Molecular Mechanism Of Curcumin Inhibiting Classical Swine Fever Virus Replication

Curcumin is a fat-soluble phenolic pigment extracted from the the Curcuma longa.It is in the form of orange crystals.Its molecular formula is C₂₁H₂₀O₆.The relative molecular weight is 368.39.The main chain is unsaturated aliphatic and aromatic groups.At present,domestic and foreign studies have confirmed that it has natural biological activities,including anti-tumor,anti-mutation,anti-oxidation,anti-bacterial and anti-virus.Because it can improve animal immunity and promote animal growth,it has also been studied for its application in animal feed additives.Classical swine fever is a highly contagious infectious disease of pigs caused by classical swine fever virus,with high morbidity and mortality which caused huge economic losses.The typical features of CSF are fever and hemorrhage.As well as the reduction of leukocytes,it can also cause abortion in pregnant sows,which is extremely harmful to the pig industry.At present,the prevention and treatment of CSF in China is still based on vaccination to reduce the economic loss by reducing the outbreaks of CSF.Although commercial vaccines are now used in the pig industry and have a certain protective effect,it is still necessary to continue to develop new vaccines or antiviral agents due to the complex and variable strains of swine fever virus.Notably,curcumin exhibits inhibitory ability against the proliferation of diverse viruses,such as human immunodeficiency virus（HIV）,Zika virus（ZIKV）,Japanese encephalitis virus（JEV）,dengue fever virus（DENV）,porcine reproductive and respiratory syndrome virus（PRRSV）,transmissible gastroenteritis virus（TGEV）and so on.A large number of research results show that curcumin have effects on different signal pathways or metabolic pathways to resist the viral infections,reducing the release of infectious virus particles,and protecting host cells.However,there is no report confirm that curcumin can inhibit CSFV.In the early stage of this experiment,the inhibitory effect of curcumin on CSFV was proved.And we tested the effect of curcumin on lipid metabolism,and then the characteristics of promoting innate immunity were verified.At last,the specific mechanism of curcumin in inhibiting CSFV replication was explained.The specific research content is as follows:1 Study On Curcumin Inhibits Classical Swine Fever Virus InfectionThe PK-15 cells were infected with CSFV and JEV and treated with different concentrations of curcumin.Then we use RT-q PCR,Western blot,IFA and other methods to confirm the inhibitory effect of curcumin on CSFV,and set a series experiments to determine the exact stage to inhibit CSFV of curcumin.The results showed that curcumin inhibited CSFV in a time-and dose-dependent manner,and mainly affected the late stage of virus replication,and had nothing to do with the process of virus adsorption and internalization.Curcumin can also affect the infectivity of CSFV after co-incubation.Curcumin can inhibit JEV in a dose-dependent manner.In short,curcumin has good antiviral activity and can inhibit the replication of CSFV and JEV,supporting the follow-up research.2 Study On Curcumin Regulates Lipid Metabolism During the Replication of Classical Swine Fever VirusIn order to explore the specific mechanism of curcumin in the CSFV replication,firstly,the changes of fatty acid synthase（FASN）and lipid droplets（LDs）in infected virus cells after curcumin treatment were detected by confocal.Regardless of normal cells or virus-infected cells（CSFV,JEV）,curcumin treatment can inhibit the production and accumulation of fatty acid synthase（FASN）and lipid droplets（LDs）.In addition,cells infected with the virus can cause endoplasmic reticulum stress,which in turn activates the unfolded protein response,and upregulates the transcription factor ATF6,which is a related factor regulating lipid metabolism.A series of experiments proved that curcumin inhibited the up-regulation of ATF6 caused by CSFV infection.After knocking down ATF6,FASN expression was decreased.At the same time,the ability of curcumin to anti-CSFV disappeared.However,in the treatment group without knocking down ATF6,curcumin still showed antiviral activity,and it also inhibited the m RNA levels and the expression levels of ATF6 and FASN.3 Study On Curcumin Enhances Innate Immunity Response of PK-15 Cells with CSFV infectionCurcumin can exert a broad-spectrum antiviral effect,which proves that curcumin affects multiple signaling pathways.In order to explore the potential mechanism of curcumin against CSFV,the effect of curcumin treatment on the innate immunity of cells was explored.The m RNA levels of IRF3,IRF7,ISG1,IFN-α1 related to the type I interferon（IFN-I）pathway were detected by RT-q PCR,and it was found that curcumin can up-regulate the m RNA levels.After knocking down ISG15,the key factor of host cell antiviral ability,RT-q PCR verified the knockout efficiency and tested the virus content after drug treatment.It was found that curcumin could still exert antiviral effect after knocking down ISG15,but the effect was weakened.Using a commercial kit to detect the content of IFN-βin the cell supernatant,we found that curcumin can up-regulate IFN-β.This proves that curcumin enhances the innate immune response after viral infection.The effect of curcumin on the NF-κB pathway was detected by RT-q PCR and Western blot with the same treatment.Curcumin has no effect on p65 and phosphorylated p65 in the NF-κB pathway.In short,curcumin can enhance the innate immunity of host cells,promote the expression of ISG15,and at the same time it can up-regulate factors related to the type I interferon（IFN-I）pathway.