MIF介导的钙拮抗剂和F2非L-型钙通道依赖地抗心肌I/R损伤机制的研究

According to scattered reports and study results at our Lab on cardiomyocytes, coronary microvasculature endothelial cells and myocardium, calcium channel blockers (CCB)and F2 (new CCB synthesized by our Lab) do have calcium channel independent effects to antagonize myocardial ischemia/reperfusion (I/R) and cellular hypoxia/reoxygenation (H/R) injury，which related to regulation on abnormal expression of MIF, in addition to L-type calcium channel blockade effect. Based on previous studies, we hypothesize that CCB and F2 have direct and indirect actions on MIF evidently increased expression in early I/R or H/R phase, and apparently decreased expression in late phase. L- type calcium channels independent inhibition of MIF over expression could reduce synthesis and release of various inflammatory factors to improve injury and protect cell function in early phase, then MIF expression will not decrease to be lower than normal level, finally to maintain its homeostasis in which MIF could retain the effects to inhibit free radical and apoptosis. The aim of the subject is to study effects of MIF systematically and comprehensively on I/R or H/R injury. We will focus on investigation on CCB and F2 indirect effects of regulating MIF expression to antagonize I/R injury, and to provide a novel target against I/R injury.

近年来零散报道和我课题组通过心肌细胞、冠脉内皮细胞和整体实验充分证实钙拮抗剂和F2（我室合成的新型钙拮抗剂）除阻断L-型钙通道外，确实存在非钙通道依赖地抗心肌缺血再灌（I/R）和细胞缺氧复氧（H/R）损伤的多方面作用，且与调控巨噬细胞移动抑制因子（MIF）的异常表达有关。 在此工作基础上，提出如下科学假说：钙拮抗剂和F2对I/R或 H/R过程中 MIF出现先"过高"后"过低"表达有"直接作用"和"间接效应"。早期通过非L-型钙通道依赖地抑制MIF高表达减少各种炎症因子的合成与释放，减轻细胞损伤，在这个过程中，细胞功能得以改善，使得MIF表达在损伤后期不会显著降低，维持其稳态平衡。从而发挥MIF抗自由基、抗凋亡等作用。 围绕这一假说，本课题拟探讨MIF在I/R或H/R损伤中变化规律及意义, 重点研究钙拮抗剂和F2调控MIF表达抗I/R损伤的"间接效应"机制，为防治I/R损伤新靶点提供依据。

本课题分别从无钙培养（3小时内）的大鼠原代心肌细胞、敲除L-型钙通道（L-VDCC）的心肌细胞株（Cacna1C-/-H9c2细胞）、无L-VDCC的大鼠心脏微血管内皮细胞和小鼠在体模型上分别观察到在以上细胞H/R和小鼠心肌I/R发展过程中，MIF表达呈现“先高后低”明显变化，且损伤表现为进行性加重，通过先期给予MIF抗体（MIF高表达期）和后期给予重组的MIF（MIF低表达期）干预，细胞H/R损伤得以改善，反之则反；小鼠在体采用MIF-/-和WT比较，MIF-/-小鼠心肌早期I/R损伤较轻。在得出MIF与细胞H/R及 心肌I/R损伤相关的基础上，观察Ver和F2对MIF表达的影响，发现Ver和F2早期给予可使MIF的 “先高后低”有所恢复，并显示良好的细胞的保护作用，在体给予“阈下剂量”Ver和F2可使心脏I/R损伤减轻，确证其作用与阻断L-型钙通道无关。在H/R和I/R的较长时间的MIF低表达期才给予Ver和F2则显示对于MIF表达及损伤均无影响，表明课题假说Ver和F2的早期直接作用及晚期的间接作用是正确的。