N-butyl Alcohol Extract Of Glycyrrhizae Radix Et Rhizoma Inhibits Dengue Virus Through Targeting Envelope Protein

Objective:Glycyrrhizae Radix et Rhizomea,a well-known herb in traditionally Chinese medicine,possess multiple pharmacological effects,such as antiviral effects.However,the inhibitory activities of Glycyrrhizae Radix et Rhizomea on dengue virus(DENV)and its potential mechanisms still have not been clearly understood.In this study,we aim to evaluate the antiviral activities of n-butyl alcohol extract of Glycyrrhizae Radix et Rhizomea(GRE)against DENV.Methods:(1)Liquid chromatography-tandem mass spectrometry was preliminary used to analyze the components in GRE.The antiviral activity of GRE on BHK-21 cells was determined by CCK-8 assay.The release level of progeny virus was detected by plaque assay.The expression of envelope protein and non-structural protein 1 was detected by quantitative real-time PCR(qRT-PCR),Western blotting and immunofluorescence assays.The inhibitory effect of GRE on different cell lines was detected by qRT-PCR.Huh7 cells were transfected with pcDNA3.1(+)-prME plasmid to detect the influence of GRE on envelope protein.The interaction between GRE and envelope protein was investigated by surface plasmon resonance(SPR)experiment.(2)The systemic infection model was established by intracranial and intrabitoneal injection of DENV-2 in 7-day-old ICR suckling mice.For the treatment of drugs,GRE(10,5,and 2.5 mg/kg)or vehicle control were administered intragastrically at 0,1,3,5 and 7 days post infection.The changes of body weight,clinical score and survival rate were monitored every day for 10 days.qRT-PCR and Western blotting assays were used to detect the viral load in brain,liver and spleen tissues.The viral load in brain was also detected by immunofluorescence assays.The expression levels of pro-inflammatory factors in brain,liver and spleen was detected by Western blotting and enzyme-linked immunosorbent assays.Hematoxylin and eosin staining was used to observe the pathological changes in brain tissue.Results:(1)Twenty-seven compounds were preliminarily identified in GRE.GRE significantly alleviated DENV-induced cell death with an IC50 of 6.24±1.16μg/mL on BHK-21 cells.Time of addition and temperature-shift assays further demonstrated that GRE inhibits the attachment process of virus replication cycle,which in turn inhibited the generation of progeny virus and viral protein expression.Besides,GRE also exerted antiviral activities in multiple cell lines,including K562 cells,Huh7 cells,Vero cells,C6/36 cells.GRE directly attenuated the expression level of viral envelope protein in transfected Huh7 cells,suggesting that E protein is a potential target of GRE.Additionally,SPR assay further demonstrated that GRE was able to bind to envelope protein domain III in a dose-dependent manner with a Kd value of 0.24 μM.(2)GRE was found to display therapeutic effect in vivo,as indicated by reducing weight loss,decreasing clinical score and prolonging survival time on DENV-2-infected ICR sucking mice.Further results showed that GRE significantly decreased viremia,reduced viral load in brain,liver and spleen tissues,inhibited the release of pro-inflammatory cytokines including tumor necrosis factor-α,interleukin-6 and interleukin-1β in brain and liver tissues,thereby alleviating the tissue lesions.Conclusion:GRE exhibited significant inhibitory activities in the adsorption stage of DENV-2 replication cycle by targeting E protein.Thus,GRE might be a promising candidate for the treatment of DENV infection.