基于自噬及其转录后调控探讨补阳还五汤干预血管衰老的机制

Arterial aging that accompanies by molecular and cellular disorder has been construed as specific risk factors for the cardiovascular diseases (CVD) , and thus might be targets of interventions designed to decrease or delay the occurrence of severe CVD. Qi deficiency and blood stasis are one of the traditional Chinese medicine (TCM) syndrome related to aging. As a classical Chinese medicine of tonifying Qi and activating blood, the role and mechanism of buyang huanwu decoction (BYHWD) on arterial aging, however, remains unknown. Our previous study considered that BYHWD protected against atherosclerosis formation through mTOR related autophagy pathway (supported by NSFC). Arteries miR microarray in our preliminary studies showed that a miRNAs disequilibrium started at mid-life and continued to be changed in later life and 15 miRNAs were differentially expressed in old Rhesus aortae. Furthermore, bioinformatics analysis found the predicted interaction between miRNAs and 3’ UTR of autophagy-associated gene. Among them, miR-34a suppressed the luciferase activity of the autophagy-associated gene compared with the negative control, and facilitated vascular smooth muscle cell senescence and regulated the expression of autophagy-related key factor mTOR. Thus, we propose that it might be one of the important mechanism of BYHWD on arterial aging to regulate the expression of miRNAs, consequently adjusting its target genes, autophagy signal transduction pathway. In this study, we will analyze the relationship between arterial aging and the syndrome of Qi deficiency and blood stasis in clinical research firstly. And then high throughput sequencing of miRNAs, qRT-PCR and dual luciferase report will be performance following intragastrically administering BYHWD to FXBN rat and treating BYHWD to vascular endothelial cells and vascular smooth muscle cells, in order to reveal more clearly the effect of BYHWD on arterial aging and the mechanism of autophagy associated transcriptional regulation. All of these are for exploring a new way of TCM's role on arterial aging and a new target of BYHWD.

伴随分子和细胞功能紊乱的血管衰老被视为心血管疾病(CVD)特定的危险因素，对其干预可减少或延缓严重CVD发生，具重要意义。改善气虚血瘀症是中医干预衰老的机制之一。补阳还五汤为益气活血经典代表方，其干预血管衰老的作用及机制远未阐明。我们前期研究表明补阳还五汤通过mTOR相关的自噬通路抗动脉粥样硬化形成(获国家自然基金资助)；预实验发现：衰老与年轻动脉间存在15个表达差异的miRNA；生物信息学分析显示自噬相关基因是多个差异miRNA的靶基因，其中对miR-34a研究发现可抑制自噬靶基因荧光酶活性，可诱导血管细胞衰老、调节mTOR表达。本项目将通过临床观察明确血管衰老与气虚血瘀中医症候的相关性；通过补阳还五汤干预的体内体外实验，应用二代测序、qRT-PCR及双荧光素酶报告等技术，解析补阳还五汤干预血管衰老的作用及其自噬相关转录后调控机制，为血管衰老中医防治提供新思路，为补阳还五汤作用提供新靶点

血管衰老是心血管疾病（CVD）特定的危险因素，对其干预可减少或延缓严重CVD发生。由于血管衰老进程复杂，目前对血管衰老有效干预及其分子机制的研究有限。改善气虚血瘀症是中医干预衰老的机制之一。补阳还五汤为益气活血经典代表方，其干预血管衰老的作用及机制远未阐明。本项目拟研究补阳还五汤干预血管衰老的作用及其自噬相关转录后调控机制，并扩展至补阳还五汤中活性成分单体“黄芪甲苷”的作用及相关机制。研究发现补阳还五汤和其活性成分单体黄芪甲苷具明确延缓血管衰老作用；同时发现补阳还五汤通过增加SIRT1表达抑制衰老相关的血管平滑肌细胞（VSMCs）迁移和侵袭；黄芪甲苷通过脂代谢组学产物溶血卵磷脂（LPC）相关的铁死亡信号发挥延缓血管内皮细胞衰老的作用。进一步探讨血管衰老的转录后调控机制发现：miRNA芯片分析示miR-665是年轻和衰老VSMCs中最差异表达的miRNA，miR-665是VSMCs衰老的重要调节因子，可通过负向调节SDC1及与lncRNA GAS5形成海绵效应发挥作用。在临床观察中通过年龄和脉搏波传导速度将人群分为年轻组、衰老组和血管衰老组，也发现lncRNA GAS5/ miR-665/ SDC1信号参与衰老及血管衰老的调节。另外正在进行的实验发现补阳还五汤可增加衰老抑制的血管中自噬，同时影响miR-665及其下游自噬相关靶基因蛋白表达；黄芪甲苷通过激活Parkin介导的线粒体自噬改善血管平滑肌细胞衰老。本项目为血管衰老中医防治提供新思路，为延缓血管衰老治疗方案精准化研究提供理论及实验依据。