| ObjectiveTuirejieduling(TRJDL) is a Compound Chinese medicine preparatio -n with anti- respirovirus virus activity.This experiment aimed to investig -ate the antiviral activity of Tuirejieduling against on Flu strain in vitro and explore the therapeutical effect of Tuirejieduling on influenza viral pneumonia and immunological mechanisms in mice.Methods1. In vitro, carrying out three different treatments on the Flu/Hep-2 cell model, including virucidal assay, drug added before or after infection assay. The antiviral effects were investigated by observing CPE and MTT colorimetric assay, plaque reducing assay for viral inhibitory rate and viral titer. In these experiments, Ribavirin and antiviral liquor were used as positive control.2. BALB/c mice were inoculated intranasally with 25LD50 A/FM/1/47 (H1N1),treated with TRJDL, antiviral liquor and ribavirin were use d as positive control.The effect of TRJDL on the weight,survival rate of FM/ infected mice after 14 days inoculation was observed.(1)Lungs were removed and weighed, then lung index was analysis -ed. The virus hemagglutination tite was detected in lungs homogenate. Routine hematoxylin-and-eosin (H&E)-stained sections were examined.(2)T lymphocyte were detected by MTT method in mice spleens proliferation. IL-2 and IFN-y sereted were detected bioactivity method.(3)Percentage of CD3+,CD4+,CD 8+ T lymphocyte subsets and CD4/CD8 ratio in mice venous blood were measured with Flow cytometr y(4)TNF-α,IL-1β, MCP-1, IL-10 gene expression were detected by real-time quantitative PCR and western-blot method in lung tissue sections(5)IFN-y, TNF-a, IL-6, IL-4, IL-10 and IL-2 were measured with Enzyme-linked immunoadsorption assay in mice sera.Results1.In vitro party:(1)Preincubation of Flu with TRJDL did protect Hep-2 or MEDCK cell against Flu. With the increase of the drug concentration, viral titers decreased correspondingly, whereas viral inhibition rate increased.The viral inhibition rate were 85.2% and plaque reduction rate were 80.1% when the drug concentration was 160μg/mL,respectively. The antiviral effect of TRJDL worked with dose depend manner between 5～160μg/mL. The IC5o value were 10.2μg/mL and the TI were 9.7 respectively. The antiviral effect was greater than antiviral liquor (P<0.05).(2)Hep-2 cell showed a CPE typical performance from Flu infection when TRJDL were present 2h before infection. MTT results showed that the inhibition rate of virus infection with the virus control group, no significant difference. With the increase of the drug concentration and the time up to 8h,viral titers decreased and and viral inhibition rate increased in the virus infection correspondingly.When the drug concentration was 160μg/mL, the viral inhibition rate were 84.3% and plaque reduction rate were.The IC50 value were 11.9μg/mL and the TI were 10.7 respectively. The antiviral effect was greater than antiviral liquor (P<0.05).(3)Hep-2 cell were optimally protected when TRJDL was added after infection. With the increase of the drug concentration, viral titers decreased correspondingly, whereas viral inhibition rate increased. When the drug concentration was 160μg/mL, the viral inhibition rate were 84.2 and plaque reduction rate were 78.2%.The IC50 value were 11.1μg/mL and the TI were 10.3 respectively. The antiviral effect was greater than antiviral liquor (P<0.05).2. In vivo party:(1)The virus control group showed lower survival rate(25%).Compar -ed with virus control group, TRJDL groups showed higher survival rate (65%,70%and 75%)and body weight, lower lung index and virus hemagglutination tite in lungs.(2)The pathomorphology results showed that lung appearance changes of virus control were consolidation and hemorrhage; the histopathology changes were serious interstitial pneumonia changes, showing bronchial epithelium shedding,alveolar interval thickening, many mononuclearcells cell infiltrating,producing consolidated regions. There were a small amount of serous effusion in alveolar cavities. Compared with virus control, the histopathology changes of TRJDL group were ameliorated obviously.(3)Compared with the control group, the ability of T-lymphocyte proliferation and secreting IL-2,IFN-y of virus control were decreased obviously.Compared with virus control, the ability of T-lymphocyte proliferation and secreting IL-2,IFN-y of TRJDL groups were improved obviously.(4)The percentage of CD3+ and CD4+ in TRJDL groups were more than nomal group and the virus control (P<0.05). The percentage of CD8+was the highest in the dose of TRJDL groups and Ribavirin group and was the lower in the high dose of group.The CD4/CD8 ratio was the highest in high dose of TRJDL group.(5)Compared with the normal control group,the virus control showed higher gene expression of TNF-α,IL-1β, MCP-1,IL-10 in lungs.Compared with virus control, gene expression of MCP-1, TNF-α, IL-βof TRJDL groups were dramatically decreased (P<0.05), while gene expression of IL-10 were significantly increased (P<0.05).(6)The levels of IFN-γ, IL-6, TNF-α,, and IL-4 in sera of virus control were more than nomal group(P<0.05),while the level of IL-2 was lower(P<0.05).The level of IL-10 was no obviously difference between virus control and nomal group. Compared with virus control, the levels of IL-6 and TNF-αof TRJDL were obviously decreased and the levels of IFN-γ, IL-2, IL-10 were significantly elevated.Conclusion1.TRJDL could directly inactivate the Flu particles, inducing durable antiviral activity in host cells, as well as inhibiting the late stage of viral replication cycle in dose dependent manne2.TRJDL has the same anti- Flu effect as the reference control expect virucidal assay and 8h before effection assay, which were stronger than antiviral liquor.(P<0.05).3.TRJDL could enhance survival rate, increase body weight and reduce inflammatory pathology in the lung of H1N1 infected mice.These indicated that TRJDL was effective for the influenza viral pneumonia. The immunological mechanisms may be:(1)TRJDL could inhibit the proliferation of the virus in lung and reduce the virus on direct damage to lung tissue(2)TRJDL could enhance T lymphocyte proliferation and secretion.T -hereby it improved the cell immune response and enhanced the ability of anti-virus(3)TRJDL could reduce the expression of MCP-1,TNF-α,IL-1βgene and increase the expression of IL-10 genes. At the same time,it could inhibit the inflammatory cytokine IL-1,IL-6,TNF-αsecretion and promote IL-10, IFN-γ, IL-2 secretion in sera. Thereby it played a role in immune regulation, reduced the immune damage...
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