丹参多酚酸盐调控Nrf2/HO-1信号通路对膜性肾病大鼠氧化应激的影响

Effect of Nrf 2/HO-1 signaling regulated by salvia miltiorrhiza polyphenols on oxidative stress in rats with membranous nephropathy

关键词:  丹参多酚酸盐  膜性肾病  氧化应激  Nrf2/HO-1信号通路

Key words:  miltiorrhiza polyphenols  membranous nephropathy  oxidative stress  Nrf 2/HO-1 signaling pathway

摘要:

[目的] 基于血清核转录因子2（Nrf2）/血红蛋白氧合酶-1（HO-1）信号通路探讨丹参多酚酸盐对膜性肾病（MN）大鼠氧化应激的影响。[方法] 80只SD雄性大鼠随机选取20只为正常对照组，其余60只大鼠均采用尾静脉注射阳离子化牛血清白蛋白（C-BSA）构建MN模型，MN模型大鼠复制成功后随机分为模型组，盐酸贝那普利组（10 mg/kg），丹参多酚酸盐分为低、中、高剂量组（16.7、33.3、66.7 mg/kg）。药物灌胃连续 4周，1 次/d，正常组和模型组予以相等体积的生理盐水灌胃。治疗后检测大鼠24 h尿蛋白定量（24 h UTP）。给药结束后，经大鼠腹主动脉取血检测血尿素氮（BUN），血清肌酐（Scr），三酰甘油（TG），总胆固醇（TC），总蛋白（TP），白蛋白（ALB）水平；过碘酸-六胺银（PASM）染色观察大鼠肾组织病理形态；免疫荧光检测肾组织免疫球蛋白G（IgG）、补体C3沉积情况；采用酶联免疫吸附测定法（ELISA）检测大鼠血清中超氧化物歧化酶（SOD）、丙二醛（MDA）表达情况；蛋白免疫印迹法（Western Blot）和实时荧光定量聚合酶链式反应（Real-time PCR）观察肾组织Nrf2、HO-1蛋白及Nrf2、HO-1 mRNA表达。[结果] 与正常组相比，模型组大鼠肾小球出现体积增大、基底膜增厚、“钉突”形成，沿毛细血管襻有补体C3、IgG弥漫性沉积，24 h UTP、血清TG、TC水平显著升高（P<0.01），TP、ALB水平显著降低（P<0.01），BUN、SCr差异无统计学意义；血清中SOD表达显著降低（P<0.01），MDA表达显著升高（P<0.01）；肾组织Nrf2、HO-1 mRNA及蛋白表达显著降低（P<0.01）。与模型组相比，各治疗组大鼠24 h UTP、血清TG、TC水平显著降低（P<0.05或P<0.01），TP、ALB 水平显著升高（P<0.01），但丹参多酚酸盐低剂量组改善不明显；各治疗组肾脏病理损害明显改善；SOD表达水平显著升高（P<0.01），MDA表达水平明显降低（P<0.01）；Nrf2、HO-1mRNA及蛋白表达显著升高（P<0.05或P<0.01）。[结论] 丹参多酚酸盐可能通过调控Nrf2/HO-1信号通路，缓解肾组织氧化应激，进而保护肾脏及延缓疾病进展。

Abstract:

[Objective] To investigate the effect of miltiorrhiza polyphenols on oxidative stress in membranous nephropathy（MN） rats based on the serum nuclear transcription factor 2/hemoglobin oxygenase-1（Nrf 2/HO-1） signaling pathway. [Methods] The 80 SD male rats were randomly selected as normal control group，and the remaining 60 rats were treated with tail vein injection of cated bovine serum albumin（C-BSA） to construct the MN model. MN rats were randomly divided into model group，benenapril hydrochloride（10 mg/kg），and polyphenols were divided into low，medium and high dose groups（16.7，33.3 and 66.7 mg/kg）. Drug gavage for 4 weeks，1 time/d，and equal volume of saline gavage in normal and model groups. Ururine protein quantification at 24 h（24 h UTP）. After the administration of the drug，blood urea nitrogen（BUN），serum creatinine（Scr），triglyceride（TG），total cholesterol（TC），total protein（TP），albumin（ALB） levels were measured；the renal histopathology was observed by silver overdate-hexidamine（PASM）；the deposition of immunoglobulin G（IgG） and complement C3 was detected by immunofluorescence；the expression of superoxide dismutase（SOD） and malondialdehyde（MDA） was detected by enzyme-linked immunosorbent assay（ELISA）；the expression of Nrf 2 and HO-1 was observed by protein immunoblotting（Western Blot）. [Results] Compared with the normal group，the glomeruli in the model group showed increased size，basement membrane thickening，the formation of “nail process”，diffuse deposition of complement C3 and IgG along the capillary haptics，UTP，serum TG and TC levels at 24 h significantly increased（P<0.01），and TP and ALB levels significantly decreased（P<0.01）. There was no significant difference between BUN and SCr；SOD expression was significantly decreased（P<0.01）；MDA expression was significantly increased（P<0.01）；Nrf 2，HO-1 mRNA and protein expression were significantly decreased（P<0.01）. Compared with the model group，24 h UTP，serum TG，TC were significantly decreased（P<0.05 or P<0.01），TP，ALB（P<0.05）；renal pathological damage was significantly improved；SOD（P<0.01），MDA（P<0.01）；Nrf 2，HO-1 mRNA and protein（P<0.05 or P<0.01）. [Conclusion] Salvia miltiorrhiza polyphenols may regulate the Nrf 2/HO-1 signaling pathway，and thus protect the kidney and delay the disease progression.