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 to assess serum inflammatory factor levels, intervertebral disc MRI imaging, and hematoxylin-eosin staining. (2) Cell experiment: Second-generation rat nucleus pulposus cells were cultured across five groups: a blank group with no added drugs, a model group with hydrogen peroxide (to establish an oxidative stress injury model), a kaempferol group treated with hydrogen peroxide for 24 hours followed by 10 μmol/L kaempferol, an autophagy inhibitor group treated with hydrogen peroxide for 24 hours followed by the addition of the autophagy inhibitor 3-methyladenine, and a kaempferol + autophagy inhibitor group treated with hydrogen peroxide for 24 hours followed by 10 μmol/L kaempferol + 3-methyladenine. After 24 hours of continuous culture, the cell survival rate was determined using the cell counting kit-8 method; protein expression of inflammatory factors and autophagy indicators was assessed via western blot; gene expression of inflammatory factors and autophagy indicators was evaluated via RT-qPCR; type II collagen expression was detected via immunofluorescence staining; and the level of reactive oxygen species was measured using a DCFH-DA fluorescent probe.
RESULTS AND CONCLUSION: (1) Animal experiment: Compared with the blank group, the levels of serum interleukin-1β, interleukin-6 and tumor necrosis factor-α in the model group increased (P < 0.05). and the levels of three inflammatory factors in each dose group of kaempferol decreased (P < 0.05), with the medium-dose kaempferol group showing a more pronounced decrease. MRI imaging and hematoxylin-eosin staining revealed that the pathological characteristics of intervertebral disc degeneration were significantly improved in each dose group of kaempferol compared with the model group. Specifically, the nucleus pulposus contour was clearer and more continuous, and the number of nucleus pulposus cells increased significantly, with the medium-dose kaempferol group exhibiting more notable improvement. (2) Cell experiment: Compared with the model group, the kaempferol group demonstrated increased cell survival rate, type II collagen expression, tensin homolog-induced protein kinase 1 expression, and parkin gene and protein expression (P < 0.05). Conversely, the kaempferol group showed decreased reactive oxygen species levels, as well as reduced gene and protein expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α (P < 0.05). Additionally, microtubule-associated protein light chain 3I/II protein expression and microtubule-associated protein light chain 3B mRNA expression were increased (P < 0.05). Notably, 3-methyladenine was found to inhibit the aforementioned effects of kaempferol. Overall, kaempferol may mitigate oxidative stress damage and delay the progression of intervertebral disc degeneration by modulating the level of mitophagy.

Key words: kaempferol, mitophagy, oxidative stress, intervertebral disc degeneration, nucleus pulposus cells, intervertebral discs