紫薯花青素调控内皮细胞老化的分子机制研究

Senescence of vascular endothelial cells (VECs) plays important roles in the development of aging-related diseases such as atherosclerosis, however, the mechanisms underlying VECs senescence are still unclear. Our previous studies suggested that caveolin-1 and integrin β4 participated in the process of VECs senescence and purple sweet potato color (PSPC) could suppress aging of endoderm in the plaques of atherosclerosis. Based on these results, we deduced that PSPC could inhibit VECs senecence and regulate atherosclerosis through caveolin-1 and integrin β4. To prove this hypothesis, we plan to study as follows: 1)utilize the model of mice with atherosclerosis and culture system of human umbilical vein endothelial cells (HUVECs) to analyze whether PSPC inhibit VECs senescence and suppress the development of atherosclerosis in vivo and in vitro; 2)detect changes of caveolin-1 and integrin β4 in VECs and plaques of atherosclerosis after PSPC administration, and investigate effects of caveolin-1 and integrin β4 overexpression or knockdown on VECs senescence regulated by PSPC; 3)analyze the relationship between caveoin-1 and integrin β4 during VECs senescence, and test the effect of PSPC on the activation and distribution of signal molecules mediated by caveolin-1 and integrin β4, to further clarify the mechanisms of VECs senescence regulated by PSPC, caveolin-1 and integrin β4. These results will supply a novel hypothesis to elucidate the mechanisms of VECs senescence and provide new theoretical targets for the application of flavonoids on VECs aging-related diseases in clinical.

血管内皮细胞的老化可以诱发动脉硬化等衰老相关疾病，但老化的分子机制尚不明确。本课题组研究表明，caveolin-1和膜整联蛋白β4参与内皮细胞的老化，紫薯花青素(PSPC)可以延缓动脉硬化斑块的内皮老化，我们推断PSPC可能通过二者调控内皮细胞的衰老。为验证这一假设，我们拟进行以下研究：1)利用动脉硬化小鼠模型和人脐静脉血管内皮细胞培养体系，检测PSPC能否抑制内皮细胞老化；2)检测PSPC对老化内皮和斑块中caveolin-1和膜整联蛋白β4的影响，分析过表达或敲除该基因后对PSPC调控作用的影响，初步判断PSPC是否通过二者调控内皮细胞老化；3)分析caveolin-1与膜整联蛋白β4在内皮老化中的相互关系，并靶向下游信号，检测PSPC对其定位及活化的影响，最终提出PSPC调控内皮细胞老化的可能分子机制。为阐明内皮细胞老化分子机制提供实验依据，同时为内皮老化相关疾病的治疗提供新的靶点。

在本项目中，我们以类黄酮物质包括紫甘薯花青素(PSPC)、槲皮素、葛根素和曲克芦丁为研究工具，探讨了内皮老化在II型糖尿病等代谢相关疾病中引发心血管疾病的发病机制。我们发现，高脂饲料喂养可以导致小鼠产生胰岛素抵抗，引发糖尿病，在这一过程中，出现动脉粥样硬化，并伴随内皮层的老化；PSPC可以通过抑制内皮的老化延缓动脉粥样硬化的发生。体外实验以高浓度的葡萄糖处理人脐静脉血管内皮细胞(HUVEC) 诱导细胞老化为模型，发现PSPC可以延缓内皮细胞的早衰，并促进内皮功能的恢复；而胞内的活性氧水平也被PSPC抑制；鉴于活性氧可以激活炎性小体并促进细胞的老化，我们进一步探讨了炎性小体在PSPC抑制内皮细胞老化中的作用。我们发现PSPC可以抑制NLRP3炎性小体的活性；敲低NLRP3或其下游的IL-1β的基因表达可以抵抗高糖引发的内皮老化；而诱导剂激活NLRP3后，PSPC不能抑制内皮的早衰，表明PSPC抑制内皮早衰是NLRP3依赖的。这一现象同样发生在槲皮素、葛根素和曲克芦丁抑制内皮老化的过程中，说明多种类黄酮可以靶向NLRP3炎性小体抑制内皮细胞老化。此外，进一步的研究表明，在PSPC抑制内皮细胞老化的过程中，细胞自噬被激活。阻断或激活老化内皮细胞自噬水平后可以干扰或增强PSPC抑制内皮老化的能力。同时NLRP3的变化与细胞自噬存在负相关性，PSPC可以通过诱导细胞自噬，降解NLRP3炎性小体来抑制内皮细胞的老化。另外，PSPC能够降低高糖引起的FAK和caveolin-1水平，但是这种变化与细胞自噬可能不存在关联。综上，我们的研究为代谢紊乱条件下靶向内皮老化来治疗动脉粥样硬化等心血管疾病提供了工具和策略。