Journal: Journal of Traditional and Complementary Medicine

Article Title: Combination therapy of Shinbaro and celecoxib improves joint pain and cartilage degradation in an osteoarthritis mouse model

doi: 10.1016/j.jtcme.2025.03.004

Figure Lengend Snippet: Fig. 7. Combined effect of Shinbaro and celecoxib on ECM components and ECM-degrading enzymes in a DMM-induced osteoarthritis model. (A–D) Representative images of immunofluorescence staining used to evaluate the expression levels of representative components of ECM. (A) COL2A1, (B) aggrecan, (C) MMP13, and (D) ADAMTS5 in the medial tibial plateau of mice after 8 weeks of oral administration. The staining shows COL2A1 in red, aggrecan in green, MMP13 in green, ADAMTS5 in green, and nuclei in blue (DAPI). Scale bar: 50 μm. (E–H) Optical density analysis of the medial tibial plateau performed to quantify the expression of (E) COL2A1, (F) aggrecan, (G) MMP13, and (H) ADAMTS5. N = 5/group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Sham; #p < 0.05, ##p < 0.01 vs. DMM. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean ± SD. DMM + Shin, DMM + Shinbaro (100 mg/kg); DMM + Cel15, DMM + celecoxib (15 mg/kg); DMM + Cel30, DMM + celecoxib (30 mg/kg); DMM + Shin + Cel15, DMM + Shinbaro (100 mg/kg) + celecoxib (15 mg/kg). COL2A1, type II collagen; MMP13, matrix metallopeptidase 13; ADAMTS5, a disintegrin and metalloproteinase with thrombospondin motifs 5.

Article Snippet: Primary antibodies against COL2A1 (mouse, 1:100, sc-52658; Santa Cruz Biotechnology Inc.), aggrecan (rabbit, 1:2000, AB313636; Abcam), MMP13 (rabbit, 1:100, 18165-1-AP; Proteintech), and ADAMTS5 (rabbit, 1:200, NBP215286; Novus Biologicals) were diluted in 1 × PBST (PBS + 0.3 % Triton X-100) supplemented with 0.3 % goat serum (Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Expressing

Journal: Scientific Reports

Article Title: Characteristics and time points to inhibit ferroptosis in human osteoarthritis

doi: 10.1038/s41598-023-49089-y

Figure Lengend Snippet: Ferroptosis occurs in OA cartilage in a region-specific manner. ( A ) Representative immunohistochemical (IHC) staining of NC (Negative Control), GPX4, ACSL4, MMP13, and COL2A1 in paired intact and damaged human OA cartilages. Scale bars: 100 μm (top) and 20 μm (bottom). ( B ) Quantification of immunohistochemical analysis (n = 3). ( C ) RT-qPCR was used to detect the mRNA expression of MMP13, COL2A1, GPX4, ACSL4 in human OA cartilage (n = 3). ( D ) Representative immunofluorescence staining and quantification analysis of GPX4 in human OA cartilage (n = 3). Scale bar: 50 μm. ( E ) Representative immunofluorescence analysis of ACSL4 in human OA cartilage (n = 3). Scale bar: 50 μm. ( F ) JC-1 staining was performed to monitor mitochondrial membrane potential (Δψm). The ratio of the red/green fluorescence indicated the degrees of mitochondrial damage (n = 3). Scale bar: 50 μm. Data are expressed as means + SD. For all experiments p-values calculated by unpaired two-tailed Student’s t test, p < 0.05, a statistically significant difference.

Article Snippet: After incubation with primary antibodies against COL2A1 (1:100; Servicebio, GB11021), MMP13 (1:100; Servicebio, GB11247), GPX4 (1:50; Zen-bio, R24461), and ACSL4 (1:20; Proteintech, 22401-1-AP), the sections were incubated with secondary antibody (Servicebio, G1215), followed by counterstaining with hematoxylin and visualized by DAB (Servicebio, G1212).

Techniques: Immunohistochemical staining, Immunohistochemistry, Negative Control, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Membrane, Fluorescence, Two Tailed Test

Journal: Scientific Reports

Article Title: Characteristics and time points to inhibit ferroptosis in human osteoarthritis

doi: 10.1038/s41598-023-49089-y

Figure Lengend Snippet: Ferroptosis increased with increasing cartilage degeneration in human OA. ( A ) Safranin O-fast green (S.O) staining of OA cartilages. ( B ) Modified Mankin score of OA cartilage. ( C ) Representative IHC staining of NC (Negative Control), GPX4, ACSL4, MMP13, and COL2A1 in human OA cartilages. Scale bars: 100 μm (top) and 20 μm (bottom). ( D ) Quantification of IHC analysis (n = 3). ( E ) RT-qPCR was used to detect the mRNA expression of MMP13, COL2A1, GPX4, ACSL4 in human OA cartilage. (n = 3). ( F ) MDA contents of human OA cartilage (n = 3). ( G ) Flow cytometry analysis of lipid peroxide level of human OA cartilage. ( H ) Representative immunofluorescence staining and quantification analysis of GPX4 in human OA cartilage (n = 3). Scale bar: 50 μm. ( I ) Representative immunofluorescence staining and quantification analysis of ACSL4 in human OA cartilage (n = 3). Scale bar: 50 μm. ( J ) Mitochondrial membrane potential was detected by JC-1 assay (n = 3). Scale bar: 50 μm. Data are expressed as means + SD. For all experiments p-values calculated by ANOVA analysis, p < 0.05, a statistically significant difference.

Article Snippet: After incubation with primary antibodies against COL2A1 (1:100; Servicebio, GB11021), MMP13 (1:100; Servicebio, GB11247), GPX4 (1:50; Zen-bio, R24461), and ACSL4 (1:20; Proteintech, 22401-1-AP), the sections were incubated with secondary antibody (Servicebio, G1215), followed by counterstaining with hematoxylin and visualized by DAB (Servicebio, G1212).

Techniques: Staining, Modification, Immunohistochemistry, Negative Control, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Membrane

Journal: Scientific Reports

Article Title: Characteristics and time points to inhibit ferroptosis in human osteoarthritis

doi: 10.1038/s41598-023-49089-y

Figure Lengend Snippet: Inhibition of ferroptosis can improve chondrocyte function in mild OA, but not moderate and severe OA. ( A ) The effects of Fer-1 (2 μM) on the viability of human normal chondrocytes after 24 h of treatment was determined by CCK-8 assay. ( B ) Human chondrocyte viability was determined by CCK-8 assay. ( C ) The viability of chondrocytes was determined by CCK-8 assay following treatment of chondrocytes of each group with Fer-1 (2 μM) for 24 h. ( D – I ) The mRNA expression levels of MMP13, COL2A1, GPX4, ACSL4, SLC7A11 and P53 were detected by RT-qPCR following treatment of chondrocytes of each group with Fer-1 (2 μM) for 24 h. ( J ) Representative immunofluorescence staining and quantification analysis of GPX4 in the chondrocytes of each group treated with Fer-1 (2 μM) for 24 h. ( K ) Representative immunofluorescence staining and quantification analysis of ACSL4 in the chondrocytes of each group treated with Fer-1 (2 μM) for 24 h. ( L ) Mitochondrial membrane potential was detected by JC-1 assay following treatment of chondrocytes of each group with Fer-1 (2 μM) for 24 h. Scale bar: 50 μm. Data are expressed as means + SD. For all experiments p-values calculated by unpaired two-tailed Student’s t test, p < 0.05, a statistically significant difference.

Article Snippet: After incubation with primary antibodies against COL2A1 (1:100; Servicebio, GB11021), MMP13 (1:100; Servicebio, GB11247), GPX4 (1:50; Zen-bio, R24461), and ACSL4 (1:20; Proteintech, 22401-1-AP), the sections were incubated with secondary antibody (Servicebio, G1215), followed by counterstaining with hematoxylin and visualized by DAB (Servicebio, G1212).

Techniques: Inhibition, CCK-8 Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Membrane, Two Tailed Test