Regulating mitochondrial dynamics balance in nucleus pulposus cells inhibits cell apoptosis

Abstract

BACKGROUND: Mitochondrial dysfunction is increasingly recognized as a key factor during intervertebral disc degeneration. Sirt3, a major mitochondrial deacetylase, mediates AMPK pathway activation by directly phosphorylating and inhibiting Drp1 activity while indirectly regulating mitochondrial function through downstream signaling. However, the specific mechanisms of Sirt3 and the AMPK/Drp1 pathway in nucleus pulposus cells during intervertebral disc degeneration remain unclear. OBJECTIVE: To investigate whether Sirt3 regulates mitochondrial dynamics balance in nucleus pulposus cells induced by tert-butyl hydroperoxide by mediating the AMPK/Drp1 pathway, thereby inhibiting cell apoptosis. METHODS: Human nucleus pulposus cells were cultured in vitro, and a degeneration model was established by oxidative damage with tert-butyl hydroperoxide. Cells were divided into the following groups: control, model, model + oe-NC, model + oe-Sirt3, model + oe-Sirt3 + Compound C (AMPK inhibitor), and Compound C alone. After 24 h of treatment, cell viability was assessed by CCK-8, apoptosis by flow cytometry, and expression of apoptosis-related proteins (Bax, Bcl2), disc degeneration-related proteins (aggrecan, collagen type II), Sirt3, mitochondrial fission proteins (Fis1, Mff), fusion proteins (Mfn1, Mfn2), and AMPK/Drp1 pathway proteins by western blot. ATP and reactive oxygen species levels were measured using kits, and mitochondrial DNA copy number was determined by RT-qPCR. RESULTS AND CONCLUSION: Compared with the control group, the model group showed significantly decreased cell viability and expression of Bcl2, aggrecan, collagen type II, and Sirt3, while apoptosis rate and Bax level were significantly increased (all P < 0.05), indicating successful establishment of the degeneration model. Additionally, ATP levels, mitochondrial membrane potential, mtDNA copy number, and Mfn1/Mfn2 expression were significantly reduced, while reactive oxygen species, Fis1, and Mff levels were elevated (all P < 0.05), indicating mitochondrial dynamics imbalance. Overexpression of Sirt3 in the model+oe-Sirt3 group inhibited TBHP-induced apoptosis, improved cell viability, restored mitochondrial dynamics balance, activated the AMPK/Drp1 pathway, and suppressed mitochondrial fission. However, the protective effects of Sirt3 overexpression were partially reversed by the AMPK inhibitor Compound C. These findings suggest that Sirt3 is a potential target for inhibiting nucleus pulposus cell apoptosis and may serve as a novel therapeutic direction for intervertebral disc degeneration.

1. Introduction

Healthy intervertebral discs consist of the inner nucleus pulposus, surrounding annulus fibrosus, and cartilaginous endplates. Intervertebral disc degeneration (IVDD) is a pathological process leading to disc deterioration, which can cause disc herniation and low back pain [1]. The molecular mechanisms underlying IVDD remain incompletely understood. Recent studies have increasingly linked disc aging and degeneration to nucleus pulposus cell apoptosis [2-3]; therefore, inhibiting apoptosis and delaying IVDD progression are of paramount importance.

Mitochondrial dynamics balance refers to the equilibrium between mitochondrial fission and fusion. Disruption of this balance leads to mitochondrial dysfunction, severely impairing energy supply in nucleus pulposus cells and contributing to IVDD pathogenesis [4-6]. The AMPK/Drp1 pathway is a classic regulator of mitochondrial dynamics. Drp1 is a fission-related protein, and AMPK can negatively regulate Drp1 to alleviate excessive fission and maintain mitochondrial dynamics balance. Sirtuin 3 (Sirt3), a major mitochondrial deacetylase, acts as an upstream activator of AMPK, promoting its phosphorylation to ameliorate mitochondrial damage [7-8]. Although the AMPK/Drp1 pathway has been studied in myocardial ischemia and neurodegenerative diseases, its specific role in IVDD remains unclear [9-10]. Sirt3 is a highly conserved NAD+-dependent deacetylase predominantly expressed in mitochondria, playing crucial roles in maintaining mitochondrial dynamics and regulating metabolism. Previous studies have established Sirt3 as a protective factor in IVDD [11-13]. This study investigates the regulatory role of Sirt3 in nucleus pulposus cell apoptosis and its molecular mechanism in maintaining mitochondrial dynamics balance via the AMPK/Drp1 pathway. Notably, by elucidating this protective regulatory network, the research not only provides novel drug targets for degenerative diseases but also lays a theoretical foundation for cell-based and tissue engineering strategies, such as Sirt3-modified stem cell transplantation and hydrogels containing AMPK activators, offering dual value for translational application.