n-6/n-3脂肪酸通过mTORC1信号通路介导血管平滑肌细胞自噬的机制研究

Vascular calcification is a major risk factor for cardiovascular morbidity and mortality, and it is prevalent in the patients with diabetes and atherosclerosis. There is no available therapy that could reverse vascular calcification at present. The osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is the cellular mechanism that mediates vascular calcification. It is reported that polyunsaturated fatty acids (PUFAs) exhibit various protective effects against vascular smooth muscle cell (VSMC) inflammation and proliferation in cardiovascular remodeling. Although the ratio of n-3 PUFA to n-6 PUFAs had recently been recognized as a risk marker of cardiovascular disease, the molecular mechanisms of PUFAs on the calcium release from VSMCs have not been clearly elucidated, and it is necessary to identify the molecular mechanism of PUFAs in the early stage of vascular calcification processes. In the prior research, we had found that endogenously produced n-3 PUFAs can attenuate vascular calcification, mainly as a consequence of keeping VSMCs steadily. In addition, it showed that one critical downstream target protein of mTORC1, p-S6 , significantly reduced in fat-1 transgenic mice . Autophagy, which is mainly regulated by mTORC1 signaling , is considered to play a pivotal role in vascular calcification. According to these results, we hypothesed that n-6/n-3 PUFAs inhibits vascular calcification by promoting autophagy via suppression of the mTORC1 signaling pathway. Here, we investigate whether n-6/n-3 PUFAs attenuates vascular calcification by regulating autophagy in VSMCs in vitro and in vivo and explore the mechanism involved. These questions to be solved will contribute to find a new target for prevention and cure of vascular calcification.

血管钙化导致严重心血管事件但防治困难。血管平滑肌细胞向成骨细胞表型分化是其重要细胞学基础。已有临床研究表明多不饱和脂肪酸（PUFAs）可降低血管钙化风险，但n-6/n-3 PUFAs在膳食和机体代谢中的比例失衡增加心血管事件发生风险，其机制不明。课题组前期研究中发现fat-1 (使n-6转化为n-3 PUFAs的酶)转基因可明显抑制小鼠血管平滑肌细胞mTORC1活性，并减少血管钙化发生。而血管平滑肌细胞自噬是血管钙化重要的内源性保护性机制，mTORC1是自噬的主要调节蛋白。据此我们推测：n-6/n-3 PUFAs通过抑制mTORC1信号通路，进而增强血管平滑肌细胞自噬水平，抑制血管钙化形成。本项目拟采用fat-1转基因小鼠、腺病毒等工具，阐明n-6/n-3 PUFAs抑制血管钙化及调控血管平滑肌细胞自噬、细胞表型转化的分子靶点和分子机制，为探寻防治血管钙化新方法提供思路和依据。

血管钙化是引发严重心血管事件高发生率和全因死亡率的主要原因之一，目前缺乏有效干预措施。临床研究表明多不饱和脂肪酸（PUFAs）可降低血管钙化风险，但n-6/n-3 PUFAs在膳食和机体代谢中的比例失衡增加心血管事件发生风险，其机制不明。本课题系统地阐明了n-6 PUFAs和n-3 PUFAs在体内的比例构成对于血管钙化发生的影响及相应的分子机理。利用前期研究中引进的fat-1转基因（FAT1）小鼠，它可将n-6 PUFAs转化成n-3 PUFAs，从而平衡二者的比例。在喂食高脂肪食物加血管钙化诱导后，FAT1小鼠主动脉血管中n-3 PUFAs含量显著升高，n-6/n-3脂肪酸比例显著降低（p＜0.01）。FAT-1组小鼠血管钙化程度较WT组显著减轻。表现为：炎症因子降低、主动脉中钙沉积显著减少。体外实验显示，n-3 PUFAs呈浓度、时间依赖性抑制高磷诱导的血管平滑肌细胞（VSMC）钙化，并可下调mTOR/ulk1信号通路活性，增强血管平滑肌细胞的自噬形成。本研究证实，增加内源性n-3 PUFAs的含量，优化n-6/n-3 PUFAs的构成比，可通过下调mTOR/ulk1信号通路活性增强细胞自噬，有效延缓血管钙化的发生，为血管钙化的防治提供新的治疗策略。围绕骨-血管对话问题，应用生物信息学cFDR方法对与出生体重和腰椎骨密度的GWAS数据进行整合，并首次鉴定出PDXDC1，LOC102724985和FAT1P1基因可能为腰椎BMD及出生体重的共同新多效性遗传易感基因。进一步研究发现PDXDC1在绝经后骨质疏松症小鼠模型中表达下调，而在FAT-1小鼠中表达上调，提示PDXDC1可能与n-3 PUFAs和n-6 PUFAs相互作用从而影响骨密度及出生体重，为血管、骨骼衰老性疾病防治提供新的见解。.