Study On The Anti-H1N1 Subtype Influenza Virus Effect Of Hypecoum Leptocarpum Hook.f.et Thoms.preparation

Influenza A virus(IAV)is capable of causing the onset of influenza and is one of the major infectious agents threatening human health.Current drug therapy for IAV consists of two main classes of anti-influenza drugs:neuraminidase inhibitors(NAIs,such as oseltamivir)and M2 channel blockers,but the latter have been discontinued due to widespread drug resistance.IAV can evolve and mutate through antigenic drift and genetic recombination,which in turn leads to the formation of new drug-resistant strains.Therefore,the development of novel anti-influenza drugs and the study of anti-influenza virus-related mechanisms are necessary.In this study,two pharmaceutical preparations,Water-extracted granule of Hypecoum leptocarpum(WGHL)and Alcohol-extracted granule of Hypecoum leptocarpum(AGHL),were prepared from our traditional Chinese medicine Hypecoum leptocarpum Hook.f.et Thoms.WGHL and AGHL were prepared and studied for their antiinfluenza viral effects.In vitro anti-H1N1 activity of drug formulations:an influenza virus infection cell model was established using MDCK cells,and the IC50 of each drug formulation was calculated using Reed-Muench’s method based on the inhibitory effect of different concentrations of WGHL and AGHL on the cytopathogenic effect of influenza virus.After establishing an influenza virus-infected cell model in MDCK cells,the cells were treated with different concentrations of AGHL at high,medium and low levels,and the effect of AGHL on virus amplification was investigated by measuring the TCID50 of cell supernatants at different times after infection and quantifying the progeny virus released into the culture medium.The results show that:both WGHL and AGHL were able to significantly inhibit MDCK cell lesions caused by H1N1 subtype AIV infection at a range of concentrations with IC50 of 12.13 mg/mL and 1.84 mg/mL,respectively.Compared with the virus control,the MDCK influenza virus-infected cell model treated with different concentrations of AGHL at high,medium and low levels was able to significantly reduce the virus titer at 12,24,36,48,60 and 72 h post-infection.Among them,high concentrations of AGHL had the strongest inhibitory effect on viral replication.In vivo anti-H1N1 activity and inflammatory inhibitory effects of pharmaceutical preparations:a mouse model of H1N1 subtype influenza virus infection was used and treated with different concentrations of AGHL administered at high,medium and low levels.The inhibitory effect of AGHL on influenza virus in IAV-infected mice and the therapeutic effect on IAV-infected mice were investigated by observing the clinical manifestations,body weight changes,survival rate,lung index,lung viral load,lung pathological changes,the content of various blood cells(leukocytes,lymphocytes,monocytes and neutrophils),and the expression levels of cytokines(TNF-α,IL-1β and IL-6)in lung tissues and serum.The results show that:IAV-infected mice treated with different concentrations of AGHL at high,medium and low levels showed delayed clinical onset,increased survival and reduced body weight loss compared to viral controls.The lung viral titers and lung indices of IAV-infected mice were reduced,lung pathological damage was alleviated,blood indices(leukocytes,lymphocytes,monocytes and neutrophils)were improved,and the expression levels of cytokines(TNF-α,IL-1β,IL-6)in lung tissues and serum were regulated.Among them,high concentrations of AGHL had the strongest in vivo anti-IAV effect.Effect of pharmacological agents on NF-κB signaling pathway:a mouse model of H1N1 subtype influenza virus infection was used and treated with different concentrations of AGHL administration.The expression levels of NF-κB p65 mRNA in the lung tissues of IAV-infected mice were detected by RT-qPCR.The protein expression of NF-κB p65 and pNF-κB p65 in lung tissues of IAV-infected mice was detected by Western-blot.Protein expression of p-NF-κB p65 in lung tissue of IAV-infected mice was detected by immunohistochemical staining.RT-qPCR assays showed that the expression levels of NFκB p65 mRNA in lung tissues of IAV-infected mice treated with different concentrations of AGHL at high,medium and low levels were reduced compared with the viral control group.Western-blot assay showed that the protein expression levels of NF-κB p65 and p-NF-κB p65 were decreased in lung tissues of IAV-infected mice.Immunohistochemical assays showed that the protein expression level of p-NF-κB p65 in the lung tissue of IAV-infected mice was decreased.Hypecoum leptocarpum Hook f.et Thoms.has good anti-IAV activity in vitro and in vivo,which can alleviate the inflammatory response in the lungs and the whole body of the animal organism by regulating the NF-κB signaling pathway,improve the indexes of the animal organism,reduce the severity of the disease caused by IAV infection,and thus produce a more effective treatment for influenza viral diseases.Hypecoum leptocarpum Hook.f.et Thoms.is a potential novel anti-influenza virus drug.