Explore Of The Anti-bacterial Activity Of Memantine Hydrochloride Against Pseudomonas Aeruginosa By Promoting Bactericidal Effect Of Neutrophils

[Background and objective]Pseudomonas aeruginosa is one of the main pathogens of nosocomial infection.The bacteremia caused by Pseudomonas aeruginosa maintains a high mortality rate due to the bacterial drug resistance.Traditional antibiotics usually target at the bacteria itself,therefore it is easy for bacteria to develop resistance to the antibiotics after long-term use.Drugs targeting host cells to block the invasion pathway of bacteria or activate the host immune system to indirectly suppress pathogens has become a new strategy for treatment of bacterial infection diseases.We have previously found that Memantine hydrochloride(MEM),a drug used to treat Alzheimer’s disease,has a good inhibitory effect on newborn mouse bacteremia and meningitis caused by Escherichia coli as well as its drug-resistant strains.However,whether the MEM could inhibit the infection casued by other pathogens,and the exact mechanism of sterilization is still unknown.Therefore,this study intends to explore whether MEM has bactericidal effect and its possible bactericidal mechanism during the infection of Pseudomonas aeruginosa or its drug-resistant strains.It probably provides a new insight for clinical treatment of Pseudomonas aeruginosa,especially the infection of its drug-resistant strains.[Methods]1.The bacterial growth curve was detected to study whether MEM could affect the growth of Pseudomonas aeruginosa and its drug-resistant strain.2.Neutrophils were isolated in vitro,and MEM,Pseudomonas aeruginosa and its drug-resistant strains were co-incubated with PMN to explore whether the drug could promote the sterilization ability of neutrophils.3.Neutrophils were isolated in vitro to explore whether the MEM has synergistic antibacterial effect with Amikacin.4.Western blotting analysis was used to detect the expression of NADPH oxidase gene after using MEM.5.Flow cytometry was used to analyze the promoting effect of MEM on the release of reactive oxygen species,and the effect of reactive oxygen species inhibitor N-acetyl-L-Cysteine(NAC)on the anti-infection effect of MEM was observed.6.Elastase assay kit and cell immunofluorescence were used to detect NETs formation after treatment with MEM.7.After the NADPH oxidase gene p67 phox was silenced,neutrophil germicidal test was conducted to determine whether the germicidal effect of MEM on Pseudomonas aeruginosa and its drug resistant bacteria was affected;flow cytometry was used to analyze whether the promoting effect of MEM on reactive oxygen species release was affected;elastase assay kit and cell immunofluorescence were used to detect whether the promoting effect of MEM on NETs formation was affected.8.After treatment with MEM,the blood,spleen,lung and liver specimens of mouse infection model were collected.Then the histopathology of the tissues was observed,and the bacterial colony count and blood inflammatory factors were detected.[Results]1.MEM has no direct bactericidal effect on pseudomonas aeruginosa and its drug-resistant strains.2.MEM could significantly promote the bactericidal action of neutrophils against pseudomonas aeruginosa and drug-resistant strains,while after the knockout of p67 phox gene,the bactericidal action of neutrophils induced by MEM was significantly weakened.3.The CI50 of MEM and Amikacin was less than 1,suggesting that they had synergistic antibacterial effect.4.Western blotting results showed that MEM could improve the expression of p67 phox gene compared with the control group.5.Flow cytometry results showed that MEM could increase the expression level of neutrophil reactive oxygen species,while after the deletion of p67 phox gene,the effect of MEM on the expression level of reactive oxygen species was significantly weakened.6.The expression of Elastase and myeloperoxidase increased after MEM treatment,indicating the formation of NETs.However,after the deletion of p67 phox gene,the effect of MEM on the improvement of elastase and myeloperoxidase was significantly weakened.7.In vivo experimental results showed that the expression level of IL-6 in the blood of the MEM group was lower than that of the untreated group,and the bacterial load in the blood and organs were also significantly lower than those of the untreated group.[Conclusion]MEM promotes the bactericidal ability of neutrophils against pseudomonas aeruginosa and its drug-resistant strains,which may be caused by promoting the expression of p67 phox gene to increase the expression level of reactive oxygen species of neutrophils,and promote the formation of NETs.In addition,MEM and Amikacin have a synergistic antibacterial effect.