Inhibitory Effect Of EGCG On Aeromonas Hydrophila And Cyprinid Herpesvirus 2

Crucian carp（Carassius auratus）is an important fish species cultured worldwide and the China Fisheries Yearbook（2021）showed that the production of crucian carp in China was about 2.75 million tons in 2020,ranking fifth among all fish production and accounting for 8.5%of the annual freshwater fish production.However,the carp production is decreasing year by year from 2015 to 2020.Aeromonas hydrophila and cyprinid herpesvirus 2（Cy HV-2）are the main pathogens responsible for the decreased yield.A large number of studies have shown green tea extract epigallocatechin gallate（EGCG）has anti-pathogenic microbial and broad-spectrum anti-infective activity.These reports provide a suitable basis for exploring the potential of EGCG as bactericide and virucide in gibel carp aquaculture and the possible mechanisms involved,which remains to be clarified.In this project,we investigated the inhibitory effect of EGCG on A.hydrophila and Cy HV-2 and the effect of EGCG on the transcriptome of C.auratus gibelio caudal fin（GiCF）cells to provide a theoretical basis for the development of EGCG fishery drugs.The results of the study are as follows.1.The mechanism of EGCG inhibiting A.hydrophilaThe minimum inhibitory concentration of EGCG against tested A.hydrophila strains ranged from 256 to 512μg/m L and the minimum bactericidal concentration was 1024μg/m L using micro-dilution method.The growth curves of treated A.hydrophila A56indicated that the concentration and treatment time of EGCG served as the two dominant factors for EGCG-mediated inhibitory effects.The images of scanning electron microscope（SEM）showed that A56 exposed to EGCG demonstrated obvious structural changes with broken,wrinkled and recessed cell walls.The results of the enzyme-labeled instrument showed that EGCG could produce H₂O₂ autoxidatively,and the yield was positively correlated with the dose and time,with a peak at 12 hours.The results of flow cytometry showed that EGCG at 128-512μg/m L could induce a significant increase in intracellular ROS in A.hydrophila A56（P<0.05）.Oxidative stress played an important role in the antibacterial effect of EGCG,because the inhibitory effect of EGCG was limited after adding the antioxidants N-Acetyl-L-cysteine（NAC）and catalase（CAT）,and the intracellular ROS of A56 was significantly reduced（P<0.01）.In addition,in the infection models established with gibel carps and A56,oral administration of EGCG significantly improved the ability of fish tissues to clear bacteria（P<0.01）.Compared with the control group,EGCG at a dose of 40 mg/kg（bw）significantly reduced the bacterial load in the tissues of gibel carps infected with A.hydrophila（P<0.01）.2.The inhibition effect of EGCG on Cy HV-2GiCF cells were used as a infected model to study the inhibitory effect of EGCG on Cy HV-2.The safe concentration of EGCG on GiCF cells was determined by MUSE cell analyzer,and the results showed that EGCG at concentrations lower than 10μg/m L was safe for GiCF and did not significantly inhibit cell viability.Through the determination of TCID₅₀,calculation of virus copy number and western blotting,it was found that EGCG targeted virus particles to exert antiviral effects,and the inactivation effect of Cy HV-2 virus treated with EGCG at 50μg/m L for 2 h could reach 99.99%,and EGCG at70μg/m L could completely inactivate Cy HV-2.EGCG might act as a disinfectant to directly inactivate Cy HV-2.Therefore,chlorine dioxide solution and compound sodium chlorite powder were selected as disinfectant controls to study the inactivation effect of chlorine-containing disinfectants on Cy HV-2.Results showed that 12.5μg/m L standard chlorine dioxide solution and 250μg/m L compound sodium chlorite solution could inactivate 99.9%the Cy HV-2 when treated for more than 30 min.EGCG inactivated Cy HV-2 better than the compound sodium chlorite powder.EGCG showed the same disinfection efficacy as chlorine dioxide.It is promising to develop new environmentally friendly disinfectants with EGCG as the main component.3.Transcriptomic analysis of EGCG on GiCF cellsTranscriptome sequencing was performed on cells treated with 10μg/m L EGCG for24 h and untreated cells to investigate the effect of EGCG on gene expression in GiCF cells.Compared with the control group,a total of 1951 significantly different genes were screened,including 1090 up-regulated genes and 861 down-regulated genes.These differential genes enriched a total of twenty-two KEGG pathways,including ten metabolism pathways such as amino acid metabolism and drug metabolism,seven genetic information processing pathways,one digestive system pathway,one malaria pathway,one cell cycle pathway and two signal transduction pathways.Two signal transduction pathways were Fox O signal transduction pathway and PI3K-Akt signal transduction pathway.In addition,nine differential genes were selected and validated:smad4、pcna、Fox O1、GADD45、cdk2、mad2、myt1、DHFR and IL-6.qRT-PCR results showed the same expression trends as the transcriptome,with three up-regulated genes:smad4,Fox O1 and GADD45;six down-regulated genes.pcna,cdk2,mad2,myt1,DHFR and IL-6.EGCG may enhance the immune response and suppress inflammation by activating the Fox O signaling pathway.The results provide a reference for the development of EGCG drugs for fisheries and the possible mechanisms involved.