Studies Of The Molecular Mechanism By Which Quercetin Suppresses BVDV Replication Through The Regulation Of HSP70 Expression And Oxidative Stress

Bovine viral diarrhea virus（BVDV）is a positive-stranded RNA virus of the genus Plague virus in the family Flaviviridae.BVDV has caused huge impact and economic losses on the cattle industry,and the use of antiviral drugs is an attractive strategy to prevent infection in newborn calves.However,very few antiviral agents are currently available that can protect against BVDV infection.The phytoflavonol quercetin has multiple health-promoting and immunomodulatory effects in humans and animals and possesses potential as an antiviral drug.Heat shock protein70（Heat shock protein70,HSP70）,one of the most abundant and typical HSPs,plays an important role in flaviviridae virus infections and is considered a logical target for viral regulation in the context of immune escape.Quercetin inhibits the synthesis of HSP in various cells,however,it is not clear whether quercetin can inhibit BVDV virus replication by regulating the expression of HSP70 and thereby.Therefore,the aim of this study was to evaluate the effect of quercetin on BVDV virus replication in vitro and in vivo by regulating HSP70 and to elucidate its mechanism of action.First,the effect of HSP 70 on BVDV（CP/NCP）replication in MDBK cells was investigated.HSP70 overexpression lentivirus and HSP70 siRNA were constructed,respectively,and transfected into MDBK cells to verify the overexpression and knockdown.Then MDBK cells were infected with BVDV（CP/NCP）,and the effect of overexpression and HSP70 knockdown on BVDV virus replication was detected at 12 h,24 h and 48 h,respectively.The results showed that the successful construction of overexpression HSP70 lentivirus（3*10⁸TU/mL）and HSP70siRNA interference plasmid.When infected with the virus,the mRNA and protein expression levels of HSP70 and virus in the overexpression group were higher than those in the control group（P<0.05）,and the expression of HSP70 and virus increased relatively with time,while the knockdown group HSP70 siRNA was the opposite（P<0.05）.Then,the molecular mechanism of quercetin regulating the replication of HSP70 to BVDV virus in MDBK cells was investigated.The interaction between quercetin protein and BVDV protein was first identified using molecular docking and protein thermal migration assays and examined quercetin to MDBK cytotoxicity by CCK8.Then quercetin was pretreated and post-treated to infect BVDV（CP/NCP）,respectively,and the mRNA and protein levels of HSP70 and BVDV were detected,and the antiviral activity was assayed by TCID₅₀.The results showed that the quercetin can bind to NADL-E2,and quercetin binds stably to the NADL-E2protein interaction.The safe concentration range of quercetin was 20-100μmol/L.After infection with the virus,TCID₅₀gradually decreased with increasing quercetin concentration（P<0.05）,while HSP70 and BVDV mRNA and protein levels also decreased in a dose-dependent manner.The mRNA and protein levels of HSP70 and BVDV in pretreated MDBK cells were significantly lower than those in post-treated group（P<0.05）.Secondly,this study examined the effects of quercetin/HSP70 siRNA on anti-oxidation genes and oxidative stress in MDBK cells.The results showed that quercetin could significantly promote the expression of antioxidant genes,while HSP70 siRNA did the opposite.After virus infection,CP-type BVDV could significantly stimulate ROS production,while NCP-type could not.At the same time,BVDV stimulated an increase in MDA,resulting in a decrease in antioxidant enzymes.When treated with quercetin and HSP70 siRNA,it relatively reduced the production of MDA and ROS and promoted the expression of antioxidant enzymes.Then,the present study examined the effect of quercetin/HSP70 siRNA on the ERK pathway,MDBK cells were treated with quercetin and HSP70 siRNA and infected with BVDV（CP/NCP）to detect ERK/P-ERK expression.The results demonstrated that quercetin inhibition and siRNA interference with HSP70 significantly decreased CP P-ERK levels（P<0.05）,while the NCP group showed no change（P>0.05）.Finally,the protective effect of quercetin in mice acutely infected with BVDV was investigated.BVDV model mice were treated with different concentrations of quercetin,and virus-infected group,blank control group and drug control group were set up.It was determined by clinical observation,viral load,expression levels of IFN-γand IL-2,antioxidant genes,oxidative stress,and antioxidant enzyme indexes,and performed pathohistological analysis.The results showed that quercetin could reduce the viral load,inhibit the expression of HSP70,relieve the immunosuppression caused by BVDV,and relieve the pathological tissue damage of spleen and duodenum in mice with acute BVDV infection.Meanwhile,quercetin treatment significantly restored the decrease in IL-2 levels of IFN-γin the serum of mice caused by BVDV（CP/NCP）infection.In addition,quercetin treatment upregulated the expression of（Nrf2）,（HO-1）and（NQO-1）,significantly reduced（ROS）and MDA production and promoted antioxidant enzyme activity.Pathological tissue analysis showed that quercetin alleviated the histopathological damage caused by BVDV.In conclusion,high HSP70 expression favors BVDV replication.Quercetin both stably binds to BVDV-NADL-E2 protein and suppresses HSP70 expression and BVDV（CP/NCP）replication at the early stage of viral replication.The use of quercetin and HSP70 siRNA significantly reduced the ROS produced by CP type BVDV induction.Meanwhile,quercetin increased the antioxidative stress level in MDBK cells and affected the expression of ERK/P-ERK,induced by CP type BVDV,by regulating HSP 70 levels.Moreover,quercetin was able to reduce the viral load in mice acutely infected with BVDV,alleviate the immunosuppression andtissue damage of spleen and duodenum by BVDV,and improve the resistance against oxidative stress in mice.Therefore,this study provides basic data to explore the mechanism of quercetin regulation of HSP70 expression on BVDV replication and oxidative stress in host cells,while providing a new strategy for drug therapy against BVDV infection.