鹰嘴豆素A通过自噬和胞外陷阱促进巨噬细胞杀灭鲍曼不动杆菌的机制研究

Acinetobacter baumannii (A. baumannii) is one of the most important pathogens of nosocomial infection. Due to the widely spread of multi-resistant strains and high mortality, it is difficult to treat A. baumannii infections. Regulation of innate immunity might provide a new direction for the treatment of bacterial infections. A. baumannii could induce autophagy by the production of isochorismatase or out memberane protein to kill intracellular bacteria in mammalian cells, while A. baumannii could not induce the release of extracellular traps. To find the medicine which can induce the clearance of A. baumannii by macrophage, we screened a variety of natural products extracted from Chinese herbal medicine and found that biochanin A (BCA) could improve the intracellular and extracellular killing of A. baumannii. Preliminary data showed that BCA could enhance the autophagy triggered by A. baumannii and induce the release of macrophage extracellular traps. It is reasonable to infer that BCA might affect the production of isochorismatase or out memberane protein to enhance A. baumannii induce autophagy, which lead to intracellular clearance of A. baumannii. In addition, BCA might induce the release of extracellular traps to facilitate extracellular killing of A. baumannii. The aim of the proposed study is to investigate the effect of BCA on the expression of isochorismatase, the production of siderophore and the expression of out memberane protein, to demonstrate the molecular mechanism of BCA enhancing the autophagic response induced by A. baumannii, to prove that BCA induced extracellular traps to kill extracellular A. baumannii and to investigate the pathway of this response by using immunofluorescence staining, western blotting and qPCR technology. Additionally, we also planned to assess the effect of BCA on the treatment of A. baumannii infection in a mouse model. This study would provide new insights for the development of novel anti-A. baumannii drugs.

鲍曼不动杆菌是院内感染最重要的致病菌，耐药率、致死率高且治疗困难，通过调节天然免疫抗感染可能成为新的治疗方向。鲍曼不动杆菌依赖异分支酸酶或外膜蛋白诱导自噬清除胞内菌，但不诱导胞外陷阱。为找到影响巨噬细胞杀菌的药物我们筛选了多种中药单体，发现鹰嘴豆素A（BCA）能促进胞内外鲍曼不动杆菌的杀灭，它能增强鲍曼不动杆菌诱导的自噬，并诱导胞外陷阱。我们推测BCA通过增强鲍曼不动杆菌异分支酸酶或外膜蛋白的表达增强自噬促进胞内杀菌，并通过诱导胞外陷阱促进胞外杀菌。因此，本研究拟应用免疫荧光染色、蛋白电泳、定量PCR等技术，观察BCA对鲍曼不动杆菌的异分支酸酶、铁载体及外膜蛋白的影响，阐明BCA增强自噬的分子机制，明确BCA诱导胞外陷阱杀伤胞外鲍曼不动杆菌的信号通路，并通过动物实验验证BCA对鲍曼不动杆菌清除的作用及机制。该研究将为新型抗鲍曼不动杆菌感染药物的研发提供新的思路。

鲍曼不动杆菌是院内感染最重要的致病菌，耐药率、致死率高且治疗困难，通过调节天然免疫抗感染可能成为新的治疗方向。我们发现鲍曼不动杆菌能够诱导通过AMPK/ERK/mTOR信号通路的Beclin-1依赖的自噬清除胞内菌，但不诱导胞外陷阱。我们已证实鹰嘴豆素A（BCA）能促进胞内外鲍曼不动杆菌的杀灭，它能增强鲍曼不动杆菌诱导的自噬，并诱导胞外陷阱。本研究通过应用免疫荧光染色、蛋白电泳、定量PCR等技术，阐明了BCA能够诱导Beclin-1依赖的自噬，并发现其信号通路是AMPK/ULK1/mTOR通路，明确BCA诱导胞外陷阱杀伤胞外鲍曼不动杆菌的信号通路是AMPK信号通路，并证明BCA诱导的胞外陷阱释放无ROS参与。我们还通过动物实验验证BCA对鲍曼不动杆菌清除的作用及机制。该研究为BCA治疗鲍曼不动杆菌感染提供合理的理论和实验依据，对临床治疗提供参考。本研究还对其他病原体感染与免疫的研究做了大量基础工作。我们发现了BCA可以杀灭巨噬细胞胞内外的沙门菌，并证实了其机制。我们发现李斯特菌诱导小胶质细胞胞外陷阱有利于杀灭其胞外菌，我们还研究了一些药物通过作用于α溶血素抗金葡菌。