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 ß-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-1ß-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-1ß 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-1ß 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-a, COX-2, and iNOS) in HNPCs treated with 10 ng/ml IL-1ß 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-1ß 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-1ß-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-1ß 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-1ß 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-1ß 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-1ß 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-1ß 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-1ß 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-1ß group. ### p < 0.001 vs. IL-1ß + 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