Efficacy And Mechanism Of Bacteroides Fragilis On The Prevention And Treatment Of Cronobacter Sakazakii-induced Necrotizing Enterocolitis

1.BackgroundCronbacter sakazakil(CS)is a gram-negative,anaerobic,sporeless bacterium,which leads to necrotizing enterocolitis(NEC)in infant.CS plays multi-faceted roles in the pathogenesis of NEC including microbial dysbiosis,gut mucosal barrier dysfunction and increased cell death.Probiotics is well-known for its roles in hindering the growth of pathogenic microbiota,maintaining the balance of gut microbiota and promoting the development of intestinal mucosa and immune cells.It has become an A-list in the prevention and treatment of NEC.In the previous study,we separate a non-toxic Bacaeroidesfragilis(ZY-312)from baby feces,as next-generation probiotics for the modulation of intestinal inflammatory diseases and diarrhea.This study will investigate whether ZY-312 could prevent or control the occurrence of NEC induced by CS and to elucidate the underlying mechanism.2.Methods(1)Frist of all,an in vitro intestinal epithelial model based on human colon adenocarcinoma cell line Caco-2 was established to assess whether ZY-312could inhibit the damage by CS and act as a protective factor.(2)We built a neonatal rat model based on "CS colonization in intestinal mucosa-disruption of gut mucosa barrier functions-trigger of NEC".We used ZY-312 to intervene and evaluate the effect of ZY-312 on preventing NEC in this unique model.(3)The mechanism of ZY-312 to prevent and control of NEC induced by CS was explored:1)To clarify the effect of ZY-312 on intestinal barrier in mice,2)To detect the regulation of ZY-312 on inflammasome NLRP3 and pyroptosis in intestinal tissue of mice,3)to validate the in vivo relevance of apoptosis inhibited by ZY-312.3.Results(1)In vitro studies,it showed that ZY-312 could inhibit CS degradation of Caco-2 secreted mucin,inhibit CS adhesion and invasion,and alleviate the destruction of CS on tight junction proteins of intestinal epithelial cells.(2)In vivo studies,it showed that ZY-312 can effectively prevent NEC from CS infection in neonatal rats and reduce mortality.(3)The mechanism of ZY-312 prevention and control of CS induced NEC was as follows:1)Enhancement of intestinal barrier function in mice,it included:?The increase expression of mucin,tight junction proteins in the intestinal epithelium and reducing the intestinal permeability altogether to enforce intestinal barrier function in neonatal rats;?Increasing the intestinal surface immunoglobulin A(Ig A)along with reducing the release of inflammatory factors to activate the immune function;?Increasing bacterial diversity,reducing the number of Proteus,increase the number of Bacteroides,realize the balances in the gut microbiota.2)ZY-312 inhibited the expression of inflammasomes NLRP3 related proteins and the release of proinflammatory factor IL-1? activated by CS to alleviate inflammation.3)ZY-312 inhibited the incidence of CS-induced pyroptosis and cell apoptosis,reduce cell death and prevent of NEC.4.ConclusionThe failure of early neonatal intestinal barrier function and the increase of cell death caused by pathogenic bacteria are the main reasons for the occurrence of NEC.In this study,we introduced Next-generation probiotics,Bacteroides fragillis ZY-312,to control CS induced NEC.The results showed that ZY-312 could regulate NEC through altering bacterial composition,repairing intestinal barrier,inhibiting the activation of inflammasomes and reducing cell death(both pyroptosis and apoptosis).Elucidation of the mechanisms that relate between ZY-312 and CS in NEC could identify therapeutic targets for preventing or modulating NEC and for boosting the efficacy of ZY-312.