Research Paper Volume 13, Issue 16 pp 20258—20276
DNMT3B decreases extracellular matrix degradation and alleviates intervertebral disc degeneration through TRPA1 methylation to inhibit the COX2/YAP axis
Figure 4. DNMT3B inhibited TRPA1 to regulate NP cell proliferation and ECM degradation. (A) The expression of DNMT3B and TRPA1 in NP cells after 24 hours of treatment with oe-DNMT3B or oe-TRPA1 was detected by RT-qPCR. (B) The expression of DNMT3B and TRPA1 in NP cells after 48 hours of treatment with oe-DNMT3B or oe-TRPA1 was detected by Western blot. (C) The proliferation of NP cells after 24 hours of treatment with oe-DNMT3B or oe-TRPA1 was detected by CCK-8. (D) The apoptosis of NP cells after 48 hours of treatment with oe-DNMT3B or oe-TRPA1 was detected by flow cytometry. (E) The expression of apoptosis-related factors Bax, Bcl-2, and caspase-3 was detected by qRT-PCR in NP cells after 48 hours of treatment with oe-DNMT3B or oe-TRPA1. (F) The expression of apoptosis-related factors Bax, Bcl-2, and caspase-3 was detected by Western blot in NP cells after 48 hours of treatment with oe-DNMT3B or oe-TRPA1. (G) The expression of collagen II, aggrecan, MMP3, and MMP9 was detected by RT-qPCR in NP cells after 48 hours of treatment with oe-DNMT3B or oe-TRPA1. (H) The expression of collagen II, aggrecan, MMP3, and MMP9 was detected by Western blot in NP cells after 48 hours of treatment with oe-DNMT3B or oe-TRPA1. (I) Immunofluorescence staining showing collagen II protein in NP cells after 48 hours of treatment with oe-DNMT3B or oe-TRPA1. (J) Inflammatory factors IL-6, TNF-α, IL-8 levels in NP cells after 24 hours of treatment with oe-DNMT3B or oe-TRPA1 were detected by ELISA. Measurement data are expressed as the mean ± standard deviation (n = 3) and analyzed using one-way ANOVA between multiple groups or using two-way ANOVA between groups at different time points. **, p < 0.01; ns, not significant.