咖啡因间歇刺激抑制环孢素A诱导的L6肌管细胞毒性研究

Intermittent Caffeine Stimulation Reduces L6 Myotube Cytotoxicity Induced by Cyclosporine A

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。

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 myo- tubes 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 re- spectively. At the end of the experimental period, L6 myotubes in each group were used to extract the pro- tein 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 experi- mental intervention, CsA resulted in the L6 myotube atrophy, and intermittent caffeine-stimulation re- duced 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 in- creased; 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 cal- pain activity （in vitro） inhibited by CsA.