Toll样受体2和4信号通路减轻失血性休克致肠黏膜屏障损伤的作用及机制研究

Previous studies revealed the activation of toll like receptor 4 (TLR4) signaling pathway in intestinal mucosa resulted in improvement of gut barrier function after intestinal ischemia and reperfusion injury. However, the underlying mechanism is still undetermined. In this study, we speculate that the protective effects of TLR4 on gut barrier function are mediated by TLR2 signaling pathway. The activation of TLR4 signaling pathway increases the transcription of TLR2 in intestinal epithelial cells (IEC). And the enhancement of TLR2 signaling pathway inhibits apoptosis of IEC and prevents tight junction between IEC from destruction after hemorrhagic shock and resuscitation(HS/R), and eventually improves gut barrier function after HS. In this study, we designed in vivo and in vitro studies to prove our hypothesis. In murine model of HS/R, we will prove the protective effects of TLR2 and TLR4 signaling pathway on gut barrier function,and explore the cross-interaction between TLR2 and TLR4 signaling pathway via studies on TLR2 and TLR4 gene knockout mice. On primary culture of murine colonic epithelial cells, we will investigate the mechanism by which the activation of TLR4 signaling pathway increases the transcription of TLR2. Finally, on colonic cells anoxia and reoxygenation model (A/R), we will study the mechanism by which the activation of TLR2 signaling pathway improves epithelial cell barrier injuried by A/R and the signaling pathway involved. These works will help us to better understand the role of TLR2 and TLR4 signaling pathway in modulating gut barrier function, and therefore ameliorate the SIRS and MODS after HS.

激活肠粘膜Toll样受体4（TLR4）信号通路可以减轻肠缺血再灌注导致的肠粘膜屏障损伤和炎症反应，但机理不明。本研究在前期研究的基础上，建立小鼠失血性休克及复苏（HS/R）模型，通过对TLR2和TLR4基因敲除鼠的研究，观察TLR4信号通路是否通过上调肠粘膜TLR2的表达，进而加强TLR2信号通的激活以减轻HS/R后肠粘膜屏障的破坏和炎症反应。同时通过对体外原代培养小鼠结肠上皮细胞（IEC）的研究，观察TLR4激活后上调TLR2的表达的相关下游信号通路。通过对IEC缺氧复氧损伤（A/R）模型的研究，观察激活TLR2信号通路减少A/R导致的上皮细胞屏障损伤的作用及相关信号通路。从而弄清TLR4激活→上调TLR2表达→增强TLR2信号通路活化→维护肠粘膜屏障→减轻HS后的炎症反应和器官损伤间的因果关系，为进一步提高HS的治疗效果奠定理论基础。

目的 脂多糖(LPS)相关信号通路可以减轻肠缺血再灌注导致的肠粘膜屏障损伤和炎症反应，但机理不明。本研究通过建立小鼠失血性休克及复苏造成肠缺血再灌注损伤模型，探讨了LPS通过激活TLR4相关信号通路增强肠上皮细胞增殖和移行以促进肠修复的作用及机制。.方法：广谱抗生素喂食小鼠4周以清除肠道共生菌。小鼠失血性休克（HS/R, hemorrhagic shock and resuscitation）造成肠缺血再灌注(IR, ischemia and reperfusion injury)损伤。喂食小鼠LPS后，检测肠、肺组织损伤及修复。体外细胞实验检测相关信号细胞通路在肠上皮细胞增殖和移行中的作用。 .结果：肠损伤在失血性休克再复苏（HS/R）4小时最严重，HS/R后72h肠黏膜修复基本完成。喂食抗生素小鼠清除了肠道共生菌，减轻肠IR损伤，但减慢了小鼠肠黏膜修复，导致休克后72h生存率无明显改善。喂食小剂量LPS增强肠上皮细胞增殖及移行，增强肠修复，减轻修复期肺损伤，并减少循环中促炎因子，从而有效改善生存率。LPS通过TLR4受体增加了肠黏膜PGE2水平，并激活NFκB、Akt、ERK1/2信号通路，进而促进肠上皮细胞增殖和移行。LPS促进TLR2基因敲除小鼠肠黏膜修复，但对TLR4敲除小鼠无促进肠黏膜修复的作用。体外实验证实LPS 通过PI3K/Akt, MEK/ERK1/2通路促进HT-29人结肠癌细胞划痕愈合。LPS通过PI3K/Akt通路促进Caco-2人结肠癌细胞增殖。 .结论： LPS通过TLR4/NFκB上调肠黏膜PGE2水平。PGE2通过激活PI3K/Akt和MEK/ERK1/2通路促进肠上皮细胞增殖和移行，进而加强肠黏膜在缺血再灌注损伤后的修复。LPS促进肠黏膜修复的作用不依赖于TLR2通路。

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