A Novel Cytoprotective Peptide for GI Epithelial Cell

描述(申请人提供)：肠屏障功能障碍可能是炎症性肠病(IBD)和肠源性脓毒症(GDS)患者发病和死亡的常见致病机制。如果是这样的话，一种通过增加连接粘膜上皮细胞的紧密和粘连连接蛋白的积累来保护粘膜屏障功能和结构的治疗策略可以用于治疗这些疾病。我们最近鉴定了一种名为AMP-18的新的18 kDa蛋白质，它是在人类和其他6种哺乳动物的胃窦粘膜上皮细胞中合成的，具有有丝分裂和运动发育特性。一种来自该蛋白中央结构域的21聚体多肽在人结肠上皮细胞(Caco-2/BBE)培养中被发现具有细胞保护作用，该细胞受到氧化剂、吲哚美辛或葡聚糖硫酸钠(DSS)的损伤，用于诱导小鼠结肠炎。细胞培养研究表明，这种AMP多肽激活p38 MAP激酶，增加特定紧密连接蛋白(occludin，ZO-1，claudin-5)、黏附连接蛋白(E-cadherin)和热休克蛋白(HSP25，Hsp72)的积聚，并保护暴露于细胞松弛素D的细胞中的肌动蛋白微丝网络。AMP肽可增加结肠粘膜中过度磷酸化的阻滞素的含量。为了确定AMP肽的这些生物学效应是否在体内提供了结肠细胞保护，我们在两个重要的屏障损害动物模型IBD和GDS上测试了它的有效性。用AMP多肽对小鼠进行预处理，可延缓给予DSS诱导结肠炎的动物的血性腹泻和体重减轻。在GDS模型中，这种多肽还可以防止在部分肝切除和术后食物剥夺的手术应激后，鞘内注射铜绿假单胞菌的小鼠死亡。我们的目标是在IBD和GDS的细胞培养和体内模型中获得额外的支持，以证明AMP肽通过增加紧密连接蛋白(如occludin)的积累来保护结肠粘膜屏障的结构和功能。证实和扩展AMP多肽的细胞保护、有丝分裂和运动生成作用的研究将进一步支持开发该分子作为治疗剂，不仅治疗IBD和GDS，而且还治疗其他由肠道紧密连接破坏和随后增加的粘膜对细菌及其产物的通透性所介导的胃肠道疾病。

DESCRIPTION (provided by applicant): Disruption of intestinal barrier function may be a common pathogenetic mechanism that mediates morbidity and mortality in subjects with inflammatory bowel disease (IBD) and gut-derived sepsis (GDS). If so, a therapeutic strategy that protects mucosal barrier function and structure by increasing accumulation of tight and adhesion junction proteins that connect mucosal epithelial cells could be used to treat these conditions. We recently characterized a novel 18-kDa protein called AMP-18, that is synthesized in epithelial cells of the gastric antrum mucosa of humans and 6 other mammals, and has mitogenic and motogenic properties. A 21-mer peptide derived from the central domain of the protein was found to be cytoprotective in human colonic epithelial cell (Caco-2/bbe) cultures subjected to injury by an oxidant, indomethacin, or dextran sulfate sodium (DSS) used to induce colitis in mice, Studies in cell culture indicate that this AMP peptide activates p38 MAP kinase, increases accumulation of specific tight junction proteins (occludin, ZO-1, claudin-5), an adherens junction protein (E-cadherin), and heat shock proteins (hsp25, hsp72), and protects the actin microfilament network in cells exposed to cytochalasin D. When given to mice, AMP peptide increases the content of hyperphosphorylated occludin in the colonic mucosa. To determine if these biological effects of AMP peptide confer colonic cytoprotection in vivo, its effectiveness was tested in two important animal models of barrier compromise, IBD and GDS. Pretreatment of mice with AMP peptide delayed the onset of bloody diarrhea and reduced weight loss in animals given DSS to induce colitis. The peptide also prevented the death of mice given intracecal Pseudomonas aeruginosa following the surgical stress of partial hepatectomy and post-operative food deprivation in a model GDS. Our objective is to obtain additional support in cell culture and in vivo models of IBD and GDS to demonstrate that AMP peptide, by increasing accumulation of tight junction proteins such as occludin, protects colonic mucosal barrier structure and function. Studies that confirm and extend the cytoprotective, mitogenic, and motogenic effects of AMP peptide would lend further support to developing this molecule as a therapeutic agent to treat not only IBD and GDS, but other GI diseases mediated by disruption of intestinal tight junctions and the subsequent increase in mucosal permeability to bacteria and their products.