Elisa

All experimental groups’ liver samples were homogenized in 9 mL of pH 7.4 PBS buffer after being weighed to 1 g. To obtain the supernatant, the liver homogenate was centrifuged (2500× g, 4 °C, 10 min). The activities of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px), ethanol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH), catalase (CAT), total protein (TP), and triglyceride (TG) in liver tissue were determined according to the manufacturer’s protocol (Shandong Biobase Biodustry, Jinan, China). The levels of hepatic CYP2E1; inflammatory cytokines, including interleukin-6 (IL-6); and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA) based on the manufacturer’s instructions (Jiangsu Meimian Industrial Co., Ltd., Jiangsu, China) [33 (link),34 (link)].

Citrate synthase (CS, Cat. No. MM-204201), Isocitrate dehydrogenase (ICD, Cat. No. MM-3280701), α-ketoglutarate dehydrogenase complex (α-KGDHC, Cat. No. MM-9176301), and Na⁺/K⁺-ATPase (Cat. No. MM-166201), were determined by the enzyme-linked immunosorbent assays (ELISA), following the manufacturer’s procedures (Jiangsu Meimian Industrial Co., Ltd., China)

The blood samples were collected in EP tubes from the internal canthal vein and centrifuged (4 °C, 3500 rpm, 15 min). The total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C), and high-density lipoprotein-cholesterol (HDL-C) were detected using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). The enzyme-linked immunosorbent assay (ELISA) was used to assess the levels of serum cytokines, including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) (Jiangsu Meimian Industrial Co., Ltd., Yancheng, China).

Plasma samples were collected into sterile enzyme-free centrifuge tubes, and then the contents of ET (MM-1626O1), IL-1β (MM-925452O1), IL-6 (MM-91624O1), TNF-α (MM-32774O1) and D-LA (MM-91646O1) in plasma and jejunum and the concentrations of secretory immunoglobulin A (sIgA, MM-91217O1), immunoglobulin G (IgG, MM-91025O1), IL-10 (MM-33637O1), IL-22 (MM-925346O1) and MUC2 (MM-1790O1) in jejunum were measured by ELISA using a commercial kit (Jiangsu Meimian Industrial Co., Ltd.).

120 healthy egg-laying shelducks (Nanjing Zhushun Biotechnology Co., Ltd., China), weighting 1.5–2 kg and six months old, negative of any DTMUV and their antibody in vivo using RT-PCR and ELISA (Jiangsu Meimian industrial Co., Ltd., China) were investigated in this experiment. The ducks were divided into one control group and 11 treatment groups (10 per group). The ducks from the treatment groups were injected into leg muscle with the dose of 10⁴ TCID₅₀ of the virus and euthanized at different times 1, 2, 6, 12 h and 1, 3, 6, 9, 12, 18, 24 days post-infection (pi). The ducks in the control group were euthanized after injection with the same volume of 0.9% NaCl. The blood samples of each duck were collected and anticoagulated with 1.5% EDTA-Na₂ and centrifuged to isolate the blood plasma. The spleen samples were fixed in 4% buffered paraformaldehyde and frozen in liquid nitrogen respectively. The virus levels in blood and spleen were detected by RT-PCR and ELISA.

The content of immunoglobulin and cytokines in each group was determined by ELISA (Jiangsu Meimian Industrial Co., Ltd., Yancheng, China).

BV2 microglia (2 × 10⁵ cells/well) were seeded in 6-well culture plate for 12 h. Cells were then pretreated with platycodigenin (0.1, 1 and 10 μM) for 12 h, then cells were incubated for 1 h in the absence or presence of 100 ng/mL LPS. After incubation, cells were washed twice with cold PBS and total protein was collected from the cells in PBS with cell scraper and sonicated for three times. The total protein was quantified using BCA method and enzyme-linked immunosorbent assay (ELISA; MeiMian, Wuhan, China) was performed for quantification of p-p65 and p-p38 according to the manufacturer’s protocol.