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. (A) Representative images of the morphology of isolated NP cells under an optical microscope. (Scale bar, 100 μm). (B) Immunocytochemical staining showing the expression of HIF-1α, HIF-1β, collagen I, collagen II in primary and second generation of NP cells from IVDD rats (scale bar, 50 μm; magnification 200 ×). (C) The expression of DNMT3B in NP cells from Sham or IVDD rats was detected by qRT-PCR. (D) Western blot showing the expression of DNMT3B in NP cells from Sham or IVDD rats. (E) The expression of DNMT3B in NP cells after 24 hours of transfection of oe-DNMT3B was detected by qRT-PCR. (F) The expression of DNMT3B in NP cells after 48 hours of transfection of oe-DNMT3B was detected by Western blot. (G) The proliferation of NP cells at 24, 48, and 72 hours, after 24 hours of transfection of oe-DNMT3B was detected by CCK-8. (H) The apoptosis of NP cells after 48 hours of treatment with oe-DNMT3B was detected by flow cytometry. (I) The expression of apoptosis-related factors Bax, Bcl-2, and caspase-3 in NP cells after 24 hours of treatment with oe-DNMT3B was detected by qRT-PCR. (J) The expression of apoptosis-related factors Bax, Bcl-2, and caspase-3 in NP cells after 48 h ours of transfection with oe-DNMT3B was detected by Western blot. (K) The expression of collagen II, aggrecan, MMP3, and MMP9 in NP cells after 24 hours of transfection with oe-DNMT3B was detected by RT-qPCR. (L) The expression of collagen II, aggrecan, MMP3, and MMP9 in NP cells after 48 hours of transfection with oe-DNMT3B was detected by Western blot. (M) Immunofluorescence staining showing the protein content of collagen II in NP cells after 48 hours of treatment with oe-DNMT3B. (N) The levels of inflammatory factors IL-6, TNF-α, IL-8 in NP cells after 24 hours of treatment with oe-DNMT3B 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 or using two-way ANOVA between groups at different time points. **, p

Figure 2. DNMT3B promoted NP cell proliferation and ECM synthesis. (A) Representative images of the morphology of isolated NP cells under an optical microscope. (Scale bar, 100 μm). (B) Immunocytochemical staining showing the expression of HIF-1α, HIF-1β, collagen I, collagen II in primary and second generation of NP cells from IVDD rats (scale bar, 50 μm; magnification 200 ×). (C) The expression of DNMT3B in NP cells from Sham or IVDD rats was detected by qRT-PCR. (D) Western blot showing the expression of DNMT3B in NP cells from Sham or IVDD rats. (E) The expression of DNMT3B in NP cells after 24 hours of transfection of oe-DNMT3B was detected by qRT-PCR. (F) The expression of DNMT3B in NP cells after 48 hours of transfection of oe-DNMT3B was detected by Western blot. (G) The proliferation of NP cells at 24, 48, and 72 hours, after 24 hours of transfection of oe-DNMT3B was detected by CCK-8. (H) The apoptosis of NP cells after 48 hours of treatment with oe-DNMT3B was detected by flow cytometry. (I) The expression of apoptosis-related factors Bax, Bcl-2, and caspase-3 in NP cells after 24 hours of treatment with oe-DNMT3B was detected by qRT-PCR. (J) The expression of apoptosis-related factors Bax, Bcl-2, and caspase-3 in NP cells after 48 h ours of transfection with oe-DNMT3B was detected by Western blot. (K) The expression of collagen II, aggrecan, MMP3, and MMP9 in NP cells after 24 hours of transfection with oe-DNMT3B was detected by RT-qPCR. (L) The expression of collagen II, aggrecan, MMP3, and MMP9 in NP cells after 48 hours of transfection with oe-DNMT3B was detected by Western blot. (M) Immunofluorescence staining showing the protein content of collagen II in NP cells after 48 hours of treatment with oe-DNMT3B. (N) The levels of inflammatory factors IL-6, TNF-α, IL-8 in NP cells after 24 hours of treatment with oe-DNMT3B 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 or using two-way ANOVA between groups at different time points. **, p < 0.01.