Background/Purpose: Inflammation is an important component of osteoarthritis (OA) pathogenesis. IL-6 is implicated in OA pathogenesis as it suppresses anabolic factors and upregulate the expression of catabolic proteins. Here, we used an in vitro model of inflammation in OA to investigate the potential of Baicalein, a natural flavonoid found in root extract of Scutellaria baicalensis, to suppress the expression of IL-6 and determined the molecular mechanism by investigating the role of Nrf2 and autophagy activation in human OA chondrocytes.

Methods: Primary human OA chondrocytes were prepared by enzymatic digestion of deidentified and discarded cartilage from donors with OA who underwent total knee arthroplasty. Autophagy activation was investigated by immunoblotting for LC3-I and LC3-II, ATG5, autophagic flux and immunofluorescence staining for LC3 puncta. Expression and activation of Nrf2 was determined by immunoblotting and by a luciferase reporter assay, respectively. mRNA and protein expression of Nrf2 regulated genes HO-1, NQO1, SOD2 was studied by qPCR and immunoblotting, respectively. Total protein levels and activation of ERK1/2 and its upstream and downstream signaling molecules was assayed by immunoblotting. For molecular docking studies using the Glide tool in Schrödinger Maestro suite, the crystal structure of Keap1 protein in complex with a small chemical compound K67 (PDB CODE: 4ZY3) was extracted from the Protein Data Bank and used as docking structure template.

Results:

OA chondrocytes showed high levels of IL-6 expression upon stimulation with IL-1β. However, pre-treatment of OA chondrocytes with Baicalein, in a dose dependent manner, abolished the IL-1β-induced upregulation of IL-6 expression. Baicalein induced macro-autophagy in OA chondrocytes in vitro as indicated by significantly (p<0.05) increased expression of LC3, ATG5, ATG3, enhanced autophagy flux and increased number of autophagic puncta in OA chondrocytes. Baicalein treated OA chondrocytes also showed enhanced activity of Nrf2/ARE as revealed by a Nrf2/ARE reporter assay. Molecular docking studies indicated that Baicalein activates Nrf2 by disrupting the Keap-1/Nrf-2 interaction by blocking the Nrf-2 binding site in the Keap-1 protein. Additionally, OA chondrocytes treated with Baicalein, and in a dose dependent manner, showed enhanced expression, both at mRNA and protein levels, of Nrf2 target genes HO-1, NQO1, and SOD2. We next determined the molecular events involved in Baicalein mediated activation of Nrf2 and it was found that treatment of OA chondrocytes with Baicalein activated MEK1/2-ERK1/2-Elk-1 signaling in a time dependent manner. Inhibition of ERK1/2 activation or inhibition of autophagy using small molecules or siRNA mediated depletion of target genes expression abolished the protective effects of Baicalein in OA chondrocytes under pathological conditions.

Conclusion: The present study indicates that Baicalein act, at least in part, by activating Nrf2/autophagy axis and increased expression of HO-1, NQO1 and SOD2 in OA chondrocytes. This property indicates that Baicalein could be developed as an effective adjunct therapy for the suppression of OA pathogenesis.

Supported by USPHS/NIH grants

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/baicalein-a-plant-derived-small-molecule-activate-nrf2autophagy-signaling-axis-via-mek12-erk12-elk1-pathway-to-suppress-the-expression-of-il-6-in-human-osteoarthritis-chondrocytes/