p38 MAPK介导的黏膜炎症影响食管痛觉敏感性的机制研究

Gastroesophageal reflux disease (GERD) is one of the most common problems encountered in clinical practice today, with a loss of quality of life and a substantial burden for national health-care system. The pathogenesis of GERD is complex. Over the past few decades, GERD has been assumed to be a caustic injury caused by damage to the squamous epitheliun by acidic contents and peptidases. In fact, molecular signs of inflammation and mucosal inflammation mediators are detected even before microscopic or macroscopic changes become apparent in those with GERD. Recently,more and more attention have been paid to the key roles of mucosal inflammation and visceral hypersensitivity in the pathogenesis of GERD. MAPKs, consisting of p38, ERK, and c-Jun N-terminal kinase (JNK), are a family of serine/threonine protein kinases that mediate many fundamental biological processes and cellular responses to external stress signals. Increased activities of p38 MAPK and its involvement in regulation of the synthesis of inflammation mediators at the level of transcription and translation make the potential targets for anti-inflammatory therapeutics. In addition, p38 MAPK is activated in primary sensory neurons, dorsal horn neurons, and spinal glial cells by nociceptive stimuli, growth factors, and inflammatory mediators, contributing to the induction and maintenance of sensitization via transcriptional, translational, and posttranslational regulation. Inhibition of MAPKs has been shown to attenuate inflammatory and neuropathic pain. We hypothesize that: mucosal inflammation caused by stimulation indirectly cause esophageal mucosal injury in GERD patients, and further influence the esophageal visceral hypersensitity. In this study, we will detect the expression levels of p38 MAPK and its related genes using human esophageal mucosal specimens and GERD animal models. In order to identify the exact role of p38 MAPK signal pathway in esoohageal mucosal inflammation, we will use a series in vitro and in vivo experiments interfered by activate the pathway and blocking the pathway. Multiple molecular biologic techniques, such as immunofluorescence, Real-time PCR, Western blot, combined with electrophysiologic methods, verify the above hypothesis. We hope to investigate the pathogenesis of GERD, and provide novel target to treat GERD.

胃食管反流病(GERD)是严重影响患者生活质量的多发慢性疾病。研究发现，食管黏膜炎症和内脏高敏感在GERD的起病和疾病发展过程中发挥关键作用。p38 MAPK信号通路在炎症和内脏高敏感中发挥重要作用，但其在GERD中的具体作用及机制尚不完全清楚。本研究提出如下假设：GERD患者通过刺激食管黏膜炎症改变间接导致食管黏膜损伤，并影响食管痛觉高敏的形成和维持。我们将采用人体食管黏膜标本及GERD动物模型观察p38 MAPK通路及相关基因的表达变化，结合离体及在体激活、拮抗剂的干预，明确p38 MAPK通路在食管黏膜炎症中的作用，研究该通路介导的黏膜炎症在内脏高敏感中的作用及机制。从人体研究、在体及离体实验等多层次，利用免疫荧光、Real-Time PCR、Western blot等分子生物学技术结合电生理等方法验证上述假设。旨在进一步探讨GERD的发病机制，为更好的认识和治疗GERD提供靶标。

胃食管反流病（GERD）发病率日益增高，该病严重影响生活质量，耗费大量卫生资源，对其发病机制的探索一直是本领域的研究热点。p38 MAPK通路是丝裂原活化蛋白激酶通路之一。.原代培养人食管上皮细胞，给予酸刺激和胰蛋白酶刺激，结果：1）食道上皮细胞中p38 MAPK的磷酸化程度随着pH值的下降和胰蛋白酶剂量的增加而增加，2）食道上皮细胞中炎性因子，如IL-8，COX2，TNFα的mRNA水平明显增加；3）给予p38 MAPK抑制剂SB203580进行预处理治疗干预，发现可以明显降低食道上皮细胞中iNOS蛋白表达和NO水平。4）通过检测上皮细胞中claudin-1和ZO-1的mRNA水平发现酸刺激和胰蛋白酶刺激激动了食道上皮细胞的p38 MAPK信号通路。此外，我们还通过胃底结扎加幽门限制法诱导形成反流性食管炎（RE）模型，结果发现：1）2周后，RE大鼠食管上皮组织中p38MAPK磷酸化水平明显增加，给予其抑制剂SB203580治疗，可降低p38MAPK磷酸化水平。2）RE大鼠食管上皮组织中claudin-1蛋白表达明显降低；上皮跨膜电阻抗明显降低，食管上皮的屏障功能明显下降。而给予p38MAPK抑制剂SB203580治疗后，食管上皮的屏障功能明显改善。3）RE大鼠食管上皮组织中MMP 3和MMP 9蛋白表达水平明显增加；给予p38MAPK抑制剂后，RE大鼠食管上皮中MMP3和MMP9蛋白表达明显降低。4）上皮组织中炎性细胞侵润增加；TNFα、IL-6、 IL-1β的mRNA水平明显增加。给予p38MAPK抑制剂SB203580治疗后，RE大鼠食管上皮中炎性细胞侵润减少（CD68表达降低），炎症因子表达水平明显降低。5）RE大鼠食管组织中诱导性一氧化氮合酶（iNOS）蛋白表达水平明显增加，给予p38MAPK抑制剂SB203580治疗后，RE大鼠食管iNOS蛋白表达降低，NO生成减少，3-硝基络氨酸水平降低。结论：在胃食管反流病中，胰蛋白酶和酸是通过激活p38 MAPK通路，诱发炎症，激动iNOS/NO，从而造成食道黏膜上皮细胞的炎性损伤。本研究从p38 MAPK通路阐述了胃食管反流病中食道黏膜损伤的潜在机制，为治疗反流性食道炎提供了一个崭新的靶点。