Wnt/β-catenin信号通路在肾缺血再灌注大鼠中的表达及ICG-001阻断对慢性肾间质纤维化的作用

Wnt/β-catenin signaling pathway in rats with ischemia reperfusion injury and its effect on renal tubulointerstitial fibrosis of blocking β-catenin by ICG-001

目的观察Wnt/β-catenin信号通路在肾缺血再灌注大鼠模型中不同时间的表达及ICG-001阻断Wnt/β-catenin信号通路对慢性肾间质纤维化的作用。方法将SD大鼠分为4组,假手术组(Sham)、IRI 7 d组、IRI 14 d组及ICG+IRI组。假手术组不夹闭左侧肾动脉;IRI 7 d组、IRI 14 d组及ICG+IRI组用无创血管夹夹闭左侧肾动脉,35 min后去除血管夹;ICG+IRI组于术后第2天开始腹腔注射ICG-001。检测4组大鼠肾功能,Western blot检测肾组织β-链蛋白(β-catenin)、纤维连接蛋白(Fibronectin)、α-平滑肌肌动蛋白(α-SMA)的蛋白表达水平,免疫组化观察β-catenin表达部位,生化染色测胶原含量,Masson染色观察病理改变。结果与假手术组相比,IRI 7 d组Fibronectin、α-SMA、胶原含量无明显增高,病理无明显肾小管间质纤维化;而IRI 14 d组Fibronectin、α-SMA的蛋白表达水平则明显增高,胶原含量增多,肾功能减退,病理显示肾小管间质纤维化。与假手术组相比,IRI 7 d组β-catenin表达增高,IRI 14 d组较IRI 7 d组增高更显著。与IRI 14 d组相比,ICG+IRI组β-catenin、Fibronectin、α-SMA的蛋白表达水平明显降低,胶原含量减少,肾功能好转,病理显示肾小管间质纤维化程度减轻。结论 Wnt/β-catenin信号通路在IRI术后7 d已激活,早于肾间质纤维化出现;ICG-001阻断Wnt/β-catenin信号通路可减轻肾缺血再灌注所致慢性肾间质纤维化。

Objective To investigate the activation of Wnt/β-catenin signaling in rats with ischemia reper- fusion injury and its effect on renal tubulointerstitial fibrosis after blocking 15-catenin by ICG-001. Method SD rats were divided into four groups ： sham group, IRI 7 d group, IRI 14 d group and ICG＋ IRI group. Ischemia reperfusion injury was achieved in rats of IRI 7 d group, IRI 14 d group and ICG ＋ IRI group. The other side kid- neys of rats in IRI 7 d group and IRI 14 d group were removed on the 6th and 13th day, and the rats were sacrified on the Ts and 14th day, respectively. The rats in ICG＋IRI group were subjected to daily intraperitoneal injections of ICG-001 from the second day after IRI. The contralateral kidneys were removed on the 13th day and were sacrified on the 14th day. The kidneys and blood were collected. Renal pathological change was observed by Masson staining assay. The location and expression of/β-catenin was detected by immunohistochemistry and Westem blot assay, respectively. The protein expression of fibronectin, α-SMA （a-smooth muscle actin） was assessed by Western blot assay. The level of collagen was checked by sirius red/fast green collagen staining. Results Compared with rats in sham group and IRI 7 d group, the protein expression of Fibronectin, a-SMA and the level of collagen in- creased significantly in rats in IRI 14 d group. Tubuloinsterstitial fibrosis was observed in rats in IRI 14 d group. The expression of β-catenin increased in rats in IRI 7 d group and increased more obviously in IRI 14 d group. Compared with rats in IRI 14 d group, the protein expressin of β-catenin, Fibronectin, α-SMA and the level of collagen decreased significantly in rats in ICG＋IRI group. Tubuloinsterstitial fibrosis was alleviated in ICG＋IRI group. Conclusion Wnt/β-catenin signaling was activated early and may play an important role in the pathologic process of renal tubuloinsterstitial fibrosis induced by ischemia reperfusion. Renal tubuloinsterstitial fibrosis could be alleviated by blocking Wnt/β-catenin signaling by using the small molecule inhibitor ICG-001.