Study On The Mechanism Of Intestinal Barrier Dysfunction Induced By Staphylococcal Enterotoxin A And EGCG Intervention

Staphylococcal enterotoxins(SEs)secreted by Staphylococcus aureus(S.aureus)are widely present in a variety of foods and have strong protein stability,posing a potential safety hazard of food poisoning caused by entering the gastrointestinal tract through "farm-to-workshop-to-table".Staphylococcal food poisoning(SFP)caused by ingestion of SEs-contaminated food has become one of the most common bacterial foodborne diseases worldwide and can cause serious food safety problems.Among many SEs,staphylococcal enterotoxin A(SEA)has become the most common and most virulent SEs in SFP outbreaks due to its extremely strong superantigen activity and emetic activity,accounting for approximately 80% of SFP events.Therefore,it is of great significance to study the target organ and toxicity mechanism of SEA and develop potential natural antitoxin compounds for the targeted prevention and control of toxintype food poisoning.Tea polyphenols have been used as food additives in China for many years.Epigallocatechin gallate(EGCG),the main active ingredient of tea polyphenols,is expected to be a promising candidate for the prevention of SEA-type foodborne diseases because of its multiple physiological functions,such as antibacterial,anti-inflammatory,antioxidant,antiviral,etc.This study investigated the induction of SEA on intestinal barrier injury and enteritis based on the "TLR4-mediated NF-κB/MAPKs-NLRP3 inflammatory cascade reaction driven by intestinal flora" and clarified the mechanism of SEA-type food poisoning.On this basis,the intervention effect of EGCG on SEA-induced intestinal damage was also investigated to provide the research paradigm and theoretical guidance for the screening of antitoxic foodborne active ingredients and the development of strategies(such as food additives,functional foods,etc.)related to the prevention and control of food safety issues.The main research contents and conclusions of this paper are as follows:(1)Preparation of SEA protein and preliminary investigation of its effect on the small intestine.First,the SEA gene was obtained by PCR amplification and the p ET-28a-SEA recombinant vector was constructed by double digestion and ligation.The sequence similarity between the p ET-28a-SEA vector and the reported SEA gene was confirmed by sequencing to be 99%.The p ET-28a-SEA recombinant vector was transferred into the E.coli BL21(DE3)expression strain.After IPTG induction,the SEA protein was purified by nickel affinity chromatography,and its purity was greater than 95%.Then,BALB/c mice models were constructed by oral administration with different concentrations of SEA(0,100,250,and 500 μg/kg).H&E staining was performed to find that SEA caused histopathological damage to the small intestine,especially villus damage of jejunum tissues.Besides,the content of LPS in the serum of mice was determined to find that SEA dose-dependently increased the content of LPS.These results preliminarily confirmed that SEA could induce small intestinal damage and increase intestinal permeability,with the potential risk of damaging intestinal barrier function.(2)Exploring the effect of SEA on intestinal barrier function and its potential molecular mechanism.Jejunum tissues of BALB/c mice and Caco-2 cells were used as research models.Western-Blot showed that SEA up-regulated the pro-apoptotic protein Bax and down-regulated the anti-apoptotic protein Bcl-2,thereby accelerating intestinal cell apoptosis.The results of Western-Blot,immunohistochemistry,and immunofluorescence showed that SEA caused intestinal barrier damage by inhibiting the expression of tight junction(TJ)proteins(ZO-1,Occludin,and Claudin-1)and mucin MUC2.Furthermore,the levels of NLRP3 inflammasome-associated proteins(NLRP3,Caspase-1 and ASC)and the release of IL-18 and IL-1β in mouse serum were detected.It was found that SEA triggered the activation of NLRP3 inflammasome,which induced intestinal inflammatory response and further aggravated intestinal barrier damage.The expression levels of TLR4 and My D88 proteins and the phosphorylation levels of p65,IκBα,p38,ERK and JNK proteins were increased with the increase of SEA concentration,confirming that SEA could activate TLR4,NF-κB and MAPKs signaling pathways.Based on this,Caco-2 cells were pretreated with inhibitors of key proteins of multiple signaling pathways(TLR4,NF-κB and MAPKs pathways and NLRP3 inflammasome),further confirming that SEA activated NLRP3 inflammasome at least partially through TLR4 receptor triggering NF-κB and MAPKs signal pathways,thereby inducing the small intestinal inflammatory response and intestinal barrier injury.(3)Clarifying the protective effect and molecular mechanism of EGCG on SEAinduced intestinal barrier injury.First,the surface plasmon resonance(SPR),isothermal titration calorimetry(ITC),and Western-Blot found that EGCG not only inhibited the secretion of SEA protein from S.aureus,but also bound to SEA protein that has been secreted outside the bacterium,indicating that EGCG has the potential to inhibit SEA toxicity.Next,BALB/c mice and Caco-2 cells were used as in vitro and in vivo models.H&E staining was employed to visually confirm that EGCG could effectively improve small intestinal injury caused by SEA.The content of LPS in serum and permeability and TEER of Caco-2 monolayers were determined to find that EGCG inhibited LPS translocation and intestinal permeability increase induced by SEA.The expression levels of apoptosis-related proteins(Bax and Bcl-2)were measured to demonstrate that EGCG alleviated the intestinal epithelial cell apoptosis induced by SEA,which was helpful to maintain the normal growth and reproduction of intestinal epithelial cells.Results of Western-Blot,immunohistochemistry and immunofluorescence showed that EGCG improved the SEA-inhibited synthesis of ZO-1,Occludin,Claudin-1 and MUC2 proteins,thus enhancing the integrity of the intestinal barrier.On this basis,the contents of IL-18 and IL-1β in serum and the expressions of NLRP3 inflammasome-related proteins in jejunum tissues and Caco-2 cells were measured.It was found that EGCG could effectively inhibit the activation of NLRP3 inflammasome induced by SEA,thus improving the inflammatory response of the small intestine.Meanwhile,Western-Blot results showed that EGCG reduced the expression levels of TLR4 and My D88 proteins and the phosphorylation levels of p65,IκBα,p38,ERK and JNK proteins elevated by SEA in vivo and in vitro,indicating that EGCG ameliorated SEA-induced inflammatory response and intestinal barrier damage in the small intestine by inhibiting TLR4-mediated NF-κB/MAPKs-NLRP3 inflammatory cascade.(4)Revealing the key role of intestinal flora in "EGCG ameliorated SEA-induced intestinal barrier damage".The composition of mouse intestinal flora was analyzed by16 S r RNA high-throughput sequencing.The results showed that SEA up-regulated the abundance of harmful bacteria and down-regulated the abundance of beneficial bacteria,while EGCG mediated a certain recovery effect on SEA-induced intestinal flora imbalance.The content of short-chain fatty acids(SCFAs)in the mouse cecum contents was also determined by GC-MS/MS,and the results showed that SEA decreased the content of acetic acid,propionic acid,isobutyric acid,valeric acid and isovaleric acid,while EGCG intervention effectively restored the metabolic levels of SCFAs inhibited by SEA.Furthermore,the correlation between intestinal flora index(microbiota abundance and its metabolites SCFAs content)and intestinal barrier index(including intestinal barrier integrity,LPS content,inflammatory factors,NLRP3 inflammasome and TLR4-NF-κB/MAPKs pathway-related proteins)was analyzed to further confirm that EGCG ameliorated SEA-induced intestinal barrier damage and enteritis by regulating the intestinal microbial composition and the content of its metabolite SCFAs.In conclusion,this study confirmed that SEA caused intestinal barrier dysfunction by TLR4-mediated NF-κB/MAPKs-NLRP3 inflammatory cascade response,elucidated the protective effect of EGCG on SEA-induced intestinal barrier injury and enteritis,and emphasized the global regulatory role of intestinal flora in "EGCG ameliorated SEA-caused intestinal barrier damage and enteritis".This provided important new research ideas and theoretical support for the mechanism analysis of toxin-type food poisoning and the development of natural compounds(e.g.EGCG)as intervention/prevention strategies for toxin-type food poisoning.