In Vitro Antiviral Efficacy And Mechanism Of Caffeic Acid Against Canine Distemper Virus

Canine Distemper?CD?is a common viral disease in dogs and carnivores.It is caused by Canine Distemper Virus?CDV?.The disease has a wide range of hosts and varied clinical symptoms,which has brought great difficulties in the diagnosis and treatment of the disease.The mechanism of the disease has not been clearly studied yet.Chinese medicine is the main component of natural medicines and it has antiviral activity.The subject of this study is coffee acid.Firstly,the activity of caffeic acid anti-CDV is studied in vitro,it's determined that it has anti-CDV effect and select the best inhibitory concentration,and then the mechanism was explored on the host cell.Secondly,explore mechanisms on the host cell.This study will investigate the effects of caffeic acid on CDV-infected cells in response to inflammation induced by caffeic acid,Nectin-4 receptor,TLR3 expression,inflammatory pathways and apoptosis in vitro.The cytotoxicity of caffeic acid and ribavirin on vero cells and the toxicity of caffeic acid on MDCK cells were measured by MTT assay to determine the half cell cytotoxicity(CC₅₀).96-well plates were incoulated with 100TCID₅₀ virus solution to determine the optimal inhibitory concentration of caffeic acid against CDV.Three modes of administration?adding the drug and then inoculating the virus,the virus and the drug at the same time,and then administering the virus?work on Vero cell for two hours,after that discard the supernatant.Add different doses of caffeic acid.According to the CPE method,the antiviral effect of caffeic acid was measured.It was found that caffeic acid had a therapeutic effect on CDV in vitro.The inhibitory virus concentrations were 23.3?g/m L and 32.3?g/m L during 1h and 2h,respectively.And the therapeutic indices were all greater than 6.Time-of-addition study to detect caffeic acid ability to resist viruses in vitro.The results showed that caffeic acid can significantly inhibit the virus progeny in both 1h and 2 h.Caffeic acid synergistic positive drug ribavirin canprevent the reproduction of CDV better.The one-step fluorescence quantitative PCR assay showed that the viral nucleic acid content was lower than that of the virus control group after treatment.It showed that caffeic acid could significantly inhibit the proliferation of CDV in vitro with increasing gradients.Useing fluorescence quantitative PCR to detect the proliferation of CDV on MDCK cells at different time points.The results showed that the virus had the highest yield in 24h and gradually decreased during 48h and 72h.75?g/m L and50?g/m L of caffeic acid can significantly inhibit the reproduction of virus progeny within 24h and 48h of CDV-infected cells.The gene expression of IL-6,IL-1?,TNF-?,IL-4,IL-10 and TGF-?produced by caffeic acid in CDV-infected MDCK cells was detected by real-time PCR.The results showed that 75?g/m L and 50?g/m L caffeic acid significantly inhibited IL-6 mRNA expression,75?g/m L,50?g/m L and 25?g/mL caffeic acid could significantly inhibit the m RNA expression of TNF-?,75?g/m L of caffeic acid significantly inhibited the expression of IL-1?mRNA.CDV can significantly increase the expression of two inflammatory cytokines,TGF-?and IL-4,and different concentrations of caffeic acid can promote the expression of anti-inflammatory cytokines.The effect of caffeic acid on the secretion of IL-6,TNF-?,IFN-?and IFN-?in CDV-infected MDCK cells was detected by ELISA;the results showed that CDV can significantly increase the secretion of IL-6,IFN-?and IFN-?.75?g/mL and 50?g/m L of caffeic acid can significantly reduce IL-6 secretion but have no significant effect on the secretion of IFN-?and IFN-?.The expression of Nectin-4 receptor and TLR3 was detected by real-time fluorescence quantitative PCR.The results showed that the expression of Nectin-4 mRNA was significantly increased in CDV-infected MDCK cells after 72h,and different concentrations of caffeic acid significantly inhibited the Nectin-4 receptor mRNA expression.CDV infected MDCK cells could significantly promote the expression of TLR3 mRNA,75?g/m L and 50?g/mL caffeic acid could significantly up-regulate the expression of TLR3.MDCK cells were infected with poly?I:C?and the expression of TLR3 in caffeic acid-infected MDCK cells was detected.The results showed that 75?g/m L caffeic acid could significantly reduce the expression of TLR3.Western blot was used to detect the expression of ERK,SRC and P65 protein.The results showed that CDV could promote the up-regulation of p-p65,50?g/m L and 25?g/m L caffeic acid could significantly down-regulate the expression of p-p65.The cells began to show apoptosis after being infected with MDCK cells for 72h with high concentration of CDV.Flow cytometry using Annexin-V,JC-1 and Caspase3 enzymes showed that after 72h of CDV infection,cells showed significant apoptosis,and different concentrations of caffeic acid could significantly promote apoptosis.Aboveall,caffeic acid has a therapeutic effect against canine distemper virus infection in vitro,and caffeic acid reduces the damage caused by inflammatory factors by modulating pro-inflammatory factors released by CDV-infected cells.Caffeic acid can upregulate the expression of TLR3 and promote the body's antiviral effect by down-regulating CDV-induced p65 expression.Caffeic acid acts as an antiviral infection by promoting apoptosis of cells infected with CDV.The results showed that caffeic acid can be used as a drug against canine distemper virus.