Abstract: 【Objective】 The aim of this study was to explore the effect of mangiferin (Man) on the inflammatory injury of duck embryo hepatocytes (DEHCs) induced by Duck hepatitis A virus-1 (DHAV-1).【Method】 The primary DEHCs were isolated from the liver tissues of 14-day-old SPF duck embryos, and cultured for 48 h with different concentrations of mangiferin (0, 5, 10, 20, 40, 80 and 160 μmol/L).Then, the safe concentration range of mangiferin was detected by CCK-8 method.The toxicity of mangiferin to DEHCs was determined by detecting the activity of lactate dehydrogenase (LDH) in the supernatant of DEHCs cultured with safe concentration of mangiferin for 12, 24 and 48 h.The DEHCs were divided into control group (Mock), model group (Model), mangiferin groups (Man10, Man20 and Man40) and ribavirin group (Rib), with three replicates in each group, and cells in all groups were cultured with serum starvation for 12 h.After that, Mock group was added with DMEM medium containing 10% FBS, Model, Man10, Man20, Man40 and Rib groups were challenged with DHAV-1 (MOI=1.0) for 2 h, and then Model group was replaced with DMEM medium containing 10% FBS, the culture medium of Man10, Man20, Man40 groups was supplemented with 10, 20 and 40 μmol/L mangiferin, respectively, and 1 μmol/L ribavirin was added to Rib group.After 48 h, cells in each group were collected, and the content of malondialdehyde (MDA) and the activities of catalase (CAT), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), total nitric oxide synthase (T-NOS) and glutathione peroxidase (GSH-Px) were detected by colorimetric method.The distribution of DHAV-1 in DEHCs cells in each group was detected by immunofluorescence (IF).Expression of NLRP3/Pro-Caspase1/IL-1β pathway proteins were detected by Western blotting method.The fresh whole blood of 2-day-old SPF ducks was extracted, and the peripheral blood mononuclear cells (duPBMCs) of the ducks were isolated.The grouping and processing of duPBMCs were the same as DEHCs.ELISA assay was used to detect the content of IL-8 and IL-1β in the supernatant of duPBMCs.【Result】 Compared with 0 μmol/L mangiferin group, the cell proliferation ability of 80 and 160 μmol/L mangiferin was significantly reduced (P<0.05), so 5-40 μmol/L mangiferin was a safe administration concentration for DEHCs.The activity of LDH in the supernatant of cells treated with 20 and 40 μmol/L mangiferin for 48 h was significantly lower than that of cells treated with 12 and 24 h (P<0.05), the activity of LDH in the supernatant of cells treated with 20 and 40 μmol/L mangiferin for 12 and 24 h was significantly higher than that treated with 5 and 10 μmol/L mangiferin (P<0.05), there was no significant difference in LDH activity in the supernatant of cells treated with mangiferin at different concentrations for 48 h (P>0.05), so 48 h was the optimum treatment time.Compared with the Mock group, the activities of CAT, T-AOC, SOD and GSH-Px in DEHCs of the Model group were significantly decreased (P<0.05), the content of MDA, the activity of T-NOS, DHAV-1 copy number, DHAV-1 positive rate, the expression of NLRP3 in DEHCs and the secretion levels of IL-8 and IL-1β in duPBMCs were significantly increased (P<0.05).Compared with Model group, the activities of CAT, T-AOC, SOD and GSH-Px in DEHCs of Man10, Man20 and Man40 groups were significantly increased (P<0.05), the content of MDA, the activity of T-NOS, DHAV-1 copy number, DHAV-1 positive rate, the expression of NLRP3 in DEHCs and the secretion levels of IL-8 and IL-1β in duPBMCs were significantly decreased (P<0.05).【Conclusion】 Mangiferin could resist the inflammatory damage caused by DHAV-1 infection of DEHCs by increasing the antioxidant capacity, reducing the replication level of DHAV-1, inhibiting the activation of NLRP3 pathway and reducing the release of pro-inflammatory cytokines.

Key words: mangiferin; Duck hepatitis A virus-1 (DHAV-1); duck embryonic hepatocytes; duck peripheral blood mononuclear cells