低氧调控EIMD后calpain活性与肌细胞膜损伤机制的探索研究

Hypoxia Regulates Calpain Activity and the Possible Mechanism of Sarcolemma Injury after EIMD

目的:观察低氧对运动性肌损伤(exercise-induced muscle damage,EIMD)后肌细胞膜损伤、钙激活中性蛋白酶(calpain)和肌细胞膜骨架蛋白(dystrophin、utrophin和dysferlin)的影响,探索低氧调控EIMD后肌细胞膜损伤的可能机制。方法:110只雄性SD健康大鼠分两批进行实验。研究Ⅰ:70只大鼠随机均分为安静对照组、常氧24 h、48 h和72 h组以及低氧24 h、48 h和72 h组,每组10只。大鼠在-16°跑台上以26.8 m/min速度持续运动5 min,运动10次,组间休息1 min。低氧和常氧各组大鼠运动后注射伊文氏蓝荧光染色剂观察膜损伤情况,用RT-q PCR和Western blot测定腓肠肌calpain和膜骨架蛋白的mRNA表达和蛋白含量。研究Ⅱ:40只大鼠随机均分为空白对照组、安慰剂24 h、48 h组和calpain抑制剂24 h、48 h组,每组8只。测定calpain抑制剂对其蛋白含量的影响,观察膜损伤情况,并验证calpain在EIMD后低氧损伤肌细胞膜中所起的作用。结果:大鼠EIMD后,1)低氧各组膜的通透性增加、完整性被破坏,阳性细胞率(positive ratio of cell,PRC)与对照组和常氧对应组相比有显著差异(P<0.01);2)低氧72 h组calpain1、calpain2的mRNA表达显著高于常氧72 h组(P<0.05),低氧各组calpain1、calpain2和calpain3的蛋白含量与常氧对应组相比无显著差异(P>0.05);3)低氧24 h组dystrophin、utrophin的m RNA表达显著低于常氧24 h组(P<0.01),但两组的蛋白含量无显著差异(P>0.05)。低氧各组dys-ferlin的mRNA表达和蛋白含量与常氧各组相比无显著差异(P> 0.05)。4)抑制calpain1、calpain2活性后,低氧24 h组calpain蛋白含量显著降低(P<0.05),低氧各组阳性细胞率显著低于安慰剂各组(P<0.01),但仍显著高于对照组(P<0.05)。结论:1) calpain1、calpain2被激活与低氧加剧大鼠EIMD后的肌细胞膜损伤有关,但calpain并非通过降解膜骨架蛋白dystrophin、utrophin和dysferlin的途径而导致膜损伤。2)抑制calpain1、calpain2活性能有效减轻但不能避免低氧加剧EIMD后的膜损伤,其中还可能存在其它的调节机制。

Objective:To observe the effects of hypoxia on sarcolemma damage,calpain activity and membrane cytoskeleton proteins after exercise-induced muscle damage(EIMD),and to explore the possible mechanism of hypoxia regulates sarcolemma damage after EIMD.Methods:One hundred ten healthy male SD rats were tested in two studies.Study I:Seventy rats were randomly divided into control,normoxia(N)24,48 and 72 h group and hypoxia(H)24,48 and 72 h groups.Rats performed downhill run at 26.8 m/min in-16°treadmill(continuous 5 min/set,intermittent interval 1min,10 sets).Observing sarcolemma injury by injecting Evan’s Blue Dye after downhill run in hypoxia and normoxia groups,and investigates mRNA and protein content of calpain and membrane cytoskeleton by RT-qPCR and Western blot method.Study II:Forty rats were randomly divided into blank,placebo(Pl-)24 h,48 h and calpain inhibitor(I-)24 h,48 h groups.To investigate changes of calpain inhibitor on the protein content of calpain and observe sarcolemma injury extent,and confirm the role of calpain on hypoxia induced sarcolemma injury after EIMD.Results:After EIMD,1)hypoxia group’s membrane permeability was increased and the integrity was decreased.There were significant differences of positive ratio of cell(PRC)in hypoxia groups compared with control and normal groups(P<0.01).2)The mRNA of calpain1 and calpain2 in the H72 group was significantly higher than N72 group(P<0.05).There was no significant difference in protein content of calpain1,calpain2 and calpain3 between hypoxia and normoxia groups(P>0.05).3)The mRNA of dystrophin and utrophin in H24 group was significantly lower than N24 group(P<0.01),but no significant difference in protein content between the two groups(P>0.05).The mRNA and protein content of dysferlin in hypoxia group were not significantly different from normoxia group(P>0.05).4)After injecting inhibitor,the protein content of calpain1 and calpain2 significantly decreased(P<0.05).The PRC in hypoxia groups was significantly lower than placebo groups(P<0.01),and was significantly higher than control group(P<0.05).Conclusion:1)Activation of calpain1 and calpain2 are involved in hypoxia exacerbation sarcolemma injury after EIMD,but not through degradation membrane cytoskeleton protein(dystrophin,utrophin and dysferlin).2)Inhibition of calpain1,calpain2 activity can effectively reduce but can not avoid sarcolemma injury exacerbated by hypoxia after EIMD.There may be other related regulatory mechanisms in calpain way.