Carvacrol Mitigates TNF-α-Driven Oxidative and Bioenergetic Impairment in L6 Rat Myoblasts

Tumor necrosis factor-alpha (TNF-α) mediates inflammatory cytotoxicity in skeletal muscle, causing mitochondrial dysfunction, bioenergetic collapse, and ATPase-dependent ion homeostasis disruption implicated in aging, cachexia, and inflammatory myopathy. Carvacrol (5-isopropyl-2-methylphenol), a monoterpenoid from Origanum vulgare and Thymus vulgaris, has antioxidant, anti-inflammatory, and membrane-stabilizing properties, but its modulation of TNF-α-induced toxicity, ROS, ATPase activities, Δψm, antioxidant enzymes, and stress-responsive gene expression remains uncharacterized. This study aimed to examine the effect of carvacrol on TNF-α-induced oxidative and bioenergetic damage in L6 rat myoblasts. L6 rat myoblasts were exposed to TNF-α (10 ng/mL, 1 h) to establish a validated model of inflammatory cytotoxicity, followed by carvacrol treatment (6.25 µg/mL, 24 h). Cytotoxicity was evaluated by MTT assay and lactate dehydrogenase (LDH) release. Intracellular ROS were quantified fluorimetrically using DCFH-DA. Oxidative stress markers included catalase and superoxide dismutase (SOD) activities, with Δψm assessed by flow cytometry (Muse MitoPotential kit). Ion-dependent ATPase activities (Na⁺/K⁺-, Ca²⁺-, Mg²⁺-dependent) were determined by inorganic phosphate release. SIRT1 and AMPK mRNA expression was quantified by RT-qPCR ( $2^{-\mathrm{\Delta \Delta }{C}_t}$ method, GAPDH-normalized). TNF-α exposure induced significant cytotoxicity, membrane damage, ROS accumulation, mitochondrial depolarization, suppression of ion-dependent ATPase activities, and dysregulation of catalase and SOD. Post-exposure carvacrol treatment significantly attenuated these effects: LDH release decreased from ~340 to ~73 U/mL, ROS generation was reduced, Δψm was partially restored, and ATPase activities and antioxidant enzyme activities were recovered toward control levels. Carvacrol significantly upregulated SIRT1 mRNA by 1.62 ± 0.18-fold and AMPK mRNA by 2.04 ± 0.23-fold relative to TNF-α-treated cells, consistent with activation of the SIRT1/AMPK metabolic stress-response axis. Carvacrol mitigates TNF-α-driven bioenergetic toxicity in L6 myoblasts through coordinated modulation of mitochondrial polarization, ROS burden, ion-dependent ATPase function, antioxidant defenses, and SIRT1/AMPK-mediated transcriptional stress responses. These findings identify carvacrol as a multi-target modulator of inflammatory skeletal muscle cell toxicity and provide mechanistic foundations for further investigation in differentiated muscle models and in vivo systems.