Effect And Mechanism Of Lactobacillus Culture Supernatant On PEDV Proliferation In Vitro

The previous study of our research group found that the abundance of Lactobacillus increased in the intestine of piglets infected with Porcine epidemic diarrhea virus（PEDV）,but the specific role and related mechanism of Lactobacillus in PEDV infection in piglets are not clear.Therefore,this study took the lactic acid bacteria isolated and identified in the early stage,aiming to explore the effect and mechanism of Lactic acid bacteria supernatant（LABS）on PEDV proliferation in vitro.The subject first explores the physiological and biochemical characteristics and morphological structure of five strains of lactic acid bacteria,the screening of high temperature,acid and bile salt resistance and the impact on the bacteriostasis of pathogenic bacteria.The effect of LABS on PEDV proliferation in vitro was determined and changes in relevant gene expression were examined.Finally,transcriptomic analysis was used to reveal the mechanism of LABS affecting PEDV proliferation in vitro.Exp.1 Study on characteristics of lactic acid bacteriaIn this experiment,five strains of lactic acid bacteria provided by Wuhan Polytechnic University were taken as the research object.Through the study on the physiological and biochemical properties,morphological and structural characteristics,high temperature resistance,acid and bile salt resistance,and their bacteriostatic effect on two pathogens,it laid a foundation for further understanding the antiviral effect of lactic acid bacteria.The results showed that:（1）through 16S r DNA sequence alignment,G1 was identified as Lactobacillus rhamnosus,G8 and G33 were Enterococcus faecium,and G64 was Lactiplantibacillus Plantarum.L1 was identified as Lactobacillus zeae by the preliminary research group.（2）The temperature sensitivity test results show that:Lactic acid bacteria can survive at 37℃,G33 and G8 can survive at 60℃,and the survival rate is high,basically reaching more than 90%.The acid tolerance test results showed that:At p H2and p H2.5,all five strains of lactic acid bacteria could not grow.At p H3 and p H4,G33and G8 grew basically the same and showed strong acid resistance,followed by G1,which was stronger than L1,and G64 was the worst.The results of the bile salt tolerance test showed that:G1,L1 and G64 could not survive at the concentration of0.2%-1.0%pig bile salt,G33 and G8 had a higher survival rate between different concentrations of pig bile salt,and the number of viable bacteria of the two strains was the highest under the culture of high concentration of bile salt（1%）.（3）The in vitro antibacterial test results showed that:G33 can effectively inhibit the growth of E.coli K88,followed by G1 and L1,G64 and G8 have weak ability to inhibit E.coli;In terms of inhibiting the growth of Salmonella,the bacteriostatic ability of several strains of lactic acid bacteria is almost the same.To sum up,the 5 strains isolated in the early stage of the research group were all lactic acid bacteria.From the perspective of high temperature acid and bile salt and antibacterial resistance,G33 and G8 strains are relatively stable and have a certain ability to withstand high temperature acid and bile salt.Exp.2 Effect of lactic acid bacteria against PEDV in vitroFirstly,the proliferation of labs on Vero cells was detected,and the maximum non-toxic dose was selected for follow-up test.The virulence of PEDV was diluted to100 TCID₅₀.Labs was filtered by 0.22?filter head and diluted by 1/2 to 1/1024times.PEDV and labs were added to cells for observation for 48h.Fluorescence observation was carried out and the expression of related genes was detected.The results showed that:（1）compared with the control group,the addition of 1/16 and 1/32 lactic acid bacteria supernatant in G64 significantly promoted the growth of Vero cells（P<0.05）,and the survival rate of 1/16 cells was the highest.Compared with the control group,the addition of 1/8,1/16 and 1/32 lactic acid bacteria supernatant in G8 significantly promoted the growth of Vero cells（P<0.05）,and the survival rate of 1/16 cells was the highest.Compared with the control group,the addition of 1/8,1/16 and 1/32 lactic acid bacteria supernatant in G33significantly promoted the growth of Vero cells（P<0.05）,and the survival rate of 1/16 cells was the highest.Compared with the control group,the addition of 1/8,1/16and 1/32 lactic acid bacteria supernatant in G1 significantly promoted the growth of Vero cells（P<0.05）,and the survival rate of 1/16 cells was the highest.Compared with the control group,the addition of 1/4,1/8,1/16 and 1/32 lactic acid bacteria supernatant in L1 significantly promoted the growth of Vero cells（P<0.05）,and the survival rate of 1/8 cells was the highest.Compared with the control group,1/2～1/128 gradient in Mrs group significantly promoted the growth of Vero cells（P<0.05）.（2）In vitro viral proliferation assay results showed that:Compared with PEDV group,the supernatant of 5 strains of lactic acid bacteria had inhibitory effect on PEDV,and gradually weakened after dilution.There was no significant difference in fluorescence between MRS group and PEDV group.（3）Genetic test results indicate that:Compared with PEDV group,add 1/8 G8LABS to PEDV M,PEDV N,IL-8,IL-1βand IL-6 genes expression were significantly down regulated（P<0.05）;PEDV N,MCP-1,TNF-α,IL-1βgenes expression in 1/16LBSA were significantly up-regulated and IL-8 gene expression was significantly down regulated（P<0.05）;PEDV M,PEDV N,IL-8,MCP-1,TNF-α,IL-1βgenes expression in 1/8 MRS were significantly up-regulated and IL-6 gene expression was significantly down regulated（P<0.05）;MCP-1,TNF-αand IL-1βgenes expression were significantly up-regulated in 1/16 MRS,significantly down regulated the gene expression of IL-8（P<0.05）.Compared with the control group,PEDV group significantly increased the genes expression of IL-8,MCP-1,TNF-α,IL-1βand the relative expression of IL-6 gene（P<0.05）.The addition of 1/8 G8 LABS significantly downregulated the relative expression of PEDV M,PEDV N,IL-8,MCP-1,TNF-α,IL-1βand IL-6 genes compared with the 1/8 MRS group（P<0.05）.Compared with the 1/16 MRS group,the addition of 1/16 G8 LABS significantly downregulated the relative expression of IL-8 and IL-6 genes（P<0.05）,and up-regulated the relative expression of MCP-1 and IL-1βgenes（P<0.05）.In G33,compared with PEDV group,the relative expression of PEDV M,PEDV N,IL-6 and IL-8 genes in 1/8 labs were significantly down regulated,MCP-1 and TNF-αgenes expression were significantly up-regulated（P<0.05）;PEDV N,PEDV M,MCP-1,TNF-αand IL-1βgenes expression in 1/16 LBSA were significantly up-regulated,and IL-8,IL-6 genes expression were significantly down regulated（P<0.05）;The genes expression of PEDV M,PEDV N,IL-8,MCP-1,TNF-αand IL-1βin1/8 MRS were significantly up-regulated and significantly down regulated the relative expression of IL-6（P<0.05）;The genes expression of PEDV M,PEDV N,MCP-1,TNF-αand IL-1βwere significantly up-regulated in 1/16 MRS,and down regulated the gene expression of IL-6（P<0.05）.Compared with the control group,PEDV group significantly up-regulated the genes expression of IL-8,MCP-1 and TNF-αand IL-6（P<0.05）.The addition of 1/8 G33 LABS significantly downregulated the relative expression of PEDV M,PEDV N,IL-8,MCP-1,TNF-α,IL-1β,and IL-6 genes compared with the 1/8 MRS group（P<0.05）.Compared with the 1/16 MRS group,the addition of 1/16 G33 LABS significantly downregulated the relative expression of PEDV M,PEDV N and IL-1βgenes（P<0.05）,and up-regulated MCP-1,IL-6 and TNF-αgenes（P<0.05）.In G64,compared with PEDV group,the addition of 1/8 labs significantly reduced the relative expression of PEDV M,IL-8 and IL-6 genes,and significantly increased the genes expression of MCP-1 and IL-1β（P<0.05）;1/16 labs significantly down regulated the relative expression of IL-8 gene and up regulated the relative expression of IL-1β,MCP-1（P<0.05）;1/8 MRS significantly up-regulated the genes expression of IL-8,IL-6 and MCP-1 and down regulated the genes expression of IL-1β（P<0.05）;The genes expression of IL-1βand IL-8 were significantly up-regulated in 1/16 MRS（P<0.05）;Compared with the control group,PEDV group significantly increased the relative genes expression of IL-8,MCP-1 and IL-6（P<0.05）.Compared with the 1/8 MRS group,the addition of 1/8 G64 LABS significantly downregulated the relative expression of PEDV M,IL-1βand IL-8 genes（P<0.05）,and up-regulated the relative expression of IL-6 and MCP-1.Compared with the 1/16 MRS group,the addition of 1/16 G64 LABS significantly downregulated the relative expression of IL-1β,IL-8,and up-regulated the relative expression of MCP-1 and IL-6 genes（P<0.05）.In G1,compared with PEDV group,the addition of 1/8 labs significantly reduced the relative expression of PEDV N,PEDV M,MCP-1,IL-8 and IL-6genes,and significantly down regulated the gene expression of IL-1β（P<0.05）;1/16labs significantly up regulated the genes expression of PEDV N,PEDV M and IL-1βand down regulated IL-8,IL-6 and MCP-1 genes expression（P<0.05）;1/8 MRS upregulated the genes expression of PEDV N,IL-1βand significantly decreased the genes expression of IL-8,IL-6 and MCP-1（P<0.05）;IL-1βgene expression was significantly up-regulated in 1/16 MRS,and were down regulated the genes expression of MCP-1 and IL-6（P<0.05）.Compared with the control group,PEDV group significantly up-regulated the genes expression of IL-8,MCP-1,IL-6 and IL-1β（P<0.05）.Compared with the 1/8 MRS group,the addition of 1/8 G1 LABS significantly downregulated the relative expression of PEDV M,PEDV N,IL-1βand IL-8 genes（P<0.05）and up-regulated the genes expression of IL-6 and MCP-1 genes（P<0.05）.Compared with the 1/16 MRS group,the addition of 1/16 G1 LABS significantly downregulated the relative expression of IL-1βand IL-8 genes（P<0.05）and up-regulated the relative expression of MCP-1 genes（P<0.05）.The above results show that lactic acid bacteria supernatant can promote the growth of Vero cells to a certain extent,and the optimal concentration gradient is 1/8.PEDV can replicate and proliferate in Vero cells.Adding a certain amount of lactic acid bacteria supernatant when infected by PEDV virus has antiviral effect.Exp.3 Transcriptome analysis to explore the effect of labs on PEDV infected Vero cellsThe single factor test method was adopted in this test.The test was divided into P（PEDV）group,K（control）group,NP1-8（PEDV+1/8 labs）group,NP1-16（PEDV+1/16 labs）group and MRS（PEDV+MRS）group.Vero was cultured to 6-well plate,100 TCID₅₀PEDV and LABS of different concentrations were added at the same time,and the sample was collected with lysate after 48 hours of observation.Bioinformatics analysis was carried out by extraction,Illumina sequencing,sequence alignment analysis,expression difference analysis and i Path metabolic pathway analysis.The results showed that:（1）compared with the control group,there were 3297differential genes screened in the PEDV group,of which 1463 were up-regulated and1834 were down regulated.It mainly focuses on the regulation of exogenous apoptosis signal pathway,GTPase activity,MAPK signal pathway,FOXO signal pathway and so on.（2）Compared with PEDV group,a total of 2382 differential genes were screened in NP1-8 group.Among them,1420 were up-regulated and 962 were down regulated.The main pathways were Protein phosphorylation,Oxidative stress,positive regulation of GTPase activity,TNF signaling pathway,MAPK signaling pathway and so on.After adding labs,PEDV infected Vero cells had a total of 60 gene callback,such as GADD45B,AMPD3,GDF15,TRAF4,GAS1,CTSK,etc.Conclusion:PEDV infection can cause changes in exogenous apoptosis regulation,GTPase regulation,MAPK and Fox O signaling.LABS leads to alterations in protein phosphorylation,oxidative stress,and positive regulatory pathways of GTPase activity.