褪黑素通过ERK/MAPK通路调控AS兔动脉内皮细胞MLCK表达

Regulation of Melatonin on Expression of Myosin Light Chain Kinase in Endothelial Cell through the Pathway of ERK/MAPK in Atherosclerosis Model Rabbit

目的探讨褪黑素(melatonin,MLT)是否可通过信号调节激酶/丝裂原活化蛋白激酶(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,分析血清中相关脂质成分的动态变化;γ-32P-ATP掺入法测定模型兔主动脉的MLCK活性;动脉经冰冻切片,免疫组化检测动脉内膜MLCK的表达,伊红(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。结果AS兔模型复制成功,实验兔在高胆固醇(cholesterol,Cho)饮食4,8,12周后,与正常对照相比,血清中相关生化指标有逐渐增加趋势(P<0.05);γ-32P-ATP掺入法显示动脉组织中MLCK的活性呈逐渐增强趋势(P<0.05),Western blot和免疫组化则显示动脉内皮细胞MLCK的表达、ERK磷酸化水平亦呈逐渐增强趋势,HE染色显示主动脉内膜逐渐增厚,细胞间隙逐渐增大;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,而泡沫细胞形成量逐渐增多,至高Cho饮食12周时则形成了明显的AS斑块,加喂MLT后,主动脉内膜病变则有一定程度减轻。结论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 expression of myosin light chain kinase（MLCK） in endothelial cell regulated by the melatonin through the pathway of ERK/MAPK in atherosclerosis（AS） model rabbit. METHODS The model rabbit of AS was established and the dynamic variability of the lipids in the serum of model rabbits was analyzed. The MLCK activity of artery tissue of model rabbits was measured by the method of y-32p-ATP incorporated.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 model rabbits was examined according to the HE stain. RESULTS The AS rabbit model was established successfully. After being fed with the high cholesterol （Cho） for 4,8,12 weeks, compared with the normal control, the related biochemistry quotas was increased obviously in the serum （P〈0.05）,the MLCK activity of artery tissue of model rabbits was increased gradually（P〈0.05）,the expression of MLCK and the phosphorylation of ERK in the artery tissue were raised gradually. At the same time, the HE staining indicated the increase of foam cells products. 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 were decreased. The aortic tunica intima symptom seem to be reliefed. CONCLUSION MLT may relief the pathological changes of plaque in the AS model rabbit through the pathway of ERK/ APK to down regulate the activity of MLCK in endothelial cell.