| Staphylococcus aureus(S.aureus)is an important zoonotic pathogen.The drug resistance of S.aureus is becoming more and more serious,which has become a public health problem of global concern.As the main virulence factor of S.aureus infection,?-hemolysin(Hl?,?-hla)is currently the most studied and the most virulent of S.aureus virulence factors,which can cause a variety of host cell damage.In addition,Hl? can be transported by the membrane vesicles(SMVs)secreted by S.aureus,which is also responsible for the diffusion of ?-hemolysin.In this study,in order to investigate the pathogenic mechanism of S.aureus Hl?,we will first explore the correlation between the secretion of SMVs of S.aureus carrying Hl? and the expression of Hl?,which will lay the foundation for our subsequent discovery of drug against the inflammation caused by S.aureus Hl?.Macrophages,the important immune effector cell,as the body's first line of defense against S.aureus pathogens,when S.aureus invasion of the body,most of macrophages react quickly to counteract the invaders.Mitogen-activated protein kinases(MAPKs)are involved in a variety of biological processes,including cell proliferation and division,and regulate the inflammatory and immune responses of macrophages.It has been reported that S.aureus can induce cell death by relying on the NLRP3-dependent cell necrosis program,leading to the release of endogenous pro-inflammatory molecules,but the specific mechanism is unclear.Intracellular activation of NF-?B and related factors is an important mechanism for the regulation of inflammatory response.In summary,MAPKs,NF-?B and NLRP3 pathways are significantly correlated with inflammation.Therefore,the mechanism of MAPKs,NF-?B and NLRP3 in S.aureus Hl?-induced inflammation will also be explored.Combination therapy is an effective strategy for the treatment of complex bacterial infections.In this study,in order to solve the problem of stubborn S.aureus and its ?-hemolysin-induced inflammation,a combined therapy method was proposed.Among the many antimicrobial drugs,Clozacillin(CXN)is a ?-lactam drug that has a strong bactericidal effect against drug-resistant S.aureus,it is known as "Staphylococcal penicillin" and is often used to treat Staphylococcal infections in animals.Tetracycline(TC)is a kind of broad spectrum antimicrobial agent and has been reported to be an autolytic inhibitor.Bacterial autolysis is the self-destructive behavior of bacterial cells.The lysosome releases the enzyme to degrade its cells.Tetracycline can significantly reduce the occurrence of bacteria autolysis,and its inhibition ability to autolysin is worth exploring.Efflux pumps is one of the important mechanisms of bacterial drug resistance.Thioridazine(TZ)is a clinical drug used in the treatment of anxiety disorders,recent studies have shown that thioridazine is also a kind of efflux pump inhibitors have been used for treatment of Mycobacterium tuberculosis infection in clinical trials.So,it is of great significance to investigate whether thioridazine can be served as S.aureus efflux pump inhibitor.In this way,we use three drugs to form a "three-drug combinations" resistance of S.aureus Hl? cause inflammation.We used cloxacillin,an anti-Staphylococcal drug,tetracycline,an autolysis agent,and thioridazine,an efflux pump inhibitor as components of three drugs.The purpose is to check whether the three-drug combinations can inhibit S.aureus,inhibit S.aureus Hl? production,inhibit the expression of S.aureus Hl? and its hemolytic activity,and inhibit the inflammatory response induced by S.aureus Hl? in vitro and in vivo.At the same time,it was also necessary to clarify whether S.aureus Hl? induced inflammation was mediated by the MAPKs/NF-?B/NLRP3 pathway,and whether the three-drug combinations we designed can inhibit the mechanism-mediated inflammation.Firstly,we examined the activity of the three-drug combinations against S.aureus,as well as the inhibition of the Hl? expression,Hl? hemolytic activity,and SMVs secretion of S.aureus carrying Hl?.On the one hand,in order to determine the in vitro test concentration of the three-drug combinations,we used the antimicrobial drug sensitivity test and the checkerboard dilution method to conduct the combined drug sensitivity test.We selected cloxacillin(0.015625 ?g/m L),thiridazine(0.25 ?g/m L)and tetracycline(0.03125 ?g/m L)as the optimal concentrations for all subsequent in vitro experiments when the three-drug combinations had a synergic effect.At this time,the mass/volume concentration ratio(or mass ratio)of cloxacillin,thiridazine and tetracycline was 1:16:2.On the other hand,we showed that the three-drug combinations significantly inhibited the expression of Hl? of S.aureus with the help of Western blot.the analysis of blood plate hemolysis and microplate reader showed that the optimal concentration of the three-drug combinations could significantly inhibit the activity of Hl? of S.aureus.Scanning electron microscopy was used to observe that the three-drug combinations significantly inhibited S.aureus secreting SMVs carrying Hl?.These results together show that the optimal concentration of the three-drug combinations inhibits the secretion of SMVs carrying Hl? by S.aureus,reduces the expression of Hl?,and thereby suppresses the activity of Hl?.Secondly,we further examined the inhibitory ability and possible mechanism of the subinhibitory three-drug combinations on the activation of macrophage-related inflammatory factors by S.aureus Hl?.We adopt Western blot technique of S.aureus 8325-4(normal ?-hemolysin gene hl?)strain and S.aureus DU1090 strain(?-hemolysin gene hl? deletion mutant)role of macrophage inflammatory protein MAPKs pathway(p-ERK,p-JNK and p-p38),NF-?B pathway(NF-?B,I?B,and IKK),inflammatory corpuscle NLRP3 pathways(ASC,Caspase-1,IL-18 and IL-1?)expression is analyzed.To further explore the upstream and downstream relationship between MAPKs pathway,NF-?B pathway and NLRP3 inflammatory bodies,we used various pathway inhibitor treatments.Results showed that S.aureus ?-hemolysin gene normal strain 8325-4 four treatment groups than DU1090 macrophages MAPKs group and the blank group,the NF-?B,NLRP3 pathways related pathways protein expression showed different degrees of the volume increase,and the optimal concentration of the three-drug combinations treatment can significantly down-regulate MAPKs pathway(p-ERK,p-JNK and p-p38),NF-?B pathway(NF-?B and IKK),inflammatory corpuscle NLRP3 pathways(ASC,Caspase-1,IL-18 and IL-1?)protein expression.In addition,MAPKs path-specific inhibitors SP600125(JNK inhibitor),PD98059(ERK inhibitor)and SB203580(P38 inhibitor)can significantly inhibit the expression of NF-?B pathway proteins and NLRP3 inflammatory body proteins,indicating that MAPKs are located in the upstream of NF-?B and NLRP3.Bay11-7082(NF-?B inhibitor)can significantly block the expression of NLRP3 inflammatory body proteins,but cannot inhibit the phosphorylation of MAPKs pathway proteins,indicating that NF-?B is located in the upstream of NLRP3.However,ac-yvad-cmk(Caspase-1 inhibitor)has no inhibitory effect on other pathways.In summary,the results showed that the optimal concentration of the three-drug combinations plays an anti-inflammatory role by inhibiting activation of the MAPKs/NF-?B/NLRP3 inflammatory pathway induced by Hl?,MAPKs were located in the upstream of NF-?B and NLRP3,NF-?B is located upstream of NLRP3 inflammation.Finally,we established a model of peritonitis in mice,and investigated the protective effect of the three-drug combinations [cloxacillin(1.6125 mg/kg)+ thiolidazine(25 mg/kg)+ tetracycline(3.125 mg/kg)] on peritonitis in mice caused by S.aureus infection in vivo.The results showed that the inflammatory pathological changes caused by S.aureus DU1090 were weaker than those of the 8325-4 strain.The three-drug combinations could significantly reduce the number of colonies infected by S.aureus in the mouse peritonitis model group,and also reduce the inflammatory pathological changes and inflammatory cytokines(Such as IL-1? and TNF-?),and inhibit the expression of MAPKs/NF-?B/NLRP3 pathway-related proteins in peritoneal macrophages.In summary,the results of the study indicated that the three-drug combinations has protective effects on mice peritonitis caused by S.aureus infection.In conclusion,this study clarifies that the three-drug combinations(cloxacillin,thioridazine and tetracycline)has good anti-S.aureus activity,can significantly inhibit the expression of S.aureus Hl? and its hemolytic activity,and inhibit the carrying of ?-hemolysis SMVs are formed,and can inhibit MAPKs/NF-?B/NLRP3 pathway to reduce S.aureus Hl? cause inflammation.It is a new drug formula against S.aureus infection,which lays a solid theoretical and material foundation for effective treatment of S.aureus infection. |
PDF Full Text Request