Cells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.