野百合碱诱导的肺动脉高压大鼠内源性硫化氢生成的变化

Change of endogenous hydrogen sulfide pathway in the monocrotaline - induced pulmonary hypertensive rats

目的研究野百合碱（MCT）诱导肺动脉高压（PAH）大鼠内源性硫化氢（H2S）生成的变化。方法14只雄性Wistar大鼠按随机数字表法随机分为对照组及MCT组，每组7只。MCT组大鼠腹腔注射MCT（60 mg/kg）1次，对照组腹腔注射同剂量的9 g/L盐水1次，常规饲养21 d。采用右心导管测定肺动脉平均压（mPAP），称量并计算右心/（左心＋室间隔）比值[RV/（LV＋SP）]。大鼠肺组织石蜡包埋切片，苏木精-伊红（HE）染色后在光学显微镜下观察血管形态学变化，测量肺动脉相对中膜厚度（RMT）和相对中膜面积（RMA）。采用自由基测定仪测定大鼠血浆及肺组织H2S水平，采用Western blot法检测大鼠肺组织H2S生成关键酶胱硫醚-γ裂解酶（CSE）的表达，采用实时聚合酶链反应（RT-PCR）法检测大鼠肺组织CSE mRNA表达。结果与对照组相比，MCT组大鼠mPAP明显升高[（49.31±3.67） mmHg比（14.31±2.07） mmHg（1 mmHg＝0.133 kPa），P〈0.01]，RV/（LV＋SP）比值升高（0.43±0.03比0.21±0.03，P〈0.01），肺动脉RMT及RMA均升高[（43.46±1.94） μm比（13.16±1.48） μm，P〈0.01；（3 321.10±318.20） μm2比（963.40±127.26） μm2，P〈0.01]。肺血管显微结构观察结果显示：对照组大鼠肺动脉管壁薄，无炎性细胞浸润，而MCT组大鼠肺动脉的管壁明显增厚，炎性细胞浸润。与对照组相比，MCT组大鼠血浆及肺组织H2S水平均显著降低[（9.28±0.94） μmol/L比（14.20±1.21） μmol/L，P〈0.01；（0.43±0.08） μmol/g蛋白比（0.87±0.17） μmol/g蛋白，P〈0.01]，肺组织CSE蛋白表达降低（0.14±0.02比0.28±0.09，P〈0.01），CSE mRNA水平降低（0.84±0.06比1.12±0.04，P〈0.01）。结论MCT诱导PAH大鼠内源性H2S生成显著下调。

Objective To investigate the production of hydrogen sulfide （H2S） in rats with monocrotaline （MCT） -induced pulmonary hypertension （PAH）. Methods Fourteen male Wistar rats were randomly divided into 2 groups （7 cases for each group） ：control group and MCT group. Rats in the MCT group were intraperitoneally injected with MCT （60 mg/kg） on day 1 while rats in the control group received only the same volume of 9 g/L saline. And then the conventional breeding was given for 21 days. Mean pulmonary artery pressure （mPAP） was evaluated via right cardiac catheterization procedure. The ratio of right ventrieular mass （RV） and left ventrieular plus septal mass （LV ＋ SP） [ RV/（ LV ＋ SP） ] was calculated. The morphological change of pulmonary artery was observed by applying hematoxylineosin （HE） staining on the lung tissue paraffin section by measuring relative media thickness （RMT） and relative media area （RMA） of pulmonary aetery. H2 S contents in serum and lung tissue rat were detected by using free radicals detector. The expression of cystathionine - γ - lyase （ CSE ）, a key enzyme catalyzing endogenous H2S generaion, in lung tissue was detected by using Western blot method. CSE mRNA level in lung was detected by adopting the real - time polymerase chain reaction （ RT - PCR） method. Results Compared with control group, mPAP of rats in MCT group was increased significantly [ （49.31 ± 3.67 ） mmHg vs （ 14.31 ± 2.07 ） mmHg （ 1 mm Hg = 0.133 kPa）, P 〈 0.01 ] and the ratio of RV/（ LV ＋ SP） was increased （0.43 ±0. 03 vs 0. 21 ± 0.03, P 〈 0.01 ）, while RMT and RMA of pulmonary artery were enlarged [（43.46 ± 1.94） μm vs （13.16 ± 1.48） μm,P 〈0.01; （3 321.10 ± 318.20） μm2 vs （963.40 ± 127.26）μm2 ,P 〈0.01 ]. The evaluation of microstructure of pulmonary artery showed that the vessel wall of pulmonary artery in control rats was thin and no inflammatory cell was observed around artery. However, the vessel wall of pulmonary artery in rats of MCT group was thickened and there was evident infiltration of inflammatory cells in perivaseular area. Compared with control group, H2S contents in serum and lung tissue of rats in MCT group were markedly decreased [ （9.28 ± 0.94） μmol/L vs （ 14.20 ± 1.21 ） μmol/L, P 〈 0.01 ; （0.43 ± 0.08 ） μmol/g protein vs （0.87±0.17） μmol/g protein,P 〈0.01 ] ,while CSE protein and mRNA expression in lung tissue of rats in MCT group were downregulated （0.14 ± 0.02 vs 0. 28 ±0.09, P 〈 0.01 ;0.84± 0.06 vs 1.12 ± 0.04, P 〈0.01 ）. Conclusion Endogenous H2S pathway is significantly downregulated in rats with monocrotaline - induced pulmonary hypertension.