Study On The Activity And Mechanism Of Nitrogenous Heterocyclic Coumarin Derivatives Against Spring Viremia Of Carp Virus

China is the largest output of aquaculture in the world,among which carp plays an important role in freshwater aquaculture,accounting for at least 60%.Spring viremia of carp(SVC)caused by spring viremia of carp virus(SVCV)is a major viral disease of cyprinidae,which is one of the animal diseases that must be reported by the Office International des Epizooties(OIE),and is listed B in Chinese animal epidemic diseases directory.SVCV infectionis highly lethal in young fish,with mortality rates up to 90%,and thus causes substantial economic losses to the aquaculture industry.Up to now,there is no commercial drug for effective control of SVC,it is difficult to eradicate the virus from affected ponds once the infection is established and elimination of the virus may require destruction of all aquatic life.Therefore,it is necessary to develop effective and low toxicity drugs against SVCV.The natural products have the advantages of low toxicity,easy degradation and environmental friendliness,it is an ideal source of lead compound in aquatic antiviral drugs.In this study,22 nitrogenous heterocyclic coumarin derivatives were designed and synthesized for evaluating the anti-SVCV activity.The main results are as follows:1.Synthesis and identification of nitrogenous heterocyclic coumarin derivatives.On the basis of previous studies,we synthesized coumarin derivatives by Wiiliamson’s etherification reaction,substitution reaction and nucleophilic reaction.The compounds were separated and purified by recrystallization,extraction and silica gel column chromatography.The structures of the compounds were identified by spectroscopic techniques.Finally,22 nitrogenous heterocyclic coumarin derivatives were obtained.2.Studies on anti-SVCV activity of derivatives in vitro.The anti-SVCV activity of 22 derivatives was determined by in vitro antiviral activity screening model.The results showed that nearly half of the derivatives were found to inhibit the expression of SVCV N protein by more than 50% through preliminary screening.Based on previous research experience,we identified these coumarins as active derivatives.After re-screening,two derivatives namely S5 and N6 inhibited the expression of SVCV N protein97.6% and 94.9%,respectively.The concentration gradient test further found that S5 and N6 inhibited the expression of G,P and M proteins of SVCV by the highest rate of more than 90%.Then,anti-SVCV activity was further confirmed by virus titer detection and observing the CPE changes induced by SVCV after S5 and N6 treatment.Additionally,the typical apoptotic characteristics of EPC cells induced by SVCV infection were observed by fluorescence staining,including apoptotic body formation,nuclear shrinkage,cell membrane rupture and cytoskeleton disintegration.In situ fluorescence staining and flow cytometry analysis showed that the number of apoptotic cells increased significantly 48 h after SVCV infection,and 78.40% of the cells were apoptotic.However,the apoptosis-related morphological characteristics were significantly inhibited after S5 and N6 treatment,and the proportion of apoptotic cells decreased to 13.25% and21.40%,respectively.The results showed that S5 and N6 had good anti-SVCV activity and could significantly inhibit the apoptosis induced by SVCV.3.The anti-SVCV study of S5 through NF-κB signaling pathway.The effect of S5 on NF-κB signaling pathway was studied to explore the anti-SVCV mechanism.The results of the ultracentrifugation showed that the amplification efficiency of SVCV N protein gene remained unchanged after 30 min co-incubation of the drug and virus,indicating that S5 could not directly act on the virion and would not reduce the early infection and proliferation of the virus.On the other hand,binding experiment confirmed that S5 also had no significant effect on the binding of virus to host cell surface receptors.The time-of-addition/removal showed that S5 played a role in the middle or late stage of infection.Subsequently,we further determined the effect of S5 on the NF-κB pathway.The results showed that the NF-κB pathway was activated rapidly within 48 h of virus infection,and the protein level of p50 and p65 proteins were significantly increased,which were up-regulated by 250% and 230%,respectively.After S5 treatment,p50 and p65 proteins in the nucleus were down-regulated,which were reduced by 32% and 28% compared with the control proteins,respectively.On the other hand,drug plus virus treatment group also showed significant effect,and there was no significant difference in NF-κB activity compared with the control group.In addition,S5 decreased the phosphorylation level of IκB,the upstream protein responsible for NF-κB activity.In conclusion,derivative S5 plays an anti-SVCV role mainly by inhibiting the initiation of NF-κB signaling pathway.4.The anti-SVCV study of N6 through p53 signaling pathway.The experiments of p53 inhibitor further proved that p53 is a key protein in SVCV-induced cell apoptosis.Inhibition of p53 showed significant antiviral effect on EPC cells,and reduced the number of the virus.By detecting antioxidant enzyme activity,it was found that SVCV infection induced oxidative stress damage in EPC cells,including decreased GSH and SOD levels,increased MDA levels,and significantly inhibited total antioxidant capacity T-AOC.In situ fluorescence staining and flow cytometry analysis showed that reactive oxygen species(ROS)were significantly increased at 48 h after infection with SVCV.Compared with 24 h control group,the fluorescence value of EPC cells after infection with SVCV increased from 86.65 ± 4.13 to 157.52± 5.23.At 48 h after infection,the fluorescence value increased from 88.93 ± 4.18 to 252.61 ± 7.72.The results of apoptosis related protein and immunofluorescence assay showed that coumarin N6 could effectively inhibit the expression of pro-apoptotic protein Bax,and promote the expression of anti-apoptotic protein Bcl-2,and effectively control the initiation of SVCV-induced apoptosis signal of caspase.In addition,it is noteworthy that SVCV upregulated the expression of total p53 protein and further regulated the activation of downstream related target proteins by promoting the phosphorylation level of Ser46,which was increased by 230% compared to the control group.By detecting the expression of p53 after N6 treatment,it was found that N6 could significantly inhibit the upregulation of p53 protein induced by the virus and inhibit the phosphorylation of p53.5.Study on the anti-SVCV activity of coumarin S5 and N6 in vivo.These results suggest that N6 plays an anti-SVCV role by inhibiting p53 phosphorylation and reducing the expression of downstream pro-apoptotic proteins.5.Study on anti-SVCV activity of S5 and N6 in vivoIn vivo experiments were conducted to investigate the anti-SVCV activity.Firstly,an in vivo experimental model was established to determine the safe dosage of the two drugs.The maximum safe dose for S5 and N6 was determined to be 10 μg/fish.Virus challenge experiment result showed that the survival rate of treat with S5 and N6 increased by 47.5% and 37.5%,respectively.At the same time,the expression of SVCV N protein in spleen and kidney was significantly decreased at 1,2 and 4 days after treatment,which reflected the excellent antiviral activity in vivo.The horizontal transmission experiment results showed that the virus content in the recipient fish treated with the drug was significantly reduced compared with the DMSO group,and S5 and N6 treatment could effectively reduce the horizontal transmission of the virus.Subsequently,we continued to detect the distribution of S5 and N6 in the body of common carp by high performance liquid chromatography.Meanwhile,HE staining confirmed that S5 and N6 could improve the tissue damage caused by SVCV infection.Thus,the derivatives S5 and N6 have certain application potential.In conclusion,coumarin derivatives S5 and N6 have strong inhibitory effects on SVCV infection in EPC cells and common carps,reduce virus-induced apoptosis,and have a direct protective effect on host.Therefore,S5 and N6 provide a theoretical basis for drug prevention and treatment of SVCV,and provide a reference for the creation of new fishery drugs with independent intellectual property rights in China.