Fig 1: Glutathione peroxidase 4 (GPX4) mediates the anti-ferroptotic effect of Transient receptor potential vanilloid 1 (TRPV1). (a) Western blot analysis of indicated proteins in mouse primary chondrocytes induced by 50 µM TBHP and/or 50 µM CPS for 8 h. (b) IF staining and quantitative analysis of Gpx4 in the indicated mice cartilage (n=6). AC, articular cavity; C, cartilage; SB, subchondral bone. (c, d) Quantification (c) and IF staining (d) of GPX4 in human OA cartilage explants treated as indicated for 7 days (n=6). (e) Cell viability (n=5) of mouse primary chondrocytes induced by 50 µM TBHP and/or 50 µM CPS for 24 h with or without pretreated Gpx4 siRNA. (f, g) S.O. staining (f) and quantitation of Osteoarthritis Research Society International (OARSI) scores and cartilage thickness (g) in Gpx4+/- mice and their littermate control mice treated as indicated (n=6). (h, i) IF staining of Col II and Mmp3 (h) and their quantification (i) in Gpx4+/- mice and their littermate control mice as treated in (f). (j, k) 3D reconstructed images and sagittal view of the medial joint compartment (j), as well as their relative quantitative analysis (k) of Gpx4+/- mice and their littermate control mice knee joints treated as in (f) (n=6). Scale bars, 50 µm (b, f, h), 200 µm (d), 1 mm (j). Two-tailed unpaired t-test (left panel of e), one-way (b, c) or two-way (right panel of e, g, i, k) ANOVA with Tukey's post-hoc test. Data are shown as mean ± SD.
Fig 2: Transient receptor potential vanilloid 1 (TRPV1) plays an anti-ferroptotic role in osteoarthritis (OA). (a, b) Safranin-O/fast green (S.O.) staining (a) and quantification (b) of mice induced by Sham or destabilization of medial meniscus (DMM) surgery with or without intraarticular injection of 50 µM capsaicin (CPS) (n=6). (c, d) Immunohistochemical (IHC) staining of Col II, immunofluorescence (IF) staining of Mmp3 (c),and corresponding quantitative analysis (d) in the articular cartilage of mice treated as in (a) (n=6). (e) IHC staining of 4-hydroxynonenal (4-HNE), IF staining of cyclooxygenase 2 (Cox2) and ferritin heavy chain 1 (Fth1) of indicated mice knee articular cartilage. (f) 3D reconstructed images of mice knee joints and the sagittal view of the medial joint compartment revealing the changes to femoral and tibial surfaces and subchondral bone plate (SBP) thickness, respectively. Red line indicates the thickness of SBP. (g) Hematoxylin & eosin (H&E) staining of mice knee and relative quantification of synovitis scores as indicated (n=6). (h) Representative images of S.O. staining, IHC staining for COL II, and IF staining for MMP3 of the cultured human OA cartilage explants treated with 50 µM TBHP and/or 50 µM CPS, or 5 µM Fer-1 for 7 days. (i, j) Representative images of IHC staining for 4-HNE, IF staining for FTH1, COX2 (i) and Tunel (j) of human OA cartilage explants as treated in (h). Scale bars, 50 µm (c, e, g), 100 µm (a, h-j) and 1mm (f). One-way ANOVA with Tukey's post-hoc test. Data are shown as mean ± SD.
Fig 3: Ferroptotic hallmarks accumulate in osteoarthritis (OA) cartilage. (a) Safranin O-Fast Green (S.O) staining of intact (I) and damaged (D) articular cartilages from OA patients. (b) Immunohistochemical (IHC) staining of Collagen II (COL II), matrix metallopeptidase 3 (MMP3) and MMP13 in human OA cartilages. (c-e) The levels of total iron (c), lipid peroxidation (LPO) (d), glutathione (GSH), GSH/GSSG (oxidized GSH) ratios and glutathione peroxidase 4 (GPX4) activities (e) in paired intact (I) and damaged (D) (n = 11) human OA cartilages. gprot, gram of protein. (f, g) IHC staining of 4-hydroxynonenal (4-HNE) and its quantification in the intact (n=12) and damaged (n=16) human OA cartilages (f), and in Sham or destabilization of medial meniscus (DMM)-induced mice (n=6) for 8 weeks (g). (h, i) Western blot analysis (h) and quantification (i) of indicated proteins in paired intact (I) and damaged (D) (n=8) human OA cartilages. P, patient. Scale bars, 50 μm (b, f, g), 200 μm (a). Two-tailed paired (c-e, i) or unpaired (f, g) t-test. Data are shown as mean ± SD.
Fig 4: Effects of CSC and ECAC on matrix metalloproteinase and collagen levels in HGECs. Levels of type I collagen (A), MMP-1 (B), and MMP-3 (C) were determined by ELISA after the cells were treated with ECAC and CSC at a nicotine concentration of 0.125 µg/mL for 24 and 48 h, respectively. Data were expressed as means ± SEM; #p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 3).
Fig 5: Effects of CS and ECA on levels of matrix metalloproteinase and collagen genes in HGECs cells. The mRNA levels of type I collagen (A), MMP-1 (B), MMP-3 (C), and COX-2 (D) were detected by RT-PCR after HGECs were treated with different kinds of ECAC and CSC at nicotine concentration of 0.125 μg/mL for 12 and 24 h, respectively. Data are expressed as means ± SEM; #p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 3).
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