静脉注射肿瘤坏死因子α对大鼠骨骼肌蛋白降解的影响及机制初探

目的　观察静脉注射重组大鼠肿瘤坏死因子α(TNFα)对大鼠骨骼肌蛋白降解的影响 ,初步探讨其与糖皮质激素的关系。　方法　Wistar大鼠随机分为 3组 :A组为对照组 ;B组一次性静脉注射重组大鼠TNFα 1× 10 6 U kg ;C组预先使用糖皮质激素受体拮抗剂RU384 86灌胃 ,2h后注射重组大鼠TNFα(条件同B组 )。用药后 12h ,测量各组大鼠体温 ,分离伸趾长肌 ,称重后进行有氧孵育。采用高效液相色谱法测定总蛋白和肌纤维蛋白的降解率 ,以RNA印迹法检测泛素mRNA(2 .4kb)和C2亚基mRNA的表达变化。　结果　用药后 12h ,体温 :B、C组大鼠均明显高于A组 (P〈0 .0 1) ;伸趾长肌重量 :B、C组均明显轻于A组 ((P〈0 .0 1) ,但C组重于B组 (P〈0 .0 5 ) ;总蛋白和肌纤维蛋白降解率 :B组较A组分别升高 4 3%和 112 % (P〈0 .0 1) ;C组较B组分别降低 16 %和 2 8% (P〈0 .0 1) ;泛素mRNA和C2亚基mRNA的表达 :B组较A组分别升高约 4 .3、3.6倍 ,C组较B组均明显降低。　结论　静脉注射大剂量重组大鼠TNFα ,能增强大鼠骨骼肌泛素 -蛋白酶体途径的活性 ,导致总蛋白 ,特别是肌纤维蛋白降解率升高 ,糖皮质激素是该效应的介导因素之一

Study on the mechanism of the effects of recombinant rat tumor necrosis factor α on the degradation of rat skeletal muscle proteins

OBJECTIVE: To investigate the mechanism and the effects of intravenously injected tumornecrosis factor alpha (TNFalpha) on skeletal muscle protein degradation in rats and its relationship with glucocorticoid. METHODS: Forty-five male Wistar rats were randomly divided into 3 groups as A (control), B (TNFalpha injection) and C (TNFalpha and glucocorticoid receptor antagonist injection) groups. TNFalpha in dose of 1x 10(6) units/kg was given to rats in B group intravenously. RU38486, a glucocorticoid receptor antagonist, was given by gavage in C group 2 hours before intravenous injection of TNFalpha in the same dose as in B group. the rat temperature was monitored 12 hours after the administration of the drugs. At the same time, the rat extensor digitorium longus muscles (EDL) were isolated, weighed and cultured under aerobic condition, and than the degradation rates of total and the myofibrillar proteins were determined with HPLC (high performance liquid chromatography), and the expression changes in C2 subunit mRNA and ubiquitin mRNA were detected by Northern blot. RESULTS: Twelve hours after the injection, the temperature of the rats in B and C group was much higher than that in A group (P < 0.01), while the weight of the extensor digitorium longus muscle in B and C groups was evidently lower than that in A group (P < 0.01) whereas that in C was higher than that in B groups (P < 0.05). The degradation rates of total and the myofibrillar proteins in B group were increased by 43% and 112%, respectively, when compared with those in A group (P < 0.01), while the rates in C group was decreased by 16% and 28%, respectively, when compared with those in B group (P < 0.01). In addition, the expressions of ubiquitin mRNA (2.4 kb) and C2 subunit mRNA in B group were increased 4.3 and 3.6 fold compared with those in A group, whereas those in C group were much lower than those in B group. CONCLUSION: Intravenous injection of recombinant TNFalpha in large dose might enhance the activity of rat skeletal muscle ubiquition-proteasome system pathway, which led to an increase in the degradation rate of rat total protein, especially the myofibrillar protein. Glucocorticoid was one of the mediating factors of that effect.