The Effect Of Alcohol Intake On Cerebrovascular Function And Construction And The Relative Impairment Mechanism

Injury cases associated with heavy drinking is becoming a serious threaten to public security. Traumatic subarachnoid hemorrhage was common to a drunken person exposed to mild hit. And evaluation of drinking involvement in this kind of injury cases and investigation of mechanisms responsible for cerebral vascular injury attributed to alcohol drinking is helpful for forensic cases identification. It can also prevent against the occurrence of medical tangle.Ethanol is the most potent agents in all alcoholic-related drinks. Associated studies have illustrated the harmful action of ethanol to central nervous system, blood coagulation and vascular function whereas it is limited to explain the development of t SAH. In addition, acetaldehyde as the main metabolite of ethanol via acetaldehyde dehydrogenase has been proposed to be a potentially vascular risk factor.Under physiological condition, maintenance of normal cerebral blood flow is preserved via cerebral auto-regulation in response to stimulus. Yet, it is not clearly understood whether acute and chronic alcoholism induces alterations of cerebral vascular structure and whether the alterations are coupled with vascular dysfunction.Alcohol dehydrogenase, NADPH oxidase and nitric oxide synthase(NOS) are expressed in cerebral vascular wall and NADPH oxidase has been reported to mediate ethanol-induced cardiovascular disease. Furthermore, reactive oxygen specious(ROS) originated from NADPH oxidase act as the downstream of renin-angiotensin system(RAS). But it is unresolved concerning the question how ethanol and acetaldehyde induce cerebral vascular dysfunction and promote t SAH in a drunken person received mild hit.The present project is designed to address the association between ethanol/acetaldehyde and t SAH. Our experiments are concerns the following descriptions:(1) Long-term effect of alcohol intake on cerebral artery function.(2) Cytotoxic effect of ethanol and acetaldehyde on endothelium and smooth muscle cells.(3) Whether reactive oxygen specious are implicated in the cytotoxic action of ethanol and acetaldehyde on vascular wall.Part I Effect of alcohol intake on cerebral artery function and constructionObjective:Heavy alcohol drinking stimulates the elevated cerebral artery pressure, vascular dilation and enhanced vascular permeability. The alteration of cerebral artery homeostasis causes the dysfunction of central nervous system. We are going to use long-term alcohol-treated rodent animal model to investigate changes of vasomotor activity and vascular wall in cerebral artery.Method:1 The animals were received 20%v/v alcoholic beverage for 12 months. Animal body weights were recorded monthly.2 Hematoxylin-eosin HE staining,masson staining and thionine staining analysis was performed for changes of cerebral vascular and neurons structures.3 Isometric changes were recorded for measurement of ACh-evoked endothelium-dependent-relaxations and endothelium-independent relaxations induced by SNP of cerebral basilar artery. Contractile response to angiotensin II(Ang II) and endothelin were also recorded.4 Radioimmunoassay was performed to measure plasma Ang II and endothelin level of alcohol-treated and untreated rats.5 Changes of reactive oxygen specious in cerebral artery by dihydroethidium(DHE) fluorescence were recorded. Immunofluorescence and western blot assay were used to analyze the changes of ROS-associated protein levels.Results:1 Chronic alcohol intake induced a marked reduction of body weights. And alcohol-treated rats were also accompanied with lags in response and dry hair.2 Long-term alcohol treated rats induced cerebral vascular remodeling: smooth muscle cell hyperplasia in tunica media and collagen deposition in tunica adventitia, enhanced media-to-lumen ratio and endothelial desquamation.3 Changes of isometric tension showed that ACh-induced endothelium-dependent relaxations were impaired in cerebral basilar artery isolated from alcohol-treated rats. Whereas SNP-evoked endothelium-independent relaxations were not altered. Contractile responses to AngⅡ(10-7 mol/L)was enhanced in basilar artery of rats treated with alcohol. By contrast, no changes were observed in endothelin-evoked contraction.4 Alcohol treated rats were followed with elevation of plasma renin activity(PRA) and Ang II level. Matched with functional study, plasma endothelin content was not changed.5 Dihydroethidium fluorescence showed that excessive reactive oxygen specious detected in cerebral basilar artery of alcohol-treated rats and elevated ROS production is accompanied with upregulation of NADPH subunit P47 phox.Summary:The well-established alcohol-treated rodent animal model used in our studies is presented with cerebral artery remodeling, cerebral vascular dysfunction. Long-term heavy alcohol intake promotes renin-angiotensin system and oxidative stress. Excessive ROS contributes largely to cerebral vascular dysfunction induced by heavy alcohol intake.Part II Cytotoxic effect of ethanol and acetaldehyde on endothelial cellsObjective:Ethanol is converted into acetaldehyde via oxidation by alcohol dehydrogenase and ethanoic acid then generated via aldehyde dehydrogenase from acetaldehyde. Excessive ethanol and acetaldehyde accumulation isharmful to central nervous and cardiovascular system. In this part, human umbilical vein endothelial cells(HUVECs) are used to evaluate the cytotoxicity property of ethanol and acetaldehyde.Method:1 Human umbilical vein endothelial cells(HUVEC) were exposed to different concentrations or duration of ethanol and acetaldehyde. MTT assay was performed for cytotoxicity study.2 Apoptotic cells attributed to ethanol and acetaldehyde exposure were determined by flow cytometry.3 Transmission electron microscope was adopted to observe ultrastructure changes in ethanol or acetaldehyde treated cells.4 Western blot assay is used to detect the level of LC3 protein, biomarker of autophagy. Changes of protein levels in PI3K-AKT-m TOR cascade and RAS-MEK-ERK pathway were also analyzed by western blot to illustrate the mechanism of acetaldehyde-stimulated autophagy. ERK inhibitor PD98059 is helpful to explain the mechanism of autophagy.5 DHE staining was adopted for measurement of ROS level in ethanol and acetaldehyde treated HUVECs. Exogenous ROS donor H2O2 and non-selective ROS scavenger NAC(N-acetyl-L-cysteine) are utilized to elaborate the involvement of ROS-ERK pathway in the development of HUVECs autophagy.Results:1 HUVEC viability test indicated cytotoxic property of ethanol and acetaldehyde and this effect is dose- and time-dependent manner. Actually, acetaldehyde shows powerful potency in cytotoxic action compared to ethanol.2 Apoptotic cells induced by 24h- and 48h-ethanol(200m M) incubation were presented with irregular nucleus, increased electron density of chromatin with half moon shape, and decreased cytoplasm. 20μM-acetaldehyde-6h treated HUVECs were presented with no-apoptosis death. And autophagosome was observed in acetaldehyde-treated HUVECs.3 Cytoskeletal derangement and Cell body shrinkage were detected in HUVECs exposed to acetaldehyde, which is also coupled with the enhancement of LC3-Ⅱ/LC3- Ⅰratio at time- and concentration-dependent manner.4 Acetaldehyde promoted the increased phosphorylation of ERK and the effect was time-dependent. And the protein level of p AKT/AKT and pm TOR/m TOR are not changed upon acetaldehyde addition. ERK inhibitor PD98059 co-treatment partially reverses the elevation of LC3-Ⅱ/LC3- Ⅰratio.5 Ethanol and acetaldehyde caused the augmented ROS production in HUVECs at different manner. Elevation of ROS level by ethanol was momentary and maximal ROS generation was detected at 30 mins exposure. Differently, Elevation of ROS level by acetaldehyde was more higher, faster and so lastingly.The maximal ROS generation was detected at 20 mins exposure, keeping a higher level for 50 min.6 H2O2 facilitated ERK phosphorylation and induced markedly elevation of LC3-Ⅱ/LC3-Ⅰratio, which was partly prevented with the addition of non-specific ROS scavenger NAC.Summary:Prolonged exposure to higher dose of ethanol induced apoptosis in HUVECs. Augmented ROS induced by acetaldehyde treatment mediated the following activation of RAS-MEK-ERK, leading cell autophagy. Actually, PI3K-AKT-m TOR cascade played minimal role in mediating acetaldehyde action.Part III Cytotoxic effect of ethanol on vascular smooth cellObjectives:Smooth muscle cells contribute largely to vascular structure and configuration. In this part, cytotoxic effect of ethanol on human brain vascular smooth cell(HBVSMC) will be illustrated.Method:1 Human brain vascular smooth cell(HBVSMC) were exposed todifferent concentrations or duration of ethanol and acetaldehyde treatment. MTT detection was performed for cytotoxicity study.2 Apoptosis evaluation by cytometry flow and TUNEL detection in ethanol-treated HBVSMC is to determinethe toxic efficacy of ethanol: apoptosis or necrosis.3 Morphologic changes in ethanol-treated HBVSMC were visualized by confocal microscopy. Specific probed F-actin and G-actin detection were used to assess the action of ethanol on cytoskeleton.4 Alterations of cytoskeleton associated proteins F-actin, G-actin, SM22 a, Cofilin and Cx43 were determined by western blot.5 ROS levels in HBVMC were determined by dihydroethidium fluorescence using confocal microscopy.Results:1Cytoactivity of HBVSMC exposed to different concentrations of ethanol(0- 800 m M) for 24 h remained unaffected. Whereas prolonged treatment to 48 h and 72 h higher 200 m M promoted the reduction of cytoactivity and the effect is dose- and time-dependent manner.2Apoptosis was observed in HBVSMC exposed to higher level of ethanol.3 Long-term treatment with excessive ethanol results from morphological alteration of HBVSMC: increased length shaped HBVSMC coupled with enhanced cell gap. In contrast, Microfilament arrangement and F-actin/G-actin ration were not changed. Western blot analysis displayed elevated expression of Cofilin, down-regulation of Cx43 and no change of SM22α.4 Ethanol treatment induced the elevation of ROS level in HBVSMC.Summary:Apoptosis was detected in HBVSMC exposed to high concentration of ethanol for a longer duration and is followed with changes of Cx43 and cofilin expressions. Yet, cytoskeletal structure was unchanged. No alterations were detected in SM22α protein level and F-actin/G-actin. Furthermore, exaggerated ROS production evoked by ethanol is involved in the subsequentapoptosis.Conclusions:1 Long-term alcohol intake induced the impairment of endotheliumdependent relaxations and stimulated renin-angiotensin system, with ROS generation enhancement. Oxidative stress serves as a key factor to alcohol-induced cerebral vascular dysfunction.2 Prolonged exposure to higher dose of ethanol induced apoptosis in HUVECs. ERK activation in HUVECs exposed to acetaldehyde was ROS-dependent and involved in mediating autophagy process.3 Augment ROS production was observed in ethanol-treated HBVSMC. Ethanol treatment for a long time caused HBVSMC apoptosis and alterations of Cofilin and Cx43 expressions, aggravated cerebrovascular damage.