H2S抑制TGF-β/Smad信号通路调节平滑肌细胞表型转化抗主动脉夹层研究

Transforming growth factor beta(TGF-β)/Smad signal pathway plays an key role in aortic dissection(AD). Our results indicated that serum H2S content in AD is lower than in normal people and H2S inhibits collagenⅠ/Ⅲ expression and phenotype swich induced by TGF-β in smooth muscle cells (SMC).This study is divided three parts. ① To further study the relationship between H2S/CSE and AD including its type through comparing H2S content of serum and aortic tissue in AD with normal people; ② First，we will construct AD model, then to explore the effects of H2S and TGF-β intervention on TGF-β/SMAD pathway, collagen and phenotype protein of SMC in animal level; ③ Two kinds of SMC were isolated and cultured from normal mice and mice with AD, TGF-β/SMAD pathway and phenotype protein of SMC were compared between these two kinds of SMC，then to explore the effects of H2S/CSE on SMAD, collagen and phenotype protein in SMC induced by TGF-β. This research will discover the effects of H2S inhibiting TGF-β/SMAD pathway on AD. Our study will be benefit to understand the molecular mechanism of H2S on AD, and it will provide new direction for the research of AD.

转化生长因子β(TGF-β)/Smad信号通路在AD的发病中起关键作用。我们近来发现AD患者血浆H2S含量明显低于正常体检者，H2S能抑制由TGF-β诱导的平滑肌细胞(SMC)由收缩型向合成型转化及胶原蛋白的表达。本项目拟:①比较AD患者和对照组的血浆和主动脉血管组织中H2S含量及CSE表达情况，明确H2S/CSE体系与AD以及AD类型的相关性；②首先建立小鼠AD模型，然后探讨H2S以及联合干预TGF-β对AD发生、TGF-β/SMAD通路以及SMC表型标志蛋白的影响；③分离培养和鉴定正常和伴AD的小鼠来源的两种主动脉SMC，检测两种SMC中TGF-β/SMAD等表达水平以及SMC表型的差异；在SMC上探讨H2S/CSE体系对TGF-β诱导的SMAD、胶原蛋白以及SMC表型转化的影响。本研究可明确H2S抑制TGF-β/Smad对AD发生的影响及机制，可为AD发病机制的研究供新的方向。

背景:CSE/H2S体系在心血管系统中发挥重要作用，但是CSE/H2S体系与AD的关系及其在AD中的作用机制尚不明确。.主要内容、重要结果和关键数据：分别观察AD时主动脉病理形态学变化、H2S含量、血清炎症因子水平；采用β-氨基丙腈（BAPN）复制大鼠AD模型，将SD大鼠分为四组：对照组、BAPN组、BAPN+NaHS组和BAPN+NaHS+氯喹（CQ，自噬抑制剂）组。比较四组大鼠AD发生率、主动脉病理形态学改变、血清H2S水平和CSE等表达变化。从人主动脉组织中分离培养主动脉VSMCs，分别转染AMPK siRNA、LV mTOR（表达质粒）和ATG5 siRNA，采用Western blot等分别观察其对AMPK、mTOR、ATG5、炎症因子水平（TNF-α、IL-6、IL-1和IL-10）、ERS（p-IRE和GRP78）的变化。结果发现，与对照组相比，AD主动脉组织CSE表达降低31.2%；与正常体检组血清相比，AD组血清H2S含量水平下降。动物学实验标明，Control组大鼠均正常，未发生AD； BAPN组AD的发生率为83.3%；与BAPN组相比，NaHS组AD的发生率为35.7%，较BAPN组明显降低（P<0.05），差异有统计学意义。与BAPN组相比，BAPN+NaHS组中大鼠主动脉壁结构相对较完整。在NaHS处理VSMC的同时分别改变AMPK、mTOR和ATG5表达水平，结果发现在NaHS处理的同时下调AMPK表达或上调mTOR表达或下调ATG5表达均可下调VSMCs自噬。进一步采用100µM的NaHS处理VSMCs 24h，然后采用RNA干扰技术以及慢病毒转染等方法分别改变AMPK、mTOR、ATG5表达和自噬水平，观察AMPK/mTOR/ATG5信号通路对VSMCs炎症因子表达和ERS的影响。结果发现100µM的NaHS处理可上调VSMCs中抑炎因子IL-10的表达，下调促炎因子TNF-α、IL-6和IL-1β的表达以及p-IRE-和GRP78的表达。.研究意义：AMPK/mTOR/ATG5可能成为防治AD的新靶点，为AD的防治提供新思路和理论依据。