Study On Murine Peritoneal Macrophages Inflammation Induced By Vibrio Harveyi Via Regulating P38 MAPK-NF-κB-NLRP3 Inflammasome Signaling Axis

Vibrio harveyi(V.harveyi)is a common marine pathogenic Vibrio that can infect marine fish,shrimp and humans.The main routes of V.harveyi infection include eating raw or uncooked aquaculture fish and shrimp,or directly contacting with seawater.A series of severe inflammatory responses will occur after infection,which seriously endanger human health.At present,the main treatment for V.harveyi infection is traditional antibiotic therapy.However,more and more evidence showed that the abuse of antibiotics will cause drug-resistant strains,which are not conducive to subsequent treatment.Innate immunity is the first line of defense against pathogen invasion.When pathogens invade the host,they will activate the pattern recognition receptors on immune cells,and then activate the downstream inflammatory-related signaling pathways to induce the secretion of inflammatory cytokines,and recruit more immune cells to reach the lesion to remove pathogens.However,the molecular mechanism of V.harveyi-induced inflammatory response remains unclear.In this study,a murine peritoneal macrophages(PMs)model infected with V.harveyi was successfully constructed,and the m RNA levels of the pattern recognition receptors Tlr2 and Nlrp3 were determined to be the most significant in V.harveyi-infected murine PMs.The role of TLR2,MAPK,NF-κB and AKT signaling pathways in V.harveyi-induced inflammation was studied by TLR2-deficient mice and inhibitor experiments.The role of NLRP3 inflammasome in V.harveyi-infected murine PMs was studied by detecting IL-1β production,Caspase1 activation,NLRP3 and ASC protein expression and using inflammasome-related pathway inhibitors.The molecular mechanism of murine PMs inflammation induced by V.harveyi was studied in order to provide a new therapeutic target for the prevention and treatment of diseases caused by V.harveyi infection.The results of this study are as follows:Establishment of murine PMs model infected with V.harveyi.The m RNA levels of inflammatory cytokines related genes were detected by q PCR.It was found that V.harveyi could up-regulate the m RNA levels of inflammatory cytokines Il-6,Il-10,Il-18,Il-1β,Tnf-α and Ifn-γ,and the m RNA levels of Il-1β were the most obvious.ELISA was used to detect the secretion of inflammatory cytokines and found that V.harveyi promoted the secretion of inflammatory cytokines IL-1β,IL-6,IL-12 and TNF-α,indicating that V.harveyi induced inflammatory response in murine PMs,which laid the foundation for the subsequent study of the molecular mechanism of V.harveyi infection.Study on the activation of p38 MAPK,NF-κB and AKT signaling pathways in V.harveyi-infected murine PMs.The role of TLR2,p38 MAPK,NF-κB and AKT signaling pathways in V.harveyi infection was investigated by using the model of TLR2-deficient murine and wild-type murine PMs infected with V.harveyi and the inhibitors of MAPK,AKT and NF-κB signaling pathway.The m RNA levels of Tlrs and inflammatory cytokines IL-1β,IL-6,IL-12 and Tnf-α were detected by q PCR,and the expression levels of phosphorylated proteins p38,ERK,JNK,AKT,p65,IKKα/β and total proteins p38,ERK,JNK,AKT,IκBα and IKKβwere detected by Western Blotting.The secretion levels of inflammatory cytokines IL-1β,IL-6,IL-12 and TNF-α were determined by ELISA,and the localization of NF-κB p65 protein in PMs of V.harveyi-infected murine was analyzed by immunofluorescence.The results showed that V.harveyi up-regulated the m RNA levels of Tlr1,Tlr2,Tlr3,Tlr5,Tlr6,Tlr7,Tlr8,Tlr9,Tlr11,Tlr12 and Tlr13,and the m RNA levels of Tlr2 were significantly up-regulated.Meanwhile,V.harveyi can activate the expression of phosphorylated proteins p38,ERK,JNK,AKT,p65 and IKKα/β,promote the secretion of inflammatory cytokines IL-1β,IL-6,IL-12 and TNF-α,and induce the translocation of NF-κB p65 protein to the nucleus.These responses were significantly inhibited in the PMs of TLR2-deficient murine.The secretion of inflammatory cytokines IL-1β,IL-6,IL-12 and TNF-α was blocked by MAPK,AKT and NF-κB signaling pathway related inhibitors.In conclusion,V.harveyi regulates the production of pro-inflammatory cytokines in murine PMs through activation of p38 MAPK,AKT and NF-κB signaling pathway in a partially TLR2-dependent manner,with the AKT signaling pathway having a negative regulatory effect.The activation of p38 MAPK and NF-κB signaling pathway can be used as the first signal of inflammasome activation,laying a foundation for further study of NLRP3 signaling pathway activated by V.harveyi.Study on the activation of NLRP3 inflammasome by V.harveyi.The m RNA levels of IL-1βin V.harveyi is up-regulated by activating p38 MAPK and NF-κB signaling pathways,but the secretion of IL-1β may also be involved in the activation of inflammasome.In this study,the role of NLRP3 inflammasome in V.harveyi infection was investigated using a murine PMs model infected with V.harveyi,as well as pan-Caspase inhibitors and NLRP3 inflammasome signaling pathway related inhibitors.The expression levels of NLRP3,ASC,GSDMD,pro-Caspase1,pro-IL-1β,Caspase1 p20 and IL-1β p17 were detected by Western Blotting,and the m RNA levels of Nlrs,Caspase1 and Il-1β were detected by q PCR.The secretion levels of inflammatory cytokines IL-1β,IL-6,IL-12 and TNF-α were detected by ELISA,and the localization of NLRP3 and ASC in murine PMs were analyzed by immunofluorescence.The results showed that V.harveyi could promote the expression of NLRP3,pro-Caspase1,pro-IL-1β,Caspase1 p20 and IL-1β p17,and induce ASC oligomerization and GSDMD-NT production.The m RNA levels of Nod1,Nod2,Nlrp1,Nlrp2,Nlrp3,Nlrp6,Nlrc4,Nlrc5,Caspase1 and Il-1β were upregulated by V.harveyi,and the m RNA levels of Nlrp3 in Nlrs were significantly upregulated.V.harveyi promotes the secretion of inflammatory cytokines IL-1β,IL-6,IL-12 and TNF-α.In V.harveyi-infected murine PMs,puncta-like NLRP3 surrounding the nucleus and ASC specks in the nucleus and cytoplasm.Pan-caspase inhibitors and NLRP3 inflammasome signaling pathway-related inhibitors block IL-1β p17 production and secretion of inflammatory cytokines IL-1β,IL-6,IL-12,and TNF-α.In conclusion,V.harveyi participates in the inflammatory response of murine PMs through activation of NLRP3 inflammasome,activation of Caspase1 and induction of IL-1β secretion in a time-dependent manner.In conclusion,the present study is the first to establish a murine PMs model infected with V.harveyi.It was found that V.harveyi can induce inflammatory responses through activation of p38 MAPK and NF-κB signaling pathways in a partially TLR2-dependent manner,and provide the first signal of NLRP3 inflammasome activation.Meanwhile,V.harveyi can also activate the second signal of NLRP3 inflammasome,activate Caspase1,promote the secretion of IL-1β,and then induce inflammatory response.This study elucidated the molecular mechanism of inflammation induced by V.harveyi in murine PMs,providing reference for prevention and treatment of murine PMs infection.There are 35 figures,14 tables and 170 references in this thesis.