Regulating mitochondrial dynamics balance in nucleus pulposus cells inhibits cell apoptosis

doi:10.12307/2026.216

Abstract

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. Each group was treated for 24 hours. Cell viability was assessed using the Cell Counting Kit-8 assay, apoptosis was detected by flow cytometry, and the expression levels of apoptosis-related proteins (Bax and Bcl2), disc degeneration-related proteins (aggrecan and type II collagen), Sirt3, mitochondrial fission-related proteins (Fis1 and Mff), mitochondrial fusion-related proteins (Mfn1 and Mfn2), and AMPK/Drp1 signaling pathway-related proteins were determined using western blot analysis. Adenosine triphosphate and reactive oxygen species levels were measured using kits, while the number of mitochondrial mtDNA copies was assessed by quantitative reverse transcription-polymerase chain reaction.
RESULTS AND CONCLUSION: (1) Compared with the control group, cell viability and expression of Bcl2, aggrecan, type II collagen, and Sirt3 were significantly decreased, while apoptosis rate and Bax expression levels significantly increased in the model group (all P < 0.05), confirming successful establishment of the nucleus pulposus cell degeneration model. (2) Compared with the control group, ATP, mitochondrial membrane potential, mtDNA copy number, and Mfn1/Mfn2 expression were significantly decreased, while reactive oxygen species, Fis1, and Mff levels were significantly increased (all P < 0.05) in the model group, indicating mitochondrial dysregulation in the nucleus pulposus cell degeneration model. In the model + oe-Sirt3 group, Sirt3 overexpression inhibited tert-butyl hydroperoxide-induced nucleus pulposus cell apoptosis, enhanced cell viability, restored mitochondrial dynamics balance, promoted AMPK/Drp1 pathway activation, and inhibited mitochondrial fission, thereby suppressing tert-butyl hydroperoxide-induced nucleus pulposus cell apoptosis. Inhibition of AMPK/Drp1 signaling partially reversed the beneficial effects of Sirt3 overexpression on tert-butyl hydroperoxide-induced nucleus pulposus cell apoptosis and mitochondrial dynamics balance in the model + oe-Sirt3 + Compound C group. These findings suggest Sirt3 as a potential therapeutic target for nucleus pulposus cell apoptosis, offering a novel therapeutic approach for intervertebral disc degeneration.

Key words: nucleus pulposus cells, mitochondrial dynamics, tert-butyl hydroperoxide, intervertebral disc degeneration, cell apoptosis

CLC Number:

Zhang Zhilong, Wang Haiying, Ma Fenghua, Hou Yanjie. Regulating mitochondrial dynamics balance in nucleus pulposus cells inhibits cell apoptosis[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(29): 7520-7528.

Figures/Tables 4

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