| Objective:Acinetobacter baumannii?A.baumannii?is a major opportunistic pathogen in hospitals,which poses a great threat to immunocompromised patients,especially in intensive care units.Due to multi-and pan-drug resistance,A.baumannii infection is a major concern worldwide due to the high mortality rate of 35%.Omp A is the main virulence factor,Previous studies have shown that Omp A affects the biofilm formation of A.baumannii,the adhesion to eukaryotic cells,and early inflammatory damage.Therefore,the pathogenic mechanism of Omp A may be a key factor in controlling A.baumannii infection.Host innate immunity system is the first defense of the body against bacterial infection,the pathogenic mechanism of Omp A was correlation with the host immunity may provide a potential immunomodulatory target.Methods:Part one:A.baumannii relied on the Omp A to trigger NLRP3inflammasome?1?Construction of Omp A knockout/supplement mutant strain by homologous recombination and the product was verified by sequencing,polymerase chain reaction?PCR?,and Western blot.?2?The cells were seeded in 6-well plates at a density of 5×104cells/well and cultured until 60–70%confluency and randomly divided into four groups:control cells group?normal alveolar epithelial cells were cultured and added 0.9%saline equivalent to the medium?,cells+Omp A-/-group?Intervention with knockout Omp A strain,multiplicity of infection,MOI=1?,cells+Omp A group?Intervention with replenishment Omp A strain,MOI=1?,cells+WT group?Intervention with Wild type bacteria,MOI=1?,persistent infection for 3 h,the cells protein and RNA was extracted for subsequent detection,The protein levels of IL-1?,IL-18,ASC,Caspase-1P10/P20 were identified by western blot,q RT-PCR were used to detect the m RNA expressions of NLRP3?NOD-Like receptor protein 3,NLRP3?,IL-1?,Caspase-1.?3?The trachea of each rat was inoculated with 0.4 m L?5×108CFU/m L,OD600was about 0.1?of bacterial suspension to establish the pneumonia models,the lung pathological changes were observed by HE staining,MPO and wet/dry specific gravity of lung tissue were evaluated for inflammatory injury,Immunohistochemical staining and Western blot were performed to observe the expression of IL-1?,IL-18,ASC,Caspase-1P10/P20 protein in lung tissue.q RT-PCR were used to detect the NLRP3,IL-1?,Caspase-1 m RNA expressions and ELISA were used to detect the IL-1?,IL-18 expressions.All methods are for detection whether the NLRP3inflammation pathway would be affected after the mutation of Omp A.The recruitment rate of neutrophils after infection reflects the severity of inflammation,which was assessed by flow cytometry of CD11b/c+Ly-6G double staining.Part two:Effects of A.baumannii infection by knock down the expression of NLRP3-ASC-Caspase-1 inflammatory corresponding proteinsThe cells were seeded in 6-well plates at a density of 5×104cells/well and cultured until 60–70%confluency,negative control plasmid?Negative?,NLRP3,ASC and Caspase-1 knockdown plasmid?NLRP3/ASC/Caspase-1-sh RNA?were transfected with 3?g plasmid and 9?l PEI/well.After transfection 6-8h,changed medium containing 2%FBS and continued culturing to 45h and randomly divided into four groups:control cells group?normal alveolar epithelial cells?,cells+knock down plasmid+Omp A-/-group?After transfection,intervention with knockout Omp A strain,MOI=1?,cells+knockdown plasmid+WT group?After transfection,intervention with wild type bacteria,MOI=1?,cells+negative control plasmid+WT group?After transfection,intervention with wild type bacteria,MOI=1?,persistent infection for 3 h,we collected protein and RNA samples after 48 hours.Western blot and q RT-PCR were used to detect the protein and m RNA expressions to evaluate the pathogenic ability of A.baumannii.Part three:Effect of MG-132 inhibiting the degradation of proteasome on A.baumannii infection?1?The cells were seeded in 6-well plates at a density of 5×104cells/well and cultured until 60–70%confluency and randomly divided into three groups:control cell group?normal alveolar epithelial cells?,cells+Omp A-/-group?intervention with knockout Omp A strain,added the same amount of 0.9%saline as MG-132 to intervene,MOI=1?,cells+Omp A-/-group+MG-132?intervention with knockout Omp A strain,MOI=1,protease inhibitor MG-132 was added at 10 m M for 3 h?.Western blot were performed to observe the expression of IL-1?,IL-18,ASC,Caspase-1P10/P20and q RT-PCR were used to detect the NLRP3,IL-1?,Caspase-1 m RNA expressions to explore the effect of MG-132-blocked proteasome degradation on bacteria-induced inflammation.?2?Then replace the infected strain with WT bacterial solution to construct an in vitro infection model,groups as before,persistent infection for 3 h,Western blot were performed to observe the expression of IL-1?,IL-18,ASC,Caspase-1P10/P20 and q RT-PCR were used to detect the NLRP3,IL-1?,Caspase-1 m RNA expressions to explore the effect of MG-132 on WT A.baumannii infection and infer the relationship between Omp A and Caspase-1.?3?The trachea of each rat was inoculated with 0.4 m L?5×108CFU/m L,OD600 was about 0.1?of bacterial suspension to establish the pneumonia models,the experimental animals were randomly divided into three groups:the control group?the trachea was slowly injected with 0.4 m L of 0.9%saline solution?,the knock-out Omp A(Omp A-/-)model group?the bacterial suspension was slowly injected into the same part of the trachea,a positive control group was constructed by continuous intraperitoneal injection of 0.4 ml of 0.9%saline for 3 days?,the MG-132 model group[the knock-out Omp A(Omp A-/-)model group(the bacterial suspension was slowly injected into the same part of the trachea,followed by immediate injection of MG-132?0.1mg/kg/d?intraperitoneal continuously for 72 h].Similarly,replace the infected strain with WT A.baumannii bacterial solution to construct an infection model.After the model construction was completed,the animals were processed according to the requirements of the ethics committee of Guizhou Medical University.The lung pathological changes were observed by HE staining,MPO and wet/dry specific gravity of lung tissue were evaluated for inflammatory injury,Immunohistochemical staining and Western blot were performed to observe the expression of IL-1?,IL-18,ASC,Caspase-1P10/P20,K48/K63/PD41 protein in lung tissue.q RT-PCR were used to detect the NLRP3,IL-1?,Caspase-1 m RNA expressions and ELISA were used to detect the IL-1?,IL-18 expressions.All methods are for detection the relationship between MG-132 and NLRP3 inflammatosome activation and the possible role of Omp A.Results:Part one:A.baumannii relied on the Omp A to trigger NLRP3inflammasome?1?Successful construction of Omp A mutant strains:The results of sequencing,PCR and Western blot showed that the homologous recombination method successfully constructed knockout/replacing mutant strains.?2?In vitro,Omp A enhanced the activation of NLRP3 inflammasome after A.baumannii infection:the expression of NLRP3 inflammasome-associated proteins was increased in all infection models?p<0.05?,especially in the Omp A and WT groups,however,albeit not significantly difference between the two groups?p>0.05?.The expression of NLRP3,IL-1?,Caspase-1 gene were measured by q RT-PCR and was found to be consistent with the protein expression.?3?The activation of NLRP3 inflammasome was consistent with in vivo and vitro:Rat pneumonia models were successfully constructed,the pathological tests of the lung tissues showed that diffuse interstitial inflammation was progressively aggravated,there is not significantly difference between in the Omp A and WT groups.In all infection groups,the MPO and lung tissue wet/dry ratios were significantly higher than the control group?p<0.05?,compared with the knockout group,the ratio of the supplementary and WT groups increased more significantly?p<0.05?.The flow cytometry of CD11b/c+Ly-6G dual expression showed the recruitment ability of neutrophils.After knockout of Omp A,the recruitment rate of neutrophil was 43.88%,indicating that the virulence of A.baumannii was weakened as compared to 83.32%of the WT group.After supplementation of Omp A gene,the recruitment ability of the neutrophils was increased to 73.6%,but not significantly as compared to the WT group?p>0.05?.The production of IL-1?and IL-18 was significantly increased with Omp A stimulation,as assessed by ELISA?p<0.05?.The expression of NLRP3inflammasome-associated proteins was markedly increased in the Omp A and WT groups?p<0.05?,however,no difference was observed between the Omp A and WT groups?p>0.05?,These results were consistent with those in vitro.Part two:Effects of A.baumannii infection by knock down the expression of NLRP3-ASC-Caspase-1 inflammatory corresponding proteins?1?After specifically knocking down the expression of NLRP3 and Caspas e-1,the expression of NLRP3 inflammatory corresponding proteins and genes i n the knockdown group was significantly lower than that in the negative contr ol group?p<0.05?.The expression of NLRP3 inflammatory body-related protei ns and genes in the Omp A-/-strain-infected group was lower than that in the wild-type strain-infected group?p<0.05?.?2?After specifically knocking down the expression of ASC,the expressio n levels of NLRP3 inflammatory corresponding proteins and gene m RNA in th e knockdown strain infection group and the knockdown wild type strain infecti on group were small?p>0.05?.The difference between the group and the neg ative control was not statistically significant?p>0.05?.Part three:Effect of MG-132 inhibiting the degradation of proteasome on A.baumannii infection?1?The in vivo/in vitro infection models constructed by the knockout strains showed that the expression of Caspase-1m RNA in each group did not change much after treatment with MG-132,that is the synthesis of Caspase-1m RNA did not increase?p>0.05?,but Caspase-1P10/P20 Protein expression was significantly increased?p<0.05?,this phenomenon may be caused by MG-132 inhibiting the degradation of Caspase-1 by proteasome.The pathological results of the in vivo model showed that the inflammation damage increased after MG-132 intervention,and the other related proteins and ubiquitin protein PD41/K48/K63 and IL-1?detected by ELISA in the MG-132 intervention model group in vivo/in vitro.The expression levels of IL-18,NLRP3 and IL-1?m RNA were significantly higher than those of the other two groups?p<0.05?;the ratio of MPO and tissue wet/dry specific gravity of MG-132 intervention model group in vivo was consistent with its protein expression results.?2?The WT strain was successfully replicated to construct an in vivo/in vitro infection model.The pathological results of the in vivo model increased after MG-132intervention.The MG-132 intervention group NLRP3 inflammation body other related proteins and ubiquitin protein PD41/K48/K63 and ELISA The detected IL-1?,IL-18,NLRP3 and IL-1?m RNA expression levels were significantly higher than those of the other two groups?p<0.05?.In vivo MG-132 intervention model group MPO and tissue wet/dry specific gravity ratio and its protein etc.The results of expression were consistent?p<0.05?,but there was no significant change in the synthesis of Caspase-1 m RNA?p<0.05?.Omp A relied on the Caspase-1 pathway to aggravate the damage to the body.Conclusions:?1?A.baumannii infection activates NLRP3 inflammasome through the Omp A to cause inflammatory damage,and may be related to the NLRP3/Caspase-1 pathway.?2?MG-132 can aggravate the inflammatory damage caused by A.baumannii infection by inhibiting the degradation of Caspase-1,to provide a new therapeutic target for A.baumannii infection.. |
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