背景:幽门螺杆菌(H.pylori)是胃炎的主要病因,并通过各种致病机制破坏胃粘膜屏障的完整性。幽门螺杆菌侵入胃粘膜后,它与固有层中的免疫细胞相互作用。巨噬细胞是炎症反应的核心角色,幽门螺杆菌刺激它们分泌多种炎症因子,导致胃粘膜的慢性损伤。因此,本研究旨在探讨巨噬细胞分泌的炎症因子引起胃黏膜损伤的机制,这可能为H.pylori相关性胃炎的发生发展提供新的机制。
方法:通过RT-qPCR检测幽门螺杆菌感染的巨噬细胞CCL3的表达和分泌。Westernblot和ELISA。通过Westernblot分析幽门螺杆菌感染的巨噬细胞培养基和CCL3对胃上皮细胞紧密连接的影响。免疫荧光和跨上皮电阻。EdU和凋亡流式细胞术检测细胞增殖和凋亡水平。使用双荧光素酶报告基因测定和染色质免疫沉淀测定来研究CCL3转录因子。最后,采用苏木精、伊红染色和免疫组织化学方法分析胃粘膜组织炎症和CCL3表达。
结果:幽门螺杆菌感染后,巨噬细胞表达和分泌的CCL3增加。幽门螺杆菌感染的巨噬细胞培养基和CCL3破坏的胃上皮细胞紧密连接,而CCL3中和抗体和CCL3受体抑制剂改善了细胞间紧密连接的破坏。此外,幽门螺杆菌感染的巨噬细胞培养基和CCL3重组蛋白刺激P38磷酸化,P38磷酸化抑制剂改善了细胞间紧密连接的破坏。此外,发现STAT1是CCL3的转录因子,幽门螺杆菌刺激巨噬细胞通过JAK1-STAT1途径分泌CCL3.最后,小鼠注射小鼠CCL3重组蛋白后,胃粘膜损伤和炎症加重,P38的磷酸化水平升高。
结论:总之,我们的研究结果表明,幽门螺杆菌感染通过JAK1-STAT1途径刺激巨噬细胞分泌CCL3.随后,CCL3通过P38的磷酸化损伤胃上皮紧密连接。这可能是幽门螺杆菌相关性胃炎胃粘膜损伤的新机制。
BACKGROUND: Helicobacter pylori (H. pylori) is the predominant etiological agent of gastritis and disrupts the integrity of the gastric mucosal barrier through various pathogenic mechanisms. After H. pylori invades the gastric mucosa, it interacts with immune cells in the lamina propria. Macrophages are central players in the inflammatory response, and H. pylori stimulates them to secrete a variety of inflammatory factors, leading to the chronic damage of the gastric mucosa. Therefore, the study aims to explore the mechanism of gastric mucosal injury caused by inflammatory factors secreted by macrophages, which may provide a new mechanism for the development of H. pylori-related gastritis.
METHODS: The expression and secretion of CCL3 from H. pylori infected macrophages were detected by RT-qPCR, Western blot and ELISA. The effect of H. pylori-infected macrophage culture medium and CCL3 on gastric epithelial cells tight junctions were analyzed by Western blot, immunofluorescence and transepithelial electrical resistance. EdU and apoptotic flow cytometry assays were used to detect cell proliferation and apoptosis levels. Dual-luciferase reporter assays and chromatin immunoprecipitation assays were used to study CCL3 transcription factors. Finally, gastric mucosal tissue inflammation and CCL3 expression were analyzed by hematoxylin and eosin staining and immunohistochemistry.
RESULTS: After H. pylori infection, CCL3 expressed and secreted from macrophages were increased. H. pylori-infected macrophage culture medium and CCL3 disrupted gastric epithelial cells tight junctions, while CCL3 neutralizing antibody and receptor inhibitor of CCL3 improved the disruption of tight junctions between cells. In addition, H. pylori-infected macrophage culture medium and CCL3 recombinant proteins stimulated P38 phosphorylation, and P38 phosphorylation inhibitor improved the disruption of tight junctions between cells. Besides, it was identified that STAT1 was a transcription factor of CCL3 and H. pylori stimulated macrophage to secret CCL3 through the JAK1-STAT1 pathway. Finally, after mice were injected with murine CCL3 recombinant protein, the gastric mucosal injury and inflammation were aggravated, and the phosphorylation level of P38 was increased.
CONCLUSIONS: In summary, our findings demonstrate that H. pylori infection stimulates macrophages to secrete CCL3 via the JAK1-STAT1 pathway. Subsequently, CCL3 damages gastric epithelial tight junctions through the phosphorylation of P38. This may be a novel mechanism of gastric mucosal injury in H. pylori-associated gastritis.