Effect And Mechanism Of Lactococcus Lactis Peptides And Flurfenicol On Inflammation In Grass Carp

Inflammation is a complex pathological process that causes serious tissue injury,which can be caused by viruses,pathogens or lipopolysaccharide(LPS).LPS can activate the immune cells of the body tissue to secrete excessive pro-inflammatory mediators or pro-inflammatory cytokines,thus leading to a series of inflammatory reactions in the body.Grass carp is one of the main freshwater economic fish in China,and it is easy to be infected by pathogens in the process of breeding,causing inflammation and economic losses.The research on inflammation of grass carp is helpful to understand the immune response mechanism of grass carp and lay a theoretical foundation for further elucidate the immune mechanism of fish.This study was conducted to investigate the effect and mechanism of lactococcus lactis peptides and florfenicol(FFC)on transcriptomics and proteomics based on the level of signal pathway regulation in grass carp(Ctenopharyngodon idella)in vivo and in vitro.The aim was to understand better the anti-inflammatory effect and mechanism of lactococcus lactis peptides and antibiotics in grass carp.The main research results are as follows:1.The effects of 11 antibiotics on the expression levels of inflammatory cytokines(TNF-α,IL-1β,IL-6,NO and PGE2)and the m RNA of nitric oxide synthase and cyclooxygenase-2 in the head kidney(HK)cells of grass carp were evaluated through an in vitro anti-inflammatory model.The results showed that1-20 μg/m L LPS could effectively increase the expression levels of inflammatory cytokines such as TNF-α,IL-1β,IL-6,NO and PGE2 in the HK cells,and the expression of these cytokines exhibted positive relationship with the concentration of LPS.High concentrations of 11 kinds of antibiotics were toxic to the HK macrophages,among which enrofloxacin and FFC were the least toxic to cells.The 11 antibiotics showed certain toxicity to HK macrophages at high concentration,and the toxicity of florfenicol was the least.25-100 μg/m L FFC and erythromycin showed inhibitory effects on inflammatory cytokines such as TNF-α,IL-1β,IL-6,NO and PGE2.Enrofloxacin,FFC,erythromycin and sulfadiazine at a concentration of 25-100μg/m L inhibited the expression levels of m RNA of i NOS and COX-2.These results indicated that FFC had low toxicity and good anti-inflammatory effects,and should be used as an anti-inflammatory drug candidate.2.The transcriptomics and proteomics of grass carp kidneys treated with or without FFC were investigated by using RNA-seq and i TRAQ-coupled LC-MS/MS.The results showed that a total of 968 differential proteins and 4689 differentially genes between the two experimental groups were observed.These differential genes or proteins were mainly attributable to signal pathways such as MAPK,Jak-STAT,Toll-like receptors,among which proteins related to the MAPK signal pathway accounted for the highest proportion.Results from RT-PCR demonstrated that the expression levels of NF-κB,P38,JNK,ERK,MEK-2,TAK-1,IKK and TNF genes in the FFC treatment group were significantly inhibited as compared with the control group(P< 0.05).Most of these genes were related to inflammation-related TLR signaling pathways.In summary,FFC may play anti-inflammatory effects by inhibiting the expression of some upstream effectors(NF-κB and MAPK)of TLR signals.3.The anti-inflammatory effects of FFC on the kidney tissue or cells of grass carp were evaluated through LPS-induced in vitro and in vivo anti-inflammatory models.Inhibitory effect of FFC on inflammatory mediators TNF-α,IL-6 and IL-1β,as well as LPS-induced NO and PGE2 production were assayed by ELISA.The expression levels of i NOS and COX-2 were investigated by RT-PCR.Expression level of TLR-related genes(TLR1,TLR2,TLR4,TLR7,TLR8),tumor necrosis factor receptor associated factor 6(TRAF6),transforming growth factor-b-activated kinase 1(TAK1),Myeloid differentiation factor 88(My D88),nucleus p65,NF-κBα(IκBα)were measured by RT-PCR after grass carp were treated.It was found that FFC dose-dependently inhibited the expression of LPS-induced inflammatory cytokines TNF-α,IL-6 and IL-1β,inflammatory factors NO and PGE2 in macrophages.In addition,i NOS and COX-2 expression levels decreased significantly as compared with LPS treated group.In vivo experiments,FFC inhibited the upregulation of IRAK4,TRAF6,p65,TAK1 and My D88 by blocking the expression of TLR2,TLR7 and TLR8.It is speculated that the anti-inflammatory activity of FFC may be realized by inhibiting the expression of i NOS,COX-2,IL-6,IL-1β and TNF-α by inhibiting the Toll/NF-κB signaling pathway.4.The effects of FFC on the expression levels of genes related to TOLL signal pathway were investigated by a LPS-induced anti-inflammatory model in the grass carp kidney cells.Results demonstrated that FFC dose-dependently promoted the expression of LPS-induced inflammatory cytokines TNF-α,IL-6and IL-1β,inflammatory factors NO and PGE2 in kidney cells.25-100 μg/m L FFC showed inhibitory effects on inflammatory cytokines such as TNF-α,IL-1β,IL-6,NO and PGE2.Under LPS induction,the three concentrations(25,50,100μg/m L)of FFC could significantly reduce the expression levels of Toll-2,Toll-7and Toll-8(P<0.05).The cell strain Sh2 TLR8 with suppressed TLR8 expression was obtained by plasmid transfection.Compared with normal CIK cells,the expression levels of NF-κB,My D88 and IκBα genes in the Sh2 TLR8 were significantly increased after LPS induction(P<0.05).However,the expression of these genes in the Sh2 TLR8 was significantly reduced after FFC treatment.These results suggested that FFC treatment might inhibit the outbreak of LPS-induced inflammation by inhibiting TLR8 gene,and then inhibiting downstream genes such as NF-κB,My D88 and IκBα.5.Head-kidney(HK)macrophages were used for the in vitro bioassay-guided isolation and the structure of the two peptide were identified by mass spectrometry analysis.Two active peptides were isolated by HPLC from the lactococcus lactis BL52,in vitro anti-inflammatory assay demonstrated that peptide ALBL1 and ALBL2dose-dependently inhibited LPS-induced inflammatory cytokines TNF-α,IL-6 and IL-1β,inflammatory factors NO and PGE2 production in macrophages(p < 0.05).In vivo,peptides ALBL1 and ALBL2 inhibit LPS-induced upregulation of TNF-α,IL-6,IL-1β,NO and PGE2.ALBL1 and ALBL2 can relieve the pathological inflammatory response induced by LPS,inhibit the expression of TLR2,and further inhibit the upregulation of nuclear NF-κB,IRAK4,TRAF6,p65,TAK1 and My D88.It is concluded that the anti-inflammatory effects of ALBL1 and ALBL2 peptides may be caused by down-regulating the Toll2/NF-κB signaling pathway and inhibiting the expression of IL-6,IL-1β,TNF-α,NO and PGE2.In conclusion,anti-inflammatory drugs were screened with inflammatory mediators TNF-α,IL-1β,IL-6,NO and PGE2 as targets,and FFC,the drug with the strongest anti-inflammatory activity and the weakest toxicity,was selected.The anti-inflammatory mechanism of FFC was studied,and it was found that FFC could reduce the inflammation induced by LPS by inhibiting the activation of Toll/NF-κB/MAPK signaling pathway.FFC mainly inhibited the downstream genes of NF-κB,My D88 and IκBα by inhibiting TLR8 gene,so as to inhibit the outbreak of LPS-induced inflammation.At the same time,two active peptides were isolated from Lactococcus lactis BL52,and grass carp treated with these peptides can reduce the LPS-induced inflammatory response by down-regulating the Toll2/NF-κB signaling pathway.This study provides a new insight into the mechanism of inhibiting inflammatory response of grass carp induced by LPS,and provides a theoretical basis for the use of lactococcus lactis peptides and FFC as inhibitors of inflammation of grass carp.