Interleukin 6 (il 6)

Mice serum preparation: orbital blood was collected and centrifuged at a speed of 3000 r min ⁻¹ for 15 min. The upper serum was taken out of a centrifuge tube and stored at – 70 ℃. Contents of IFN-γ (Lot No.228070832, Multi Science), TGF-β (Lot No.228170744, Multi Science), IL-4 (Lot No.220471131, Multi Science), IL-13 (Lot No.221371135, Multi Science), IL-6 (Lot No.220671132, Multi Science), IL-10 (Lot No.221070945, Multi Science), TNF- α (Lot No.218280443, Multi Science) in serum of mice were detected by sandwich ELISA assay according to the Kit instructions.

Participants were seated, and approximately 5 mL of venous blood was drawn from the anterior cubital vein by venipuncture. The blood sample was collected in a heparin anticoagulant tube and centrifuged at 2000 g for 10 min at 4 °C. Then, the upper plasma layer was extracted and stored in a refrigerator at -80 °C. The total ghrelin (Millipore, Burlington, USA; % coefficient of variation (CV): 6.2%) and peptide YY (PYY) (Millipore, Burlington, USA; %CV: 4.8%) in the extracted sample were determined using radio-immunoassays, while glucagon-like peptide-1 (GLP-1) (Cusabio; %CV: 5.7%) and high sensitivity interleukin-6 (IL-6) (Multi science, Hangzhou, China; %CV: 6.7%) was measured using ELISA.

Andrographolide (C₂₀H₃₀O₅) was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). The sample consisted of a white powder with a molecular weight of 350.45 and a purity of above 98% determined by HPLC analysis. OVA was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). The aluminum hydroxide gel was purchased from Thermo Fisher Scientific (Watham, MA, USA). The IL-6, IL-17A, and IL-17F ELISA kits were purchased from Multi Sciences (Hangzhou, China). Primary antibodies and HRP-conjugated anti-rabbit IgG were purchased from Bioworld Technology (Minnesota, USA), Cell Signaling Technology (Beverly, MA, USA), Affinity Bioscience (Shanghai, China), and Proteintech Group (Wuhan, China), respectively. SuperSignal West Femto Stable Peroxide Substrate and AlexaFluor594-labeled goat anti-rabbit IgG antibodies were obtained from Thermo Scientific (San Jose, CA, USA).

Nine times the volume of PBS (pH 7.4) were added to the heart tissue to prepare a 10% buffered homogenate, and the supernatants of the cardiac homogenate samples were collected after centrifugation at 3,000 rpm for 10 min. Detected based on the ELISA kits procedures were the inflammatory cytokines tumor necrosis factor-α (TNF-α) (Thermo Fisher Scientific Inc., Massachusetts, America; Catalog number: 88–7324) and interleukin-6 (IL-6) (Multi Sciences, Biotech, Co., Ltd, Hangzhou, China; Catalog number: EK206/3-01), in the supernatant of tissue homogenate.