脓毒症中上皮细胞-DC转分化致肠道损伤的作用及机制研究

The intestine injury induced by sepsis is a research focus in critical care medicine. Our previous study showed that the epithelial-dendritic cell (DC) transformation in intestine played an important role in the occurrence of intestine injury in hemorrhagic hypotension rats. The results of our preliminary experiment showed that the intestinal epithelium of sepsis mouse also could express dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin(DC-SIGN), so we guess that epithelial-dendritic cell (DC) transformation could also take place in the intestine of sepsis mouse and plays an important role in the occurrence of intestine injury. Our previous study suggested that hypoxia induced epithelial-DC transformation by suppressing glycogen synthase kinase 3β (GSK-3β) activity and in turn decreasing the expression level of E-cadherin. But we also observed that hypoxia suppressed GSK-3β activity transiently, that cannot explain the continuous decreasing of E-cadherin expression. There must be other factors to suppress E-cadherin expression. A large amount of High mobility group protein 1 (HMGB1) was secreted in Sepsis. HMGB1 has the function of inhibiting E-cadherin expression and promoting DC mature, so we speculate that it can start epithelial-DC transformation. We plan to investigate whether epithelial-DC transformation could be induced by sepsis and took important role in the occurrence of intestine injury, by observing the changes of the phenotype and function of intestinal epithelial cells in a mouse sepsis model. We also plan to investigate if HMGB1 could participate in epithelial-DC transformation and the possible mechanism by using specific neutralizing antibody and siRNA, cDNA transfection techniques. This project will provide a new direction for the research on the mechanism by which sepsis induce MODS, and provide a theoretical basis for organ protection measures in sepsis.

脓毒症引发肠损伤是危重症研究热点。我们前期研究发现小肠上皮-树突状细胞（DC）转分化在失血性低血压大鼠肠损伤中起重要作用，预实验显示脓毒症小鼠小肠上皮也表达DC标志物——树突状细胞特异性细胞间粘附分子非整合素蛋白-3（DC-SIGN），故推测脓毒症也能通过小肠上皮-DC转分化导致肠损伤。前期研究示上皮-DC转分化与糖原合成酶激酶-3 （GSK-3β）活性下降导致E-钙粘蛋白（E-cadherin）表达降低有关，但仍不能完全解释转分化机制。脓毒症大量分泌的高迁移率族蛋白1 （HMGB1），有抑制E-cadherin、促进DC成熟的作用，推测其也能启动上皮-DC转分化。拟建立脓毒症小鼠模型，观察小肠上皮细胞表型及功能变化，明确是否存在上皮-DC转分化及在肠损伤中的作用；使用siRNA干扰、cDNA转染等技术，探讨HMGB1对上皮-DC转分化的作用。本项目将为脓毒症致MODS防治研究提供新方向。

脓毒症是危重症常见的并发症，进一步发展可导致脓毒性休克、多器官功能障碍综合征。肠道是脓毒症的“中心器官”，小肠上皮-DC转分化在失血性低血压诱发SIRS-MODS的过程中起到重要作用，其在脓毒症中是否存在及作用如何尚需探讨。本研究以小鼠及小肠上皮细胞FHs74Int为研究对象，以CLP法建立体内脓毒症模型、LPS刺激体外模拟脓毒症环境。采用DC-SIGN-siRNA预处理降低DC-SIGN在体内和体外的表达。免疫印迹和免疫组化检测DC-SIGN的表达。western blot检测ERK1/2和NF-kB/p65的表达及磷酸化。ELISA法测定血清和培养上清中细胞因子的含量。对感染小鼠的存活率和器官损伤进行了评估。结果显示：1、在脓毒症发病早期，肠道发生上皮-DC转分化现象，且DC-SIGN 表达水平与全身炎症反应、组织病理损伤、疾病严重度密切相关；2、DC-SIGN siRNA抑制肠道DC-SIGN 表达后，可显著减轻脓毒症诱导的急性肠损伤，并改善肠外脏器肺、肝、肾损伤及功能障碍，减少全身炎症反应， 提高脓毒症小鼠生存率；3、DC-SIGN 通过激活下游通路“ERK1/2-NF-κΒ/p65”磷酸化，促进TNF-α、IL-1β、 IL-6 等细胞因子合成释放，参与调控脓毒症引发的“急性肠损伤-SIRS-MODS” 过程。本研究结果为脓毒症致肠道及全身多脏器损伤的机制研究提供新方向，也为脓毒症的临床治疗开拓了新思路。