚,足突排列紊乱、融合率显著升高,FK组上述病变有所减轻;(4)免疫组化结果:DM组Nephrin表达较NC组明显减少,而FK506干预后Nephrin表达上调;(5)Western blot结果显示,DM组肾组织NOX4表达较NC组明显增多(P0.05),FK组中NOX4的表达较DM组明显减少(P0.05)。(6)Pearson相关性分析提示,NOX4/β-actin蛋白与Nephrin半定量含量成负相关(r=㧟0.65,p0.01)。结论:他克莫司减少DKD大鼠蛋白尿,其机制可能是通过减少NOX4的表达,抑制肾组织氧化应激,减少足细胞损伤,从而起到延缓DKD进程的作用。
[Abstract]:Aim: to observe the effect of tacrolimus (FK506) on oxidative stress of podocytes in type 2 diabetes mellitus (T2DM) rats. Methods: forty normal male wistar rats were randomly divided into normal control group (NC group) and control group (NC group). The remaining 30 rats were fed with high fat and high sugar (10.0% lard 20.0% sucrose 2.5% cholesterol 1.0% cholate 1.0% conventional diet) for 8 weeks. A rat model of type 2 diabetes was established by intraperitoneal injection of low dose streptozotocin (30mg/kg). The 21 successful rats were randomly divided into diabetic group (DM group), 10 FK506 treatment group (FK group), 11 FK506 treated group (FK group), and FK506 0.5mg/ (kg d) intragastric perfusion group (NC group) and DM group (DM group) with the same amount of citric acid buffer intragastric perfusion. Fasting blood glucose (FBG), endogenous creatinine clearance (CCR), total cholesterol (TG), triglyceride (TC), urinary creatinine (UCr), renal mass / body weight (KW / BW), blood pressure (SBP), alanine transaminase (alt), aspartate aminotransferase (AST) and white blood cell (WBC) were measured 1 day before and 8 weeks after the intervention. Urinary albumin (UAL) was measured by enzyme-linked immunosorbent assay (Elisa). Urinary albumin excretion rate (UAER) was expressed as UAL (mg/24h) / UCr (mg/24h) ratio, and endogenous creatinine clearance (CCR) was calculated by Ucr-SCR. The pathological changes of renal tissue were observed under optical microscope and electron microscope, and the expression of NOX4 protein in renal tissue was detected by Western-blot Western blot. Results of the 30 wistar rats, 21 were successful. (1) compared with NC group, fasting blood glucose (FBG), tail systolic blood pressure (SBP), endogenous creatinine clearance (CCR), total cholesterol (TG), urinary albumin excretion rate (UAER) and renal hypertrophic fat (KW / BW) in DM rats were significantly higher than those in DM group (P0.05). Compared with FK group, serum creatinine (SCR), alanine aminotransferase (alt), aspartate aminotransferase (AST) and white blood cell (WBC) were significantly decreased (P0.05). Compared with NC group, the glomerular basement membrane thickened and the foot process was disordered in the FK group, and the pathological changes in the FK group were significantly less than those in the DM group, (3) compared with the NC group, the glomerular basement membrane was thickened and the basement membrane was thickened in the FK group. The expression of Nephrin in FK group was significantly lower than that in NC group, and the expression of Nephrin in FK group was lower than that in NC group. (5) the expression of NOX4 in renal tissue in DM group was significantly higher than that in NC group (P0.05) and the expression of NOX4 in FK group was significantly lower than that in DM group (P0.05). (6) Pearson correlation analysis showed that NOX4 / 尾 -actin protein was negatively correlated with the semi-quantitative content of Nephrin in DM group (P 0.05 p0.01). Conclusion: tacrolimus reduces proteinuria in DKD rats by reducing the expression of NOX4, inhibiting oxidative stress in renal tissue and reducing damage of podocytes, which may delay the progression of DKD.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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