Butein Activates Autophagy Through AMPK/TSC2/ULK1/mTOR Pathway to Inhibit IL-6 Expression in IL-1β Stimulated Human Chondrocytes

Abstract

Background/Aims: Butein (2’,3,4,4’-Tetrahydroxychalcone), a polyphenol produced by several plants including Butea monoserpma, has been reported to exert potent anti-inflammatory activity but the mechanism remains unknown. In the present work we investigated the mechanism of Butein-mediated suppression of IL-6 expression in normal and human osteoarthritis (OA) chondrocytes under pathological conditions. Methods: Expression level of interleukin-6 (IL-6) protein in OA cartilage was analyzed by immunohistochemistry using a validated antibody. Chondrocytes derived from normal or OA cartilage by enzymatic digestion were pretreated with Butein followed by stimulation with interleukin-1β (IL-1β) and the levels of IL-6 mRNA were quantified by TaqMan assay and the protein levels were measured by Western immunoblotting. Autophagy activation was determined by Western blotting and confocal microscopy. Autophagy was inhibited by siRNA mediated knockdown of ATG5. Results: Expression of IL-6 protein was high in the OA cartilage compared to smooth cartilage from the same patient. OA chondrocytes and cartilage explants stimulated with IL-1β showed high level expression of IL-6 mRNA and protein. Butein increased the phosphorylation of AMPKα^Thr-172, TSC2^Ser-1387 and ULK1^Ser-317 and inhibited the phosphorylation of mTOR^Ser-2448 and its downstream target p70S6K and increased autophagy flux that correlated with the suppression of the IL-1β mediated expression of IL-6 in normal and OA chondrocytes. In OA chondrocytes with siRNA-mediated knockdown of ATG5 expression, treatment with Butein failed to activate autophagy and abrogated the suppression of IL-1β induced IL-6 expression. Conclusion: Our findings demonstrate for the first time that Butein activate autophagy in OA chondrocytes via AMPK/TSC2/ULK1/mTOR pathway. Additionally, activation of autophagy was essential to block the IL-1β-induced expression of IL-6 in OA chondrocytes. These data support further studies to evaluate the use of Butein or compounds derived from it for the management of OA.