单 位：
广州体育学院运动医学室;广州越秀区第三人民医院
摘 要：
为观察咖啡因间歇刺激是否减轻环孢素A所致的细胞毒性及对骨骼肌细胞的泛素-蛋白酶体途径和钙蛋白酶活性的影响,将分化的L6肌管细胞分为4组,即对照组、咖啡因组、环孢素A组和咖啡因+环孢素A组。咖啡因(5mmol/L)和环孢素A(10μmol/L)每天刺激5h,连续刺激5d,然后收集细胞。荧光实时定量PCR检测FoxO1、MAFbx、MuRF1基因表达;Western blot法检测FoxO1、MAFbx、MuRF1和20S蛋白含量;荧光定量法检测20S蛋白酶和钙蛋白酶的体外活性。结果表明,加环孢素A的两组细胞大小明显小于另外两组,且咖啡因间歇刺激可以减轻环孢素A诱导的肌管细胞萎缩;间歇刺激下,加咖啡因的两组FoxO1mRNA表达下调;MuRF1和MAFbx两个基因的表达在各组之间无差异,且咖啡因和环孢素A对FoxO1/MuRF1/MAFbx和20S蛋白酶体蛋白含量皆无主效应。荧光定量检测蛋白酶活性结果显示,咖啡因和环孢素A不影响20S蛋白酶体的活性,但对钙蛋白酶体外活性有交互影响。环孢素组钙蛋白酶体外活性低于正常组;环孢素A+咖啡因组低于咖啡因组,但高于环孢素组。说明咖啡因间歇刺激可减轻环孢素A所致的肌管萎缩,且咖啡因间歇刺激改可善环孢素A抑制的钙蛋白酶体外活性。
译 名：
Intermittent Caffeine Stimulation Reduces L6 Myotube Cytotoxicity Induced by Cyclosporine A
作 者：
LIAO Ba-gen1,YANG Min1,LUO Xing-hua1,XIONG Xi2(1.Department of Sports Medicine,Guangzhou Sports University,Guangzhou,Guangdong,510076,China; 2.The Third People's Hospital of Yuxiu District In Guangzhou,Guangzhou,Guangdong,510095,China)
关键词：
caffeine;cyclosporine A;forkhead box O1;calpain
摘 要：
To investigate whether intermittent caffeine stimulation reduces cyclosporine A(CsA)-induced cytotoxicity in L6 myotubes as well as related proteolytic signaling pathway,the differentiated L6 myotubes were divided into 4 groups: control group,caffeine group,CsA group and caffeine+ CsA group.Caffeine(5 mmol/L) and CsA(10 μmol/L) were treated to intermittently stimulate for 5 h/day,5 days respectively.At the end of the experimental period,L6 myotubes in each group were used to extract the protein and nucleic acid.FoxO1,MAFbx,MuRF1 expression of the genes were assayed by real time PCR,protein content of FoxO1,MAFbx,MuRF1 and 20 S by Western blot analysis,and the in vitro activity of 20 S proteasome and calpain by fluorescent quantitative assay.Results showed that after 5 days of experimental intervention,CsA resulted in the L6 myotube atrophy,and intermittent caffeine-stimulation reduced CsA-induced myotubes atrophy.Intermittent caffeine-stimulation downregulated L6 myotube FoxO1 mRNA,but not MuRF1 mRNA and MAFbx mRNA.Both CsA and caffeine had no main effect on the protein content of FoxO1/ MuRF1/MAFbx and 20 S proteasome.Compared with the other two groups,the cytosolic calpain activities in both CsA group and the CsA +caffeine group were significantly reduced.Compared with the CsA group,the calpain activity of CsA + caffeine group was significantly increased;The activity of cytosolic 20 S proteasome had no significant changes among groups.In conclusion,the intermittent stimulation of caffeine reduces CsA-induced L6 myotube atrophy,and increases the calpain activity(in vitro) inhibited by CsA.
