Il 1β

IL-6 ELISA kit (PI335, Beyotime, China) and IL-1β (ml02783) and TNF-α ELISA kit (ml001696, Mlbio, China) were used to detect. Achilles tendon tissues at the edge of the healing site were triturated and lysed, and the supernatant was collected by centrifugation (1,000 x g, 4°C). Into each 96-well plate, 100 μl of sample was added. Then, 100 μl enzyme-labeled reagent was added and incubated at 37°C for 1 h. Subsequently, 50 μl of chromogenic reagent A was added into each well and an equal volume of chromogenic reagent B was added. The mixture was shaken gently to mix the two stains evenly in the dark. After 15 min, 50 μl stop solution was added to the reaction to halt color development. It was observed that the sample color changed from blue to yellow. Subsequently, the absorbance was detected. A blank well was used for zero adjustment and the detection wavelength used was 450 nm (680, Bio-Rad, USA). A standard curve was used to determine the concentrations of IL-6, IL-1β, and TNF-α.

Plasma and ileum contents were centrifuged at 3000 rpm for 30 s at 4 °C, and the supernatant was transferred to a 1.5 mL centrifuge tube. ELISAs were performed according to the instructions of the respective kits (IL-1β, TNF-α, IL-6, cholecystokinin (CCK), ghrelin, GLP-1, and peptide YY (PYY)) (Mlbio, Shanghai, China). Absorbance was measured at 450 nm using an enzyme marker, and the contents of IL-1β, TNF-α, IL-6, CCK, ghrelin, GLP-1, and PYY in the samples were calculated using the respective standard curves.

The cytokine concentrations (IL-6, MCP-1, TNFα, IL-1β, and TGF-β1) in rat kidney tissues and cell culture supernatant were measured. We determined the total protein concentration of tissues using BCA before the assay in rat kidney tissues. The cytokine concentration in rat kidney tissues was expressed using the cytokine content per gram of tissue homogenate protein. The concentration of NGAL, and KIM-1 in serum were measured. Assay kits for interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) (primary and mature forms), and transforming growth factor β1 (TGF-β1) were purchased from MlBio (Shanghai, China), while those for NGAL and KIM-1 were purchased from Elabscience (Wuhan, China). We measured IL-1β (ml037361), Pro-IL-1β (ml027415), TNF-α (ml002859), IL-6 (ml102828), MCP-1 (ml002960), TGF-β1 (ml002856), NGAL (E-EL- R0662c), and KIM-1 (E-EL-R3019) using the double antibody sandwich method and calculated the concentrations as per manufacturer's protocol.

IFN-α/β, IL-1β, and IL-18 ELISA kits were purchased from Shanghai mlbio. Caspase-1, caspase-3, caspase-8 activity assay kits, Total Nitric Oxide (NO) assay kit, Enhanced ATP assay kit, Enhanced mitochondrial membrane potential assay kit, and PI3K inhibitor (LY294002) were purchased from Beyotime Institute of Biotechnology. Recombinant chicken IFN-α was purchased from Cloud clone crop (CCC, USA). Lipofectamine 3000 was purchased from Invitrogen. The following antibodies were used for immunoblot analysis: anti-ALV-J envelope protein JE9 (kindly provided by Dr. Aijian Qin, Yangzhou University, Yangzhou, China), anti-Flag (AF5051, Beyotime), FITC-labeled goat anti-rabbit IgG (H+L) (A0562, Beyotime), anti-STAT1 (70R-51641, Fitzgerald), anti-phosphorylated STAT1 (15H13L67, Invitrogen), anti-Akt (10176-2-AP, Proteintech), anti-phosphorylated Akt (D9E, Cell Signaling), anti-mTOR (bs-1992R, Bioss), anti-phosphorylated mTOR (D9C2, Cell Signaling), anti-IKKα/β (bs-10123R, Bioss), anti-phosphorylated IKK (bs-3229R, Bioss), anti-β-actin (AF5003, Beyotime), Goat anti-rabbit IgG/HRP (bs-0295G, Bioworld), Goat anti-mouse IgG/HRP (bs-0296G, Bioworld).

Freund’s complete adjuvant (FCA) was purchased from Sigma Aldrich Chemie Gmbh (St. Louis, MA, USA). All the enzyme-linked immunosorbent assay (ELISA) kits (IL-1β, IL-6, PGE2, and TNF-α) were purchased from Shanghai Mlbio Biotechnology Co., Ltd. (Shanghai, China). Rutin was obtained from Guizhou Dida Technology Co., Ltd. (Guiyang, China); the purity of these reagents was higher than 98% as determined by HPLC. Rheumatoid bone pain capsules (Batch No. 180304) were purchased from Sinopharm Group Jingfang (Anhui) Pharmaceutical Co., Ltd (Xuancheng, China). Acetonitrile and methanol of HPLC grade were obtained from Tedia Co., Inc. (Fairfield, OH, USA). Ultrapure water was used for all solutions and dilutions (ultrapure water was prepared by the subject TM-D24UV ultrapure water machine). Other chemicals and solvents were of analytical or HPLC grade.

Freund’s complete adjuvant (FCA) was purchased from Sigma Aldrich Chemie Gmbh (St. Louis, MA, USA). All the enzyme-linked immunosorbent assay (ELISA) kits (IL-1β, IL-6, PGE2, and TNF-α) were purchased from Shanghai Mlbio Biotechnology Co., Ltd. (Shanghai, China). Rutin was obtained from Guizhou Dida Technology Co., Ltd. (Guiyang, China); the purity of these reagents was higher than 98% as determined by HPLC. Rheumatoid bone pain capsules (Batch No. 180304) were purchased from Sinopharm Group Jingfang (Anhui) Pharmaceutical Co., Ltd (Xuancheng, China). Acetonitrile and methanol of HPLC grade were obtained from Tedia Co., Inc. (Fairfield, OH, USA). Ultrapure water was used for all solutions and dilutions (ultrapure water was prepared by the subject TM-D24UV ultrapure water machine). Other chemicals and solvents were of analytical or HPLC grade.