Pinus koraiensis leaf extract and lambertianic acid attenuate fatigue and improve endurance capacity via PI3K-mediated regulation of oxidative stress and mitochondrial biogenesis

Abstract Pinus koraiensis leaves (PKL) are phytochemically rich agricultural by-products that remain largely underutilized despite containing diverse bioactive constituents. Although our previous study demonstrated the anti-fatigue potential of PKL, the underlying molecular mechanisms have not been fully elucidated. This study investigated the anti-fatigue effects and mechanisms of a 50% ethanol extract of PKL and its marker compound lambertianic acid (LA) using integrated in vitro, in vivo, and in silico approaches. In H₂O₂-challenged C2C12 myotubes, PKL dose-dependently reduced intracellular ROS levels, suppressed NF-κB and IL-6 expression, and enhanced SOD activity, thereby preserving myotube morphology and mitochondrial integrity. LA exerted comparable protective effects. Mechanistically, these responses were associated with activation of the PI3K/NRF2/HO-1 antioxidant axis and upregulation of the SIRT1/PGC-1α/NRF1 mitochondrial biogenesis pathway. Network pharmacology analysis identified PI3K/Akt/mTOR as the central signaling hub, and molecular docking supported favorable binding of LA to an allosteric site on PI3K p110α. In a lipopolysaccharide (LPS)-induced immunological fatigue mouse model, oral administration of PKL and LA significantly improved grip strength and forced-swimming endurance, restored antioxidant enzyme activities and energy metabolism markers, and upregulated mitochondrial biogenesis regulators (SIRT1, PGC-1α, TFAM) in skeletal muscle. Notably, PKL also restored LPS-suppressed PI3K complex expression (p85, p110α), consistent with the in vitro findings. Collectively, these findings provide mechanistic evidence that PKL and LA ameliorate fatigue through PI3K-mediated regulation of oxidative stress and mitochondrial biogenesis, supporting the valorization of PKL as a functional material for fatigue management.