一氧化碳对脂多糖诱导大鼠肺巨噬细胞损伤的影响

摘要： 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-2 100 μmol/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 mRNA 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 mRNA 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 mRNA 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。

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

Abstract: 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-2 100 μmol/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 downregulation of Drp1 and amelioration of mitochondrial function.