Mechanism by which kaempferol inhibits intervertebral disc degeneration in rats by regulating mitophagy levels

Abstract

BACKGROUND: Oxidative stress is a primary factor accelerating the progression of intervertebral disc degeneration. Mitophagy plays a crucial role in mitigating oxidative stress and preventing mitochondrial dysfunction and associated diseases. Prior investigations have demonstrated that kaempferol enhances the proliferative capacity of degenerating nucleus pulposus cells, diminishes inflammation, and decelerates the progression of intervertebral disc degeneration in rat models. OBJECTIVE: To investigate the effects of kaempferol on mitophagy in degenerated intervertebral disc tissue. METHODS: Fifteen Sprague-Dawley rats were randomly allocated into five intervention groups: a blank group (n=3), a model group (n=3), a low-dose kaempferol group (n=3), a medium-dose kaempferol group (n=3), and a high-dose kaempferol group (n=3). A coccygeal disc degeneration model was established in all groups except the blank group via full-thickness annulus fibrosus puncture. Commencing 4 weeks post-surgery, the blank and model groups received normal saline via gavage, while the low-dose, medium-dose, and high-dose kaempferol groups were administered 25, 50, and 100 mg/kg kaempferol via gavage, respectively, once daily for 8 consecutive weeks. Following the final administration, samples were collected for serum inflammatory cytokine level measurement, MRI imaging, and hematoxylin-eosin staining. In cell experiments, second-passage rat nucleus pulposus cells were divided into five groups: blank group (no treatment), model group (H2O2-induced oxidative stress), kaempferol group (H2O2 for 24 h then 10 μmol/L kaempferol), autophagy inhibitor group (H2O2 for 24 h then 3-methyladenine), and kaempferol + autophagy inhibitor group (H2O2 for 24 h then 10 μmol/L kaempferol plus 3-methyladenine). After 24 h of further culture, cell viability was assessed by CCK-8 assay, protein expression of inflammatory cytokines and autophagy markers by western blot, gene expression by RT-qPCR, type II collagen expression by immunofluorescence staining, and reactive oxygen species levels by DCFH-DA fluorescent probe. RESULTS AND CONCLUSION: In animal experiments, compared with the blank group, serum levels of interleukin-1β, interleukin-6, and tumor necrosis factor-α were elevated in the model group (P < 0.05), while kaempferol treatment at all doses reduced these levels (P < 0.05), with the medium dose showing the most pronounced effect. MRI and hematoxylin-eosin staining revealed that kaempferol treatment significantly ameliorated the pathological features of disc degeneration, with clearer and more continuous nucleus pulposus contours and increased cell numbers, particularly in the medium-dose group. In cell experiments, compared with the model group, kaempferol increased cell viability, type II collagen expression, and the gene and protein expression of PTEN-induced putative kinase 1 and Parkin (P < 0.05), while decreasing reactive oxygen species levels and the expression of inflammatory cytokines (P < 0.05). Kaempferol also increased the LC3-II/LC3-I ratio and LC3B mRNA expression (P < 0.05). 3-Methyladenine inhibited these effects of kaempferol. These findings indicate that kaempferol may alleviate oxidative stress injury and delay intervertebral disc degeneration by regulating mitophagy.

1. Introduction

Intervertebral disc degeneration (IVDD) is a degenerative process involving structural and functional decline of the intervertebral disc, characterized by apoptosis of nucleus pulposus cells and annulus fibrosus cells, as well as degradation of the extracellular matrix. It is commonly triggered by multiple mechanisms including cellular senescence, matrix degradation, inflammatory responses, and oxidative stress, leading to reduced disc function and pain. IVDD is a major cause of chronic low back pain, with approximately 40% of cases directly associated with disc degeneration. With societal progress and increased life expectancy, the prevalence of IVDD continues to rise.

Oxidative stress, an imbalance between oxidants and antioxidants resulting in excessive accumulation of reactive oxygen species (ROS) or reactive nitrogen species, is a key factor accelerating IVDD progression by inducing cellular apoptosis. Mitophagy, a selective autophagic pathway that eliminates damaged or excess mitochondria, plays a critical role in maintaining mitochondrial quality and cellular homeostasis, thereby mitigating oxidative stress and preventing mitochondrial dysfunction. Previous studies have shown that kaempferol, a natural flavonoid with antioxidant and anti-inflammatory properties, can improve the proliferative activity of degenerated nucleus pulposus cells and reduce inflammation, thereby delaying IVDD progression in rats. However, the specific effects of kaempferol on mitophagy in degenerated disc tissue remain unclear. This study aims to investigate the impact of kaempferol on mitophagy in a rat model of IVDD and in H2O2-treated nucleus pulposus cells, providing insights into its mechanism of action.