硫化氢供体对动脉粥样硬化大鼠内皮素-1生成的影响

Suppressive effect of hydrogen sulfide donor on endothelin-1 production in aorta of atherosclerotic rats

目的 探讨硫化氢（H2S）供体NaHS对动脉粥样硬化（AS）大鼠血浆及主动脉内皮素-1（ET-1）的影响.方法 30只雄性SD大鼠随机分为AS组、AS＋ NaHS组及对照组,每组10只.采用维生素D3腹腔注射及高脂饲料喂养的方法制备AS大鼠模型.AS＋NaHS组大鼠腹腔注射NaHS56μmol/（kg·d）,余两组以生理盐水代替.8周后麻醉后处死大鼠,分别分离并留取血浆、主动脉及冠状动脉组织,以油红O染色观察大鼠主动脉根部和冠状动脉AS斑块的变化;硫电极法测定血浆H2S含量;放射免疫分析法检测血浆和主动脉ET-1含量,免疫组织化学法对主动脉ET表达进行定位检测;比色法测定血浆一氧化氮合成酶（NOS）及其亚型内皮型NOS（eNOS）、诱导型NOS （iNOS）的活性.结果 AS组、AS＋ NaHS组及对照组大鼠主动脉根部AS面积百分比分别为（11.6±3.3）％,（1.6±1.1）％,（0.0±0.1）％,组间比较差异有统计学意义（F=97.675,P＜0.05）.三组大鼠冠状动脉AS面积百分比分别为（21.4±5.7）％,（4.8±2.5）％,（0.0±0.0）％,组间比较差异有统计学意义（F=97.519,P＜0.05）.AS组大鼠血浆H2S含量低于AS＋ NaHS组和对照组[（22.0±3.1）比（33.3±6.2）、（27.9±1.0）μmol/L,P均＜0.05].AS组和AS＋ NaHS组大鼠血浆ET-1含量均高于对照组[（89.6±14.2）和（93.1±15.5）比（70.0±10.7）ng/L,P均＜0.05];AS组与AS＋ NaHS组差异无统计学意义（P＞0.05）.AS组和AS＋ NaHS组大鼠主动脉ET-1含量均高于对照组[（11.9±4.9）和（8.2±2.5）比（3.8±1.2） ng/g,P均＜0.05];AS＋ NaHS组低于AS组（P＜0.05）.免疫组织化学结果显示对照组与AS＋ NaHS组大鼠主动脉内皮细胞胞浆ET-1表达较弱,而AS组则为强阳性表达;AS组、AS＋ NaHS组、对照组大鼠血浆NOS总活性分别为（51.8±10.0）,（27.6±6.5）,（25.4±5.6）U/ml、eNOS活性分别为（4.5±2.7）,（8.7±3.9）,（15.3±6.2） U/ml、iNOS活性分别为（47.3±10.7）,（19.0±5.2）,（9.9±4.0）U/ml,三组间比较差异均有统计学意义（NOS总活性：F =37.231,P＜0.05、eNOS活性：F=14.600,P＜0.05及iNOS活性：F =72.131,P＜0.05）.结论 H2S供体NaHS抑制AS大鼠中AS斑块的形成,上述效应可能与其保护血管内皮细胞,减少ET-1生成有关.

Objective To examine the effect of H2S donor,sodium hydrosulfide （NaHS）,on ET-1 level in plasma and aorta in rats with atherosclerosis （AS）.Method Thirty male rats,weighting 200-220 g,were randomly divided into AS,AS ＋NaHS and control groups,n =10 in each group.Rats were given a single dose of vitamin D3 （700 000 U/kg） in the first three days and fed with a high-cholesterol diet for 8 weeks to induce AS.Rats in AS ＋ NaHS group were intraperitoneally injected with an H2S donor NaHS,at a dose of 56 μmol/（kg · d） for 8 weeks.At the end of the experiment for 8 weeks,all the rats were sacrificed.The plasma was collected and the aorta and coronary tissues were isolated.The atherosclerotic lesions in both aorta and coronary arteries were detected using oil red 0 method.H2S concentration in plasma was determined with sulfide-sensitive electrode method.ET-1 levels in plasma and aorta were calculated by radioimmunoassay kit and the localization of ET-1 in the aorta was detected by immunohistochemistry.Plasma nitric oxide synthase （NOS）,endothelial NOS （eNOS）,inducible NOS （iNOS） were detected with colorimetry.Result AS plaque area in root of aorta of rats in AS group,AS ＋ NaHS group and control group were （11.6 ± 3.3） %,（1.6 ± 1.1） %,（0.0 ± 0.1） % respectively.The difference in AS plaque area in root of aorta among the three groups was statistically significant （F =97.675,P 〈 0.05）.AS plaque area in coronary artery of rats in AS group,AS ＋ NaHS group and control group were （21.4 ± 5.7） %,（4.8 ± 2.5） %,（0.0 ± 0.0） % respectively.The difference in AS plaque area in coronary artery among the three groups was statistically significant （F =97.519,P 〈 0.05）.Plasma H2S level in rats of AS group （（22.0 ±3.1） μmol/L） was significantly lower than that of control group （（27.9 ± 1.0） μmol/L） and AS ＋ NaHS group （（33.3 ± 6.2） μmol/L,all P 〈 0.05）.Compared with control group （（70.0 ± 10.7） ng/L）,plasma ET-1 in rats of AS group （（89.6 ± 14.2） ng/L） and AS ＋ NaHS group （（93.1 ± 15.5） ng/L,P both 〈0.05） were increased.However,there was no significant difference in plasma ET-1 content in rats between AS ＋ NaHS group and AS group （P 〉 0.05）.Compared with control group （（3.8 ± 1.2） ng/g）,ET-1 content in aorta in rats of AS group （（11.9 ±4.9） ng/g） and AS ＋ NaHS group （（8.2 ±2.5） ng/g,both P 〈 0.05） were increased,and ET-1 content in aorta in rats of AS ＋ NaHS group was decreased compared with AS group （P 〈 0.05）.Immunochemistry results showed that ET expression in cytoplasm in aortic endothelial cells in rats of AS group was strengthened,while ET expression in rats of control group and AS ＋ NaHS group was weak.NOS activity of rats in control group,AS group and AS ＋ NaHS group was （25.4±5.6）,（51.8±10.0）and （27.6±6.5） U/ml,eNOS activity （15.3 ±6.2）,（4.5 ±2.7） and （8.7± 3.9） U/ml,and iNOS activity （9.9 ± 4.0）,（47.3 ± 10.7） and （19.0 ± 5.2） U/ml,respectively.Differences among the three groups were statistically significant （NOS activity：F =37.231,P 〈0.05,eNOS activity：F=14.600,P〈0.05,and iNOS activity：F=72.131,P〈0.05）.Conclusion H2S donor NaHS reduced the AS plaque in AS rats.The mechanisms might involve the protective effect of H2S on the vascular endothelial cell,decreasing ET-1 production in aortal endothelium of atherosclerotic rats.