同型半胱氨酸和硫化氢的动态平衡与肺动脉高压的相关性
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摘要
研究背景：肺动脉高压是先天性心脏病常见的并发症,尤其在左向右分流(体循环-肺循环)的患者中。肺血管持续暴露于增加的肺血流将导致肺血管结构重塑和功能障碍,引起肺血管阻力增加,最终导致分流不可逆并产生艾森曼格综合症。由此可见,在肺动脉高压的形成过程中,肺血管内皮细胞的功能障碍是一个重要的因素。目前,反应肺动脉高压严重程度的生化标记物包括贫血、低氧血症、尿酸水平的升高及C反应蛋白的水平,然而它们都是非特异性的生化标记物。当前指南推荐脑利钠肽和N端-脑利钠肽作为替代,但它们也只是在反映心衰严重程度方面优于肺动脉高压,在肺动脉高压的早期预防中没有起到主要的作用。由于缺乏合适的生化标记物,关于肺动脉高压的最优治疗策略目前还不明确,因此用于诊断和治疗肺动脉高压的新的生化标记物不容忽视。高同型半胱氨酸血症被认为是心血管事件独立的危险因子,与冠心病,心肌梗死,脑卒中,动脉粥样硬化及外周血管等疾病密切相关。相关研究表明,血浆中升高的同型半胱氨酸能够导致心肌细胞的凋亡,内皮细胞的增殖及促进炎症反应的产生,尽管大量的临床试验已证实高同型半胱氨酸血症是心血管疾病的致病因子,但是还没有相关证据表明在肺动脉高压的形成中,同型半胱氨酸是肺动脉高压的致病因子。血浆中升高的同型半胱氨酸的水平主要与同型半胱氨酸的代谢有关,在体内经两种代谢途径,一方面在亚甲基四氢叶酸还原酶的催化下生成S-腺苷甲硫氨酸,参与甲基化反应；另一方面通过转硫作用在胱硫醚-p-合成酶及胱硫醚-r-裂解酶的催化下生成硫化氢气体,它是继一氧化氮(NO)和一氧化碳(CO)后第三种气体信号分子。在早期,曾被认为是一种具有臭鸡蛋气味的有毒气体,因为它能阻滞细胞色素C氧化酶。最近的研究表明,硫化氢气体具有心血管系统的保护作用,如舒张血管平滑肌,降低血压,抑制血管平滑肌细胞的增殖,等等。然而硫化氢气体与肺动脉高压的相关性目前也未明确,并且对于凋亡的影响,研究不多,有学者认为,硫化氢抑制凋亡是由于它对线粒体的保护功能,还有学者认为,在凋亡的线粒体途径, caspase-3及MAP激酶被激活的过程中,它产生了促凋亡的效应。因此本研究的目的是探讨肺动脉高压的患儿血浆中同型半胱氨酸和硫化氢的变化水平及找到可能的生化标记物,研究同型半胱氨酸与硫化氢代谢通路中相关基因单核苷酸的多态性与肺动脉高压的相关性,寻找疾病的易感基因,同时在细胞水平上探讨同型半胱氨酸是否会诱导肺动脉内皮细胞的凋亡及相关机制,以及硫化氢气体的保护作用。
方法：选择2012.9-2013.4广东省心血管病研究所心儿科住院患儿158例(81例男性,77例女性；平均年龄3.32±4.44岁；年龄范围1月-19岁),包括17例房间隔缺损、122例室间隔缺损、8例动脉导管未闭、7例艾森曼格综合症、4例特发性肺动脉高压。入选标准为患有单一先天性心脏病(如房间隔缺损、室间隔缺损、动脉导管未闭)或特发性肺动脉高压及患有艾森曼格综合症的患儿,无G6PD病史、无遗传代谢疾病病史及肿瘤病史。排除标准为1)伴有心脏瓣膜疾病；2)伴有心肌病；3)伴有凝血功能障碍。入选的患儿分为病例组和对照组,其中对照组为具有单一先天性心脏病(如房间隔缺损、室间隔缺损、动脉导管未闭)无肺动脉高压的患儿81例,病例组为具有单一先天性心脏病(如房间隔缺损、室间隔缺损、动脉导管未闭)伴肺动脉高压的患儿或特发性肺动脉高压及患有艾森曼格综合症的患儿77例。肺动脉高压的诊断依据2009年欧洲心脏病学会(ESC)的指南,该指南定义为以右心导管检查静息状态下肺动脉的平均压力(mPAP)≥25mmHg,其中肺动脉高压组根据肺血管的阻力指数(PVRI)分为两个亚组,当肺血管的阻力指数>10wood被定义为梗阻性肺动脉高压,当肺血管的阻力指数>3wood而<10wood时被定义为动力性肺动脉高压。所有的患儿需行标准的右心导管的检查。荧光免疫偏振分析用来检测所有患儿血浆中同型半胱氨酸的水平,敏感的银硫电极法检测血浆中硫化氢的浓度。酶联免疫吸附试验检测代谢通路中相关代谢酶(亚甲基四氢叶酸还原酶、胱硫醚-p-合成酶、胱硫醚-r-裂解酶)的活性表达情况。放射免疫法测定血浆中叶酸和维生素B12的水平。选择同型半胱氨酸和硫化氢代谢途径中相关代谢酶亚甲基四氢叶酸还原酶、胱硫醚-p-合成酶及胱硫醚-r-裂解酶的基因：rs1021737、rs482843、rs1801131、rs1801133. rs121964962,五个SNP采用时间飞行质谱技术对SNP基因分型。根据关联分析评价基因型与肺动脉高压的相关性,同时采用单因素方差分析各组基因型与代谢酶活性表达情况的关系。在细胞水平上,对肺动脉内皮细胞采用慢病毒转染的方法及干扰RNA技术使胱硫醚-p-合成酶(CBS)基因和胱硫醚-r-裂解酶(CSE)基因沉默,在沉默的和非沉默的肺动脉内皮细胞中加入不同浓度的同型半胱氨酸和NaHS,采用CCK-8试剂盒检测肺动脉内皮细胞的生长活力,同时采用敏感的银硫电极法检测基因沉默的肺动脉内皮细胞和非基因沉默的肺动脉内皮细胞培养基中硫化氢的浓度水平。Hoechst33258、 Annexin V-PE及流式细胞仪检测同型半胱氨酸预处理的沉默组和非沉默组肺动脉内皮细胞的凋亡情况,同时在肺动脉内皮细胞中采用Western blot法检测凋亡途径的内质网应激途径中GRP78和CHOP的蛋白表达水平。在给予外源性硫化氢气体的供体(NaHS)后,再次检测沉默组和非沉默组肺动脉内皮细胞的凋亡情况及内质网应激途径中GRP78和CHOP的蛋白表达水平。
结果：1)在先天性心脏病的患儿中肺动脉高压组与无肺动脉高压组比较,两组的同型半胱氨酸的浓度、硫化氢气体的浓度、叶酸的含量、维生素B12的含量、血浆中胱硫醚-r-裂解酶的含量及亚甲基四氢叶酸合成酶的含量差异有统计学意义(所有P<0.05),而血浆中胱硫醚-p-合成酶的含量差异无统计学意义(P>0.05)。在肺动脉高压组中,患儿的同型半胱氨酸的含量比无肺动脉高压组高,硫化氢的浓度在肺动脉高压组中降低,两者呈负相关。同型半胱氨酸浓度的升高与代谢中辅助因子叶酸的含量升高,维生素B12的含量降低及代谢途径中亚甲基四氢叶酸还原酶(MTHFR)的活性降低有关。硫化氢气体浓度的降低与胱硫醚-r-裂解酶(CSE)的活性降低有关,与胱硫醚-p-合成酶(CBS)的活性无明显相关性。根据ROC曲线,以同型半胱氨酸的浓度诊断肺动脉高压有显著意义,ROC曲线下面积为0.799；面积的95%可信区间为(0.728,0.871)。同时在动力性肺动脉高压和梗阻性肺动脉高压的亚组分析中,用ROC曲线预测动力性和梗阻性肺动脉高压的指标,以同型半胱氨酸的浓度诊断梗阻性肺动脉高压有显著意义,ROC曲线下面积为0.849；面积的95%可信区间分别为(0.767,0.932)。2)对同型半胱氨酸和硫化氢代谢通路中相关代谢酶的基因单核苷酸多态性的关联分析表明,SNP rs482843(CSE)与rs1801133(MTHFR)与肺动脉高压有关联,P分别为0.034,0.006,并且在突变的rs482843(CSE)中,基因型GG/AA的OR值为4.493。SNP rs1021737和rs1801131与肺动脉高压没有关联。rs121964962没有发生基因突变。双变量相关分析析表明SNP rs1021737和SNP rs482843与血浆中CSE的表达水平有关；而SNP rs1801131和SNP rs1801133与血浆中的MTHFR的表达水平无关。3)Hoechst33258, Annexin V-PE和CCK-8实验证实了在沉默了胱硫醚-p-合成酶(CBS)基因和胱硫醚-r-裂解酶(CSE)基因和非沉默基因的肺动脉内皮细胞中,随着同型半胱氨酸浓度的升高,肺动脉内皮细胞的生长活力逐渐下降,而加入外源性的硫化氢气体供体(NaHS)后,肺动脉内皮细胞生长活力受抑制情况明显改善,表明同型半胱氨酸能诱导肺动脉内皮细胞产生凋亡,并且内生的硫化氢气体及外源性的硫化氢气体供体(NaHS)对肺动脉内皮细胞具有保护作用,其凋亡机制是通过内质网应激途径中CHOP蛋白上调实现的,蛋白免疫印迹实验证实CHOP及GRP78蛋白的表达水平上调,而硫化氢气体可以抑制CHOP及GRP78蛋白的表达。同时同型半胱氨酸对肺动脉内皮细胞的破坏与硫化氢气体对肺动脉内皮细胞的保护作用与同型半胱氨酸的浓度密切相关,当同型半胱氨酸处于低浓度时,同型半胱氨酸对肺动脉内皮细胞的破坏与硫化氢气体对肺动脉内皮细胞的保护作用处于平衡状态或者硫化氢气体对肺动脉内皮细胞的保护作用大于同型半胱氨酸对肺动脉内皮细胞的破坏作用,反之当同型半胱氨酸处于高浓度时,同型半胱氨酸对肺动脉内皮细胞的破坏作用大于硫化氢气体对肺动脉内皮细胞的保护作用。
结论：1)先天性心脏病的肺动脉高压患儿中,同型半胱氨酸的浓度升高,硫化氢的含量降低,两者呈负相关,与代谢途径中的亚甲基四氢叶酸还原酶及胱硫醚-r-裂解酶的活性降低有关,也与血浆中叶酸和维生素B12的水平有关。同型半胱氨酸的浓度可以作为预测肺动脉高压的诊断指标,同时亚组分析表明以同型半胱氨酸的浓度预测梗阻性肺动脉高压的敏感性和特异性最好。2)SNPrs482843(CSE)与rs1801133(MTHFR)与肺动脉高压有关联,并且rs482843(CSE)的基因型为GG可能是肺动脉高压的危险因素。3)同型半胱氨酸通过内质网应激途径中的CHOP途径诱导肺动脉内皮细胞的凋亡,并且硫化氢气体对肺动脉内皮细胞具有保护作用。临床研究表明在先天性心脏病造成的肺动脉高压的患儿中血浆中同型半胱氨酸的浓度小于30μmol/L,因此细胞学实验对于临床的意义在于该浓度范围的同型半胱氨酸并不会直接对肺动脉内皮细胞产生严重的破坏作用,而是由于硫化氢对肺动脉内皮细胞的保护作用下降,才引起同型半胱氨酸对肺动脉内皮细胞的破坏作用变得明显。
Background:Pulmonary hypertension (PH) is a frequent complication of congenital heart disease (CHD), particularly in patients with left-to-right (systemic-to-pulmonary) shunts. Persistent exposure of the pulmonary vasculature to increased blood flow and pressure may result in vascular remodeling and dysfunction. This leads to increased pulmonary vascular resistance (PVR) and, ultimately, to reversal of the shunt and development of Eisenmenger syndrome. Thus, pulmonary vascular endothelial dysfunction is an important factor in the development of pulmonary hypertension. Currently, emerging biomarkers reflecting the severity of PH include anemia, hypocarbia, elevated uric acid, and C-reactive protein levels. However, they are nonspecific markers. Current guidelines recommend brain natriuretic peptide and N-terminal fragment of pro-brain natriuretic peptide instead although they only reflect heart failure, which has no major role in the early prevention. Due to the lack of adaptive markers, optimal therapeutic intervention with combination therapy remains unclear. The need for novel biomarkers for prognosis and therapy cannot be overlooked, especially in the management of children with pulmonary hypertension. Hyperhomocysteinemia is an independent risk factor for cardiovascular events, especially atherosclerosis, ischemia-reperfusion injury and high blood pressure. It can lead to impaired endothelial function, inflammation, free radical formation, impaired angiogenesis, atherosclerosis and increased risk of stroke. A large number of experimental studies demonstrated that elevated homocysteine levels can lead to cardiomyocyte apoptosis, and promotion of endothelial cell proliferation and inflammatory response. Although, some clinical trials have demonstrated that hyperhomocysteinemia is the pathogenic factor involved in cardiovascular diseases. However, there is lack of clinical evidence whether hyperhomocysteinemia was a risk factor in pulmonary hypertension with congenital heart disease. In vivo, homocysteine can produce hydrogen sulfide through the transsulfuration pathway under the catalysis of cystathionine β-synthase (CBS) and cystathionine gamma-lyase(CSE).It is the third gas signaling molecule after NO and CO and considered as a rotten egg smell of toxic gas because of blockade of cytochrome C oxidase. Recent studies indicate that hydrogen sulfide plays an important role in cardiovascular protection, including vascular smooth muscle relaxation, reduced blood pressure, and inhibition of vascular smooth muscle proliferation. However, there are fewer studies of its effect on cell apoptosis, and the mechanism of apoptosis is unclear. Some authors consider that hydrogen sulfide inhibits apoptosis due to its protective effect on mitochondrial function. Others reported that hydrogen sulfide produces a proapoptotic effect during activation of the mitochondrial pathway of apoptosis induction, caspase-3, and MAP kinase family. Thus, the aim of the present study was to investigate the changes of homocysteine and hydrogen sulfide levels and find potential biomarkers for early prevention and treatment, the possible associations between single nucleotide polymorphisms involving methylenetetrahydrofolate reductase, cystathionine beta-synthase, and cystathionine gamma-lyase genes mediating the metabolic pathways of homocysteine and hydrogen sulfide, and pulmonary hypertension caused by congenital heart disease, and determine if homocysteine can induce apoptosis of pulmonary artery endothelial cells and related mechanisms. We also evaluated the role of hydrogen sulfide on inhibiting apoptosis and the potential molecular signaling pathways.
Methods:A total of158pediatric patients with congenital heart disease were enrolled in, including17with atrial septal defect (ASD);122with ventricular septal defect (VSD);8with patent ductus arteriosus (PDA);7with Eisenmenger syndrome; and4with idiopathic pulmonary hypertension at the Department of Pediatric Cardiology of Guangdong Provincial Cardiovascular Disease Research Institute, from September2012to April2013(81male,77female; mean age,3.32±4.44years; age range,1month to18years). Exclusion criteria included:1) cardiomyopathy;2) valvular heart disease; and3) coagulation dysfunction. The diagnosis of pulmonary hypertension fulfilled the criteria of the European Society of Cardiology (ESC) guidelines in2009.Pulmonary hypertension (PH) was defined as mean pulmonary artery pressure (PAPm)≥25mmHg The pulmonary hypertension group was divided into two subgroups, based on pulmonary vessel resistance index (PVRI). When the PVRI was more than10Wood units, it was defined as obstructive pulmonary hypertension. If the PVRI was more than3Wood units but less than10Wood units, it was defined as dynamic pulmonary hypertension. Standard right heart catheterizations were performed in all cases. The levels of homocysteine and hydrogen sulfide were detected with the techniques of fluorescence polarization immunoassay and sensitive silver-sulphur electrode, respectively. Enzyme-linked immunosorbent assay was used to determine the expressions of methylenetetrahydrofolate reductase (MTHFR), cystathionine β-synthase (CBS) and cystathionine gamma-lyase (CSE). Radioimmunoassays were used to obtain folic acid and vitamin B12levels. Time-of-flight mass spectrometry was used to genotype five single nucleotide polymorphisms:rs1021737, rs482843, rs1801131, rs1801133, rs121964962in the metabolic pathway of homocysteine and hydrogen sulfide. Multilinear regression and correlation analyses were used to correlate above genes and pulmonary hypertension. In the cell experiments, Hoechst33258, Annexin V-PE and Cell Counting Kit-8were used to detect the apoptotic morphological appearance of HPAECs and the role of hydrogen sulfide after homocysteine and NaHS treatment. Permanent CBS and CSE gene silencing with the method of lentivirus transfection in the HPAECs was also used to discuss the apoptotic morphological appearance of HPAECs and the role of hydrogen sulfide after homocysteine and NaHS treatment. Western blot was used to detect the expression levels of GRP78and CHOP.
Results:1) The Hcy, folic acid, vitamin B12, H2S levels between the PH and control groups showed statistical significance (all P<0.05).The OD value of MTHFR and CSE in the PH and case groups had statistical significance (P=0.002and0.002, respectively). However, the OD value of CBS had no statistical significance. Increased levels of homocysteine and decreased levels of hydrogen sulfide were significantly negatively correlated in PH with CHD. The underlying mechanism involved the decreased expressions of MTHFR and CSE along with vitamin B12deficiency. Based on the ROC curve, the Hcy showed significant difference in predicting pulmonary hypertension. The areas under the ROC curve were0.799, corresponding to the area of the95%CI (0.728,0.871).The ROC curve was also used to predict dynamic and obstructive pulmonary hypertension. Significant prognostic indicators in obstructive pulmonary hypertension was Hcy.. The areas under the ROC curve were0.849and the ranges of the95%CI (0.767,0.932).2) Correlation analysis showed that rs482843and rs1801133were significantly associated with pulmonary hypertension (P=0.034and P=0.006), while rs1021737and rs121964962, rs1801131were not (P>0.05). In the mutation of rs482843, the OR for genotype GG/AA was4.493. The rs1021737and rs482843were associated with the cystathionine gamma-lyase concentration (all P<0.05),while the rs1801131and rs1801133were not associated with the methylenetetrahydrofolate reductase concentration.3) Hoechst33258, Annexin V-PE and Cell Counting Kit-8demonstrated the apoptotic morphological appearance of HPAECs and the protection of hydrogen sulfide after homocysteine and NaHS treatment. Permanent CBS and CSE gene silencing with the method of lentivirus transfection in the HPAECs also demonstrated hydrogen sulfide had protective effects on HPAECs and can inhibit the damage of homocysteine to HPAECs. Interestingly, it is associated with the concentrations of homocysteine, when the concentrations of homocysteine were low level, the damage of homocysteine to HPAECs and protective effects of hydrogen sulfide maybe in a state of balance or protective effects of hydrogen sulfide was larger than the damage of homocysteine to HPAECs, on the contrary, the damage function of homocysteine to HPAECs was larger than the protective effect of hydrogen sulfide on HPAECs. Western blot also confirmed that endoplasmic reticulum stress pathway was significantly activated after exposing HPAECs to homocysteine. Homocysteine treatment caused a significant increase of the expressions of GRP78and CHOP, after adding NaHS, the expressions of GRP78and CHOP were reduced.
Conclusions:1) Increased levels of homocysteine and decreased levels of hydrogen sulfide were significantly negatively correlated in PH with CHD. The underlying mechanism involved the decreased expressions of MTHFR and CSE along with vitamin B12deficiency. Homocysteine is a potential biomarker to predict PH.2) The rs482843and rs1801133were associated with pulmonary hypertension. The rs482843for GG genotype significantly increased the risk of pulmonary hypertension caused by congenital heart disease.3) Homocysteine induced HPAECs apoptosis via the CHOP endoplasmic reticulum stress signal pathway and hydrogen sulfide has protective effects on HPAECs. Clinical data showed that homocysteine concentrations in patients with pulmonary hypertension caused by congential heart disease were about≤30μmol/L. The decrease in hydrogen sulfide's protective effects caused development of pulmonary hypertension, while homocysteine did not directly damage HPAECs.
方法：选择2012.9-2013.4广东省心血管病研究所心儿科住院患儿158例(81例男性,77例女性；平均年龄3.32±4.44岁；年龄范围1月-19岁),包括17例房间隔缺损、122例室间隔缺损、8例动脉导管未闭、7例艾森曼格综合症、4例特发性肺动脉高压。入选标准为患有单一先天性心脏病(如房间隔缺损、室间隔缺损、动脉导管未闭)或特发性肺动脉高压及患有艾森曼格综合症的患儿,无G6PD病史、无遗传代谢疾病病史及肿瘤病史。排除标准为1)伴有心脏瓣膜疾病；2)伴有心肌病；3)伴有凝血功能障碍。入选的患儿分为病例组和对照组,其中对照组为具有单一先天性心脏病(如房间隔缺损、室间隔缺损、动脉导管未闭)无肺动脉高压的患儿81例,病例组为具有单一先天性心脏病(如房间隔缺损、室间隔缺损、动脉导管未闭)伴肺动脉高压的患儿或特发性肺动脉高压及患有艾森曼格综合症的患儿77例。肺动脉高压的诊断依据2009年欧洲心脏病学会(ESC)的指南,该指南定义为以右心导管检查静息状态下肺动脉的平均压力(mPAP)≥25mmHg,其中肺动脉高压组根据肺血管的阻力指数(PVRI)分为两个亚组,当肺血管的阻力指数>10wood被定义为梗阻性肺动脉高压,当肺血管的阻力指数>3wood而<10wood时被定义为动力性肺动脉高压。所有的患儿需行标准的右心导管的检查。荧光免疫偏振分析用来检测所有患儿血浆中同型半胱氨酸的水平,敏感的银硫电极法检测血浆中硫化氢的浓度。酶联免疫吸附试验检测代谢通路中相关代谢酶(亚甲基四氢叶酸还原酶、胱硫醚-p-合成酶、胱硫醚-r-裂解酶)的活性表达情况。放射免疫法测定血浆中叶酸和维生素B12的水平。选择同型半胱氨酸和硫化氢代谢途径中相关代谢酶亚甲基四氢叶酸还原酶、胱硫醚-p-合成酶及胱硫醚-r-裂解酶的基因：rs1021737、rs482843、rs1801131、rs1801133. rs121964962,五个SNP采用时间飞行质谱技术对SNP基因分型。根据关联分析评价基因型与肺动脉高压的相关性,同时采用单因素方差分析各组基因型与代谢酶活性表达情况的关系。在细胞水平上,对肺动脉内皮细胞采用慢病毒转染的方法及干扰RNA技术使胱硫醚-p-合成酶(CBS)基因和胱硫醚-r-裂解酶(CSE)基因沉默,在沉默的和非沉默的肺动脉内皮细胞中加入不同浓度的同型半胱氨酸和NaHS,采用CCK-8试剂盒检测肺动脉内皮细胞的生长活力,同时采用敏感的银硫电极法检测基因沉默的肺动脉内皮细胞和非基因沉默的肺动脉内皮细胞培养基中硫化氢的浓度水平。Hoechst33258、 Annexin V-PE及流式细胞仪检测同型半胱氨酸预处理的沉默组和非沉默组肺动脉内皮细胞的凋亡情况,同时在肺动脉内皮细胞中采用Western blot法检测凋亡途径的内质网应激途径中GRP78和CHOP的蛋白表达水平。在给予外源性硫化氢气体的供体(NaHS)后,再次检测沉默组和非沉默组肺动脉内皮细胞的凋亡情况及内质网应激途径中GRP78和CHOP的蛋白表达水平。
结果：1)在先天性心脏病的患儿中肺动脉高压组与无肺动脉高压组比较,两组的同型半胱氨酸的浓度、硫化氢气体的浓度、叶酸的含量、维生素B12的含量、血浆中胱硫醚-r-裂解酶的含量及亚甲基四氢叶酸合成酶的含量差异有统计学意义(所有P<0.05),而血浆中胱硫醚-p-合成酶的含量差异无统计学意义(P>0.05)。在肺动脉高压组中,患儿的同型半胱氨酸的含量比无肺动脉高压组高,硫化氢的浓度在肺动脉高压组中降低,两者呈负相关。同型半胱氨酸浓度的升高与代谢中辅助因子叶酸的含量升高,维生素B12的含量降低及代谢途径中亚甲基四氢叶酸还原酶(MTHFR)的活性降低有关。硫化氢气体浓度的降低与胱硫醚-r-裂解酶(CSE)的活性降低有关,与胱硫醚-p-合成酶(CBS)的活性无明显相关性。根据ROC曲线,以同型半胱氨酸的浓度诊断肺动脉高压有显著意义,ROC曲线下面积为0.799；面积的95%可信区间为(0.728,0.871)。同时在动力性肺动脉高压和梗阻性肺动脉高压的亚组分析中,用ROC曲线预测动力性和梗阻性肺动脉高压的指标,以同型半胱氨酸的浓度诊断梗阻性肺动脉高压有显著意义,ROC曲线下面积为0.849；面积的95%可信区间分别为(0.767,0.932)。2)对同型半胱氨酸和硫化氢代谢通路中相关代谢酶的基因单核苷酸多态性的关联分析表明,SNP rs482843(CSE)与rs1801133(MTHFR)与肺动脉高压有关联,P分别为0.034,0.006,并且在突变的rs482843(CSE)中,基因型GG/AA的OR值为4.493。SNP rs1021737和rs1801131与肺动脉高压没有关联。rs121964962没有发生基因突变。双变量相关分析析表明SNP rs1021737和SNP rs482843与血浆中CSE的表达水平有关；而SNP rs1801131和SNP rs1801133与血浆中的MTHFR的表达水平无关。3)Hoechst33258, Annexin V-PE和CCK-8实验证实了在沉默了胱硫醚-p-合成酶(CBS)基因和胱硫醚-r-裂解酶(CSE)基因和非沉默基因的肺动脉内皮细胞中,随着同型半胱氨酸浓度的升高,肺动脉内皮细胞的生长活力逐渐下降,而加入外源性的硫化氢气体供体(NaHS)后,肺动脉内皮细胞生长活力受抑制情况明显改善,表明同型半胱氨酸能诱导肺动脉内皮细胞产生凋亡,并且内生的硫化氢气体及外源性的硫化氢气体供体(NaHS)对肺动脉内皮细胞具有保护作用,其凋亡机制是通过内质网应激途径中CHOP蛋白上调实现的,蛋白免疫印迹实验证实CHOP及GRP78蛋白的表达水平上调,而硫化氢气体可以抑制CHOP及GRP78蛋白的表达。同时同型半胱氨酸对肺动脉内皮细胞的破坏与硫化氢气体对肺动脉内皮细胞的保护作用与同型半胱氨酸的浓度密切相关,当同型半胱氨酸处于低浓度时,同型半胱氨酸对肺动脉内皮细胞的破坏与硫化氢气体对肺动脉内皮细胞的保护作用处于平衡状态或者硫化氢气体对肺动脉内皮细胞的保护作用大于同型半胱氨酸对肺动脉内皮细胞的破坏作用,反之当同型半胱氨酸处于高浓度时,同型半胱氨酸对肺动脉内皮细胞的破坏作用大于硫化氢气体对肺动脉内皮细胞的保护作用。
结论：1)先天性心脏病的肺动脉高压患儿中,同型半胱氨酸的浓度升高,硫化氢的含量降低,两者呈负相关,与代谢途径中的亚甲基四氢叶酸还原酶及胱硫醚-r-裂解酶的活性降低有关,也与血浆中叶酸和维生素B12的水平有关。同型半胱氨酸的浓度可以作为预测肺动脉高压的诊断指标,同时亚组分析表明以同型半胱氨酸的浓度预测梗阻性肺动脉高压的敏感性和特异性最好。2)SNPrs482843(CSE)与rs1801133(MTHFR)与肺动脉高压有关联,并且rs482843(CSE)的基因型为GG可能是肺动脉高压的危险因素。3)同型半胱氨酸通过内质网应激途径中的CHOP途径诱导肺动脉内皮细胞的凋亡,并且硫化氢气体对肺动脉内皮细胞具有保护作用。临床研究表明在先天性心脏病造成的肺动脉高压的患儿中血浆中同型半胱氨酸的浓度小于30μmol/L,因此细胞学实验对于临床的意义在于该浓度范围的同型半胱氨酸并不会直接对肺动脉内皮细胞产生严重的破坏作用,而是由于硫化氢对肺动脉内皮细胞的保护作用下降,才引起同型半胱氨酸对肺动脉内皮细胞的破坏作用变得明显。
Background:Pulmonary hypertension (PH) is a frequent complication of congenital heart disease (CHD), particularly in patients with left-to-right (systemic-to-pulmonary) shunts. Persistent exposure of the pulmonary vasculature to increased blood flow and pressure may result in vascular remodeling and dysfunction. This leads to increased pulmonary vascular resistance (PVR) and, ultimately, to reversal of the shunt and development of Eisenmenger syndrome. Thus, pulmonary vascular endothelial dysfunction is an important factor in the development of pulmonary hypertension. Currently, emerging biomarkers reflecting the severity of PH include anemia, hypocarbia, elevated uric acid, and C-reactive protein levels. However, they are nonspecific markers. Current guidelines recommend brain natriuretic peptide and N-terminal fragment of pro-brain natriuretic peptide instead although they only reflect heart failure, which has no major role in the early prevention. Due to the lack of adaptive markers, optimal therapeutic intervention with combination therapy remains unclear. The need for novel biomarkers for prognosis and therapy cannot be overlooked, especially in the management of children with pulmonary hypertension. Hyperhomocysteinemia is an independent risk factor for cardiovascular events, especially atherosclerosis, ischemia-reperfusion injury and high blood pressure. It can lead to impaired endothelial function, inflammation, free radical formation, impaired angiogenesis, atherosclerosis and increased risk of stroke. A large number of experimental studies demonstrated that elevated homocysteine levels can lead to cardiomyocyte apoptosis, and promotion of endothelial cell proliferation and inflammatory response. Although, some clinical trials have demonstrated that hyperhomocysteinemia is the pathogenic factor involved in cardiovascular diseases. However, there is lack of clinical evidence whether hyperhomocysteinemia was a risk factor in pulmonary hypertension with congenital heart disease. In vivo, homocysteine can produce hydrogen sulfide through the transsulfuration pathway under the catalysis of cystathionine β-synthase (CBS) and cystathionine gamma-lyase(CSE).It is the third gas signaling molecule after NO and CO and considered as a rotten egg smell of toxic gas because of blockade of cytochrome C oxidase. Recent studies indicate that hydrogen sulfide plays an important role in cardiovascular protection, including vascular smooth muscle relaxation, reduced blood pressure, and inhibition of vascular smooth muscle proliferation. However, there are fewer studies of its effect on cell apoptosis, and the mechanism of apoptosis is unclear. Some authors consider that hydrogen sulfide inhibits apoptosis due to its protective effect on mitochondrial function. Others reported that hydrogen sulfide produces a proapoptotic effect during activation of the mitochondrial pathway of apoptosis induction, caspase-3, and MAP kinase family. Thus, the aim of the present study was to investigate the changes of homocysteine and hydrogen sulfide levels and find potential biomarkers for early prevention and treatment, the possible associations between single nucleotide polymorphisms involving methylenetetrahydrofolate reductase, cystathionine beta-synthase, and cystathionine gamma-lyase genes mediating the metabolic pathways of homocysteine and hydrogen sulfide, and pulmonary hypertension caused by congenital heart disease, and determine if homocysteine can induce apoptosis of pulmonary artery endothelial cells and related mechanisms. We also evaluated the role of hydrogen sulfide on inhibiting apoptosis and the potential molecular signaling pathways.
Methods:A total of158pediatric patients with congenital heart disease were enrolled in, including17with atrial septal defect (ASD);122with ventricular septal defect (VSD);8with patent ductus arteriosus (PDA);7with Eisenmenger syndrome; and4with idiopathic pulmonary hypertension at the Department of Pediatric Cardiology of Guangdong Provincial Cardiovascular Disease Research Institute, from September2012to April2013(81male,77female; mean age,3.32±4.44years; age range,1month to18years). Exclusion criteria included:1) cardiomyopathy;2) valvular heart disease; and3) coagulation dysfunction. The diagnosis of pulmonary hypertension fulfilled the criteria of the European Society of Cardiology (ESC) guidelines in2009.Pulmonary hypertension (PH) was defined as mean pulmonary artery pressure (PAPm)≥25mmHg The pulmonary hypertension group was divided into two subgroups, based on pulmonary vessel resistance index (PVRI). When the PVRI was more than10Wood units, it was defined as obstructive pulmonary hypertension. If the PVRI was more than3Wood units but less than10Wood units, it was defined as dynamic pulmonary hypertension. Standard right heart catheterizations were performed in all cases. The levels of homocysteine and hydrogen sulfide were detected with the techniques of fluorescence polarization immunoassay and sensitive silver-sulphur electrode, respectively. Enzyme-linked immunosorbent assay was used to determine the expressions of methylenetetrahydrofolate reductase (MTHFR), cystathionine β-synthase (CBS) and cystathionine gamma-lyase (CSE). Radioimmunoassays were used to obtain folic acid and vitamin B12levels. Time-of-flight mass spectrometry was used to genotype five single nucleotide polymorphisms:rs1021737, rs482843, rs1801131, rs1801133, rs121964962in the metabolic pathway of homocysteine and hydrogen sulfide. Multilinear regression and correlation analyses were used to correlate above genes and pulmonary hypertension. In the cell experiments, Hoechst33258, Annexin V-PE and Cell Counting Kit-8were used to detect the apoptotic morphological appearance of HPAECs and the role of hydrogen sulfide after homocysteine and NaHS treatment. Permanent CBS and CSE gene silencing with the method of lentivirus transfection in the HPAECs was also used to discuss the apoptotic morphological appearance of HPAECs and the role of hydrogen sulfide after homocysteine and NaHS treatment. Western blot was used to detect the expression levels of GRP78and CHOP.
Results:1) The Hcy, folic acid, vitamin B12, H2S levels between the PH and control groups showed statistical significance (all P<0.05).The OD value of MTHFR and CSE in the PH and case groups had statistical significance (P=0.002and0.002, respectively). However, the OD value of CBS had no statistical significance. Increased levels of homocysteine and decreased levels of hydrogen sulfide were significantly negatively correlated in PH with CHD. The underlying mechanism involved the decreased expressions of MTHFR and CSE along with vitamin B12deficiency. Based on the ROC curve, the Hcy showed significant difference in predicting pulmonary hypertension. The areas under the ROC curve were0.799, corresponding to the area of the95%CI (0.728,0.871).The ROC curve was also used to predict dynamic and obstructive pulmonary hypertension. Significant prognostic indicators in obstructive pulmonary hypertension was Hcy.. The areas under the ROC curve were0.849and the ranges of the95%CI (0.767,0.932).2) Correlation analysis showed that rs482843and rs1801133were significantly associated with pulmonary hypertension (P=0.034and P=0.006), while rs1021737and rs121964962, rs1801131were not (P>0.05). In the mutation of rs482843, the OR for genotype GG/AA was4.493. The rs1021737and rs482843were associated with the cystathionine gamma-lyase concentration (all P<0.05),while the rs1801131and rs1801133were not associated with the methylenetetrahydrofolate reductase concentration.3) Hoechst33258, Annexin V-PE and Cell Counting Kit-8demonstrated the apoptotic morphological appearance of HPAECs and the protection of hydrogen sulfide after homocysteine and NaHS treatment. Permanent CBS and CSE gene silencing with the method of lentivirus transfection in the HPAECs also demonstrated hydrogen sulfide had protective effects on HPAECs and can inhibit the damage of homocysteine to HPAECs. Interestingly, it is associated with the concentrations of homocysteine, when the concentrations of homocysteine were low level, the damage of homocysteine to HPAECs and protective effects of hydrogen sulfide maybe in a state of balance or protective effects of hydrogen sulfide was larger than the damage of homocysteine to HPAECs, on the contrary, the damage function of homocysteine to HPAECs was larger than the protective effect of hydrogen sulfide on HPAECs. Western blot also confirmed that endoplasmic reticulum stress pathway was significantly activated after exposing HPAECs to homocysteine. Homocysteine treatment caused a significant increase of the expressions of GRP78and CHOP, after adding NaHS, the expressions of GRP78and CHOP were reduced.
Conclusions:1) Increased levels of homocysteine and decreased levels of hydrogen sulfide were significantly negatively correlated in PH with CHD. The underlying mechanism involved the decreased expressions of MTHFR and CSE along with vitamin B12deficiency. Homocysteine is a potential biomarker to predict PH.2) The rs482843and rs1801133were associated with pulmonary hypertension. The rs482843for GG genotype significantly increased the risk of pulmonary hypertension caused by congenital heart disease.3) Homocysteine induced HPAECs apoptosis via the CHOP endoplasmic reticulum stress signal pathway and hydrogen sulfide has protective effects on HPAECs. Clinical data showed that homocysteine concentrations in patients with pulmonary hypertension caused by congential heart disease were about≤30μmol/L. The decrease in hydrogen sulfide's protective effects caused development of pulmonary hypertension, while homocysteine did not directly damage HPAECs.
引文
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[24]Janosik M, Sokolova J, Janosikova B, et al. Birth prevalence of homocystinuria in Central Europe:frequency and pathogenicity of mutation c.1105C>T (p.R369C) in the cystathionine beta-synthase gene[J]. J Pediatr.2009,154(3):431-437.
[25]Kohli U, Arora S, Kabra M, et al. Prevalence of MTHFR C677T polymorphism in north Indian mothers having babies with Trisomy 21 Down syndrome[J]. Downs Syndr Res Pract.2008,12(2):133-137.
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[29]Yin M, Dong L, Zheng J, et al. Meta analysis of the association between MTHFR C677T polymorphism and the risk of congenital heart defects[J]. Ann Hum Genet. 2012,76(1):9-16.
[30]Li X H, Du JB, Bu D F, et al. Sodium hydrosulfide alleviated pulmonary vascular structural remodeling induced by high pulmonary blood flow in rats[J]. Acta Pharmacol Sin.2006,27(8):971-980.
[31]张欣,王颖,王燕飞,等.肺动脉高压新生儿血浆硫化氢变化的意义[J].实用儿科临床杂志.2008,23(6)：423-424.
[32]Mustafa A K, Gadalla M M, Sen N, et al. H2S signals through protein S-sulfhydration[J]. Sci Signal.2009,2(96):a72.
[33]Humbert M, Montan iD, Perros F,et al. Endothelial cell dysfunction and cross talk between endothelium and smooth muscle cells in pulmonary arterial hypertension [J].Vascul Pharmacol.2008,49(4-6):113-118
[34]Wen YD,Wang H,Kho SH,et al. Hydrogen sulfide protects HUVECs against hydrogen peroxide induced mitochondrial dysfunction and oxidative stress[J].PLoS One.2013;8(2):e53147.
[35]Yang G, Sun X, Wang R.Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen-activated protein kinases and caspase-3[J].FASEB.2004;18(14):1782-4.
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[1]Kocer B, Engur S, Ak F, et al. Serum vitamin B12, folate, and homocysteine levels and their association with clinical and electrophysiological parameters in multiple sclerosis[J]. J Clin Neurosci.2009,16(3):399-403.
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[24]Janosik M, Sokolova J, Janosikova B, et al. Birth prevalence of homocystinuria in Central Europe:frequency and pathogenicity of mutation c.1105C>T (p.R369C) in the cystathionine beta-synthase gene[J]. J Pediatr.2009,154(3):431-437.
[25]Kohli U, Arora S, Kabra M, et al. Prevalence of MTHFR C677T polymorphism in north Indian mothers having babies with Trisomy 21 Down syndrome[J]. Downs Syndr Res Pract.2008,12(2):133-137.
[26]Bhargava S, Ali A, Parakh R, et al. Higher incidence of C677T polymorphism of the MTHFR gene in North Indian patients with vascular disease[J]. Vascular.2012, 20(2):88-95.
[27]A Z C, Yang Y, Zhang S Z, et al. Single nucleotide polymorphism C677T in the methylenetetrahydrofolate reductase gene might be a genetic risk factor for infertility for Chinese men with azoospermia or severe oligozoospermia[J]. Asian J Androl. 2007,9(1):57-62.
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[31]张欣,王颖,王燕飞,等.肺动脉高压新生儿血浆硫化氢变化的意义[J].实用儿科临床杂志.2008,23(6)：423-424.
[32]Mustafa A K, Gadalla M M, Sen N, et al. H2S signals through protein S-sulfhydration[J]. Sci Signal.2009,2(96):a72.
[33]Humbert M, Montan iD, Perros F,et al. Endothelial cell dysfunction and cross talk between endothelium and smooth muscle cells in pulmonary arterial hypertension [J].Vascul Pharmacol.2008,49(4-6):113-118
[34]Wen YD,Wang H,Kho SH,et al. Hydrogen sulfide protects HUVECs against hydrogen peroxide induced mitochondrial dysfunction and oxidative stress[J].PLoS One.2013;8(2):e53147.
[35]Yang G, Sun X, Wang R.Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen-activated protein kinases and caspase-3[J].FASEB.2004;18(14):1782-4.
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