Mechanisms Of Resistin-and P38 MAPK/Wnt Pathway-mediated Cell Damage Caused By Haemophilus Parasuis

Haemophilus parasuis(HPS)causes acute systemic inflammation and sudden death in pig,which threatens the stability and productivity of the pig industry worldwide.HPS leads to the Gl(?)sser's disease in piglets characterized by a mass of fibrinous exudation in the pleuroperitoneal cavity.However,the molecular mechanism of Gl(?)sser's disease is still unclear.In this study,to investigate the mechanism of endothelial cell dysfunction in exudative inflammation caused by HPS,we tested the expression and secretion level of resistin in 3D4/21 cells and explored the influences of resistin on the tight junction of co-cultured porcine vascular endothelial cell(PVEC).Moreover,we have also analysis the epithelial-mesenchymal transition(EMT)occurring in PK-15 and NPTr cells to reveal the damage of epithelial cells during HPS infection.The mean results were as follow:1.Secretion of resistin by 3D4/21 cells destroyed the tight junction of PVEC during HPS infectionIn the present study,the transcription factor Ets2 was proved to regulate the expression of resistin in 3D4/21 cells during HPS infection.Moreover,inhibition of p38 MAPK pathway decreased the expression of Ets2 and resistin,which demonstrated that p38 MAPK pathway activated the expression and secretion of resistin via mediating the expression of Ets2 gene in 3D4/21 cells during HPS infection.The results of transendothelial electrical resistance(TEER)experiment showed that knockdown of resistin gene in 3D4/21 cells recovered the monolayer PVEC permeability induced by HPS infection,which confirmed that resistin could increase monolayer PVEC permeability during HPS infection.In addition,resistin inhibited the expression of claudin-5 and occludin as well as increased monolayer PVEC permeability by LKB1/AMPK/mTOR pathway.2.Out membrane lipoprotein LPPA of HPS regulated the expression of resistin and the disruption of PVEC tight junctionThe results of TEER test showed that only high-virulence HPS strain,not non-virulence HPS strain,could increase the secretion of resistin and the monolayer permeability of PVEC.We have screened out 32 genes that were significantly distributed in high-virulence strains and demonstrated that out membrane lipoprotein lppA gene played an important part in regulating the expression of claudin-5 and occludin.Compared gene-deletion strain ?lppA and complement strain C-lppA,with wild type HPS-SH0165 strain,the results shown that LPPA of HPS regulated the expression of resistin via inducing p38 MAPK/ Ets2 activation.At the same time,LPPA of HPS regulated the activation of LKB1/AMPK/mTOR pathway to influence the tight junction and permeability of PVEC.3.P38 MAPK pathway regulated the EMT which was initialized by Wnt/?-catenin pathway in epithelial cells during HPS infectionThe results of this study showed that Wnt/?-catenin signaling pathway,as well as the target genes MMP7,COX2 and PAI-1,contributed to the initiation of EMT during HPS infection.Moreover,in the present study,p38 MAPK was found to be involved in the activation of Wnt/?-catenin pathway during HPS infection in PK-15 and NPTr cells.Knockout of dickkofp-1(DKK-1),a negative regulator of Wnt/?-catenin signaling,led to the dysfunction of p38 MAPK in regulating Wnt/?-catenin signaling activity in HPS-infected PK-15 and NPTr cells.In addition,the inhibition of p38 MAPK decreased the activation of Wnt/?-catenin pathway and recovered the EMT process induced by HPS.These results indicated that p38 MAPK pathway regulated the EMT process which was initialized by Wnt/?-catenin pathway in epithelial cells during HPS infection.In summary,the present study firstly confirmed that secretion of resistin induced by HPS in 3D4/21 cells destroyed the tight junction of PVEC,at the same time,p38 MAPK and Wnt/?-catenin pathway regulated the EMT process initialized by HPS in epithelial cells.Dysfunction of epithelial and endothelial cells might contribute to the exudative inflammation caused by HPS.These results provided a preliminary theoretical framework for the pathogenesis of Gl(?)sser's disease caused by HPS.