Fig 2: YAP over-expression significantly reversed Cant-induced decline in RA-FLS cell migration and invasion. (A) The expression of YAP in RA-FLS cells transfected with a lentiviral vector carrying YAP (OE-YAP) or an empty vector (OE-NC) were measured by qPCR and Western blot assay. (B,C) Transwell assays showed the capacity of vertical migration and invasion of Cant-treated OE-NC and OE-YAP RA-FLS cells. (D–G) The effects of Cant on the expression of MMP1, MMP3, IL-6, and IL-1β in OE-NC and OE-YAP RA-FLS cells were measured by qPCR. (H–J) The OE-NC and OE-YAP cells were pre-treated with Cant for 1 h and stimulated with TNF-α (50 ng/mL) for 24 h. Then, the expressions of MMP1 and MMP3 were analyzed by Western blot. (K) IL-6 in the cell culture supernatant was tested by ELISA. (L,M) Western blot detection of proteins in OE-NC and OE-YAP RA-FLS cells after 24 h of Cant stimulation. All experiments were performed independently three times, and data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with OE-NC RA-FLS group.

Fig 3: CircRNA-CIDN retarded compression-induced IDD ex vivo. (a) qRT-PCR showed that the downregulated circRNA-CIDN level in the compression treated IVDs was rescued by the injection of circRNA-CIDN. (b) The RNA level of miR-34a-5p was measured by qRT-PCR. (c) Hematoxylin-eosin (HE) and Safranin O-fast green (SO) staining of the IVDs samples. Scale bar: 1 mm. (d) The histological scores of the IVDs according to histological grading scale. (e) The protein levels of SIRT1, aggrecan, collagen II, MMP-3, MMP-13, Bax, Bcl-2 and c-caspase 3 in the NP tissues of IVDs were measured by western blotting. Proteins were normalized to GAPDH expression levels. (f) Immunohistochemical analysis showed the expression of SIRT1, collagen II, MMP-13 and cleaved-caspase3 proteins in the NP tissues. Scale bar: 40 μm. (g) TUNEL staining and fluorescence microscope analysis were used to detected apoptosis in the IVDs samples. The green fluorescence indicated TUNEL positive cells. Scale bar:100 µm. *p < 0.05 versus control group, #p < 0.05 versus circRNA-CIDN-mut group, n = 6 [Mann–Whitney U test]. Data presented as means with error bars representing standard errors of the mean (SEM).

Fig 4: The effect of exogenous hyaluronan (HA) on inhibition of interleukin (IL)-1β by 4-methylumbelliferone (4-MU). (A) The HA concentrations in media (ng/mL) were determined using the HA ELISA (n = 6). Exogenous HA (1.0 mg/mL) was added to the cultured anterior cruciate ligament (ACL)-derived cells during incubation without or with 0.1 ng/mL IL-1β in the presence or absence of 1.0 mM 4-MU. After a 12 h incubation, the cells were lysed and analyzed by (B) real-time qRT-PCR (n = 6) and (C) capillary western immunoassay for determining the MMP-1, MMP-3, and IL-6 mRNA and matrix metalloproteinase (MMP)-1, MMP-3, and IL-6 protein expression, respectively. (D) Statistical quantification of each protein expression (n = 3). The values are presented as mean ± SD. *p < 0.05; **p < 0.01. n.s., not significant

Fig 5: The upregulation of senescence-associated biomarkers was increased following extended cell passages, determined by ELISA assay.ELISA demonstrates that IL-6, IL-8, CCL5, TNF-α and MMP3 (A–E, respectively) were all increased with extended cell passage (P3, P12, and P21), and showed substantially enhanced responsiveness to IL-1β. Following IL-1β stimulation, a two-factor ANOVA analysis revealed a significant upregulation of five SASP components: IL-8 (p < 0.01), IL-6 (p < 0.001), CCL5 (p < 0.05), MMP3 (p < 0.01), and TNF-α (p < 0.001). For a more detailed description of the Scheffé Test analysis results, please refer to the Results section. Subtle but similar trends were noted under control conditions with increased biomarker concentrations in prolonged passage ESC. Error bars indicate SD of triplicate determinations.