| Research background:Acinetobacter baumannii has increasingly allocated an important situation as a hospital-acquired pathogen in worldwide,causing skin and soft tissue infections and respiratory diseases.With the increase of clinical invasive operation opportunities and the widespread applications of antibiotics,the detection rate of multi-drug resistant strains have virually increased and the distribution rate is the highest in various provinces and cities,resulting in more difficulty in treatment.During the later years,studies have discovered that bacterial drug-resistance is not completely related to its virulence.OmpA plays an essential role in the pathogenesis of A.baumannii,besides,the bacteria secrete OMVs carrying OmpA remotely to damage host cells.Nowadays,there is no rapid clinical detection method for OmpA and its specific pathogenic mechanism is still unclear.Short-chain fatty acids（SCFAs）,produced by some anaerobes fermenting dietary fibre,can be involved in regulating the inflammatory microenvironment and improving the patient’s mucosal immunity.This study investigates the pathogenesis of OmpA and the immunomodulatory of SCFAs to explore non-invasive treatment in regulating A.baumannii-induced inflammatory damage and its mechanism,providing a theoretical basis and new ideas for regulating lung inflammation in patients with acute lung injury（ALI）and acute respiratory distress syndrome（ARDS）.Objective:1.Analyze the relation between drug resistance-related genes and drug resistance phenotypes of clinical strains;2.To study A.baumannii virulence factor OMVs and its main outer membrane protein OmpA in inducing autophagy and inflammatory factor release in immune cells（THP-1 cells）,as well as its pathogenic mechanism;3.Comprehensive analyze A.baumannii induce inflammatory damage to immune cells（THP-1 cells）and epithelial cells（A549 cells）,and SCFAs（butyric acid,propionic acid and acetic acid）effects on inflammation,autophagy and its specific mechanism.Methods:1.Susceptibility identification of drug and drug resistance gene detectionK-B method and VITEK 2-Compact instrument method were used to detect the sensitivity of A.baumannii to clinically commonly used antibiotics.The PCR method was used to detect the carbapenemase-related genes of the collected A.baumannii strains,including class Aβ-lactamase genes(bla TEM、bla PER,bla CARB)and class B β-lactamase genes(bla IMP,bla VIM),Class Cβ-lactamase genes(bla ADC,bla DHA),Class Dβ-lactamase genes(bla OXA-23,bla OXA-24,bla OXA-51,bla OXA-58);active efflux pump related genes include ade B、ade J、mac B、emr B、emr A、abe S、abe M and cra A.2.Detection of cytotoxicity of OMVs and recombinant OmpA protein on THP-1 cellsProkaryotic expression technology to prepare and purify recombinant OmpA protein,immunize rabbits to prepare polyclonal antibodies to detect clinical strains,and establish rapid detection technology.Different concentrations of OMVs（5,50 μg/m L）or recombinant OmpA protein（1,10μg/m L）stimulated THP-1 cells for 1 h,3h,and 6 h,respectively.The expression of LC-3 and Beclin-1 tested by WB; qRT-PCR method was processed to analyse the mRNA level of NLRP3,Caspase-1and IL-1βin cells;Cytometry detects the production of reactive oxygen species in cells.3.Analysis of the influence of SCFAs on A.baumannii induced damage in THP-1 cells and A549 cellsBefore A.baumannii stimulates THP-1 cells or A549 cells,the cells were incubated with butyric acid,propionic acid and acetic acid for 24 h.LC-3,Beclin-1,inflammasomes related proteins NLRP3,Caspase-1（p20）;GSDMD（p30）and NF-κB p65 protein expression were analyzed like before;flow cytometry to detect cell reactive oxygen generation;qRT-PCR method to detect NLRP3 and Caspase-1 and immune balance factors IL-1β,IFN-γ,IL-4,IL-10,TGF-βand IL-17A.Results:1.The resistance rate of A.baumannii to imipenem is 83%;all carries the bla OXA-51gene;the carrying rate of bla OXA-23 gene in the imipenem resistance group is 98.3%,and not detected in the sensitive group（P<0.05）;the ade B gene carrying rate in resistant strains was 98.3%,and 41.7%in sensitive ones（P<0.05）.2.A.baumannii OMVs can induce THP-1 cell autophagy,reactive oxygen species release and NLRP3 inflammasome activation in vitro.With the increase of OMVs concentration and time,the quantity of LC3-Ⅱprotein,NLRP3 and Caspase-1 mRNA in THP-1 cells gradually up-regulated at 1 h and 3 h（P<0.05）;after 6 h infection,the activation of NLRP3 inflammation was inhibited（P<0.05）.3.Establish a rapid detection technology for A.baumannii OmpA based on PCR and Western blotting;at 3 h and 6 h,the quantity of LC3-Ⅱand Beclin-1 proteins in THP-1 cells were related with the concentration of recombinant OmpA protein.The10μg/m L recombinant OmpA protein up-regulated NLRP3 and Caspase-1 mRNA at3 h,and decreased rapidly at 6 h;while the reactive oxygen content began to decrease at 3 h.The rate of apoptosis showed a certain time and concentration dependence（P<0.05）.4.In THP-1 cells,NaB and NaPc significantly inhibited NLRP3 and Caspase-1mRNA levels and GSDMD（p30）expression,and the mRNA expression of TLR-2significantly decreased;pre-incubation of NaB could significantly increase LC3-Ⅱand Beclin-1 expressions;under the effect of SCFAs,the mRNA expression of IL-4,IL-10,TGF-βand IL-17A cytokines are up-regulated,and the mRNA expression of IL-1βand TNF-αis significantly inhibited;in addition,The three kinds of SCFAs all inhibited the nuclear effect of NF-κB p65 protein and the release of reactive oxygen species（P<0.05）.5.In A549 cells,the three SCFAs can down-regulate NLRP3 mRNA as well as the Caspase-1 p20 fragment;NaPc and NaB incubation can suppress NF-κB p65 protein enterence to nucleus,level-down Caspase-1 mRNA and increase LC3-Ⅱ;NaB can enhance the expression of Beclin-1,and at the same time significantly inhibit the expression of activated GSDMD p30 fragment and the release of ROS;three kinds of SCFAs pre-incubated,IL-1βand IL-6 mRNA expression Obviously inhibited,only NaB up-regulated the anti-inflammatory factor TGF-β（P<0.05）.Conclusion:The drug-resistance of A.baumannii in the sputum of patients with severe pneumonia in our hospital is severe,and the resistance to imipenem is as high as 83%,which requires clinical attention;Imipenem resistance of A.baumannii is closely related to the carrying of bla OXA-23gene and active efflux pump ade B gene.OmpA and OMVs are important virulence factors of A.baumannii,which can regulate the autophagy of THP-1 cells through the ROS/NLRP3 signaling pathway,activate NLRP3 inflammasomes,and induce mitochondrial damage to release reactive oxygen species.The enhancement of macrophage autophagy can inhibit the activation of NLRP3 inflammasome to a certain extent.In the inflammatory injury of A549 and THP-1 cells induced by A.baumannii,three SCFAs,especially NaB,can enhance the autophagy through the TLR-2/NF-κB/ROS/NLRP3 pathway to inhibit the activation of NLRP3 inflammasomes and cell"pyrolysis",as well as regulate the immune balance of Th1/Th2 and Th17/Treg. |
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