TGF-β1 Inhibits Osteoclast Differentiation and Abnormal Angiogenesis in Intervertebral Disc Degeneration: Evidence from RNA Sequencing and Animal Studies

Abstract

Objective: Mechanisms involved in developing intervertebral disc degeneration (IDD) are poorly understood, thus making developing effective therapies difficult. This study aimed to suggest a possible molecular mechanism, based on transcriptome sequencing-identified transforming growth factor (TGF-β), underlying the effects on bone homeostasis in IDD.

Methods: A mouse model for IDD was established. Transcriptome sequencing of nucleus pulposus tissue from mice (n = 3) identified differentially expressed mRNAs and key genes impacting bone homeostasis. A protein-protein interaction network pinpointed core genes. GO and KEGG analysis revealed gene functions. Expression levels of TGF-β1, tartrate-resistant acid phosphatase (TRAP), and cathepsin K (CTSK) were measured. Micro-CT evaluated vertebral structures and vascular imaging. Western Blot measured expression levels of Vegf, Opn, MMP3, and MMP13. Safranin O-Fast Green and TRAP staining were performed on intervertebral discs and endplates.

Results: Transcriptomic analysis found 1790 differentially expressed mRNAs in IDD mice. Twenty-eight genes related to bone homeostasis in IDD were identified. TGF-β1 was confirmed as the core gene. GO and KEGG showed TGF-β1 regulates osteoclast markers like CTSK and TRAP through pathways including NF-κB and MAPK. Experimental validation revealed lower TGF-β1 expression in IDD mice than controls, and increased TRAP and CTSK expression. Micro-CT showed decreased bone mass and intervertebral disc space in IDD mice. Vascular imaging showed increased vascular volume in IDD cartilaginous endplates. Western blot displayed increased VEGF and OPN levels, but decreased MMP3 and MMP13 in IDD mice. Safranin O-fast green staining revealed severe IDD degeneration. However, TGF-β1 injection improved bone parameters in IDD mice. In vitro experiments confirmed TGF-β1 inhibits bone marrow macrophages differentiation into osteoclasts.

Conclusion: From our data, we conclude that TGF-β1 repressed osteoclast differentiation and aberrant bone-associated angiogenesis in cartilage endplates (EPs) to alleviate IDD, which may be instrumental for the therapeutic targeting of IDD.

Keywords: Angiogenesis; Bone homeostasis; Cartilage endplate; Intervertebral disc degeneration; Osteoclast differentiation; TGF-β1; Transcriptome sequencing.