Perillaldehyde protect chondrocytes from mitophagy-associated apoptosis and NLRP3-mediated inflammation by regulating ALOX5/NF-kB signaling in osteoarthritis

Background: Osteoarthritis (OA) is an age-related progressive joint disease characterized by loss of cartilage and subsequent inflammation. Perillaldehyde is a compound extracted from Perilla, which has multiple pharmacological activities including anti-inflammatory, and anti-apoptosis. However, it still remains unknown whether and how perillaldehyde could regulate chondrocyte Apoptosis and inflammation in OA.

Methods: The interleukin-1beta (IL-1β)-treated chondrocytes and destabilized medial meniscus (DMM) -induced rats were used as in vitro and in vivo models of OA. Cell viability, proliferation, and Apoptosis were investigated by cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. The mitochondrial membrane potential was analyzed by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining. Light chain 3 (LC3) location, and expression of NOD-like Receptor thermal protein domain-associated protein 3 (NLRP3) and apoptosis-associated speck-like protein (ASC) were detected by immunofluorescence staining. Relative protein levels were measured via western blotting. Tumor necrosis factor-alpha (TNF-α), IL-6 and IL-8 levels were measured by enzyme-linked immunosorbent assay (ELISA). The cartilage injury in rats was investigated via hematoxylin-eosin (HE) staining.

Results: Perillaldehyde attenuated IL-1β-induced inhibition of viability (from 44.45 % to 92.72 %, p < 0.05) and proliferative potential of chondrocytes (p < 0.05). Perillaldehyde mitigated mitophagy-associated Apoptosis of chondrocytes by enhancing Mitophagy (p < 0.05) and reducing cell Apoptosis (from 32.36 % to 8.00 %, p < 0.05). Perillaldehyde attenuated NLRP3-mediated inflammation through reducing NLRP3, ASC, TNF-α, IL-6 and IL-8 levels (p < 0.05). ALOX5 expression was upregulated in OA (fold change: 2.61, p < 0.05), and decreased by perillaldehyde treatment (fold change: 1.34, p < 0.05). Perillaldehyde inhibits p65 nuclear factor kappa B (NF-κB) signaling activation (fold change: 3.19 to 1.33, p < 0.05) by regulating ALOX5 expression. ALOX5 overexpression reversed the effects of perillaldehyde on mitophagy-associated Apoptosis and NLRP3-mediated inflammation (p < 0.05), and these roles were mitigated due to NF-κB inactivation (p < 0.05). Perillaldehyde attenuated cartilage injury in OA rats (p < 0.05).

Conclusion: Perillaldehyde attenuated IL-1β-induced mitophagy-associated Apoptosis and NLRP3-mediated inflammation through decreasing ALOX5 and inactivating NF-κB signaling, indicating the potentially protective potential of perillaldehyde in osteoarthritis.

ALOX5; Mitophagy; NLRP3; Osteoarthritis; Perillaldehyde.