卡拉胶协同内毒素引起肠道炎性损伤的分子机制研究

To extend our former project "Research on the influence factors and mechanism of harmful inflammatory effect of carrageenan", the present research is designed to elucidate the mechanism of intestinal tract inflammation induced by carrageenans which are important food additives. In the former project, we found that the immune responses of human colon epithelial cells and macrophage cells exposed to carrageenans were relatively weak, however, carrageenans showed the ability to enhance the effect of lipopolysaccharide (LPS) to induce the secretion of inflammatory cytokines. Moreover, when differentiated human colonic epithelial cells were cocultured with macrophage cells, the epithelial monolayers were significantly and easily damaged by carrageenans. Based on these information, we propose a hypothesis that carrageenans are potential conditional inflammatory agents. When the intestinal tract was under a "unhealthy" state, the carrageenans can synergistically increase the inflammation responses with LPS, and induce signal communication between immune cells and intestinal epithelial cells which will cause the damage of intestinal tract. Therefore, in this research, we propose to utilize human colon epithelial cells (Caco-2) and macrophage cells (Raw264.7 and THP-1) as our cell model. The immune responses of cells elicited by carrageenans cooperated with LPS will be analyzed by CBA and ELISA assay. To understand the underlying molecular mechanism of the synergia between carrageenans and LPS, we will detect the gene expression profile by microarray, and find the binding receptor of carrageenans and LPS by focusing on the TLR4 pathway. Furthermore, we will establish a coculture model of Caco-2 and THP-1 cells to detect the dose and time points that carrageenans caused damage to epithelial monolayers, and also to investigate the immune communication between these two cells in order to clarify the reason for the intestinal tract damage induced by carrageenans. Then, we will establish an animal model by inducing slight intestinal tract infection or increasing the microbial population in tract, to observe the synergistic enhancement of inflammatory damage of carrageenans on intestinal tract of animals. Meanwhile, we will try to validate the damage reasons discovered in cell experiments by pathologic and immunohistochemical assays in animal model . The significance of this research is to clarify the molecular mechanism of inflammatory damage of intestinal tract induced by carrageenan. Our results can help to reevaluate the safty of carrageenans as food additives, and provide scientific instructions for use as inflammatory agent.

本研究是前一个基金"卡拉胶非安全性因素及其作用方式的分子方式研究"的延续并深化。为解决卡拉胶这种重要的食品添加剂引起肠道炎症的机制。在前研究发现卡拉胶对细胞具低免疫调节性，但上调内毒素LPS的炎性因子分泌，在免疫细胞存在时易引起肠上皮层破损的基础上，本项目提出如下假说：卡拉胶具条件致炎性，即在肠道亚健康状态下，能协同LPS致炎,并引起免疫和上皮细胞的信号交流从而造成粘膜层破损。以结肠上皮细胞和巨噬细胞为肠道炎症研究模型，通过CBA和ELISA技术观察卡拉胶与LPS的协同作用，并以TLR4信号通路为主线，结合受体寻找和数字表达谱分析来探索协同机理；采用双细胞共培养模型，研究两类细胞间的免疫交互作用，阐明分化上皮层易受损的原因；再建立动物模型，重新审视动物结肠受损的条件、程度并验证其原因。本项目将为卡拉胶在食品添加剂行业的重新定位和作为致炎模型分子的使用提供明确的依据和方法。

为解决卡拉胶这种重要的食品添加剂引起肠道炎症的机制，项目提出如下假说：卡拉胶具条件致炎性，即在肠道亚健康状态下，能协同LPS致炎,并引起免疫和上皮细胞的信号交流从而造成粘膜层破损。本研究以结肠上皮细胞和巨噬细胞为肠道炎症研究模型，通过CBA和ELISA技术观察卡拉胶与LPS的协同作用，并以TLR4信号通路为主线，结合受体寻找和基因芯片分析来探索协同机理；采用双细胞共培养模型，研究两类细胞间的免疫交互作用，阐明分化上皮层易受损的原因；再建立动物模型，重新审视动物结肠受损的条件、程度并验证其原因。.通过量构效关系研究，建立起卡拉胶协同模式识别分子LPS的构效和量效关系。构效：针对肠上皮细胞，最易协同破坏肠道上皮细胞的是食品级的κ-卡拉胶；针对免疫细胞，分子量在10-40kDa的λ-卡拉胶，在浓度10μg/ml以上即可协同LPS引起显著的炎症因子上升。量效：卡拉胶的安全量效低限是10μg/ml。时效，卡拉胶作用10h以上，会引起炎症的级联反应，即安全时效是10h。明确卡拉胶的协同致炎机理，对免疫细胞，λ-10-40k能通过增加LPS的结合受体TLR4的表达，以及激活TLR4-ERK/JNK-AP-1途径，使下游免疫因子的分泌出现协同增加，从而增强LPS所引起的机体炎症反应。对肠上皮细胞，κ-卡拉胶强化了LPS诱导的Bcl10, p-IκBα和NF-κB的表达并且增加了LPS诱导的NF-κB的转录活性。通过共培养模型发现，κ卡拉胶可以破坏上层细胞层的完整性，增加细胞因子的过度表达，一旦上皮细胞被损伤，肠道中的细菌和内毒素可以直接进入身体中，破坏机体本身细胞因子与抗炎因子之间的平衡，使免疫调剂发生紊乱。从动物模型证实，κ-卡拉胶具有协同致炎的特点，会协同增加小鼠的溃疡性结肠炎和克罗恩病，使小鼠体重下降，炎症死亡率增加，结肠损伤加重。κ-卡拉胶能增加炎症因子表达，抑制Tregs的活性，提高小鼠肠道组织中TLR4 和NF-κB的转录和表达，p-ERK1/2，JNK 和c-Jun的表达增加。基因芯片结果也证实了Toll-like信号通路基因的激活。.本项目为卡拉胶在食品添加剂行业的重新定位和作为致炎模型分子的使用提供明确的依据和方法。