| Glaesserella parasuis(GPS),often causes Gl(?)sser’s disease in farms.GPS is one of the main bacteria that harm the breeding industry in the world,with symptoms such as high fever,dyspnea,peritonitis,pericarditis and meningitis.Under the global trend of banning anti-antibiotics in feed,and the effect of vaccines is unstable,it is urgent to find a new antibiotic alternative.GPS can cause vascular damage and inflammation,but the mechanism of vascular damage is not yet fully understood.It has been reported that lncRNAs(long non-coding RNA)and miRNAs(micro RNA)can regulate the inflammatory immune response.Baicalin is the main active ingredient of Scutellaria baicalensis Georgi.It has the functions of antibacterial,antiviral,antioxidant,anti-tumor,treatment and prevention of diabetes,protection of heart and brain vessels and nerves,protection of liver,enhancement of immunity and suppression of allergic reactions.Therefore,this experiment is to explore the effect of piglet vascular endothelial cells on the expression of lncRNAs and miRNAs after infected by GPS,explore the regulation effect of baicalin on the expression of lncRNAs and miRNAs.Through RNA-seq and lncRNA sequencing,miRNA sequencing,it is found that baicalin can regulate the expression of lncRNAs and miRNAs after GPS infection,thereby inhibiting vascular inflammation.1.The effect of baicalin on the RNA-seq and lncRNA expression of piglet vascular endothelial cells induced by GPSThe experiment set up 3 groups:control group,GPS group,and GPS+baicalin group.3 repetitions per group.Isolate the primary vascular endothelial cells,resuspend them in 10%fetal bovine serum M-199 without Penicillin-Streptomycin,count and dilute them to make the cell concentration in the system 1×10~6 cells/m L,and add 1 m L of cell suspension for each 1 m L of medium without Penicillin-Streptomycin,500μg/m L baicalin 0.25 m L was added to the baicalin treatment group.After 1 h of pretreatment,the baicalin group and GPS group were added with 0.25 m L of GPS diluted to 4×10~6 CFU/m L in the logarithmic phase.The control group was supplemented with medium to 2.5 m L and co-cultured After 12hours,the cells were collected,RNA was extracted,and sequenced using the Illumina Hiseq 2000 platform.Transcriptome results showed that after GPS stimulated vascular endothelial cells,4,067 genes were differentially expressed(Fold Change>2,P<0.05),of which1499 genes were up-regulated and 2568 genes were down-regulated.KEGG analysis showed that differential genes were mainly enriched in Toll-like pathway and tumor necrosis factor pathway.Compared with the GPS infection group,after GPS+baicalin treatment,492 genes were differentially expressed(Fold Change>2,P<0.05),of which 260 genes were up-regulated and 232 genes were down-regulated;KEGG analysis,enriched pathways Mainly focus on cytokine-cytokine receptor interaction,tumor necrosis factor signaling pathway,etc.Through the analysis of PPI diagram,it is found that TLR2,CCL5,CXR4,CD40 and other genes are located in the center of PPI.KEGG enrichment analysis of the 30 most connected genes in the PPI network revealed that these genes are mainly involved in cytokine-cytokine receptor interaction,Toll-like receptor signaling pathway,and chemokine signaling pathway.Q-RT PCR results showed that the expression of CCL5,GBP1,SAMHD1 was up-regulated after GPS was infected with vascular endothelial cells,while the gene expression was down-regulated after baicalin treatment.Therefore,we speculate that baicalin can reduce the infection of GPS by changing the expression of CCL5,GBP1,SAMHD1 and other genes,inhibiting the cytokine and cytokine receptor,TNF pathway and other signaling pathways involved in the gene.The lncRNA results showed that 584 lncRNAs were differentially expressed after GPS infection,of which 294 were up-regulated and 290 were down-regulated(Fold Change>2,P<0.05).Compared with the GPS infection group,the expression of27 lncRNAs was significantly different after baicalin treatment,11 of which were up-regulated and 16 were down-regulated.In this experiment,lncRNA target genes were predicted by cis and trans.A total of 18407 target genes were obtained,of which264 target genes were predicted by cis and 18167 target genes were predicted by trans.KEGG analysis of the target genes of differential lncRNAs found that after GPS infects porcine vascular endothelial cells,the corresponding target genes are mainly involved in the tumor necrosis factor signaling pathway,apoptosis,NF-κB signaling pathway,and N-polymerization.Sugar biosynthesis.Compared with the GPS group,after baicalin treatment,the corresponding target genes are mainly involved in propionic acid metabolism,pyrimidine metabolism,and tumor necrosis factor signaling pathways.The results of Q-RT PCR verification of the interaction between lncRNAs and target genes showed that the expression levels of lncRNA ALDBSSCT0000001677,ALDBSSCT0000001353,MSTRG.10724.2 and ALDBSSCT0000010434 were significantly up-regulated after GPS infection.After baicalin treatment,these 4 lncRNAs were significantly increased.The expression levels were all significantly down-regulated,which was consistent with the sequencing results.We speculate that baicalin may act on GBP1,SAMHD1,CCL5 and other target genes by regulating the expression of lncRNA ALDBSSCT0000001677,ALDBSSCT0000001353,MSTRG.10724.2 and ALDBSSCT0000010434,and reduce piglet vascular endothelial cells inflammation caused by GPS infection.2.The effect of baicalin on the expression of miRNAs in piglet vascular endothelial cells induced by GPSAccording to the same experimental treatment,the cells were collected for miRNA expression profile analysis.427 known miRNAs and 155 new miRNAs were obtained in 3 groups(control group,GPS infection group and baicalin treatment group).After infection by GPS,22 miRNAs were significantly differentially expressed in vascular endothelial cells.Among them:19 miRNAs were up-regulated and 3 miRNAs were down-regulated.The expression levels of ssc-mi R-375,ssc-mi R-155-3p and ssc-mi R-221-5p in piglet vascular endothelial cells infected with GPS were higher than those in the control group.Compared with the GPS group,54miRNAs were significantly differentially expressed in vascular endothelial cells after baicalin treatment,among which 4 miRNAs were up-regulated and 50 miRNAs were down-regulated.Compared with the GPS infection group,the expression of ssc-mi R-375,ssc-mi R-122-5p and ssc-mi R-200b in the baicalin treatment group were down-regulated the most.Using mi Randa to predict the possible target genes of miRNA,it was found that there were 5 differentially expressed miRNAs in the GPS infection group:ssc-mi R-221-5p,novel.114,novel.1,ssc-mi R-219a,ssc-mi R-146b,a total of 5868 predicted target genes were obtained,the main difference target genes are:ENDPD8,COL4A2,etc.KEGG analysis of differential target genes found that the main signal pathways involved in them are:actin cytoskeleton regulation,focal adhesion,ECM-receptor interaction,bacterial invasion of epithelial cells,adhesion and connection,etc.One differentially expressed miRNA in the baicalin group:ssc-mi R-192,99 predicted target genes were obtained,the main target genes are:EIF4E,MSN,etc.KEGG analysis of the target genes of miRNA:It is found that the main signal pathways involved in the target genes are:mismatch repair,peroxisome,oxidative phosphorylation,DNA replication and ABC transporter.In summary,GPS can change the expression of lncRNAs and miRNAs in vascular endothelial cells after infecting porcine vascular endothelial cells.Baicalin acts on SAMHD1 and CCL5 by regulating the expression of lncRNA ALDBSSCT0000001677,ALDBSSCT0000001353,MSTRG.10724.2 and ALDBSSCT0000010434.It can reduce the influence of GPS and other target genes;mainly by regulating miRNAs such as ssc-mi R-192 and its target genes EIF4E,MSN,etc.,reducing the vascular inflammation induced by GPS. |
