硫化氢在大鼠急性支气管哮喘模型中的变化及意义

The regulatory effect of endogenous hydrogen sulfide on acute asthma

目的观察卵白蛋白(OVA)诱导的大鼠急性支气管哮喘(简称哮喘)模型,内源性硫化氢(H_2S)生成的变化以及应用外源性硫氢化钠(NaHS,H_2S 供体)处理对哮喘大鼠的影响,探讨气体信号分子 H_2S 在哮喘发病中的作用。方法 24只健康 SD 大鼠按随机数字表法分为正常对照组、哮喘组和 NaHS 干预组,每组8只。致敏后28 d 测定所有大鼠肺功能并观察大鼠支气管周围炎性细胞浸润程度并进行评分;采用敏感硫电极测定血浆及肺组织 H_2S 的生成量;采用酶促反应法测定大鼠肺组织匀浆中胱硫醚-γ-裂解酶(CSE)活性;用 Western blot 法测定大鼠肺组织中 CSE 蛋白含量(每组3只)。结果哮喘组大鼠呼气峰流量(PEF)、血浆及肺组织中 H_2S 分别为(2.90±0.70)L/s、(10±3)、(4.9±1.3)μmol/L,对照组分别为(6.50±0.10)L/s、(54±10)、(24.1±8.0)μmol/L,NaHS 干预组大鼠分别为(5.70±0.50)L/s、(17±4)、(15.3±4.0)μmol/L,3组间比较差异有统计学意义(F 值分别为112.13、110.10、27.34,P 均<0.01);哮喘组大鼠肺组织匀浆每毫克蛋白中 CSE 活性和肺组织匀浆中 CSE 蛋白含量[用相对吸光度(A)值表示]分别为(1.00±0.10)nmol·min^(-1)·mg^(-1)、0.20±0.10,正常对照组分别为(1.80±0.10)nmol·min^(-1)·mg^(-1)、0.90±0.30,NaHS 干预组大鼠分别为(1.60±0.20)nmol·min^(-1)·mg^(-1)、1.10±0.20,3组间比较差异有统计学意义(F 值分别为79.39、12.28,P 均<0.05);光镜下支气管周围炎性细胞浸润程度评分[用中位数(四分位数)]表示,正常对照组为1(0～1)分,哮喘组为3(2～4)分,NaHS 干预组为1(1～2)分,3组间比较差异有统计学意义(H=16.93,P<0.01);哮喘组肺组织 H_2S 含量与 PEF 呈正相关(r=0.74,P<0.01);与光镜下支气管周围炎性细胞浸润程度评分呈负相关(r=-0.64,P<0.01)。结论内源性 H_2S 参与了大鼠急性哮喘发病过程,外源性 NaHS 可以减轻哮喘气道炎症,对哮喘急性发病起到保护作用。

Objective To study the changes of endogenous hydrogen sulfide （H2 S） and the effect of exogenously applied H2S on ovalbumin-induced acute asthma. Methods Twenty-four Male SD rats were randomly divided into a control group （ n = 8 ）, an asthma group （ n = 8 ） and a Naris group （ n = 8 ）. Pulmonary function was measured, and the pulmonary pathology changes, the content of H2 S in lung tissue and plasma and the activity of H2S generating enzymes in lung tissue were detected at the 28 th day after ovalbumin administration. Western blotting was used to detect the endothelial cystathionine-γ-lyase CSE protein in the lung tissues. Results In the asthma group, the peak expiratory flow （PEF） was （2.90 ± 0. 70） L/s, the contents of H2S in the plasma was （10±3） μmol/L, in the lung tissue was （4. 9 ± 1.3） μmol/L. The H2S generating enzyme activity in the lung tissue of the asthma group was （ 1.00 ± 0. 10 ） nmol · min^ -1· mg^-1 pro, and the lung CSE content of the asthma group was significantly lower than that of the control group （6. 50 ± 0. 10） L/s, （54 ± 10）, （24. 1 ± 8.0） μmol/L, （ 1.80 ± 0. 10） nmol · min^ -1· mg^-1 ,F=112.13,110. 10 , 27. 34 , 79. 39 ,12. 28 , all P〈0.05）. The pulmonary pathology score of the asthma group was 3（2-4）, significantly higher than that of the control group [ 1 （0 - 1 ） ,H = 16. 93 ,P 〈 0. 01 ]. In the NariS group, the PEF was （5.70 ±0. 50） L/s, the content of H2S in the plasma was（ 17 ± 4） μmol/L, in the lung tissue was（ 15. 3± 4. 0） μmol/L, the H2S generating enzyme activity in the lung tissue was （1.60 ± 0. 20） nmol · min^-1 · mg^-1 pro, the lung CSE content of the NariS group was significantly higher than that of the asthma group （ F = 112. 13,110. 10,27. 34,79. 39,12. 28, all P 〈0. 05 ）. The pulmonary pathology score of the NariS group was 1 （ 1 - 2 ）, significantly lower than that of the asthma group [ 3 （2 - 4 ） score, H = 16. 93, P 〈 0. 01 ]. There was a significantly positive correlation between the content of the content of H2S in lung tissue and PEF （ r = 0. 74, P 〈 0. 01 ）. There was a significantly negative correlation between the content of H2S in lung tissue and the pulmonary pathology score （r = -0.64, P 〈 0. 01 ）. Conclusion Endogenous H2S is involved in the pathogenesis of asthma in this animal model. Exogenously applied H2S can attenuate inflammation of asthma and exert protective effect from asthma.