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

ABSTRACT 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 + ‐, Ca 2+ ‐, Mg 2+ ‐dependent) were determined by inorganic phosphate release. SIRT1 and AMPK mRNA expression was quantified by RT‐qPCR ( 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.