Mitophagy Reprograms Lactate Metabolism to Suppress THBS1 via H3K18la Reduction, Alleviating Intervertebral Disc Degeneration

Abstract

Mitophagy alleviates intervertebral disc degeneration (IVDD) by suppressing cGAS-STING and NLRP3 inflammasome-mediated pyroptosis pathways; however, its metabolic regulatory mechanism remains unexplored. Herein, we discovered that mitophagy activator TJ0113 drives metabolic reprogramming characterized by substantially reduced lactate production in senescent nucleus pulposus (NP) cells. This decline directly diminishes histone H3 lysine 18 lactylation (H3K18la), consequently suppressing transcription of the pro-inflammatory gene thrombospondin-1 (THBS1) and blocking downstream inflammatory cascades in IVDD progress. Through combined genetic silencing of THBS1 and pharmacological inhibition of lactate generation, we establish the lactate-H3K18la-THBS1 axis as the essential mechanism mediating mitophagy's anti-inflammatory effects. Our work provides the first evidence that mitophagy orchestrates a metabolic-epigenetic regulatory axis (lactate-H3K18la-THBS1), unveiling novel therapeutic targets for IVDD and paving the way for epigenetic therapies against disc degeneration.

Fig. 1.

Intervertebral disc degeneration (IVDD) is associated with mitochondrial dysfunction. (A) Representative T2-weighted magnetic resonance imaging (MRI) images were assessed using the Pfirrmann grading system. Grade I or II was classified as the nondegenerated group. Grade IV was classified as the degenerated group. (B) Schematic of cell extraction and culture. (C) Representative SA-β-gal staining images of NP cells. Scale bar, 20 μm. (D) Hierarchical clustering revealed prominent separation between young and aged samples. (E) KEGG pathway enrichment analysis of RNA-seq data (n = 3) was conducted to compare inflammation- and senescence-related differentially expressed genes (DEGs) between young and aged NP cells. (F) Hierarchical clustering analysis revealed major differences between young and degenerated groups. (G) KEGG enrichment analysis of RNA sequencing (n = 3) was conducted to compare inflammation- and senescence-related DEGs between young and degenerated groups. (H) Representative images of JC-1 staining. Scale bar, 50 μm. (I and J) Seahorse metabolic analysis (OCR) of NP cells of different groups. Results represent the mean ± SD of at least 3 independent experiments. Significance levels are shown within the graphs.

Fig. 2.

Pharmacological activation of mitophagy delays IVDD in rat models. (A) Schematic illustration of the experimental design. (B) Representative images of micro-CT, H&E staining, and SO&FG staining of rat coccygeal IVDs. Scale bar, 1 mm. (C) Pfirrmann degenerative grades (n = 6) and histological score (n = 6) of rat IVDs. (D) Representative immunofluorescence (IF) images of SASP factors in rat coccygeal IVDs at 4 weeks after treatment. Scale bar, 500 μm. (E and F) Quantification of interleukin-1β (IL-1β) and IL-6 in rat coccygeal IVDs at 4 weeks after treatment. Results represent the mean ± SD of at least 3 independent experiments. Significance levels are shown within the graphs.

Fig. 3.

Mitophagy activation reduces SASP factors and improves mitochondrial quality in senescent NP cells. (A) IF analysis and quantification of MitoTracker and LysoTracker in aged NP cells treated with or without TJ0113 (5 μM) after knocking down MCL1. Scale bars, 10 μm. (B) Western blot analysis of SASP factors (IL-1β, IL-6, and MMP3) and MCL1 in young and aged NP cells treated with TJ0113 or not (5 μM, 48 h) after knocking down MCL1. (C) Quantitative reverse transcription PCR (qRT-PCR) analysis of SASP factors in young and aged NP cells treated with TJ0113 or not (5 μM, 48 h; n = 3) after knocking down MCL1. (D and E) Seahorse metabolic analysis (OCR) of aged NP cells treated with TJ0113 or not (5 μM, 48 h). Young group served as control. (F and G) Representative transmission electron microscopy (TEM) images and quantitative analysis of healthy mitochondria numbers (5 μM). Young NP cells served as control. Scale bar, 5 μm. (H) Western blot analysis of mitochondrial dynamic markers and mitochondrial membrane proteins in aged and degenerated NP cells treated with TJ0113 or not (5 μM, 48 h). Results represent the mean ± SD of at least 3 independent experiments. Significance levels are shown within the graphs.

Fig. 4.

Metabolic reprogramming represents a key response to mitophagy activation. (A) RNA-seq analysis of aged NP cells treated with or without TJ0113 (5 μM, 48 h; n = 3). KEGG pathway enrichment analysis of RNA-seq data (n = 3) was conducted to compare inflammation-related DEGs. (B and C) GSEA showed significant enrichment of the “TNF signaling pathway” and “NF-κB signaling pathway” in TJ0113-treated aged NP cells. (D) GSEA showed significant enrichment of the “glycolytic process” in TJ0113-treated aged NP cells versus controls. (E) Heatmap of LC-MS data showing metabolite changes in glycolysis and tricarboxylic acid (TCA) cycle between TJ0113-treated and untreated aged NP cells (n = 5). Glycolytic metabolites (blue) were markedly reduced in TJ0113-treated aged NP cells versus controls. (F to H) Seahorse metabolic analysis (ECAR) of aged NP cells treated with TJ0113 or not (5 μM, 48 h). Young NP cells served as control. (I to K) Seahorse metabolic analysis (ECAR) of aged and degenerated NP cells treated with or without urolithin A (UA; 10 μM, 48 h). Results represent the mean ± SD of at least 3 independent experiments. Significance levels are shown within the graphs.

Fig. 5.

Down-regulation of lactate production and histone lactylation is driven by mitophagy activation. (A) Lactate levels of the young and aged NP cells treated with or without TJ0113 (5 μM, 48 h; n = 5) after knocking down MCL1. (B) Western blot analysis of PAN-KLA in young and aged NP cells treated with or without TJ0113 (5 μM, 48 h) after knocking down MCL1. (C and D) Western blot analysis of lysine lactylation markers (H3K9la, H3K14la, H3K18la, H4K12la) treated with TJ0113 or not (5 μM, 48 h). Histone H3/H4 served as loading controls. (E) Heatmap depicting H3K18la genomic occupancy profiles [±3 kb flanking transcription start sites (TSSs)] treated with TJ0113 or not (5 μM, 48 h). Genes are ranked vertically by descending H3K18la signal intensity. (F) Comparative pie charts showing genome-wide differential H3K18la distribution patterns at annotated genomic regions in aged NP cells treated with TJ0113 (5 μM, 48 h) compared to untreated controls. (G) KEGG enrichment analysis of H3K18la-marked genes. (H) Venn diagram depicting genes down-regulated in RNA-seq analysis of aged NP cells treated with TJ0113 (5 μM, 48 h) compared to untreated controls and the down-regulated target genes bound by H3K18la under identical treatment conditions. (I) Normalized read densities for H3K18la at the THBS1 genes. (J) ChIP-qPCR assay in aged NP cells. Results represent the mean ± SD of at least 3 independent experiments. Significance levels are shown within the graphs.

Fig. 6.

The mitophagy-driven metabolic–epigenetic axis suppresses SASP factors in senescent NP cells. (A) Western blot analysis of SASP factors and thrombospondin-1 (THBS1) in young and aged NP cells. (B) Schematic illustration of the experimental design. (C) Western blot analysis of SASP factors and THBS1 in aged NP cells treated with vehicle, TJ0113 (5 μM), FX-11(20 μM), or both. (D) Western blot analysis of SASP factors and THBS1 in aged NP cells treated with vehicle, TJ0113 (5 μM), L-Lac (10 μM), or both. (E) qRT-PCR analysis of SASP factors in aged NP cells with treatment as (B) and (C) (n = 3). (F) Western blot analysis of SASP factors and THBS1 in control and THBS1 knockdown aged NP cells treated with or without TJ0113 (5 μM, 48 h). (G) qRT-PCR analysis of SASP factors in control and THBS1 knockdown aged NP cells treated with TJ0113 or not (5 μM, 48 h) (n = 3). (H) Western blot analysis of SASP factors and THBS1 in control and THBS1-treated (0.1/1 μg/ml, 48 h) aged NP cells under treatment with or without TJ0113 (5 μM, 48 h). (I) qRT-PCR analysis of SASP factors in control and THBS1-treated (0.1/1 μg/ml, 48 h) aged NP cells under treatment with or without TJ0113 (5 μM, 48 h) (n = 3). Results represent the mean ± SD of at least 3 independent experiments. Significance levels are shown within the graphs.

Fig. 7.

Targeting the lactate–H3K18la–THBS1 axis attenuates IVDD. (A) Schematic illustration of the experimental design. (B) Representative images of H&E and SO&FG staining of rat coccygeal IVDs at 12 weeks after treatment. Scale bar, 1 mm. (C) histological score (n = 6) of rat coccygeal IVDs. (D) Representative immunohistochemistry images of H3K18la in rat coccygeal IVDs at 12 weeks after treatment. Scale bar, 200 μm. (E) Representative IF images of THBS1 in rat coccygeal IVDs at 12 weeks after treatment. Scale bar, 500 μm. (F and G) Quantification of SASP factors in rat coccygeal IVDs. Results represent the mean ± SD of at least 3 independent experiments. Significance levels are shown within the graphs.

Fig. 8.

Molecular mechanism of mitophagy activation in alleviating IVDD via metabolic–epigenetic cascade.