参苓白术散通过抑制内质网应激调控肠上皮细胞自噬-凋亡治疗炎症性肠病的机制研究

When the intestinal environment changes, the excessive endoplasmic reticulum stress(ERS) of intestinal epithelial cells(IECs) causes intestinal mucosal barrier dysfunction, which induced IBD. The apoptosis and autophagy induced by ERS depend on the action intensity and time of the inductive agent. The autophagy and apoptosis induced by ERS are closely related to PERK/eIF2a/CHOP. Therefore, the interaction of ERS, autophagy and apoptosis promote genesis and development of IBD. Therefore, the interaction of endoplasmic reticulum stress (ERS), autophagy and apoptosis process together to promote the occurrence and development of IBD. Spleen qi deficiency, dampness connotation is IBD potential pathogenesis and earlier stage of the project found that spleen qi, dampness zhixie formulas of Shenlingbaizhu powder in the treatment of IBD by maintaining intestinal epithelial cell homeostasis and regulation of intestinal epithelial autophagy and inhibition of apoptosis related. The project intends to the basis of the result of the pre National Natural Science Foundation of China, through a combination of IBD animal and cell models that intestinal epithelial cells endoplasmic reticulum stress caused by closely and causation between apoptosis / autophagy and PERK/eIF2a/CHOP pathway and to elucidate the regulatory mechanism of Shenlingbaizhu powder in the treatment of IBD and maintaining intestinal homeostasis and for the treatment of IBD provides new ideas.

肠道内环境改变时，引起肠上皮细胞（IECs）过度内质网应激(ERS)导致肠粘膜屏障功能受损，介导IBD发生。ERS同时引起细胞凋亡与自噬的发生，该作用取决于诱导剂作用强度和时间，ERS引起细胞自噬、凋亡与PERK/eIF2a/CHOP通路密切相关。因此，ERS、细胞自噬与凋亡过程相互影响共同促进IBD发生、发展。脾气亏虚，湿邪内蕴是IBD潜在病机，前期课题发现益气健脾、渗湿止泻经方参苓白术散通过改善肠粘膜屏障功能、调控IECs自噬水平、抑制IECs细胞凋亡而治疗IBD。本项目拟在前期国家自然基金基础上，通过IBD动物与细胞模型相结合，研究参苓白术散治疗IBD与抑制肠上皮细胞内质网应激引起的细胞凋亡、自噬调节与PERK/eIF2a/CHOP通路的紧密性及因果关系，阐明肠上皮细胞内质网应激、细胞自噬与凋亡之间的关系及其信号转导途径在IBD发生发展中的重要性，为IBD的治疗提供新思路。

给予BALB/C小鼠5 % DSS自由饮水7天建立IBD小鼠模型。与正常组比较，模型组小鼠出现明显腹泻、血便、体重下降等症状，DAI疾病评分显著上升，小鼠结肠出现溃疡，长度明显缩短，肠壁结构破坏存在大量炎症细胞浸润；模型组小鼠IL-6、IL-8、IL-1β含量、结肠中IL-18和MPO、TNF-α含量显著升高，血清中IL-10显著性降低，内质网应激相关GRP78、ATF-6、PERK、P-IRE1、P-EIF2α、CHOP、P-JUK蛋白表达量上升，IRE1蛋白表达量下降；自噬相关LC3BⅡ/Ⅰ、Beclin1 蛋白表达水平显著降低，P62蛋白表达水平显著性升高。与模型组相比，不同剂量的参苓白术散降低DAI评分，改善结肠组织炎症，降低促炎因子、升高抑炎因子含量，调控内质网应激-自噬相关蛋白表达。.在培养的IEC-6细胞中，30ug/ml LPS引起IEC-6细胞轻度ERS，100ug/mlLPS引起IEC-6细胞重度ERS，诱发细胞不同程度凋亡。LPS诱发IEC-6细胞ERS与IRE1-XBP1、PERK-eIF-2a-ATF-4、ATF-6信号通路激活有关。参苓白术散含药血清抑制IEC-6细胞ERS与调控IRE1-XBP1、PERK-eIF-2a-CHOP途径有关，通过Caspase12、Bcl-2、Fas-Fasl途径调控IEC-6细胞在ERS情况下出现的细胞凋亡，升高轻度、重度ERS时IEC-6细胞内自噬水平，改善受到阻碍的自噬流，促进细胞自噬，抑制ERS的过度激活及细胞过度凋亡。沉默IEC-6细胞CHOP基因后，参苓白术散仍抑制IEC-6细胞ERS引起的炎症，与调控GRP78、IRE1-XBP1、PERK-eIF-2α-ATF4-CHOP通路蛋白表达、上调细胞自噬水平、抑制细胞凋亡密切相关。.本研究证实参苓白术散治疗IBD与调控巨噬细胞自噬-内质网应激-凋亡密切相关。..