DNMT3a-mediated methylation of PPARγ promote intervertebral disc degeneration by regulating the NF-κB pathway

Abstract

Intervertebral disc degeneration (IVDD) is a common chronic musculoskeletal disease that causes chronic low back pain and imposes an immense financial strain on patients. The pathological mechanisms underlying IVDD have not been fully elucidated. The development of IVDD is closely associated with abnormal epigenetic changes, suggesting that IVDD progression may be controlled by epigenetic mechanisms. Consequently, this study aimed to investigate the role of epigenetic regulation, including DNA methyltransferase 3a (DNMT3a)-mediated methylation and peroxisome proliferator-activated receptor γ (PPARγ) inhibition, in IVDD development. The expression of DNMT3a and PPARγ in early and late IVDD of nucleus pulposus (NP) tissues was detected using immunohistochemistry and western blotting analyses. Cellularly, DNMT3a inhibition significantly inhibited IL-1β-induced apoptosis and extracellular matrix (ECM) degradation in rat NP cells. Pretreatment with T0070907, a specific inhibitor of PPARγ, significantly reversed the anti-apoptotic and ECM degradation effects of DNMT3a inhibition. Mechanistically, DNMT3a modified PPARγ promoter hypermethylation to activate the nuclear factor-κB (NF-κB) pathway. DNMT3a inhibition alleviated IVDD progression. Conclusively, the results of this study show that DNMT3a activates the NF-κB pathway by modifying PPARγ promoter hypermethylation to promote apoptosis and ECM degradation. Therefore, we believe that the ability of DNMT3a to mediate the PPARγ/NF-κB axis may provide new ideas for the potential pathogenesis of IVDD and may become an attractive target for the treatment of IVDD.

Keywords: DNMT3a; PPARγ; intervertebral disc degeneration; low back pain.

FIGURE 1

Expression of DNMT3a and PPARγ in human NP tissues; (A) MRI T2 images of human NP tissue according to the Pfirrmann grades; (B) Haematoxylin and eosin staining (HE) and toluidine blue staining of early and late human NP tissues; (C) Western blotting analysis of DNMT3a and PPARγ in early and late human NP tissues; (D, E) Relative quantitative protein levels of DNMT3a and PPARγ; (F) Expression of DNMT3a and PPARγ by immunochemical staining in early and late human NP tissues; (G, H) Quantitative levels of immunohistochemical staining of DNMT3a and PPARγ in early and late human NP tissues; Three biological replicates were contained in each experiment. All data are expressed as mean ± standard deviation (SD); **p < 0.01, ***p < 0.01.

FIGURE 2

IL‐1β treatment can cause increased DNMT3a expression and decreased PPARγ expression in NP cells. (A–F) Western blotting was used to measure the expression of DNMT3a and PPARγ and the relative quantitative protein levels of DNMT3a and PPARγ in NP cells treated with different IL‐1β concentrations and times; (G, H) IL‐1β treated DNMT3a and PPARγ representative immunofluorescence staining in NP cells at different times; Three biological replicates were contained in each experiment. All data are expressed as mean ± standard deviation (SD); *p < 0.05, **p < 0.01, ***p < 0.01.

FIGURE 3

DNMT3a inhibition attenuates IL‐1β‐induced apoptosis and ECM degradation. (A–E) Western blotting and quantitative analyses show western blot levels of DNMT3a, Bax, Bcl‐2, and Caspase‐3 in rat NP cells in different groups; (F, G) TUNEL staining and apoptotic cell ratio quantification. (H–L) Western blotting and quantitative analyses show levels of Collagen II, Aggrecan, Adamts‐4, and MMP‐3 in different groups; (M–O) immunofluorescence and quantitative analysis showed changes in Collagen II and MMP‐3. Three biological replicates were contained in each experiment. All data are expressed as mean ± standard deviation (SD); *p < 0.05, **p < 0.01, ***p < 0.01, ns p > 0.05.

FIGURE 4

DNMT3a inhibits PPARγ expression by modifying PPARγ promoter methylation. (A) The island of C‐phosphate‐G (CpG) in the PPARγ promoter region was found on the UCSC website; (B, C) methylation and quantification of PPARγ promoters in NP cells by MSP detection 24 h after transfection of shRNA‐DNMT3a; (D, E) Western blotting and quantification analyses show levels of PPARγ after IL‐1β and shRNA‐DNMT3a treatment; (F, G) immunofluorescence and quantitative analyses show changes in PPARγ. Three biological replicates were contained in each experiment. All data are expressed as mean ± standard deviation (SD); *p < 0.05, **p < 0.01, ***p < 0.01, ns p > 0.05.

FIGURE 5

DNMT3a inhibits PPARγ to promote apoptosis in NP cells. (A) CCK‐8 was used to determine the cytotoxic effect of different concentrations of T0070907 on NP cells for 24 h; (B) Western blotting and quantitative analyses show changes in PPARγ, Bax, Bcl‐2, and Caspase‐3 in rat NP cells after T0070907 pretreatment; (C–F) Western blotting quantification of PPARγ, Bax, Bcl‐2, and Caspase‐3; (G, H) immunofluorescence and quantitative analyses show changes in PPARγ; (G, I) TUNEL staining and apoptotic cell ratio quantification. Three biological replicates were contained in each experiment. All data are expressed as mean ± standard deviation (SD); *p < 0.05, **p < 0.01, ***p < 0.01.

FIGURE 6

DNMT3a inhibits PPARγ to promote ECM degradation in NP cells. (A–E) Western blotting and quantitative analysis showed changes in Collagen II, Aggrecan, Adamts‐4, and MMP‐3 expression levels in rat NP cells after T0070907 pretreatment; (F–H) immunofluorescence and quantitative analysis showed changes in Collagen II and MMP‐3; Three biological replicates were contained in each experiment. All data are expressed as mean ± standard deviation (SD); *p < 0.05, **p < 0.01, ***p < 0.01.

FIGURE 7

DNMT3a regulates the activity of the PPARγ/NF‐κB axis. (A–C) Western blotting and quantitative analyses show levels of p‐P65, P65, p‐IkBα, and IkBα in rat NP cells following IL‐1β and shRNA‐DNMT3a treatment; (D–F) Western blotting and quantitative analyses showed p‐P65, P65, p‐IkBα, and IkBα levels in rat NP cells after T0070907 pretreatment; Three biological replicates were contained in each experiment. All data are expressed as mean ± standard deviation (SD); *p < 0.05, **p < 0.01, ***p < 0.01, ns p > 0.05.

FIGURE 8

PPARγ regulates apoptosis and ECM degradation by inhibiting the NF‐κB pathway. (A–C) Western blotting and quantitative analysis showed p‐P65, P65, p‐IkBα, and IkBα levels in rat NP cells after treatment with GW1929 and Diprovocim; (D–K) western blotting and quantitative analyses show Bax, Bcl‐2, Caspase‐3, Collagen II, Aggrecan, Adamts‐4 and MMP‐3 levels treated with GW1929 and Diprovocim. Three biological replicates were contained in each experiment. All data are expressed as mean ± standard deviation (SD); *p < 0.05, **p < 0.01, ***p < 0.01.

FIGURE 9

DNMT3a inhibition ameliorated puncture‐induced IVDD in vivo. (A) Representative X‐ray image and MRI T2 image of the intervertebral disc at 8 weeks after shRNA‐DNMT3a injection in rats; (B) the variation of the disc height index is calculated based on X‐ray images; (C) Pfirrmann grade scores calculated from MRI T2 images; (D, E) HE, Safranine O staining images and histological scores; (F–K) IHC staining and quantitative analysis of DNMT3a, PPARγ, Collagen II, MMP‐3 and Caspase‐3 in intervertebral disc samples from different groups of rats. Three biological replicates were contained in each experiment. All data are expressed as mean ± standard deviation (SD); **p < 0.01, ***p < 0.01 and ns p > 0.05.