牛樟芝及其单体通过调控乙醛脱氢酶2抗心肌纤维化作用的研究

Aldehyde dehydrogenase 2 (ALDH2) is a new target in intervention for cardiovascular diseases. However, currently there are no new cardiovascular drugs on the market that aim at targeting ALDH2. Antrodia cinnamomea (AC) is a kind of fungus grown in Taiwan, and there are no reported studies about its protective effects on cardiovascular diseases. Preliminary results from our study showed that the inhibitory effects of AC on both alcoholic fatty liver disease and nonalcoholic fatty liver disease were all dependent on the existence of ALDH2 gene, and AC exhibited such effects by upregulating the enzyme activity of ALDH2, indicating that ALDH2 could be the probable therapeutic target of AC. In addition, AC was found having inhibitory effects on myocardial fibrosis in wild type mice, which was accompanied by upregulation of ALDH2 enzyme activity, decrease of 4-hydroxynonenal, suppression of mRNA expression of genes related to oxidative stress, as well as downregulation of mRNA expression of AP-1, TGF-β1 and Samd3. These phenomena suggest that AC probably upregulated the enzyme activity of its potential target, ALDH2, to scavenge reactive oxygen species, therefore inhibited activation of the TGF-β1 pathway induced by mitochondrial oxidative stress, finally led to the prevention of myocardial fibrosis. In this research project, we will use angiotensin II to induce myocardial fibrosis in both wild type and ALDH2 knockout mice, as well as in primary cardiac fibroblasts. The inhibitory effects and related mechanisms of AC and its active compounds on myocardial fibrosis, and the role of ALDH2 therein will be investigated on these in vivo and in vitro myocardial fibrosis models. Moreover, the molecular mechanisms behind the regulation of ALDH2 enzyme activity by the active compounds in AC will be revealed. Our study will be of great significance to the discovery of new cardiovascular drugs that target ALDH2.

乙醛脱氢酶2(ALDH2)是心血管疾病干预的新靶点，目前尚未有针对此靶点的新药上市。牛樟芝(AC)是产于台湾地区的真菌，尚未见AC防治心血管疾病的报道。我们研究发现AC抗脂肪肝的作用依赖于ALDH2基因，通过上调ALDH2酶活性实现，表明AC的作用靶点可能是ALDH2；AC具有抑制野生型小鼠心肌纤维化的作用，伴随心脏ALDH2酶活性上调，4-HNE水平减低，氧化应激抑制，AP-1、TGF-β1和Samd3表达下调，表明AC可能通过上调靶点ALDH2的酶活性促进活性氧清除，抑制线粒体氧化应激对TGF-β1通路的激活而抗心肌纤维化。本课题采用Ang II在野生型和ALDH2敲除小鼠及心脏成纤维细胞上建立心肌纤维化体内和体外模型，研究AC及其单体抗心肌纤维化的作用及机制与ALDH2的关系，阐明AC中单体调控ALDH2酶活性的分子机制。本研究将对发现调控ALDH2靶点的心血管新药具有重要意义。

乙醛脱氢酶2（ALDH2）是心血管疾病干预的新靶点。牛樟芝（AC）是产于台湾地区的真菌，尚未见AC抗心脏重构作用的报道。我们分别采用异丙肾上腺素、主动脉缩窄术和血管紧张素II（Ang II）造小鼠心脏重构模型，发现AC三萜富集物（ACT）在野生型（WT）小鼠中可抑制三种模型造成的心脏肥大和纤维化，抑制MAPK和TGF-β/Samd信号通路；而对ALDH2 KO小鼠的心脏重构无抑制作用，不能调控以上信号通路。在WT小鼠心脏组织中，ACT可上调ALDH2的mRNA和蛋白表达量及酶活性，降低4-HNE含量，减轻炎症和氧化应激，对线粒体的融合、分裂和自噬的损伤具有修复作用；在ALDH2-/-小鼠上，ACT无以上作用。以上表明ACT抗心脏重构作用由ALDH2介导，与其上调ALDH2酶活性抗炎、抗氧化、调控心肌线粒体稳态有关。在Ang II刺激乳大鼠原代心肌细胞和成纤维细胞建立的肥大和纤维化细胞模型上，发现单体DSA具有与ACT一致的抗肥大和纤维化相关基因表达的作用；采用Ad-shALDH2和Ad-ALDH2分别敲低和高表达ALDH2，可显著影响ACT/DSA抗肥大和纤维化的作用；DSA可上调ALDH2表达量和酶活性、抗炎、抗氧化、调控细胞线粒体稳态。以上均表明DSA是AC抗心脏重构作用的活性单体。在ACT/DSA调控ALDH2的机制研究中，发现ACT/DSA可增加ALDH2蛋白的热稳定性；以ALDH2小分子激动剂ald-1为对照，SPR实验发现DSA不直接结合ALDH2蛋白。将DSA与ALDH2共结晶或Soaking，在蛋白晶体结构中没有发现有DSA分子结合；通过靶点钩钓技术，从可与DSA结合的蛋白中寻找到可能调控ALDH2转录和翻译的靶标，目前正在进行靶点验证。本项目研究将对发现AC抗心脏重构的作用与机制，及其临床转化用于防治心血管疾病具有重要意义。