Mitochondrial Dynamics Disorder Drives Nucleus Pulposus Cell Senescence in Lumbar Scoliosis of Aging Bipedal Rats Under Asymmetric Force.

Objective: The purpose of this study was to investigate the effect of mitochondrial dynamics disorder driving nucleus pulposus cell (NPC) senescence in lumbar scoliosis of aging bipedal rats under asymmetric force. Methods: A rat model of lumbar scoliosis with asymmetric force was established using nickel-titanium springs and anchorage screws. The condition of lumbar scoliosis was observed by X-ray. MicroCT was used for 3D reconstruction of the microporous structure of the lumbar endplates. Histopathological changes in the L4/5 intervertebral disc were observed using Hematoxylin and Eosin (H&E) staining and Safranin O-Fast Green staining. Immunohistochemical staining was used to observe the expression of IL-8, IL-6, MMP-3, and MMP-13 in the cells of the L4/5 intervertebral disc tissue. Western blot was performed to analyze the protein expression of p53, p21, p16, p-p65, NLRP3, mitofusin 2 (Mfn2), dynamin-related protein 1 (Drp1), OPA1, and Fis1. The mitochondrial morphology in NPCs of the L4/5 intervertebral disc was observed by transmission electron microscopy (TEM). The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and adenosine triphosphate (ATP) in the nucleus pulposus tissue of lumbar scoliosis were measured using commercial assay kits. Reactive oxygen species (ROS) content in the nucleus pulposus tissue was quantified by flow cytometry. Results: X-ray and MicroCT-3D revealed that rats in the asymmetric group exhibited significant scoliosis deformity, accompanied by marked reductions in bone mineral density (BMD), tissue mineral density (TMD), bone volume fraction (BV/TV), and trabecular thickness (Tb.Th). H&E staining and Safranin O-Fast Green staining demonstrated that asymmetric force significantly exacerbated pathological changes in the intervertebral disc, particularly damage to the cartilage endplate, chondrocyte necrosis, and hyperplasia. Immunohistochemical results indicated a significant increase in the positive expression of IL-8, IL-6, MMP-3, and MMP-13 in the asymmetric group, suggesting a synergistic effect of aging and asymmetric force in amplifying inflammatory responses and matrix degradation. Western blot analysis showed that the expression of senescence-associated proteins (p53, p21, and p16) and inflammation-related proteins (p-p65, NLRP3) was significantly upregulated in the asymmetric group, indicating that asymmetric force accelerated the senescence of NPCs and activated inflammatory pathways. TEM revealed that asymmetric force markedly aggravated mitochondrial swelling and structural damage in the L4/5 intervertebral disc, with the most severe mitochondrial injury observed in the 48-week asymmetric group. Western blot further demonstrated that the expression of mitochondrial fusion proteins (Mfn2, OPA1) was significantly decreased, while mitochondrial fission proteins (Drp1, Fis1) were significantly increased in the asymmetric group, indicating disrupted mitochondrial fission-fusion balance. Additionally, the asymmetric loading group exhibited significantly reduced SOD activity and ATP levels, along with elevated MDA content, suggesting oxidative stress-induced mitochondrial dysfunction. Flow cytometry results confirmed a significant increase in ROS levels in the asymmetric group, highlighting that asymmetric force intensified oxidative stress in the nucleus pulposus tissue, and this effect was further exacerbated with aging. Conclusion: Asymmetric force drives the NPC senescence by disrupting mitochondrial fission-fusion balance, triggering oxidative stress and inflammatory responses, ultimately leading to the progression of degenerative lumbar scoliosis. This pathological cascade is significantly exacerbated in the context of aging.