硫化氢在内毒素休克大鼠急性肺损伤中的作用及其与一氧化氮、一氧化碳的关系

目的 探讨硫化氢(H2S)在内毒素休克(ES)大鼠急性肺损伤(ALI)中的作用及一氧化氮(NO)、一氧化碳(CO)与其作用机制的关系.方法 将64只雄性SD大鼠随机分为对照组(经尾静脉注射生理盐水0.5 ml/kg)、脂多糖组(经尾静脉注射脂多糖复制ES模型)、脂多糖+硫氢化钠NaHS(外源性H2S供体)]组、脂多糖+炔丙基甘氨酸(PPG)组,每组16只.给药后连续监测平均动脉压(MAP)的变化,6 h后处死,测定肺系数;光镜观察肺组织形态学改变并观测肺损伤的定量评价指标(IQA);化学法检测血浆H2S含量、肺组织丙二醛、NO和CO含量、髓过氧化物酶(MPO)、胱硫醚-γ-裂解酶(CSE)、一氧化氮合酶(NOS)和血红素氧合酶(HO)活性;用免疫组织化学法和Western印迹检测肺组织诱生型NOS(iNOS)和HO-1蛋白表达.结果 脂多糖组大鼠MAP明显低于对照组,肺组织出现明显的结构损伤,脂多糖组大鼠IQA、肺系数、肺组织丙二醛含量、MPO活性、血浆H2S含量和肺组织CSE活性均明显高于对照组(P<0.05或P<0.01);脂多糖+NaHS组大鼠血浆H2S含量和肺组织CSE活性均高于脂多糖组,而血压低于脂多糖组,且大鼠肺损伤加重;脂多糖+PPG组大鼠血压高于脂多糖组,且大鼠肺损伤减轻(均P<0.05);脂多糖组肺组织中eNOS活性明显低于对照组(5.26±0.25)U·mg-1·prot-1vs(6.45±0.42)U·mg-1·prot-1],而iNOS活性和NO含量均高于对照组(12.6±0.6)U·mg-1·prot-1vs(10.5±0.7)U·mg-1·prot-1、(144±25)μmol/L vs(68±5)μmol/L,P<0.05或P<0.01];脂多糖+PPG组肺组织中eNOS活性明显高于脂多糖组,iNOS活性(10.2±0.4)U·mg-1·prot-1]、蛋白表达和NO含量(74±5)μmol/L]均明显低于脂多糖组(P<0.05或P<0.01),而脂多糖+NaHS组肺组织中eNOS活性(4.81±0.23)U·mg-1·prot-1]明显低于脂多糖组,iNOS活性(14.6±0.4)U·mg-1·prot-1]、蛋白表达和NO含量(217±18)μmol/L]均明显高于脂多糖组(P<0.05或P<0.01);脂多糖组肺组织中HO活性(173±31)pkat/g]、蛋白表达和CO含量(3.63±0.24)%]均明显高于对照组(125±22)pkat/g、(2.48±0.33)%,均P<0.05]和脂多糖+PPG组(88±17)pkat/g、(2.98±0.23)%,均P<0.05],而脂多糖+NaHS组肺组织中HO活性(263±37)pkat/g]、蛋白表达和CO含量(4.35±0.32)%]均明显高于脂多糖组(均P<0.05).结论 H2S生成增多参与了ES大鼠肺组织炎症损伤的发生,此作用与其引起的eNOS活性下降,iNOS活性升高及随后产生的大量NO有关;H2S可上调ES大鼠肺组织HO-1/CO体系,这可能是机体针对损伤产生的内源性的代偿性保护反应.

The role of hydrogen sulfide in acute lung injury during endotoxic shock and its relationship with nitric oxide and carbon monoxide

Objective To explore the role of hydrogen sulfide (H2S) in acute lung injury (ALI) during endotoxic shock (ES) and its relationship with nitric oxide (NO) and carbon monoxide (CO). Methods Sixty-four adult male SD rats were randomly divided into 4 equal groups: control group injected with normal saline via the caudal vein, lipopolysaccharide (LPS)-treated group injected with LPS to establish ES model, LPS + NariS group injected with LPS and sodium hydrosulfide ( NaHS, an exogenous H2S donor], and LPS + PPG group injected with LPS and polypropylene glycol ( PPG, a H2S synthase inhibitor). The mean artery pressure (MAP) was measured via apolyethylene catheter in the right common carotid artery for 6 h. Then the rats were sacrificed with their lungs taken out to determine the lung water content, lung tissue melonyldialdehyde (MDA), NO, and CO contents, as well as lung tissue cystathionine-γ-lyase ( CSE), myeloperoxidase ( MPO), nitric oxide synthase ( NOS), and heine oxygenase (HO) activities. The H2S content in blood plasma was detected also. Morphological changes of the lung tissues were observed under light microscope and the index of quantitative assessment (IQA) of lung injury was calculated. Immunohistochemistry and Western blotting were used to detect the lung tissue inducible NOS (iNOS) and HO-1 protein expression. Results Compared with the control group, the MAP of the LPS group was significantly lower, the pathological changes in lung tissue was more obvious, and the IQA, lung water content, lung MDA content, lung MPO and CSE activities as well as plasma H2S content were all significantly higher(P0. 05 or P 0. 01 ). Compared to the LPS group, the plasma H2S and lung CSE activity of the LPS + NariS group were higher, the lung injury was more severe, and the MAP was lower. And compared to the LPS group, the MAP of the LPS + PPG group was higher, and the lung injury was milder ( both P0. 05 ). The eNOS activity in the lung tissue of the LPS group was (5.26 ± 0. 25 ) U · mg-1 · prot-1, significantly lower than that of the control group (6. 45 ± 0. 42)U · mg-1 · prot-1> ] ; and the iNOS activity and NO content of the LPS group were ( 12.6 ± 0. 6) U · mg-1 · prot-1 and ( 144 ± 25)μmol/L respectively, both higher than those of the control group ( 10. 5±0. 7)U ·mg-1 · prot-1 and (68±5)mol/L respectively] ( P 0. 05 or P 0. 01 ). Compared with the LPS group, the lung tissue eNOS activity of the LPS + PPG group was significantly higher, and the iNOS activity ( 10. 2 ± 0.4) U· mg-1 · prot-1], iNOS protein expression, and NO content ( 74 ± 5 ) μmol/L] were all significantly lower (P0.05 or P 0. 01 ). Compared with the LPS group, the lung tissue eNOS activity of the LPS + NaHS group (4. 81 ± 0. 23)U · mg-1 · prot-1] was significantly lower, and the iNOS activity (14. 6 ±0. 4) U · mg-1 · prot-1 ], iNOS protein expression, and NO content ( 217 ± 18 ) μmol/L] were significantly higher (P0. 05 or P0. 01 ). The lung tissue HO activity (173 ± 31 )pkat/g], HO protein expression, and CO content (3.63 ± 0. 24) % ] of the LPS group were all significantly higher than those of the control group(125±22)pkat/g,(2.48±0.33)% ,both P0.05 ] , and the LPS +PPG group(88 ± 17) pkat/g, ( 2. 98 ± 0. 23 ) %, both P 0. 05 ]. Compared to the LPS group, the lung tissue HO activity (263 ± 37 ) pkat/g ], HO protein expression, and CO content ( 4. 35 ± 0. 32 ) % ] of the LPS + NaHS group were all significantly higher ( all P0. 05 ). Conclusion The increase of H2S generation participates in the lung tissue injury during ES and this event is related to eNOS activity decrease, iNOS activity increase that causes the production of large amount of NO. H2S up-regulates the HO-1/CO system in the lung tissues during ES, which may be the endogenous compensatory response against the injury.