脂多糖经TGF-β/Smad3通路促进内皮间质转化参与脓毒症心肌病的机制研究

Sepsis is common cause of death in hospital, which is closely associated with the myocardial depression effects from sepsis. Enhanced infiltration of inflammatory cells and excessive expression and secretion of inflammatory cytokines are pivotal causes of sepsis-induced myocardial depression. And the disturbance of endothelial barrier is the important structure basis. However, the mechanisms are still unclear. It was found that endothelial cells’ phenotypes would be lost and replaced by the fibroblasts’ phenotypes, which was called as endothelial-mesenchymal transformation (EndMT). During the cause of EndMT, cell-cell junction and their polarity were gradually weakened, cell-cell gap became wider, and endothelial barrier was destroyed, which meant EndMT could provide the structure basis for sepsis-induced myocardial depression. I was found in our experiment that lipopolysaccharide (LPS) could increase the expression of TGF-β and Smad3 in human aortic endothelial cells, and induce EndMT, which could be inhibited by Smad3 inhibitor or SiRNA of Smad3. So we speculated that LPS could induce EndMT in endothelial cells through activate TGF-β/Smad3, which would result in endothelial barrier disturbance, and enhance inflammatory cells infiltration followed by myocardial depression. The present study aimed to determine the related mechanisms of LPS-induced EndMT in endothelial cells, and provide the theory base for further understanding septic cardiomyopathy and searching the possible new therapy targets.

脓毒症是常见的院内死亡原因，这与脓毒症的心肌抑制作用密切相关。心肌炎症细胞浸润及炎症因子的高度表达、分泌是脓毒症心肌抑制的重要原因。而内皮屏障破坏则是其重要结构基础，但其机制尚不明确。研究发现，内皮细胞在某些因素刺激下内皮细胞表型消失，代之以成纤维细胞表型，称为内皮间质转化（EndMT）。该过程中，内皮细胞间连接和细胞极性减弱，细胞间隙增宽，内皮屏障破坏，提示EndMT可为脓毒症心肌抑制提供结构基础。我们发现，脂多糖能使人主动脉内皮细胞TGF-β及Smad3表达增加，并发生EndMT，而以Smad3抑制剂或SiRNA干预均可抑制EndMT。为此，我们推测脂多糖激活TGF-β/Smad3通路使内皮细胞发生EndMT，导致内皮屏障破坏，炎症细胞浸润及心肌抑制增加。本项目旨在阐明脂多糖诱导内皮细胞EndMT及其对心肌抑制的分子机制，为进一步理解脓毒症心肌病发生机制及探索其新的治疗靶点奠定基础。

脓毒症是常见的院内死亡原因，这与脓毒症的心肌抑制作用密切相关。心肌炎症细胞浸润及炎症因子的高度表达、分泌是脓毒症心肌抑制的重要原因。而内皮屏障破坏则是其重要结构基础，但其机制尚不明确。研究发现，内皮细胞在某些因素刺激下内皮细胞表型消失，代之以成纤维细胞表型，称为内皮间质转化（EndMT）。该过程中，内皮细胞间连接和细胞极性减弱，细胞间隙增宽，内皮屏障破坏，提示EndMT可为脓毒症心肌抑制提供结构基础。我们发现，脂多糖能使人主动脉内皮细胞TGF-β及Smad3表达增加，并发生EndMT，而以Smad3抑制剂或SiRNA干预均可抑制EndMT。基于这些，在该研究项目中，我们在LPS诱导的内毒素血症C57BL/6J小鼠中，观察LPS对各组小鼠心功能、心脏血管内皮通透性、心肌炎症细胞浸润的影响及它们与EndMT的关系，并明确TGF-β/Smad3通路是否为其中的关键调控通路。细胞实验中比较脂氧素A4、TGF-β中和抗体、Smad3抑制剂、Smad3-SiRNA等各干预组和空白组人主动脉内皮细胞在LPS刺激后的EndMT情况和内皮细胞单层通透性，并明确LPS诱导血管内皮发生EndMT的分子机制及脂氧素A4的保护作用。结果显示：1）LPS诱导的内毒素血症小鼠的心功能显著下降，心脏血管内皮通透性增加，心肌组织巨噬细胞浸润明显增加，促纤维化因子表达显著增加。2)LPS促进血管内皮细胞发生EndMT，并增加内皮通透性，脂氧素A4可抑制LPS诱导的EndMT，且可能是过激活Wnt-β-catenin通路发挥作用。本项目旨在阐明脂多糖诱导内皮细胞EndMT及其对心肌抑制的分子机制，为进一步理解脓毒症心肌病发生机制及探索其新的治疗靶点奠定基础。