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

DNMT3B promoted NP cell proliferation and ECM synthesis via COX2-YAP axis. (A) DNMT3B, TRPA1, COX2, and YAP expression in NP cells after 48 hours of treatment with oe-DNMT3B and oe-COX2 was detected by RT-qPCR. (B) DNMT3B, TRPA1, COX2, and YAP expression in NP cells after 48 hours of treatment with oe-DNMT3B and oe-COX2 was detected by Western blot. (C) DNMT3B, TRPA1, COX2, and YAP expression in NP cells after 48 hours of treatment with oe-DNMT3B and oe-YAP was detected by RT-qPCR. (D) DNMT3B, TRPA1, COX2, and YAP expression in NP cells after 48 hours of treatment with oe-DNMT3B and oe-YAP was detected by Western blot. (E) The proliferation of NP cells after 48 hours of treatment with oe-DNMT3B or oe-YAP was detected by CCK-8. (F) The apoptosis of NP cells after 48 hours treatment with oe-DNMT3B or oe-YAP was detected by flow cytometry. (G) The expression of apoptosis-related factors Bax, Bcl-2, and caspase-3 was detected by RT-qPCR in NP cells after 48 hours of treatment with oe-DNMT3B or oe-YAP. (H) 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-YAP. (I) 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-YAP. (J) 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-YAP. (K) Immunofluorescence staining showing collagen II protein in NP cells after 48 hours of treatment with oe-DNMT3B or oe-YAP. (L) Inflammatory factors IL-6, TNF-α, IL-8 levels in NP cells after 48 hours of treatment with oe-DNMT3B or oe-YAP 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

Figure 7. DNMT3B promoted NP cell proliferation and ECM synthesis via COX2-YAP axis. (A) DNMT3B, TRPA1, COX2, and YAP expression in NP cells after 48 hours of treatment with oe-DNMT3B and oe-COX2 was detected by RT-qPCR. (B) DNMT3B, TRPA1, COX2, and YAP expression in NP cells after 48 hours of treatment with oe-DNMT3B and oe-COX2 was detected by Western blot. (C) DNMT3B, TRPA1, COX2, and YAP expression in NP cells after 48 hours of treatment with oe-DNMT3B and oe-YAP was detected by RT-qPCR. (D) DNMT3B, TRPA1, COX2, and YAP expression in NP cells after 48 hours of treatment with oe-DNMT3B and oe-YAP was detected by Western blot. (E) The proliferation of NP cells after 48 hours of treatment with oe-DNMT3B or oe-YAP was detected by CCK-8. (F) The apoptosis of NP cells after 48 hours treatment with oe-DNMT3B or oe-YAP was detected by flow cytometry. (G) The expression of apoptosis-related factors Bax, Bcl-2, and caspase-3 was detected by RT-qPCR in NP cells after 48 hours of treatment with oe-DNMT3B or oe-YAP. (H) 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-YAP. (I) 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-YAP. (J) 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-YAP. (K) Immunofluorescence staining showing collagen II protein in NP cells after 48 hours of treatment with oe-DNMT3B or oe-YAP. (L) Inflammatory factors IL-6, TNF-α, IL-8 levels in NP cells after 48 hours of treatment with oe-DNMT3B or oe-YAP 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.