线粒体超离子与血管紧张素Ⅱ介导肾间质细胞外基质纤维化的研究

Mitochondria superoxides mediates angiontension Ⅱ-induced synthesis of extracelluar matrix

目的探索线粒体超氧离子(superoxides,O^-_2)是否参与血管紧张素Ⅱ(AngⅡ)介导的细胞外基质纤维化。方法培养大鼠肾脏成纤维细胞(NRK-49F),AngⅡ刺激24 h后进行活细胞线粒体荧光染色(mitosox)以检测细胞内的线粒体O^-_2;采用酶标仪和流式细胞术检测线粒体超氧离子含量和分布;利用Real-time PCR、Western blot及免疫荧光检测AngⅡ对细胞外基质胶原蛋白-Ⅰ(collagen-Ⅰ,Col-Ⅰ)、胶原蛋白-Ⅲ(collagen-Ⅲ,Col-Ⅲ)和纤维连接蛋白(fibronectin,FN)表达的影响;并进一步检测NADPH氧化酶抑制剂罗布麻宁(apocynin,APO)对下游超氧离子合成以及ColⅠ、ColⅢ和FN表达的影响。结果 AngⅡ明显促进大鼠肾脏成纤维细胞O^-_2的生成以及细胞外基质Col-Ⅰ、Col-Ⅲ和FN的表达。Apo可显著抑制AngⅡ所引起的肾成纤维细胞中O^-_2表达,而AngⅡ诱导的Col-Ⅰ、Col-Ⅲ和FN的合成作用亦被APO阻断。结论线粒体O^-_2介导了AngⅡ诱导的肾脏成纤维细胞的细胞外基质蛋白的合成,采用APO抑制O^-_2的表达,可使纤维化程度减轻。

Objective To investigate the effects of mitochondria superoxides on synthesis of extracelluar matrix induced by angiontension Ⅱ（Ang Ⅱ） in NRK-49 F cells.Methods Cultured rat renal fibroblast NRK-49 F cells were stimulated with Ang Ⅱ and / or apocynin（APO） for 24 h,then stained with mitochondrial mitosox fluorescence method,the contents and distribution of mitochondrial O2^- was detected by enzyme-labeling method and flowcytometry.The mRNA and protein levels of collagen Ⅰ,Ⅲ and fibronectin were detected by Real time PCR,immunofluorescence and Western blot,respectively.Results Compared with the control group,Ang Ⅱ increased the level of O2^- in NRK-49 F mitochondria.In Ang Ⅱ ＋ APO group O2^- significantly decreased compared with Ang Ⅱgroup,which was consistent with the results of flowcytometry and enzyme-labeling measurement.Compared with the control group,mRNA level of collagen Ⅰ,Ⅲ and fibronectin in Ang Ⅱ group was increased significantly;while that in Ang Ⅱ ＋ APO group decreased compared with Ang Ⅱ group.The collagen Ⅰ,Ⅲ,and fibronectin protein expression in Ang Ⅱ group was significantly increased,compared with control group;while that in Ang Ⅱ ＋ APO group was decreased compared with Ang Ⅱgroup.Conclusion M itochondria superoxide may mediate the Ang Ⅱ-induced matrix protein synthesis in rat renal fibroblast NRK-49 F cells.