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 5. DNMT3B regulated NP cell proliferation and ECM degradation by inhibiting TRPA1-COX2 axis. (A) The expression of COX2 in the intervertebral disc tissue of IVDD and Sham rats was detected by RT-qPCR (n = 10). (B) Western blot showing the expression of COX2 in the intervertebral disc tissue of IVDD and Sham rats. (C) mRNA expression of DNMT3B, TRPA1, and COX2 in NP cells after 24 hours of treatment with oe-DNMT3B and oe-TRPA1 was detected by qRT-PCR. (D) Protein expression of DNMT3B, TRPA1, and COX2 in NP cells after 48 hours of treatment with oe-DNMT3B, oe-TRPA1 or oe-NC was detected by Western blot. (E) DNMT3B, TRPA1, and COX2 mRNA expression in NP cells after 24 hours of treatment with oe-DNMT3B, oe-COX2 or oe-NC detected by RT-qPCR. (F) DNMT3B, TRPA1, and COX2 protein expression in NP cells after 48 hours of treatment with oe-DNMT3B, oe-COX2 or oe-NC detected by Western blot. (G) The proliferation of NP cells after 24 hours of treatment with oe-DNMT3B, oe-COX2 or oe-NC was detected by CCK-8 assay. (H) The apoptosis of NP cells after 48 hours of treatment with oe-DNMT3B or oe-COX2 was detected by flow cytometry. (I) The expression of apoptosis-related factors Bax, Bcl-2, and caspase-3 was detected by qRT-PCR in NP cells after 24 hours of treatment with oe-DNMT3B or oe-COX2. (J) 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-COX2. (K) The expression of collagen II, aggrecan, MMP3, and MMP9 was detected by qRT-PCR in NP cells after 24 hours of treatment with oe-DNMT3B or oe-COX2. (L) 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-COX2. (M) Immunofluorescence staining showing collagen II protein in NP cells after 48 hours of treatment with oe-DNMT3B or oe-COX2. (N) Inflammatory factors IL-6, TNF-α, IL-8 levels in NP cells after 24 hours of treatment with oe-DNMT3B or oe-COX2 were detected by ELISA. Measurement data are expressed as the mean ± standard deviation (n = 3) and analyzed using independent sample t-tests between two groups, using one-way ANOVA between multiple groups, or using two-way ANOVA between groups at different time points. **, p < 0.01.