目的 探讨CORM-2通过p38分裂原激活蛋白激酶(p38 MAPK)信号通路对脂多糖(LPS)刺激大鼠肺巨噬细胞中线粒体分裂蛋白Fis1的影响.方法 将传代培养的大鼠肺泡巨噬细胞以2×105/mL密度接种于96孔板.培养24 h后,采用随机数字表法将其分为4组:正常对照组(C组)、LPS组(L组)、CO释放剂CORM-2+ LPS组(LC组)、p38MAPK抑制剂SB203580+CORM-2+ LPS组(LCS组).细胞孵育24h时,应用试剂盒测定线粒体MDA含量及SOD活力,应用Western blot测定HO-1、线粒体相关分裂蛋白Fis1和p38的表达.应用RT-PCR测定HO-1和Fis1 mRNA含量的表达.结果 与C组比较,L组MDA[2.43±0.12vs.3.59±0.07]含量、HO-1[1.31 ±0.27 vs.1.65 ±0.41]、Fis1 [1.27 ±0.23vs.1.65 ±0.41]、p38[1.01 ±0.24 vs.1.36±0.17]表达水平升高,SOD[81.7±1.62vs.54.7±1.62]活力降低(P<0、05);与L组比较,LC组MDA[3.59±0.07 vs.3.08±0.52]含量、Fis1 [2.01 ±0.35 vs.1.48±0.39]表达水平降低,SOD [54.7±1.62vs.67.4±1.32]活力、HO-1[1.65±0.41 vs.2.25 ±0.18]、p38[1.36±0.17vs.1.78±0.23]表达水平升高(P<0、05);与LC组比较,LCS组MDA[3.08±0.52vs.4.16 ±0.19]含量、Fis1[1.48 ±0.39 vs.1.96±0.31]表达水平升高,SOD [67.4±1.32vs.45.9±1.52]活力、HO-1 [2.25 ±0.18vs.1.78 ±0.19]、p38[1.78±0.23 vs.1.12±0.29]表达水平降低(P<0、05).结论 HO-1/CO抑制LPS刺激大鼠肺巨噬细胞中线粒体分裂蛋白Fis1表达机制与p38MAPK信号通路有关.
Objective To investigate the effects of CORM-2 via p38 mitogeu-activated protein kinase (p38MAPK) signaling pathway on the expression of the mitochondrial fission protein 1 (Fisl) in lipopolysaccharide (LPS)-induced mouse pulmonary macrophages.Methods The rat subculture alveolar macrophages were seeded on 96 well plates with 2 × 105/ml densities.After 24 hours of culture,it was divided into 4 groups by random number table method:normal control group (group C),group LPS (group L),CO releasing agent CORM-2 + LPS group (group LC),p38MAPK inhibitor SB203580 + CORM-2 + LPS group (group LCS).When the cells were incubated for 24 hours,the mitochondrial MDA content and SOD activity were determined by ELISA kit,the levels of HO-1、mitochondrial fission protein Fis1 and p38 were determined by Western blot,the expressions of HO-1 and mitochondrial fission protein Fis1 were detected by RT-PCR.Results Compared with the C group,the levels of MDA [(2.43 ±0.12) vs.(3.59 ±0.07)],HO-1 [(1.31±0.27) vs.(1.65±0.41)],Fis1 [(1.27±0.23) vs.(1.65±0.41)] andp38 [(1.01 ±0.24) vs.(1.36 ±0.17)] in group L were increased,and the activity of SOD [(81.7 ± 1.62) vs.(54.7 ± 1.62)] was decreased (P < 0.05);Compared with the group L,the MDA content [(3.59 ± 0.07) vs.(3.08 ±0.52)] and the level of Fis1 [(2.01 ±0.35) vs.(1.48 ±0.39)] in group LC were down-regulated,and the levels of SOD [(54.7 ± 1.62) vs.(67.4 ± 1.32)]、and the expressions of HO-1 [(1.65±0.41)vs.(2.25±0.18)] andp38 [(1.36±0.17) vs.(1.78±0.23)] wereup-regulated (P <0.05).Compared with the group LC,the MDA content [(3.08 ±0.52) vs.(4.16 ±0.19)] and the expression of Fis1 [(1.48 ±0.39) vs.(1.96 ±0.31)] in group LCS were increased,and the level of SOD [(67.4±1.32)vs.(45.9±1.52)]、and the expressions of HO-1 [(2.25±0.18)vs.(1.78± 0.19)] and p38 [(1.78 ±0.23) vs.(1.12 ±0.29)] were decreased (P <0.05).Conclusions HO-1/CO system inhibits the expression of Fis1 in LPS-induced lung macrophages,which may be regulated by p38MAPK signaling pathway.