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目的明确法舒地尔(fasudil)能否阻断Rho相关卷曲螺旋形成蛋白激酶1(ROCK1)激活导致的C2C12成肌细胞呼吸功能异常介导的肌肉萎缩。方法体外培养C2C12成肌细胞,给予2%的马血清诱导分化为成熟肌小管细胞。然后根据给予不同处理将其分成4组:Ad-GFP组,仅感染GFP腺病毒载体;Ad-ROCK1组,感染ROCK1腺病毒载体诱导细胞过表达ROCK1;Ad-GFPF组,感染GFP腺病毒载体,并给予10μmol/L法舒地尔刺激;Ad-ROCK1F组,感染ROCK1腺病毒载体诱导细胞过表达ROCK1,并给予10μmol/L法舒地尔刺激。通过Seahorse能量代谢分析仪评估不同刺激条件下C2C12成肌细胞的细胞耗氧率(OCR)及胞外酸化率(ECAR),以明确ROCK1过表达和法舒地尔刺激对C2C12成肌细胞呼吸功能的影响。采用MitoTracker○R红色荧光探针测定线粒体裂变情况。用蛋白质印迹法检测ROCK1、线粒体动力相关蛋白1(Drp1)及其磷酸化蛋白p-Drp1、肌肉萎缩相关蛋白E3泛素连接酶肌肉环指蛋白1(MuRF1)和肌肉萎缩F-box(MAFbx,又称Atrogin1)的表达。结果 Seahorse分析结果显示,与Ad-GFP组比较,AdROCK1组C2C12成肌细胞OCR及ECAR明显增加,细胞的基础呼吸、最大呼吸及偶联ATP产生所需的呼吸增加,差异均有统计学意义(P<0.01);MitoTracker○R荧光探针结果显示Ad-ROCK1组细胞线粒体裂变增加,线粒体大小频数分布左移。Ad-ROCK1F组C2C12成肌细胞OCR和ECAR较Ad-ROCK1组明显减少(P<0.05),基础呼吸和最大呼吸也降低(P<0.05)。Ad-ROCK1F组p-Drp1/Drp1比例、ROCK1、MuRF1和Atrogin1的表达均较Ad-ROCK1组减少(P<0.05)。结论法舒地尔作为ROCK1的抑制剂,可阻断体外培养C2C12成肌细胞ROCK1过表达导致的细胞呼吸异常,并减少线粒体动力相关蛋白的活性和肌肉萎缩相关蛋白的表达。
Objectives To determine whether Fasudil blocks the muscular atrophy mediated by Rho-associated activation of ROCK1-induced C2C12 myoblast dysfunction. Methods C2C12 myoblasts were cultured in vitro and were induced to differentiate into mature myotubes by 2% horse serum. The cells were divided into 4 groups according to different treatment: Ad-GFP group, only GFP-infected adenovirus vector; Ad-ROCK1 group, infected with ROCK1 adenovirus vector induced cell overexpression of ROCK1; Ad-GFPF group, infected GFP adenovirus vector, The cells were stimulated with 10μmol / L Fasudil. Ad-ROCK1F cells were infected with ROCK1 adenovirus vector to induce cell overexpression of ROCK1 and stimulated with 10μmol / L fasudil. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of C2C12 myoblasts under different stimulation conditions were evaluated by Seahorse energy metabolism analyzer in order to confirm that ROCK1 overexpression and fasudil stimulate the respiratory function of C2C12 myoblasts Impact. Mitochondrial fission was measured using a MitoTracker R red fluorescent probe. Western blotting was used to detect the expression of ROCK1, mitochondrial DRP1 and its phosphorylated protein p-Drp1, muscular atrophy-associated protein E3 ubiquitin ligase muscle ring finger protein 1 (MuRF1) and muscle atrophy F-box (MAFbx, Also known as Atrogin1) expression. Results Seahorse analysis showed that compared with Ad-GFP group, the OCR and ECAR of C2C12 myoblasts in AdROCK1 group were significantly increased, and the basal respiration, maximal respiration and respiration required for coupling ATP were increased in AdROCK1 group (P <0.05) P <0.01). The results of MitoTracker ○ R fluorescent probe showed that mitochondrial fission increased and mitochondrial size distribution shifted to the left in Ad-ROCK1 group. OCR and ECAR of C2C12 myoblasts in Ad-ROCK1F group were significantly lower than those in Ad-ROCK1 group (P <0.05), and basal respiration and maximal respiration were also decreased (P <0.05). The expressions of p-Drp1 / Drp1, ROCK1, MuRF1 and Atrogin1 were decreased in Ad-ROCK1F group compared with Ad-ROCK1 group (P <0.05). Conclusion Fasudil, an inhibitor of ROCK1, blocks the cellular resorption induced by ROCK1 overexpression in cultured C2C12 myoblasts and decreases the expression of mitochondria-related protein and muscle-associated protein.