急性力竭运动恢复期内大鼠骨骼肌内质网应激水平变化研究

Changes of endoplasmic reticulum stress level in skeletal muscle during recovery acute exhaustive exercise

目的:研究急性力竭运动恢复期内不同时间点内质网应激关键基因表达水平,探究急性运动干预对大鼠骨骼肌内质网应激信号通路及细胞凋亡的影响。方法:将48只雄性大鼠随机分为对照组(C)、力竭运动后即刻组(E)、力竭运动后恢复3h组(E3)、力竭运动后恢复8h组(E8)、力竭运动后恢复15h组(E15)及力竭运动后恢复24h组(E24),每组8只。在不同时间点取大鼠血液及腓肠肌组织,血液指标检测乳酸脱氢酶(LDH)、肌酸激酶(CK)及C反应蛋白(CRP)含量,提取骨骼肌总RNA,采用荧光定量PCR法检测大鼠骨骼肌内质网应激基因mRNA水平,包括免疫球蛋白结合蛋白(Bip/GRP78),内质网膜上跨膜蛋白激活转录因子6(ATF-6)、PER样内质网激酶(PERK)、真核生物起始因子2(eIF2α)以及CCAAT/增强子结合蛋白同源蛋白(CHOP)等,采用ELISA检测骨骼肌Bip含量,Western Blot法检测Bip、CRT、CHOP蛋白表达水平。结果:与对照组相比:①E组血液CK、CRP显著升高(P<0.05),E3组血液LDH显著升高(P<0.05);②E组Bip、ATF6、PERK、eIF2α等内质网应激基因转录水平均显著上调,其中内质网应激关键基因Bip mRNA水平及蛋白表达水平在恢复3h时达到峰值;③E组CRT与E24组CHOP蛋白表达水平显著升高(P<0.05)。结论:①急性力竭运动后恢复期骨骼肌内质网应激关键蛋白Bip表达水平及下游相关基因mRNA水平均呈一过性上调趋势,在运动后即刻至3h间出现峰值,随着恢复期时间的延长,Bip蛋白表达水平及各基因转录水平逐渐恢复正常;②急性力竭运动后24h引发细胞凋亡,其在内质网应激效应后发生,具有一定的延迟性。

Objective:To detect the expression levels of key genes of endoplasmic reticulum stress at different time points during the recovery period of acute exhaustive exercise,and to explore the effects of acute exercise intervention on endoplasmic reticulum stress signal pathway and cell apoptosis in rat's skeletal muscle.Method:Forty eight male rats were randomly divided into control group(C),immediately after exhaustive exercise group(E),3-hour recovery group(E3),8-hour recovery group(E8),15-hour recovery group(E15)and 24-hour recovery group(E24),with 8 rats in each group.At different time points,rat blood and skeletal muscle gastrocnemius tissue were taken,serum lactate dehydrogenase(LDH),creatine kinase(CK)and C-reactive protein(CRP)were detected,the total RNA of skeletal muscle was extracted,the mRNA level of endoplasmic reticulum stress gene in skeletal muscle of rats was detected by fluorescence quantitative PCR,including glucose-regulated protein 78(Bip/GRP78),transmembrane protein ATF-6 of endoplasmic reticulum、PERK、eukaryotic initiation factor 2(eIF2α)and CCAAT/enhancer binding protein homologous protein(CHOP).The content of Bip in skeletal muscle was detected by ELISA,and the expression of Bip,CRT and CHOP were detected by Western Blot.Result:Compared with the control group:①Serum CK and CRP in group E increased significantly(P<0.05),and LDH in serum in group E3 increased significantly(P<0.05);②The transcriptional levels of ER stress genes in group E such as Bip、ATF-6、PERK、eIF2αsignificantly upregulated,and the mRNA and protein expression of key ER stress genes Bip reached the peak at 3 hours after exhaustive exercise;③The expressions of CRT and CHOP protein significantly increased in group E and in group E24,respectively(P<0.05).Conclusion:①The expression of endoplasmic reticulum stress key protein Bip and mRNA level of downstream related genes in skeletal muscle showed a transient up-regulation trend during the recovery period after exhaustive exercise.The peak appeared between immediately and 3h after exercise,with the prolongation of the recovery period,the expression level of Bip protein and the transcription level of each gene gradually returned to normal;②Apoptosis was induced 24 hours after acute exhaustive exercise,which occurred after endoplasmic reticulum stress effect,with a certain delay.