丁酸盐靶向自噬-溶酶体通路清除幽门螺杆菌的机制研究

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.

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。

幽门螺杆菌（Helicobacter pylori，Hp）是一种常见的人类致病细菌，与慢性胃炎、消化性溃疡和癌症密切相关。从核内体到溶酶体的内吞途径是宿主清除包括Hp在内的胞内细菌的重要途径。然而，对于Hp如何溶酶体功能，从而避免自身被免疫清除并在宿主细胞内存活，我们知之甚少。.本研究发现，Hp感染可以抑制自噬-溶酶体通路从而易于在宿主细胞内定植，而丁酸盐通过靶向修复溶酶体酸化环境，可以修复进一步激活自噬-溶酶体降解通路，最终介导Hp的清除。在此基础上，我们进一步研究了Hp感染导致溶酶体受损的机制，发现Hp毒力因子CagA发现Hp毒力因子CagA直接诱导MCOLN3转录抑制，导致溶酶体降解功能受损。恢复内源性 MCOLN3 蛋白表达或重新激活MCOLN通道活性后，可成功修复溶酶体降解功能，从而达到细菌根除效果。此外，我们指出Hp感染导致的MCOLN3蛋白水平下调是由与MCOLN3 mRNA的3’非翻译区结合的microRNA介导的。.总体来说，本研究提出了Hp介导溶酶体功能障碍，从而在人类正常胃上皮细胞内持续定值的一种新机制。此外，研究发现丁酸盐和粘脂蛋白激活剂可恢复受损的溶酶体功能，从而根除细胞内Hp。因此，本研究结果将为开发新的Hp感染及其相关疾病开辟了新的治疗途径。