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 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

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.