硫化氢抗As血管重构的新机制：巯基化修饰NFIL3抑制MEST介导的内皮间质转化

Hydrogen sulfide (H2S) has been found to inhibit abnormal shear stress- induced atherosclerosis(As)-related vascular remodeling. However, the exact mechanism is not fully elucidated. Recent studies have found that endothelium-to-endothelin transition (EndMT) is a shear stress-sensitive biological process that mediates and promotes vascular remodeling. The previous studies in our group have shown that H2S inhibits abnormal shear stress-induced EndMT and down-regulates expression of imprinted gene MEST. Bioinformatics analysis revealed that the MEST-specific inhibitor NFIL3 has three potential thiolated sites. Based on this, this project proposed that H2S can thiolate and activate NFIL3, down-regulates the expression of MEST, thereby negatively regulates EndMT induced by abnormal shear stress to inhibit As-related vascular remodeling. In order to confirm this hypothesis, human carotid plaque, the apoE-/- mouse model of local carotid artery ligation and parallel plate flow cavity system, as well as some techniques including CRISPR/Cas9, amino acid site-directed mutation, mass spectrometry, etc. will be used to explore the effect of H2S on the thiolation of NFIL3, MEST expression, EndMT as well as the shear stress-induced As vascular remodeling. The project will provide new theoretical and experimental basis for the mechanism and prevention of As-related vascular remodeling.

硫化氢(H2S)抑制异常剪切应力引起的动脉粥样硬化(As)血管重构，但其调控机制尚未阐明。新近研究发现血管内皮间质转化(EndMT)为剪切应力敏感的生物学过程，参与并促进血管重构。本组前期发现，H2S抑制异常剪切应力诱导的EndMT，下调印记基因MEST表达；生物信息学分析发现MEST特异性抑制因子NFIL3含有3个潜在的巯基化修饰位点。基于此，本项目提出“H2S巯基化修饰并激活NFIL3，下调MEST表达，负性调控异常剪切应力诱导的EndMT和As血管重构”。为证实该假说，拟利用人颈动脉斑块、颈总动脉局部结扎的apoE-/-小鼠As模型以及平行平板流动腔系统，结合CRISPR/Cas9、氨基酸定点突变、质谱分析等技术探讨H2S对NFIL3的巯基化修饰、MEST表达、EndMT的影响及其在异常剪切应力引起的As血管重构中的作用，为As血管重构性病变的发生机制和防治提供新的理论及实验依据。

本项目紧紧围绕“血管稳态与重构的动态调控网络与关键节点”这一核心科学问题，采用临床标本、动物和细胞模型，靶向血管重构中的重要环节和关键节点-内皮间质转化（EndMT)，探讨内源性气体信号分子硫化氢（H2S） 抑制异常剪切力诱导动脉粥样硬化（As）血管重构的调控机制。.重要进展：.1.低剪切应力诱导EndMT并上调印记基因MEST的表达，干扰MEST的表达可以抑制低剪切应力诱导的EndMT，H2S通过抑制MEST的表达从而抑制低剪应力诱导的EndMT。.2.低剪切应力上调ApoE-/-小鼠主动脉MEST表达；NaHS下调ApoE-/-小鼠模型中低剪切应力诱导的MEST表达上调；.3.干预MEST抑制EndMT，延缓As的进程， MEST为低剪切应力诱导EndM及As中的关键分子。.4.机制研究表明，H2S通过巯基化修饰上游转录因子NFIL3，但不影响其NFIL3的核转位，抑制低剪切应力诱导的MEST上调与EndMT。.5.ApoE-/-小鼠主动脉As斑块与尸检标本主动脉As病变中E3连接酶家族家族成员TRIM65表达上调，TRIM65通过其SPRY结构域与VCAM-1特异性结合，诱导其K48位与K63位赖氨酸的多聚泛素化修饰，通过泛素-蛋白酶体途径介导VCAM-1的泛素化降解；.6.建立了TRIM65-/-/apoE-/-双敲除的小鼠模型，敲除TRIM65抑制apoE-/-小鼠As病变以及斑块内脂质的蓄积；TRIM65通过激活PI3K/Akt/mTOR信号通路促进PDGF-BB诱导的VSMCs由收缩型向合成型转化。.科学意义：.本项目的研究结果表明H2S通过巯基化修饰转录因子NFIL3、负性调控印记基因MEST表达，从而抑制EndMT以及As血管重构；此外，研究结果表明泛素连接酶TRIM65是介导动脉粥样硬化病变的关键调节分子，这些结果将为As血管重构性病变的发生机制和防治提供新的理论及实验依据。