The Antiviral Activity And Mechanisms Of Salvianolic Acid A And Cryptotanshinone From Salvia Miltiorrhiza On Zika Virus

Background and objectives:Zika virus(ZIKV)is mainly transmitted by Aedes mosquitoes,and belong to the genus Flavivirus of the family Flaviviridae.Most ZIKV-infected people are asymptomatic or develop a dengue fever-like,self-limiting febrile disease.The Zika outbreak in Brazil in 2015 indicated that ZIKV infection may cause serious complications such as neonatal microcephaly and Guillain-Barre syndrome.The diverse and specific transmission patterns of ZIKV present many challenges for drug and vaccine development.Currently,no specific therapeutic drugs or vaccines have been approved clinically.Therefore,there is an urgent need to develop effective antiviral drugs with low toxicity."Compendium of Materia Medica" recorded that Salvia miltiorrhiza has effects such as promoting blood circulation,removing blood stasis,dredging meridian and relieving pain.Salvia miltiorrhiza contains many active ingredients,some of which have been reported to have antiviral effects,but few have an effect on zika virus.Salvianolic acid A and cryptotanshinone are respectively one of the water-soluble and fat-soluble representative components of Salvia miltiorrhiza,and both have biological activities such as antibacterial,anti-inflammatory,anti-tumor and neuroprotection.In this study,we will investigate the antiviral activity and mechanisms of salvianolic acidA and cryptotanshinone against ZIKV,and further study the effects of salvianolic acid A and cryptotanshinone on innate immune signaling pathways,with a view to discovering safe and effective antivirals from natural products,and provide an option to solve the current problem of no drugs available for zika virus.Methods:1.MTT assay was applied to evaluate the cytotoxicity of the compounds on Vero cells,compounds with anti-zika virus activity were screened by CPE inhibition and ELISA assays.2.The effects of salvianolic acid A and cryptotanshinone on zika virus replication were detected by qRT-PCR,western blot,immunofluorescence microscopy and plaque assays,respectively.3.Time of drug addition assay was used to explore which periods of ZIKV-infection were disturbed by salvianolic acid A and cryptotanshinone;the effects of salvianolic acid A and cryptotanshinone on virus attachment and entry were respectively detected by anti-attachment and anti-entry assays;and virucidal assay was adopted to study whether salvianolic acid A and cryptotanshinone can directly inactivate the zika virus.4.Western blot was ultilized to detect the effects of salvianolic acid A and cryptotanshinone on signaling pathways of JAK/STAT and PI3K/Akt;qRT-PCR was applied to evaluate the effects of salvianolic acid A and cryptotanshinone on the expression of inflammatory cytokines and ISGs.5.MTT,qRT-PCR,western blot and plaque assays were used to detect the inhibitory ability of salvianolic acid A and cryptotanshinone against other viruses.Results:1.Five compounds with anti-zika virus activity were screened out,which were salvianolic acid A,salvianolic acid C,cryptotanshinone,tanshinone I,dihydrotanshinone I;salvianolic acid A is the water-soluble monomer of Salvia miltiorrhiza with the largest selectivity index,with CC50 and IC50 values of 125.90±7.15 μM and 12.94±2.07 μM,respectively;cryptotanshinone is the lipid-soluble monomer of Salvia miltiorrhiza with the largest selectivity index,with CC50 and IC50 values of 35.09±1.65 μM and 5.49±0.40 μM,respectively.2.Salvianolic acid A and cryptotanshinone inhibited the expression of zika virus genes and proteins and reduce the number of progeny viruses.3.Salvianolic acid A had inhibitory effect on multiple stages of the virus life cycle,which can directly inactivate ZIKV,inhibited the attacnment and entry of ZIKV,and inhibited the replication stage of post-infection;cryptotanshinone inhibited the replication stage of post-infection.4.Zika virus infection induced cells to produce IFN-β and activated the JAK/STAT signaling pathway,salvianolic acid A and cryptotanshinone significantly downregulated STAT1/2 phosphorylation and reduced expression of ISGs;futher more,salvianolic acid A inhibited zika virus via down-regulation of the PI3K/Akt pathway.5.In addition to zika virus,salvianolic acid A could also inhibited the replication of IAV,HIV-1 and HSV-1/HSV-2;cryptotanshinone had an inhibitory effect on DENV-2 and IAV.Conclusions:Salvianolic acid A and cryptotanshinone effectively inhibited zika virus replication in vitro;salvianolic acid A showed an inhibitory effect on multiple stages of the viral life cycle,while cryptotanshinone inhibited virus replication post entry;salvianolic acid A and cryptotanshinone down-regulated the JAK/STAT signaling pathway;salvianolic acid A reduced zika virus replication via down-regulating PI3K/Akt signaling pathway;salvianolic acid A and cryptotanshinone had inhibitory activity on a variety of viruses and had the potential to become broad-spectrum antiviral drugs.