The Research Of HBXIP-mediated Autophagy Regulation And Antibacterial Mechanism

Hepatitis B X-interacting protein（HBXIP）is a type of cellular protein that is present in mammals,It is named for its specific binding to the C-terminus of the hepatitis B virus-encoded protein HBx.Current studies related to HBXIP mainly focus on the replication of hepatitis B virus,proliferation and apoptosis of cells,replication of centrosomes,and migration and metastasis of tumor cells.However,there have been no relevant studies on the role and mechanism of host antibacterial immunity.Staphylococcus aureus is a highly pathogenic gram-positive bacterium and it is one of the most difficult-to-treat pathogens in the world.Bacterial resistance has also become an increasingly serious global public health issue,so we urgently need to develop new antibacterial infection strategies.In recent years,studies have shown that autophagy plays an important role in the body’s resistance to intracellular infections.Therefore,the purpose of this study was to investigate whether HBXIP is involved in the body’s antibacterial immunity.In this study,in combination with mouse in vivo experiments and in vitro cell experiments,the changes of inflammation-related and autophagy-related signaling pathways were detected from the molecular level and protein level,respectively,explore the possible antibacterial mechanism of HBXIP on host antibacterial immunity.The main research results of this research are as follows:（1）Mouse peritoneal macrophages and mouse mononuclear macrophage RAW264.7 were infected with S.aureus（USA300）.The results showed that HBXIP could be induced by USA300,indicating that HBXIP may be involved in antibacterial immunity.（2）The results in vivo in mice showed that the survival rate of HBXIP^-/+mice was higher under USA300 infection,and the bacterial load was greater in the lung tissue;the expression of inflammatory factors was higher in the mouse bronchoalveolar lavage fluid（BALF）;the alveolar structure of lung tissue was severely disrupted with a large number of inflammatory factors infiltrating.These results showed that HBXIP can reduce the inflammatory response in mice.（3）HBXIP overexpression and low expression transient system constructed in vitro were used to detect the expression of inflammatory cytokines TNF-α,IL-6 and IL-1β,and NF-κB and MAPKs inflammation pathways after stimulation with USA300.These results showed that over-expression of HBXIP can inhibit the expression of inflammatory factors and inhibit the activation of signaling pathways,indicating that HBXIP is indeed involved in the inflammatory response of the host.（4）Bacterial phagocytosis assays,colony dilution plate counts,and slide-through experiments were used to detect the biological function of macrophages.The results showed that HBXIP did not affect the phagocytic ability of macrophages,but could enhance the killing ability of macrophages.（5）Detection of expression of mTOR signaling pathway-related proteins,autophagy marker protein LC3,lysosomal membrane protein LAMP1,and transcription factor TFEB in HBXIP overexpressing or underexpressing cells stimulated by USA300;qPCR was used to detect the expression of autophagy-related genes;the expression of the autophagic vacuole was detected by immunofluorescence technique;the biological activity of lysosome was detected by acridine orange and Lyso-Tracker Red staining.These results showed that HBXIP could enhance cells autophagy.（6）HBXIP over-expressing cells were treated with autophagy-related agonists（Torin1）,inhibitors（3-MA）,and（Baf-A1）,followed by immunofluorescence techniques to detect changes in autophagosomes;colony plate count assay were used to detect the bacterial load;the expression of inflammatory factors was detected by ELISA.These results showed that autophagy helps to increase the bactericidal capacity of the body,which in turn inhibits the inflammatory response.In summary,HBXIP is indeed involved in the anti-bacterial and anti-inflammatory reactions of the body.On the one hand,HBXIP inhibits the activation of the mTOR pathway and regulates the initiation of autophagy;on the other hand,HBXIP promotes the entry of TFEB into the nucleus and regulates the genes associated with autophagy and lysosomal biological functions,therefore enhances the cells autophagy to regulate the host’s antibacterial immunity.This study opens a new field of HBXIP molecular research and provides new directions for the prevention and treatment of inflammation-related diseases.