世界中医药

作者	单位
夏青松1，周俪珊2，吴凡1，巩静3，邹欣1，徐丽君1，董慧1，陆付耳1	1 华中科技大学同济医学院附属同济医院中西医结合研究所，武汉，430030； 2 华中科技大学同济医学院附属儿童医院中医科，武汉，430016； 3 华中科技大学同济医学院附属同济医院中西医结合科，武汉，430030

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中文摘要:

目的：探讨胡芦巴丸及其单味药抗氧化应激治疗大鼠糖尿病肾病（DKD）的分子机制。方法：采用高糖高脂饮食和尾静脉小剂量链脲佐菌素（STZ）的方法建立大鼠糖尿病肾病模型，将大鼠随机分为模型组、胡芦巴组、补骨脂组、胡芦巴丸组、卡托普利组，另设正常对照组，给予相应药物灌胃治疗16周后，检测血糖、血脂、血尿素氮（BUN）、血肌酐（SCr）、尿24 h总蛋白（UTP）、尿24 h尿白蛋白（UALB），光镜下观察肾脏形态学改变（HE染色、PAS染色、Masson染色）、电镜下观察肾脏超微结构改变，DHE染色评估肾脏组织超氧阴离子水平，浓度比色法检测肾组织烟酰胺腺嘌呤二核苷酸磷酸（NADPH）活性，Western Blotting检测肾脏组织磷酸化蛋白激酶C-α（PKC-α、NADPH氧化酶p47phox亚基、纤黏蛋白（Fibronectin）的表达水平，实时荧光定量PCR（RT-PCR）检测肾脏组织p47phox、PKC-α的mRNA表达水平。结果：与正常组比较，模型组大鼠血糖升高、血脂紊乱，24 h尿总蛋白及白蛋白水平均明显升高（P<0.001），肾小球体积增大、肾小球纤维化、细胞外基质增生、足细胞足突融合；与模型组比较，胡芦巴、补骨脂、胡芦巴丸组大鼠血糖水平下降，血脂紊乱改善，24 h尿总蛋白及白蛋白水平均明显下降（P<0.01），上述肾脏病理学改变明显减轻。与正常组比较，模型组大鼠肾组织DHE染色荧光强度明显升高，肾组织NADPH氧化酶活性、磷酸化PKC-α、p47phox、Fibronectin蛋白均明显升高（P<0.001）；与模型组比较，胡芦巴、补骨脂、胡芦巴丸组大鼠肾组织DHE染色荧光强度明显降低，肾组织NADPH氧化酶活性、磷酸化PKC-α、p47phox、Fibronectin蛋白均明显降低（P<0.01），p47phox、PKC-α的mRNA表达水平降低（P<0.01）。与单味药比较，胡芦巴丸复方组在抗氧化应激，改善肾小球纤维化，降低血脂血糖优于胡芦巴组和补骨脂组。结论：胡芦巴丸通过抑制PKC-α/NADPH通路抗氧化应激以治疗糖尿病肾病。

English Summary:

To investigate the molecular mechanism of hu-lu-ba-wan and its single components in the anti-oxidative stress treatment of the diabetic kidney disease(DKD) in rats.Methods:A high-sugar and high-fat diet with intravenous injection of small dose of streptozotocin(STZ) was used to establish the diabetic kidney disease(DKD) model in rat.The rats were randomly divided into model group,TFG group,PC group,and Hu-lu-ba-wan group,captopril group,and a normal control group was set to compare.After 16 weeks of intragastric treatment with corresponding drugs,blood glucose,blood lipids,blood urea nitrogen(BUN),blood creatinine(SCr),and 24-hour urine total protein(UTP),24-hour urine albumin(UALB) were examined.HE staining,PAS staining,Masson staining were used to observe renal morphological changes and electron microscope was applied to observe kidney ultrastructural changes.The assess of the level of superoxide anion in kidney tissue was conducted by DHE staining and the activity of nicotinamide adenine dinucleotide phosphate(NADPH) in kidney tissue was detected using the concentration colorimetric method.Western Blotting was used to detect the expression level of phosphorylated PKC-α,NADPH oxidase p47phox subunit,Fibronectin protein in kidney tissue,and the mRNA expression levels of p47phox and PKC-α in kidney tissues were detected by real-time fluorescent quantitative PCR(RT-PCR).Results:Compared with the normal group,the rats in the model group had elevated blood sugar and dyslipidemia,and the 24 h urine total protein and albumin levels were significantly increased(P<0.001),and showed increased glomerular volume,glomerular fibrosis,and extracellular matrix proliferation and podocyte foot process fusion.Compared with the model group,the blood glucose levels decreased,dyslipidemia were improved,and 24-hour urine total protein and albumin levels significantly decreased in rats of the TFG group,PC group,and hu-lu-ba-wan group were significantly lower than the model group(P<0.01).Besides,the above-mentioned renal pathological changes were obviously reduced.Compared with normal group,the fluorescence intensity of DHE staining in the kidney tissue of the model group was significantly increased,and the NADPH oxidase activity,phosphorylated PKC-α,p47phox,and Fibronectin protein in the kidney tissue were distinctly enhanced in the model group(P<0.001).In comparison of model group,the fluorescence intensity of DHE staining in the kidney tissue of rats was significantly reduced and the NADPH oxidase activity,phosphorylated PKC-α,p47phox,and Fibronectin protein in the kidney tissue were significantly reduced(P<0.01),and mRNA expression levels of p47phox and PKC-α were reduced in the TFG group,PC group,and hu-lu-ba-wan group(P<0.01).Compared with the single components,the hu-lu-ba-wan group was better than the TFG group and the PC group in anti-oxidative stress,improving glomerular fibrosis,and lowering blood lipids and blood glucose.Conclusion:Hu-lu-ba-wan improves diabetic kidney disease by inhibiting the oxidative stress of PKC-α/NADPH pathway.

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