Journal: Arthritis Research & Therapy

Article Title: Ketogenic diet ameliorates inflammation by inhibiting the NLRP3 inflammasome in osteoarthritis

doi: 10.1186/s13075-022-02802-0

Figure Lengend Snippet: Evaluation of cartilage degeneration in the knee joint. A Expression of MMP13 and COL2 was detected using immunohistochemistry. Scale bar, 20 μm. B , C Quantitative analysis of the expression of MMP13 and COL2 in immunohistochemical results. n = 3 per group, with a total of 9 animals used. # P < 0.05 versus the sham group, * P < 0.05 versus the SD group

Article Snippet: The specimens were incubated with primary antibodies against NLRP3 (1:100, Abcam, MA, USA), ASC (1:100, Abcam, MA, USA), Caspase-1 p20 (1:100, Bioworld Technology, MN, USA), IL-1β (1:100, Bioworld Technology, MN, USA), IL-18 (1:100, Bioworld Technology, MN, USA), MMP13 (1:100, Abcam, MA, USA), and COL2 (1:100, Bioworld Technology, MN, USA) at 4 °C overnight.

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Catabolic mediators from TLR2-mediated proteoglycan aggrecan peptide-stimulated chondrocytes are reduced by Lactobacillus -conditioned media

doi: 10.1038/s41598-024-68404-9

Figure Lengend Snippet: Effects of TLR2-blockade on expression levels of chondrocyte development and cartilage degradation-related genes in PG peptide-stimulated chondrocytes. Knee OA chondrocytes (N = 10) were treated with 50 ng/ml of IFNγ and 10 µg/ml of PG peptides (p16-31, p263-280 or p2379-2394), the aggrecan 32-mer peptide or Col2 peptide in the presence of 20 ng/ml of anti-TLR2 antibodies and expression levels of chondrocyte development and cartilage degradation-related genes were determined by quantitative realtime PCR. Comparison of expression levels of chondrocyte differentiation and development (HOXA11, SOX5 and RUNX2) ( A ), cartilagenous matrix (COL2A1) ( B ), and cartilage degradation (IL-6) ( C ) -related genes from chondrocytes stimulated with IFNγ and PG peptides, aggrecan 32-mer peptide and Col2 peptide with and without anti-TLR2 treated chondrocytes were normalized to GAPDH and interpreted as relative expression levels (fold change) (Y-axis). Data was shown as mean ± SEM and statistical significance was calculated by two-way ANOVA with bonferroni post hoc test (*, p < 0.0500; **, p < 0.0100; ***, p < 0.0010; ****, p < 0.0001).

Article Snippet: Isolated chondrocytes were cultured in a 48-well plate (1 × 10 5 cells/well) and stimulated with 50 ng/ml IFNγ (R&D System) with either 10 μg/ml p16-31, p263-280, p2379-2394, 32-mer or Col2 peptides (GenScript) for 30 min with or without anti-TLR2 antibodies or LCM- LS- B60.

Techniques: Expressing, Comparison

Journal: Scientific Reports

Article Title: Catabolic mediators from TLR2-mediated proteoglycan aggrecan peptide-stimulated chondrocytes are reduced by Lactobacillus -conditioned media

doi: 10.1038/s41598-024-68404-9

Figure Lengend Snippet: Effects of TLR2 blockade on cartilage-degrading enzyme production in PG peptide-stimulated chondrocytes. Knee OA chondrocytes (N = 10) were treated with 50 ng/ml of IFNγ and 10 µg/ml of PG peptides (p16-31, p263-280 or p2379-2394), the aggrecan 32-mer peptide or Col2 peptide in the presence of 20 ng/ml of anti-TLR2 antibodies. Cartilage degrading- enzymes (MMP-1, MMP-9, MMP-13, ADAMTS-4 and ADAMTS-5) and IL-6 production were determined by ELISA. ( A ) Bar graphs showing comparison of MMP-1, MMP-9, MMP-13, ADAMTS-4 and ADAMTS-5 production from IFNγ and PG peptide, aggrecan 32-mer peptide and Col2 peptide stimulation with ( +) and without (-) anti-TLR2 antibodies. Y-axis represents cartilage-degrading enzyme concentrations (pg/ml). ( B ) Bar graphs showing percentage of inhibition of MMP-1, MMP-9, MMP-13, ADAMTS-4 and ADAMTS-5 production after TLR2 blockade in each stimulation condition. ( C ) Bar graph showing comparison of IL-6 production from IFNγ and PG peptide stimulation with ( +) and without (-) anti-TLR2 antibody. Y-axis represents IL-6 concentration (pg/ml). ( D ) Bar graph showing the percentage of inhibition of IL-6 production after TLR2 blocking in IFNγ and PG peptide-stimulated chondrocytes. Data was shown as mean ± SEM and significant difference of cartilage degrading factor production was calculated by two-way ANOVA with bonferroni post hoc test, while significant difference of percentage of inhibition was calculated by one-way ANOVA with post-hoc Turky HSD (*, p < 0.0500; **, p < 0.0100; ***, p < 0.0010; ****, p < 0.0001).

Article Snippet: Isolated chondrocytes were cultured in a 48-well plate (1 × 10 5 cells/well) and stimulated with 50 ng/ml IFNγ (R&D System) with either 10 μg/ml p16-31, p263-280, p2379-2394, 32-mer or Col2 peptides (GenScript) for 30 min with or without anti-TLR2 antibodies or LCM- LS- B60.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Inhibition, Concentration Assay, Blocking Assay

Journal: Scientific Reports

Article Title: Catabolic mediators from TLR2-mediated proteoglycan aggrecan peptide-stimulated chondrocytes are reduced by Lactobacillus -conditioned media

doi: 10.1038/s41598-024-68404-9

Figure Lengend Snippet: Inhibition effect of Lactobacillus- conditioned medium on cartilage degraded mediators released from chondrocytes after IFNγ with PG peptides treatment. Chondrocytes isolated from OA patients (N = 4) were treated with 10% of Lactobacillus -conditioned medium (LCM), L. gasseri- L10 (LG-L10), L. rhamnosus-L34 (LR-L34), L. casei -L39 (LC-L39), L. salivarius -B60 (LS-B60) and L. plantarum -XB7 (LP-XB7) before stimulating with IFNγ and either PG peptides (p16-31, p263-280 and p2379-2394) or control peptides (32-mer and Col2). The cultured medium was collected for cartilage degraded mediators measurement by ELISA. ( A ) Comparison of MMP-1, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5 and IL-6 production from IFNγ and PG peptides stimulated chondrocytes between LCM-treated and untreated conditions ( B ) Bar graphs show the MMP-1 and MMP-13 production in 10% LCM-treated chondrocytes (without IFNγ and PG peptides stimulation) compared with that of chondrocytes treated with 10% of heated LCM. ( C ) Bar graphs show the MMP-1 and MMP-13 production in 10% LCM-treated chondrocytes (without IFNγ and PG peptides stimulation) compared with that of chondrocytes treated with 10% of proteinase K-treated LCM. Data was shown as mean ± SEM and statistical significance was calculated by two-way ANOVA with Bonferroni post hoc test (*, p < 0.0500; **, p < 0.0100; ***, p < 0.0010; ****, p < 0.0001).

Article Snippet: Isolated chondrocytes were cultured in a 48-well plate (1 × 10 5 cells/well) and stimulated with 50 ng/ml IFNγ (R&D System) with either 10 μg/ml p16-31, p263-280, p2379-2394, 32-mer or Col2 peptides (GenScript) for 30 min with or without anti-TLR2 antibodies or LCM- LS- B60.

Techniques: Inhibition, Isolation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Scientific Reports

Article Title: Catabolic mediators from TLR2-mediated proteoglycan aggrecan peptide-stimulated chondrocytes are reduced by Lactobacillus -conditioned media

doi: 10.1038/s41598-024-68404-9

Figure Lengend Snippet: Effects of TLR2 blockade and Lactobacillus- conditioned media treatment on intracellular signaling activation in PG peptide-stimulated chondrocytes. Chondrocytes isolated from OA patients (N = 5) were treated with 20 μg/ml of anti-TLR2 antibodies or 10% of LCM, L. salivarius -B60 for 1 h before stimulating with IFNγ and PG peptides (p16-31, p263-280 and p2379-2394) or control peptides (32-mer and Col2 peptides). Intracellular phosphorylated proteins; p-STAT3, p-IkBα, p-ERK1/2, p-p38 and p-MAPK9 (JNK2) were determined by flow cytometry. ( A ) Bar graph showing the fold change of mean fluorescent intensity of phosphorylated proteins of stimulated chondrocyte which was divided by that of unstimulated chondrocytes. Statistical significance was calculated by one-way ANOVA with post-hoc Turky HSD. ( B ) Comparison of mean fluorescence intensity (MFI) of phosphorylated protein expression after stimulating with IFNγ and PG peptides between no blocking and anti-TLR2 antibody treatment (upper panel) or between no blocking and L. salivarius -B60 LCM treatment in each individual. Statistical significance was calculated by paired-T test. ( C ) Bar graphs showing the comparison of percentage of phosphorylated protein- expressing chondrocytes in no blocking, anti-TLR2 antibody treatment and L. salivarius -B60 LCM treatment conditions after stimulation with IFNγ and PG peptides or control peptides. Data was shown as mean ± SEM and statistical significance was calculated by two-way ANOVA with Bonferroni post hoc test (*, p < 0.0500; **, p < 0.0100; ***, p < 0.0010; ****, p < 0.0001).

Article Snippet: Isolated chondrocytes were cultured in a 48-well plate (1 × 10 5 cells/well) and stimulated with 50 ng/ml IFNγ (R&D System) with either 10 μg/ml p16-31, p263-280, p2379-2394, 32-mer or Col2 peptides (GenScript) for 30 min with or without anti-TLR2 antibodies or LCM- LS- B60.

Techniques: Activation Assay, Isolation, Control, Flow Cytometry, Comparison, Fluorescence, Expressing, Blocking Assay