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  

Fasudil blocks muscle atrophy and C2C12 myoblasts respiratory dysfunction triggered by ROCK1

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作  者:郭织运 张杰[1] 董睿[1] 许静[1] 

机构地区:[1]第二军医大学长海医院肾内科,上海200433

出  处:《第二军医大学学报》2017年第6期734-738,共5页Academic Journal of Second Military Medical University

基  金:国家自然科学基金(81470970)~~

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(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。Objective To confirm whether fasudil can block C2C12 myoblasts respiration dysfunction triggered by Rho-associated coiled-coil containing protein kinase 1(ROCK1),and whether it can block the occurrence of muscle atrophy.Methods C2C12 myoblasts were cultured in vitro,and 2% horse serum was used to induce cell differentiation and maturation.The obtained mature muscle tubule cells were divided into four groups according to the different stimuli:Ad-GFP group,only transfected GFP-adenovirus vector(Adv)in C2C12 myoblasts;Ad-ROCK1 group,transfected ROCK1-Adv in C2C12 myoblasts to induce ROCK1overexpression;Ad-GFPF group,transfected GFP-Adv and given10μmol/L fasudil in C2C12 myoblasts;and Ad-ROCK1 F group,transfected ROCK1-Adv and given 10μmol/L fasudil in C2C12 myoblasts.The oxygen consumption rate(OCR)and extracelluar acidification rate(ECAR)of C2C12 myoblasts under different stimulation conditions were evaluated by cell energy metabolism analyzer(Seahorse),so as to determine the effect of ROCK1 overexpression and fasudil stimulation on the respiratory function of C2C12 myoblasts.Mitochondrial fission was measured by MitoTracker○Rred fluorescent probes.The expressions of ROCK1,mitochondrialrelated protein 1(Drp1)and phosphorylated p-Drp1,E3 ubiquitin ligase muscle RING finger-1protein(MuRF1)and muscle atrophy F-box(MAFbx,Atrogin 1)was measured by Western blotting analysis.Results Seahorse analysis showed that the OCR,ECAR,basal respiration,maximal respiration and respiration required for coupling ATP of C2C12 myoblasts in the Ad-ROCK1 group were significantly increased compared with those in the Ad-GFP group(P〈0.01);Meanwhile,MitoTracker○Rstaining showed that the mitochondrial fission was increased and the mitochondrial size frequency distribution shifted left in the Ad-ROCK1 group.After exposed to fasudil,the OCR and EACR of C2C12 myoblasts in the Ad-ROCK1 Fgroup were significantly decreased versus the Ad-ROCK1 group,and the basal respiration and maximal respiration were s

关 键 词:肾疾病 萎缩性肌疾病 细胞呼吸 RHO相关激酶类 法舒地尔 

分 类 号:R692[医药卫生—泌尿科学]

 

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