Gamma-oryzanol alleviates osteoarthritis development by targeting Keap1-Nrf2 binding to interfere with chondrocyte ferroptosis.

Osteoarthritis (OA) is a prevalent joint disorder pathologically correlated to chondrocyte ferroptosis. Gamma-oryzanol (γ-Ory), as a first-line drug for autonomic disorders, aroused our interest because of its antioxidant, lipid-lowering, and hypoglycemic potential. The purpose of this study was to investigate the potential impact and mechanism of γ-Ory in treating OA. And the inhibition of γ-Ory in extracellular matrix molecule (ECM) degradation, ferroptosis, and Keap1-Nrf2 binding in IL-1ß-exposed chondrocytes was detected via immunoblotting, immunofluorescence, and co-immunoprecipitation. Micro-CT, SO staining, and immunofluorescence have been conducted to assess the impact of γ-Ory treatment on ACLT-mediated OA in rats at both imaging and histological stages. We found that γ-Ory dose-dependently suppressed IL-1ß-induced ECM deterioration and chondrocyte ferroptosis. Our animal experiments revealed that γ-Ory delayed ACLT-mediated OA development. Mechanistically, γ-Ory interfered with the binding of Keap1 to Nrf2 to promote the latter's nuclear import, thereby increasing the expression of detoxification enzymes. Summarily, our works support γ-Ory's potential as a candidate drug for the treatment of OA.

[Preparation, characterization and potential application of monoclonal antibody 18A4 against AGR2].

AIM: To prepare mouse monoclonal antibodies (mAb) against AGR2 (human homolog of xenopus anterior gradient 2), to characterize these antibodies' properties, and to develop potential applications. METHODS: BALB/c mice were immunized with AGR2-MBP (maltose binding protein) fusion protein. The mAb was prepared by hybridoma technique and purified by protein G affinity chromatography. The titer and specificity of the mAb was determined by ELISA and Western blot respectively. The mAb was then further characterized by immunoprecipitation, immunofluorescent staining and tumor cell inhibition assay. RESULTS: One clone of hybridoma, 18A4, secreting specific mAb against AGR2, was obtained. The Ig subclass of the mAb was IgG1 (kappa). The titer of the mAb was 1 x 10(-6);. Western blot analysis showed specific binding of 18A4 with both recombinant and native AGR2 in cell extract. Immunofluorescent staining using 18A4 mAb demonstrated specific staining of AGR2 in the cytoplasma of Breast cancer cell line MCF7. Immunoprecipitation assay confirmed that this mAb could specifically bind and effectively precipitate the native AGR2. mAb 18A4 could also inhibit the growth of breast cancer cell MCF7. CONCLUSION: The mAb anti-AGR2 with high titer and specificity has been obtained. This mAb, 18A4, can be used in most molecular biology studies including Western blot, immunostaining, immunoprecipitation. It also inhibited the growth of cancer cells and therefore is a potential therapeutic starting point. It is a useful tool for the functional study of AGR2 and for the diagnosis and potential treatment of certain cancers.