Study On Realgar Nano-particles Inhibiting The Replication Of Adenovirus And Respiratory Syncytial Virus At The Cellular Level

Respiratory syncytial virus and adenovirus are the common pathogens that infect the respiratory tract of young children. These two viruses, which cause an outbreak of no-socomial infection, are virulent and with high incidence rates. The drugs for them are still in short. Although there are many papers discribing clinical treatments of viral diseases, experimental study of realgar anti-virus is not witnessed. In this study, respi-ratory syncytial virus and adenovirus are selected as research objects to observe realgar nanoparticles antiviral effect. Respiratory syncytial is the non-stage single negative stra-nd RNA virus, and adenovirus is the double-stranded DNA virus. At early stage of HA-dV-3infection, adenovirus inhibit apoptosis infection to breed and spread to generate en-ough number of progeny virus and avoid host immune surveillance system's supervision before apoptosis. This thesis described mechanism of the realgar anti-virus and reveal-ed targets of realgar nanoparticles in anti-adenovirus by observing the effects of realgar nanoparticles on the PI3-K/AKT pathway and result of realgar nanoparticles that indu-ce the apoptosis of adenovirus-infected cells.Objective1Illustrating the anti-viral effect of realgar nanoparticles. The cytopathic effect (CPE) of cells by modeling RSV and HAdV-3infecting Hep-2cells in vitro is observ-ed. The effect of realgar on the expression of RSV and HAdV-3by using fluorescent quantitative PCR and PCR method is detected.2Discussing the effect of realgar nanoparticles on the apoptosis of cells infected by HAdV-3at early stage. The intervention of realgar nanoparticles on apoptosis of adenovirus-infected cells at early stage is noticed by using laser scanning confocal mi-croscope. The impact of realgar nanoparticles on protein expression of adenoviral infecte-d cells on AKT, p-AKT, NF-κB p65is tested by western-blot. The effect of realgar nan-oparticles on gene expression of adenoviral-infected cells on Fas L mRNA, Bcl-2mRNA, bax mRNA and p53mRNA is tested by using RT-PCR assay. Discussing the role of re algar nano-particles intervention adenovirus induced apoptosis through the PI3-K pathway.Method1Realgar nanoparticles on the replication of adenovirus and respiratory syncytial virus: Using CPE inhibition assay, we observe the effect of realgar nano-particles reducing He-p-2cell infected by adenovirus and respiratory syncytial virus, through prophylaxis, treat-ment and direct inactivation of administration in three ways.Using fluorescence quantitati-ve PCR and quantitative RT-PCR method to observe the adenovirus and respi- reatory syncytial virus gene expression in order to explore the role of realgar nanoparticl-es of anti-DNA virus and RNA viruses.2The result of realgar nanoparticles effect on Hep-2cell activity infected by aden-ovirus. When Hep-2cell was infected by adenovirus on24h,48h,72h and120h, dif-ferrent concentrations of realgar nanoparticles on Hep-2cell was observed by using M-TT method.3Realgar nanoparticles induce adenovius infected Hep-2cell apoptosis. Using Anne-xinV/PI laser scanning confocal microscope to observe high-dose and low-dose realgar nanoparticles inducing Hep-2cell apoptosis when it infected by adenovirus24h.4Effect of realgar nanoparticles on PI3-K pathway of adenovius infected Hep-2cell. Exploring the impact of realgar nanoparticles on PI3-K pathway with the use of west-ern-blot method to observe high-dose and low-dose realgar nanoparticles effect on AKT, P-AKT,NF-kB p65protein expression of Hep-2cells infected by adenovius.5The result of realgar nanoparticals on gene expression of apoptosis regulatory protein (FasL,p53,Bcl-2and Bax mRNA) of adenovius infected Hep-2cell. When Hep-2cells infected by adenovius on24h, observe the effect of high-dose and low-dose re-algar nanoparticles on regulatory protein gene(FasL, p53and Bcl-2and Bax mRNA) on apoptosis expression by using real-time RT-PCR.Result1Realgar nanoparticles can reduce the extent CPE of HAdV-3and RSV infection of Hep-2cells through prevention, treatment and direct inactivation of three delivery meth-ods. The median effective concentration (IC50) of realgar nano-particle anti-HAdV-3, were0.255μg/mL,0.142μg/mL,0.117μg/mL, and therapeutic index (TI) were2.55,4.57and5.55, respectively. The median effective concentration (IC50) of realgar nano-p-article anti-RSV, were0.20μg/mL,0.13μg/mL,0.16μg/mL and therapeutic index (TI) were3.18,4.99and4.11respectively. There is a dose-effect relationship between realgar nanoparticles inhibit the CPE of HAdV-3and RSV infection of Hep-2cells.2The results of Real-time PCR and RT-PCR testing HAdV-3and RSV viral repli-cation display the Hep-2cells group has no amplification curve, CT>35, which illustrate HAdV-3and RSV pathogen detection are negative.Realgar nanoparticles (plus adenovirus) group, ribavirin (plus adenovirus) group and the adenovirus model group amplification curve of CT values were29.30±0.08,33.05±1.29,26.01±0.25, adenovirus pathogen detection are positive. Compared with the adenovirus group(66,699,932±23.85), the viral copy amount of realgar nanoparticles (plus adenovirus) group (912,435.44±16.57) and ribavirin (plus adenovirus) group (459,124.84±12.82) were lower (P<0.05). The amplify-cation curve of CT values of Realgar nanoparticles (plus respiratory syncytial virus) group, the ribavirin group (plus respiratory syncytial virus) and respiratory syncytial virus group were25.58±0.19,27.42±5.74,17.59±0.1,and respiratory syncytial virus pathogen detection was positive.Compared with respiratory syncytial virus (70.952million±46.14), the viral replication copy quantity of realgar nanoparticles (plus respiratory syncytial virus) group (1.1235million±20.25) and ribavirin (plus respiratory syncytial virus) group (426,593.78±49.02) were lower, with significant differences (P<0.05).3The results of MTT assay and confocal laser microscopy testing realgar induced HAdV-3infection of Hep-2cell apoptosis show that0.367μg/mL,0.184μg/mL,0.092μg/mL,0.046μg/mL realgar nanoparticles can induce HAdV-3infection of Hep-2cell apoptosis when24h. Confocal laser scanning microscope show that realgar high-dose group induce apoptosis is better than the low-dose group. The effection of realgar nano-particles group reducing CPE of HAdV-3infection of Hep-2cell was significantly better than the adenovirus control group when48h and the role of anti-HAdV-3can continue to120h.4Realgar nano-particles can effect on PI3-K-mediated signaling pathway, through reducing the level of phosphorylation of AKT protein and NF-k B p65protein expression,so as to increasing the expression of pro-apoptotic protein P53, FasL and Bax mRNA and reducing the antiapoptotic protein bcl-2mRNA expression induced HAdV-3infection of Hep-2cell apoptosis at the early stage of infection.Conclusion1This study demonstrated that realgar nanoparticles have the role of anti-adenoviru-s and respiratorysyncytial virus,and comply the the realgar antiviral experimental study;2At early stages of adenovirus infection, realgar nanoparticles can reduce the phosp-horylation of AKT, promote the gene expression of apoptosis protein of P53, FasL an-d Bax mRNA and inhibit expression of apoptosis protein Bcl-2mRNA by acting on PI3-K pathway.