Effects And Mechanisms Of MafK In Salmonella Infection

Salmonella is the most common foodborne pathogen with it substanial morbidity and mortality which can affect human and most animal species.Salmonellosis could cause huge losses in animal husbandry and human health.At present,antibiotics are mainly used for the treatment of salmonellosis which will bring bacterial resistance and medicine residue as well.Much safer and more efficiency treatment strategies are urgently needed for salmonellosis.The integrity of intestinal epithelial barrier is very important to defence the invasion of pathogens.V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog K(MafK),a member of the small Maf proteins in the Maf family.Studies have shown that the expression of MafK is increased in patients with UC,suggesting that MafK may play a role in UC.However,the role and mechanism of MafK in intestinal barrier,and whether it could promote or limit Salmonella infection by regulating intestinal barrier function has not been reported.In our study MafK overexpressed(MafK Tg)mice,wild type(WT)mice and Caco-2 cells infection models were constructed by Salmonella and were used to analyze the role of MafK in Salmonella typhimurium infected inflammation.The colitis model infected by Salmonella in WT mice and MafK Tg mice was used to analysis the effect of MafK overexpression in the susceptibility of host Salmonella infection.Following mucosal infection of Salmonella,MafK Tg mice displayed a significant higher mortality and more body weight loss.It was found that MafK Tg mice showed severe splenomegaly and cecal weight loss,and the histopathological score showed goblet cells lose,submucosal edema,polymorphonuclear leucocytes infiltration after Salmonella infected 48 h.The protein levels of proinflammatory mediators were examined by ELISA and western blot.It was found that MafK Tg mice showed more pro-inflammatory mediator production after Salmonella infected 48 h.The loads of Salmonella in cecum and mesenteric lymph nodes(MLN),liver and spleen at 48 h p.i.were evaluated.It was found the overexpression of MafK accelerated the colonization and dissemination of Salmonella.Collectively,the results suggested that the MafK overexpressed mice suffered a more severly Salmonella mucosal infection.To investigate whether MafK Tg mice suffered a more severly Salmonella mucosal infection through regulating the integrity of intestinal mucosal barrier function.The expression of tight junction proteins were examined in WT mice and MafK Tg mice by immunofluorescence,western blot and q-PCR.After Salmonella infection for 48 hours,the m RNA and protein expression of claudin3 in MafK Tg mice was decreased than in WT mice,while the m RNA level of occludin and ZO-1 had no difference.The expression of mucin2(MUC2)was examined in cecal by immunohistochemical.The results showed the expression of MUC2 in MafK Tg mice was significantly decrease than WT mice at 48 h p.i.The intestinal epithelial cells(IECs)proliferation and apoptosis were examined by immunofluorescence and TUNEL staining.The results showed that the proliferation of IECs MafK Tg mice didn't have significant difference between MafK Tg mice and WT mice at 48 h p.i.,while the apoptosis of IECs in MafK Tg mice presented significantly increased.MafK Tg mice had a higher level of cleaved caspase-3 expresssion than WT mice at 48 h p.i.,that's maybe the reason why MafK Tg mice suffered excessive inflammation.Before obvious inflammation and tissue-associated pathologic changes were observed in mice,the expresssion of cleaved caspase-3 was further confirmed.The results showed that MafK Tg mice had increased level of cleaved caspase-3 expresssion than WT mice at 6 h p.i.The intestinal flora was detected by high-throughput sequencing in WT and MafK Tg mice.The results showed that there was no significant difference in the diversity of intestinal flora between WT and MafK Tg mice.These results indicated that MafK may affect the function of intestinal barrier through regulate the apoptosis of IECs.The mechanism of MafK in regulate the function of intestinal barrier was further researched in vitro.The nuclear translocation of NF-?B p65,interactions between MafK and NF-?B p65 and the expression of cleaved caspase-3 were examined by western blot and CO-IP in MafK overexpressed Caco-2 cells and si RNA interrupted MafK Caco-2 cells during Salmonella infection.It was found that the nuclear translocation of NF-?B p65 was increased in MafK overexpressed Caco-2 cells during Salmonella infection.MafK bound more NF-?B p65 in MafK overexpressed Caco-2 cells during Salmonella infection.And the expression of cleaved caspase-3 was significantly increased in MafK overexpressed Caco-2 cells during Salmonella infection.On the other hand,when MafK was interrupted in Caco-2 cells,the nuclear translocation of NF-?B p65,interactions between MafK and NF-?B p65 and the expression of cleaved caspase-3 were decreased in MafK interrupted Caco-2 cells.These results suggested that MafK could promote the entry of p65,and increase increasing the activation of caspase-3,induce the apoptosis of intestinal epithelial cells.Collectively,theses results indicated MafK disrupts the intestinal barrier by promoting apoptosis of intestinal epithelial cells through NF-?B p65 pathway and the activation of caspase-3.Thus leading MafK overexpressed mice more susceptible to inflammation induced by Salmonella.That will provide a target for the prevention and treatment of Salmonellosis.