高血压与脑血管病—RAS成份与缺血性脑卒中的关联
南京医科大学附属脑科医院 (100029)
高血压是脑血管病的首要危险因素。在中国，80%的脑血管发生与高血压有关，其中86%的脑出血和71%的脑梗死患者都有高血压病史；而无症状性高血压，发生脑血管病的机率是血压正常者的4倍。而且，无论是收缩压或舒张压升高，脑血管病发生的危险性都明显增大。
肾素-血管紧张素系统(Renin-Angiotensin-System, RAS)是机体重要的激素调节系统，广泛存在于人体内，其中约15%存在于血液循环中，85%存在于组织如血管壁、心脏、中枢、肾脏等中。在正常情况下，它对正常的心血管系统发育，维持心血管功能稳态、电解质和体液平衡以及血压调节起重要的作用。
RAS的链式反应包括：肾素(Ren)把血管紧张素原(Ang)转化为血管紧张素I(AT-I)，再由血管紧张素转化酶(ACE)将后者转化为血管紧张素II(AT-II)，然后通过和血管紧张素II受体Ⅰ亚型(ATⅠR)结合而发挥作用。当高血压时，RAS被过度激活，整个链式反应的产物AT-II生成过多，对于高血压及高血压相关疾病的发生发展产生有害作用，并可能直接对各组织、器官损害。
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为此，我们对RAS各成份与缺血性脑血管病发生之间联系进行了系列研究。
一、资料与方法
根据1995年全国第四届“脑血管疾病会议修订的诊断标准”和“脑卒中患者临床神经功能缺损程度评分标准”，经头颅CT或MRI检查确诊，收集发病72 h内的脑卒中患者包括动脉粥样硬化性血栓性脑梗死(atherothrombotic cerebral infarction, ACI)患者87例，男45例，女42例，平均 (68.9±6.4) 岁；腔隙性梗死(lacunar infarction, LI)患者102例，男60例，女42例，平均 (66.8±9.1) 岁；并收集67例血管性痴呆(vascular dementia, VaD)患者，男37例，女30例，平均 (65.7±6.9) 岁；住院和门诊原发性高血压(essential hypertension, EH)患者93例，男44例，女39例，平均年龄 (62.4±8.3) 岁；同时选择收集江苏省体检中心的年龄及性别匹配的健康体检者95例作为正常对照(healthy controls, HC)组，男54例，女41例，平均年龄 (65.7±8.4) 岁，均排除心脑血管病、糖尿病、肝肾疾病、肿瘤、炎症及血液病等。各组受试者年龄及性别组间比较无显著性差异。
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以放射免疫法检测受试者血浆Ang和AT-II浓度(对患者急性期为发病3 d内，缓解期为发病3周后分别时间段取血检测)。
以高压毛细管电泳法测定受试者血浆ACE活性水平。
以多聚酶链反应-限制性片段长度多态性(PCR-RFLP)方法分析Ang基因第2外显子T704C及C521T碱基变异多态性、及AT1R基因3’非翻译区(untranslating region, UTR)的A1166C碱基变异的多态性；对ACE基因2个定量性状位点(quantitative trait loci, QTLs)：16内含子插入/缺失(I/D)片段多态性和3’非翻译区(UTR)4656(CT)2/3多态性分别以多聚酶链反应-扩增片段长度多态性PCR-AFLP(PCR-AFLP)方法和以多聚酶链反应-变性梯度凝胶电泳(PCR-DGGE)方法分析。
二、结果
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(一)RAS各成份血水平在缺血性卒中的变化
1. 急性期ACI患者血浆Ang浓度显著增高：
急性期血浆Ang浓度(194.47±43.83ng/L)明显高于对照组(159.34±45.24ng/L) (t=12.34，P<0.001)及恢复期(163.45±42.64ng/L) (t=11.06，P<0.001)，而恢复期与对照组差异无显著性意义(t=0.96，P>0.05)。
2. ACI患者急性期血浆AT-Ⅱ浓度显著增高：
患者急性期血浆AT-Ⅱ浓度(37.52±12.11ng/L)显著高于其恢复期(29.70±8.70ng/L)及健康对照者(21.34±4.76ng/L)(均P<0.05)，恢复期AT-Ⅱ浓度仍高于对照组(P<0.05)；
, 百拇医药 3. ACI患者血浆Ang浓度和AT-Ⅱ浓度增高程度与其病情轻重呈正相关：
ACI患者入院时血浆Ang浓度随病情轻重而增高，轻(0-15分)、中(16-30分)和重度(31-45分)时血浆Ang浓度分别为173.84±33.87ng/L、215.83±30.19ng/L和236.91±48.53ng/L；且血浆AT-Ⅱ浓度与患者神经功能缺损程度呈直线正相关(r=0.63，P<0.001)。
4. 受试者血浆AT-Ⅱ浓度及与Ang浓度呈直线正相关：
ACI患者及正常对照组血浆两种指标浓度均呈直线正相关(r=0.68，P<0.05；r=0.61，P<0.05)。
5. 急性ACI患者血浆ACE活性水平显著降低，而慢性脑血管病的血管性痴呆(VaD)患者血ACE活性水平降低不明显：
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与健康对照者(22.62±5.44 U/L)比较，ACI患者血清ACE活性(19.26±5.11 U/L)显著降低(P<0.05)，尤其是伴发高血压患者(18.42±5.20 U/L)；而VaD患者血ACE活性(21.95±7.19 U/L)虽亦稍低于HC受试者，但无统计学意义。
(二)RAS各成份基因多态性与缺血性脑卒中的关联
1. Ang基因多态性
1.1 Ang基因第2外显子704CC基因型及C等位基因频率在ACI及EH受试者显著高于对照人群(表1)；ACI患者中伴或不伴高血压与Ang基因第2外显子T704C碱基变异无关(表2)：
1.2 Ang基因第2外显子521位点T等位基因频率在LI患者中显著增高(表3)：
1.3 Ang基因第2外显子704位点C等位基因决定血浆Ang高浓度，而C521T位点碱基变异与血浆Ang浓度变化无关：
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对照者中，2外显子704位点CC基因型的血浆Ang浓度(196.34±44.37ng/L)显著高于CT型(170.41±43.64ng/L)(t=2.74，P<0.01)及TT型(146.45±21.43ng/L) (t=3.47，P<0.01)；而C521T位点CC基因型的血浆Ang浓度(185.53±44.37ng/L)与CT型(170.41±42.84ng/L)相比无显著差异(t=0.785，P>0.05)。
2. ACE基因多态性
2.1 ACI患者中ACE基因第16内含子D等位基因频率显著增高(表4)：
2.2 本组受试者中未发现ACE基因3’UTR4656位点(CT)2/3多态性。
2.3 不同ACE基因型者血浆ACE活性及AT-Ⅱ浓度比较：
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各ACE基因型者血浆ACE活性比较表明D等位基因决定血ACE活性高水平：依次为DD型(27.61±5.32U/L)>D/I型(23.24±4.94U/L)>II型(19.17±3.69U/L)；而血浆AT-Ⅱ浓度依次为DD型(32.04±12.90ng/L)>D/I型(24.91±9.08ng/L)>II型(17.18±10.48ng/L)。
3. ATⅠR基因多态性
3.1 AT1R基因3’UTR的A1166C基因型及等位基因频率在受试者中的分布中，仅EH患者的C等位基因频率显著增高：
与对照组比较，原发性高血压组AC基因型频率56.8%及C等位基因频率28.4%明显高于对照组，见表5。
3.2 ACI患者(恢复期)A1166C基因型与血浆AT-Ⅱ浓度的关系：
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AA基因型的血浆AT-Ⅱ浓度(28.41±8.73ng/L)与AC型(25.80±9.62ng/L)相比无显著差异(t=0.43，P>0.05)。
3.3 脑血管病组中AT1R基因A1166C变异的相关分析
以AT1R基因为完全等级离散变量，采用Spearman相关分析方法及双尾检验，结果未发现AT1R基因与收缩压、舒张压、Glu、TG、Cho、HDL及LDL之间有相关性。
三、结论
1. ACI患者急性期时，血浆Ang和AT-Ⅱ浓度可应急性增高，Ang是通过AT-Ⅱ发挥作用；且此成份改变与缺血性脑损伤程度有关，可反映病情轻重程度，有急性反应物作用。
2. Ang基因第2外显子704位点C等位基因与ACI发病存在内在联系。而此联系并非简单由于C等位基因决定血浆Ang高水平所致，因为ACI恢复期时血浆Ang水平降至正常；尽管C等位基因频率在EH患者中增高，但C等位基因与ACI患者伴否高血压无关。
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3. Ang基因第2外显子C521T多态性对血浆Ang浓度变化无影响作用，但却与LI发病存在内在联系，而此与是否致高血压无关。
4. 同许多研究相似，本研究也得出ACE基因第16内含子D等位基因与ACI发病密切关联，是ACI的危险因素。虽然D等位基因决定血浆ACE高活性，但ACI急性期时血浆ACE活性并不增高，反明显降低，且与脑组织损伤程度有关。此表明D等位基因与ACI发病的内在联系并非简单缘于ACE在RAS中作用，可能与脑内RAS独立作用或通过其他途径所致。而ACI急性期血ACE活性降低的原因可能与损伤脑组织释放如IL、TNF等细胞因子对ACE活性抑制以及脑、体循环RAS差异等因素有关，这也为ACI急性期ACE拮抗剂的使用带来新思考。
5. 作为定量性状位点的ACE基因3’非翻译区4656(CT)2/3多态性在中国人中并明显。
6. AT1R基因与RAS主要活性物质AT-Ⅱ血内水平无关联性，与缺血性卒中发生与与否无关，但与高血压发病有相关性。
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7. 综合以上结论，似表明RAS成份中Ang、ACE分子水平与缺血性卒中存在潜在联系，而此联系并非通过RAS对血管缩舒功能的影响作用实现。RAS成份分子水平的变化与缺血性卒中的易感性有关。
Hypertention and stroke
—The association of RAS components with ischemic stroke
ZHANG Yingdong
Nanjing Brain Hospital, Nanjing Medical University
It is widely accepted that hypertension is the major risk factor for stroke. In China, 80% the occurrence of stroke associates with the hypertension. The epidemic studies showed that the 86% patients with cerebral hemorrhage and 71% patients with cerebral infarction have the histories of hypertension. Both systolic and diastolic hypertension will augment the stroke risk.
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RAS, as an important system of hormone regulation, exists in the body, 15% in the blood circulation and 85% in the tissues such as vessel walls, heart, CNS and kidney, etc. In the normal condition, RAS plays important roles in the development and function of cardiovascular system, and in the regulation of blood pressure.
In the chain-reaction of RAS components, angiotensinogen (Ang) is changed firstly to angiotensin-I (AT-I) by the action of rennin (Ren), and then angiotensin-I converting enzyme (ACE) converts AT-I to AT-II. The binding of AT-II to angiotensinⅡ typeⅠreceptor (ATⅠR) plays the vasoactive effect of RAS. When high blood pressure occurring, RAS is over activated, so excessive AT-II is produced, and is associated with the development of hypertension and its related disorders, and may be harmful to some tissues and organs.
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Therefore, we will show our series of studies on the association of the components of RAS with ischemic stroke as follows.
Materials and Methods
According to the “stroke diagnostic criteria” and “clinical score of neurological deficits” formulated by the 4th National Conference of Cerebrovascular Disorders in 1995, and their CT/MRI features, 87 patients with atherothrombotic cerebral infarction (ACI), 102 patients with lacunar infarction (LI), 67 patients with vascular dementia (VaD) and 93 patients with essential hypertension (EH) were recruited consecutively in this study. Meanwhile, 95 healthy unrelated subjects were selected as the healthy control (HC) group. The ages and genders among the every group were matched and not of significant difference.
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The plasma Ang and AT-II concentrations were measured by means of radioimmunoassay (RI).
The high-voltage capillary-electrophoresis (CEP) was employed to analyze the plasma ACE activity.
The polymorphisms of T704C and C521T base-variation in the 2nd exon of Ang gene, of A1166C base-variation in 3’ untranslating region (UTR) were determined by the methods of polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP). The 2 quantitative trait loci (QTLs): insertion/deletion (I/D) polymorphism in the 6th intron and 4656(CT)2/3 polymorphism in the 3’ UTR of ACE gene were determined by means of polymerase chain reaction – amplifying fragment length polymorphism (PCR-AFLP) and polymerase chain reaction –denaturing gradient gel electrophoresis (PCR-DGGE), respectively.
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RESULTS
1. The change of RAS components in ischemic stroke
1.1 The plasma Ang concentration was increased significantly in the acute phase of ACI patients:
The plasma Ang concentration (194.47±43.83ng/L) in the acute phase (within 72 hours after ictus)of ACI patients was significantly higher than that in the healthy controls(159.34±45.24ng/L)(t=12.34, P<0.001) and that in the recovery phase (3 weeks later after ictus) of ACI cases (163.45±42.64ng/L) (t=11.06, P<0.001).
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1.2 Plasma AT-Ⅱ concentration was increased significantly in the acute phase of ACI patients:
The plasma AT-Ⅱ concentration in the acute phase of ACI patients ((37.52±12.11ng/L) markedly higher than that in the recovery phase of the patients (29.70±8.70ng/L) and that the healthy controls (21.34±4.76ng/L) (both P<0.05). That concentration in the recovery phase was also higher than that in the controls (P<0.05).
1.3 The plasma Ang and AT-Ⅱ concentrations correlated with the degree of neurological deficits in the ACI patients:
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At the admission of ACI patients, their plasma Ang concentrations were 173.84±33.87ng/L when in mild degree (score of 1-15), 215.83±30.19ng/L when in moderate degree (score 16-30) and 236.91±48.53ng/L when in severe degree (score 31-45), respectively. And the plasma AT-Ⅱ concentrations related positively to the clinical scores of the patients (r=0.63, P<0.001).
1.4 The plasma AT-Ⅱ concentration was linear correlated positively with the plasma Ang concentration in both ACI cases and controls (r=0.68，P<0.05；r=0.61，P<0.05, respectively).
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1.5 The plasma ACE activity decreased significantly in the acute phase of ACI patients, but did not changed in the VaD cases, a kind of chronic cerebrovascular disorders.
Compared to that in the controls (22.62±5.44U/L), the plasma ACE activity in the acute ACI cases (19.26±5.11U/L) was significantly reduced (P<0.05), especially in the patients complicated with hypertension (18.42±5.20U/L), while did not changed significantly in the VaD patients (21.95±7.19U/L).
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2. The gene polymorphisms of RAS components in ischemic stroke
2.1 Ang gene polymorphism
2.1.1 The frequencies of 704CC genotype and C allele of Ang gene exon 2 in ACI cases and EH cases were significantly higher than those in the controls (Table 1). There was, however, no difference between the ACI cases complicated with and without hypertension (table 2).
2.1.2 The frequencies of 521T allele of Ang gene exon 2 in LI cases were significantly higher (table 3).
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2.1.3 The C allele at 704 locus of Ang gene exon 2 determined increased plasma Ang concentration, while the base-variation of C521T had no effect on the plasma Ang concentration in this study:
In the controls, the plasma Ang concentration in the subjects with CC genotype at 704 locus (196.34±44.37ng/L) markedly higher than that with CT genotype (170.41±43.64ng/L) (t=2.74, P<0.01) and that with TT genotype (146.45±21.43ng/L) (t=3.47, P<0.01). However, the plasma Ang concentration in the subjects with CC genotype at 521 locus (185.53±44.37ng/L) was not different significantly from that with CT genotype (170.41±42.84ng/L) (t=0.785, P>0.05).
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2.2 ACE gene polymorphism
2.2.1 The frequency of D allele at ACE gene 16th intron was significantly increased in the ACI patients (table 4).
2.2.2 The (CT)2/3 polymorphism at ACE gene 3’UTR locus was not found in the subjects of this study.
2.2.3 The comparisons of plasma ACE activity and AT-Ⅱ concentration among the different ACE genotypes:
The comparison of plasma ACE activity between the subjects with different ACE genotypes indicated that D allele determined the increased activity of plasma ACE: i.e. DD genotype (27.61±5.32U/L) >D/I genotype (23.24±4.94U/L) > II genotype (19.17±3.69U/L) in order. As to the plasma AT-Ⅱ concentration, DD genotype (32.04±12.90ng/L) >D/I genotype (24.91±9.08ng/L) > II genotype (17.18±10.48ng/L) in order.
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2.3 ATⅠR gene polymorphism
2.3.1 The frequency of C allele at ATⅠR gene 3’UTR A1166C locus was significantly increased in EH patients compared to that in other subjects:
Comparison to those in other groups, the frequencies of AC genotypes (56.8%) and C allele (28.4%) in the EH cases increased significantly (table 5).
2.3.2 The association of ATⅠR gene polymorphism with plasma AT-Ⅱ concentration:
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The plasma AT-Ⅱ concentration in the ACI patients with AA genotype (28.41±8.73ng/L) was similar to that with AC (25.80±9.62ng/L) (t=0.43, P>0.05).
2.3.3 The correlation analysis ATⅠR gene A1166C polymorphism in the patients with stroke:
With ATⅠR gene as whole ranking discrete variable, Spearman correlation analysis and double-tail analysis have shown no correlation of ATⅠR gene with systolic pressure, diastolic pressure, glucose, triglyceride, cholesterol, HDL and LDL.
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Conclusions
1. At the acute phase of ACI, plasma Ang and AT-Ⅱ can increase responsively, as the reactants. Those changes are related to the degree of the ischemic cerebral injuries, and can reflect the severity of cerebral infraction.
2. The 704 C allele at Ang gene exon 2 relates potentially to the occurrence of ACI. This association may be not attributed to the determination of C allele on the increased level of plasma, since the plasma Ang level will be back to the normal level when ACI in the recovery phase. The C allele have no relation to the complication of ACI patients with hypertension or not, although its frequency is increased in EH cases.
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3. The C521T polymorphism of Ang gene exon 2 has no effect on plasma Ang level, but potentially associate with lacunar infarction, which is independent on its effects on blood regulation.
4. Similar to many other studies, the D allele of ACE gene 16th intron is closely associated with ACI, and regarded as the risk factor for ischemic stroke. Although D allele determines the increased plasma ACE activity, the plasma ACE activity is not increased, but reduced in the acute phase of ACI, and is associated with the degree of cerebral injury, which indicates that the association of D allele with ACI can not be attributed simply to the ACE effect on RAS, but to the independent role in cerebral RAS or other way of RAS. The reason of the reduction of plasma ACE activity in the acute phase of ACI maybe relates to the increased release of interleukins (Ils) and tumor necrosis factor (TNF), which inhibit the activity of ACE, from the ischemic cerebral lesions, and the difference between brain and body RASs, etc. These results bring about a new view of ACE inhibitor administration in acute phase of ACI.
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5. The polymorphism of 4656(CT) 2/3 in ACE gene 3’UTR, one of ACE gene quantitative trait loci, maybe not exist in the Chinese population.
6. AT1R gene may be not of association with plasma AT-Ⅱ level, the major active compound, and not of ischemic stroke, but to hypertension at all.
7. On the whole, this serial study shows that the RAS components Ang and AT-Ⅱ associate potentially at molecular level with ischemic stroke, the effect of which is not dependent of the role of RAS on vascular function. The molecular variation of RAS components may be the susceptible for the ischemic stroke., http://www.100md.com(张颖冬)
高血压是脑血管病的首要危险因素。在中国，80%的脑血管发生与高血压有关，其中86%的脑出血和71%的脑梗死患者都有高血压病史；而无症状性高血压，发生脑血管病的机率是血压正常者的4倍。而且，无论是收缩压或舒张压升高，脑血管病发生的危险性都明显增大。
肾素-血管紧张素系统(Renin-Angiotensin-System, RAS)是机体重要的激素调节系统，广泛存在于人体内，其中约15%存在于血液循环中，85%存在于组织如血管壁、心脏、中枢、肾脏等中。在正常情况下，它对正常的心血管系统发育，维持心血管功能稳态、电解质和体液平衡以及血压调节起重要的作用。
RAS的链式反应包括：肾素(Ren)把血管紧张素原(Ang)转化为血管紧张素I(AT-I)，再由血管紧张素转化酶(ACE)将后者转化为血管紧张素II(AT-II)，然后通过和血管紧张素II受体Ⅰ亚型(ATⅠR)结合而发挥作用。当高血压时，RAS被过度激活，整个链式反应的产物AT-II生成过多，对于高血压及高血压相关疾病的发生发展产生有害作用，并可能直接对各组织、器官损害。
, 百拇医药
为此，我们对RAS各成份与缺血性脑血管病发生之间联系进行了系列研究。
一、资料与方法
根据1995年全国第四届“脑血管疾病会议修订的诊断标准”和“脑卒中患者临床神经功能缺损程度评分标准”，经头颅CT或MRI检查确诊，收集发病72 h内的脑卒中患者包括动脉粥样硬化性血栓性脑梗死(atherothrombotic cerebral infarction, ACI)患者87例，男45例，女42例，平均 (68.9±6.4) 岁；腔隙性梗死(lacunar infarction, LI)患者102例，男60例，女42例，平均 (66.8±9.1) 岁；并收集67例血管性痴呆(vascular dementia, VaD)患者，男37例，女30例，平均 (65.7±6.9) 岁；住院和门诊原发性高血压(essential hypertension, EH)患者93例，男44例，女39例，平均年龄 (62.4±8.3) 岁；同时选择收集江苏省体检中心的年龄及性别匹配的健康体检者95例作为正常对照(healthy controls, HC)组，男54例，女41例，平均年龄 (65.7±8.4) 岁，均排除心脑血管病、糖尿病、肝肾疾病、肿瘤、炎症及血液病等。各组受试者年龄及性别组间比较无显著性差异。
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以放射免疫法检测受试者血浆Ang和AT-II浓度(对患者急性期为发病3 d内，缓解期为发病3周后分别时间段取血检测)。
以高压毛细管电泳法测定受试者血浆ACE活性水平。
以多聚酶链反应-限制性片段长度多态性(PCR-RFLP)方法分析Ang基因第2外显子T704C及C521T碱基变异多态性、及AT1R基因3’非翻译区(untranslating region, UTR)的A1166C碱基变异的多态性；对ACE基因2个定量性状位点(quantitative trait loci, QTLs)：16内含子插入/缺失(I/D)片段多态性和3’非翻译区(UTR)4656(CT)2/3多态性分别以多聚酶链反应-扩增片段长度多态性PCR-AFLP(PCR-AFLP)方法和以多聚酶链反应-变性梯度凝胶电泳(PCR-DGGE)方法分析。
二、结果
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(一)RAS各成份血水平在缺血性卒中的变化
1. 急性期ACI患者血浆Ang浓度显著增高：
急性期血浆Ang浓度(194.47±43.83ng/L)明显高于对照组(159.34±45.24ng/L) (t=12.34，P<0.001)及恢复期(163.45±42.64ng/L) (t=11.06，P<0.001)，而恢复期与对照组差异无显著性意义(t=0.96，P>0.05)。
2. ACI患者急性期血浆AT-Ⅱ浓度显著增高：
患者急性期血浆AT-Ⅱ浓度(37.52±12.11ng/L)显著高于其恢复期(29.70±8.70ng/L)及健康对照者(21.34±4.76ng/L)(均P<0.05)，恢复期AT-Ⅱ浓度仍高于对照组(P<0.05)；
, 百拇医药 3. ACI患者血浆Ang浓度和AT-Ⅱ浓度增高程度与其病情轻重呈正相关：
ACI患者入院时血浆Ang浓度随病情轻重而增高，轻(0-15分)、中(16-30分)和重度(31-45分)时血浆Ang浓度分别为173.84±33.87ng/L、215.83±30.19ng/L和236.91±48.53ng/L；且血浆AT-Ⅱ浓度与患者神经功能缺损程度呈直线正相关(r=0.63，P<0.001)。
4. 受试者血浆AT-Ⅱ浓度及与Ang浓度呈直线正相关：
ACI患者及正常对照组血浆两种指标浓度均呈直线正相关(r=0.68，P<0.05；r=0.61，P<0.05)。
5. 急性ACI患者血浆ACE活性水平显著降低，而慢性脑血管病的血管性痴呆(VaD)患者血ACE活性水平降低不明显：
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与健康对照者(22.62±5.44 U/L)比较，ACI患者血清ACE活性(19.26±5.11 U/L)显著降低(P<0.05)，尤其是伴发高血压患者(18.42±5.20 U/L)；而VaD患者血ACE活性(21.95±7.19 U/L)虽亦稍低于HC受试者，但无统计学意义。
(二)RAS各成份基因多态性与缺血性脑卒中的关联
1. Ang基因多态性
1.1 Ang基因第2外显子704CC基因型及C等位基因频率在ACI及EH受试者显著高于对照人群(表1)；ACI患者中伴或不伴高血压与Ang基因第2外显子T704C碱基变异无关(表2)：
1.2 Ang基因第2外显子521位点T等位基因频率在LI患者中显著增高(表3)：
1.3 Ang基因第2外显子704位点C等位基因决定血浆Ang高浓度，而C521T位点碱基变异与血浆Ang浓度变化无关：
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对照者中，2外显子704位点CC基因型的血浆Ang浓度(196.34±44.37ng/L)显著高于CT型(170.41±43.64ng/L)(t=2.74，P<0.01)及TT型(146.45±21.43ng/L) (t=3.47，P<0.01)；而C521T位点CC基因型的血浆Ang浓度(185.53±44.37ng/L)与CT型(170.41±42.84ng/L)相比无显著差异(t=0.785，P>0.05)。
2. ACE基因多态性
2.1 ACI患者中ACE基因第16内含子D等位基因频率显著增高(表4)：
2.2 本组受试者中未发现ACE基因3’UTR4656位点(CT)2/3多态性。
2.3 不同ACE基因型者血浆ACE活性及AT-Ⅱ浓度比较：
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各ACE基因型者血浆ACE活性比较表明D等位基因决定血ACE活性高水平：依次为DD型(27.61±5.32U/L)>D/I型(23.24±4.94U/L)>II型(19.17±3.69U/L)；而血浆AT-Ⅱ浓度依次为DD型(32.04±12.90ng/L)>D/I型(24.91±9.08ng/L)>II型(17.18±10.48ng/L)。
3. ATⅠR基因多态性
3.1 AT1R基因3’UTR的A1166C基因型及等位基因频率在受试者中的分布中，仅EH患者的C等位基因频率显著增高：
与对照组比较，原发性高血压组AC基因型频率56.8%及C等位基因频率28.4%明显高于对照组，见表5。
3.2 ACI患者(恢复期)A1166C基因型与血浆AT-Ⅱ浓度的关系：
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AA基因型的血浆AT-Ⅱ浓度(28.41±8.73ng/L)与AC型(25.80±9.62ng/L)相比无显著差异(t=0.43，P>0.05)。
3.3 脑血管病组中AT1R基因A1166C变异的相关分析
以AT1R基因为完全等级离散变量，采用Spearman相关分析方法及双尾检验，结果未发现AT1R基因与收缩压、舒张压、Glu、TG、Cho、HDL及LDL之间有相关性。
三、结论
1. ACI患者急性期时，血浆Ang和AT-Ⅱ浓度可应急性增高，Ang是通过AT-Ⅱ发挥作用；且此成份改变与缺血性脑损伤程度有关，可反映病情轻重程度，有急性反应物作用。
2. Ang基因第2外显子704位点C等位基因与ACI发病存在内在联系。而此联系并非简单由于C等位基因决定血浆Ang高水平所致，因为ACI恢复期时血浆Ang水平降至正常；尽管C等位基因频率在EH患者中增高，但C等位基因与ACI患者伴否高血压无关。
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3. Ang基因第2外显子C521T多态性对血浆Ang浓度变化无影响作用，但却与LI发病存在内在联系，而此与是否致高血压无关。
4. 同许多研究相似，本研究也得出ACE基因第16内含子D等位基因与ACI发病密切关联，是ACI的危险因素。虽然D等位基因决定血浆ACE高活性，但ACI急性期时血浆ACE活性并不增高，反明显降低，且与脑组织损伤程度有关。此表明D等位基因与ACI发病的内在联系并非简单缘于ACE在RAS中作用，可能与脑内RAS独立作用或通过其他途径所致。而ACI急性期血ACE活性降低的原因可能与损伤脑组织释放如IL、TNF等细胞因子对ACE活性抑制以及脑、体循环RAS差异等因素有关，这也为ACI急性期ACE拮抗剂的使用带来新思考。
5. 作为定量性状位点的ACE基因3’非翻译区4656(CT)2/3多态性在中国人中并明显。
6. AT1R基因与RAS主要活性物质AT-Ⅱ血内水平无关联性，与缺血性卒中发生与与否无关，但与高血压发病有相关性。
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7. 综合以上结论，似表明RAS成份中Ang、ACE分子水平与缺血性卒中存在潜在联系，而此联系并非通过RAS对血管缩舒功能的影响作用实现。RAS成份分子水平的变化与缺血性卒中的易感性有关。
Hypertention and stroke
—The association of RAS components with ischemic stroke
ZHANG Yingdong
Nanjing Brain Hospital, Nanjing Medical University
It is widely accepted that hypertension is the major risk factor for stroke. In China, 80% the occurrence of stroke associates with the hypertension. The epidemic studies showed that the 86% patients with cerebral hemorrhage and 71% patients with cerebral infarction have the histories of hypertension. Both systolic and diastolic hypertension will augment the stroke risk.
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RAS, as an important system of hormone regulation, exists in the body, 15% in the blood circulation and 85% in the tissues such as vessel walls, heart, CNS and kidney, etc. In the normal condition, RAS plays important roles in the development and function of cardiovascular system, and in the regulation of blood pressure.
In the chain-reaction of RAS components, angiotensinogen (Ang) is changed firstly to angiotensin-I (AT-I) by the action of rennin (Ren), and then angiotensin-I converting enzyme (ACE) converts AT-I to AT-II. The binding of AT-II to angiotensinⅡ typeⅠreceptor (ATⅠR) plays the vasoactive effect of RAS. When high blood pressure occurring, RAS is over activated, so excessive AT-II is produced, and is associated with the development of hypertension and its related disorders, and may be harmful to some tissues and organs.
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Therefore, we will show our series of studies on the association of the components of RAS with ischemic stroke as follows.
Materials and Methods
According to the “stroke diagnostic criteria” and “clinical score of neurological deficits” formulated by the 4th National Conference of Cerebrovascular Disorders in 1995, and their CT/MRI features, 87 patients with atherothrombotic cerebral infarction (ACI), 102 patients with lacunar infarction (LI), 67 patients with vascular dementia (VaD) and 93 patients with essential hypertension (EH) were recruited consecutively in this study. Meanwhile, 95 healthy unrelated subjects were selected as the healthy control (HC) group. The ages and genders among the every group were matched and not of significant difference.
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The plasma Ang and AT-II concentrations were measured by means of radioimmunoassay (RI).
The high-voltage capillary-electrophoresis (CEP) was employed to analyze the plasma ACE activity.
The polymorphisms of T704C and C521T base-variation in the 2nd exon of Ang gene, of A1166C base-variation in 3’ untranslating region (UTR) were determined by the methods of polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP). The 2 quantitative trait loci (QTLs): insertion/deletion (I/D) polymorphism in the 6th intron and 4656(CT)2/3 polymorphism in the 3’ UTR of ACE gene were determined by means of polymerase chain reaction – amplifying fragment length polymorphism (PCR-AFLP) and polymerase chain reaction –denaturing gradient gel electrophoresis (PCR-DGGE), respectively.
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RESULTS
1. The change of RAS components in ischemic stroke
1.1 The plasma Ang concentration was increased significantly in the acute phase of ACI patients:
The plasma Ang concentration (194.47±43.83ng/L) in the acute phase (within 72 hours after ictus)of ACI patients was significantly higher than that in the healthy controls(159.34±45.24ng/L)(t=12.34, P<0.001) and that in the recovery phase (3 weeks later after ictus) of ACI cases (163.45±42.64ng/L) (t=11.06, P<0.001).
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1.2 Plasma AT-Ⅱ concentration was increased significantly in the acute phase of ACI patients:
The plasma AT-Ⅱ concentration in the acute phase of ACI patients ((37.52±12.11ng/L) markedly higher than that in the recovery phase of the patients (29.70±8.70ng/L) and that the healthy controls (21.34±4.76ng/L) (both P<0.05). That concentration in the recovery phase was also higher than that in the controls (P<0.05).
1.3 The plasma Ang and AT-Ⅱ concentrations correlated with the degree of neurological deficits in the ACI patients:
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At the admission of ACI patients, their plasma Ang concentrations were 173.84±33.87ng/L when in mild degree (score of 1-15), 215.83±30.19ng/L when in moderate degree (score 16-30) and 236.91±48.53ng/L when in severe degree (score 31-45), respectively. And the plasma AT-Ⅱ concentrations related positively to the clinical scores of the patients (r=0.63, P<0.001).
1.4 The plasma AT-Ⅱ concentration was linear correlated positively with the plasma Ang concentration in both ACI cases and controls (r=0.68，P<0.05；r=0.61，P<0.05, respectively).
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1.5 The plasma ACE activity decreased significantly in the acute phase of ACI patients, but did not changed in the VaD cases, a kind of chronic cerebrovascular disorders.
Compared to that in the controls (22.62±5.44U/L), the plasma ACE activity in the acute ACI cases (19.26±5.11U/L) was significantly reduced (P<0.05), especially in the patients complicated with hypertension (18.42±5.20U/L), while did not changed significantly in the VaD patients (21.95±7.19U/L).
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2. The gene polymorphisms of RAS components in ischemic stroke
2.1 Ang gene polymorphism
2.1.1 The frequencies of 704CC genotype and C allele of Ang gene exon 2 in ACI cases and EH cases were significantly higher than those in the controls (Table 1). There was, however, no difference between the ACI cases complicated with and without hypertension (table 2).
2.1.2 The frequencies of 521T allele of Ang gene exon 2 in LI cases were significantly higher (table 3).
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2.1.3 The C allele at 704 locus of Ang gene exon 2 determined increased plasma Ang concentration, while the base-variation of C521T had no effect on the plasma Ang concentration in this study:
In the controls, the plasma Ang concentration in the subjects with CC genotype at 704 locus (196.34±44.37ng/L) markedly higher than that with CT genotype (170.41±43.64ng/L) (t=2.74, P<0.01) and that with TT genotype (146.45±21.43ng/L) (t=3.47, P<0.01). However, the plasma Ang concentration in the subjects with CC genotype at 521 locus (185.53±44.37ng/L) was not different significantly from that with CT genotype (170.41±42.84ng/L) (t=0.785, P>0.05).
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2.2 ACE gene polymorphism
2.2.1 The frequency of D allele at ACE gene 16th intron was significantly increased in the ACI patients (table 4).
2.2.2 The (CT)2/3 polymorphism at ACE gene 3’UTR locus was not found in the subjects of this study.
2.2.3 The comparisons of plasma ACE activity and AT-Ⅱ concentration among the different ACE genotypes:
The comparison of plasma ACE activity between the subjects with different ACE genotypes indicated that D allele determined the increased activity of plasma ACE: i.e. DD genotype (27.61±5.32U/L) >D/I genotype (23.24±4.94U/L) > II genotype (19.17±3.69U/L) in order. As to the plasma AT-Ⅱ concentration, DD genotype (32.04±12.90ng/L) >D/I genotype (24.91±9.08ng/L) > II genotype (17.18±10.48ng/L) in order.
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2.3 ATⅠR gene polymorphism
2.3.1 The frequency of C allele at ATⅠR gene 3’UTR A1166C locus was significantly increased in EH patients compared to that in other subjects:
Comparison to those in other groups, the frequencies of AC genotypes (56.8%) and C allele (28.4%) in the EH cases increased significantly (table 5).
2.3.2 The association of ATⅠR gene polymorphism with plasma AT-Ⅱ concentration:
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The plasma AT-Ⅱ concentration in the ACI patients with AA genotype (28.41±8.73ng/L) was similar to that with AC (25.80±9.62ng/L) (t=0.43, P>0.05).
2.3.3 The correlation analysis ATⅠR gene A1166C polymorphism in the patients with stroke:
With ATⅠR gene as whole ranking discrete variable, Spearman correlation analysis and double-tail analysis have shown no correlation of ATⅠR gene with systolic pressure, diastolic pressure, glucose, triglyceride, cholesterol, HDL and LDL.
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Conclusions
1. At the acute phase of ACI, plasma Ang and AT-Ⅱ can increase responsively, as the reactants. Those changes are related to the degree of the ischemic cerebral injuries, and can reflect the severity of cerebral infraction.
2. The 704 C allele at Ang gene exon 2 relates potentially to the occurrence of ACI. This association may be not attributed to the determination of C allele on the increased level of plasma, since the plasma Ang level will be back to the normal level when ACI in the recovery phase. The C allele have no relation to the complication of ACI patients with hypertension or not, although its frequency is increased in EH cases.
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3. The C521T polymorphism of Ang gene exon 2 has no effect on plasma Ang level, but potentially associate with lacunar infarction, which is independent on its effects on blood regulation.
4. Similar to many other studies, the D allele of ACE gene 16th intron is closely associated with ACI, and regarded as the risk factor for ischemic stroke. Although D allele determines the increased plasma ACE activity, the plasma ACE activity is not increased, but reduced in the acute phase of ACI, and is associated with the degree of cerebral injury, which indicates that the association of D allele with ACI can not be attributed simply to the ACE effect on RAS, but to the independent role in cerebral RAS or other way of RAS. The reason of the reduction of plasma ACE activity in the acute phase of ACI maybe relates to the increased release of interleukins (Ils) and tumor necrosis factor (TNF), which inhibit the activity of ACE, from the ischemic cerebral lesions, and the difference between brain and body RASs, etc. These results bring about a new view of ACE inhibitor administration in acute phase of ACI.
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5. The polymorphism of 4656(CT) 2/3 in ACE gene 3’UTR, one of ACE gene quantitative trait loci, maybe not exist in the Chinese population.
6. AT1R gene may be not of association with plasma AT-Ⅱ level, the major active compound, and not of ischemic stroke, but to hypertension at all.
7. On the whole, this serial study shows that the RAS components Ang and AT-Ⅱ associate potentially at molecular level with ischemic stroke, the effect of which is not dependent of the role of RAS on vascular function. The molecular variation of RAS components may be the susceptible for the ischemic stroke., http://www.100md.com(张颖冬)