COPD大鼠骨骼肌泛素-蛋白酶体降解通路与IL-10相关性的研究

目的：研究 COPD 大鼠模型的骨骼肌泛素(Ub)-蛋白酶体降解途径中 E2-14K、MAFbx、Ub 蛋白表达,肿瘤坏死因子α(TNF-α)和炎症抑制因子 IL-10在大鼠血清和骨骼肌中含量变化,探讨 IL-10在 COPD 骨骼肌蛋白高分解过程中的作用及调控机制。方法成年雄性 SD 大鼠45只,其中分为模型组30只、对照组15只,采用反复熏香烟+气管内注入脂多糖法复制 COPD 大鼠动物模型。蛋白质印迹法分别检测大鼠膈肌、腓肠肌和肋间肌中 E2-14K、MAFbx、Ub 蛋白表达。用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测大鼠血清、膈肌、腓肠肌和肋间肌中 IL-10和 TNF-α的含量。结果模型组膈肌、肋间肌、腓肠肌中 E2-14K、MAFbx、Ub 蛋白的相对表达量显著增加。在模型组中,血清和骨骼肌肌内 TNF-α较对照组显著升高,IL-10表达均下降血清(44.68±8.67)vs (75.96±10.59),膈肌(50.55±7.41)vs (80.06±9.22),肋间肌(47.47±8.98)vs (78.10±7.21),腓肠肌(48.60±10.88)vs (76.77±8.92),P <0.01]。血清、膈肌、肋间肌和腓肠肌中 IL-10和 TNF-α水平呈负相关(血清 r =-0.67,膈肌 r =-0.78,肋间肌 r =-0.80,腓肠肌 r =-0.77,P <0.05)。血清、膈肌、肋间肌和腓肠肌中 IL-10都与中性粒细胞百分比、E2-14K、MAFbx、Ub 蛋白相对表达量呈负相关(血清 r =-0.80、r =-0.73、r =-0.69、r =-0.81；膈肌 r =-0.83、r =-0.81、r =-0.75、r =-0.79；肋间肌 r =-0.81、r =-0.80、r =-0.74、r =-0.54；腓肠肌 r =-0.74、r =-0.69、r =-0.71、r =-0.62,P <0.05)。血清 IL-10与骨骼肌IL-10呈显著正相关(膈肌 r =0.83、肋间肌 r =0.80、腓肠肌 r =0.73,P <0.01)。结论 IL-10的下调在 COPD 炎症中起重要作用,可能参与骨骼肌 Ub-蛋白酶体降解途径降解机制的调控。

Skeletal muscle protein degradation mechanism in COPD rats and relationship with IL-10

Objective To investigate the expressions of ubiquitin-proteasome markers,including E2-14K,MAFbx,Ub,tumor necrosis factor-α(INF-α),in skeletal muscle of COPD rats,and their relationship with IL-10 level in diaphragm and serum in order to elucidate the potential mechanism of diaphragm atrophy.Methods Forty-five adult male SD rats were randomly divided into a COPD model group(n =30)and a normal control group(n =1 5).The COPD rat model was established by instillation of lipopolysaccharide(LPS) and exposure to cigarette smoke for 28 days.The protein levels of E2-14K, MAFbx,Ub in diaphragm,gastrocnemius and intercostal muscle were measured by western blot.The concentration of IL-10 and TNF-α in serum,diaphragm,gastrocnemius and intercostal muscle was measured by ELISA.Results In the model group,the protein expressions of E2-14K,MAFbx,Ub protein in diaphragm,intercostal muscle,gastrocnemius increased significantly.In the model group,the serum and skeletal muscle of TNF-α increased significantly compared with the control group,but the expression of IL-10 decreasedserum (44.68 ± 8.67) vs (75.96 ± 10.59),diaphragm (50.55 ± 7.41) vs (80.06 ± 9.22),intercostal muscle(47.47±8.98)vs(78.10±7.21),gastrocnemius(48.60±10.88)vs(76.77± 8.92),respectively,P < 0.01].The level of IL-10 in diaphragm,intercostal muscle and gastrocnemius was negatively correlated with TNF-α(serum r = -0.67,diaphragm r = -0.78,intercostal muscle r =-0.80,gastrocnemius r =-0.77,P < 0.05).The level of IL-10 in diaphragm,intercostal muscle and gastrocnemius was negatively correlated with the percentage of neutrophils,levels of E2-14K,MAFbx,Ub (serum r =-0.80,r =-0.73,r =-0.69,r =-0.81;diaphragm r =-0.83,r =-0.81,r =-0.75,r =-0.79;intercostal muscle r =-0.81,r =-0.80,r =-0.74,r =-0.54,gastrocnemius r =-0.74,r =-0.69,r =-0.71,r = -0.62;P < 0.05).The IL-10 levels in serum and skeletal muscle were also positively correlated each other(diaphragm r =0.83,intercostal muscle r =0.80,gastrocnemius r =0.73, P <0.01).Conclusions In COPD rats,down regulation of IL-10 plays an important role in inflammation, may be involved in skeletal muscle protein ubiquitin-proteasome degradation pathway of degradation mechanism of regulation.