| Aim:Herpes Simplex Virus（HSV）belongs to the family of Herpesviridae and are common pathogens.Depending on the serotype,herpes viruses include two subtypes:HSV-1 and HSV-2.HSV-1 can be transmitted by close contact between skin,respiratory tract and mucous membrane and plays a key role in varieties of diseases including recurrent cold sores,keratoconjunctivitis and encephalitis in humans.HSV-2 can be transmitted by sexual contact and cause genital herpes.Both herpes viruses are characterized by rapid pathological progression,short replication cycle,and lifelong latent infection.Treatments currently directed against HSV infections are acyclovir and other nucleoside analogs that target viral DNA polymerase.However,extensive use of these drugs has led to the emergence of drug-resistant viral strains,particularly in immunocompromised patients.The discovery of new drugs to treat HSV infection has become an important goal of drug development.Chinese herbal medicines play an important role in antiviral infection.Rhubarb is one of the most commonly used Chinese medicines.Studies have found that rhubarb has a wide range of pharmacological activities,including antioxidant,anti-inflammatory,anti-microbial,anti-tumor and antiviral activities,but the utilization of raw materials is not sufficient and the dosage form is simple.Based on our previous research,in this study,nanotechnology was applied to the antiviral study of Rhubarb and we evaluated the antiviral efficacy of Rheum tanguticum nanoparticles against HSV in vitro and in vivo and its possible mechanism.These may provide experimental evidence for the development of new anti-herpes virus drugs.Methods:1)The preparation and characterization of R.tanguticum nanoparticles R.tanguticum nanoparticles was produced using R.tanguticum and ultrafine pulverizing technology.R.tanguticum roots were grounded to produce coarse powders and they were smashed to cell breaking as nanoparticles using the high pressure homogenizer.The content of main active components of R.tanguticum nanoparticles was performed by high-performance liquid chromatography（HPLC）.The morphology of R.tanguticum nanoparticles were characterized by a transmission electron microscope.The particle size and zeta potential of R.tanguticum nanoparticles were measured via dynamic light scattering（DLS）technique.2)The antiviral activity and mechanism of R.tanguticum nanoparticles against HSV in vitro HEp-2 and Vero cells were treated with serial dilutions of R.tanguticum nanoparticles and the cytotoxicity of R.tanguticum nanoparticles was performed by MTT assay.The plaque reduction assay（PRA）was used to evaluate the antiviral activity of various concentrations R.tanguticum nanoparticles on HSV-1 and HSV-2 in different antiviral modes.Time-of-addition assay was conducted to investigate the activity of R.tanguticum nanoparticles was interfering with HSV-1and HSV-2 infection cycle.Real-time PCR（q RT-PCR）was used to investigate the effect of R.tanguticum nanoparticles on HSV-1 IE gene ICP4,E gene ICP8 and L gene g D expression,HSV-2 IE gene ICP27,E gene ICP8 and L gene g D expression.The protein expression levels of HSV-1 ICP4 and ICP8,HSV-2 ICP8 and g D were detected with Western blot analysis.Meanwhile,the expression levels of HSV-1 ICP4and ICP8 proteins were further detected with immunofluorescence assay（IFA）.Moreover,q RT-PCR was used to detect the expression level of TNF-α、IL-8、IFN-αand IFN-βin the HSV-2-infected HEp-2 cells.3)The anti-HSV-1 efficacy of R.tanguticum nanoparticles in vivo Furthermore,the in vivo efficacies of these nanoparticles were investigated with a mouse model of HSV-1 encephalitis.Survival rate,median survival time,viral titer and brain pathological changes were used to evaluate the effect of different concentrations of R.tanguticum nanoparticles against HSV-1 infection in vivo.The levels of viral genes and protein expression in HSV-1 infected mice were also tested to determine the anti-HSV-1efficacy of the R.tanguticum nanoparticles.Results:1)The identification and characterization of R.tanguticum nanoparticles The HPLC method results indicated that the chemical components of R.tanguticum nanoparticles are similar with the R.tanguticum.And the content in R.tanguticum nanoparticles was in accord with the quality standard of Chinese Pharmacopoeia.Transmission electron microscopy（TEM）images showed that the R.tanguticum nanoparticles were distributed in spherical shape and the mean particle size was around 123.2 nm.The DLS results indicated that the zeta potential was about-19.93m V which may tend to aggregate in aqueous state.2)Inhibitory effect of R.tanguticum nanoparticles on HSV infection in vitro The 50%cytotoxic concentration(CC50)of R.tanguticum nanoparticles on HEp-2 and Vero cells were 415.3μg/ml and 649.8μg/ml.R.tanguticum nanoparticles could inhibit HSV-1 replication and the EC50 of R.tanguticum nanoparticles towards HEp-2and Vero cells were 194.1μg/ml and 209.8μg/ml.In addition,R.tanguticum nanoparticles inhibited both the inactivation,attachment and penetration processes of HSV-1 and the viral activities were dramatically reduced.The result of time of addition assay indicated that the inhibitory effect was significant when R.tanguticum nanoparticles were added at 0-15 h.p.i..R.tanguticum nanoparticles could significantly reduce the m RNA levels of HSV-1 IE gene ICP4,E gene ICP8 and L gene g D.Moreover,the HSV-1-induced ICP4 and ICP8 protein expression was also strongly suppressed by R.tanguticum nanoparticles.R.tanguticum nanoparticles inhibited HSV-2 replication and the EC50 values of R.tanguticum nanoparticles on HSV-2 infected HEp-2 and Vero cells were 188.1μg/ml and 228.7μg/ml.The results of the inactivation,attachment and penetration assays suggested that R.tanguticum nanoparticles also show effect on directly inactivate HSV-2 particles,prevent viral adsorption and block HSV-2 entry into cells in a dose-dependent manner.Time-of-addition results showed that R.tanguticum nanoparticles could effectively inhibit the virus during 0-12 h.R.tanguticum nanoparticles showed a significant inhibitory effect on the expression levels of the IE gene ICP27,E gene ICP8 and L gene g D compared with the virus-infected group.Similarly,the protein expression levels of HSV-2-induced ICP8 and g D was also found to be significantly suppressed.Moreover,the transcription levels of cytokines,TNF-α、IL-8、IFN-αand IFN-βof the drug treated group were reduced.3)The protective efficacy of R.tanguticum nanoparticles against HSV-1infection in vivo.In vivo,mice injected intracerebrally with HSV-1 showed lack of appetite hyperactivity,a hunched posture,inactivity and death.Orally administered with serial dilutions of R.tanguticum nanoparticles reduced the mortality of infected mice,prolong the survival time and alleviated clinical signs.Additionally,the protective rate of the drug at 625 mg/kg/d and 312.5 mg/kg/d was shown to be 60%and 30%.Oral gavage by R.tanguticum nanoparticles in 625 mg/kg/d and acyclovir significantly reduced the virus titers compared to the virus control group.The pathological changes in mice brains showed that post-treatment with R.tanguticum nanoparticles（625 mg/kg/d）and acyclovir can effectively alleviate the virus-induced inflammation and the severity of the brain damage in the virus-infected mice.And the m RNA expression levels of both ICP4 and ICP8 as well as the ICP4 protein level could be down-regulated in brain tissues of mice compared to virus control.Conclusion:1)The characteristics of R.tanguticum nanoparticles are consistent with the R.tanguticum.2)R.tanguticum nanoparticles have effectively antiviral activity against HSV-1 and HSV-2 in vitro.R.tanguticum nanoparticles present the anti-HSV activity mainly by affect virus replication.It can inhibit both the virus key gene and protein expression and also modulate the productions of cytokines.3)R.tanguticum nanoparticles can protect the HSV-1 infected mice from death,prolong the survival time and alleviate the brain lesion.It has potential therapeutic effect on herpes encephalitis caused by HSV-1. |
