运动对骨骼肌泛素蛋白酶体途径活性的影响及机制
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摘要
目的:一次长时间大强度运动结合PDTC、Trolox药物注射,观察骨骼肌蛋白降解、泛素蛋白酶体途径(UPP)活性在运动后的变化特点,探讨氧化应激、NF-κB信号对UPP活性的调节,揭示UPP在运动骨骼肌蛋白降解中的重要作用及机制。
方法:雄性SD大鼠进行一次大强度运动(25m/min,5%坡度,1h)。研究一:36只SD大鼠分成6组(每组6只),安静组、运动半小时组、运动1小时组、运动后1、2、6小时组。检测腓肠肌3-MH含量、泛素化蛋白含量、26S蛋白酶体活性,泛素、MuRF1、MAFbx、26S蛋白酶体C2亚基mRNA表达的变化。研究二:60只雄性SD大鼠分为三组,对照组24只,PDTC注射组和Trolox注射组各18只。安静时取材对照组6只为安静水平,然后三组分别在运动后即刻、运动后1、6小时各取材6只。除检测研究一的指标外,还检测胞质ⅠκBα含量、核NF-κB结合DNA能力,以及肌肉、血清MDA、血清CK活性的变化。
结果:(1)运动半小时、运动后6小时腓肠肌3-MH含量、26S蛋白酶体活性增加(P＜0.05)。(2)运动后6小时泛素化蛋白含量明显增加(P＜0.01)。(3)运动后MAFbx、MuRF1 mRNA表达升高(P＜0.05)。(4)运动时、运动后泛素、C2mRNA低于安静水平,运动后6小时C2 mRNA表达显著高于安静时(P＜0.01)。(5)Trolox减缓运动引起的腓肠肌、血清MDA的增加(P＜0.05)。(6)PDTC和Trolox都能减缓运动对胞质ⅠκBα的降解和核NF-κB信号的激活(P＜0.05)。(7)PDTC和Trolox都能减缓运动对腓肠肌3-MH含量、26S蛋白酶体活性和MAFbx、MuRF1、泛素、C2mRNA表达的影响(P＜0.05)。
结论:(1)一次长时间大强度运动使骨骼肌UPP活性升高,促进收缩蛋白降解。(2)运动中骨骼肌UPP活性升高的主要原因是蛋白泛素化相关的酶和26S蛋白酶体酶活性的增强;运动后UPP活性升高的主要原因是MAFbx、MuRF1、C2mRNA表达以及26S蛋白酶体活性的增加。(3)抑制运动诱导的骨骼肌氧化应激,可以减少骨骼肌NF-κB的激活,并通过对UPP成分基因转录的不同调节,降低UPP的活性,减少收缩蛋白降解。
Purpose:In this study,we investigated the effect of ubiquitin-proteasome pathway(UPP)activity with or without injection of PDTC or Trolox on proteolysis after an acute bout of physical exercise in rat skeletal muscle,and revealed roles of oxidative stress and NF-kB signal in UPP activity after exercise.
Method:The male Sprague-Dawley rats run on the treadmill at 25 m/min, 5%grade,for 1 h after familiarization with the treadmill by 10-15 min on 3 days.In Study 1,six of thirty six rats were killed respectively at rest,immediately after half an hour and an hour exercise,and 1 h, 2h,6h post-an exercise.Changes of 3-MH content,ubiquitinated protein content,26S proteasome activity,and ubiquitin,MuRF1,MAFbx,26S proteasome C2 subunit mRNA expression were detected in gastrocnemius muscle.In study 2,sixty rats were divided into three groups,control group(n=24),PDTC and Trolox injection group(n=18,respectively).Six rats of control group were killed at rest,then six rats of the three groups separately were killed immediately,1 and 6 hours after exercise in the three groups.Besides of the indexs in study 1,IκBαprotein content in myofibrillar cytoplasm,activity of NF-κB binding DNA in nucleus,as well as MDA content in muscle and serum,serum CK dynamic changes were dfetected.
Results:(1)3-MH content and 26S proteasome activity increased immediately after half an hour exercise and at 6h post-exercise in rat gastrocnemius muscle(P＜0.05).(2)Ubiquitined protein content increased significantly at 6h post-exercise(P＜0.01).(3)There was a increasing trend of expression of ubiquitin ligase MAFbx,MuRF1 mRNA after exercise (P＜0.05).(4)Expression of ubiquitin and 26S proteasome C2 subunit mRNA were low than the resting during and after exercise,but expression of C2 mRNA increased Sharply at 6h post-exercise(P＜0.01).(5)Trolox slowed down MDA content in gastrocnemius muscle and serum after exercise (P＜0.05).(6)PDTC and Trolox slowed down the degradation of IκBαin cytoplasm,and inhibited exercise-induced activation of NF-κB signal in nucleolus(P＜0.05).(7)PDTC and Trolox slowed down effects of exercise on 3-MH content,26S proteasome activity and gene expression of MAFbx, MuRF1,ubiquitin,C2 subunit(P＜0.05).
Conclusions:(1)The increase of UPP activity enhanced contractile protein degradation in skeletal muscle during and after a strenuous exercise.(2)The increase of protein ubiquitination-related enzymes activity and 26S proteasome activity resulted in the increase of UPP activity during a strenuous exercise,and the increase of components gene expression in UPP resulted in the increase of its activity after a strenuous exercise.(3)Inhibition of exercise-induced oxidative stress in skeletal muscle could reduce the activation of NF-κB signal,and reduce the activity of UPP by regulation gene transcription of the components of UPP,which could reduce the contractile protein degradation.
方法:雄性SD大鼠进行一次大强度运动(25m/min,5%坡度,1h)。研究一:36只SD大鼠分成6组(每组6只),安静组、运动半小时组、运动1小时组、运动后1、2、6小时组。检测腓肠肌3-MH含量、泛素化蛋白含量、26S蛋白酶体活性,泛素、MuRF1、MAFbx、26S蛋白酶体C2亚基mRNA表达的变化。研究二:60只雄性SD大鼠分为三组,对照组24只,PDTC注射组和Trolox注射组各18只。安静时取材对照组6只为安静水平,然后三组分别在运动后即刻、运动后1、6小时各取材6只。除检测研究一的指标外,还检测胞质ⅠκBα含量、核NF-κB结合DNA能力,以及肌肉、血清MDA、血清CK活性的变化。
结果:(1)运动半小时、运动后6小时腓肠肌3-MH含量、26S蛋白酶体活性增加(P＜0.05)。(2)运动后6小时泛素化蛋白含量明显增加(P＜0.01)。(3)运动后MAFbx、MuRF1 mRNA表达升高(P＜0.05)。(4)运动时、运动后泛素、C2mRNA低于安静水平,运动后6小时C2 mRNA表达显著高于安静时(P＜0.01)。(5)Trolox减缓运动引起的腓肠肌、血清MDA的增加(P＜0.05)。(6)PDTC和Trolox都能减缓运动对胞质ⅠκBα的降解和核NF-κB信号的激活(P＜0.05)。(7)PDTC和Trolox都能减缓运动对腓肠肌3-MH含量、26S蛋白酶体活性和MAFbx、MuRF1、泛素、C2mRNA表达的影响(P＜0.05)。
结论:(1)一次长时间大强度运动使骨骼肌UPP活性升高,促进收缩蛋白降解。(2)运动中骨骼肌UPP活性升高的主要原因是蛋白泛素化相关的酶和26S蛋白酶体酶活性的增强;运动后UPP活性升高的主要原因是MAFbx、MuRF1、C2mRNA表达以及26S蛋白酶体活性的增加。(3)抑制运动诱导的骨骼肌氧化应激,可以减少骨骼肌NF-κB的激活,并通过对UPP成分基因转录的不同调节,降低UPP的活性,减少收缩蛋白降解。
Purpose:In this study,we investigated the effect of ubiquitin-proteasome pathway(UPP)activity with or without injection of PDTC or Trolox on proteolysis after an acute bout of physical exercise in rat skeletal muscle,and revealed roles of oxidative stress and NF-kB signal in UPP activity after exercise.
Method:The male Sprague-Dawley rats run on the treadmill at 25 m/min, 5%grade,for 1 h after familiarization with the treadmill by 10-15 min on 3 days.In Study 1,six of thirty six rats were killed respectively at rest,immediately after half an hour and an hour exercise,and 1 h, 2h,6h post-an exercise.Changes of 3-MH content,ubiquitinated protein content,26S proteasome activity,and ubiquitin,MuRF1,MAFbx,26S proteasome C2 subunit mRNA expression were detected in gastrocnemius muscle.In study 2,sixty rats were divided into three groups,control group(n=24),PDTC and Trolox injection group(n=18,respectively).Six rats of control group were killed at rest,then six rats of the three groups separately were killed immediately,1 and 6 hours after exercise in the three groups.Besides of the indexs in study 1,IκBαprotein content in myofibrillar cytoplasm,activity of NF-κB binding DNA in nucleus,as well as MDA content in muscle and serum,serum CK dynamic changes were dfetected.
Results:(1)3-MH content and 26S proteasome activity increased immediately after half an hour exercise and at 6h post-exercise in rat gastrocnemius muscle(P＜0.05).(2)Ubiquitined protein content increased significantly at 6h post-exercise(P＜0.01).(3)There was a increasing trend of expression of ubiquitin ligase MAFbx,MuRF1 mRNA after exercise (P＜0.05).(4)Expression of ubiquitin and 26S proteasome C2 subunit mRNA were low than the resting during and after exercise,but expression of C2 mRNA increased Sharply at 6h post-exercise(P＜0.01).(5)Trolox slowed down MDA content in gastrocnemius muscle and serum after exercise (P＜0.05).(6)PDTC and Trolox slowed down the degradation of IκBαin cytoplasm,and inhibited exercise-induced activation of NF-κB signal in nucleolus(P＜0.05).(7)PDTC and Trolox slowed down effects of exercise on 3-MH content,26S proteasome activity and gene expression of MAFbx, MuRF1,ubiquitin,C2 subunit(P＜0.05).
Conclusions:(1)The increase of UPP activity enhanced contractile protein degradation in skeletal muscle during and after a strenuous exercise.(2)The increase of protein ubiquitination-related enzymes activity and 26S proteasome activity resulted in the increase of UPP activity during a strenuous exercise,and the increase of components gene expression in UPP resulted in the increase of its activity after a strenuous exercise.(3)Inhibition of exercise-induced oxidative stress in skeletal muscle could reduce the activation of NF-κB signal,and reduce the activity of UPP by regulation gene transcription of the components of UPP,which could reduce the contractile protein degradation.
引文
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