β-Mangostin Attenuates TET2-Mediated DNA Demethylation of Prkcg in the Prevention of Intervertebral Disc Degeneration

Abstract

Intervertebral disc degeneration (IDD) induced lower back pain is a main cause of disability, resulting in a substantial workforce loss worldwide and placing a substantial burden on the global economy and healthcare systems. However, no effective disease-modifying therapies presently exist for IDD or its related pathologies. Single-cell sequencing analyses reveal progressive M1 macrophage polarization in NP cells correlating with IDD severity, underscoring the therapeutic imperative for dual-targeting agents addressing both inflammatory dysregulation and matrix homeostasis. β-mangostin (β_Man) is screened to be proven to possess potential therapeutic effects in alleviating IDD. β_Man possesses anti-inflammatory capabilities, which include remodeling the homeostasis of the extracellular matrix, regulating macrophage polarization, and inhibiting apoptosis in the nucleus pulposus. TET2-Prkcg exerts significant regulatory functions downstream of β_Man. Mechanically, β_Man mediated reduction of TET2 maintains the DNA methylation of Prkcg rather than hydroxymethylation, which promotes mitophagy and alleviates the inflammatory microenvironment. β_Man represents a promising novel therapeutic strategy for IDD treatment. The TET2-Prkcg axis emerges as a novel therapeutic target for IDD treatment.

Keywords: Prkcg; TET2; demethylation; intervertebral disc degeneration; β‐mangostin.

Figure 1

ECM metabolic disruption and macrophage infiltration in degenerated NP. A) Collection of NP sample and experimental workflow diagram (created using BioRender.com). B) UMAP map of the following 5 cell types: T cells, NPCs, Neutrophils, progenitor NP cells (Pro_NPC), M1 and M2 macrophages. C) The proportion of various cell types in different degenerated NP. D) PCR detection for ECM metabolic changes in NP (n = 5). E) Representative IF images of Aggrecan, CD11b, CD206, and CD86 (n = 5, scale bar: 100 µm). Data is presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 2

β_Man is a potential drug candidate for the treatment of IDD. A) Drug screening flowchart (created using BioRender.com). B) PCR analysis of mRNA expression for related genes (n = 4). C) Comparison of the effects of different drugs.

Figure 3

β_Man effectively restrains IDD progression in a rat tail needle puncture model. A) Schematic of the in vivo experimental workflow (created using BioRender.com). B) CT and MRI imaging of IVD in weeks 4 and 8 (S: superior, I: inferior). C) DHI calculations were performed using CT at 4 and 8 weeks. D) Pfirrmann grading was used to assess disc degeneration at 4 and 8 weeks. E) Changes in DHI at different times. F) Histological analysis of IVD (scale bar: 1 mm). G) Heatmap illustrating changes in histological scores across groups. Data is presented as mean ± SD, n = 5. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, no significance.

Figure 4

β_Man improves NPCs ECM metabolism, alleviates cellular senescence, and reduces apoptosis. A–C) PCR analysis of ECM metabolism and inflammation in NPCs (n = 4). D) Western blot shows the protein expression of the above genes (n = 3). E–G) Quantitative analysis of Western blot results. H) Quantitative analysis of live/dead staining results. I) Live/dead staining of NPCs (n = 3, scale bar: 250 µm). J) Flow cytometry of NPCs apoptosis under different treatments (n = 3). K) Flow cytometry of JC‐1 (n = 3). L) Safranin O, Alcian Blue, and β‐gal staining (n = 3, scale bar: 250 µm). M) JC‐1 staining of NPCs (n = 3, scale bar: 10 µm). N,O,P) IF staining of Collagen‐2α1, MMP‐13, INOS in NPCs (n = 3, scale bar: 50 µm). Q) Quantitative analysis of IF. R) Quantitative analysis of JC‐1 results. Data is presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, no significance.

Figure 5

β_Man protects NPCs by inhibiting Prkcg expression. A) Venn diagram showing common gene expression in NPCs with IL‐1β and β_Man (n = 5). B) Hierarchical clustering between the three NPC groups. C) IF of Prkcg in different treatments (n = 3, scale bar: 50 µm). D) The heatmap illustrates differential gene expressions (n = 5). E) GO enrichment analysis of differentially expressed genes. F) KEGG pathway enrichment analysis. G) Western blot analysis of ECM metabolism expression in NPCs under different treatments (IL‐1β, β_Man, siPrkcg, NT siRNA, OE Prkcg, pcDNA3.1(0); (n = 3). H) Quantitative analysis of Western blot. Data is presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, no significance.

Figure 6

Prkcg knockdown under inflammation enhances mitophagy in NPCs. A) Venn diagram showing common gene expression between NPCs treated with NT siRNA and siPrkcg (n = 3). B) Heatmap of differentially expressed genes (n = 3). C) KEGG enrichment analysis. D) GO enrichment analysis. E) Western blot of LC3B, SQSTM1, Parkin, and Pink1 expression in NPCs treated with NT siRNA, siPrkcg, and IL‐1β (n = 3). F,G) Quantitative analysis of Western blot results. H) Quantitative analysis of IF for Pink1 and Parkin. I) IF of Pink1 and Parkin expression (n = 3, scale bar: 20 µm). J) Colocalization analysis of MitoTracker and LysotTacker (n = 3, scale bar: 10 µm). K) Quantitative analysis of MitoTracker and LysoTracker colocalization. L) TEM of NPCs from different treatment groups (n = 3, scale bar: 400 nm). M) IHC of Prkcg in human NP with Pfirrmann grade II and grade IV (n = 5, scale bar: 100 µm). N) IHC of Prkcg in control and defect groups of IVD in rats (n = 5, scale bar: 500 µm, 100 µm). Data is presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, no significance.

Figure 7

Knockout of Prkcg effectively prevents IDD Progression in vivo. A) Schematic of the experimental workflow in vivo (created using BioRender.com). B) Representative CT and MRI images of rat IVD at 4 and 8 weeks. C) Statistical analysis of CT. D) Statistical analysis of MRI. E) Histological staining (H&E, Safranin O/Fast Green, and Sirius Red combined with polarized microscopy) of IVD (scale bar: 1 mm). F) Heatmap illustrating changes in histological scores G) IHC of Collagen‐2α1, MMP‐13, and INOS (scale bar:100 µm). H) Statistical analysis of IHC. I) IF of Pink1 expression (scale bar: 50 µm). J) IF of Parkin expression (scale bar: 50 µm). Data is presented as mean ± SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.

Figure 8

β_Man enhances Prkcg DNA methylation by reducing TET2. A) RNA sequence reveals changes in TET2 (n = 5). B) IF of TET2 expression under different treatments (n = 3, scale bar: 50 µm). C) Quantitative analysis of IF results. D) PCR results showing changes in Prkcg mRNA expression after TET2 silencing (n = 4). E) Western blot analysis of TET2 and Prkcg protein expression following TET2 silencing (n = 3). F) Statistical analysis of Western blot results. G) IHC of TET2 in IVD of rats (n = 5, scale bar: 500 µm, 100 µm). H) IHC of TET2 in human NP (n = 5, scale bar:100 µm). I) Quantitative analysis of IHC results. J) Diagram of Prkcg DNA CpG island locations. K) DMRs of Prkcg in NPCs treated with IL‐1β or β_Man (n = 3). L) DMRs of Prkcg with TET2 silencing (n = 3). M) Differential hydroxymethylation regions (DhMRs) of Prkcg following TET2 silencing (n = 3). N) DMs of Prkcg in NPCs treated with IL‐1β or β_Man (n = 3). O) DMs of Prkcg with TET2 silencing (n = 3). P) Differential hydroxymethylation sites (DhMs) of Prkcg with TET2 silencing (n = 3). Data is presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.

Figure 9

Overexpression of TET2 attenuated the effect of β_Man in preventing IDD. A) Schematic of the in vivo experimental workflow (created using BioRender.com). B) Histological analysis of IVD (n = 5, scale bar:1 mm). C) IHC and IF of Collagen‐2α1, MMP‐13, INOS, and Prkcg in different groups of rats (n = 5, scale bar: 100 µm, 70 µm).

Figure 10

Schematic representation of the mechanisms that β_Man therapeutic effects in treating IDD. β_Man inhibits M1 polarization of macrophages and improves ECM metabolism disorder of NPCs by inflammation‐induced. In NPCs, β_Man mitigates inflammation‐induced inhibition of Prkcg methylation by suppressing TET2‐mediated demethylation, which decreases Prkcg and increases mitophagy of NPCs and inhibits the progression of IDD.