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

目的 探索线粒体超氧离子(superoxides, O-2)是否参与血管紧张素Ⅱ(Ang Ⅱ)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24h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 Ang Ⅱ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Ang Ⅱ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 angiontensionⅡ(Ang Ⅱ) in NRK-49F cells. Methods Cultured rat renal fibroblast NRK-49F cells were stimulated with AngⅡand/or apocynin (APO) for 24h, then stained with mitochondrial mitosox fluorescence method, the contents and distribution of mitochondrial O-2 was detected by enzyme-labeling method and flow cytometry. 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, AngⅡincreased the level of O-2 in NRK-49F mitochondria. In AngⅡ+APO group O-2 significantly decreased compared with AngⅡ group, which was consistent with the results of flow cytometry and enzyme-labeling measurement. Compared with the control group,mRNA level of collagen Ⅰ, Ⅲ and fibronectin in AngⅡ group was increased significantly; while that in AngⅡ + APO group decreased compared with AngⅡ group. The collagen Ⅰ, Ⅲ, and fibronectin protein expression in AngⅡ group was significantly increased, compared with control group; while that in AngⅡ+APO group was decreased compared with AngⅡ group. Conclusion Mitochondria superoxide may mediate the AngⅡ-induced matrix protein synthesis in rat renal fibroblast NRK-49F cells