Effect of Luteolin on LPS Induced Chondrocyte Injury by Regulating Nrf2/HO-1 Pathway
PAN Yongli1, Rehemujiang muhetaer1, Tayier palati1, LIU Liqiong2*
This study investigated the protective effect of LPS (Luteolin） on lipopolysaccharide-induced chondrocyte injury by regulating the Nrf2 (nuclear factor erythroid 2-related factor 2)/HO-1 (heme oxygenase-1) pathway. Human chondrocyte line C28/I2 cells were assigned into blank group, LPS group (10 μg/mL), low dose Luteolin (25 μmol/L) group, medium dose Luteolin (50 μmol/L) group, high dose Luteolin (100 μmol/L) group, and Luteolin (100 μmol/L)+Nrf2 inhibitor (5 μmol/L ML385) group. Except for the blank group, all other groups were induced cell injury with LPS. The LDH (lactate dehydrogenase) method was used to detect cytotoxicity. CCK-8 (cell counting kit-8) method was used to detect cell viability. TUNEL staining was used to detect cell apoptosis. ELISA (enzyme linked immunosorbent assay) kits were used to detect the concentrations of TNF-α (tumor necrosis factor-α), iNOS (inducible nitric oxide synthase), and IL-6 (interleukin-6). The colorimetric method was used to detect oxidative stress factors T-SOD (total superoxide dismutase) and CAT (catalase). The probe DCFH-DA was used to detect the generation of ROS (reactive oxygen species). Western blot was used to detect the protein expression of Nrf2, HO-1, MMP-13 (matrix metalloproteinase-13), and a disintegrin and metalloproteinase with ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motif 5) in cells. Compared with the blank group, the cell LDH content, TUNEL positivity rate, TNF-α, iNOS and IL-6 concentrations, ROS level, MMP-13 and ADAMTS-5 protein expression levels increased in LPS group (P<0.05), while the D450 value, CAT and T-SOD activities, Nrf2 and HO-1 protein expression levels reduced (P<0.05). Compared with the LPS group, the cell LDH content, TUNEL positivity rate, TNF-α, iNOS and IL-6 concentrations, ROS level, MMP-13 and ADAMTS-5 protein expression levels reduced in 25, 50, and 100 μmol/L Luteolin groups (P<0.05), while the D450 value, CAT and T-SOD activities, Nrf2 and HO-1 protein expression levels increased (P<0.05). Compared with the 100 μmol/L Luteolin group, the cell LDH content, TUNEL positivity rate, TNF-α, iNOS and IL-6 concentrations, ROS level, MMP-13 and ADAMTS-5 protein expression levels increased in Luteolin+ML385 group (P<0.05), while the D450 value, CAT and T-SOD activities, Nrf2 and HO-1 protein expression levels reduced (P<0.05). Luteolin inhibites LPS-induced chondrocyte injury by activating Nrf2/HO-1 pathway.
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