骨骼肌心磷脂酰基转移酶1的运动强度变化特征及其对氧化应激和线粒体自噬的影响

Changes of Exercise Intensity of Acyl-Co A:Lysocardiolipin Acyltransferase-1 in Skeletal Muscle and Its Effects on Oxidative Stress and Mitochondrial Autophagy

目的:探讨骨骼肌心磷脂酰基转移酶1(acyl-CoA:lysocardiolipin acyltransferase-1,ALCAT1)表达变化的运动强度特征及不同强度运动对骨骼肌氧化应激和PINK1/Parkin介导的线粒体自噬的影响,为运动抑制ALCAT1重塑的强度筛选和耐力训练诱导线粒体自噬的发生机制提供实验依据。方法:雄性C57BL/6J野生型小鼠,随机分为安静对照组(C组)、60% VO2max运动强度组(SE组)、76% VO2max运动强度组(ME组)和85% VO2max运动强度组(HE组),每组8只。SE组以8 m/min、ME组以12 m/min、HE组以18 m/min进行跑台运动,坡度为0°,60 min/天,5天/周×4周。4周运动结束24 h后取材,分离腓肠肌,RT-qPCR检测骨骼肌ALCAT1 mRNA表达,Western blotting检测骨骼肌ALCAT1、MFN2、PINK1、Parkin、LC3、P62、SOD1和SOD2蛋白表达,酶活性试剂盒检测骨骼肌氧化应激指标,线粒体呼吸仪测定骨骼肌线粒体呼吸功能。结果:1)与C组比较,SE、ME和HE组小鼠骨骼肌ALCAT1 mRNA与蛋白表达和P62蛋白表达均显著降低,MDA含量、MFN2蛋白表达、LC3-Ⅱ/Ⅰ比值、ST3和RCR值均显著升高;2)与C组比较,SE和ME组小鼠骨骼肌PINK1、Parkin、SOD1、SOD2的蛋白表达以及T-AOC含量、T-SOD活性均显著增加,HE组不具有显著性;3)与ME组比较,HE组小鼠骨骼肌ALCAT1 mRNA与蛋白表达和MDA含量均显著升高,PINK1、Parkin、SOD1、SOD2蛋白表达和T-AOC含量、T-SOD活性以及ST3、RCR值均显著降低。结论:1)骨骼肌ALCAT1表达变化呈现明显的运动强度特征,4周不同强度运动均可抑制骨骼肌ALCAT1表达,提高线粒体呼吸功能,其中76% VO2max运动强度效果更显著;2)60% VO2max和76% VO2max运动强度可以激活骨骼肌氧化-抗氧化系统,提高PINK1/Parkin介导的线粒体自噬,85% VO2max运动强度可显著增加骨骼肌氧化应激水平,但对骨骼肌抗氧化能力和PINK1/Parkin介导的线粒体自噬无显著影响。

Objective:To investigate the changes of acyl-CoA:lysocardiolipin acyltransferase-1(ALCAT1)expression in skeletal muscle and the effects of different intensities of exercise on oxidative stress and PINK1/Parkin-mediated mitochondrial autophagy in skeletal muscle,and to provide experimental basis for the intensity selection of exercise inhibiting ALCAT1 remodeling and the mechanism of mitochondrial autophagy induced by endurance training.Methods:Male C57BL/6J wild-type mice were randomly divided into quiet control group(C),60%V.O2max exercise intensity group(SE),76% VO2max exercise intensity group(ME)and 85% VO2max exercise intensity group(HE),with 8 mice in each group.Treadmill exercise was performed at 8m/min in the SE group,12 m/min in the ME group,and 18 m/min in the HE group,slope of 0°,60 min/d,5 d/wk×4wk.The gastrocnemius muscle was isolated 24 hours after 4 weeks of exercise.The mRNA expression of skeletal muscle ALCAT1 was detected by RT-qPCR.The protein expression of skeletal muscle ALCAT1,MFN2,PINK1,Parkin,LC3,P62,SOD1 and SOD2 was detected by Western blotting.The oxidative stress index of skeletal muscle was detected by enzyme activity kit.Mitochondrial respiratory function of skeletal muscle was measured by mitochondrial respiration apparatus.Results:1)Compared with group C,skeletal muscle ALCAT1 mRNA and protein expression and P62 protein expression were significantly decreased in SE,ME and HE groups,while MDA content,MFN2 protein expression,LC3-Ⅱ/Ⅰratio,ST3 and RCR value were significantly increased;2)compared with group C,protein expressions of PINK1,Parkin,SOD1 and SOD2 and T-AOC content and T-SOD activity were significantly increased in SE and ME groups,but not in HE group;3)compared with ME group,ALCAT1 mRNA and protein expression and MDA content in skeletal muscle of HE group were significantly increased.PINK1,Parkin,SOD1,SOD2 protein expression and T-AOC content,TSOD activity and ST3,RCR value were significantly decreased.Conclusion:1)The expression of ALCAT1 in skeletal muscle showed obvious characteristics of exercise intensity.Four weeks of exercise with different intensity could inhibit the expression of ALCAT1 in skeletal muscle and improve mitochondrial respiratory function.76% VO2max exercise intensity had more significant effect;2)60% VO2max and 76% VO2max exercise can activate skeletal muscle oxidation-antioxidant system and improve PINK1/Parkin-mediated mitochondrial autophagy,while 85% VO2max exercise can significantly increase the level of skeletal muscle oxidative stress,but it has no significant effect on skeletal muscle antioxidant capacity and PINK1/Parkin-mediated mitochondrial autophagy.