基于线粒体氧化损伤探究埃克替尼对肾纤维化的保护作用

Investigating the protective effect of Icotinib on renal fibrosis based on mitochondrial oxidative damage

关键词:  埃克替尼  肾纤维化  上皮-间质转化  氧化应激  线粒体损伤

Key words:  Icotinib  renal fibrosis  epithelial-mesenchymal transition  oxidative stress  mitochondrial damage

摘要:

探究埃克替尼（ICO）对肾纤维化（RF）的作用及其机制。方法 采用结扎左输尿管建立小鼠单侧输尿管梗阻（UUO）模型。将小鼠随机分为：假手术（Sham）组、UUO组及ICO 20和40 mg·kg-1剂量组，每组8只。采用血尿素氮（BUN）和血肌酐（Scr）及肾损伤分子1（KIM-1）评价肾功能。HE及Masson染色评价肾纤维化程度。免疫组化检测肾组织表皮生长因子受体（EGFR）磷酸化水平。投射电镜观察肾组织线粒体超微结构。体外培养肾小管上皮细胞，细胞实验设Control组、TGF-β1组及TGF-β1+ ICO（5、10、20 μmol·L-1）三个剂量组。JC-1荧光探针检测细胞线粒体膜电位。DCFH-DA荧光探针检测细胞ROS水平。免疫荧光和（或）western blotting检测相关蛋白的表达情况。结果 体内实验，ICO抑制了UUO诱导的小鼠肾功能损伤（BUN、Scr和KIM-1水平降低），减轻了肾纤维化（胶原沉积及I、III型胶原表达降低），逆转了上皮-间质转化（EMT）（E-cad表达显著降低而α-SMA和FN表达显著升高），抑制了EGFR的磷酸化及Snail的表达，减轻了线粒体损伤（SIRT3的表达明显升高而UCP2的表达明显下调）。体外实验，ICO显著抑制了TGF-β1诱导的EGFR的磷酸化及Snail的表达、逆转了肾小管上皮细胞EMT，降低了细胞ROS和MDA水平，减轻了线粒体损伤（线粒体膜电位升高及SIRT3的表达明显升高而UCP2的表达明显下调）。结论 本研究表明埃克替尼具有缓解肾纤维化作用，机制可能与其抑制氧化应激介导的线粒体损伤，进而抑制肾小管上皮细胞EMT有关。

Abstract:

OBJECTIVE To explore the effect and mechanism of Icotinib (ICO) on renal fibrosis (RF). METHODS A unilateral ureteral obstruction (UUO)model was induced by ligating the left ureter of mice. The mice were randomly divided into: sham group, UUO group, ICO (20 mg· kg-1)-treated UUO group and ICO (40 mg· kg-1)-treated UUO group, eight mice in each group. The renal function was evaluated by blood urea nitrogen (BUN), serum creatinine (Scr) and kidney injury molecule 1(KIM-1). The degree of renal ?brosis was assessed by HE and Masson staining. The level of epidermal growth factor receptor (EGFR) phosphorylation in kidney tissues was detected by immunohistochemistry. The ultrastructure of renal mitochondria was observed by transmission electron microscopy. In vitro, the renal tubular epithelial cells were cultured and divided into control group, TGF-β1group, TGF-β1combined with ICO (5, 10, 20 μmol·L-1) groups. Detection of mitochondrial membrane potential by JC-1 fluorescent probe. DCFH-DA fluorescent probe was used to detect ROS levels in cells. Immunofluorescence and (or) western blotting were used to detect the expression of proteins. RESULTS In vivo, ICO inhibited UUO-induced renal damage(the levels of BUN, Scr and KIM-1 were reduced) , alleviated renal fibrosis (collagen deposition and type I and III collagen expression were decreased), reversed epithelial-mesenchymal transition (EMT), inhibited EGFR phosphorylation and Snail expression, and alleviated mitochondrial damage (the expression of SIRT3 was significantly up-regulated while the expression of UCP2 was significantly down-regulated) in mice. In vitro, ICO significantly inhibited TGF-β1-induced EGFR phosphorylation and Snail expression, repressed EMT, decreased the levels of ROS and MDA, and alleviated mitochondrial damage (mitochondrial membrane potential was increased and SIRT3 expression was significantly increased while UCP2 expression was significantly decreased) in renal tubular epithelial cells. CONCLUSION This study shows that Icotinib can alleviate renal fibrosis，and anti-?brotic mechanism is related to inhibit oxidative stress-mediated mitochondrial damage and thus the reversal of renal tubular epithelial EMT. and reverse EMT of renal tubular epithelial cells.