褪黑素通过ERK/MAPK通路调控动脉粥样硬化模型兔动脉内皮细胞MLCK的表达
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摘要
目的:探讨褪黑素(melatonin,MLT)是否可通过ERK/MAPK通路调控动脉粥样硬化(atherosclerosis,AS)模型兔动脉内皮细胞肌球蛋白轻链激酶(myosin light chain kinase ,MLCK)的表达。方法:复制兔AS模型,分析血清中各种脂质成分及相关生化指标的动态变化;γ-32P-ATP掺入法测定模型兔主动脉的MLCK活性;动脉经O.C.T包埋、冰冻切片,免疫荧光检测动脉内膜通透性的变化,同时用免疫组化检测动脉内膜MLCK的表达,HE染色观察动脉壁的形态结构; Western blot检测AS兔动脉组织MLCK的表达和ERK磷酸化水平;ox-LDL作用于体外培养的人脐静脉内皮细胞(HUVEC),分析MLCK转录和表达。结果:动脉粥样硬化兔模型复制成功,新西兰大耳白兔在高胆固醇饮食1周、2周、3周、4周后,与正常对照相比血清中甘油三酯(triglyceride,TG),胆固醇(cholesterol,CHO),高密度脂蛋白(high—density lipoprotein,HDL)和低密度脂蛋白(low-density lipoprotein,LDL)含量显著增加(P<0.05,P<0.01,P<0.05,P<0.01);而高胆固醇饮食8周时,测定的血清TG、CHO、HDL、LDL含量和4周相比增加不明显;高胆固醇饮食12周时,测定的血清TG、CHO和LDL含量和8周相比有所下降;高胆固醇饮食8周加喂MLT,与高胆固醇饮食组相比血清中TG、CHO和LDL含量下降。模型兔高胆固醇饮食1周、2周、3周、4周、8周、12周后,免疫荧光显示主动脉内膜通透性与正常对照相比呈逐渐增强趋势,Western blot和免疫组化则显示动脉内皮细胞MLCK的表达、ERK磷酸化水平与正常对照相比呈逐渐增强趋势,γ-32P-ATP掺入法显示动脉组织中MLCK的活性与正常对照相比呈逐渐增强趋势(P<0.05),而加喂MLT后,动脉组织MLCK的表达、ERK磷酸化水平及MLCK的活性和主动脉内膜通透性均有所下降。HE染色显示,高胆固醇饮食1周、2周、3周、4周、8周的兔主动脉内膜逐渐增厚,细胞间隙逐渐增大,高胆固醇饮食4周开始出现有少量泡沫细胞形成,高胆固醇饮食8周时泡沫细胞的量增多,至高胆固醇饮食12周时则形成了明显的动脉粥样硬化斑块,斑块表面有一层纤维帽,而加喂MLT后,主动脉内膜病变则有一定程度减轻。结论:提示在AS发生发展过程中,动脉内膜屏障完整性的改变可能与MLCK活性增强相关;AS模型兔主动脉组织中MLCK表达与ERK磷酸化呈现一致性。MLT可能通过ERK/MAPK通路下调模型兔动脉内皮细胞MLCK活性而减轻AS斑块的病变程度。
Objective To study the possibility of the expression of myosin light chain kinase(MLCK) in endothelial cell is regulated by the melatonin through the pathway of ERK/MAPK in atherosclerosis model rabbit. Methods To establish the model rabbit of atherosclerosis(AS) and analyze the dynamic variability of the lipids and other related biochemistry quotas in the serum of model rabbits. The MLCK activity of artery tissue of model rabbits was measured by the method ofγ-32P-ATP incorporated and the endothelial permeability was detected by immunofluorescent. The expression of MLCK in the endothelial cell was detected by immunohistochemistry and western blot, meanwhile, the phosphorylation of ERK in the endothelial cell was also detected by western blot. The change of morphology of artery wall in modle rabbits was examined according to the HE stain. The human umbilical vein endothelial cells (HUVECs) were treated by ox-LDL to analyze the levels of transcription and translation of MLCK. Results The AS rabbit model was established successfully. After being fed with the high cholesterol(CHO) for 1,2,3,4 weeks,the triglyceride(TG), CHO, high-density lipoprotein(HDL) and low-density lipoprotein(LDL) increased obviously in the serum, there is significantly statistical difference compared with the normal control( P<0.05,P<0.01,P<0.05,P<0.01). After being fed with high CHO for 8 weeks, the TG, CHO, HDL and LDL increased little in the serum and no statistical difference is found compared with the model group of 4 weeks. While after being fed with high CHO for 12 weeks,the content of the TG, CHO and LDL degraded compared with the model group of 8 weeks. In group fed with high CHO and melatonin, the concentrations of the TG, CHO and LDL decreased compared with the model group which fed with high CHO. After feeding with high CHO for 1,2,3,4,8,12 weeks, the MLCK activity of artery tissue of model rabbits increased gradually,and there is significantly statistical difference when compared with normal control(P<0.05). The permeability of arterial intima, the expression of MLCK and the phosphorylation of ERK in the artery tissue were raised gradually after the rabbits were fed with high CHO for 1,2,3,4,8,12 weeks. At the same time,the HE staining indicated that there are a lot of foam cells products gradually. When melatonin(MLT) was added into the high CHO food,the activity of MLCK,the permeability of arterial wall,the expression of MLCK and the phosphorylation of ERK in artery tissue degraded all,the aortic tunica intima symptom seem to be reliefed. Conclutions With the development of AS, the changes of endarterium integrity may be related with the augmented activity of MLCK. The expression of MLCK and the phosphorylation of ERK present consistency in artery tissue of AS model rabbit. MLT may relief the pathological changes of plaque in the AS model rabbit through the pathway of ERK/MAPK to down regulation the activity of MLCK in endothelial cell.
Objective To study the possibility of the expression of myosin light chain kinase(MLCK) in endothelial cell is regulated by the melatonin through the pathway of ERK/MAPK in atherosclerosis model rabbit. Methods To establish the model rabbit of atherosclerosis(AS) and analyze the dynamic variability of the lipids and other related biochemistry quotas in the serum of model rabbits. The MLCK activity of artery tissue of model rabbits was measured by the method ofγ-32P-ATP incorporated and the endothelial permeability was detected by immunofluorescent. The expression of MLCK in the endothelial cell was detected by immunohistochemistry and western blot, meanwhile, the phosphorylation of ERK in the endothelial cell was also detected by western blot. The change of morphology of artery wall in modle rabbits was examined according to the HE stain. The human umbilical vein endothelial cells (HUVECs) were treated by ox-LDL to analyze the levels of transcription and translation of MLCK. Results The AS rabbit model was established successfully. After being fed with the high cholesterol(CHO) for 1,2,3,4 weeks,the triglyceride(TG), CHO, high-density lipoprotein(HDL) and low-density lipoprotein(LDL) increased obviously in the serum, there is significantly statistical difference compared with the normal control( P<0.05,P<0.01,P<0.05,P<0.01). After being fed with high CHO for 8 weeks, the TG, CHO, HDL and LDL increased little in the serum and no statistical difference is found compared with the model group of 4 weeks. While after being fed with high CHO for 12 weeks,the content of the TG, CHO and LDL degraded compared with the model group of 8 weeks. In group fed with high CHO and melatonin, the concentrations of the TG, CHO and LDL decreased compared with the model group which fed with high CHO. After feeding with high CHO for 1,2,3,4,8,12 weeks, the MLCK activity of artery tissue of model rabbits increased gradually,and there is significantly statistical difference when compared with normal control(P<0.05). The permeability of arterial intima, the expression of MLCK and the phosphorylation of ERK in the artery tissue were raised gradually after the rabbits were fed with high CHO for 1,2,3,4,8,12 weeks. At the same time,the HE staining indicated that there are a lot of foam cells products gradually. When melatonin(MLT) was added into the high CHO food,the activity of MLCK,the permeability of arterial wall,the expression of MLCK and the phosphorylation of ERK in artery tissue degraded all,the aortic tunica intima symptom seem to be reliefed. Conclutions With the development of AS, the changes of endarterium integrity may be related with the augmented activity of MLCK. The expression of MLCK and the phosphorylation of ERK present consistency in artery tissue of AS model rabbit. MLT may relief the pathological changes of plaque in the AS model rabbit through the pathway of ERK/MAPK to down regulation the activity of MLCK in endothelial cell.
引文
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3. Kharitonova MA, Levina CM, Rovenskii IA. Cytoskeletal control of cell length regulation[J],Ontoqenez.2002;33(1):50-59.
4. Dudek SM, Garcia JG. Cytoskeletal regulation of pulmonary vascular permeability[J], J Appl Physiol.2001;91(4):1487-1500.
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6. Parathyroid hormone stimulates endothelial expression of atherosclerotic parameters through protein kinase pathways[J],Am J Physiol Renal Physiol.2007;292(4):F1215-1218.
7. Moses S, Franzen A, Lovdahl C,et al.Injury-induced osteopontin gene expression in rat arterial smooth muscle cells is dependent on mitogen-activated protein kinases ERK1/ERK2[J],Arch Biochem Biophys. 2001;396(1):133-137.
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