一种小分子热休克蛋白αB-Crystallin肠道抑炎作用及其在炎症性肠病中的调控机制研究

Immune dysfunction-mediated intestinal inflammation is a vital factor in the pathogenesis and progression of inflammatory bowel disease (IBD). Alpha B-crystallin (CRYAB), one key member of the superfamily of small heat shock proteins, has been identified as a negative regulator of immune-related inflammatory disorders. In our previous studies, we found that: ① CRYAB expression was significantly decreased in the THP1 macrophage inflammatory model, dextran sodium sulfate-induced colitis model mice, and colon tissue samples of IBD patients. ② Over-expression of CRYAB could significantly inhibit the phosphorylation of NF-κB and MAPK signaling pathway, and reduce the levels of TNF-α , IL-6 and other inflammatory cytokines. Accordingly, we propose the hypothesis that “CRYAB plays a negative regulatory effect in inflammatory process of IBD through mediating NF-κB and MAPK-p38/JNK signaling pathways”.Therefore, the project is intended to clarify molecular mechanism of CRYAB in IBD intestinal inflammation and identify related target genes by involving CRYAB-/- transgenic mouse model, cell lines cultured in vitro and IBD subjects, using the methods of TAT-protein transduction, lentiviral vector transfection, CRISPR/cas9-mediated gene knockout, and immunoprecipitation coupled to MALDI-MS. The study aims to discuss the mechanism of the pathogenesis and investigate for novel therapeutic strategies of IBD.

免疫异常介导的肠道炎症损伤是炎症性肠病（IBD）发生和进展的核心因素，小分子热休克蛋白αB-晶状体蛋白（CRYAB）已被证实是免疫相关炎症的负性调节因子。预实验结果发现：①THP1巨噬细胞炎症模型、结肠炎模型小鼠和IBD患者结肠中CRYAB表达均明显降低；②过表达CRYAB可抑制NF-κB和MAPK等信号通路的蛋白磷酸化水平，显著降低TNF-α、IL-6等炎症因子表达。据此我们提出“CRYAB通过介导NF-κB、MAPK-p38/JNK途径在IBD肠道炎症过程中发挥负性调控效应”的假说，并以CRYAB-/-转基因小鼠、体外培养细胞系和IBD患者手术标本为研究对象，采用TAT-蛋白质转导技术、慢病毒载体转染、CRISPR/cas9基因敲除、免疫共沉淀联合质谱分析等方法，探讨CRYAB在IBD肠道炎症中的作用与分子机制，筛选并验证相关靶基因，为完善IBD发病机制并开发新的治疗策略提供新思路。

炎症性肠病（IBD）是一种累及全结直肠黏膜的炎症性疾病，以病因不明、病情反复发作、病程迁延不愈为特点，严重影响病人的生活质量（OQL）。尽管 IBD 的治疗以内科治疗为主，但是仍有 25- 30%的病人因药物治疗失败或并发症而最终需要接受外科手术治疗。因此，寻找新的肠道炎症的治疗靶点治疗 IBD 显得尤为重。其在中枢神经系统炎症中发挥着重要的抗炎作用。然而，CRYAB 在肠道炎症的作用仍不清楚。课题组研究发现，CRYAB表达在IBD患者炎症组织和葡聚糖硫酸钠（DSS）诱导的小鼠肠炎中表达显著降低，且与炎症因子TNF-α、IL-6呈负相关 CRYAB过表达可与IKK复合物结合抑制IKK复合物的形成，从而显著抑制炎症因子水平，而敲除CRYAB则显著增加炎症反应。注射TAT-CRYAB重组蛋白可显著治疗DSS和三硝基苯磺酸（TNBS）诱导的小鼠急性肠炎，并可保护肠屏障完整性。因此，本研究首次提出CRYAB可通过抑制IKKβ介导的炎症通路减弱肠道炎症反应，为IBD的治疗提供新的途径和方法。