新型气体信号分子硫化氢在大鼠肺纤维化发病中的作用

Effect of hydrogen sulfide on Bleomycin-induced pulmonary fibrosis in rats

目的:在博来霉素(Bleomycin,BLM)诱导的肺纤维化大鼠模型上,观察内源性胱硫脒-γ-裂解酶／硫化氢 (CSE／H2S)体系的变化以及应用外源性H2S供体硫氢化钠(NaHS)对肺纤维化的影响,以探讨内源性H2S在肺纤维化发病中的病理生理意义。方法:(1)雄性Wistar大鼠48只,随机分为对照组、肺纤维化组(经气管内滴入 BLMA5 5 mg／kg)、肺纤维化+NaHS低剂量组(腹腔注射NaHS 1．4μmol／kg,每日两次)和肺纤维化+NaHS高剂量组(腹腔注射NaHS 7μ1ol／kg,每日两次)。分别于第7天、第28天取肺组织行HE染色和Masson染色,用生化法测定丙二醛(MDA)、羟脯氨酸含量,采用敏感硫电极测定血浆H2S的生成量及亚甲基蓝法测定CSE活性,用半定量RT-PCR方法测定肺组织CSE的mRNA水平。(2)以羟自由基发生系统Fe2+(5mmoL／L)／H2O2(25 mmoL／L)单独及与不同浓度NaHS(10-5、5×10-5、10-4、10-3 mmol／L)共同孵育离体大鼠肺组织,测定MDA含量。结果:(1) 第7天纤维化组大鼠血浆H2S含量较对照组下降46％,第28天时增加25％(均P<0．01);肺组织CSE活性在第7 天时比对照组降低26％(P<0．01),第28天时CSE活性恢复,与对照组相比差异无统计学意义(P>0．05);第7 天和第28天纤维化组肺组织CSE mRNA含量较对照组分别升高34％和144％(P<0．01);第7天及第28天纤维化组肺组织MDA、羟脯氨酸含量均较对照组明显升高(P<0．01),NaHS高剂量组和低剂量组的MDA、羟脯氨酸含量均较纤维化组显著降低,高、低剂量组间差异无统计学意义(P>0．05)。(2)Fe2+／H2O2孵育组肺组织MDA含量较对照组明显升高,经给予不同浓度NaHS处理后,MDA含量均显著下降。结论:内源性CSE／H2S体系参与了大鼠肺纤维化的病理生理过程,外源性补充H2S可能通过减轻氧化应激抑制肺纤维化的发生。

Objective ： To explore the change of endogenous hydrogen sulfide and the effect of exogenously applied H2S on Bleomycin-induced pulmonary fibrosis in rats. Methods： （ 1 ） Forty-eight male Wistar rats were randomly divided into control group, fibrosis group, fibrosis ＋ NaHS-low group （ 1.4 μmol/kg, bid intraperitoneally） and fibrosis ＋ NaHS-high group （ 7.0 μmol/kg, bid intraperitoneally）. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin . The control group and pulmonary fibrosis group were injected with the same volume of normal saline. Pulmonary pathology changes were observed , the plasma H2S concentration, the activity of CSE in lung tissue and the contents of MDA, hydroxyproline were detected on the 7th and 28th days respectively after bleomycin administration. CSE mRNA was also measured using quantitative RT-PCR. （2） Isolated lung tissues were exposed to Fe^2＋~/H2O2 without or with different concentrations of NaHS （ 10^-5,5 × 10^-5,10^-4,10^-3 mmol/L） at 37℃ for 10 minutes , and the MDA content was assayed by biochemical method. Results： （1）Compared with rats in control group, the concentration of plasma H2S and the activity of CSE in lung tissues in fibrosis group decreased on day 7 （ all P 〈 0.01 ） ; On the 28th day, the level of plasma H2S increased clearly （P 〈 0.01 ） and the activity of CSE was ascendant without significant difference compared with that of control group. Relative CSE mRNA amounts in lung tissues increased 34% , 144% , respectively （ all P 〈0.01 ） in rats of fibrosis group compared with those of control group. The contents of MDA and hydroxyproline increased significantly in fibrosis group compared with those of control group on days 7, 28 （all P 〈0.01 ）. In NaHS-low group and NaHS-high group, the contents of MDA and hydroxyproline decreased markedly（ all P 〈0. O1 ）. （2）The contents of MDA in incubation of lung tissues at different concentrations of NaHS were all decreased as compared with those in incubation with Fe^2＋/H2O2 alone（all P 〈0.01 ）. When NaHS was at 10^-4 mol/L, the MDA content reversed to the lowest value, which had the highest value at 10^-3 mol/L of NaHS. Conclusion： Endogenous CSE / H2S pathway was involved in the pathogenesis of rat pulmonary fibrosis. Exogenously applied H2S could attenuate the process of pulmonary fibrosis possibly because of reducing oxidative stress.