低浓度一氧化碳在防止大鼠小肠脂多糖诱导损伤中的保护作用

Low-concentration carbon monoxide protects against lipopolysaccharide-induced rat small intestine injury

目的:观察低浓度一氧化碳(CO)吸入和腹腔给予在防止脂多糖(LPS)诱导大鼠小肠损伤中的作用,探讨其可能的信号转导机制.方法:6组SD大鼠ip 5 mg/kg体质量LPS或等容量生理盐水1 h后,对照组(A)吸入室内空气,CO组(B)吸入体积分数为2.5×10^(-4)CO,CO腹腔组(C)腹腔通入体积分数为2.5×10^(-4)CO,LPS组(D)吸入室内空气,LPS+CO组(E)吸入体积分数为2.5×10^(-4)CO,LPS+CO腹腔组(F)腹腔通入体积分数为2.5×10^(-4)CO.观察3 h放血处死,取小肠,酶联免疫吸附法测定血小板活化因子(PAF)、细胞间黏附分子-1(ICAM-1)水平:化学比色法测定丙二醛(MDA)含量及髓过氧化物酶(MPO)活性;流式细胞仪测定细胞凋亡率;半定量逆转录聚合酶链反应测定血红素氧合酶-1(HO-1)mRNA,蛋白印迹法测定磷酸化p38 MAPK表达;光镜观察形态学变化.结果:E和F组PAF、ICAM-1、MDA、MPO的表达(0.87±0.18 ng/g,0.82±0.16 ng/g vs 1.15±0.21 ng/g;2.96±0.39 ng/g,2.69±0.23 ng/g vs 3.48±0.36 ng/g;1.74±0.17 mmol/g,1.71±0.24 mmol/g vs 2.75±0.76 mmol/g;35.34±14.67μkat/g,33.01±12.84μkat/g vs 68.01±18.67μkat/g;P<0.05)以及细胞凋亡率均明显低于D组(30.56%±6.33%,34.45%±5.77%vs 41.52%±3.36%;P<0.05),而HO-1 mRNA及磷酸化p38 MAPK的表达显著高于D组(6.29±1.56,7.21±1.78 vs 3.97±1.16,14219±1724,13774±1886 vs 10227±1312;P<0.05),E和F组小肠损伤较D组减轻,组间比较,差异无统计学意义.结论:低浓度CO吸入和腹腔给予通过抗氧化、抗炎、抑制细胞凋亡及上调HO-1表达,在防止大鼠小肠避免LPS诱导损伤中的保护作用;p38 MAPK可能参与CO保护作用的信号转导.

AIM： To observe the effects of the inhalation or intraperitoneal infusion of a low concentration of carbon monoxide （CO） on lipopolysaccharide （LPS）-induced rat small intestine injury, and to identify the roles of the p38 mitogen-activated protein kinase （p38 MAPK） pathway in these effects. METHODS： Sprague-Dawley rats with small intestine injury induced by a 5 mg/kg LPS intravenous injection or an equal volume of NS were divided into six groups： A, control group （inhalation of room air）; B, CO group （inhalation of 2.5 ×10^-4 V/V CO ）; C, CO intraperitoneal infusion group （intraperitoneal infusion of 2.5 ×10^-4 V/V CO）; D, LPS group （inhalation of room air）; E, LPS ＋ CO group （inhalation of 2.5 ×10^-4 V/V CO）; and F, LPS ＋ CO intraperitoneal infusion group （intraperitoneal infusion of 2.5 ×10^-4 V/V CO）. Rats were sacrificed by exsanguination and small intestinal tissues were homogenized for testing. The levels of platelet activator factor （PAF） and intercellular adhesion molecule-1 （ICAM-1） were determined by enzyme-linked immunosorbent assay. Maleic dialdehyde （MDA） content and myeloperoxidase （MPO） activity were determined by chemical methods. The extent of cell apoptosis was determined by flow cytometry. The expression level of the heme oxygenase-1 （HO-1） gene was analyzed by semiquantitative reverse transcription-polymerase chain reaction and the level of phosphorylated p38 mitogen-activated protein kinase （MAPK） activity was determined by Western blot. Pathology was determined by light microscopy. RESULTS： The levels of PAF, ICAM -1, MDA and MPO （P 〈 0.05）, and the rates of apoptosis, were lower in groups E （0.87 ±0.18 ng/g, 2.96 ±0.39 ng/g, 1.74 ±0.17 mmol/g, 35.34 ±14.67 μkat/g, 30.56% ±6.33%） and F （0.82 ±0.16 ng/ g, 2.69 ±0.23 ng/g, 1.71 ± 0.24 mmol/g, 33.01 ± 12.84 μkat/g, 34.45% ± 5.77%） than in group D （1.15 ±0.21 ng/g, 3.48 ±0.36 ng/g, 2.75 ± 0.76 mmol/g, 68.01 ± 18.67 μkat/g, 41.52% ＋ 3.36%, P 〈 0.05）. The levels of HO-1 mRNA and phosphorylated p38 MAPK were higher in groups E （6.29 ±1.56. 14 219 ± 1724） and F （7.21 ±1.78. 13 774 ±1886） than in group D （3.97 ±1.16. 10 227 ±1312; P 〈 0.05）. In contrast to group D rats, the small intestine injury in rats in groups E and F was ameliorated. There were no significant differences between groups E and F. CONCLUSION： Low-concentration CO inhalation and intraperitoneal infusion exert similar protection against LPS-induced rat small intestine injury via anti-oxidant, anti-inflammatory and antiapoptotic mechanisms, as well as through the upregulation of HO-1 expression. This may involve the p38 MAPK pathway.