Figure 4.

Exogenous CST restored the homeostasis of cultured NP cells and NP tissues stimulated with TNF-α. (A) The expression of IL-1β in human NP cell culture media following stimulation with TNF-α with or without additional treatment with exogenous CST, as detected by ELISA (n=5). (B) Western blot analysis of MMP-13, ADAMTS-5, Col 2 and Aggrecan in human NP cells (n=5). (C) Immunofluorescence of MMP-13 in human NP cells (n=5). Nuclei were stained with DAPI. Scale bar, 20 µm. (D) Safranin O staining of ex vivo-cultured murine IVD tissues (n=5). Murine IVD tissues were cultured under TNF-α stimulation in the presence or absence of CST. Scale bar, 150 µm. (E) Expression of IL-1β in the culture media of murine IVD tissues (n=5). (F-G) The mRNA expression of MMP-13, ADAMTS-5, Col 2 and Aggrecan, as measured by real-time PCR. Total mRNA was collected from the murine IVD tissues of each indicated group, and real-time PCR was performed (n=5). Normalized values were calibrated against the control group and given a value of 1. (H) Western blot analysis of Col 2 and Aggrecan in murine IVD tissues (n=5). (I) Representative images showing Safranin O staining of ex vivo-cultured human IVD tissues (n=5). Human IVD tissues were cultured under TNF-α stimulation in the presence or absence of CST. Scale bar, 100 µm. (J) The release of GAG in the culture media of each group of ex vivo-cultured human IVD tissues (n=5). (K) Immunohistochemistry to detect MMP-13 and ADAMTS-5 in ex vivo-cultured human IVD tissues (n=5). Scale bar, 100 µm. (L) Relative mRNA expression of MMP-13 and ADAMTS-5 in ex vivo-cultured human IVD tissues, as measured by real-time PCR (n=5). (M) Western blot analysis of Col 2 and Aggrecan in ex vivo-cultured human IVD tissues (n=5). (N) The expression of IL-1β in the culture media of groups of ex vivo-cultured human IVD tissues, as detected by ELISA (n=5). *p<0.05, **p<0.01 and ***p<0.001 vs. the indicated control group. Data are presented as the mean ± SD.