白藜芦醇对VDAC1的翻译后修饰在心肌缺血再灌注损伤中的作用研究

The effect of resveratrol's protection against myocardial ischemia/reperfusion, is tightly related to its inhibition to mitochondrial permeability transition pore (mPTP) opening. As we previously demonstrated, voltage dependent anion channel 1(VDAC1), a channel that located in the outer membrane of mitochondrial, is involved in the effects of resveratrol on mPTP. Our preliminary experiments also found that both agonist of p38 mitogen-activated protein kinase (MAPK) and antagonist of SIRT1 abolish the protective effects of resveratrol. Combined with literature, we presume that resveratrol regulates VDAC1 activity by means of reduces VDAC1 phosphorylation via inhibiting p38 MAPK pathway, and cooperates with deacetylation on VDAC1 by activating SIRT1. mPTP opening is then blocked, and as a final effect, resveratrol protect caridomyocytes from ischemia/reperfusion injury. In this project, we planed to use gene cloning, RNA interference and recombinant adenovirus vector to construct corresponding transgenic cells and aniamals. A variety of molecular biology techniques, combines with classical functional evaluations to make a full illustration about the role of p38 MAPK mediated phosphorylation and/or SIRT1 mediated deacetylation modification of VDAC1 in the mitochondrial mechanism of resveratrol in myocardial protection. The research work provides new ideas to investigate natural nutrients and plant compounds in prevention and treatment of cardiovascular diseases.

白藜芦醇抗心肌缺血再灌注损伤作用与防止mPTP开放有关。我们前期研究证实白藜芦醇对mPTP的影响是由线粒体外膜上的VDAC1介导；预实验中又发现p38MAPK通路激动剂和SIRT1抑制剂均可取消白藜芦醇的心肌保护作用。结合文献，我们假设心肌缺血再灌注时，白藜芦醇通过抑制p38MAPK通路减少VDAC1磷酸化，且又通过激活SIRT1途径对VDAC1去乙酰化，两种方式协同调节VDAC1表达与活性，防止mPTP开放，进而保护心肌细胞。为此，拟用基因克隆、RNAi等重组相应蛋白的腺病毒表达载体，构建相应的转基因细胞与动物，将多种细胞分子生物学技术与经典功能学评价方法有机结合，从心肌细胞及整体动物水平，阐明p38MAPK介导的磷酸化和/或SIRT1介导的去乙酰化修饰VDAC1，在白藜芦醇心肌保护的线粒体机制的作用及其意义，为正确诠释、研究开发防治心血管疾病的天然营养素、植物化合物开辟新的思路。

本课题建立了RNA干扰、腺病毒构建等研究的技术平台。本研究发现，H9c2心肌细胞经缺氧/复氧（A/R）处理后，电压依赖性阴离子通道（VDAC1）乙酰化及磷酸化水平明显增高。而白藜芦醇预处理可激活SIRT1和Akt-GSK3β通路，抑制VDAC1的乙酰化及磷酸化，从而抑制VDAC1与Bax的结合，但提高VDAC1与Bcl-2和HK2的亲和力。通过对VDAC1翻译后修饰的影响，白藜芦醇可有效抑制ROS生成，稳定线粒体膜电位，防止mPTP开放，最终减少细胞凋亡，保护心肌细胞。动物实验与细胞水平结果一致。大鼠心脏经缺血/再灌注损伤后，VDAC1磷酸化及乙酰化水平均升高。然而白藜芦醇喂食大鼠SIRT1和Akt-GSK3β通路活性均明显提高，可抑制VDAC1的磷酸化及乙酰化，从而对抗心肌缺血再灌注损伤。本课题全面完成研究计划，发表学术论文5篇，培养硕士研究生4名。