食醋及其功能因子——四甲基吡嗪心肌保护作用中mPTP/VDAC1/ClICR通路的意义

In light of previous work by us and others, we hypothesize: vinegar and its functional factor, tetramethylpyrazine (TMP) directly acts on mitochondrion mPTP as does DIDS, inhibiting Cl- entering the VDAC1, offsetting “original trigger” of ClICR, preventing Ca2+ overload, and thereby playing a protective role in myocardium. Therefore, such factors as Cl-, mPTP, VDAC1, ClICR, mCICR will be comsidered in studying the cause and effect relationship between the protective role of vinegar, TMP and ClICR/Ca2+ overloading from a new angle. Adenovirus infection, plasmid and RNAi transfection will be used to altergene expression. Once the protective role of vinegar, TMP have been shown, these protective effect against cardiomyocytes A/R injury influenced by different levels of VDAC1 and variation of Ca2+, Cl- in intracellular microenvironment will be further examined. Moreover, the underlying mechanisms will be investigated at different levels, such as mitochondrial membrane potential, ROS, mPTP opening; Finally the above finding will be tested in vivo; ous purpore is to explore the molecular mechanisms involving in the protective role of vinegar and TMP in myocardium and provide theoretical and experimental rationale for scientific evaluation of health quality for food such as vinegar and wine, improvement of food quality, and perfection of dietary pattern.

在总结前人与自身工作基础上，假设：心肌细胞急性损伤时，食醋及其功能因子——四甲基吡嗪（TMP）可像DIDS直接作用于线粒体mPTP，抑制Cl-通过VDAC1，取消ClICR的“起始触发”作用，防止Ca2+超载，发挥心肌保护作用。拟将Cl-/mPTP/VDAC1/ClICR/mCICR有机结合，从全新思路探讨食醋和TMP心肌保护作用与ClICR/Ca2+超载的因果关系。用重组腺病毒载体、基因转染和RNAi技术，构建相应的转基因细胞，在确认已产生保护作用的基础上，观察不同VDAC１表达水平及改变细胞微环境中Ca2+ 、Cl-对其抗心肌细胞A/R损伤能力的影响，从线粒体膜电位、ROS系统活性、mPTP开放等角度探讨其作用机制；最后在整体动物上进行比较论证；旨在用现代科学理论与方法探讨食醋和TMP心肌保护作用的分子机制，为食醋、酒等相关食品保健功能科学评估等，完善人们膳食结构提供理论与实验依据。

本项目将Cl-/mPTP/VDAC1/ClICR/mCICR相结合，从一个全新思路探讨了TMP/食醋心肌保护作用与ClICR/Ca2+超载的因果关系。首先完成了6种市售食醋中TMP含量测定，5种含有TMP，含量与储存时间成正比。构建了pAD/VDAC1及pAD/VDAC1-shRNA及转染、测定了H9C2细胞相应蛋白；预处理给予TMP/Vinegar孵育24 h并经A/R损伤后，细胞存活率增加，LDH、CPK活性降低，呈剂量依赖性；VDAC1表达下调，caspases-3活性/凋亡比例降低，细胞OCR/ECAR与储备能力/ATP生成增加，细胞酸中毒改善；SOD/GSH-Px/Catalas活性增加、MDA含量/ROS生成减少；线粒体膜电位稳定，mPTP开放以及cyt c释放显著减少；VDAC1高表达以及细胞外液Ca2+ free 或 Cl- free可逆转TMP/Vinegar相关保护作用。接着完成了整体小鼠验证实验：喂食0.1 ml/10g/d Vinegar及6 mg/kg/d TMP，持续6 w，可有效改善左心功能、降低LDH、CPK与caspases-3活性，减小凋亡指数和心梗面积，抑制氧化应激，具有良好的预适应保护作用，上调VDAC1表达可逆转之。综上所述，TMP/Vinegar体内/体外预处理，均可诱导良好的预适应心肌保护作用，机制与下调VDAC1表达、抑制氧化应激、维护线粒体功能有关；TMP是食醋的主要活性成分。我们还证实：TMP可上调血管内皮14-3-3γ表达，促进Bad磷酸化，帮助Bcl-2定位线粒体，维持线粒体功能；促进eNOS磷酸化/NO生成，对抗阿霉素/铁过载诱发的内皮功能衰竭；相关机制可成为干预药物分子靶点，为类似药物研发奠定理论与实验基础。本课题全面完成研究计划，发表学术论文14篇（标注资助），并培养博士生2名，硕士生4名。