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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 investigate whether melatonin (MLT) regulates the expression of myosin light chain kinase (MAPK) in arterial endothelial cells of atherosclerosis (AS) model by the signal regulated kinase / mitogen activated protein kinase (ERK / MAPK) (myosin light chain kinase, MLCK) expression. Methods Rabbit AS models were replicated and the changes of serum lipid components were analyzed. The MLCK activity was measured by γ-32P-ATP incorporation assay. The arterial sections were stained with frozen sections and immunohistochemistry to detect the expression of MLCK. The morphology of arterial wall was observed by HE staining. The expression of MLCK and ERK phosphorylation were detected by Western blot. Results The rabbit model of AS was successfully replicated. After 4, 8 and 12 weeks of cholesterol (Cho) diet, the serum biochemical parameters showed a trend of increase (P <0.05) compared with the control. The level of γ-32P -ATP incorporation method showed that the activity of MLCK in arterial tissue was gradually increased (P <0.05). Western blot and immunohistochemistry showed MLCK expression in endothelial cells and the phosphorylation of ERK also showed a gradually increasing trend. HE staining showed The thickening of the intima of the aorta and the gradual increase of the interstitial space of the aorta increased the expression of MLCK, the phosphorylation of ERK, the activity of MLCK and the intima aortic intima in MLT, Formation gradually increased, to high Cho diet 12 weeks when the formation of a significant AS plaque, fed with MLT, aortic intimal lesions have a certain degree of relief. Conclusion MLT may reduce the severity of AS plaque by down-regulating the MLCK activity of the model rabbit arterial endothelial cells through ERK / MAPK pathway.