Antiviral Activity Of Arbidol Hydrochloride Against Coxsackievirus In Vitro And In Vivo

PartⅠAntiviral Activity of Arbidol Hydrochloride against Coxsackievirus in vitro and in vivoCoxsackievirus is a non-enveloped, single positive-strand RNA virus, which can cause numerous diseases such as myocarditis, pericarditis, aseptic meningitis, and hand, foot, and mouth disease. Coxsackie B viruses (CB) are major etiologic agents of acute and chronic myocarditis, especially for CB3 and CB5. The likely mechanisms of CB-induced viral myocarditis involve direct virus-induced myocyte damage and immune-mediated pathogenesis. There are several proposed treatment strategies, including early application of antiviral drugs and immune regulator. Arbidol hydrochloride (ARB) is a new immunomodulator. It can activate phagocytic activity of macrophages and enhance the immune system. It has the ability to elicit broad-spectrum antiviral activity against some enveloped RNA viruses, namely influenza virus, respiratory syncytial virus, and Hantaan virus. The aim of the present study was to evaluate the anti-CB5 activity of ARB in vitro and in vivo.Objective:To investigate the antiviral activity of ARB against CB5 in vitro and to evaluate ARB’s effect against viral myocarditis induced by CB5 in mice.Methods:Cell culture technique was used in HEp-2 cells. The inhibition of the virus-induced cytopathic effect (CPE) was scored by light microscopy and measured by MTT assay. And the therapeutic index (TI=CC50/IC50) was utilized as a comprehensive criterion to evaluate the antiviral activity of ARB. CB5 mRNA was measured by semi-quantitative reverse transcriptase polymerase chain reaction after treatment with ARB and interferon-β(IFN-β) level in the supernatant was determined by ELISA. In the 2nd stage, BALB/c mice were inoculated intraperitoneally with CB5 and ARB at a dose of 0.025, or 0.050g·kg-1·d-1, or a placebo was orally administered to the mice for 6 days beginning 24 h post-virus exposure. Eight animals were killed on day 7, myocardial histopathology changes were observed and viral load of target organs were determined. The mice weight and survival rate were also utilized for assessment of the anti-CB5 activity of ARB in vivo.Results:1. ARB displayed antiviral activity against CB5 when added during, or after viral infection, with 50% inhibitory concentration (IC50) of 2.66±0.4μg/mL and 6.62±1.1μg/mL. With the increase of the drug dose, the degree of CPE decreased correspondingly, whereas viral inhibitory rates increased.2. CB5 mRNA levels treated with ARB at concentrations of 5 or 10μg/mL showed a significant reduction in comparison with the untreated controls (p<0.05).3. IFN-βlevel in the supernatant treated with ARB at concentrations of 2-16μg/mL for 24h didn’t increase in comparison with the untreated controls (p>0.05).4. Eight animals of each group were sacrificed on day 7 post viral exposures。Microscopically it was shown that the hearts of the viral control animals had denaturation of cardic muscle cell. Tissue samples of the rest animals did not have changes except the lungs of animals received 0.025g·kg-1·d-1 ARB showed local hemorrhage. Oral administration of ARB for 6 days significantly decreased the virus titers of mice heart homogenates. In the group treated with arbidol at 0.025, or 0.050g·kg-1·d-1, the mean virus yields were reduced to 103 and 102PFU respectively, whereas the yields in placebo controls were 105PFU.5. On 7 day post challenge the animals in viral control group began to exhibit regular death and all of them died on 18 day. Orally administered ARB prevent CB5-induced death in a dose-dependent manner. For the groups treated with arbidol at a dose of 0.025, or 0.050g·kg-1·d-1, the survival rates were 62.5% and 100%, respectively.Conclusion:1. ARB has antiviral properties against CB5 manifested at several levels including directly inactivating of viral particles, as well as inhibiting the late stage of viral replication cycle. 2. Orally administered ARB at 0.025, or 0.050g·kg-1·d-1 dose 24 h after infected with CB5 could significantly increase the mice weight and survival rates, and reduce the mean heart virus titers.PartⅡNeutrophil responses to injury or inflammation impair peripheral gustatory functionThe adult peripheral taste system is capable of extensive functional plasticity after injury. Unilateral chorda tympani (CT) nerve sectioning leads to a rapid decrease in neurophysiological taste responses to sodium in the contralateral, intact CT nerve. Normal taste function recovers by day 2 in control-fed animals. Generally these far-reaching effects initiated by neighboring injury are not widely appreciated. The role of immune responses to nerve injury has been widely explored, while immune cells have beneficial roles in clearing bacteria from injured tissurs, their effects on intact neurons are generally undesirable. We hypothesized that the immune responses to nerve injury mediate early functional deficits in the intact CT. Polymorphonuclear neutrophils (PMNs), are the first leukocytes to arrive at damaged tissue and play an important role during injury and inflammation. The role of PMN recruitment in neural injury has been widely explored. However, the mechanisms responsible for adverse effects of PMN on neurons are not fully understood. We hypothesized that PMNs infiltrate injured neural tissues and play a role in modulating neural responses in the uninjured CT. We tested whether PMN responses to injury or inflammation impair peripheral gustatory function.Objective:Using taste system as an excellent model to study the complex role of immune activity after neural injury. To investigate whether neutrophils impair taste function, and if injury is a prerequisite to neural plasticity in the adult taste system. These studies provide needed insight to gustatory-immune interactions and highlight the ability of the innate immune system to elicit plasticity in the adult peripheral taste system.Methods: 1. To determine the dynamics of the PMN response to CT sectioning, separate groups of rats received the following treatments:1) CT section alone; 2) Unilateral CT section and dietary sodium restriction; 3) Dietary sodium restriction alone; 4) Sham section alone (control for the surgical incision); 5) Normal controls. Rats were sacrificed and tongues were dissected at 12 hr,24 hr,48 hr, or 72 hr after CT sectioning. The PMN response was examined in coronal frozen sections using immunohistochemistry assay. The primary antibody used was against myeloperoxidase (MPO), a marker for PMN. PMN counts were performed blinded to condition with a computer imaging system equipped with MetaMorph software.2. We depleted PMNs systemically to determine their influence on peripheral taste function. Rats were injected with a rabbit anti-polymorphonuclear antibody or normal rabbit serum one day before CT sectioning. On day 1 post-sectioning, neurophysiological responses to taste stimuli were recorded.3. To stimulate lingual neutrophil infiltration, rats received subcutaneous injections of 10μg lipopolysaccharide (LPS, E. coli 026:B6) in 10μl sterile PBS. Controls received PBS alone. Taste responses were recorded from CT nerves ipsilateral and contralateral to the injection site 24 hr after LPS injections.Results:1. Quantitative analyses confirmed the effects of nerve sectioning and diet on PMNs. At 12 hr post-sectioning, PMNs were increased on both the sectioned (p<0.05) and intact (p<0.05) sides of the anterior region of the tongue. At day 1 after nerve section, the number of PMNs subsided in control-fed animals and the absolute number MPO+cells approached baseline at day 2 after sectioning (p>0.05). The PMN response was even more striking in sodium-deficient animals. In this group, the number of PMNs increased dramatically on both sides of the anterior region at 12 hr post-sectioning (p<0.05). The PMN response peaked at 24 hr post-injury (p<0.001) and was still bilaterally increased in each lingual region by day 2 post-injury (p< 0.05).2. Injection of anti-PMN serum dramatically reduced PMN infiltration of the tongue after CT nerve sectioning. There was a significant decrease in the mean number of PMNs on both the denervated (p<0.05) and intact (p<0.001) sides of the tongue. Responses to NaCl were restored to normal levels when the neutrophil response was inhibited. Specifically, responses to 0.10 M (p=0.021),0.25 M (p=0.003), and 0.50 M NaCl (p=0.010) were significantly higher in anti-PMN-treated animals compared to controls. Responses to 0.10M-0.50M NaAc were also significantly increased in Cut+anti-PMN compared to Cut+NRS treated rats (p= 0.026-0.048). Furthermore, responses to non-sodium stimuli, including 0.05-0.50M KCl,1M sucrose,0.01M quinine,0.01N HC1, and 0.3M MSG were similar in neutrophil-depleted and control groups after contralateral CT sectioning (p>0.05). Application of ENaC antagonist-amiloride to the tongue reduced mean responses to NaCl to the same magnitude in each group (p> 0.05).3. We used immunohistochemistry assay to confirm that lingual LPS injection stimulated lingual PMN infiltration. MPO+neutrophils were present in the lingual mucosa, lamina propria, and fungiform papillae after lingual LPS injection. Quantitative analyses of PMNs demonstrated a significant increase ipsilateral (p= 0.005) and contralateral (p= 0.011) to the ventral LPS injection site. The number of neutrophils was also elevated bilaterally in the mid and caudal regions of the fungiform field in LPS-injected vs. control rats (p=0.0001-.0.006). Neural responses to 0.10-0.50M NaCl were significantly reduced in LPS-injected animals vs. PBS injected controls (p< 0.001-0.05). Sodium taste function was inhibited in CT nerves ipsilateral and contralateral to the LPS injection site. In contrast, responses to non-sodium stimuli such as a concentration series of KCl,0.10 and 0.50M MSG,1.0M sucrose, and 0.01M quinine (p> 0.05) were not significantly different between groups.Conclusion:1. Nerve sectioning increased the number of PMNs on both the denervated and uninjured sides of the tongue. The low-sodium diet amplified and extended the bilateral neutrophil response to injury, in parallel with the persistent changes in sodium taste function.2. Blocking the PMN response to injury restores normal neural responses to NaCl and NaAc.3. PMNs responding to a local inflammatory stimulus, in the absence of nerve injury, also induce deficits in sodium taste function.