肌球蛋白轻链激酶对早期实验性动脉粥样硬化兔动脉内皮功能的影响及机制探讨

Atherosclerosis (AS) threat to human health, tends to younger age of onset. Early AS is characterized by intimal thickening, elastic membrane rupture, medial smooth muscle cell proliferation and migration of pathological changes, and endothelial dependent diastolic function (EDD) decreased. our previous researchs demonstrated that when fed a high cholesterol diet for 1 weeks, there was no obvious change of structure in arterial intimal wall and muscle layer, but the myosin light chain kinase (MLCK) expression and activity increased significantly, and increase along with the cholesterol diet feeding time increase. The expression and activity of MLCK can cause arterial muscle contraction, MLCK inhibitor ML-7 can significantly increase the experimental AS rabbit vascular EDD. It is suggested that the decrease of EDD maybe related with increase of MLCK expression, activity and enhance of systolic function of arterial wall in hypercholesterolemia and early AS. This project aims to reproduce high cholesterol and AS rabbit model, to analyze the changes of EDD using high frequency ultrasound; to detect the endothelial function using artery ring analysis and to observe the expression of diastolic function related protein in arterial endothelial cells cultured with hyperlipemia serum, finally, to investigate the effect the expression and activity of rabbit arterial wall MLCK on EDD in development of early AS and its regulation mechanism.

动脉粥样硬化(AS)严重威胁人类健康，发病年龄趋于年轻化。早期AS以内膜增厚、弹力膜断裂、中层平滑肌细胞增生和迁移等病理变化为特征，且动脉内皮依赖性舒张功能（EDD）下降。我们先前研究表明在喂饲高胆固醇饮食1周时动脉壁（包括内膜、肌层）未出现明显结构变化，但肌球蛋白轻链激酶（MLCK）的表达和活性出现明显升高，并随胆固醇饮食喂饲时间的增加而增强，而MLCK表达和活性的升高可引起动脉肌层的收缩增强，MLCK抑制剂ML-7可明显增加实验性AS兔血管EDD。提示在高胆固醇血症和早期AS时，EDD降低可能与动脉壁MLCK表达和活性增加、收缩功能增强有关。本项目拟复制高胆固醇血症和AS兔模型，用高频超声检测EDD变化；动脉环离体实验检测内皮舒张功能；并观察高脂血清等对动脉内皮细胞舒张功能相关蛋白表达等影响，探讨实验性AS发生发展过程中兔动脉壁MLCK的表达及其活性的变化对EDD的影响及其调控机制。

本研究成功复制了实验性早期动脉粥样硬化兔模型，采用经皮超声法检测腹主动脉血管内皮功能。结果表明高胆固醇血症模型兔血胆固醇、LDL-c明显升高，升高幅度在15倍以上。大体标本显示实验性高胆固醇血症兔主动脉未见明显动脉粥样硬化斑块，高脂饲料喂饲4周时兔主动脉及其分支处可见早期动脉粥样硬化改变，主要变化是可见脂质条纹和微小斑块，喂食高胆固醇食物8周时兔主动脉及其分支处可见明显动脉粥样硬化。高脂喂饲增加MLCK抑制剂ML-7 8周后与仅喂饲高脂饲料兔比较，主动脉脂质沉积或斑块明显减少。随着喂饲高脂饲料时间的增加，正常对照组动脉内膜通透性变化不大，喂饲高脂兔主动脉内膜通透性从4周开始明显增加，而ML-7组主动脉内膜通透性明显低于高脂组。正常对照组动脉中MLCK的表达较少，在高脂血症组动脉MLCK的表达急骤增强，ML-7干预组MLCK的表达量较高脂组有所下降。所有周次兔动脉高脂组和ML-7组pp38和pJNK表达明显高于对照组，与高脂组相比，ML-7组pp38和pJNK表达明显下调。实验性早期动脉粥样硬化兔离体和在体动脉舒张功能均明显降低，而ML-7可增加实验性早期AS兔离体和在体动脉内皮依赖性舒张功能。体外实验表明oxLDL抑制动脉内皮细胞一氧化氮合酶活性，降低细胞内NO含量。上述结果提示在高胆固醇饮食诱导的实验性兔AS形成过程中，当血浆胆固醇浓度升高，血液中oxLDL抑制一氧化氮合成酶的活性，使得血管壁NO浓度减少；激活MAPK（p38和JNK），通过信号转导促进MLCK的表达，MLCK表达和活性增加可磷酸化MLC，导致血管平滑肌细胞收缩。MLC磷酸化升高而NO降低而引起动脉EDD的降低，同时动脉内皮细胞向心性收缩、通透性增加，血液中致动脉粥样硬化因子，如oxLDL等进入动脉内膜，被移行的平滑肌细胞和巨噬细胞吞噬形成泡沫细胞，促进早期动脉粥样硬化形成。