沙利度胺通过抑制NF-κB诱导的Hedgehog信号通路对克罗恩病肠黏膜愈合的作用

Crohn’s disease (CD) often presents in early age and commonly manifests with chronic relapsing debilitating symptoms and severe complications which result in up to 70% CD patients need for abdominal surgeries. With continuously increasing incidence in China, CD becomes one of the most common diseases in the gastroenterology field. However, no cure for CD until now and the current treatments are difficult to achieve and maintain remission. Anti-TNF monoclonal antibodies therapy is effective in partial patients but so expensive that most families can’t afford especially in the developing countries. Therefore, it remains a great need for new therapies that are both effective and economically feasible. Our previous clinical studies have reported that thalidomide is effective in treating CD including mucosal healing. However, few studies have been focus on the mechanism by which thalidomide induces mucosal healing. Based on the previous studies, thalidomide inhibits Nuclear Factor-κB (NF-κB) pathway and in turn play immunomodulatory role in CD. It has been shown that Hedgehog proteins is a target gene of transcription factor of NF-κB. And these two pathways have been identified to play important roles in the pathogenesis of CD and mucosal homeostasis. However, no study has built the crosstalk between these two in mucosal healing of CD. Whether thalidomide has effects in the regulation of the crosstalk also need to be identified. Therefore, based on our previous data, we proposed to test the mechanism that thalidomide-induced mucosal healing is regulated by the inhibition of NF-κB mediated Hedgehog signaling. To test our hypothesis, first we will oral gavage thalidomide to trinitrobenzesulfonic acid (TNBS)-induced colitis mice models and analyze the NF-κB by western blot and separate epithelium with mesenchyme and test the expression of Hh signaling by qRT-PCR. Then by the co-culture system of murine-derived intestinal organoids with different mesenchymal cells, we are going to dissect the signaling transduction and targeted cell types. Specific deletion of Smo from mesenchymal cells will then support our hypothesis. Last, we will collect data and samples from CD patients who received thalidomide treatment and test the roles of thalidomide in promoting histological and ultrastructural mucosal healing and the proposed mechanism. In all, our research will study the mechanism by which thalidomide mediates mucosal healing therefore expand the clinical therapy of CD. And the detection of target cells can help us to limit additional effects and search new therapeutic targets in CD.

克罗恩病（CD）发病率上升快，主要累及青少年，致残率高。传统治疗效果不理想，部分有效的生物制剂价格昂贵，多数患者无法承受。我们前期工作证实沙利度胺能够促进黏膜愈合改善CD患者的临床结局，被认为是CD治疗的新希望。目前缺乏对黏膜愈合机制的探索。沙利度胺抑制NF-κB通路，而Hedgehog是NF-κB通路转录因子的下游产物。且这两者均被证明参与CD病理生理过程及黏膜稳态调控。因此我们推测沙利度胺通过抑制NF-κB诱导的Hedgehog（Hh）信号通路促进CD肠黏膜愈合。首先以结肠炎小鼠模型为载体应用免疫染色、蛋白印迹、PCR等方法验证沙利度胺对NF-κB及 Hh的调控。然后应用微小肠与间质细胞共培养模型深入解析沙利度胺作用的靶向细胞。最后应用临床标本验证沙利度胺促黏膜愈合作用及机制。本研究对沙利度胺促黏膜愈合机制的探索，对开拓有效经济的CD药物及探索潜在的治疗靶点具有重要的科学意义。

克罗恩病（CD）发病率上升快，主要累及青少年，致残率高。传统治疗效果不理想，部分有效的生物制剂价格昂贵，多数患者无法承受。我们前期工作证实沙利度胺能够促进黏膜愈合改善CD患者的临床结局，被认为是CD治疗的新希望。本研究通过探索沙利度胺促进黏膜愈合机制，拓展沙利度胺对CD的治疗意义，从而降低CD患者并发症及手术率，因此具有重要的临床意义。.主要的研究内容和结果：.1.通过对CD患者肠黏膜损伤进行了细胞水平分析发现，CD患者肠黏膜存在肠上皮细胞增殖及凋亡增加，伴随着肠上皮细胞分化的异常，体现为分泌型细胞增多而吸收性细胞数目减少。.2.应用CD患者肠镜或手术标本分离肠隐窝结构离体构建三维立体多细胞类器官微小肠，并应用qRT-PCR、免疫染色等验证该模型表达肠黏膜中所有细胞系，最大程度接近CD患者病生理环境，为进一步探索机制奠定基础。.3.随访沙利度胺治疗的CD患者结合临床资料对沙利度胺安全性及治疗作用尤其是促黏膜愈合、防治CD并发症肛周病变的作用进行评估。.4.在验证沙利度胺治疗作用的基础上进一步验证其机制。应用结直肠上皮细胞培养、CD患者来源的微小肠模型及结肠炎小鼠模型验证，沙利度胺治疗通过抑制Hedgehog（Hh）蛋白释放因子Scube2的表达，从而抑制Hh通路的激活。. 因此，本项目从CD治疗中棘手的问题出发，验证沙利度胺的治疗作用及安全性，并从机制上验证了沙利度胺通过抑制Scube2产生从而抑制Hh通路促进CD肠黏膜愈合。