The Protective Effect Of Short Chain Fatty Acids On Klebsiella Pneumoniae Infection In Mice By Metabolic Receptor GPR43

ObjectiveThe model of Klebsiella pneumoniae pneumonia infection in mice was preliminarily established,and the protective effects of different types of short-chain fatty acids on mice were evaluated by this model,which laid a theoretical foundation for the treatment of Klebsiella pneumoniae pneumonia by short-chain fatty acids.To investigate the antibacterial activity of short-chain fatty acids against Klebsiella pneumoniae in vitro.The effects of short-chain fatty acids on macrophages in Klebsiella pneumoniae infection were investigated from the perspectives of phagocytosis and secretion of inflammatory factors.To further explore the role of the short-chain fatty acid receptor GPR43 on macrophages in Klebsiella pneumoniae pneumonia.Materials and MethodsBacterial strainsThe standard strain of Klebsiella pneumoniae ATCC 43816,was preserved by anhui provincial bacterial resistance monitoring center.Methods1. Mice can be divided into four groups,each group only 6-8,sterile water feeding respectively（negative control）,sodium acetate（150 m M）,sodium propionate（150m M）,sodium butyrate（150 m M）for five days,Klebsiella pneumoniae was infected by nasal drops 12 hours later,remove the mouse left and right pulmonary respectively,the left lung was placed in formalin for pathological examination,grinding coated plate of the upper lobe,in 37℃incubator 16 to 18 hours,record the number of bacterial colonies.The middle and lower lobes of the right lung were milled and the amount of inflammatory factors in the lung tissue was detected by flow cytometry.Mice pretreated with short-chain fatty acids for 5 days and untreated by nasal drip infection were recorded in real time within 7 days,and the survival curve of the mice was plotted.2.Klebsiella pneumoniae was placed in different concentrations of short-chain fatty acids to observe whether the growth rate of bacteria changed.In vitro with different concentration of short chain fatty acid RAW 264.7 cell incubation 12hours ahead of time,to the bacteria:the proportion of cells（100:1）to add CFSE labeled cells Klebsiella pneumoniae,collecting cells after 3 hours,by flow cytometry to test different cell average fluorescence intensity of short chain fatty acid treatment,then observe the changes of the bacteria can swallow a bite force;Mouse alveolar macrophages treated with 150m M for 5 days were collected and adherent cultured in DMEM medium.Alveolar macrophages were isolated and the changes in their phagocytosis ability were detected in the same way.RAW 264.7was stimulated by different concentrations of short-chain fatty acids and Klebsiella pneumoniae.The ratio of bacteria to cells was 10:1.Macrophages in the 6-well plate were collected after 6 hours of infection.3.Changes in GPR43 receptor expression on alveolar macrophages under stimulation of different concentrations of short-chain fatty acids and GPR43receptor expression on macrophages under co-stimulation of Klebsiella pneumoniae and short-chain fatty acids were detected.Gpr43^-/-cells were obtained by CRISPR/Cas9 gene knockout on macrophages,and CFSE-labeled Klebsiella pneumoniae was added to gpr43^-/-cells in the proportion of bacteria:cells（100:1）to observe the changes in their phagocytosis ability to bacteria.Sodium acetate,sodium propionate and sodium butyrate were incubated to observe their phagocytosis ability to bacteria.Klebsiella pneumoniae was added to gpr43knockout cells at a ratio of 10:1（bacteria:cells）.Cells were collected 6 hours later and cell RNA was extracted to detect m RNA expression levels of inflammatory cytokines IL-6,TNF-αand MCP-1.Results1. Mice pretreated with 150m M sodium acetate,sodium propionate and sodium butyrate had different survival curves after infection with Klebsiella pneumoniae compared with mice treated with oral sterile water.After 12 hours of Klebsiella pneumoniae infection,the bacterial count in the lung tissues of mice pretreated with sodium acetate,sodium propionate and sodium butyrate decreased,the cytokines IL-6,MCP-1 and TNF-αlevels decreased,and the pulmonary histological score significantly reduced.2. There was no difference in the growth curve of Klebsiella pneumoniae in sodium acetate,sodium propionate,sodium butyrate and PBS.Compared with untreated RAW 264.7 cells,RAW 264.7 cells pretreated with sodium acetate,sodium propionate,sodium butyrate showed increased average fluorescence after infection with CFSE-labeled Klebsiella pneumoniae.Compared with mice taking oral sterile water,alveolar macrophages in mice taking oral sodium acetate,sodium propionate,sodium butyrate showed increased fluorescence intensity after infection with CFSE-labeled Klebsiella pneumoniae.Compared with RAW 264.7 cells stimulated by Klebsiella pneumoniae alone,RAW 264.7 cells stimulated by sodium acetate,sodium propionate,sodium butyrate and Klebsiella pneumoniae alone showed increased m RNA expression of inflammatory cytokines IL-6,MCP-1 and TNF-α.3. Enhanced expression of GPR43 m RNA on RAW 264.7 cells incubated with sodium acetate,sodium propionate,sodium butyrate;RAW 264.7 cells were stimulated by Klebsiella pneumoniae with sodium acetate,sodium propionate,sodium butyrate.Compared with wild-type RAW 264.7 cells,the average fluorescence intensity of CFSE-labeled Klebsiella pneumoniae decreased after infection with gpr43^-/-cells.Compared with wild-type RAW 264.7 cells,gpr43^-/-cells showed decreased m RNA expression of inflammatory cytokines IL-6,MCP-1and TNF-αwhen stimulated by Klebsiella pneumoniae.Compared with Klebsiella pneumoniae alone,gpr43^-/-cells were stimulated by sodium acetate,sodium propionate,sodium butyrate in combination with Klebsiella pneumoniae,and m RNA expression of inflammatory cytokines IL-6,MCP-1 and TNF-αwere not significantly changed.Conclusions1.Oral SCFAs（sodium acetate,sodium propionate and sodium butyrate）reduced the bacterial count,cytokines IL-6,MCP-1 and TNF-αin the lung tissues of mice infected by Klebsiella pneumoniae.Oral administration of sodium acetate,sodium propionate and sodium butyrate reduced pulmonary histological scores and decreased mortality in infected mice.SCFAs had protective effects on mice infected with Klebsiella pneumoniae.2.SCFAs could not affect the growth of Klebsiella pneumoniae in vitro.SCFAs could enhance the phagocytosis ability of RAW 264.7 cells to Klebsiella pneumoniae.Oral administration of SCFAs can enhance the phagocytosis ability of alveolar macrophages to Klebsiella pneumoniae.SCFAs can enhance the expression of inflammatory cytokines IL-6,MCP-1 and TNF-αm RNA in macrophages infected by Klebsiella pneumoniae.3. SCFAs can enhance GPR43 expression on RAW 264.7 cells;RAW 264.7 cells were stimulated by Klebsiella pneumoniae and SCFAs,and GPR43 expression was significantly increased.The phagocytosis ability of gpr43^-/-cells to Klebsiella pneumoniae was decreased.After gpr43 gene was knocked out,the effect of SCFAs to enhance phagocytosis ability of macrophages disappeared.Gpr43 gene plays a regulatory role in the m RNA expression of inflammatory cytokines IL-6,MCP-1and TNF-α.The gpr43 gene plays a key role in the m RNA expression of inflammatory cytokines IL-6,MCP-1 and TNF-αin macrophages that enhance Klebsiella pneumoniae infection.