Il 10

Isolated hippocampi were homogenized, and aliquots were used for the following determinations using the ELISA technique: IL-10, TNF-α, and IL-1β, levels (MyBioSource, USA). Each sample’s protein content was measured using a colorimetric technique, and all parameters were represented as pg/mg protein.

Ethanol was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Normal saline (0.9 % w/v) was prepared from the Samen® pharmacy factory, Iran. Xylazine, acepromazine, and ketamine were prepared from Alfasen, Woerden, Holland. Also, enzyme-linked immunosorbent assay (ELISA) kits, including VEGF, IL-6, TGF-β, IL-10, and TNF-α, were obtained from MyBioSource, USA with catalog number respectively; MBS3015758, MBS8244673, MBS2701296, MBS2707969, MBS175904. Dichloromethane (DCM) was purchased from Caledon, Canada, for HPLC grade. In addition, deionized water and Ethanol (96 %) were obtained from Abtin Co. (Iran), Kian Kaveh Azma Co. (Iran), and Taghtir Khorasan Co. (Iran), respectively. Furthermore, 6-gingerol was purchased from Golexir Pars ® Co., Iran, as an internal standard. Other chemicals and reagents were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

The IL-10, IL-6, tumor necrosis factor-α (TNF-α), IFN-α, IFN-β, transforming growth factor-β (TGF-β), and CXCL13 enzyme linked immunosorbent assay (ELISA) kits were purchased from MyBioSource (Vancouver, DC, USA), while the IL-2, IL-17, IL-18, IL-1β, and CXCL1 ELISA kits were purchased from Cloud Clone Corp (Wuhan, China). The level of cytokines in the serum was measured according to the manufacturer’s instructions.

Blood was collected from the mice in each group and subsequently centrifuged at 4000 rpm for 10 min. The levels of MCP-1, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the obtained sera were measured utilizing ELISA sets (Becton Dickinson).
In the case of reactive oxygen species (ROS), IL-17, IL-10 and Foxp3, ELISA kits were used: ROS (MyBiosource, San Diego), IL-17 (R & D Systems, Minneapolis), IL-10 (Chondrex, Redmond), Foxp3 (Cloud-Clone, Houston) These assays were conducted according to the protocols provided by the above manufacturers.

LPS (Escherichia coli 055:B5) were obtained from Aladdin (Beijing, China). De Man, Rogosa, and Sharpe (MRS) was from Solarbio Ltd. (Solarbio, Beijing, China). Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), ALT, AST, MDA, T-AOC, SOD, GSH-Px, CAT, TNF-α, transforming growth factor (TGF)-β, IL-1β, IL-6, IL-17, IL-10, IL-22, and LPS kits were obtained from MY BioSource Co., Ltd. (San Diego, CA, USA).

Fifteen days post-immunization, whole blood obtained from hamsters immunized with rLS1-HN-RBD, rLS1-S1-F, and rLS1-HN-RBD/rLS1-S1-F or allantoic fluid (mock) was centrifuged at 1000×g for 20 min at 4 °C to obtain the serum, which was duly aliquoted, frozen, and stored at − 80 °C until analysis. For the quantitative ELISA, several kits for the accurate quantitative detection of hamster cytokines such as, TNFα, IFNγ, IL-2, IL-4, and IL-10 were purchased from MyBioSource, Inc., San Diego, CA. Cytokine quantifications were performed following the manufacturer's instructions. The plates were read in the EON spectrophotometer (Biotek, USA) at 450 nm. The level of cytokines (pg/mL) detected in the serum of the animals vaccinated with rLS1-HN-RBD, rLS1-S1-F, and rLS1-HN-RBD/rLS1-S1-F were compared with mock vaccinated animals.