Journal: Journal of Pharmaceutical Analysis

Article Title: Screen of FDA-approved drug library identifies vitamin K as anti-ferroptotic drug for osteoarthritis therapy through Gas6

doi: 10.1016/j.jpha.2024.101092

Figure Lengend Snippet: Growth arrest-specific 6 (Gas6) is identified as the target of vitamin K. (A) Venn diagram showing the numbers of differentially expressed genes by RNA-sequence identified in chondrocytes treated with RSL3 (0.5 μM), vitamin K1 (VK1, 10 μM), vitamin K2 (VK2, 10 μM) and vitamin K3 (VK3, 1 μM) as indicated for 24 h. (B) Heatmap showing the 64 common differentially expressed genes. Top 12 upregulated genes were displayed. (C) Cell viability was detected by Cell Counting Kit-8 (CCK-8). Chondrocytes were transfected with small interfering RNA (siRNA) or negative control siRNA (si-NC). Two siRNAs against per gene, #1, #2, #3. A total of 24 h after transfection, chondrocytes were treated with RSL3 (0.5 μM) and VK1 (10 μM) for 24 h. (D) Cell viability was detected by CCK-8. Chondrocytes were transfected with Gas6 siRNA #2 (si-Gas6) or si-NC for 24 h. A total of 24 h after transfection, chondrocytes were treated with RSL3 (0.5 μM) and VK1 (10 μM) for 24 h. (E–G) The mRNA and protein levels of Gas6 were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (E) and Western blotting analysis (F, G). (H) Intracellular malondialdehyde (MDA) analysis of chondrocytes. (I) Reduced glutathione/oxidized glutathione (GSH/GSSG) ratio of chondrocytes. (J) Lipid peroxidation evaluated by BODIPY 581/591 C11 staining of chondrocytes (green: oxidized lipids; red: lipids; and blue: Hoechst). (K) The protein levels of glutathione peroxidase 4 (GPX4) were detected by Western blot analysis. (L) The mRNA levels of Mmp13 , Adamts4 , Col2a1 and Acan in chondrocytes were evaluated by RT-qPCR. (M) The protein expression levels of matrix metalloproteinase 13 (MMP13), ADAMTS4, collagen type II (COL II) and Aggrecan in chondrocytes were detected by Western blot analysis. (N) Immunofluorescence of MMP13, ADAMTS4, COL II and Aggrecan in chondrocytes. Data are the mean ± standard deviation (SD) from three independent experiments with one-way analysis of variance (ANOVA) with Dunnett's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01; ns: not significant. Ctrl: Control.

Article Snippet: For IHC, sections were treated with primary antibodies against matrix metalloproteinase 13 (MMP13; 1:100, ab39012, Abcam, Cambridge, UK), COL II (1:100, ab34712, Abcam), GPX4 (1:200, ab125066, Abcam) and Gas6 (1:100, A00608-1, BOSTER Biological Technology, Wuhan, China) at 4 °C overnight.

Techniques: Sequencing, Cell Counting, CCK-8 Assay, Transfection, Small Interfering RNA, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Expressing, Immunofluorescence, Standard Deviation, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: Screen of FDA-approved drug library identifies vitamin K as anti-ferroptotic drug for osteoarthritis therapy through Gas6

doi: 10.1016/j.jpha.2024.101092

Figure Lengend Snippet: Vitamin K attenuates destabilization of the medial meniscus (DMM)-induced mice osteoarthritis development. (A) Representative images of hematoxylin-eosin staining (H&E) staining, safranin O fast green (Safranin-O) staining and toluidine blue staining of cartilage sections in the five study groups at 8 weeks after surgery. (B) Osteoarthritis Research Society International (OARSI) score of cartilage based on Safranin-O staining of the five groups after treatment for 8 weeks. n = 8. (C) Representative three-dimensional (3D) reconstructed micro-computed tomography (micro-CT) images in the five study groups at 8 weeks after surgery. (D) Quantitative analysis of structural parameters of subchondral bone by micro-CT analysis: thickness of subchondral bone plates (SBP), total bone volume (BV) and trabecular pattern factor (Tb. Pf). n = 8. (E) The mRNA levels of catabolic enzymes ( Mmp13 and Adamts4 ) and matrix components ( Col2a1 and Acan ) in articular cartilage obtained from four study groups. n = 3. (F) The mRNA levels of glutathione peroxidase 4 ( Gpx4 ) and growth arrest-specific 6 ( Gas6 ) in articular cartilage obtained from four study groups. n = 3. (G) Representative images of immunohistochemistry (IHC) of matrix metalloproteinase 13 (MMP13), collagen type II (COL II), GPX4 and Gas6 in mice cartilage sections. (H) Quantitative analysis of MMP13, COL II, GPX4 and Gas6 expression with IHC. n = 3. Data are the mean ± standard deviation (SD) with one-way analysis of variance (ANOVA) with Dunnett's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01; ns: not significant. VK1: vitamin K1; Fer-1: Ferrostatin-1; AAV-NC: adeno-associated virus-negative control; AAV-shGas6: adeno-associated virus-Gas6 short hairpin RNA.

Article Snippet: For IHC, sections were treated with primary antibodies against matrix metalloproteinase 13 (MMP13; 1:100, ab39012, Abcam, Cambridge, UK), COL II (1:100, ab34712, Abcam), GPX4 (1:200, ab125066, Abcam) and Gas6 (1:100, A00608-1, BOSTER Biological Technology, Wuhan, China) at 4 °C overnight.

Techniques: Staining, Micro-CT, Immunohistochemistry, Expressing, Standard Deviation, Virus, Negative Control, shRNA

Journal: Journal of Pharmaceutical Analysis

Article Title: Screen of FDA-approved drug library identifies vitamin K as anti-ferroptotic drug for osteoarthritis therapy through Gas6

doi: 10.1016/j.jpha.2024.101092

Figure Lengend Snippet: AXL receptor tyrosine kinase (AXL)/phosphatidylinositol 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) pathway mediates the anti-ferroptotic effects of growth arrest-specific 6 (Gas6) and vitamin K. (A) Evaluation of anti-ferroptotic effect of Gas6 in chondrocytes. Chondrocytes were pretreated with indicated Gas6 concentrations for 2 h followed by treated with RSL3 (0.5 μM) for 24 h. (B) The protein expression levels of TAM receptors (AXL, TYRO3) in chondrocytes were detected by Western blot analysis. Chondrocytes were treated with RSL3 (0.5 μM, 24 h), vitamin K1(VK1, 10 μM, 24 h) or Gas6 (100 ng/mL, 2 h). (C) Cell viability was detected by Cell Counting Kit-8 (CCK-8). Chondrocytes were transfected with AXL small interfering RNA (si-AXL, #1, #3), TYRO3 small interfering RNA (si-TYRO3, #2, #3) or negative control siRNA (si-NC). A total of 24 h after transfection, chondrocytes were pretreated with Gas6 (100 ng/mL) for 2 h followed by treatment with RSL3 (0.5 μM) for 24 h. (D, E) Intracellular malondialdehyde (MDA) analysis (D) and reduced glutathione/oxidized glutathione (GSH/GSSG) (E) ratio of chondrocytes. Chondrocytes were transfected with si-AXL #1 or si-NC for 24 h. A total of 24 h after transfection, chondrocytes were pretreated with Gas6 (100 ng/mL) for 2 h followed by treatment with RSL3 (0.5 μM) for 24 h. (F) Intracellular ferrous levels detected by FerroOrange probe and lipid peroxidation evaluated by BODIPY 581/591 C11 staining of chondrocytes (green: oxidized lipids; red: lipids; and blue: Hoechst). (G) The mRNA levels of Mmp13 , Adamts4 , Col2a1 and Acan in chondrocytes were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). (H) The protein expression levels of matrix metalloproteinase 13 (MMP13), ADAMTS4, collagen type II (COL II) and Aggrecan in chondrocytes were detected by Western blot analysis. (I) The phosphorylation of AXL (P-AXL) was detected by Western blot analysis. After pretreated with R428 (AXL inhibitor, 5 μM) for 15 min, chondrocytes were treated with Gas6 (100 ng/mL) for 2 h followed by treatment with RSL3 (0.5 μM) for 2 h. (J) Cell viability were detected by CCK-8. Chondrocytes were pretreated with R428 (5 μM) for 15 min followed by treated with Gas6 (100 ng/mL, 2 h) and RSL3 (0.5 μM, 24 h). (K) The P-AKT was detected by Western blot analysis. After pretreated with GDC-0941 (PI3K inhibitor, 5 μM) for 15 min, chondrocytes were treated with Gas6 (100 ng/mL) for 2 h followed by treatment with RSL3 (0.5 μM) for 2 h. (L) Cell viability were detected by CCK-8. Chondrocytes were pretreated with GDC-0941 (5 μM) for 15 min followed by treatment with Gas6 (100 ng/mL, 2 h) and RSL3 (0.5 μM, 24 h). (M) Cell viability was detected by CCK-8. Chondrocytes were transfected with si-AXL, si-TYRO3 or si-NC. A total of 24 h after transfection, chondrocytes were treated with VK1 (10 μM) and RSL3 (0.5 μM) for 24 h. (N) Cell viability were detected by CCK-8. Chondrocytes were pretreated with R428 (5 μM) or GDC-0941 (5 μM) for 15 min followed by treated with VK1 (10 μM) and RSL3 (0.5 μM) for 24 h. (O) Graphic abstract of our study indicating how vitamin K and Gas6 protects against osteoarthritis through inhibiting ferroptosis. Data are the mean ± standard deviation (SD) from three independent experiments with one-way analysis of variance (ANOVA) with Dunnett's post hoc test. ∗∗ P < 0.01; ns: not significant. PBS: phosphate buffered saline;Ctrl: Control.

Article Snippet: For IHC, sections were treated with primary antibodies against matrix metalloproteinase 13 (MMP13; 1:100, ab39012, Abcam, Cambridge, UK), COL II (1:100, ab34712, Abcam), GPX4 (1:200, ab125066, Abcam) and Gas6 (1:100, A00608-1, BOSTER Biological Technology, Wuhan, China) at 4 °C overnight.

Techniques: Expressing, Western Blot, Cell Counting, CCK-8 Assay, Transfection, Small Interfering RNA, Negative Control, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Phospho-proteomics, Standard Deviation, Saline, Control