The Effects Of Baicalin On TLRs/NF-κB And NLRP3/caspase-l Signal Pathways In Porcine Aortic Vascular Endothelial Cells After Haemophilus Parasuis Infection

Haemophilus parasuis（HPS）,is a constant bacteria colonized in the upper respiratory tract of swines,and cause Gl?sser’s disease,which has brought huge economic losses in the pig industry.The typical characteristics of this disease is meningitis,serositis and arthritis,when severe infection will cause septicemia and result in sudden death of the pig.The characteristics of Haemophilus parasuis disease is cellulitis,and the bacteria can lead to vascular endothelial cell damage and systemic inflammatory response.In this study,the primary porcine vascular endothelial ce lls were successfully isolated and the model of cell infection of swine vascular endothelial cells infected with Haemophilus parasuis was established.The effect of baicalin on the inflammatory response of Haemophilus parasuis infected with porcine vascular endothelial cells,and the role of baicalin on inhibition the inflammatory responses in TLRs/NF-κB and NLRP3/caspase-1 signaling pathway after porcine vascular endothelial cells infected Haemophilus parasuis were explored.1.Isolation and culture of primary porcine vascular endothelial cells and establishment of cell model of porcine vascular endothelial cells infected by Haemophilus parasuis in vitro.The porcine aorta was sterilized and the porcine aorta was digested with typeⅠcollagenase.The porcine vascular endothelial cells were cultured for one week,and when the cells were covered,use trypsin digested the cells.Set different infection concentration of Haemophilus parasuis and interaction time,the release of inflammatory cytokines TNF-α,IL-1βand IL-18 from the cell curture supernatant were measured by ELISA.The results showed that,the MOI at 1:1 was considered as the infection model of inflammatory response triggered by Haemophilus parasuis.2.Effect of baicalin on TLRs/NF-κB signaling pathway of porcine vascular endothelial cells infected by Haemophilus parasuis.The experiment was set up the control group,Haemophilus parasuis model group,NAC drug-treated group,baicalin drug-treated groups（12.5,25,50,100μg/mL）.Drug-treated groups were treated with the appropriate concentrations of baicalin and NAC for 1 h,and then the final concentration of 10⁵ CFU/mL Haemophilus parasuis was added into the plates and co-cultured for 12 h.The productions of inflammatory cytokines（IL-6,IL-8,IL-10,PGE₂,and COX-2）from the cell culture supernatants were measured by ELISA assays.Q-RT PCR was used to determine the mRNA expression of intracellular inflammatory cytokine（IL-6,IL-8,IL-10）and inflammatory mediators（COX-2）and nuclear transcription factor NF-κB;fluorescence immunoassay was used to determine the nuclear transcription factor NF-κB.The results showed that the nuclear transfer of NF-κB p65 was significantly increased compared with the model group,and the nuclear transfer of NAC-treated group and baicalin-treated groups were significantly decreased after vascular endothelial cells infected by Haemophilus parasuis.In Haemophilus parasuis model group,the release of inflammatory cytokines IL-6,IL-8,IL-10,inflammatory mediators PGE₂ and intracellular inflammatory mediators COX-2 and intracellular mRNA expression levels of IL-6,IL-8,IL-10,COX-2 in the cell culture supernatant significantly increased compared with the control group,and in baicalin drug-treated groups and NAC drug-treated group,the release of inflammatory cytokines IL-6,IL-8,IL-10,inflammatory mediators PGE₂ and intracellular inflammatory mediators COX-2 and intracellular mRNA expression levels of IL-6,IL-8,IL-10,COX-2 in the cell culture supernatant decreased or significantly decreased compared with the control group.3.Effect of baicalin on NLRP3/caspase-1 signaling pathway of porcine vascular endothelial cells infected by Haemophilus parasuis.The experiment set up the control group,Haemophilus parasuis model group,NAC drug-treated group,baicalin drug-treated groups（12.5,25,50,100μg/mL）.Detecting intracellular ROS generation and cell apoptosis.The release of inflammatory cytokines（TNF-α,IL-1βand IL-18）from the cell culture supernatants,and the mRNA expression level of inflammatory factors and inflammatory corpuscle receptor,and the expression levels of cleaved caspase-1 protein,and to investigate the effect of Haemophilus parasuis on the activation of NLRP3 pathway and the effec t of baicalin.The results showed that Haemophilus parasuis significantly increased the generation of intracellular ROS and cell apoptosis（p<0.01）;protein expression levels of TNF-α,IL-1βand IL-18 in cell supernatants were significantly increased;At the same time the levels of mRNA expression of IL-1β,IL-18,TNF-αand NLRP3 were significantly increased;Cleaved caspase-1 p20 protein expression level was also up-regulated（p<0.01）.Baicalin concentrations at 12.5,25,50 and 100μg/mL were able to down-regulate the mRNA expression of IL-1β,IL-18,TNF-αand NLRP3,reduce the secretion of TNF-α,IL-1βand IL-18 in cell supernatant and Cleaved caspase 1-p20 protein expression and inhibit the release of intracellular ROS and cell apoptosis（p<0.05）.Taken together,our results demonstrated that Haemophilus parasuis could activate TLRs/NF-κB and NLRP3/caspase-1 signaling pathway in porcine vascular endothelial cells,while baicalin could reverse the inflammatory effect initiated by Haemophilus parasuis and possesses significant immunosuppression activity,which may represent a promising therapeutic agent for treatment of Haemophilus parasuis infection.