The Effects Of Baicalin On PKC-MAPK Singal Pathway In Porcine Vascular Endothelial Cells Infected By Haemophilus Parasuis

Haemophilus parasuis(HPS)is a common colonizing bacterium in the upper respiratory tract of pigs and can cause Gl?sser’s disease,causing huge economic losses to the pig industry.The main pathological features of the disease are fibrinous polyserositis,meningitis,and arthritis.Severe infections cause sepsis and lead to the death of pigs.In this experiment,the vascular endothelial cells infected with Haemophilus parasuis was used as a model,and baicalin was used as an intervention drug to study the effects of baicalin on apoptosis and vascular injury after infection with porcine vascular endothelial cells by Haemophilus parasuis.The role of PKC-MAPK signaling pathway in baicalin-induced apoptosis of vascular endothelial cells infected by Haemophilus parasuis was preliminarily explored.In addition,we performed transcriptome sequencing of H.parasuis-infected vascular endothelial cells to explore other molecular mechanisms that may be associated with vascular injury.1.Effect of baicalin on PKC-MAPK signaling pathway in porcine vascular endothelial cells infected by Haemophilus parasuis.The experiment was conducted in control group,Haemophilus parasuis infection model group,HPS and acetylcysteine(NAC)drug control group and HPS and baicalin drug treatment group(12.5,25,50,100μg/mL).The drug group was pretreated with 1 mM NAC and baicalin at a corresponding concentration for 1 h,then vascular endothelial cells were infected with Haemophilus parasuis at a final concentration of 4×10~6 CFU/mL,and cultured for 12h before Western blot analysis for PKC-MAPK signaling pathway related proteins(PKC-α,p-PKC-α,PKC-δ,p-PKC-δ,p38,p-p38,ERK,p-ERK,JNK,p-JNK,Cleaved Caspase-3)expression levels;Membrane receptor Rage,apoptotic gene(Bax,Bcl-xl,C-myc,Fasl)and nuclear transcription factor AP-1(c-fos,c-jun)mRNA expression level were measured by Q-RT PCR method;Nuclear transfer factor AP-1 nuclear metastasis was observed by immunofluorescence single marked method;Cellular deformation and HPS infection patterns were observed by transmission electron microscopy.The results showed that PKC-MAPK signaling pathway protein(p-PKC-α,p-PKC-δ,p-p38,p-ERK,p-JNK,Cleaved Caspase-3)expression levels after infection with vascular endothelial cells by Haemophilus parasuis and mRNA expression levels of membrane receptor Rage,apoptotic genes(Bax,Bcl-xl,C-myc,Fasl)and nuclear transcription factor AP-1(c-fos,c-jun)were significantly up-regulated(p<0.01).Nuclear transcription factor AP-1(p-c-jun fluorescent labeling)nuclear transfer phenomenon and cell deformation are very obvious.HPS infection of vascular endothelial cells may be attached to the cell membrane and invade intracellular both.After drug intervention,50 and 100μg/mL baicalin significantly down-regulated PKC-MAPK signaling pathway proteins(p-PKC-α,p-PKC-δ,p-p38,p-ERK,p-JNK,Cleaved Caspase-3)expression level and mRNA expression level of membrane receptor Rage,apoptotic gene(Bax,Bcl-xl,C-myc,Fasl),nuclear transcription factor AP-1(c-fos,c-jun)(p<0.01),but also can significantly inhibit the nuclear transfer of AP-1 and improve cell deformation.2.Effects of baicalin on cell cycle,reactive oxygen species release and apoptosis of porcine vascular endothelial cells infected by Haemophilus parasuis.The experiment was conducted in control group,Haemophilus parasuis infection model group,HPS and acetylcysteine(NAC)drug control group and HPS and baicalin drug treatment group(12.5,25,50,100μg/mL).The drug group was pretreated with 1 mM NAC and baicalin at a corresponding concentration for 1 h,and then added to the vascular endothelial cells at a final concentration of 4×10~6 CFU/mL of Haemophilus parasuis,and the cells were cultured for 12 h using flow cytometry detection of cell cycle,release of reactive oxygen species,and apoptosis.The results showed that the infection of vascular endothelial cells by Haemophilus parasuis caused a significant S phase arrest(p<0.01),increased release of reactive oxygen species,and significant apoptosis(p<0.01).After intervention with drugs,baicalin at all concentrations can significantly alleviate the S phase arrest(p<0.01).All of them can significantly inhibit the release of reactive oxygen species and significantly improve the occurrence of apoptosis(p<0.01).3.Effect of Baicalin on Cell Adhesion Molecules Infected with Vascular Endothelial Cells of Haemophilus Parasuis.The experiment was conducted in control group,Haemophilus parasuis infection model group,HPS and acetylcysteine(NAC)drug control group and HPS and baicalin drug treatment group(12.5,25,50,100μg/mL).The drug group was pretreated with 1 mM NAC and baicalin at a corresponding concentration for 1 h,then vascular endothelial cells were infected with Haemophilus parasuis at a final concentration of 4×10~6 CFU/mL,and cultured for 12h.ELISA detected release of cell adhesion molecules(E-selectin,ICAM-1,VCAM-1)in the fluid;mRNA expression of cell adhesion molecules(E-selectin,ICAM-1,VCAM-1)was measured by Q-RT PCR.The results showed that the release of E-selectin,ICAM-1,VCAM-1 and the expression levels of E-selectin,ICAM-1 and VCAM-1 in cell culture medium after infection with vascular endothelial cells by Haemophilus parasuis all significantly up-regulated(p<0.01).With the addition of drug intervention,100μg/mL baicalin significantly inhibited the release of E-selectin,ICAM-1,VCAM-1 in cell culture medium and the mRNA expression level of E-selectin,ICAM-1,and VCAM-1 in cells.4.Transcriptome sequencing analysis of porcine vascular endothelial cells infected with Haemophilus parasuis.Two treatment groups,the control group and the HPS model group,were set up in the experiment,with 3 replicates in each group.The vascular endothelial cells were infected with Haemophilus parasuis at a final concentration of 4×10~6 CFU/mL.After 12 hours of co-culture,RNA samples were extracted and subjected to transcriptome sequencing.The 12 differentially expressed genes were verified by Q-RT PCR.The results showed that 281 genes were significantly different(p<0.05)after H.parasuis infection of vascular endothelial cells,of which 236 genes were up-regulated and 45 genes were down-regulated.Among the 12 genes selected,compared with RNA-seq data,9 genes showed similar expression levels,and 1 gene showed no significant changes in expression levels.FoxO signaling pathway was very active and involved in the process of bacterial infection of vascular endothelial cells.In summary,Haemophilus parasuis infection of porcine vascular endothelial cells can activate PKC-MAPK signaling pathway and induce apoptosis,and damage endothelial cells through multiple pathways.Baicalin may exert its anti-apoptotic effect by inhibiting PKC-MAPK signaling pathway.The results provide a new strategy for the prevention and treatment of Gl?sser’s disease.