Study On The Mechanism Of Emodin On The Inhibition Of Aeromonas Hydrophila In Vitro

Megalobrama amblycephala(M amblycephala)is one of the most important freshwater fishes in China,with an annual yield of about 700,000 tons.In the process of culture,M.amblycephala is easy to be infected with bacterial diseases,which leads to a large number of deaths.Bacterial disease is one of the main reasons for the outbreak of aquatic diseases,which has caused serious losses to aquaculture.Aeromonas hydrophila(A.hydrophila)is a main pathogen causing bacterial hemorrhagic disease in aquatic animal.It has strong infection,high incidence rate and high lethality rate,especially on M.amblycephala.The pathogenicity of A.hydrophila is related to virulence factors.At present,the main virulence factors are secretion system,biofilm formation,adhesin,hemolytic toxin and extracellular protease.Emodin(1,3,8-trihydroxy-6-methylanthraquinone)is a kind of anthraquinone derivative,which mainly comes from Chinese herbal medicines such as rhubarb,Polygonum cuspidatum,Polygonum multiflorum,aloe and cassia seed.It has many pharmacological activities,such as antiviral,antiviral,anti-inflammatory,anti-oxidation,anti-osteoporosis,anti-tumor,nerve protection,liver and gallbladder protection,etc.Previous studies have shown that emodin can inhibit the growth of A.hydrophila,but its specific mechanism is not clear.The mechanism of action of Chinese medicine has become a hot topic.The mechanism of Chinese medicine regulating human immune function is one of the ten important scientific questions introduced by the China Science and Technology Association in 2020.Therefore,elucidating the specific molecular mechanism of emodin against A.hydrophila and searching for potential targets are of great significance for the prevention and treatment of bacterial diseases caused by A.hydrophila.In view of this,we studied the mechanism of A.hydrophila on M.amblycephala infection based on proteomics.Emodin was used as the research object to study the effect of emodin on A.hydrophila.The genes and proteins differentially expressed in A.hydrophila induced by emodin were identified by transcriptome and proteome techniques by modern molecular biological methods,and the antibacterial mechanism of emodin against A.hydrophila was elucidated at the molecular level.Molecular docking and molecular dynamics simulation were used to analyze the binding interaction between emodin and potential targets,which provided theoretical basis for the exploration of interaction targets and laid the foundation for the development of anti A.hydrophila drugs.1.Comparative proteomic analysis of the infection mechanism of Aeromonas hydrophila on Megalobrama amblycephala0.85%normal saline was used as the control group,and a half-lethal concentration of A.hydrophila of 106 CFU/mL was used as the infection group.Liver samples of M.amblycephala were collected at 24 h after the challenge.The protein expression profiles of the two groups were sequenced by proteomics technology.A total of 30 protein spots with significant changes(Fold Change≥ 2 times)were detected.Among them,9 were up-regulated and 21 were down-regulated,representing 18 different proteins.In order to explore the pathogenic mechanism of A.hydrophila on M.amblycephala,and provide theoretical basis for clarifying the body damage of M.amblycephala.According to bioinformatics analysis,the identified liver proteins of M amblycephala were divided into 9 categories,including 7 carbohydrate metabolism proteins(5 unique,accounting for 23%),6 energy metabolism proteins(2 unique,accounting for 20%),6 antioxidant proteins(3 unique,accounting for 20%),5 cytoskeleton proteins(2 unique,accounting for 17%),2 chaperone proteins(2 unique,accounting for 7%),1 nucleotide metabolic protein(1 unique,accounting for 3%),cofactor and life support One protein(1 unique,accounting for 3%),one signal transduction(1 unique,accounting for 3%)and one(1 unique,accounting for 3%)of lipid metabolism in M amblycephala,indicating that A.hydrophila mainly affected the metabolism,cell and intercellular information transmission and other life processes of M.amblycephala.The functional annotation showed that 13 differential proteins were closely related to immune response and regulated each other directly.Among them,11 proteins participated in metabolic process and 3 proteins participated in immune process.Candidate biomarkers such as ENO3,ApoA1,CAT,FASN,MDH,ALDOB and RSP12 were found.2.Transcriptome analysis of the antibacterial effect of emodin on Aeromonas hydrophilaUsing DMSO as control group and emodin as experimental group,A.hydrophila was cultured for 6 h.The two groups were sequenced by high-throughput sequencing technology.Based on bioinformatics analysis,the differentially expressed genes were mined in order to explore the key regulatory pathway of emodin induced A.hydrophila damage,and provide theoretical basis for elucidating the antibacterial mechanism.The results showed that 4187 differentially expressed genes were identified,including 2141 up-regulated genes and 2046 down-regulated genes.281 differentially expressed genes were screened with log2(Fold Change)≥ 1.2,P<0.05,including 147 down-regulated genes and 134 up-regulated genes.Go enrichment analysis showed that the differentially expressed genes were mainly enriched in cell,catalytic activity,cell part,binding,cellular process and metabolic process.KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in metabolism(metabolic pathways,microbial metabolism in different environments,purine metabolism,carbon metabolism,etc.),virulence system(two component system,bacterial chemotaxis,flagella assembly,etc.),energy transport(ABC transporters,protein export,etc.)and biosynthesis(biosynthesis of antibiotics,biosynthesis of secondary metabolites).The TCS/BIF/FA pathway plays a regulatory role in the inhibition of the growth of A.hydrophila by emodin.3.Comparative transcriptomic and proteomic analysis of the antibacterial activity of emodin on Aeromonas hydrophilaITRAQ technology was used to sequence the proteome of the control group and the infection group,and bioinformatics analysis was used to mine the differentially expressed proteins,and the joint analysis with the transcriptome data in the previous chapter was conducted to explore the transcription and protein level changes of A.hydrophila induced by emodin,and provide a theoretical basis for further elucidating its antibacterial mechanism.The results showed that there were 2012 proteins were quantified,of which 237 were 2-fold differentially expressed proteins.The results of transcriptome and proteome analysis showed that 99 genes and their corresponding proteins expressed differently at gene and protein levels.Go enrichment analysis showed that the differentially expressed genes and proteins were mainly enriched in cellular process,biological process,organic substance metabolic process,cell,cell part,molecular function and binding.KEGG enrichment analysis showed that the differentially expressed genes and proteins were mainly concentrated in metabolic pathways,ribosomes,microbial metabolism in diverse environments,biosynthesis of secondary metabolites,oxidative phosphorylation,biosynthesis of antibiotics,carbon metabolism and ABC transporters.Emodin inhibited the growth of A.hydrophila by inducing oxidative damage and inhibiting the synthesis of ribosomes,amino acids and fatty acids.4.Molecular docking between emodin and key proteins of Aeromonas hydrophilaOn the basis of the above experiments,five key proteins,RstB,CRR,FlgG,SecB and MetK,were selected to explore whether emodin could bind to them and how they bind by homologous modeling and molecular docking technology.The protein RstB,CRR,FlgG,SecB and MetK were modeled by SWISS-MODEL,and the model quality was evaluated by SAVES V5.0 online software.Molecular docking was performed using discovery Studio software.The results showed that emodin could bind to RstB,CRR,FlgG,SecB and MetK.Emodin has hydrogen bonds with RstB amino acid residues GLN214,ARG323,LEU215 and SER397,has a hydrophobic interaction with residues ALA218,etc.,and has van der Waals force with SER211.Emodin has hydrogen bonds with Crr amino acid residues SER12,GLU160,LYS9 and LYS59,has a hydrophobic interaction with residue LYS9,and has van der Waals force with ARG116.Emodin has a hydrogen bond with FlgG amino acid residue SER92,has a hydrophobic interaction with residue LEU 122,etc.,and has van der Waals force with PRO 144.Emodin has a hydrogen bond with SecB amino acid residues SER122 and VAL19,has a hydrophobic interaction with residue LYS123,and has van der Waals force with TYR20.Emodin has hydrophobic interactions with MetK residues LYS165,PHE231,HIS14 and ALA40,and has van der Waals force with ASP239.5.Molecular dynamics of emodin and Aeromonas hydrophila SecB and MetK and effect of QseC deletion on virulence function genesOn the basis of the above experiments,SecB and metk proteins were further selected for molecular dynamics simulation and binding free energy calculation,to explore the specific action mechanism of emodin with SecB and metk.The results showed that the volatility of SecB and MetK residues decreased after binding with emodin,and hydrogen bonds were formed between residues and ligands.The sum and energy of emodin,SecB and MetK were-83.80 kcal/mol and-3.77 kcal/mol,respectively.The binding effect of emodin with SecB was better than that of MetK.Emodin combined with TYR112,GLU115,THR21,LYS123,VAL19,ALA116,SER118 and TYR20 of the main amino acid residues of SecB,resulting in the inhibition of its activity.Emodin combined with ASP 118,ILE233,ARG161,GLY235,GLU8,LYS46,VAL10,HIS14 and GLY234 of MetK and inhibited the active sites.Through the determination of SecB and MetK expression in theΔQseC-deficient strain,it is found that the effect of emodin on SecB and MetK was related to the QseBC two-component regulatory system.