口服分泌表达HMGB1 A box的重组乳酸杆菌治疗实验性结肠炎

The incidence of ulcerative colitis (UC) is continuously increasing in China. Non-specific inflammation is the primary pathological characteristics of UC. Intracolonic microbiota imbalance is also involved in the pathogenesis of UC. Most of the clinical therapeutic agents are aimed to suppress the inflammation by targeting some of the inflammation mediators: Aminosalicylates effect through inhibiting the conversion of arachidonic acid into leukotrienes (LTs) and prostaglandins (PGs). Steroids induce inhibitory κB (IκB) and stabilize the cell membrane to reduce the release of proinflammatory cytokines. Immunosuppressants non-specifically suppress the immune response in the colonic mucosa. And the most recently available bioactive agents such as infliximab, adalimumab and etanercept are all tumor necrosis factor-α scavengers. Probiotics such as Lactobacillus and Bifidobacteria are also reported to be beneficial for the remission of UC. However, these measures are less than ideal for their insufficient effects and undesired adverse effects. Recently, High mobility group box 1 (HMGB1) has been recognized as an initiator of innate immune response and an important proinflammatory cytokine when it is released actively or passively from the damaged cells and immune-related cells. HMGB1 can induce the expression and secretion of a variety of proinflammatory cytokines especially TNF-α and vice versa. The level of HMGB1 is increased in both the circulation and the feces of UC patients and experimental colitic rodents. Inhibiting the release of HMGB1 by ethyl pyruvate or neutralizing the released HMGB1 by specific antibodies could alleviate experimentally induced colitis, suggesting that HMGB1 plays an essential role in the pathogenesis of UC. Ablation of extracellular HMGB1-mediated inflammation might represent a promising therapeutic approach for UC. In preliminary experiments, we confirmed that the native antagonist of HMGB1, HMGB1 A box, had a protective effect against TNBS-induced colitis in mice. Therefore, we reason that orally administered recombinant Lactobacillu casei secreting HMGB1 A box, will exert a therapeutic effect by alleviating inflammation and improving the intracolonic microbiota. In this project, we plan to construct Lactobacillu casei-compatible plasmid carrying the coding sequence of human HMGB1 A box led by a SPusp45 signal peptide, generate the recombinant Lactobacillu casei-HMGB1 A box. After the verification of the secretion and the bioactivity of HMGB1 A box, we will test the preventive and therapeutic effects of orally administered Lactobacillu casei-HMGB1 A box on DSS and TNBS-induced acute and chronic colitis, respectively, in rats and attempt to explore the possible machanisms underlying the therapeutic effect by examining the proinflammatory cytokines, proliferation and apoptosis in the colonic mucosa. Collectively, the fulfill of this project might provide a novel therapeutic approach for the treatment of UC.

非特异性炎症和肠道微生态失调是溃疡性结肠炎（UC）的主要病理特征。晚期致炎因子高迁移率族蛋白B1（HMGB1）在UC的发生和持续过程中起着关键的作用。我们前期研究已初步证实腺相关病毒介导HMGB1的天然拮抗物HMGB1 A box在结肠黏膜分泌表达可有效抑制TNBS诱导的小鼠结肠炎。在此基础上，我们提出以口服乳酸杆菌介导HMGB1 A box肠道分泌表达发挥抗炎及调节微生态的双重作用治疗UC的新思路。本课题拟制备可分泌表达HMGB1 A box的食品级重组乳酸杆菌；检测以此制剂灌胃后在大鼠肠内定植并分泌表达HMGB1 A box的能力；通过检测肠黏膜通透性、炎症细胞浸润、炎症调节因子表达、肠道黏膜细胞增殖与凋亡及肠壁纤维化等的变化，观察其对DSS和TNBS诱导的大鼠急、慢性结肠炎的抑制作用并初步阐明其治疗机理。旨在探讨口服重组乳酸杆菌介导 HMGB1 A box表达治疗UC的可行性。

溃疡性结肠炎（ulcerative colitis，UC）为原因不明的以大肠黏膜坏死和炎症为主要病理表现的疾病。晚期致炎因子高迁移率族蛋白B1（HMGB1）在UC的发生发展过程中起着关键的作用。本研究通过体外实验验证了乳酸杆菌可介导有活性HMGB1 A box的分泌表达；通过体内实验验证由乳酸杆菌介导HMGB1 A box肠道的分泌表达对UC的治疗作用并探讨其可能机制。结果发现，重组L. casei-HMGB1 A box可有效抑制急慢性UC大鼠结肠炎症，各实验组大鼠疾病活动指数明显下降，免疫组化染色显示炎症细胞浸润减少，肠黏膜上皮损伤减轻，隐窝破坏减少。对各实验组大鼠肠道黏膜炎症相关细胞因子检测结果表明，重组L. casei-HMGB1 A box组大鼠结肠黏膜内促炎细胞因子TNF-α及 IL-6表达下降，抗炎细胞因子IL-10表达升高，IL-4表达则无明显变化。治疗组大鼠肠道黏膜中炎症氧化损伤指标MDA含量下降而SOD活性升高，表明重组L. casei-HMGB1 A box可通过增加SOD的活性增强机体清除氧自由基的能力，抑制脂质过氧化反应从而保护肠道黏膜免受氧化损害。信号转导芯片及全基因组表达芯片结果提示HMGB1 A box可能通过NF-κB信号转导通路发挥作用，HMGB1 A box的高表达可引起大量与HMGB1 A box生物学功能相关的基因表达发生改变并上调生长相关因子表达。这些研究结果提示，口服重组L. casei-HMGB1 A box对大鼠急慢性UC均具有治疗作用，其可能作用机制为调节炎症因子表达、增加机体清除氧自由基的能力从而减轻氧化损伤、上调多种生长相关因子表达促进受损结肠上皮的再生和修复。本实验为UC的治疗提供了一种全新的方向。