法舒地尔阻断ROCK1触发C2C12成肌细胞呼吸功能异常介导的肌肉萎缩

发布时间：2018-04-01 19:08

本文选题：肾疾病　切入点：萎缩性肌疾病　出处：《第二军医大学学报》2017年06期

【摘要】：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(P0.01);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(P0.05),基础呼吸和最大呼吸也降低(P0.05)。Ad-ROCK1F组p-Drp1/Drp1比例、ROCK1、MuRF1和Atrogin1的表达均较Ad-ROCK1组减少(P0.05)。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。
[Abstract]:Aim to determine whether fasudil can block the respiratory dysfunction of C2C12 myoblasts induced by the activation of Rho-associated crimp-forming protein kinase 1-ROCK1.Methods C2C12 myoblasts were cultured in vitro and differentiated into mature myotubule cells with 2% horse serum.Then they were divided into 4 groups according to the different treatments, only infected with GFP adenovirus vector Ad-ROCK1, infected with ROCK1 adenovirus vector ROCK1, infected with GFP adenovirus vector, and treated with 10 渭 mol/L fastidil to stimulate Ad-ROCK1F group.Adenovirus vector infected with ROCK1 induced overexpression of Rock1 and was stimulated by 10 渭 mol/L faridil.The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of C2C12 myoblasts under different stimulation conditions were evaluated by Seahorse Energy Metabolism Analyzer to determine the effects of ROCK1 overexpression and fasudil stimulation on the respiratory function of C2C12 myoblasts.Mitochondrial fission was determined by MitoTracker0R red fluorescence probe.The expressions of rock 1, mitochondrial motility associated protein 1 (mtp1) and its phosphorylated protein p-Drp1, muscle dystrophy associated protein E3 ubiquitin ligase muscle ring ligase protein 1 MuRF1) and muscle atrophy F-box MAFbx (also known as Atrogin1) were detected by Western blotting.Results compared with Ad-GFP group, OCR and ECAR of C2C12 myoblast in AdROCK1 group were significantly increased, and the basic respiration, maximum respiration and the respiration required by coupling ATP were increased in AdROCK1 group.The difference was statistically significant (P 0.01). The results of Mito Tracker0R fluorescence probe showed that mitochondrial fission increased in Ad-ROCK1 group.
【作者单位】：第二军医大学长海医院肾内科;
【基金】：国家自然科学基金(81470970)~~
【分类号】：R692

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