罗沙司他抑制HIF-1α/Notch-1信号通路缓解单侧输尿管梗阻大鼠肾间质纤维化的研究

Study on Roxadustat Attenuates Renal Interstitial Fibrosis Through Inhibiting HIF-1α/Notch-1 Signaling Pathway in Rats of Unilateral Ureteral Obstruction

关键词:  罗沙司他  肾间质纤维化  低氧诱导因子1α  Notch-1  上皮-间质转分化

Key words:  roxadustat  renal interstitial fibrosis  hypoxia-inducible factor 1α  Notch-1  epithelial-mesenchymal transition

摘要:

目的 研究罗沙司他(roxadustat,ROX)改善单侧输尿管梗阻(unilateral ureteral obstruction,UUO)大鼠肾间质纤维化(renal interstitial fibrosis,RIF)的机制。方法 48只♂SD大鼠随机分为假手术组、UUO组、UUO+ROX (25,50 mg·kg^-1)剂量组,每组12只。左侧输尿管结扎制备RIF大鼠模型,造模后第3天开始每天灌胃给药,连续给药21 d。测定大鼠血肌酐(Scr)、血尿素氮(BUN)水平;HE染色、Masson染色观察肾组织病理变化及胶原沉积情况;免疫组化法检测肾组织转化生长因子-β1(TGF-β1)表达情况。体外培养近曲小管上皮细胞,分为对照组、TGF-β1(5 ng·mL^-1)组、TGF-β1+二甲基亚砜(DMSO)组和TGF-β1+ROX (10,20,40 μmol·L^-1)组,细胞先用ROX或DMSO预处理2 h,再用TGF-β1(5 ng·mL-1)处理48 h。Western blotting检测肾组织和细胞内I型胶原(collagen Ⅰ)、Ⅲ型胶原(collagen Ⅲ)、E-钙黏蛋白(E-Cadherin)、纤维连接蛋白(Fibronectin)、α-平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)、低氧诱导因子1α(HIF-1α)、Notch-1、Notch-1胞内域(NICD)和Hes1的表达。结果 动物实验结果显示,与UUO组相比,ROX给药组大鼠Scr和BUN水平明显降低,肾组织胶原沉积明显减少,collagen Ⅰ和collagen Ⅲ的蛋白表达明显降低,RIF程度明显减轻;肾组织上皮细胞标志物E-Cadherin的表达明显升高而间质细胞标志物Fibronectin、α-SMA和Vimentin明显降低(P<0.05或P<0.01);同时肾组织HIF-1α、Notch-1、NICD和Hes1的表达明显下调(P<0.05或P<0.01)。细胞实验结果表明,与TGF-β1组相比,不同剂量ROX均能明显降低HIF-1α、Notch-1、NICD和Hes1的表达;显著上调E-Cadherin的表达,明显下调Fibronectin、α-SMA和Vimentin的表达(P<0.05或P<0.01)。结论 本研究结果提示ROX可减轻UUO大鼠RIF,其作用机制可能与抑制HIF-1α/Notch-1信号通路、逆转肾小管上皮细胞上皮-间质转分化有关。

Abstract:

OBJECTIVE To study the mechanism of roxadustat(ROX) in improving renal interstitial fibrosis(RIF) in rats with unilateral ureteral obstruction(UUO). METHODS Forty-eight ♂ rats were randomly divided into sham operation group, UUO group, UUO+ROX(25, 50 mg·kg^–1) group, twelve rats in each group. The rat model of RIF was established by ligation of the left ureter. The rats were given intragastric administration for 21 d from the third day after modeling. The serum creatinine(Scr) and blood urea nitrogen(BUN) was measured. HE staining and Masson staining were used to observe the pathological changes of renal tissue and collagen deposition. The expression of TGF-β1 in renal tissue detected by immunohistochemistry. The human proximal tubular epithelial cells were cultured in vitro, divided into control group, TGF-β1 (5 ng·mL^–1) group, TGF-β1+DMSO group and TGF-β1+ROX(10, 20, 40 μmol·L^–1) group. The cells were pre-treated with DMSO or ROX for 2 h, and then subjected to TGF-β1(5 ng·mL^–1) for 48 h. The expression of collagen Ⅰ, collagen Ⅲ, E-Cadherin, Fibronectin, α-smooth muscle actin(α-SMA), Vimentin, hypoxia-inducible factor 1α(HIF-1α), Notch-1, Notch-1 intracellular domain(NICD) and Hes1 were assessed by Western blotting. RESULTS The results of animal experiments showed that, compared with UUO group, ROX significantly reduced the levels of BUN and Scr, obviously attenuated collagen deposition and expression of collagen Ⅰ and collagen Ⅲ, and reduced the degree of RIF. The expression of epithelial cell marker E-Cadherin in renal tissue increased significantly, while interstitial cell marker Fibronectin, α-SMA and Vimentin decreased significantly(P<0.05 or P<0.01). At the same time, the expressions of HIF-1α, Notch-1, NICD and Hes1 in renal tissue were significantly down regulated(P<0.05 or P<0.01). The results of cell experiments showed that, compared with the TGF-β1 group, different doses of ROX could significantly reduce the expression of HIF-1α, Notch-1, NICD and Hes1 induced by TGF-β1, the expression of E-Cadherin was significantly up-regulated and the expression of Fibronectin, α-SMA and Vimentin was significantly down-regulated(P<0.05 or P<0.01). CONCLUSION These results suggest that ROX can alleviate UUO-induced RIF in rats, which may be involved in blocking HIF-1α/Notch-1 signaling pathway and reverse tubular epithelial cell epithelial-mesenchymal transition.