GABA及其受体在心肌梗死缺血再灌注损伤中的心肌保护机制研究

Myocardial preservation mechanism of GABA and its receptors in ischemia-reperfusion injury from myocardial infarction

目的探讨GABA及其受体在心肌梗死缺血再灌注损伤过程中的心肌保护机制。方法取自SD大鼠的原代心室肌细胞,根据不同干预分为6组:空白对照组(BC组)无任何干预措施;空白模型组(BM组)模拟缺血再灌注模型,缺糖缺氧培养6 h后,恢复正常含糖及氧浓度培养3 h;干预组包括:在缺糖缺氧及复糖复氧开始时给予GABA 100μmol/L(GBAB组)、GABABR激动剂巴氯芬100μmol/L(Baclofen组)、TLR4拮抗剂E5564 100μmol/L(E5564组)以及GABABR拮抗剂CGP35348 100μmol/L(CGP35348组)。MTT方法检测心肌细胞活力,RT-qPCR方法检测心肌细胞GABABR及TLR4基因表达。ELISA法检测上清液TNF-α、CKMB。结果缺血再灌注损伤前后,心肌细胞活力严重受损,重要保护因子GABABR基因表达显著下调(0.72 vs 0.23,P <0.001),而关键促炎因子TLR4基因表达显著升高(1.40 vs 1.60,P <0.05),CKMB、TNF-α均显著升高(4.70±3.08 vs 34.10±13.75,25.50±9.78vs 68.75±12.97,P <0.001)。与BM组比较,Baclofen组GABABR、TLR4基因表达变化均无统计学差异,其炎性指标CKMB、TNF-α均显著下降(19.46±8.43 vs 34.10±13.75,47.31±14.79 vs 68.75±12.97,P <0.01);CGP35348组因失去GABABR保护,其结果与BM组基本一致。结论 GABA及其受体通过抑制促炎细胞因子来减轻炎症反应,从而在心肌梗死缺血再灌注损伤过程中起到心肌保护作用。TLR4通路在该心肌保护机制中可能发挥重要调节作用。

Objective To investigate the myocardial preservation mechanism of GABA and its receptors in ischemiareperfusion injury from myocardial infarction. Methods Primary ventricular myocardium cells derived from SD rats were randomly assigned to six groups based on different treatments: blank control( BC) group in which there was not any intervention,and blank model( BM) group in which ischemia-reperfusion injury was simulated with glycoprival and anaerobic cultivation for 6 h,followed with resumption of normal saccharic and aerobic cultivation for 3 hs,intervention groups composed of subgroups: E5564 group treated with 100 μmol/L GABABR group with 100 μmol/L GABA B receptor agonist Baclofen,E5564 group with 100 μmol/L Toll-like receptor antagonist E5564,and CGP35348 group with GABA B receptor antagonist CGP35348 during glycoprival and anaerobic cultivation and saccharic and aerobic cultivation,respectively. The activity of the myocardium cells was detected by MTT. The gene expression of GABA B receptors and Toll-like receptor 4 was measured by RT-qPCR. Supernatant solution was used to detect the content of TNF-α and CKMB by ELISA technique. Results During ischemia-reperfusion injury,the vitality of myocardium cells was seriously damaged. GABA B receptors,the critical myocardial protectin,were remarkably down-regulated in mRNA level( 0. 72 vs. 0. 23,P < 0. 001),while Toll-like receptor 4,the important proinflammatory factor,was significantly up-regulated( 1. 40 vs. 1. 60,P < 0. 05). The content of CKMB and TNF-α was also significantly increased in the cultural supernatant( 4. 70 ± 3. 08 vs. 34. 10 ± 13. 75,25. 50 ± 9. 78 vs.68. 75 ± 12. 97,P < 0. 001). When preconditioned with Baclofen,the gene expression of GABA B receptors and Toll-like receptor 4 showed no significant difference before and after ischemia-reperfusion injury. The content of CKMB and TNF-α was significantly decreased compared with BM group( 34. 10 ± 13. 75 vs. 19. 46 ± 8. 43;68. 75 ± 12. 97 vs. 47. 31 ± 14. 79,P <0. 001). The results of CGP35348 group were consistent with BM group. Conclusions By way of relieving inflammatory reaction through inhibiting proinflammatory cytokine,GABA and its receptors play a protective role in myocardial cells during ischemia-reperfusion injury. This protective mechanism may play a critical role in myocardial preservation mechanism via Toll-like receptor 4 pathway.