乙醇对内皮细胞黏着斑激酶的影响及其细胞毒性作用
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摘要
我国酒文化源远流长，但随着饮酒在社会交往活动中更加频繁，饮酒所带来的社会问题也日益凸显。近年来饮酒后伤害案件屡见不鲜，加之案情复杂，给检案人员造成很大压力。早在1963年有国外法医提出外伤性蛛网膜下腔出血（traumatic subarachnoid hemorrhage，tSAH）与酗酒有关，实际检案过程发现酒后受到轻微外力作用，颅内出血的发病率和死亡率明显增高。虽然已知饮酒可以调节心率和局部血流，改变血管通透性，影响血管舒缩功能，破坏凝血、抗凝血平衡，但这些改变尚不足以解释饮酒后外力作用易于引发颅内出血的原因。此外，长期困扰法医工作者的难题是：如何界定饮酒和外力各自的参与度，进而判定案件性质。所以更深入地研究饮酒易于造成脑血管破裂出血机制，有助于指导法医实践、改进检案思路和明确判定结果。
乙醇（ethanol，EtOH）又名酒精，是酒类饮品中的主要成分。饮酒后乙醇可以以原型形式进入血液，血乙醇浓度（blood alcohol concentration,BAC）快速升高，乙醇随血流遍布全身。内皮细胞（endotheilial cell，EC）位于血管腔面最内侧，直接接触血液中乙醇及其代谢产物；另一方面内皮细胞生物功能活跃，拥有乙醇代谢相关酶体参与乙醇代谢，这就决定了内皮细胞可能遭受乙醇的直接和间接毒性作用，成为乙醇首当其冲的损伤靶点。
由于血管内皮承担着重要的血管调节功能，在物质交换、血管张力调控、凝血、血管发生、白细胞游走等多种生物过程中发挥不可替代的作用；并可以“感知”血流切应力、周期应力等各种机械应力及各种血液成分改变，协调其他细胞共同作出相应反应，所以乙醇引起的内皮细胞损伤会危及到整个血管系统。
其中脑血管具有其他部位血管不可比拟的自主调控能力以保持稳定的脑灌流量，对内皮细胞损伤愈发敏感。本课题以内皮细胞作为主要研究对象，探索乙醇的血管毒性作用，为进一步研究饮酒后轻微外力引发蛛网膜下腔出血（subarachnoid hemorrhage，SAH）的机制做好铺垫。
血管内皮以单层细胞形式分布，其结构、功能完整性特别依赖于与细胞外基质（extracellular matrix，ECM）的连接、交流。黏着斑复合体是细胞与ECM连接处的复合结构，实现了细胞骨架与ECM的连接。一方面它可以将细胞外的信号传入胞内，通过“引导”应力纤维分布实现对细胞形态、功能的调整；另一方面将细胞生命活动产生的细胞内牵引力（tractionforce）传递到ECM，进而转化为生物信号传递给周围细胞，使机体能作出及时的宏观调整。
黏着斑激酶（focal adhesion kinase，FAK）是黏着斑复合体重要的组成成分。一方面FAK受到integrin聚集、生长因子受体连接、切应力等多种因素调节活化；另一方面FAK可以连接并激活其他黏着斑结构蛋白，参与黏着斑复合体形成与成熟，调节细胞粘附、迁移等基本功能，还可以招募多种功能蛋白，参加到不同的信号通路调节中去。现已证实FAK在内皮细胞中发挥着举足轻重的作用。用特异失活内皮细胞内FAK的方法研究胚胎发育，发现FAK失活的胚胎虽然早期发育正常，但是在胚胎发育晚期出现胚胎、卵黄囊和胎盘的血管发育异常，内皮细胞死亡增多，血管破裂进而出现出血、水肿。多种疾病中也发现内皮细胞FAK表达、活化异常。对于过量乙醇刺激对内皮细胞FAK的影响及相应的生物学意义尚缺乏研究。本研究将着眼于过量乙醇对内皮细胞FAK的影响，探讨乙醇血管毒性的相关机制。
第一部分：乙醇对内皮细胞的毒性作用
目的：本部分将从动物整体水平和体外细胞水平观察乙醇对血管的毒性作用，全面研究乙醇对内皮细胞结构和功能的影响。
方法：
1采用灌胃器给酒制备大鼠饮酒模型，检测饮酒后BAC变化。运用Masson三色染色和透射电子显微镜（transmission electron microscope，TEM）技术观察不同饮酒时间大鼠脑实质毛细血管和基底动脉形态改变；
2给予人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）不同浓度、不同时间的乙醇刺激，利用MTT检测HUVEC存活率，反映乙醇的细胞毒性；
3利用流式细胞术观测乙醇诱导的HUVCE凋亡；
4用光镜、扫描电子显微镜（scanning electron microscope，SEM）和TEM观察乙醇引起的内皮细胞形态结构改变；
5待乙醇干预结束后用胰酶替代物消化HUVEC，重新接种于纤维连接蛋白（fibronectin，FN）包被的培养板中，测量1-2h内的粘附率和粘附过程中细胞铺展的面积以评价内皮细胞粘附能力；利用特异阻断抗体预处理细胞实现整合素（integrin）阻断后，进行粘附实验，观察integrin在其中作用；
6给予乙醇干预后，利用划痕实验检验乙醇对HUVEC迁移能力的影响。
结果：
1短期饮酒大鼠脑内既可见明显血管源性水肿，毛细血管周围间隙明显增大，内皮细胞微绒毛减少，胞质内线粒体空泡化；
2长期饮酒大鼠基底动脉中膜平滑肌成分与外膜胶原成分增多，壁/腔比明显增大（p<0.05），并可见内皮细胞脱落；
3乙醇使内皮细胞生存率呈剂量、时间依赖性下降，与对照组相比出现较多早期凋亡细胞（p<0.05），这些凋亡细胞在TEM下呈现出细胞核形态不规则，核染色质边集呈半月状且电子密度增高，细胞胞浆减少等征象；
4光镜、SEM、TEM观察可见乙醇刺激使HUVEC失去正常形态，胞内细胞器表现出空泡化等损伤征象；
5乙醇明显降低HUVEC粘附率（p<0.05）和平均细胞铺展面积（p<0.05）。特异integrin β1抗体预处理后可以降低正常组细胞粘附率，但对乙醇组影响不明显；
6乙醇明显降低HUVEC单细胞层划痕的愈合速度（p<0.05）。
小结：在体和细胞实验均证明过量乙醇具有明显的内皮毒性作用，影响内皮细胞形态、降低生存率、抑制其与ECM粘附及粘附后铺展，以及降低内皮细胞迁移能力；乙醇作用后HUVEC粘附力下降可能与integrin信号通路有关。
第二部分：过量乙醇对内皮细胞黏着斑激酶的影响及其产生机制
目的：本部分将系统研究过量乙醇对内皮细胞中FAK分布、蛋白表达及磷酸化的影响，并探讨相关机制。
方法：
1用细胞免疫荧光标记细胞粘附、铺展和迁移过程中的磷酸化的FAK酪氨酸397位点（phosphorylated FAK at Tyr397，pFAK Y397），观察黏着斑复合体分布变化；
2利用western blot检测乙醇作用对FAK总蛋白水平和磷酸化水平的影响；
3给予HUVEC特异乙醇脱氢酶（alcohol dehydrogenase，ADH）抑制剂4-甲基吡唑（4-methyle-pyrazole，4MP）预孵或给予乙醇主要的代谢产物乙醛（acetaldehyde，Ach）刺激，研究乙醇代谢在FAK变化中的作用；
4利用活性氧簇（reactive oxygen species，ROS）特异探针DCFH-DA标记ROS观察乙醇、乙醛引起的氧化应激变化；
5给予外源性过氧化氢（H2O2）或非特异性内源性ROS清除剂N-乙酰-L-半胱氨酸（N-acetyl-L-cysteine，NAC），研究氧化应激在FAK变化中的作用。
结果：
1乙醇并未影响integrin β1的表达和分布；
2在铺展过程中的对照组细胞内pFAK Y397形成较大的片状分布，尤以细胞边缘明显，迁移过程中细胞迁移方向的前缘亦可形成大片状pFAK Y397表达；而过量乙醇干预过的HUVEC中形成的片状结构小而少；
3乙醇增加FAK磷酸化，且呈剂量、时间依赖性（p<0.05，p<0.01），但并不影响FAK总蛋白水平；
4给予4MP可以削弱过量乙醇引起的FAK磷酸化增高（p<0.05）；给予乙醛增加pFAK Y397（p<0.01）；
5乙醇、乙醛刺激均可激发内皮细胞中ROS产生；
6给予外源性H2O2可以激发FAK磷酸化（p<0.01）；给予NAC可部分降低乙醇、乙醛引起的FAK磷酸化水平（p<0.05）。
小结：过量乙醇一方面干扰了FAK参与的内皮细胞黏着斑成熟，不能有效“引导”F-actin骨架，导致内皮细胞生存、粘附、迁移功能受损；另一方面增加pFAK水平，且乙醇和（或）其代谢产生的乙醛、氧化应激是引起FAK酪氨酸磷酸化的重要因素之一；过量乙醇并不影响FAK总蛋白水平。
第三部分：黏着斑激酶磷酸化在过量乙醇引起的内皮细胞毒性中的作用
目的：本部分实验将以PI3K/AKT信号通路着手研究过量乙醇干预后FAK磷酸化增高在内皮细胞一氧化氮（nitric oxide，NO）内稳态破坏中的作用。
方法：
1利用NO特异探针DAF-FM检测内皮细胞NO产生，并利用eNOS特异抑制剂N-硝基-L-精氨酸甲酯盐酸盐（ω-nitro-L-argine methyl ester，L-NAME）、 iNOS特异抑制剂L-canavanine和FAK自主磷酸化抑制剂PF573228（PF228）研究NO来源；
2在给予HUVEC乙醇刺激前给予PI3K/AKT通路抑制剂wortmannin和PF228预处理研究eNOS活化的相关通路；
3用western blot检测硝基酪氨酸（nitrotyrosine，NT）产生量。
结果：
1乙醇引起NO明显增高（p<0.05）；L-NAME或PF228可以减弱乙醇引起的NO增加（p<0.05），而L-canavanine作用不明显；
2给予wortmannin和PF228均可明显减弱乙醇引起AKT和eNOS磷酸化（p<0.05）；
3200mM乙醇干预24h后明显增加内皮细胞NT生成量（p<0.05）。
小结：过量乙醇刺激引起的FAK磷酸化通过PI3K/AKT通路促进的eNOS活化，产生过量NO，生成具有细胞毒性的NT，造成内皮细胞损伤。
结论：
过量乙醇可以引起脑血管内皮细胞损伤，其机制主要表现在两个方面：
1乙醇干扰黏着斑复合体成熟，影响内皮细胞功能；
2乙醇及其代谢产生的乙醛、氧化应激通过增加FAK磷酸化水平，激活PI3K/AKT通路，上调eNOS/NO系统，使NT过量生成，造成内皮细胞损伤。
Drinking has a long history in China, and with the development of socialactivities, drinking is becoming more and more common. The related socialproblems have been serious. In recent years, injury cases after drinking havebeen increasing, and the related cases are always complicated to judge. In1963, some researchers indicated that traumatic subarachnoid hemorrhage(tSAH) was related with heavy drinking. In forensic medicine cases, drinkingleads to high incidence and mortality of intracranial hemorrhage with a slightexternal force. Although some mechanisms have been studied, such as chagesof heart rate and blood flow, vascular permeability，vasomotor function, andthe imbalance between coagulation and anticoagulation, it is still difficult toexplain the reasons. There are some challenges in forensic practice: how toevaluate the contribute degree of external force and alcohol and how toevaluate the case nature.
Ethanol (EtOH) is the most important component of alcoholic beverages.Aftering drinking, the ethanol is taken into blood and the blood alcoholconcentration (BAC) increases rapidly. With blood stream, ethanol is taken toorgans in the body. Vascular endothelial cells (EC) lining the blood vesselsform the interface between the bloodstream and the vessel wall and as suchthey are continuously subjected to ethanol and metabolites from the flowingblood after drinking. And EC take part in ethanol metabolism. As result, ECare susceptible to ethanol totoxicity.
Endothelial cells (EC) are the substaintial constitution of vessels, whotake responsibility to material exchange, angiotasis regulation, bloodcoagulation, angiogenesis and leukocyte migration. In addition, EC ‘sense’and transmit the biosignals from shear stress, cycle tension and bloodcomponents and coordinate diverse cells to make biological response. In summary, the toxicity of ethanol to EC will lead injury of vascular disorder.
Cerebral vessels are charactered with autonomic regulation, so they aresensitive to EC injury. The current study was designed to study toxicity ofethanol to cerebral vessels, which will be helpful to futher study aboutmechanisms of tSAH with slight blow after drinking.
EC line the wall of all blood vessels with monolayer of cells, so thedelicate balance between cell-cell interaction and EC-matrix adhesion areimportant to endothelial function. Focal adhesions (FAs) are complexstructures that assemble at the plasma membrane in discrete regions ofintegrin-mediated recognition of extracellular matrix (ECM) components.They connect the extracellular filamentous meshwork to the intracellularcytoskeleton controlling or mediating bidirectional signal transductionthrough endothelial cell.
Focal adhesion kinase (FAK) is an important member in FA pathwaytransducting in-outside or outside-in signals. FAK is a non receptor proteintyrosine kinase (PTK), taking part in composition of focal adhesion (FA)complex and regulation of cytoskeleton. In the one hand, several factors, suchas integrin clustering, growth factor receptors and shear stress, regulate FAK.In another hand, FAK is a scaffolding protein binding with other proteins. SoFAK is involved in multiple pathways. There is evidence that FAK is relatedwith adhesion, migration, proliferation and mechanotransduction. Inpathological conditions, FAK related signal pathways become disorder. Thecurrent study was designed to observe the changes of FAK induced byexcessive ethanol, and to explore the mechanisms.
Part I: The cytotoxicity of ethanol to endothelial cells
Objective: To observe the cytotoxicity of excessive ethanol to vessels invivo and in vitro and to study systematically the effects of ethanol on structureand functions of endothelial cells.
Methods:
1The rats were given alcoholic beverage with a blunt tipped needle, andBAC was detected after drinking. Masson’s trichrome staining and transmission electron microscope (TEM) were used to observe themorphological changes of capillaries and basilar arteries (BA) from alcoholicrats with different duration of drinking;
2Human umbilical vein endothelial cells were given differentconcentrations or different duration of ethanol treatment, and the viability wasdetected with MTT, representing the cytotoxicity of ethanol;
3Apoptotic cells were determined by Annexin V/PI apoptosis detectionkit with flow cytometry;
4The morphological changes of the endothelial cell after ethanol treatedwere observed with light microscope, scanning electron microscope (SEM)and transmission electron microscope (TEM);
5After the ethanol treatment, the cells were harvested by TrypLE Express, and allowed to adhere in plates coated with fibronectin (FN) for1-2h.The adhesion was detected with MTT and the average areas were measured byimage soft; the role of integrin pathway in adhesion was observed by blockingintegrin with specific blocking antibody;
6Wound healing assay was used to observe the migration of HUVECafter ethanol treatment.
Results:
1The rats with short-time drinking appeared changes in capillaries:obvious vasogenic edema, enlarged perivascular space, microvillus decreasedand vacuolus in mitochondria;
2The rats with long-time drinking appeared vascular remodeling: SMChyperplasia in tunica media and collagen deposition in tunica adventitia, thewall: lumen ratio increasing (p<0.05), and endothelial desquamation;
3Excessive ethanol treatment decreased the HUVEC viability in a dose-and time-dependent manner. Compared to the control group, there were moreearly apoptotic cells (p<0.05) showing unregular nucleus, increased electrondensity of chromatin with half moon shape, and decreased cytoplasm;
4HUVEC after excessive treatment lost normal shape, even withorganelle injury.
5Excessive ethanol treatment decreased adhesion rate (p<0.05) andspreading areas of HUVEC (p<0.05). The antibody to block integrin β1reduced adhesion rate of control EC, without affects on ethanol treated EC.
6Excessive ethanol treatment decreased migration of HUVEC in woundhealing assay.
Summary: Excessive ethanol caused endothelial injury, includingmorphological changes, viability decreasing, and interference of adhesion,spreading, and migration of EC. The adhesion injury induced by ethanol mightbe related with integrin pathway.
Part Ⅱ: Changes of FAK and the related mechanisms in EC caused byexcessive ethanol
Objective: This part was designed to study the distribution, proteinexpression and phosphorylation of FAK after excessive ethanol treatment, andto explore the related metablisms.
Methods:
1Distribution of pFAK Y397in spreading and migarating EC wasobserved with immunofluorenscence.
2Total protein level and phosphorylation level of FAK were detectedwith western blot.
3To study the relationship between the metabolism of ethanol and theFAK changes, specific ADH inhibitor4MP and ethanol metaboliteacetaldehyde (Ach) were given to HUVEC.
4The oxidative stress from ethanol or Ach was detected with specificprobe DCFH-DA.
5To study the relationship between oxidative stress caused by themetabolism of ethanol and the FAK changes, exogenous H2O2and nonspecific ROS scanvenger N-acetyl-L-cysteine (NAC) were given to HUVEC.
Results:
1In control group, pFAK397appeared as large pelltes, especially at edgeof cells during spreading and in parapodia during migrating. In ethanol group,pFAK397appeared as small dots;
2Ethanol increased pFAK in a dose-and time-dependent manner(p<0.05，p<0.01), without affect on total FAK protein;
3Increased pFAK induced by ethanol was attenuated by4MP (p<0.05);Ach increased pFAK397significantly (p<0.05);
4Both ethanol and Ach increased ROS level of HUVEC;
5Exogenous H2O2incresased FAK phosphorylation (p<0.01); IncreasedpFAK induced by ethanol or Ach was attenuated by NAC (p<0.05).
Summary：Ethanol disordered the distribution of pFAK, and increasedthe level of pFAK; disordered distribution of pFAK was involved in FA mature,leading to injury in EC survival, adhesion and migration; Ach and oxidativestress induced by ethanol metabolism was one of important factors of FAKphosphorylation.
Part Ⅲ: The role of FAK phosphorylation on cytotoxicity of excessiveethanol to EC
Objective：This part was designed to explore the relationship betweenincrease of pFAK and NO-mediated homeostasis, and to evaluate whetherPI3K/AKT pathway was involved in this process.
Methods:
1NO production was detected with specific probe DAF-FM; eNOSinhibitor L-NAME, iNOS inhibitor L-NAME L-canavanine and inhibitor ofFAK autophosphorylation PF228were employed to assess the origin of NO;
2NT production was dectected by western blot;
3The pathways involving in eNOS activation were vertified withPI3K/AKT inhibitor wortmannin and PF228.
Results:
1Ethanol increased NO production (p<0.05)，which was attenuated bypretreatment of L-NAME or PF228(p<0.05), but not by L-canavanine.
2NT production was significantly increased after treated with200mMethanol for24h (p<0.05).
3Increase of AKT and eNOS phosphorylation induced by ethanol wasattenuated by both wortmannin and PF228(p<0.05).
Summary: Excessive ethanol increased NO generation and toxic NTproduction; FAK phosphorylation induced by ethanol promote the activationof eNOS via PI3K/AKT, which broke the NO-mediated endothelialhomeostasis.
Conclusions:
1Short-time drinking leads to microvasular morphological and structuralchanges, while long-time drinking causes vascular remodeling of cerebralarteries. Endothelial cells are the injury target of ethanol cytotoxicity.
2Ethanol disorders the distribution of FA, inducing decrease of survivalrate, and down-regulation of adhesion and migration ability.
3Metabolites and oxidative stress from ethanol increase FAKphosphorylation, which up-regulates FAK/PI3K/AKT/eNOS pathway.Overproduction of NO and ROS generates NT, taking part in endothelialinjury.
乙醇（ethanol，EtOH）又名酒精，是酒类饮品中的主要成分。饮酒后乙醇可以以原型形式进入血液，血乙醇浓度（blood alcohol concentration,BAC）快速升高，乙醇随血流遍布全身。内皮细胞（endotheilial cell，EC）位于血管腔面最内侧，直接接触血液中乙醇及其代谢产物；另一方面内皮细胞生物功能活跃，拥有乙醇代谢相关酶体参与乙醇代谢，这就决定了内皮细胞可能遭受乙醇的直接和间接毒性作用，成为乙醇首当其冲的损伤靶点。
由于血管内皮承担着重要的血管调节功能，在物质交换、血管张力调控、凝血、血管发生、白细胞游走等多种生物过程中发挥不可替代的作用；并可以“感知”血流切应力、周期应力等各种机械应力及各种血液成分改变，协调其他细胞共同作出相应反应，所以乙醇引起的内皮细胞损伤会危及到整个血管系统。
其中脑血管具有其他部位血管不可比拟的自主调控能力以保持稳定的脑灌流量，对内皮细胞损伤愈发敏感。本课题以内皮细胞作为主要研究对象，探索乙醇的血管毒性作用，为进一步研究饮酒后轻微外力引发蛛网膜下腔出血（subarachnoid hemorrhage，SAH）的机制做好铺垫。
血管内皮以单层细胞形式分布，其结构、功能完整性特别依赖于与细胞外基质（extracellular matrix，ECM）的连接、交流。黏着斑复合体是细胞与ECM连接处的复合结构，实现了细胞骨架与ECM的连接。一方面它可以将细胞外的信号传入胞内，通过“引导”应力纤维分布实现对细胞形态、功能的调整；另一方面将细胞生命活动产生的细胞内牵引力（tractionforce）传递到ECM，进而转化为生物信号传递给周围细胞，使机体能作出及时的宏观调整。
黏着斑激酶（focal adhesion kinase，FAK）是黏着斑复合体重要的组成成分。一方面FAK受到integrin聚集、生长因子受体连接、切应力等多种因素调节活化；另一方面FAK可以连接并激活其他黏着斑结构蛋白，参与黏着斑复合体形成与成熟，调节细胞粘附、迁移等基本功能，还可以招募多种功能蛋白，参加到不同的信号通路调节中去。现已证实FAK在内皮细胞中发挥着举足轻重的作用。用特异失活内皮细胞内FAK的方法研究胚胎发育，发现FAK失活的胚胎虽然早期发育正常，但是在胚胎发育晚期出现胚胎、卵黄囊和胎盘的血管发育异常，内皮细胞死亡增多，血管破裂进而出现出血、水肿。多种疾病中也发现内皮细胞FAK表达、活化异常。对于过量乙醇刺激对内皮细胞FAK的影响及相应的生物学意义尚缺乏研究。本研究将着眼于过量乙醇对内皮细胞FAK的影响，探讨乙醇血管毒性的相关机制。
第一部分：乙醇对内皮细胞的毒性作用
目的：本部分将从动物整体水平和体外细胞水平观察乙醇对血管的毒性作用，全面研究乙醇对内皮细胞结构和功能的影响。
方法：
1采用灌胃器给酒制备大鼠饮酒模型，检测饮酒后BAC变化。运用Masson三色染色和透射电子显微镜（transmission electron microscope，TEM）技术观察不同饮酒时间大鼠脑实质毛细血管和基底动脉形态改变；
2给予人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）不同浓度、不同时间的乙醇刺激，利用MTT检测HUVEC存活率，反映乙醇的细胞毒性；
3利用流式细胞术观测乙醇诱导的HUVCE凋亡；
4用光镜、扫描电子显微镜（scanning electron microscope，SEM）和TEM观察乙醇引起的内皮细胞形态结构改变；
5待乙醇干预结束后用胰酶替代物消化HUVEC，重新接种于纤维连接蛋白（fibronectin，FN）包被的培养板中，测量1-2h内的粘附率和粘附过程中细胞铺展的面积以评价内皮细胞粘附能力；利用特异阻断抗体预处理细胞实现整合素（integrin）阻断后，进行粘附实验，观察integrin在其中作用；
6给予乙醇干预后，利用划痕实验检验乙醇对HUVEC迁移能力的影响。
结果：
1短期饮酒大鼠脑内既可见明显血管源性水肿，毛细血管周围间隙明显增大，内皮细胞微绒毛减少，胞质内线粒体空泡化；
2长期饮酒大鼠基底动脉中膜平滑肌成分与外膜胶原成分增多，壁/腔比明显增大（p<0.05），并可见内皮细胞脱落；
3乙醇使内皮细胞生存率呈剂量、时间依赖性下降，与对照组相比出现较多早期凋亡细胞（p<0.05），这些凋亡细胞在TEM下呈现出细胞核形态不规则，核染色质边集呈半月状且电子密度增高，细胞胞浆减少等征象；
4光镜、SEM、TEM观察可见乙醇刺激使HUVEC失去正常形态，胞内细胞器表现出空泡化等损伤征象；
5乙醇明显降低HUVEC粘附率（p<0.05）和平均细胞铺展面积（p<0.05）。特异integrin β1抗体预处理后可以降低正常组细胞粘附率，但对乙醇组影响不明显；
6乙醇明显降低HUVEC单细胞层划痕的愈合速度（p<0.05）。
小结：在体和细胞实验均证明过量乙醇具有明显的内皮毒性作用，影响内皮细胞形态、降低生存率、抑制其与ECM粘附及粘附后铺展，以及降低内皮细胞迁移能力；乙醇作用后HUVEC粘附力下降可能与integrin信号通路有关。
第二部分：过量乙醇对内皮细胞黏着斑激酶的影响及其产生机制
目的：本部分将系统研究过量乙醇对内皮细胞中FAK分布、蛋白表达及磷酸化的影响，并探讨相关机制。
方法：
1用细胞免疫荧光标记细胞粘附、铺展和迁移过程中的磷酸化的FAK酪氨酸397位点（phosphorylated FAK at Tyr397，pFAK Y397），观察黏着斑复合体分布变化；
2利用western blot检测乙醇作用对FAK总蛋白水平和磷酸化水平的影响；
3给予HUVEC特异乙醇脱氢酶（alcohol dehydrogenase，ADH）抑制剂4-甲基吡唑（4-methyle-pyrazole，4MP）预孵或给予乙醇主要的代谢产物乙醛（acetaldehyde，Ach）刺激，研究乙醇代谢在FAK变化中的作用；
4利用活性氧簇（reactive oxygen species，ROS）特异探针DCFH-DA标记ROS观察乙醇、乙醛引起的氧化应激变化；
5给予外源性过氧化氢（H2O2）或非特异性内源性ROS清除剂N-乙酰-L-半胱氨酸（N-acetyl-L-cysteine，NAC），研究氧化应激在FAK变化中的作用。
结果：
1乙醇并未影响integrin β1的表达和分布；
2在铺展过程中的对照组细胞内pFAK Y397形成较大的片状分布，尤以细胞边缘明显，迁移过程中细胞迁移方向的前缘亦可形成大片状pFAK Y397表达；而过量乙醇干预过的HUVEC中形成的片状结构小而少；
3乙醇增加FAK磷酸化，且呈剂量、时间依赖性（p<0.05，p<0.01），但并不影响FAK总蛋白水平；
4给予4MP可以削弱过量乙醇引起的FAK磷酸化增高（p<0.05）；给予乙醛增加pFAK Y397（p<0.01）；
5乙醇、乙醛刺激均可激发内皮细胞中ROS产生；
6给予外源性H2O2可以激发FAK磷酸化（p<0.01）；给予NAC可部分降低乙醇、乙醛引起的FAK磷酸化水平（p<0.05）。
小结：过量乙醇一方面干扰了FAK参与的内皮细胞黏着斑成熟，不能有效“引导”F-actin骨架，导致内皮细胞生存、粘附、迁移功能受损；另一方面增加pFAK水平，且乙醇和（或）其代谢产生的乙醛、氧化应激是引起FAK酪氨酸磷酸化的重要因素之一；过量乙醇并不影响FAK总蛋白水平。
第三部分：黏着斑激酶磷酸化在过量乙醇引起的内皮细胞毒性中的作用
目的：本部分实验将以PI3K/AKT信号通路着手研究过量乙醇干预后FAK磷酸化增高在内皮细胞一氧化氮（nitric oxide，NO）内稳态破坏中的作用。
方法：
1利用NO特异探针DAF-FM检测内皮细胞NO产生，并利用eNOS特异抑制剂N-硝基-L-精氨酸甲酯盐酸盐（ω-nitro-L-argine methyl ester，L-NAME）、 iNOS特异抑制剂L-canavanine和FAK自主磷酸化抑制剂PF573228（PF228）研究NO来源；
2在给予HUVEC乙醇刺激前给予PI3K/AKT通路抑制剂wortmannin和PF228预处理研究eNOS活化的相关通路；
3用western blot检测硝基酪氨酸（nitrotyrosine，NT）产生量。
结果：
1乙醇引起NO明显增高（p<0.05）；L-NAME或PF228可以减弱乙醇引起的NO增加（p<0.05），而L-canavanine作用不明显；
2给予wortmannin和PF228均可明显减弱乙醇引起AKT和eNOS磷酸化（p<0.05）；
3200mM乙醇干预24h后明显增加内皮细胞NT生成量（p<0.05）。
小结：过量乙醇刺激引起的FAK磷酸化通过PI3K/AKT通路促进的eNOS活化，产生过量NO，生成具有细胞毒性的NT，造成内皮细胞损伤。
结论：
过量乙醇可以引起脑血管内皮细胞损伤，其机制主要表现在两个方面：
1乙醇干扰黏着斑复合体成熟，影响内皮细胞功能；
2乙醇及其代谢产生的乙醛、氧化应激通过增加FAK磷酸化水平，激活PI3K/AKT通路，上调eNOS/NO系统，使NT过量生成，造成内皮细胞损伤。
Drinking has a long history in China, and with the development of socialactivities, drinking is becoming more and more common. The related socialproblems have been serious. In recent years, injury cases after drinking havebeen increasing, and the related cases are always complicated to judge. In1963, some researchers indicated that traumatic subarachnoid hemorrhage(tSAH) was related with heavy drinking. In forensic medicine cases, drinkingleads to high incidence and mortality of intracranial hemorrhage with a slightexternal force. Although some mechanisms have been studied, such as chagesof heart rate and blood flow, vascular permeability，vasomotor function, andthe imbalance between coagulation and anticoagulation, it is still difficult toexplain the reasons. There are some challenges in forensic practice: how toevaluate the contribute degree of external force and alcohol and how toevaluate the case nature.
Ethanol (EtOH) is the most important component of alcoholic beverages.Aftering drinking, the ethanol is taken into blood and the blood alcoholconcentration (BAC) increases rapidly. With blood stream, ethanol is taken toorgans in the body. Vascular endothelial cells (EC) lining the blood vesselsform the interface between the bloodstream and the vessel wall and as suchthey are continuously subjected to ethanol and metabolites from the flowingblood after drinking. And EC take part in ethanol metabolism. As result, ECare susceptible to ethanol totoxicity.
Endothelial cells (EC) are the substaintial constitution of vessels, whotake responsibility to material exchange, angiotasis regulation, bloodcoagulation, angiogenesis and leukocyte migration. In addition, EC ‘sense’and transmit the biosignals from shear stress, cycle tension and bloodcomponents and coordinate diverse cells to make biological response. In summary, the toxicity of ethanol to EC will lead injury of vascular disorder.
Cerebral vessels are charactered with autonomic regulation, so they aresensitive to EC injury. The current study was designed to study toxicity ofethanol to cerebral vessels, which will be helpful to futher study aboutmechanisms of tSAH with slight blow after drinking.
EC line the wall of all blood vessels with monolayer of cells, so thedelicate balance between cell-cell interaction and EC-matrix adhesion areimportant to endothelial function. Focal adhesions (FAs) are complexstructures that assemble at the plasma membrane in discrete regions ofintegrin-mediated recognition of extracellular matrix (ECM) components.They connect the extracellular filamentous meshwork to the intracellularcytoskeleton controlling or mediating bidirectional signal transductionthrough endothelial cell.
Focal adhesion kinase (FAK) is an important member in FA pathwaytransducting in-outside or outside-in signals. FAK is a non receptor proteintyrosine kinase (PTK), taking part in composition of focal adhesion (FA)complex and regulation of cytoskeleton. In the one hand, several factors, suchas integrin clustering, growth factor receptors and shear stress, regulate FAK.In another hand, FAK is a scaffolding protein binding with other proteins. SoFAK is involved in multiple pathways. There is evidence that FAK is relatedwith adhesion, migration, proliferation and mechanotransduction. Inpathological conditions, FAK related signal pathways become disorder. Thecurrent study was designed to observe the changes of FAK induced byexcessive ethanol, and to explore the mechanisms.
Part I: The cytotoxicity of ethanol to endothelial cells
Objective: To observe the cytotoxicity of excessive ethanol to vessels invivo and in vitro and to study systematically the effects of ethanol on structureand functions of endothelial cells.
Methods:
1The rats were given alcoholic beverage with a blunt tipped needle, andBAC was detected after drinking. Masson’s trichrome staining and transmission electron microscope (TEM) were used to observe themorphological changes of capillaries and basilar arteries (BA) from alcoholicrats with different duration of drinking;
2Human umbilical vein endothelial cells were given differentconcentrations or different duration of ethanol treatment, and the viability wasdetected with MTT, representing the cytotoxicity of ethanol;
3Apoptotic cells were determined by Annexin V/PI apoptosis detectionkit with flow cytometry;
4The morphological changes of the endothelial cell after ethanol treatedwere observed with light microscope, scanning electron microscope (SEM)and transmission electron microscope (TEM);
5After the ethanol treatment, the cells were harvested by TrypLE Express, and allowed to adhere in plates coated with fibronectin (FN) for1-2h.The adhesion was detected with MTT and the average areas were measured byimage soft; the role of integrin pathway in adhesion was observed by blockingintegrin with specific blocking antibody;
6Wound healing assay was used to observe the migration of HUVECafter ethanol treatment.
Results:
1The rats with short-time drinking appeared changes in capillaries:obvious vasogenic edema, enlarged perivascular space, microvillus decreasedand vacuolus in mitochondria;
2The rats with long-time drinking appeared vascular remodeling: SMChyperplasia in tunica media and collagen deposition in tunica adventitia, thewall: lumen ratio increasing (p<0.05), and endothelial desquamation;
3Excessive ethanol treatment decreased the HUVEC viability in a dose-and time-dependent manner. Compared to the control group, there were moreearly apoptotic cells (p<0.05) showing unregular nucleus, increased electrondensity of chromatin with half moon shape, and decreased cytoplasm;
4HUVEC after excessive treatment lost normal shape, even withorganelle injury.
5Excessive ethanol treatment decreased adhesion rate (p<0.05) andspreading areas of HUVEC (p<0.05). The antibody to block integrin β1reduced adhesion rate of control EC, without affects on ethanol treated EC.
6Excessive ethanol treatment decreased migration of HUVEC in woundhealing assay.
Summary: Excessive ethanol caused endothelial injury, includingmorphological changes, viability decreasing, and interference of adhesion,spreading, and migration of EC. The adhesion injury induced by ethanol mightbe related with integrin pathway.
Part Ⅱ: Changes of FAK and the related mechanisms in EC caused byexcessive ethanol
Objective: This part was designed to study the distribution, proteinexpression and phosphorylation of FAK after excessive ethanol treatment, andto explore the related metablisms.
Methods:
1Distribution of pFAK Y397in spreading and migarating EC wasobserved with immunofluorenscence.
2Total protein level and phosphorylation level of FAK were detectedwith western blot.
3To study the relationship between the metabolism of ethanol and theFAK changes, specific ADH inhibitor4MP and ethanol metaboliteacetaldehyde (Ach) were given to HUVEC.
4The oxidative stress from ethanol or Ach was detected with specificprobe DCFH-DA.
5To study the relationship between oxidative stress caused by themetabolism of ethanol and the FAK changes, exogenous H2O2and nonspecific ROS scanvenger N-acetyl-L-cysteine (NAC) were given to HUVEC.
Results:
1In control group, pFAK397appeared as large pelltes, especially at edgeof cells during spreading and in parapodia during migrating. In ethanol group,pFAK397appeared as small dots;
2Ethanol increased pFAK in a dose-and time-dependent manner(p<0.05，p<0.01), without affect on total FAK protein;
3Increased pFAK induced by ethanol was attenuated by4MP (p<0.05);Ach increased pFAK397significantly (p<0.05);
4Both ethanol and Ach increased ROS level of HUVEC;
5Exogenous H2O2incresased FAK phosphorylation (p<0.01); IncreasedpFAK induced by ethanol or Ach was attenuated by NAC (p<0.05).
Summary：Ethanol disordered the distribution of pFAK, and increasedthe level of pFAK; disordered distribution of pFAK was involved in FA mature,leading to injury in EC survival, adhesion and migration; Ach and oxidativestress induced by ethanol metabolism was one of important factors of FAKphosphorylation.
Part Ⅲ: The role of FAK phosphorylation on cytotoxicity of excessiveethanol to EC
Objective：This part was designed to explore the relationship betweenincrease of pFAK and NO-mediated homeostasis, and to evaluate whetherPI3K/AKT pathway was involved in this process.
Methods:
1NO production was detected with specific probe DAF-FM; eNOSinhibitor L-NAME, iNOS inhibitor L-NAME L-canavanine and inhibitor ofFAK autophosphorylation PF228were employed to assess the origin of NO;
2NT production was dectected by western blot;
3The pathways involving in eNOS activation were vertified withPI3K/AKT inhibitor wortmannin and PF228.
Results:
1Ethanol increased NO production (p<0.05)，which was attenuated bypretreatment of L-NAME or PF228(p<0.05), but not by L-canavanine.
2NT production was significantly increased after treated with200mMethanol for24h (p<0.05).
3Increase of AKT and eNOS phosphorylation induced by ethanol wasattenuated by both wortmannin and PF228(p<0.05).
Summary: Excessive ethanol increased NO generation and toxic NTproduction; FAK phosphorylation induced by ethanol promote the activationof eNOS via PI3K/AKT, which broke the NO-mediated endothelialhomeostasis.
Conclusions:
1Short-time drinking leads to microvasular morphological and structuralchanges, while long-time drinking causes vascular remodeling of cerebralarteries. Endothelial cells are the injury target of ethanol cytotoxicity.
2Ethanol disorders the distribution of FA, inducing decrease of survivalrate, and down-regulation of adhesion and migration ability.
3Metabolites and oxidative stress from ethanol increase FAKphosphorylation, which up-regulates FAK/PI3K/AKT/eNOS pathway.Overproduction of NO and ROS generates NT, taking part in endothelialinjury.
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