摘要
目的 了解外源性一氧化碳释放分子2（CORM2）对脓毒症时Janus激酶/信号转导和转录激活子（JAK/STAT）信号通路活化的抑制作用.方法 将RAW264.7细胞分为正常对照组、LPS（10 μg/mL LPS,浓度下同）组、LPS＋无活性CORM-2组、LPS＋小剂量CORM-2（50 μmol/LCORM-2）组、LPS＋大剂量CORM-2（100μmol/L CORM-2）组,ELISA法检测细胞上清液中TNF-α的水平及蛋白质印迹法检测JAK1、JAK3分子磷酸化水平.另将35只雄性BALB/c小鼠按随机数字表法分为正常对照组、盲肠结扎和穿孔术（CLP）组、CLP＋无活性CORM-2（8.0 mg/Kg）组和CLP＋CORM-2（8.0 mg/kg）组.CLP＋CORM-2组除伤后使用CORM-2外,其他处理同CLP组.于伤后24 h按上述方法检测小鼠血浆TNF-α、IL-1β的表达水平以及肝组织JAK1、JAK3分子磷酸化水平.对数据行t检验.结果 与正常对照组[（1.9±0.3）pg/mL]比较,LPS组细胞的TNF-α水平明显升高[（8.2±2.7）pg/mL,t=2.844,P〈0.01],磷酸化JAK1、JAK3蛋白水平也升高;2种剂量LPS＋CORM-2组细胞的TNF-α,水平分别为（5.7±1.4）、（3.2±0.9）pg/mL,较LPS组明显下降（t值分别为2.104、2.363,P值均小于0.05）,JAK1、JAK3分子的磷酸化水平呈浓度依赖性下降.与正常对照组比较,CLP组血浆TNF-α、IL-1β水平明显升高（t值分别为2.916、2.796,P值均小于0.01）,小鼠肝组织JAK1、JAK3分子的磷酸化水平亦明显升高.CLP＋CORM-2组TNF-α、IL-1β血浆水平显著降低（t值分别为2.115、2.398,P值均小于0.05）,肝组织JAK1、JAK3蛋白的磷酸化得到有效抑制.结论 CORM-2能明显抑制JAK分子磷酸化,继而抑制JAK/STAT信号通路的活化,减少下游相关细胞因子的表达,有效防止严重感染时炎症反应的级联反应.
Objective To study the inhibitive effect of exogenous carbon monoxide-releasing molecules 2 （ CORM-2） on the activation of Janus kinase/signal transducer and activator of transcription （JAK/ STAT） pathway in sepsis. Methods RAW264. 7 cells were divided into normal control group, LPS group （10 mg/mL LPS, the same concentration below） , LPS ＋ inactive CORM-2 （iCORM-2） group, LPS ＋50 mmol/L CORM-2 group, and LPS ＋ 100 mmol/L CORM-2 group. TNF-α level in the supernatant was determined with EL1SA, and the phosphorylation levels of JAK1 and JAK3 were determined with Western blot. Thirty-five male BALB/c mice were divided into normal control group, cecal ligation and puncture （CLP） group, CLP ＋ iCORM-2 （8.0 mg/kg） group and CLP ＋ CORM-2 group （8.0 mg/kg） according to the random number table. Mice in CLP ＋ CORM-2 group were treated the same as mice in CLP group except for administration of CORM-2 after CLP. The plasma levels of TNF-α, IL-1β, and the phosphorylation levels of JAK1 , JAK3 in liver tissue were determined with ELISA 24 hours post CLP. Data were processed with t test. Results Compared with that of normal control group [ （ 1. 9 ± 0. 3 ） pg/mL ] , the TNF-α level [（8.2 ± 2.7） pg/mL, t =2.844, P 〈0.01] and phosphorylation levels of JAK1 , JAK3 in LPS group increased significantly; while TNF-α levels in LPS ＋50 mmol/L CORM-2 and LPS ＋ 100 mmol/L CORM-2 groups decreased obviously as compared with that of LPS group [（5.7 ± 1.4）, （3.2±0.9） pg/mL, with t value respectively 2. 104 and 2. 363, P values all below 0. 05 ] , and it was the same with phosphorylation levels of JAK1 , JAK3 in a dose-dependent manner. Compared with those of normal control group, plasma levels of TNF-α and 1L-1β and phosphorylation levels of JAK1 , JAK3 in liver tissue significantly increased in CLP group （with t value respectively 2. 916 and 2. 796, and P values all below 0. 05） ; while plasma levels of TNF-α and IL-1β and the phosphorylation levels of JAK1 , JAK3 in liver tissue decreased significantly in CLP ＋ CORM-2 group （ with t value respectively 2.115 and 2. 398 , and P values all below 0. 05 ） . Conclusions Exogenous CORM-2 can obviously inhibit the phosphorylation of JAKs molecules and then inhibit the activation of JAK/STAT signal pathway in sepsis, and decrease the expression of downstream cytokines to effectively prevent cascade reaction in the inflammatory response after severe infection.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2010年第2期100-103,共4页
Chinese Journal of Burns
基金
国家自然科学基金（30772256）
江苏省自然科学基金面上项目（BK2008237）
