LPS激活TLR4/NF-κB通路诱导炎症小体形成在反流性食管炎中的作用及其相关机制研究

Reflux esophagitis (RE) caused by gastroesophageal reflux (GER) is a risk factor for esophageal adenocarcinoma. About 40% GERD patients failed to show response to acid suppressors, which challenges the conventional pathogenesis of acid-mediated mechanism. Recent studies have raised an alternative concept that chemokines mediate inflammatory damage of esophageal tissue and that lipopolysaccharides(LPS) released by Gram-negative microbiomes in distal esophagus causes expression of inflammatory cytokines. Our preliminary study recently found that LPS can induce cytokine production and pyroptosis. We further discovered that LPS induced production of pro-inflammatory cytokines via TLR4/NF-κb signaling and at the same time activated inflammasome, resulting in incision of pro-inflammatory cytokines and cytokines secretion. Furtherly, we found that caspase-1 inhibitor could suppress inflammatory cytokine secretion and protected esophageal epithelial cells. Therefore we hypothesize that LPS activates TLR4/NF-κb signaling and inflammasome, releasing inflammatory cytokines and inducing pyroptosis, finally resulting in inflammatory damage of esophageal tissue. Inhibition of caspase-1 might by a new target for RE therapy. In this present study, we will collect and detect the biopsy samples from RE paitients, perform experiments in vivo and in vitro using qPCR, western-blot, interference and over-expression, etc. Once our hypothesis gets proved, we are going to contribute to the treatment of RE and the prevention of dysplasia and carcinogenesis.

胃食管反流引起的反流性食管炎（RE）是食管腺癌的危险因素。约40%胃食管反流病抑酸治疗迁延不愈，挑战传统酸损伤机制。最新研究提出细胞因子介导的炎性损伤是RE的发病机制，食管微生物群释放的脂多糖（LPS)促进促炎因子表达。我们前期实验发现：LPS可刺激RE患者上皮细胞分泌炎症因子并引起细胞焦亡；LPS还可激活TLR4/NF-κb通路上调炎症因子前体，并激活炎症体切割炎症因子前体致炎症因子分泌；且caspase-1抑制剂可抑制LPS所致炎症因子释放保护食管上皮细胞。因此提出：难治性RE患者中LPS激活TLR4/NF-κb通路与炎症体并释放炎症因子，导致食管上皮细胞焦亡，最终引起食管损伤。在本项目中，我们将收集并检测临床RE患者活检标本、构建RE细胞与动物模型，采取QPCR、WB、慢病毒干扰与过表达等技术手段，进一步阐明假设，为治愈RE、预防其炎性增生和癌变提供新思路。

胃食管反流引起的反流性食管炎（RE）是食管腺癌的危险因素。然而约40%胃食管反流病抑酸治疗迁延不愈，这对酸反流导致黏膜化学损伤的传统机制是极大的挑战。根据我们前期的实验结果，革兰氏阴性菌细胞壁主要组分LPS可激活RE原代食管上皮细胞的炎症体并释放炎症因子，导致食管上皮细胞焦亡，最终引起食管损伤。按照实验计划，我们对RE患者和健康志愿者的食管黏膜进行菌群16s rRNA测序从而探索食管LPS的来源，最后我们发现以革兰氏阴性菌（G-）比例升高为特征的食管菌群失调是食管LPS含量上升的原因。然而，以上结果并不能阐明菌群失调和反流的因果关系，我们因此进一步采集非糜烂性胃食管反流病和功能性食管病患者食管黏膜进行菌群测序，随后发现食管菌群失调并非只出现在RE患者中，而是出现在所有具有反流症状的患者中（无论是否存在病理性酸暴露和黏膜破损），这告诉我们食管菌群失调不是酸反流造成的后果。为明确食管菌群失调的反流症状患者的意义，我们对不同亚型的反流症状患者进行了食管黏膜转录组测序，寻找GERD和功能性食管病患者共同变化的差异基因，并通过细胞模型初步验证通路，最后发现食管菌群失调可能通过LPS激活TLR2-p38通路诱发上皮炎症和破坏食管上皮屏障，从而参与胃食管反流症状的发生。食管菌群失调有望成为抑酸治疗无效的难治性反流症状患者的新的治疗靶点。