| Background:Enterovirus 71(EV71)is one of the main pathogens of hand-foot-and-mouth disease.It is a neurotropic enterovirus with a high capacity for transmission.Severe cases of EV71 in children have a high disability rate and mortality rate,posing a serious threat to their health and lives.Traditional broad-spectrum antiviral drugs such as ribavirin and interferon-a have significant side effects on young children,so it is essential to search for new antiviral drugs.Bidens Pilosa,a commonly used Chinese herbal medicine in the Lingnan region,is sweet,light,bitter,and slightly cold in nature.It is often used to treat sore throat,diarrhea,dysentery,boils,snake bites,and other diseases.Studies have found that Bidens Pilosa has a series of pharmacological effects,such as anti-inflammatory,antibacterial,antitumor,and hypotensive effects,and also exhibits activity against influenza A and B viruses,herpes simplex virus,pseudorabies virus,and so on.However,the activity of Bidens Pilosa and its components against EV71 and their effects on the mediated inflammatory response remain unclear.Objectives:This study aims to use experimental methods to establish a method for preparing aqueous extract of Bidens Pilosa and analyze its effects on EV71 virus activity,as well as investigate its effects on the inflammatory response induced by EV71 virus infection.The results of this study can provide new ideas and methods for developing new antiviral drugs against EV71.Methods:The Bidens Pilosa used in this study were purchased from Guangzhou Caizhi Pharmacy.First,a water extraction method was used to prepare the BB water extract.A method for determining the total flavonoid content was established using ultraviolet spectrophotometry.Second,the cytotoxicity of the Bidens Pilosa water extract was detected using the MTT assay,and its in vitro effects on EV71 virus activity were measured using the CPE assay,MTT assay,and real-time quantitative PCR(qRT-PCR).Finally,qRT-PCR was used to investigate the inhibitory effect of Bidens Pilosa aqueous extract on inflammatory factors induced by EV71,and Western Blot was used to measure the effect of Bidens Pilosa aqueous extract on EV71-mediated NF-κB/P65 protein phosphorylation.Results:The method for preparing Bidens Pilosa aqueous extract established in this study has good precision.The determination coefficient(R2)of the standard curve was 0.9973,the relative standard deviation(RSD%)was 0.04%,and the average recovery rate was 97.08%.The average total flavonoid content in the Bidens Pilosa aqueous extract extracted by the established method was about 9.2mg/mL.Under three different treatment conditions(cells not infected with EV71 virus,cells infected with EV71 virus,and cells treated simultaneously with EV71 virus and BB water extract),The water extracts of Bidens bipinnata all have anti EV71 virus effects.The selection indices(SI)under the three treatment conditions were 4.5,35.32,and 21.54,respectively.And the water extract of Bidens bipinnata was added within 8 hours after the virus entered the cell,both of which had an inhibitory effect on the virus.In addition,the Bidens Pilosa aqueous extract had a significant inhibitory effect on the expression of inflammatory factors(IL-6 and IL-10)induced by EV71,and it could inhibit the phosphorylation of NF-κB/P65 protein mediated by EV71.Conclusions:The study established a method for preparing Bidens Pilosa aqueous extract and demonstrated its potential as an anti-EV71 agent.The aqueous extract showed low toxicity to cells and significantly inhibited EV71 replication in vitro.The extract also demonstrated anti-inflammatory effects by reducing the production of inflammatory cytokines(IL-6 and IL-10)and inhibit the phosphorylation of NF-κB/P65 protein.These findings suggest that Bidens Pilosa aqueous extract has potential as a therapeutic agent for EV71 infection. |
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