NF-κB在电刺激引起C2C12肌管线粒体功能低下时的作用

The Protective Role of NF-κB Protein on the Mitochondria Dysfunction of C2C12 Myotubes Induced by Electrical Stimulation

目的:探讨NF-κB在不同电刺激状态下C2C12肌管粒体功能低下时的作用。方法:采用TY-C型电刺激器(强度为45 V、20 ms、5 Hz)刺激分化6天的C2C12肌管来建立耐力运动时的神经冲动模型,对照组无任何电刺激,实验组分为刺激即刻组、孵育组和对照组,刺激即刻组的刺激时间分别为60分钟、75分钟、90分钟、120分钟、150分钟和180分钟,孵育组刺激120分钟后放入培养箱中继续培养,时间分别为30分钟、60分钟、120分钟和180分钟。测定各组细胞pIKK-α、NF-κB及MnSOD活性变化。结果:与对照组相比,电刺激即刻组C2C12细胞NF-κB活性显著升高(P<0.01),且刺激120分钟和150分钟增加明显(P<0.05),到180分钟时,略有下降。孵育组中孵育120分钟时NF-κB活性较孵育组其它细胞显著增加(P<0.01);与对照组相比,刺激即刻各组细胞内pIKK-α表达显著增加(P<0.05),但不同时间电刺激各组之间无显著差别。孵育组中,孵育120分钟细胞pIKK-α蛋白表达与孵育组其他细胞相比显著升高(P<0.01);与对照组相比,刺激即刻组刺激150分钟时细胞线粒体MnSOD活性显著增加(P<0.01),孵育组孵育120分钟时C2C12细胞线粒体MnSOD活性显著增加(P<0.01)。结论:(1)在电刺激引起C2C12细胞产生氧化应激,导致细胞线粒体功能的下降中,内源性ROS的产生会激活NF-κB的表达,同时激活IKK-α的磷酸化,且在电刺激强度一定时,NF-κB的活性对电刺激的应答有一定的时相性。(2)随着刺激时间的不同,细胞线粒体MnSOD的活性变化有差异,且线粒体MnSOD活性的变化与刺激时间有关,推测NF-κB的表达上调了细胞内抗氧化物酶MnSOD的表达,这在一定程度上对细胞起到保护作用。

Objective To study the effect of NF-κB on the mitochondria dysfunction of C2C12 myotubes induced by electrical stimulation （ES）. Methods C2C12 myotubes contraction was stimulated by ES with 20ms-5Hz and 45V in vitro to induce mitochondrial dysfunction. Experimental groups consisted of electrical stimulation （ES）,incubation,and control groups. Group ES was stimulated electrically immediately and at 60min, 75min ,90min, 120min, 150min ,and 180min thereafter. Incubation group was stimulated for 120min and then incubated for 30min, 60min,75min, 90min, 120min, 150min, and 180min,respective/y. Rats in control group received no any stimulation. The changes in plKK-α,NF-κB activity and MnSOD activity were detected. Results In the group ES,the NF-κB activity increased immediately（P 〈 0.01 ）,at 120min and 150min（P 〈 0.05） as compared to the control group. In the incubation group,the NF-κB activity increased significantly at 120min（P 〈 0.01 ） as compared to the control group. Higher levels of cell pIKK-ct in the group ES was seen immediately after ES as compared to the control group （P 〈 0.05）. There was also higher level of cell pIKK-α in incubation group 120min after incubation as compared to the control group. Mitochondria MnSOD activity increased more significantly and transiently in ES group than in control group,and significantly than baseline at 150min and 180min after ES （P 〈 0.05）. Mitochondria MnSOD activity increased significantly 120min after incubation（P 〈 0.01 ）. Conclusion 1）ES leads to oxidative stress,inducing mitochondria dysfunction. 2） The level of mitochondria MnSOD activity relates to the duration of ES and upregulation of MnSOD probably has protective effect on the cells.