Myricetin Inhibits Pseudorabies Virus Infection By Regulating The CGAS/STING Signal Pathway

Pseudorabies virus（PRV）,also known as porcine herpesvirus type I,belongs to the family Herpesviridae,subfamily Herpesvirus A and genus Varicella.PRV is widely spread and its main host is the pig.At present,due to the emergence of PRV variants,the immune protection capacity of traditional vaccines is significantly reduced,so it is important to develop therapeutic drugs for PRV infection.c GAS/STING signalling pathway,the main intracellular DNA sensor,recognizes PRV infection and triggers a natural immune response,inducing type I interferon production,PRV inhibits this pathway and thus enables immune escape.Flavonoids have a variety of pharmacological activities and are an important source of potential anti-herpesvirus drugs.In this study,the anti-PRV activity of flavonoids and the regulatory effect on the correlated factors of the c GAS/STING pathway were screened to obtain candidate compounds,and then the regulatory effect on the c GAS/STING signaling pathway was verified in vivo and in vitro.The main research contents are as follows:1.Screening of flavonoids to modulate the c GAS/STING signalling pathwayCCK-8 was used to detect half of the toxic concentration of CC₅₀ and the inhibition rate of PRV of 25 flavonoids against PK-15 cells,and calculated the half inhibitory concentration IC₅₀.The results showed that the detection of kaempferol(IC₅₀=25.57μM),baicalin(IC₅₀=291.47μM),silymarin(IC₅₀=203.26μM),myricetin(IC₅₀=23.24μM),luteolin(IC₅₀=36.87μM),dihydromyricetin(IC₅₀=161.34μM),naringenin(IC₅₀=269.43μM)and liquiritigenin(IC₅₀=323.09μM)showed good inhibition of PRV activity after CCK-8.The effect of the above antiviral active compounds on the expression level of c GAS/STING signaling pathway-related genes（c GAS,STING,IRF3,IFN-β）after PRV infection was detected by real-time PCR.The results showed that PRV could significantly inhibit the expression of cytokines in this pathway（P<0.01）.Naringenin,baicalein,luteolin,and myricetin could significantly promote the m RNA expression level of c GAS（P<0.001）.Baicalein（P<0.01）,luteolin（P<0.001）,and myricetin（P<0.001）can significantly increase the m RNA level of STING.Myricetin can increase the expression levels of m RNA in IRF3 and IFN-β（P<0.001）.The above results preliminarily show that myricetin can regulate the c GAS/STING pathway to inhibit PRV infection.2.The modulation of myricetin on the c GAS/STING signaling pathway in PRV-infected cellsThe activation of myricetin on c GAS/STING signaling pathway was determined by detecting the content of the second messenger cyclic avianynylate（c GAMP）in cells,the m RNA expression level of relevant genes and effector factors in the c GAS/STING signaling pathway,the phosphorylation of IRF3 into the nucleus,and the expression of IRF3 and p-IRF3 proteins.The results showed that myricetin increased c GAMP content（P<0.001）;The m RNA expression levels of pathway-related genes（c GAS,STING,TBK1,IRF3 and IRF7）,pathway effector factors（IFN-β）,and related interferon-stimulatory genes（ISG54 and ISG56）were upregulated 2 h,6 h,and 12 h after virus infection（P<0.01）.Immunofluorescence tests showed that myricetin could increase the entry of p-IRF3 protein into the nucleus（P<0.01）;The Western blot test showed that myricetin increased the expression level of the p-IRF3 protein（P<0.01）.The above results show that myricetin can activate the c GAS/STING signaling pathway after PRV infection,thereby inhibiting the proliferation of PRV.3.Confirmation of myricetin regulating the c GAS/STING signaling pathway after PRV infectionA single c GAS/STING pathway cell model was constructed by overexpressing c GAS-HA and STING-flag on HEK-293T cells,and the expression of c GAS-HA,STING-flag,TBK1,IRF3 and p-IRF3 proteins was detected by Western blot method,and the expression level of IFN-βpromoter was detected by the double luciferase reporter gene detection system.The results showed that myricetin in 293T cells could promote the activation of the pathway by increasing the expression of c GAS,STING and downstream proteins TBK1 and p-IRF3（P<0.01）,and further enhance the transcription and synthesis of effector factor IFN-β（P<0.001）.Finally,in PRV-infected mice,the expression of genes and proteins related to the c GAS/STING signaling pathway in various organ tissues after myricetin treatment was detected,and the amount of IFN-βwas measured.The results showed that myricetin could upregulate the transcription level of the above c GAS/STING pathway-related genes in PRV-infected mice,increase the expression of TBK1 and p-IRF3 proteins（P<0.001）,and promote the production of IFN-β.The above results further confirm the regulatory effect of myricetin on the c GAS/STING signaling pathway after PRV infection.In summary,this study confirmed that the flavonoid myricetin can activate the c GAS/STING signaling pathway inhibited by PRV,improve the body’s natural immune response and thereby inhibit PRV infection,and the results elucidate the mechanism of action of myricetin against PRV replication,which provides a new idea for finding the prevention and control methods of PRV infection from flavonoids.