OBJECTIVE To study the effects and mechanisms of serum containing Wenjing Tongluo Decoction on interleukin-1β (IL-1β)-induced mouse primary chondrocyte injury.
METHODS The IL-1β-induced primary chondrocyte injury model was established, and the mRNA expression of inflammatory factors IL-1β, IL-6, TNF-α, cartilage degradation-related proteases matrix metalloproteinase 3 (MMP3), MMP9, MMP13, ADAM metallopeptidase containing thrombospondin 1 motif 4 (ADAMTS4), and glycolysis-related enzymes lactate dehydrogenase A (LDHA), M2 pyruvate kinase (PKM2), reduced coenzyme Ⅱ oxidase 2 (NOX2) and reduced coenzyme Ⅱ oxidase 4 (NOX4) in the cells was detected; the intracellular MMP3, MMP13, P65, IκB-ζ, hypoxia-inducible factor 1α (HIF-1α) and LDHA protein expression levels were determined; the concentrations of nitric oxide (NO), malondialdehyde (MDA) and lactic acid in the cell supernatant were further detected, and the ratio of the reduced state (NADH) and oxidized state (NAD+) of intracellular coenzyme Ⅰ (NAD), as well as intracellular reactive oxygen species (ROS) levels were assayed.
RESULTS Serum containing Wenjing Tongluo Decoction significantly inhibited the mRNA expression of IL-1β, IL-6, and TNF-α in IL-1β chondrocytes (P < 0.01), reduced the NO concentration in the cell supernatant (P < 0.05, P < 0.01), and downregulated the protein expression of IκB-ζ (P < 0.05) and P65 (P < 0.05, P < 0.01). Serum containing Wenjing Tongluo Decoction significantly downregulated the mRNA expression of MMP3, MMP9, MMP13 and ADAMTS4 (P < 0.01), and inhibited the protein expression of MMP13 (P < 0.05, P < 0.01) and MMP3 (P < 0.05). In terms of oxidative stress, it significantly suppressed the production of ROS in chondrocytes, increased the NADH/NAD+ ratio, reduced the MDA concentration in the supernatant, and downregulated the expression of HIF-1α protein (P < 0.01). The serum containing Wenjing Tongluo Decoction significantly reduced the lactic acid concentration, downregulated the expression of LDHA, PKM2, NOX2 and NOX4, and reduced the level of intracellular glycolysis (P < 0.05, P < 0.01).
CONCLUSION The serum containing Wenjing Tongluo Decoction can alleviate IL-1β-induced mouse primary chondrocyte injury by inhibiting inflammation, cartilage degradation, oxidative stress and glycolysis, which may be related to the regulation of IκB-ζ/HIF-1α/LDHA axis.
DownLoad: