Anti-HSV-1 Activity And Mechanism Of Pinostrobin Obtained From Pigeonpea[Cajanus Cajan(L.)Millsp.]Leaves

In the present work,the HSV-1 infection mice mode was set up to study anti-HSV-1 activity of pinostrobin obtained from Pigeonpea(Cajanus cajan(L.)Millsp.)leaves in vivo.The in vitro anti-viral activity of pinostrobin against herpes simplex virus-1(HSV-1)was investigated by MTT assay.Furthermore,AFM,FCM,SDS-PAGE,Western blotting and PCR technology were used to investigate the anti-HSV-1 activity mechanism of pinostrobin.With the in silico simualtions,an in-depth and systemetic investaigation was carried out on the interacting mechanisms between pinostrobin and HSV-1 envelope glycoprotein D(gD)protein.Conclusions were summarized below:1.The HSV-1 infection mice mode was set up to investingate anti-HSV-1 activity of pinostrobin in vivo for the first time.The LD50 value of pinostrobin was determined as>100 mg/kg weight by acute toxicity assay.This means that the toxicity of pinostrobin was small in the range of dosage administration.Pathological section assay showed that pinostrobin has obvious effect on HSV-1 induced neutrophil and macrophage increasing,tissue loosen and cell vacuolation in both liver and spleen.The percent death of mice observed at day 14 after infection were 16.7%and 0%for the 20 mg/kg weight pinostrobin and 50 mg/kg weight pinostrobin,respectively.Overall,the results indicated that pinostrobin is a potential anti-HSV-1 agent for HSV-1 infection treatment in vivo.2.The antiviral activity,mode of antiviral activity and different time addition of pinostrobin against herpes simplex virus-1(HSV-1)were investigated by MTT assay in vitro for the first time.The cytotoxicity of pinostrobin on Vero cells were investigated and were expressed as CC50 and TD0.Pinostrobin showed low cytotoxicity(CC5o>100 μg/mL,TD0=95.37±7.14 μg/mL),which indicates that pinostrobin did not affect the growth of Vero cells.The IC50 value of pinostrobin was determined as 22.71±1.72μg/mL.The SI value of pinostrobin was 4.40(>4),which indicated pinostrobin can be judged to have significant anti-HSV-1 activity.Pinostrobin showed the maximum antiviral activity when added at a concentration of 100 μg/mL during the pretreatment virus period with inhibition of the viral replication of 85.69%i2.59%for HSV-1.The inhibition of pretreatment cell phase,adsorption phase and pretreatment virus phase were 61.19±1.58%,68.48±0.91%and 74.22±3.40%,respectivly.Differently from pinostrobin,acyclovir only showed antiviral activity at a concentration of 100 μg/mL during the replication period with inhibition of the viral replication of 95.11%±3.98%.However,no significant effect on viral replication was detected when acyclovir was used for pretreatment of cells or viruses or when acyclovir was only added during the adsorption phase.These results suggested that free virus is very sensitive to pinostrobin,the inhibition of HSV appears to occur before entering the cell but also moderately occur after penetration of the virus into the cell.This indicated that different from acyclovir,pinostrobin process an all-sided anti-HSV-1 activity.Different time of pinostrobin addition assay showed that the anti-HSV-1 activity of pinostrobin was decreased to 50.12±4.37%within 12 h,and then reached stably.But anti-HSV-1 activity of acyclovir was decreased with the time increased.It seemed that pinostrobin inhibited HSV-1 replication both in the early and late stage.3.Atomic force microscopy(AFM),flow cytometry,SDS-PAGE,western blotting and PCR method were used to dectect morphology and size of HSV-1,cell cyle and mitochondrial membrane potential of host cell,HSV-1 protein synthesis and UL30 expression to further analysis the anti-HSV-1 mechanism of pinostrobin for the first time.When HSV-1 treated with pinostrobin for 9 h,the length,width and height of virion were only half of the normal ones.When HSV-1 was treated with 900 μg/mL pinostrobin,virion was destroyed to small particles, w,ith the length ranged of 12.60±3.77-34.74±9.39 nm,Width ranged of 7.15±3.02-28.76±6.91 nm,and height ranged of 15.13±3.71-32.40±2.45 nm,respectively.With increasing time and concentration,the envelope was shedded and damaged,finally leading to virus inactivation.HSV-1 exerted growth-inhibitory effects via G1 phase arrest and depolarization of △Φm which could be inhibited by pinostrobin in a concentration and time-dependent manner.Furthermore,pinostrobin showed obviously inhibition against ICP27and gD protein of HSV-1.When HSV-1 was treated with 12.5,25 and 50μg/mL pinostrobin,the content of gD和ICP27 was 86.25%,50.09%,18.64%and 62.35%、8.56%、0 compared with the untreated HSV-1.It indicated that pinostrobin could inhibit not only HSV-1 glycoprotein such as gD binding to the receptor of cell surface,but also ICP27 and gD expression,which caused infection inhibitory of HSV-1.Moreover,pinostrobin possesses significant inhibition against UL30 in a concentration-dependent manner.When HSV－1 was treated with 12.5、25 and 50 μg/mL pinostrobin,UL30 expression decreased to 79.46±4.11%,27.37±2.07%and 16.30±2.18%compared with HSV-1 control.It indicated that both pinostrobin and acyclovir can obiously inhibited DNA polymerase of HSV-1,thus inhibited its DNA synthesis.4.With the in silico simualtions,an in-depth and systemetic investaigation was first carried out on the interacting mechanisms between pinostrobin and gD protein.Results showed that interaction energy between pinostrobin and gD was-66.11 kcal·mol-1.Thereinto,vdW plays the leading role,possessed 86.62%of the total energy(-57.29 kcal mol-1).Besides,pinostrobin could bind to gD protein residues-Leu28,Arg130,Glnl32,Pro133,Trp135,Val1444 Asp147,Asnl48,Gln228 and Arg229,especially to Gln228 and Arg229,the Esum was 9.48 and-5.86 kcal·mol-1,respectively.Results showed that pinostrobin could inhibit gD function by binding to gD protein residue(Gln228 and Arg229 et al)which inhibited HSV-1 adsorption.