The Intervention Effect And Mechanism Of Theaflavin-3’-gallate On ETEC Infected IPEC-J2 Cells

Enterotoxigenic Escherichia coli(ETEC)is one of the important causes of diarrhea in newborn piglets.ETEC can colonize the small intestinal epithelium through adhesin and produce large amounts of enterotoxins that cause diarrhea in the host.The current clinical use of antibiotics is limited by problems such as bacterial resistance and antibiotic residue,so it is crucial to find alternative drugs.Targeting drugs to bacterial virulence factors instead of bacterial survival is one of the effective strategies to control drug-resistant bacterial infections.Reducing the survival pressure of bacteria by antagonizing or regulating virulence factors without killing bacteria can prevent the development of bacterial resistance.More and more studies have been conducted on the prevention and treatment of diarrhea in piglets due to the advantages of small side effects,no residue and no resistance to drugs in traditional Chinese medicine and its active ingredients.Theaflavin-3’-gallate(TF2B)in black tea has a variety of biological activities such as antioxidant,anti-inflammatory,antibacterial and anti-cancer,and has antibacterial activity against a variety of bacteria.The team previously targeted the heatsensitive enterotoxin LTB of Escherichia coli.TF2 B was found to be a potential toxin inhibitor with good effect.There are few reports on the effect and mechanism of TF2 B in the treatment of diarrhea caused by ETEC in piglets.Polymyxin B(PMB)is one of the main drugs used in the veterinary clinic for the treatment of yellow and white dysentery in piglets.It can destroy the integrity of the outer membrane phospholipids of Gram-negative bacteria and play a bactericidal role.In this study,the porcine intestinal epithelial cells(IPEC-J2)were cultured in vitro to construct an ETEC infected IPEC-J2 cell model,and to investigate the antagonistic effect and mechanism of TF2 B alone or in combination with PMB on ETEC infected IPEC-J2 cells.The aim is to obtain alternatives to antibiotic additives and to discover new strategies for the prevention and treatment of infections caused by drug-resistant bacteria.The details are as follows:On the basis of measuring the safe concentration range of drugs on IPEC-J2 cells and the best infective concentration and the best action time of ETEC on IPEC-J2 cells,a group comparison experiment was conducted.TF2 B was used as the test drug,and the enterotoxin inhibitors Glycyrrhizic acid(GL)and PMB were used as control drugs.First,the effective concentration of the test drugs against ETEC infected IPEC-J2 cells was determined.Then three administration modes were set up(adding drugs first and then adding bacteria,adding drugs and bacteria at the same time,adding bacteria first and then adding drugs).Lactate dehydrogenase activity assay,crystal violet staining,bacterial adhesion rate assay,LT toxin and c AMP release assay,real-time fluorescence quantitative PCR,Western blot and ELISA were used to detect the changes of the bacteria.The cytotoxicity of ETEC infected cells,pathogenic process of ETEC,cell inflammation,cell barrier and other related indicators of different treatments were compared and analyzed,and the antagonistic effect of TF2 B on ETEC infected IPEC-J2 cells was comprehensively evaluated.In addition,the minimal inhibitory concentration(MIC)of TF2 B against ETEC was determined by broth microdilution method to determine its antibacterial activity in vitro.Finally,the outer membrane permeability test,biofilm detection,real-time fluorescence quantitative PCR and molecular docking were used to analyze the changes of bacterial outer membrane permeability,biofilm and virulence related genes expression in different treatment groups,and the virtual binding ability of TF2 B to LTB to explore the anti-ETEC mechanism of TF2 B.Results of the trial study were as follows:By constructing an ETEC infected IPEC-J2 cell model,TF2 B was found to protect against the damage of ETEC to IPEC-J2 cells by antagonizing the toxicity of ETEC infection,inhibiting the adhesion process of ETEC to IPEC-J2 cells and the inflammatory response of cells,and repairing the cell barrier of ETEC infection.Its antagonism is a comprehensive action of multiple pathways.CCK-8 assay showed that the maximum safe concentrations of TF2 B,GL,and PMB for IPecs were 100 μM(71.66 μg/m L),100 μM(82.29 μg/m L),and >100 μg/m L,respectively.The results of cytotoxicity test,adhesion rate test and crystal violet staining showed that when the ratio of bacteria to cell number(MOI)was 50 and the infection time was2 h,ETEC had high cytotoxicity and adhesion rate to IPEC-J2 cells,which was determined to be the best infection condition for the subsequent experiment of ETEC infection in IPEC-J2 cell model.TF2B(20 μM,14.332 μg/m L),GL(20 μM,16.4586 μg/m L),and PMB(2 μg/m L)were determined as the experimental doses of the tested drugs against ETEC infected IPEC-J2 cells according to the results of effective concentration determination.The results of cytotoxicity,ETEC pathogenic process,cell inflammation,cell barrier and other related indicators showed that compared with the blank control group,the death rate,bacterial adhesion rate and c AMP content of IPEC-J2 cells in the ETEC infection group were significantly increased(P<0.01).The expression of inflammatory related factors was significantly(P<0.05)or extremely significantly(P<0.01)up-regulated,and the expression of tight junction protein was significantly(P<0.05)or extremely significantly(P<0.01)downregulated.Compared with the ETEC infection group,the bacterial adhesion rate of the ETEC+TF2B group was significantly decreased by adding bacteria first and bacteria at the same time(P<0.01),but there was no significant change in the bacterial adhesion rate of the ETEC+TF2B group by adding bacteria first and then adding bacteria(P>0.05).The death rate and c AMP content of IPEC-J2 cells in ETEC+TF2B group were significantly decreased(P<0.01),the cell damage was significantly improved,and the expression levels of inflammatory related factors IL-6,IL-1β and TNF-α m RNA and protein were significantly decreased(P<0.01).The m RNA and protein expression levels of tight junction proteins ZO-1,Occludin,and Claudin-1 were significantly(P<0.05)or extremely significantly(P<0.01)increased.Compared with ETEC+TF2B and ETEC+PMB groups,the expression levels of IL-6 m RNA and protein in ETEC+TF2B+PMB group were significantly decreased(P<0.01).The expression levels of ZO-1 m RNA and protein in ETEC+TF2B+PMB group were significantly higher than those in the control group(P<0.01).Therefore,TF2 B combined with PMB had a synergistic effect in alleviating the inflammatory response of ETEC and repairing the damage of cell barrier.The MIC assay showed that TF2 B had no significant antibacterial activity against ETEC.Compared with the control group,TF2 B significantly inhibited the formation of ETEC biofilm(P<0.01)and down-regulated the expression of bss S and fae G genes(P<0.01),as measured by bacterial outer membrane permeability assay,biofilm detection assay and virulence gene expression assay.The expression level of elt A gene in the TF2B+PMB group was significantly lower than that in the TF2 B and PMB groups(P<0.01),and the expression level of bss S gene in the TF2B+PMB group was significantly lower than that in the TF2 B group(P<0.05).It was significantly lower than that in PMB group(P<0.01).These results suggested that TF2 B could inhibit the biofilm formation of ETEC,and the inhibition of bss S gene and pilus subunit expression was the main mechanism of TF2 B against ETEC infection.The combination of TF2 B and PMB had A synergistic effect on inhibiting the expression of biofilm-related genes bss S and LT toxin subunit A.The results of virtual molecular docking showed that TF2 B could bind to the active site of LTB,and formed hydrogen bonds and van der Waals forces with the amino acid residues of the binding site,which made the conformation of TF2 B bound to LTB more stable,suggesting that the binding of TF2 B to LTB protein may be one of the mechanisms of TF2 B against ETEC infection.In summary,TF2 B exerted anti-ETEC infection effect in vitro by reducing the toxicity of ETEC to IPEC-J2 cells,bacterial adhesion rate,cell c AMP level,inhibiting cell inflammation and repairing cell barrier damage caused by ETEC infection.TF2 B had no significant antibacterial activity against ETEC.The main mechanisms of TF2 B against ETEC infection in vitro were inhibiting the formation of ETEC biofilm,the expression of biofilm related genes bss S and fimbriae subunit,and directly binding to LTB toxin protein.The results of this study provide a theoretical basis for further evaluation of the in vivo efficacy of TF2 B and the development of effective drugs for the prevention and treatment of ETEC induced diarrhea in piglets.