过量酒精加重脑卒中损伤的机制研究

发布时间：2018-09-03 13:36

【摘要】：脑卒中又称脑中风，脑血管意外，是一种突然起病的脑血液循环障碍性疾病，包括蛛网膜下腔出血、脑栓塞和脑出血等。脑卒中给人类健康和生命造成极大威胁，具有高发病率、高致残率（高达75%）、高死亡率及高复发率等特点。脑卒中发病后的幸存者中约30%带有永久性残疾，，20%生活无法自理，不仅让罹患者本人极度痛苦，更累及家人和社会。中国每年因脑卒中死亡的患者约有160多万，在全球范围内，每年有460万人死于此病。并且近年来其发病率仍然不断上升，且好发于中老年人，尤其是60岁以上人群。流行病学研究表明长期大剂量饮酒是导致各种类型脑卒中的主要风险因素，并且会加重脑缺血对大脑的损伤作用。酒精的摄入量与脑卒中之间有密切关联，适度的酒精摄入可以起到保护作用，而大量酒精摄入则会产生危害作用。长期过量饮酒人群脑神经细胞活性降低，大脑功能也会随之衰退，最常见死因就是与心脑血管疾病相关的并发症。然而，摄入大量酒精是否影响中风患者的预后及未来的存活率还不清楚，其分子机制还有待阐明。一些研究表明长期过量摄入酒精导致的缺血性中风的发病机理包括:抑制兴奋性氨基酸转运蛋白和增加NMDA（N-methyl-D-aspartic acid receptor）兴奋性等。另一些研究认为，长期摄入酒精的危害可通过增加氧化应激或者降低肝脏中脑苷磺基转移酶的表达以降低血清中硫苷含量来实现。虽然这些理论都可以解释过量酒精的危害，但具体的致病分子靶点仍不清楚。酒的主要成分乙醇在人体内的代谢主要是通过乙醇脱氢酶（Alcohol dehydrogenase，ADH）和乙醛脱氢酶（Aldehyde dehydrogenase，ALDH）。乙醇脱氢酶将乙醇分解为乙醛，乙醛脱氢酶可以进一步催化乙醛生成无毒的乙酸，并最终生成二氧化碳和水排出体外。如果这两种酶在人体内含量高，就可以快速分解酒精，酒精的中枢神经系统毒性作用就较轻；而乙醛脱氢酶缺少的人，乙醛大量积累，导致产生醉酒的不适症状。乙醛脱氢酶2（Aldehyde dehydrogenase2，ALDH2），是酒精代谢过程中的一种酶，对脑卒中的作用报道相对较少。我们实验室先前研究结果证明激动ALDH2能够减轻缺血性脑损伤。4-羟基-2-壬烯醛（4-HNE）被证实是评估脑卒中的危险因子，在易卒中高血压大鼠(Stroke-Prone Spontaneously Hypertensive Rats，SHR-SP)血清中有显著的高表达。ALDH2这种保护作用正是通过清除脑内过量的4-HNE来实现的。但是，过量饮酒可导致体循环中的乙醛水平突然上升。乙醛比乙醇的毒性大，这可能会相对地影响酗酒后ALDH2的功能。为了更好地理解ALDH2在酗酒导致的中风发病机理中的作用，我们利用ALDH2抑制剂/激活剂或基因敲除/过表达等方法，探讨其可能的机制，提出假说：长期大剂量饮酒能够加重脑卒中损伤，且这种损伤作用是通过积累有毒的醛类物质，抑制醛脱氢酶活力，进而调节其下游信号通路实现的。我们设计了相关实验并取得了以下实验结果。（1）大剂量酒精加重脑缺血的损害作用 SD大鼠长期饲喂大剂量酒精，能够增加线栓法MCAO的梗死面积（34.4%±4.8%vs27.0%±4.6%in vehicle, P 0.01）；大剂量酒精能够明显缩短易卒中型高血压大鼠SHR-SP的存活时间，使其更早发生脑卒中而死亡。（2）大剂量酒精改变脑内ALDH2等电点分布，降低脑内酶活性使用二维电泳技术检测长期饲喂大剂量酒精SD大鼠脑内蛋白，与正常对照组相比，实验组动物脑内ALDH2等电点分布发生改变，活性受到抑制（62%±21%vs100%±17%in vehicle, P 0.01），但是肝脏内的活性并没有被抑制。并且在脑内和肝脏内两组酶的表达量并无差异。（3）大剂量酒精使脑缺血动物脑内醛类物质积累加重脑损伤利用顶空气相色谱耦合火焰离子化检测器检测各组动物血清及脑组织中乙醇和乙醛含量。用ELISA、Western Blotting、免疫组织荧光等方法检测醛类物质如4-HNE、MDA的含量。正常大鼠长期饲喂酒精饲料后，大鼠血清中乙醛、4-HNE和MDA含量并未发生改变。但在脑缺血后，这些物质含量均大幅上升，并且酒精加重了这些有毒物质的积累。乙醛能够直接损伤神经元，增大脑梗死面积（46.0%±3.6%vs29.7%±3.2%, P 0.01）。（4）激活ALDH2可取消大剂量酒精对脑缺血的损伤作用原代培养神经元OGD12h可诱导神经元凋亡，经用TUNEL检测凋亡率。乙醇（300μM）使凋亡损伤加重。ALDH2的激动剂Alda1（10μM）减轻损伤作用达30%，并且取消了乙醇的对细胞凋亡的进一步加重。拮抗剂Cya（1mM）作用与Alda相反（64.4%±4.82%vs45.4%±4.32%withethanol, P 0.05）。流式细胞仪检测结果趋势同上。对SD大鼠进行线栓法脑梗，在梗死面积和神经功能评分上也验证了上述结果。（5）过表达ALDH2取消酒精对脑缺血的损伤作用 SD大鼠脑立体定位注射慢病毒过表达ALDH2，3周后取脑冰冻切片扫描脑片确认转染效率。病毒过表达后能够使ALDH2蛋白上调约63%，基因沉默后蛋白下调73%。成功感染病毒的大鼠常规给予6周的大剂量酒精液体饲料，在注射病毒同侧实施MCAO手术。乙醇可以增加脑梗死面积，过表达ALDH2能缩小梗死面积并取消过量酒精对脑梗损伤的加重作用。RNA干扰实验结果也与过表达相符合。（6）大剂量乙醇可以减少ALDH2与PKC epsilon的结合 PKC epsilon是ALDH2上游重要蛋白之一，乙醇能够激活PKC eps ilon使之磷酸化并且在一定范围内与剂量和时间都成正比。但是大剂量乙醇可以减少ALDH2与PKC epsilon的结合。结论：我们的研究结果显示，大剂量乙醇通过增加醛的积累，抑制ALDH2活性同时减少ALDH2与PKC epsilon结合加重脑损伤。
[Abstract]:Stroke, also known as stroke, cerebrovascular accident, is a sudden onset of cerebral blood circulation disorders, including subarachnoid hemorrhage, cerebral embolism and cerebral hemorrhage. Stroke poses a great threat to human health and life, with high incidence, high disability rate (as high as 75%), high mortality and high recurrence rate. About 30% of the survivors are permanently disabled and 20% of them are unable to take care of themselves, which not only makes them extremely painful, but also affects their families and society. Epidemiological studies have shown that long-term high-dose drinking is a major risk factor for various types of stroke and may exacerbate the brain damage caused by cerebral ischemia.
Alcohol intake is closely associated with stroke. Moderate alcohol intake can protect against the effects of high alcohol intake, which can be harmful. Long-term excessive drinking reduces the activity of brain neurons and leads to deterioration of brain function. The most common cause of death is complications associated with cardiovascular and cerebrovascular diseases. Whether high alcohol intake affects the prognosis and future survival of stroke patients remains unclear, and its molecular mechanisms remain to be elucidated. Other studies suggest that long-term exposure to alcohol can be achieved by increasing oxidative stress or decreasing the expression of brain glycoside sulfotransferase in the liver to reduce glucosinolate levels in the serum. Although these theories can explain the harm of excessive alcohol, the specific molecular targets for the pathogenesis of alcohol are still unclear. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the main metabolites in the body. Alcohol dehydrogenase decomposes ethanol into acetaldehyde. Aldehyde dehydrogenase can catalyze acetaldehyde to produce nontoxic acetic acid and eventually produce carbon dioxide and water to be excreted from the body. Alcohol can be quickly decomposed at high levels, and the central nervous system of alcohol has less toxic effects; while aldehyde dehydrogenase deficient people, acetaldehyde accumulated in large quantities, resulting in drunken symptoms.
Aldehyde dehydrogenase 2 (ALDH2), an enzyme in the process of alcohol metabolism, has relatively few reports on the role of stroke. Previous studies in our laboratory have shown that activating ALDH2 can alleviate ischemic brain damage. 4-hydroxy-2-nonenoaldehyde (4-HNE) has been shown to be a risk factor for assessing stroke in stroke-prone hypertension. The protective effect of ALDH2 is achieved by eliminating excessive 4-HNE in the brain. However, excessive alcohol consumption can lead to a sudden increase in acetaldehyde levels in the systemic circulation. Acetaldehyde is more toxic than ethanol, which may have a relative effect on ALD after alcoholism. The function of H2.
In order to better understand the role of ALDH2 in the pathogenesis of alcoholism, we used ALDH2 inhibitor/activator or gene knockout/overexpression methods to explore its possible mechanisms and hypothesized that long-term high-dose drinking could aggravate stroke injury, which was inhibited by accumulation of toxic aldehydes. The activity of aldehyde dehydrogenase is regulated and its downstream signal pathway is realized.
(1) high dose of alcohol aggravates the damage of cerebral ischemia.
Long-term high-dose alcohol could increase the infarct size of MCAO (34.4%+4.8% vs 27.0%+4.6% in vehicle, P 0.01), and high-dose alcohol could significantly shorten the survival time of SHR-SP in stroke-prone hypertensive rats and make them die earlier.
(2) high dose alcohol changed the distribution of ALDH2 isoelectric point in brain and reduced the activity of brain enzymes.
Compared with the control group, the distribution of ALDH2 isoelectric point in the brain of SD rats fed with high dose alcohol for a long time was changed and the activity of ALDH2 isoelectric point was inhibited (62%+21% vs 100%+17% in vehicle, P 0.01), but the activity in the liver was not inhibited. There was no difference in the amount of expression.
(3) large doses of alcohol aggravate brain damage by increasing the aldehyde accumulation in the brain of animals with cerebral ischemia.
The contents of ethanol and acetaldehyde in serum and brain tissues of rats were detected by headspace gas chromatography coupled with flame ionization detector. The contents of aldehydes such as 4-HNE and MDA were detected by ELISA, Western Blotting and immunohistofluorescence. However, after cerebral ischemia, the contents of these substances increased significantly, and the accumulation of these toxic substances was aggravated by alcohol. Acetaldehyde could directly damage neurons and increase the area of cerebral infarction (46.0% + 3.6% vs 29.7% + 3.2%, P 0.01).
(4) activation of ALDH2 can abolish the effect of high-dose alcohol on cerebral ischemia.
The primary cultured neurons were induced to apoptosis by OGD for 12 hours, and the apoptosis rate was detected by TUNEL. Ethanol (300 mu M) aggravated the damage of apoptosis. Alda1 (10 mu M), an agonist of ALDH2, attenuated the injury by 30% and eliminated the further aggravation of apoptosis by ethanol. The antagonist Cya (1mM) had the opposite effect as Alda (64.4% + 4.82% vs 45.4% + 4.32% withethan). The results of flow cytometry showed the same trend. The infarct size and neurological function score of SD rats were also validated.
(5) overexpression of ALDH2 abolished the effect of alcohol on cerebral ischemia.
The transfection efficiency was confirmed by stereotactic injection of lentivirus overexpressing ALDH2 into the brain of SD rats. After overexpression of the virus, the ALDH2 protein was up-regulated by 63% and the protein was down-regulated by 73% after gene silencing. Ethanol can increase the area of cerebral infarction. Overexpression of ALDH2 can reduce the area of infarction and cancel the aggravating effect of excessive alcohol on cerebral infarction.
(6) high dose ethanol can reduce the combination of ALDH2 and PKC epsilon.
PKC EPS ilon is one of the important proteins in the upstream of ALDH2. Ethanol can activate PKC EPS ilon and phosphorylate it in a certain range in direct proportion to the dose and time. However, high dose ethanol can reduce the binding of ALDH2 to PKC EPS ilon. Conclusion: Our results show that high dose ethanol can inhibit ALDH2 activity by increasing aldehyde accumulation. Sex at the same time reduced the combination of ALDH2 and PKC epsilon to aggravate brain damage.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3

【共引文献】