一氧化碳对脂多糖诱导大鼠肠道损伤的影响

Effects of Carbon Monoxide on Lipopolysaccharide Induced Rat Intestinal Tract Injury

目的观察250 ml/m^3一氧化碳(CO)吸入和腹腔给予对脂多糖(LPS)诱导大鼠肠道损伤的影响,以及影响过程中丝裂原活化蛋白激酶p38(p38 MAPK)表达的变化及意义。方法108只SD大鼠按随机数字表法均分为对照、CO吸入、CO腹腔、LPS注入、LPS+CO吸入及LPS+CO腹腔6组,后3组静脉注入5 mg/kg体质量LPS,前3组静脉注入等量生理盐水;1h后,对照及LPS注入组吸入室内空气,CO吸入及LPS+CO吸入组吸入250 ml/m^3 CO,CO腹腔及LPS+CO腹腔组以2 L/min腹腔通入250 ml/m^3 CO。观察1、3、6h后,批次(每批6只)放血处死大鼠,取回盲部小肠。酶联免疫吸附法测定细胞间黏附分子-1(ICAM-1)、血小板活化因子(PAF)及白细胞介素-10 (IL-10)水平;硫代巴比妥酸法测定丙二醛(MDA)含量;化学比色法测定髓过氧化物酶(MPO)活性;羟胺法测定超氧化物歧化酶(SOD)活性;流式细胞仪检测细胞凋亡率;蛋白印迹法测定磷酸化p38 MAPK表达;光镜观察组织形态学变化。结果LPS组ICAM-1、PAF、MDA、MPO、细胞凋亡率及磷酸化p38 MAPK表达均显著高于对照、CO吸入及CO腹腔组(P<0.05或0.01),而IL-10和SOD明显低于该3组(均P<0.05),肠损伤严重;组内各时间点比较,差异无统计学意义。与相应时间点的LPS组比较,LPS+CO吸入组及LPS+CO腹腔组ICAM-1、PAF、MDA、MPO及细胞凋亡率明显降低(均P<0.05),而IL-10和SOD显著升高(均P<0.05),肠损伤减轻,但磷酸化p38 MAPK表达进一步增强(P<0.05);LPS+CO吸入组及LPS+CO腹腔组间及2组内各时间点比较,差异无统计学意义。结论250 ml/m^3 CO吸入和腹腔给予以非时间依赖方式抑制LPS诱导的大鼠肠道过氧化物和促炎细胞因子产生、减少细胞凋亡,增强抗氧化酶、抗炎细胞因子表达而起相似的保护作用;p38 MAPK信号转导通路可能参与了这一过程。

Objective To investigate the effects of 250 ml/m3 carbon monoxide （CO） inhalation or intraperitoneal infusion on lipopolysaccharide （LPS） induced rat intestinal tract injury, and to detect the roles of p38 mitogen-activated protein kinase （MAPK） pathway during CO administration. Methods After received 5 mg/kg LPS or an equal volume of normal saline by intravenous injection, 108 male SD rats were randomly divided into 6 groups： control group, CO inhalation （250 ml/m^3） group, CO intraperitoneal infusion （250 ml/m^3 at a rate of 2 L/min） group, LPS （5 mg/kg） group, LPS （5 mg/kg） ＋CO inhalation （250 ml/m^3 ） group and LPS （5 mg/kg） ＋CO intraperitoneal infusion （250 ml/m^3 at a rate of 2 L/min） group. The animals were differently sacrificed at 1, 3 and 6 h for the observation, and the ileum tissues were homogenized for determination the levels of platelet activator factor （PAF）, intercellular adhesion molecule-1 （ICAM-1） and interlukin-10 （IL-10） with enzyme-lined immunosorbent assay, the content of maleic dialdehyde （MDA） with thiobarbitric acid, the activity of myeloperoxidase （MPO） with chemical method, the activity of superoxide dismutase （SOD） with hydroxylamine, the activity of phosphorylated p38 MAPK with Western blot, the pathology with light microscope, and the extents of cell apoptosis were showed by the ratio of the apoptotic cells which had less DNA to the total cells of a cell-suspension sample by using the flow cytometry after being stained with propidium iodide. Results Compared with both control, CO inhalation and intra- peritoneal infusion group at the same time point, the levels of PAF, ICAM-1, MDA, MPO, cell apoptosis rate and the phosphorylated p38 MAPK protein in LPS group were increased, while IL-10 and SOD were decreased （P〈0.05 or 0.01）, and accompanied by severe intestinal tract injury. There were no statistics differences at the different time point in the same group. PAF, ICAM-1, MDA, MPO and cell apoptosis rate in both LPS-FCO inhalation group and LPS-FCO intraperitoneal infusion group were lower, while IL-10 and SOD were higher than the corresponding value in LPS group at the same time point （all P〈0.05）, with ameliorate injury too, but the expression of phosphorylated p38 MAPK was further up-regulated than that of LPS group （all P〈0.05）. However, there were no significant differences in these parameters between LPS＋ CO inhalation group and LPS＋ CO intraperitoneal infusion group. Conclusion 250 ml/m^3 CO inhalation and intraperitoneal infusion exerts the similar protection against LPS induced rat intestinal tract injury via anti-oxidant, anti-inflammation, and anti-apoptosis. This may involve the p38 MAPK pathway.