Il 6 elisa kit

Male Sprague-Dawley rats (Shanghai Laboratory Animal Center, China) aged 7–8 weeks and weighing 250–300 g were used in the experiments. The rats were housed under standard laboratory conditions at a temperature of 20–22°C and 12 h light-dark cycle and given access to food and water. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. The study protocol was approved by the local Ethics Committee of the 117th Hospital of PLA. Rapamycin, AG490, and curcumin were purchased from Sigma-Aldrich (St. Louis, MO, USA). A TNF-α ELISA Kit, IL-1β ELISA Kit, and IL-6 ELISA Kit were purchased from Beyotime Biotechnology (Haimen, China). Antibodies against HMGB1, JAK2, p-JAK2, STAT3, p-STAT3, and the internal standard GAPDH (all from Affinity Biosciences, Cincinnati, OH, USA) were used for western blot analysis and immunohistochemical (IHC) analysis. The secondary antibody was a ready-to-use goat-anti rabbit (or goat-anti-mouse, or donkey-anti-goat) HRP-IgG dilution purchased from Beyotime Biotechnology (Haimen, China).

The cell supernatant was collected by centrifugation at 12,000 x g for 10 min at 4˚C and the concentrations of inflammatory cytokines were measured using interleukin (IL)-1β ELISA kit (cat. no. PI305; Beyotime Institute of Biotechnology), IL-6 ELISA kit (cat. no. PI330; Beyotime Institute of Biotechnology) and tumor necrosis factor (TNF)-α kit (cat. no. PT518; Beyotime Institute of Biotechnology) according to the manufacturers' protocols. All experiments were repeated in triplicate.