Fig 1: In vitro COL2A1 immunofluorescence staining of the chondrocyte sheets in different groups. Fluorescence microscopy imaging (bar 1000 μm) of the nucleus (blue) and COL2A1 (green) (n=3).
Fig 2: TFDG protects the cartilage matrix of chondrocytes. (a and b) The gene levels of Mmp3, Mmp13, and Adamts5 and Col2a1, Sox9, and ACAN were analyzed by RT-qPCR. (c–j) The protein expressions of COL2, SOX9, aggrecan, MMP13, MMP3, and ADAMTS5 were analyzed by western blotting. The grey values normalized with β-actin and control group were quantified by ImageJ. The bar graph shows the mean ± SD of data (n = 4). ∗p < .05, ∗∗p < .01, ∗∗∗p < .001, and ∗∗∗∗p < .0001. (k and l) COL2 and MMP13 protein expressions were detected by immunofluorescence. Scale bar: 100 μm.
Fig 3: Effect of LPS-pre EVs on cartilage protection and protein expression in vivo. a Animal experiment model diagram. b Safranin-O and Fast Green and immunofluorescence staining of each group of mouse knee section (n = 6, one-way ANOVA). c OARSI scores in each group (n = 6, one-way ANOVA). d Protein immunofluorescence intensity of aggrecan, COL2A1, and ADAMTS5 in each group (n = 6, one-way ANOVA). c, d Values are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA
Fig 4: Effects of ATG on the viability, apoptosis, and expressions of ECM‐related genes and inflammatory factors in IL‐1β‐induced HNPCs. (A) The chemical structural formula of ATG. (B) After HNPCs were treated with different concentrations of ATG (0, 2, 5, 10, 20, and 50 μmol/L) for 24 h or 48 h, cell viability was detected by CCK‐8. (C) The viability of HNPCs treated with 10 ng/ml IL‐1β and/or ATG (10, 50 μmol/L) for 24 h was detected by CCK‐8. (D) The apoptosis of HNPCs treated with 10 ng/ml IL‐1β and/or ATG (10, 50 μmol/L) for 24 h was detected by flow cytometry. (E,F) The mRNA levels of ECM‐related genes (MMP3, MMP13, COL2A1, and Aggrecan) and inflammatory factors (IL‐6, TNF‐α, COX‐2, and iNOS) in HNPCs treated with 10 ng/ml IL‐1β and/or ATG (10, 50 μmol/L) for 24 h were detected by qRT‐PCR. GAPDH was used as the internal control. Quantified values were described as mean ± standard deviation of at least three independent experiments. *** p < 0.001 vs. control group. ^ p < 0.05, ^^^ p < 0.001 vs. IL‐1β group. ATG, arctigenin; CCK‐8, cell counting kit 8; COL2A1, collagen type II alpha 1; COX‐2, cyclooxygenase‐2; ECM, extracellular matrix; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HNPCs, human nucleus pulposus cells; IL, interleukin; iNOS, inducible nitric oxide synthase; MMP, matrix metalloproteinase; qRT‐PCR, quantitative real time polymerase chain reaction; TNF, tumor necrosis factor
Fig 5: MiR‐483‐3p inhibitor reversed the regulation of ATG on IL‐1β‐induced HNPC viability, apoptosis, and expressions of ECM‐related genes and apoptosis factors. (A) The expression of miR‐483‐3p in HNPCs treated with 10 ng/ml IL‐1β and/or ATG (10, 50 μmol/L) for 24 h was detected by qRT‐PCR. U6 was used as the internal control. (B) The expression of miR‐483‐3p in HNPCs transfected with miR‐483‐3p inhibitor/inhibitor control and treated with 10 ng/ml IL‐1β and/or ATG (50 μmol/L) for 24 h was detected by qRT‐PCR. U6 was used as the internal control. (C) HNPCs were transfected with miR‐483‐3p inhibitor/inhibitor control and then treated with 10 ng/ml IL‐1β and/or ATG (50 μmol/L) for 24 h, and cell viability was tested by CCK‐8. (D) After HNPCs received miR‐483‐3p inhibitor/inhibitor control transfection and 10 ng/ml IL‐1β and/or ATG (50 μmol/L) treatment, cell apoptosis was detected by flow cytometry. (E) qRT‐PCR was conducted to quantify MMP3, MMP13, COL2A1, and Aggrecan expressions in HNPCs after miR‐483‐3p inhibitor/inhibitor control transfection and 10 ng/ml IL‐1β and/or ATG (50 μmol/L) treatment. GAPDH was used as the internal control. (F–G) the protein levels of MMP‐3, MMP‐12, COL2A1, Aggrecan, Bcl‐2, Bax, and cleaved caspase 3 in HNPCs transfected with miR‐483‐3p inhibitor/inhibitor control and treated with 10 ng/ml IL‐1β and/or ATG (50 μmol/L) for 24 h were detected by Western blot. Quantified values were described as mean ± standard deviation of at least three independent experiments. *** p < 0.001 vs. control group. ^^^ p < 0.001 vs. IL‐1β group. ### p < 0.001 vs. IL‐1β + IC group. && p < 0.01, &&& p < 0.001 vs. ATG 50 + IC group. ATG, arctigenin; COL2A1, collagen type II alpha 1; ECM, extracellular matrix; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HNPCs, human nucleus pulposus cells; I, inhibitor; IC, inhibitor control; IL, interleukin; MMP, matrix metalloproteinase; qRT‐PCR, quantitative real time polymerase chain reaction
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