一氧化碳对脂多糖诱导大鼠肺巨噬细胞损伤的影响

Effects of carbon monoxide on lipopolysaccharide induced damage in rat alveolar macrophages

目的探讨一氧化碳(CO)对脂多糖(LPS)诱导大鼠肺巨噬细胞损伤的影响及可能机制。方法用含有10%胎牛血清的细胞培养基,在37℃、5%CO2细胞培养孵箱内培养大鼠肺巨噬细胞,采用随机数字表法将其分为4组(n=10):空白对照组(C组)、CO组、LPS组、LPS+CO组。CO组加入体外一氧化碳释放分子-2(CORM-2)100μmol/L孵育,LPS组加入LPS 10 mg/L,LPS+CO组加入CORM-2 100μmol/L预处理1 h后加入LPS 10 mg/L孵育,C组加入等量PBS液作为对照。每组细胞处理完毕后继续孵育24 h,采用MTT法测定细胞活力;流式细胞仪测定细胞凋亡率和线粒体膜电位;ATP酶含量试剂盒测定细胞内ATP含量;RT-PCR法测定细胞中线粒体分裂相关蛋白Drp1的m RNA含量;Western blot法测定Drp1的蛋白表达。结果与C组相比,LPS组和LPS+CO组细胞活力、ATP含量和线粒体膜电位下降,细胞凋亡率、线粒体分裂蛋白Drp1 m RNA及蛋白表达增加(P<0.05),CO组上述指标比较差异无统计学意义;与LPS组比较,LPS+CO组细胞活力、ATP含量和线粒体膜电位增加(P<0.05),细胞凋亡率、Drp1 m RNA及蛋白表达减少(P<0.05)。结论 CO可减轻LPS诱导的大鼠肺巨噬细胞损伤,其机制与下调线粒体分裂相关蛋白Drp1和改善线粒体功能有关。

Objective To evaluate effects of carbon monoxide (CO) on lipopolysaccharide (LPS) induced damage and possible mechanism in rat alveolar macrophages. Methods Rat alveolar macrophages were cultured in DMEM containing 10%fetal bovine serum with 5%CO2 at 37℃in Heraeus sepatech. The cells were divided into four groups using random number table (n=10): control group (group C),CO group, LPS group and LPS+CO group. The CO release molecule-2 (CORM-2) 100 μmol/L was added into CO group,LPS 10 mg/L was added into LPS group, cells were pretreated with CORM-2 100μmol/L for 1 h then LPS 10 mg/L was added into LPS+CO group, the same amount of PBS was added to group C. Proliferation was measured by MTT assay. Apoptosis and mitochondrial membrane potential were detected with flow cytometer. The content of ATP was tested by ATP content kit. Drp1 mRNA was measured by RT-PCR, and Drp1 expression was determined by Western blot assay. Results Compared with group C, the cell vitality, content of ATP and mitochondrial membrane potential were decreased in LPS group and LPS+CO group,and cell apoptosis rate, Drp1 mRNA and protein expression were increased (P<0.05). There were no significant changes were found in CO group. Compared with LPS group, the cell vitality, content of ATP and mitochondrial membrane potential were increased in LPS+CO group,and the cell apoptosis rate, Drp1 mRNA and protein expression were decreased (P<0.05). Conclusion Carbon monoxide can alleviate LPS-induced damage in rat alveolar macrophages, which is related with down-regulation of Drp1 and amelioration of mitochondrial function.