Protective Effect Of Quercetin On PRRSV-induced Inflammatory Response In Swines

Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which seriously endangers the pig industry.Since PRRSV has been epidemic in China,it has caused severe economic losses to pig farms.The inflammatory response plays a very important role in the pathogenicity of PRRSV.After PRRSV infection,the inflammatory cytokines will be overexpressed,resulting in damage to multiple tissues and organs,or even death in pigs.Quercetin(Qct)is a common polyhydroxy flavonoid compound that has many functions such as anti-inflammation,anti-virus,anti-oxidation,and anti-bacteria.However,research about the effect and mechanism of quercetin on improving PRRSV-induced porcine inflammatory response has not yet been reported.Therefore,in this study,we will explore the effect of quercetin on improving PRRSV-induced inflammatory response in vitro and in vivo,and screen the key metabolites in vivo through non-targeted metabolomics,thus clarify its mechanism whereby quercetin improves inflammatory response through regulating key metabolites.The main research contents and results are as follows:1.Study on the inhibitory effect of quercetin on PRRSV-induced inflammation in vitro.The 3D4/21 porcine alveolar macrophages were taken as the research object,and the drug toxicity test results by CCK-8 method showed that the maximum safe concentration of quercetin in 3D4/21 cells was 200 μmol/L;the half-toxic concentration of the virus(TCID50)calculated by Reed-Muench method was 10-5.79/100 μL;q RT-PCR was used to determine the changes of IL-6 and TNF-α m RNA expression at different time points after PRRSV infection in 3D4/21 cells,the changes of IL-6 and TNF-α m RNA expression after PRRSV infection in 3D4/21 cells with different titers,and the changes of IL-6 and TNF-α m RNA expression in PRRSV-infected 3D4/21 cells treated with different concentrations of quercetin.The results of q RT-PCR showed that PRRSV infection in 3D4/21 cells could increase the expression of TNF-α and IL-6 m RNA,and the optimal concentration was 1 MOI,and the optimal time was 3 h;200,100,50 and 25 μmol/L of quercetin could significantly suppress the PRRSV-induced expression of IL-6 and TNF-α m RNA in 3D4/21 cells.2.Effect of quercetin on inflammation of weaned piglets infected with PRRSVIn clinical trials,piglets infected with PRRSV were fed with quercetin at 12.5mg/kg,25 mg/kg and 50mg/kg basal diet.A blank control group and a virus control group were set up to determine the effect of quercetin on the growth of piglets.The changes of serum IL-1,IL-6,IL-8,TNF-α and other inflammatory cytokines were detected by ELISA.The relative expression levels of IL-1β,IL-6 and IL-10 m RNA in lung tissues were determined by q RT-PCR.And to observe pathological change of lung tissue with paraffin section.The results showed that medium and high doses of quercetin could reduce the levels of IL-1,IL-6,IL-8 and TNF-α cytokines in serum of infected piglets.Low,medium and high doses of quercetin could reduce the levels of IL-1β and IL-6 m RNA in the lung tissue of infected piglets.Indicating that quercetin had the effect of improving PRRSV-induced inflammation.At the same time,low,medium and high doses of quercetin could reduce the relative expression of IL-10 m RNA in the lung tissue of infected piglets,indicating that quercetin might promote the immune response in lung tissue by reducing the secretion of immunosuppressive cytokines.The histopathological examination showed that the lung tissues of the piglets in PRRSV group were characterized by interstitial pneumonia with a large number of inflammatory cell infiltration,and a small number of hemorrhagic spots on the surface of the lung.Quercetin improves lung pathological changes induced by PRRSV infection in piglets.3.The mechanism of quercetin inhibits PRRSV-induced inflammation based on metabonomics.The serum of the control group,PRRSV group,and the quercetin medium dose group were collected and detected using the metabonomics technology based on ultra-performance liquid chromatography-tandem triple and quadruple time-of-flight mass spectrometry(UHPLC-Triple-TOF-MS).The principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were used to analyze the serum metabolic profiles of the three groups.Important differential metabolites were screened based on VIP value of principal component of OPLS-DA model and P value of t test.Results show: Non-targeted metabolomics finally identified 13 key endogenous differential metabolites of quercetin against PRRSV,namely,Dopamine,Glycerol 3-phosphate,2-Methoxyestradiol-3-methylether,5-Hydroxyindoleacetic acid,Prostaglandin H2,Bisdiphosphoinositol tetrakisphosphate,Lyso PC(O-18:0/0:0),3-Dehydroquinic acid,Ethyl(2E,6Z)-dodecadienoate,(Z)-8-Tetradecenal,trans-and cis-2,4,8-Trimethyl-3,7-nona-dien-2-ol,Nnal-N-oxide,3-(3,5-Diiodo-4-hydroxyphenyl)lactate.Among them,the most important differential metabolite is glycerol-3-phosphate,and enrichment analysis of metabolic pathways revealed that the main pathways for quercetin to improve PRRSV-induced inflammatory response are glycerol-phospholipid metabolism and arachidonic acid metabolism.In summary,quercetin can improve the inflammatory response by inhibiting the inflammatory cytokines induced by PRRSV.Moreover,the main pathways of quercetin improving the inflammatory response induced by PRRSV are glycerophospholipid metabolism and arachidonic acid metabolism.This study provides a theoretical basis for the development of quercetin as an anti PRRSV inflammatory agent.