硫化氢抗大鼠动脉粥样硬化作用研究

Effect of hydrogen sulfide on anti-atherosclerosis in rats

目的:探讨硫化氢(H2S)对大鼠动脉粥样硬化(AS)的作用及其机制。方法:体重(210±10)g的健康雄性SD大鼠125只,随机分为:对照组、AS模型组、AS+低剂量NaHS(2.8μmol/(kg.d))组、AS+中剂量NaHS(14μmol/(kg.d))组及AS+高剂量NaHS(28μmol/(kg.d))组。采用高脂饲料加大剂量VitD3注射复制大鼠AS模型。NaHS腹腔注射,连续用药12周。分别在喂养前及喂养后3、6、9、12周各处死动物。用生化分析仪检测血脂,去蛋白法检测血浆硫化氢,HE染色观察血管病理损伤程度及病变评分,免疫组化法检测血管组织中血管内皮生长因子(VEGF)的表达。结果:与相同时期的对照组相比,AS模型组在喂养后3、6、9、12周,血清甘油三脂(TG)和胆固醇(TC)均明显升高;主动脉病变评分从第6周到12周明显增加(P<0.01),并出现明显的动脉粥样硬化病变,表现为阳性区域的脂质斑块;血清H2S浓度明显降低,从喂养前的(44.98±2.06)μmol/L到第3、6、9、12周分别为(38.56±2.26),(32.96±2.38),(28.63±0.92),(23.55±0.92)μmol/L,并分别低于同时期各对照组的(44.72±0.85),(43.71±0.59),(41.96±0.97),(39.87±1.25)μmol/L(P<0.01);血管组织中VEGF的表达明显增强(P<0.01)。与模型组比较,低剂量NaHS组,各指标均无明显变化;中剂量NaHS组大鼠血清H2S含量于第6周开始明显高于模型组(36.13±0.73)vs(32.96±2.38)μmol/L,P<0.05;于9、12周时,分别为(33.07±1.14)vs(28.63±0.92)μmol/L,(30.16±0.62)vs(23.55±0.92)μmol/L,P<0.01;高剂量NaHS组大鼠血中H2S浓度于第3周开始到12周,分别为:(41.25±0.80),(38.71±0.46),(35.31±0.62),(33.38±0.78)μmol/L,均明显高于模型组(P<0.01);中、高剂量NaHS组血清TC均从第3周开始到12周明显降低(P<0.01),TG分别从第3、第6周开始到12周明显降低(P<0.05,P<0.01),血管组织病变评分与VEGF的表达均于第6周开始到12周明显降低(P<0.05)。相关分析显示血清中硫化氢的浓度与动脉粥样硬化的病变评分及血管VEGF的表达呈明显的负相关(r=-0.917,P<0.01,r=-0.885,P<0.01),而与血清甘油三脂和胆固醇之间无显著相关性。结论:动脉粥样硬化病变的形成与发展与内源性硫化氢的降低密切相关,补充外源性H2S可提高动脉粥样硬化大鼠血清中硫化氢浓度,减轻血管损伤程度,抑制VEGF的表达。

Objective：To investigate the effect of hydrogen sulfide（H2S） on artherosclerosis（AS） and its mechanism in rats.Mothods：125 healthy male SD rats of the weight （210 ±10）g were randomly divided into 5 groups：control group,AS model group,AS＋low-dose NaHS（2.8 μmol/（kg.d）） group,AS＋middle-dose（14 μmol/（kg.d）） NaHS group,AS＋ high-dose NaHS（28 μmol/（kg.d）） group.The atherosclerotic model was established by feeding high grease food and injecting large doses of VitD3.The rats were using NaHS by peritoneal injection for 12 weeks.5 rats were executed in each group before the experiment and in the weeks of 3,6,9,12 after the experiment,respectively.The blood fat was analyzed by automatic biochemistry analysator.H2S content in serum was detected by the method of deproteinization.The pathological damage of vessels was observed and scored by HE stain.The expression of VEGF in the vessel tissue was detected by immunohistochemistry staining.Results：Compared with the control group at contemporaneity,both serumal triglyceride（TG） and cholesterol（TC） increased significantly in the AS model group after rat feeded 3,6,9,12 weeks,and scores of the artery pathological damage also increased obviously from the 6th week to the 12th week（P〈0.01）,as well as artherosclerosis plaque appeared,displaying as lipid plaque in the positive part.The serumal H2S concentration decreased obviously,from （44.98±2.06）μmol/L of before feeding to （38.56±2.26）,（32.96±2.38）,（28.63±0.92）,（23.55±0.92）μmol/L of after feeding3,6,9,and 12 weeks,respectively,and lower than that of control at contemporaneity（44.72±0.85）,（43.71±0.59）,（41.96±0.97）,（39.87±1.25）μmol/L,respectively（P〈0.01）,and VEGF expression of the vascular tissue also increased （P〈0.01）.Compared with the AS model group,all of above indexes in rat of the low-dose of NaHS group did not appear any obvious change.The serumal H2S concentration in rat of the middle-dose NaHS began increase at the 6th week after rat feeded（36.13±0.73 vs 32.96±2.38 μmol/L,P〈0.05）,and continuously increased at the 9th and the 12th week（33.07±1.14 vs 28.63±0.92 μmol/L,30.16±0.62 vs 23.55±0.92 μmol/L;P〈0.01,respectively）.The serumal H2S concentration in high-dose NaHS groups,increased from the 3th to the 12th week （41.25±0.80,38.71±0.46,35.31±0.62,33.38±0.78 μmol/L,respectively,P〈0.01）.The rat serumal TC in both middle and high-dose NaHS groups,decreased from the 3th to the 12th week（P〈0.01）,and TG began decrease from the 3th and the 6th week to the 12th week after rat feeded,respectively（P〈0.05,P〈0.01）.Both of the pathological damage scores and the expression of VEGF decrease from the 6th week to the 12th week（P〈0.05）.The correlation analysis showed that H2S in serum had a negative correlation with both pathological damage scores（r=-0.917,P〈0.01） and the expression of VEGF（r=-0.885,P〈0.01）.But it had no obvious correlation with serumal TG and TC.Conclusion：The formation and development of artherosclerosis has a close correlation with the depressing of endogenous H2S.Administration of exogenous H2S could raise the H2S concentration of serum in artherosclerosis,which might improve the damage of vessels and inhibit the expression of VEGF.