Academic Journal of Naval Medical University
Academic Journal of Naval Medical University
To explore the regulatory effect of corilagin (COR) on Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome activation and chondrocyte pyroptosis induced by lipopolysaccharide (LPS) combined with nigericin (NIG). Primary chondrocytes isolated from C57BL/6J mice were cultured to passage 3 for experiments. Cells were divided into control group, LPS group, LPS＋NIG group, and LPS＋NIG＋ COR (low-, medium-, and high-dose) groups. The chondrocytes were pre-sensitized with LPS for 4 h. Then the cells were treated with COR at different concentrations (10, 20, and 40 μmol/L) for 30 min, and finally NIG (10 μmol/L) was supplemented for 1 h. Control cells were cultured in DMEM/F-12 medium supplemented with 1% FBS. Cell counting kit 8 (CCK-8) was used to detect the effect of COR at different concentrations (10, 20, 40 μmol/L) on chondrocyte viability. Propidium iodide (PI) and Hoechst 33342 staining and lactate dehydrogenase (LDH) release assay were used to analyze the effect of COR on chondrocyte death induced by LPS and NIG. Western blotting was used to detect the expression of the NLRP3 inflammasome activation marker cysteine aspartic acid specific protease 1 (caspase 1) p20 in the cell supernatant and NLRP3, apoptosis-associated speck-like protein (ASC), caspase 1, interleukin-1β precursor (pro-IL-1β), and pyroptosis execution protein gasdermin D (GSDMD) in the cell lysate. Enzyme-linked immunosorbent assay was used to detect the level of IL-1β in cell culture supernatant. Reactive oxygen species (ROS) fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) staining was used to observe the effect of COR on ROS production, and Western blotting was used to detect the expression of intracellular glycolysis-related proteins hexokinase 2 (HK2), glucose transporter 1 (GLUT1), and lactate dehydrogenase A (LDHA). COR exhibited slight effect on chondrocyte viability at the concentration ≤ 40 μmol/L. COR (10-40 μmol/L) reduced the proportion of PI-positive cells (all P＜0.05) and the release of LDH (all P＜ 0.01) stimulated by LPS and NIG, inhibited the expression of GSDMD N-terminus domain in chondrocytes, and reduced the release of caspase 1 p20 and IL-1β from chondrocytes (all P＜0.01). Furthermore, COR (40 μmol/L) reduced the production of ROS (compared with the control group, P＜0.01) and inhibited the expression of glycolysis-related proteins HK2, GLUT1, and LDHA (all P＜0.05). COR can inhibit NIG-induced glycolysis/ROS/NLRP3 signaling, thereby preventing NLRP3 inflammasome activation and chondrocyte pyroptosis.
