白藜芦醇抑制肾纤维化减轻单侧输尿管梗阻诱导的肾损伤

Resveratrol Inhibits Renal Fibrosis and Reduces Kidney Damage Caused by Unilateral Ureteral Obstruction

目的研究白藜芦醇抑制肾纤维化对单侧输尿管梗阻诱导的肾损伤的减轻作用及相关机制。方法以Sprague-Dawley(SD)大鼠和成纤维细胞NRK-49F为实验对象,分别进行体内研究和体外验证,体内:SD大鼠行单侧输尿管梗阻诱导建立TIF大鼠模型,将大鼠随机分为4组(假手术+空白组、假手术+白藜芦醇组、模型+空白组和模型+白藜芦醇组),每组12只大鼠。体外:将NRK-49F细胞用重组TGF-β1(5 ng/mL)或白藜芦醇(10和100μmol/L)处理后随机分为4组(对照组、TGF-β1组、TGF-β1+白藜芦醇低剂量组和TGF-β1+白藜芦醇高剂量组)。Masson’s染色检测肾脏中总胶原蛋白沉积;免疫组织化学染色检测组织中E-cadherin、GFAP和Ki67阳性细胞的表达;免疫荧光染色检测细胞中Ki67阳性细胞的表达;蛋白质印迹检测组织中E-cadherin、Vimentin、N-cadherin、Rac1、c-Myc、Bcl-2、Cyclin D1、α-SMA、Ⅰ型胶原和Ⅲ型胶原的表达。结果与对照组比较,白藜芦醇可以抑制大鼠体内单侧输尿管梗阻(unilateral ureteral obstruction,UUO)肾脏中总胶原蛋白的过度沉积,下调α-SMA阳性成肌纤维细胞减少波形蛋白、Ⅰ型和Ⅲ型胶原,抑制Rac1、GFAP和N-cadherin的上调以及E-cadherin的下调;与假手术组比较,白藜芦醇可显著降低UUO大鼠中Ki67阳性细胞比率,并抑制c-Myc,Bcl-2和cyclin D1的表达;白藜芦醇抑制体内增殖相关途径Wnt/β-catenin的激活;体外实验表明白藜芦醇治疗可减少成纤维细胞和TECs的增殖,从而抑制TGF-β1介导的表型转化和ECM积累。结论白藜芦醇通过抑制Wnt/β-catenin通路的活性,抑制成肌纤维细胞的积累,并减轻了肾小管间质纤维化。

Objective To investigate the effect of resveratrol on renal fibrosis and kidney damage induced by unilateral ureteral obstruction and its related mechanism.Methods Sprague-dawley(SD)rats and NRK-49F fibroblasts were used as experimental subjects for in vivo study and in vitro validation.In vivo,TIF rat model was establish by surgery to induce the unilateral ureteral obstruction in SD rats,and the rats were then randomly divided into 4 groups:sham operation+blank group,sham operation+resveratrol group,model+blank group and model+resveratrol group,12 rats in each group.In vitro:NRK-49F cells were treated with recombinant TGF-β1(5 ng/mL)or resveratrol(10 and 100μmol/L)and randomly divided into 4 groups:control group,TGF-β1 group,TGF-β1+resveratrol low-dose group and TGF-β+resveratrol high-dose group.Masson’s staining was used to detect total collagen deposition in the kidney.Immunohistochemical staining was used to detect the expression of E-cadherin,GFAP and Ki67 in the tissues.Immunofluorescence staining was used to detect the expression of Ki67 in cells.Western blotting was used to detect the expression level of Vimentin,N-cadherin,Racl,c-Myc,Bcl-2,Cyclin D1,α-SMA,type Ⅰ collagen and type Ⅲ collagen,E-cadherin.Results Resveratrol could inhibit the excessive deposition of total collagen in kidneys of UUO rats,down-regulateα-SMA-positive myofibroblasts,reduce the expression of vimentin,type Ⅰ and type Ⅲ collagen,and inhibit the up-regulation of Rac1,GFAP and down-regulation of N-cadherin.Resveratrol could significantly reduce the ratio of Ki67-positive cells in UUO rats and inhibit the expression level of c-Myc,Bcl-2 and cyclin D1.Resveratrol inhibited the activation of proliferation-related pathway Wnt/β-catenin.The in vitro experimentsshowed that resveratrol treatment could reduce the proliferation of fibroblasts and TECs,thereby inhibiting TGF-β1-mediated phenotypic transformation and ECM accumulation.Conclusion Resveratrol inhibits the activity of Wnt/β-catenin pathway and the accumulation of myofibroblasts,and reduces renal tubulointerstitial fibrosis.