iPLA2β-LPC系统:钙拮抗剂抗心肌I/R损伤新靶点与新机制的研究

Calcium independent phospholipase A2β (iPLA2β) has a variety of physiological functions. Its over expression and high activity are closely related to myocardial ischemia/reperfusion (I/R) injury, which opens the store-operated calcium channels (SOC) on endothelial cells, and voltage-dependent L-type calcium channels (L-VDCC) on cardiomyocytes, and lead to calcium overload. Our result also indicated in the Cacna1C-/- H9c2 cells and cardiac microvascular endothelial cells which have no L-type calcium channel, higher expression of iPLA2β also aggravate cell injury under cardiomyocytes hypoxia-reoxygenation. These showed that myocardial I/R injury by iPLA2β mediated have calcium channel-dependent and calcium channel-independent mechanism. Our other results have confirmed that calcium channel blockers (CCB) can protect against cardiomyocyte H/R injuries through calcium channel-independent mechanisms. In view of the above, we propose that CCB have a “calcium channel-dependent” and “calcium channel-independent”hypothesis against myocardial I/R injury through iPLA2β regulation. Based on this hypothesis, we plan to study CCB which not only inhibit the opening of L-VDCC and SOC by suppressing the expression and activity of iPLA2β, but also regulate iPLA2β to protect against myocardial I/R injuries through calcium channel-independent mechanisms. We further explore the effects of CCB on iPLA2β gene transcription, identify the promoter sites that can be intervened. The results from the study will provide novel targets and new molecular mechanisms against myocardial I/R injury with CCB.

钙非依赖磷脂酶A2β（iPLA2β）有多样生理功能，其表达和活性增强与心肌缺血/再灌（I/R）损伤密切相关，可致心肌细胞膜L-型钙通道（L-VDCC）和内皮细胞膜SOC通道开放，导致钙超载。我们结果显示在敲除L-VDCC的心肌细胞和缺失L-VDCC心脏微血管内皮细胞制作缺氧/复氧（H/R）模型，iPLA2β异常表达仍可致细胞损伤加重，表明iPLA2β介导损伤有钙通道与非钙通道依赖机制。在我们观察到钙拮抗剂具有非钙通道依赖抗细胞H/R损伤的基础上，提出钙拮抗剂对iPLA2β异常表达的调控发挥抗心肌I/R损伤具有“钙通道”和“非钙通道依赖”假说。本课题拟研究钙拮抗剂既通过iPLA2β对L-VDCC和SOC开启的调节，拮抗钙超载，又有不依赖对钙通道作用抗心肌I/R损伤机制，通过探寻作用于iPLA2β启动子位点，找到调控iPLA2β的转录调节的直接证据，探讨钙拮抗剂抗心肌I/R损伤新靶点及新机制。

本课题组前期发现钙拮抗剂(CCB)呈现了非L型钙通道（L-VDCC）阻断抗心肌缺血再灌注（I/R）损伤的作用，利用基因芯片表达谱差异筛选，发现钙非依赖磷脂酶(iPLA2β) 可能是CCB抗心肌I/R损伤的新靶点与新机制。本项目在敲除L-VDCC的CacnalC-/-H9c2细胞、无L-VDCC心脏微血管内皮细胞（CMECs）以及无细胞外钙条件下原代心肌细胞（NRVMs）缺氧复氧（H/R）模型及心肌I/R整体动物模型上，借助于基因敲除或转染等技术进行研究，发现iPLA2β高表达是小鼠心肌I/R损伤的重要原因；H/R可引起iPLA2β表达增加，活性增高，进而导致花生四烯酸（AA）以及溶血磷脂酰胆碱（LPC）浓度增加, 最终导致心肌细胞及CMECs损伤。敲除iPLA2β基因后能明显减轻I/R导致的氧化应激增加，炎症以及心肌组织凋亡水平，改善小鼠心肌I/R损伤。经典钙拮抗剂维拉帕米（Ver）、硝苯地平（Nif）、地尔硫卓（Dil）以及我们实验室创新合成钙拮抗剂F2可通过抑制非L-VDCC依赖的 iPLA2β/LPC/PKA/CREB/c-Fos通路拮抗心脏来源细胞H/R损伤和心肌I/R损伤。同时发现阈下剂量（不影响血流动力学）Nif和Ver能显著降低I/R后小鼠血清中AA的浓度，Nif和F2能显著降低I/R后小鼠血清中LPC的浓度。此外F2可通过钙依赖方式抑制SOC离子通道STIM1，Orai1/Orai2的mRNA表达。总之，我们从三种细胞体外模型，整体动物模型上说明CCB可通过非L-VDCC调控iPLA2β表达及活性，进而减少AA和LPC的生成，发挥其抗心肌I/R损伤以及心肌细胞H/R损伤作用，是对CCB拮抗心肌I/R损伤作用机制的重要补充。