Hydrogen Peroxide (H2O2) Induced Oxidative Stress in DEHs

A 100 μL volume of H₂O₂ (serially diluted by twofold in MM from 1 mol/L) was added to each well of the 96-well culture plate containing the DEHs monolayer, thus obtaining 6 concentrations for each. At the same time, the cell control (CC) group was set. The plate was cultured for 2 or 4 h. Then, the H₂O₂ solution was removed and the monolayer was washed three times with D-Hank’s. Subsequently, 100 μL of MM was added to each well and the cells were cultured for another 48 h, the MTT method was used to determine the DEHs viability.

As in the in vivo experiments, the treatments are divided into two groups: the normal groups and the intervention groups. In the normal groups (CC, VC, ICA and pICA), 100 μL of DHAV was added into the wells containing the monolayer of DEHs, while in the cell control (CC, not contain DHAV) and virus control (VC, contain DHAV) wells were reserved. Then, the plate was incubated for 2 h, and subsequently the virus solution was removed and the plate was washed three times with D-Hank’s. A 100 μL dilution of ICA and pICA was separately added into the test wells (6 wells in parallel). The plate was further incubated for 48 h. In the intervention groups (H₂O₂, VC-i, ICA-i, pICA-I group), 100 μL of H₂O₂ was added into the wells containing the monolayer DEHS. After 2 h incubation, the H₂O₂ was discarded and the wells were washed three times with D-Hank’s, 100 μL DHAV was added into the wells containing the monolayer of DEHS, while the H₂O₂ treatment control (H₂O₂, not contain DHAV) wells were reserved. Then, the plate was incubated for 2 h and subsequently the virus solution was removed and the plate was washed three times with D-Hank’s, the virus control was reserved for the intervention treatment (VC-i). A 100 μL dilution of ICA (ICA-i) and pICA (pICA-i) was then separately added into the test wells (6 wells in parallel). The plate was further incubated for 48 h. The MTT assay was used to determine the cell viability of each DEHs group.

In the normal groups (CC, VC, ICA and pICA), 400 μL of DHAV was added into the wells of a 24-well culture plate containing the monolayer DEHs, while the cell control (CC, not contain DHAV) and virus control (VC, contain DHAV) wells were reserved. After being incubated for 2 h, the wells containing the DEHs monolayer were washed three times with D-Hank’s. Then, both ICA and pICA, at the most effective concentrations, were added into wells of the ICA and pICA groups; MM was added into the wells of the CC and VC groups, three wells each group and 400 μL per well. Subsequently, the plate was incubated for 30 h. Then, the 24-well plate was washed three times with D-Hank’s to remove the remaining drugs and 400 μL of MM was added into each well. Afterward, the plate was further incubated for 24 h. In the intervention groups (H₂O₂, VC-i, ICA-i, pICA-i group), 400 μL H₂O₂ was added into the wells of the 24-well culture plate containing the monolayer DEHs. Following a 2 h incubation period, and the wells were washed three times with D-Hank’s, the rest of the process was the same as the treated normal groups. The corresponding groups were renamed as H₂O₂, VC-i, ICA-i and pICA-i. Also, the DHAV from each well was collected sterilely. The 50% tissue culture Infective Dose (TCID₅₀) of the DHAV of each group was measured using the Reed–Muench assay.

A 1.6 mL volume of DHAV was added into the 6-well culture plate containing the DEHs monolayer, while the cell control (CC, not contain DHAV) and virus control (VC, contain DHAV) wells were reserved. The plate was incubated for 2 h, then the virus solution was removed, and the plate was washed three times with D-Hank’s. Subsequently, 1.6 mL of the ICA and pICA dilutions were separately added into the test well (three wells in parallel), as the most effective antiviral concentration. The 6-well culture plate was incubated for 48 h and following this incubation, all samples were trypsinized and collected for testing the levels of SOD, MDA, CAT, GSH and GSH-PX.

Duck embryonic hepatocytes were harvested and suspended into 80 μL of lysis buffer containing protease inhibitors (Beyotime, China) and the concentration of the protein extract was determined using the BCA protein assay kit (Beyotime). Sixty micrograms of protein were diluted in sample loading buffer and heated at 95°C for 5 min. The denatured proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 40 min at room temperature in tris-buffered saline containing 5% BSA and 0.1% tween 20, followed by overnight incubation at 4°C, separately, with specific primary antibodies (anti SOD, anti GSH-px, anti JNK, anti p-JNK, anti ERK1/2, anti p-ERK1/2, anti-p38, anti p-p38, or anti β-actin), which detect proteins involved in various cell signaling pathways. The membranes were washed and incubated with the appropriate secondary antibody (Thermo Pierce, Rockford, IL, United States) at room temperature for 1 h. Blots were visualized using a standard enhanced chemiluminescence system (Bio-Rad Labs., Hercules, CA, United States).