基于Nrf2/HO-1通路探讨金雀异黄酮对糖尿病肾病大鼠肾损伤的影响及机制

Effect and mechanism of genistein on renal injury in diabetic nephropathy rats via Nrf2/HO-1 pathway

目的探讨金雀异黄酮对糖尿病肾病大鼠肾组织损伤的影响及其可能的分子机制。方法取雄性SD大鼠55只,随机选择10只作为正常组,其余大鼠采用一次性腹腔注射链脲佐菌素60 mg/kg的方法复制糖尿病肾病模型。将40只成模大鼠随机分为模型组、金雀异黄酮组、鸦胆子苦醇组和金雀异黄酮+鸦胆子苦醇组,每组10只。金雀异黄酮组给予30 mg/kg金雀异黄酮灌胃,鸦胆子苦醇组给予鸦胆子苦醇2 mg/kg灌胃,金雀异黄酮+鸦胆子苦醇组给予30 mg/kg金雀异黄酮和鸦胆子苦醇2 mg/kg灌胃,均1次/d,连续灌胃6周。检测各组大鼠空腹血糖(FPG)水平及肾功能指标[血清肌酐(SCr)、尿素氮(BUN)和24 h尿微量白蛋白(24h U-mAlb)],计算肾脏指数,HE染色、PAS染色、Masson染色观察肾组织形态,透射电子显微镜观察肾细胞超微结构,分光光度法检测肾组织中总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量,ELISA法检测肾组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-8(IL-8)含量,Western blot法检测肾组织中E2相关因子2(Nrf2)、p-Nrf2、血红素加氧酶1(HO-1)、醌氧化还原酶1(NQO1)、总核因子κB(NF-κB)、胞核NF-κB蛋白表达情况。结果与正常组比较,模型组大鼠FPG、SCr、BUN、24hU-mAlb水平和肾脏指数均明显升高(P均<0.05);肾小球肥大、硬化,炎性浸润,胶原沉积,肾小球细胞可见足突肿胀或消失、基底膜弥漫性增厚、线粒体嵴断裂或消失;肾组织中T-SOD、CAT活性均明显降低(P均<0.05),MDA、TNF-α、IL-1β、IL-8含量均明显升高(P均<0.05);肾组织中p-Nrf2、HO-1、NQO1蛋白相对表达量及p-Nrf2/Nrf2比值均明显降低(P均<0.05),胞核NF-κB蛋白相对表达量及胞核NF-κB/总NF-κB比值均明显升高(P均<0.05)。与模型组比较,金雀异黄酮组和金雀异黄酮+鸦胆子苦醇组大鼠FPG、SCr、BUN、24hU-mAlb水平和肾脏指数均明显降低(P均<0.05);肾组织病理改变和肾小球细胞超微结构病变均明显减轻;肾组织中T-SOD、CAT活性均明显升高(P均<0.05),MDA、TNF-α、IL-1β、IL-8含量均明显降低(P均<0.05);肾组织中p-Nrf2、HO-1、NQO1蛋白相对表达量及p-Nrf2/Nrf2比值均明显升高(P均<0.05),胞核NF-κB蛋白相对表达量及胞核NF-κB/总NF-κB比值均明显降低(P均<0.05)。鸦胆子苦醇组上述指标变化趋势与模型组相同且更加严重,鸦胆子苦醇可明显逆转金雀异黄酮对上述指标的调控作用。结论金雀异黄酮能够抑制糖尿病肾病大鼠肾组织氧化应激和炎症反应,减轻肾组织病变和肾细胞超微结构病变,其机制可能与促进Nrf2/HO-1通路活化、抑制NF-κB核转位有关。

Objective It is to explore the effects of genistein on renal tissue injury in rats with diabetic nephropathy and its possible molecular mechanisms.Methods Fifty-five male SD rats were taken,10 of which were randomly selected as the normal group,and the rest were used to replicate the diabetic nephropathy models by one-time intraperitoneal injection of streptozotocin 60 mg/kg.The 40 modeled rats were randomly divided into model group,genistein group,brusatol group and genistein+brusatol group,with 10 rats in each group.The rats in the genistein group were gavaged with 30 mg/kg of genistein,the rats in the brusatol group were gavaged with 2 mg/kg of brusatol,and the rats in the genistein+brusatol group were gavaged with 30 mg/kg of genistein and 2 mg/kg of genistein+brusatol,all of which were administered once daily,continuously treated for 6 weeks.The levels of fasting plasma glucose(FPG)and renal function indexes[serum creatinine(SCr),urea nitrogen(BUN)and 24 h urinary microalbumin(24hU-mAlb)]of rats in each group were detected,and their renal index was calculated,and the morphology of renal tissues was observed by HE staining,PAS staining,and Masson staining,and the ultrastructure of renal cells was observed by transmission electron microscope,the activities of total superoxide dismutase(T-SOD),catalase(CAT)and the contents of malondialdehyde(MDA)in renal tissues were detected by spectrophotometry,the contents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-8(IL-8)in renal tissues were detected by ELISA,and the expressions of E2-related factor 2(Nrf2),p-Nrf2,heme oxygenase 1(HO-1),quinone oxidoreductase 1(NQO1),total nuclear factorκB(NF-κB),and cytosolic NF-κB proteins in renal tissues were detected by Western blot.Results Compared with the normal group,the levels of FPG,SCr,BUN,24hU-mAlb and renal index of rats in the model group were significantly increased(all P<0.05);the renal tissue presented pathological changes such as glomerular hypertrophy,matrix hyperplasia,mesangial expansion,inflammatory infiltration,glomeruloscler-osis,collagen deposition;the lomerular cells presented ultrastructural changes such as feet process swelling or disappearance,diffuse thickening of basement membrane,mitochondrial swelling,ridge rupture or disappearance;the activities of T-SOD,CAT in renal tissue were significantly decreased(all P<0.05),the contents of MDA,TNF-α,IL-1β,IL-8 were significantly increased(all P<0.05);the relative expressions of p-Nrf2,HO-1,and NQO1 proteins and the ratio of p-Nrf2/Nrf2 in renal tissues were significantly reduced(all P<0.05),and the relative expression of cytosolic NF-κB proteins and the ratio of cytosolic NF-κB/total NF-κB were significantly increased(all P<0.05).Compared with the model group,the levels of FPG,SCr,BUN,24hU-mAlb and renal index of rats in the genistein group and genistein+brusatol group were significantly reduced(all P<0.05);the renal histopathological changes and glomerular cell ultrastructural lesions were significantly improved;the activities of T-SOD and CAT in the renal tissues were significantly elevated(all P<0.05),and the contents of MDA,TNF-α,IL-1βand IL-8 were significantly reduced(all P<0.05);the relative expressions of p-Nrf2,HO-1,and NQO1 proteins in renal tissues and the p-Nrf2/Nrf2 ratio were significantly increased(all P<0.05),the relative expressions of cytosolic NF-κB proteins and the cytosolic NF-κB/total NF-κB ratio were significantly reduced(all P<0.05).The trend of changes in the above indexes in the brusatol group was the same as that in the model group and was more serious,and brusatol could significantly reverse the regulatory effect of genistein on the above indexes.Conclusion Genistein can inhibit oxidative stress and inflammation in renal tissues of rats with diabetic nephropathy,and attenuate renal tissue lesions and ultrastructural lesions of renal cells,and its mechanism may be related to the promotion of the activation of the Nrf2/HO-1 pathway and the inhibition of NF-κB nuclear translocation.