| Food borne disease caused by bacterial pollution is one of the biggest problems affecting human health and food safety.Preservatives are added during food manufacturing process to inhibit growth of pathogens to avoid food safety problems.However,chemical preservatives may be harmful to human health.Therefore,it is important to develop green and safe natural antibacterial and preservative agent to inhibit microbial contamination to ensure public food safety.Epigallocatechin gallate(EGCG)is one of the most abundant polyphenols in green tea.It has many biological properties,such as antioxidant,anti-cancer,anti-inflammatory activities and so on.It also has inhibitory effect on many microorganisms.Staphylococcus aureus(S.aureus)is a common foodborne pathogen,which causes food poisoning events with high frequency all over the world.?-Hemolysin(Hla)is an extracellular pore forming toxin coded by S.aureus hla gene,which plays an important role in S.aureus infection.The effect of EGCG on inflammatory response induced by Hla has not been reported.Therefore,this study aims to investigate the inhibitory effect of EGCG on the NLRP3 inflammasome activation induced by Hla and to elucidate the molecular mechanism.In vitro,the drug sensitivity assay and growth curve showed that EGCG had a good inhibitory effect on S.aureus,and the MICs of EGCG on S.aureus 8325-4 and DU1090(hla gene knock-out mutant of 8325-4)both were 128 ?g/m L.Western blot,oligomerization assay and hemolysis test showed that EGCG(< 128 ?g/m L)could inhibit the hemolysis of S.aureus on rabbit red blood cells by inhibiting secretion of Hla and interfering with the formation of heptatomers of Hla.It was found that Hla can induce decline of cell viability and apoptosis.EGCG can enhance cell viability and weaken cell apoptosis,showing protective effect on Hla-induced cell injury.On this basis,in order to explore the molecular mechanism of EGCG inhibiting Hla-induced NLRP3 inflammasome activation from the protein level,the production of reactive oxygen species(ROS)in THP-1 cells was measured by flow cytometry in vitro,the phosphorylation level of MAPK signal pathway and the expression level of NLRP3 inflammasome were detected by Western blot assay.The results showed that Hla could activate NLRP3 inflammasome by inducing ROS production and MAPK signal pathway activation.EGCG can inhibit ROS production and MAPK signal pathway activation induced by Hla,and thus inhibit NLRP3 inflammasome activation mediated by ROS/MAPK.The results showed that EGCG interfere with the activation of inflammasome through inhibition of Hla.The inhibitory effect of EGCG on the activation of NLRP3 inflammatsome induced by Hla was investigated in mice infected with S.aureus.It was found that Hla secreted by S.aureus promoted expression of NLRP3,ASC,and caspase-1 and secretion of cytokines IL-18 and IL-1?.However,EGCG down regulated expression of NLRP3 inflammasome as well as production of IL-18 and IL-1?.The results in vivo were consistent with above results in vitro,indicating that EGCG can inhibit activation of NLRP3 inflammasome caused by Hla both in vitro and in vivo.This paper demonstrated that EGCG inhibited the secretion of Hla by S.aureus and the Hla-induced NLRP3 inflammasome activation,and elucidated its molecular mechanism.The results show that EGCG has the function of anti-hemolysin,and has great potential in preventing inflammatory response caused by Hla in food,which lays a foundation for the development of EGCG as a green and safe preservative agent. |
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