一氧化碳吸入对急性肺损伤大鼠肺组织细胞凋亡的影响及其机制

The mechanisms of carbon monoxide inhalation on the apoptosis of pulmonary cells in rats with acute lung injury induced by lipopolysaccharide

目的观察吸入低浓度一氧化碳(CO)对脂多糖(LPS)致急性肺损伤(ALI)大鼠肺组织细胞凋亡的影响,探讨可能机制。方法18只雄性SD大鼠随机均分为3组。ALI组静脉滴注LPS5mg/kg,正常对照组注入生理盐水,CO吸入组在LPS诱导肺损伤后持续吸入体积分数为2.5×10-4CO。观察3h后放血处死,取肺组织,半定量逆转录聚合酶链反应(RT PCR)测定血红素氧化酶1(HO1)表达,酶联免疫吸附测定(ELISA)法检测肿瘤坏死因子α(TNFα)、白细胞介素6(IL6)和IL10的含量;化学比色法测定丙二醛(MDA)含量、髓过氧化物酶(MPO)和超氧化物歧化酶(SOD)活性;光镜观察并盲法评分比较肺组织学;流式细胞仪检测细胞凋亡。结果ALI组HO1mRNA、TNFα、IL6、IL10、MDA、MPO、SOD、细胞凋亡与正常对照组[1.002±0.004、(0.47±0.06)pg/mg蛋白、(0.49±0.12)pg/mg蛋白、(0.42±0.08)pg/mg蛋白、(0.79±0.14)nmol/mg蛋白、(6.0±1.0)U/mg蛋白、(74±7)U/mg蛋白、(0.12±0.03)%]比较差异均有统计学意义(P<0.05或<0.01),肺损伤严重。CO吸入组TNFα、IL6、MDA、MPO和细胞凋亡[(0.91±0.25)pg/mg蛋白、(0.64±0.05)pg/mg蛋白、(1.02±0.23)nmol/mg蛋白、(7.2±1.6)U/mg蛋白、(1.60±0.34)%]显著低于ALI组[(1.48±0.23)pg/mg蛋白、(1.16±0.26)pg/mg蛋白、(1.27±0.33)nmol/mg蛋白、(8.2±1.5)U/mg蛋白、(3.18±0.51)%,P均<0.05];HO1mRNA、IL10和SOD表达[5.433±0.921、(0.26±0.07)pg/mg蛋白、(60±10)U/mg蛋白]显著高于ALI组[3.081±0.823、(0.15±0.03)pg/mg蛋白、(51±7)U/mg蛋白,P均<0.05],肺损伤减轻。结论吸入CO通过调控氧化/抗氧化、促炎/抗炎反应、抑制过度细胞凋亡和上调HO1表达,可能对LPS诱导ALI起保护作用。

Objective To investigate the effects of carbon monoxide （CO） inhalation on the apoptosis of pulmonary cells in rats with acute lung injury（ALI） induced by lipopolysaccharide（LPS） and to investigate its mechanisms. Methods Eighteen male SD rats were randomly divided into three groups. The ALI group received LPS 5 mg/kg intravenously, while the control group received normal saline, and the CO inhalation group received an inhalation of 2. 5 × 10 ^-4 CO（V/V） after ALI induced by LPS 5 mg/kg. The animals were sacrificed by exsanguinations and lung tissue was harvested at 3 h of observation for determination of the heme oxygenase-1 （ HO-1 ） mRNA with semiquantitative reverse transcription-polymerase chain reaction （ RT-PCR）, the level of tumor necrosis factor-α（ TNF-α）, interlukin-6 （ IL-6 ） and IL-10 with enzyme-linked immunosorbent assay （ELISA） and the maleic dialdehyde （MDA） concentration, myeloperexidase（MPO） and superoxide dismutase（SOD） activity with chemical methods. The extent of cell apoptosis was observed by using the flow cytometric method. Results The change of the levels of HO-1 mRNA,TNF-α, IL-6, IL-10, MDA, MPO, SOD and apoptotic cells of the ALI group was significant as compared with the control group[ 1. 002 ± 0. 004, （0. 47 ± 0. 06） pg/mg prot, （0. 49 ± 0. 12） pg/mg plot, （0. 42 ± 0. 08 ） pg/mg plot, （ 0. 79 ± 0. 14 ） nmol/mg prot, （ 6. 0 ± 1.0 ） U/mg prot, （ 74 ± 7 ） U/mg plot, （0. 12 ±0. 03 ）% , P 〈 0. 05 or 〈 0. 01 ], and lung injury was severe. The levels of TNF-α, IL-6, MDA, MPO,and apoptotic cells of the CO inhalation group[ （0. 91 ±0. 25） pg/mg prot, （0. 64 ±0. 05） pg/mg plot, （ 1.02 ± 0. 23 ） nmol/mg plot, （ 7. 2 ± 1.6 ） U/mg plot, （ 1.60 ± 0. 34） % ] were decreased, while HO-1, IL- 10 and SOD expression [ 5. 433 ± 0. 921, （0. 26 ± 0. 07 ） pg/mg prot, （60 ± 10） U/rag prot ] were increased compared with the ALI group[ （ 1.48± 0. 23 ） pg/mg prot, （ 1.16 ± 0. 26） pg/mg prot, （ 1.27 ± 0. 33 ） nmol/ mg prot,（8. 2 ± 1.5）U/mg prot, （3. 18 ±0.51）% ,3.081 ±0. 823,（0. 15 ±0. 03）pg/mg prot,（51±7） U/ mg prot, P〈0.05 or 〈0.01] ,and lung injury was attenuated. Conclusion CO inhalation protects lung from injury by regulating oxidative/anti-oxidative and inflammatory/anti-inflammatory disorders, inhibiting excessive cell apoptosis and up-regulating HO-1 expression, which may play an important role in the pathogenesis of LPS-induced ALI.