丁酸盐靶向自噬-溶酶体通路清除幽门螺杆菌的机制研究

幽门螺杆菌（Hp）慢性感染的分子机制尚不明确。申请者前期研究结果证明：Hp慢性感染与其对自噬-溶酶体通路的抑制相关,丁酸盐能有效降低Hp在细胞内和小鼠胃内定植，这一过程可能与靶向激活自噬-溶酶体通路相关，但具体分子机制不明。故本项目拟开展：(1)采用免疫穿透电镜观察丁酸盐对Hp感染后胃上皮细胞内自噬体、自噬溶酶体及溶酶体超微结构的改变；(2)采用分子探针研究丁酸盐对Hp感染后细胞氢质子泵和溶酶体的酸化情况；（3）基因表达谱及转录因子网络分析技术，揭示丁酸盐对细胞自噬-溶酶体通路靶向修复的分子机制；(4) 在小鼠Hp感染模型中，通过激活/抑制靶标基因，验证丁酸盐对Hp的清除作用及对自噬-溶酶体的靶向修复作用。本研究成果将首次阐明丁酸钠通过靶向自噬-溶酶体通路清除Hp的新机制, 为如何激活机体自主防御系统清除Hp的认知带来新突破，同时对开发新的根除Hp感染方案有着深远的科学与实践意义。

The molecular pathogenesis of chronic Helicobacter pylori infection is unclear. Emerging evidence demonstrated that some bacterial species could devise strategy to promote their intracellular survival through interfering with host cells’ lysosomal acidification after being sequestered by autophagosomes, leading to chronic infection. Our preliminary data showed that H. pylori promoted autophagosome formation with concomitant inhibition of lysosomal acidification so as to sustain their intracellular survival. In this connection, sodium butyrate reduced the intracellular survival and colonization of H. pylori in vitro and in vivo as well as restored lysosomal acidification. Nevertheless, the molecular mechanism underlying the protective action of sodium butyrate is unknown. Herein, we proposed (1) using immunoelectron microscopy to study the effect of sodium butyrate on the ultrastructure of lysosomes in H. pylori-infected gastric epithelial cells; (2) using molecular probes to assay the effect of sodium butyrate on vacuolar proton pump and lysosomal acidification in H. pylori-infected cells; (3) combining transcriptomics with proteomic analysis of lysosomes isolated from H. pylori-infected gastric epithelial cells to identify novel lysosome-regulatory pathway of sodium butyrate; (4) using pharmacological activators and inhibitors of lysosomes to corroborate the lysosome protective effect of sodium butyrate in H. pylori-infected cultured gastric epithelial cells and murine stomachs. The proposed project will not only elucidate the mechanism underlying the rectifying effect of sodium butyrate on lysosomal acidification in H. pylori infection, but may also open up novel therapeutic avenues for modulating host immune response to achieve infection clearance. The results from this study will also help formulate regimens for more effective H. pylori eradication.