J Vis Exp. 2026 Sep 22;(235). doi: 10.3791/72823.
ABSTRACT
The application of tourniquets during limb surgery can lead to ischemia-reperfusion (IR) injury, a pathological process characterized by excessive oxidative stress, increased inflammatory signaling, and progressive muscle fiber damage. Hesperidin, a bioactive flavonoid derived from citrus fruits, is known for its antioxidant and anti-inflammatory properties. This study evaluated the protective effects of hesperidin against lower-limb skeletal muscle IR injury and investigated the molecular pathways potentially associated with these effects. Male Wistar rats were allocated to four groups: control, hesperidin alone, IR (2 h of ischemia followed by 2 h of reperfusion), and IR + hesperidin (100 mg/kg administered 30 min before ischemia). Histological examination of the gastrocnemius muscle was performed, and plasma markers of muscle injury, including creatine kinase (CK), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP), were measured. Oxidative stress and antioxidant parameters (MDA, SOD, CAT, and GPx), components of the Nrf2/HO-1/NQO1 pathway, inflammatory mediators (NF-κB, TNF-α, IL-6, and IL-1β), pyroptosis-associated markers (NLRP3 and caspase-1), and apoptosis-associated proteins (Bax and caspase-3) were also assessed. IR resulted in pronounced muscle fiber disruption, elevated plasma injury markers, increased lipid peroxidation, and reduced antioxidant capacity. IR also decreased Nrf2, HO-1, and NQO1 levels while increasing NF-κB, NLRP3, caspase-1, pro-inflammatory cytokines, and apoptosis-associated proteins. Hesperidin pretreatment markedly attenuated these changes by preserving muscle structural integrity, reducing plasma injury markers and lipid peroxidation, restoring antioxidant enzyme activity, increasing Nrf2/HO-1/NQO1-associated antioxidant responses, and reducing NF-κB-associated inflammatory, NLRP3/caspase-1-associated pyroptotic, and apoptotic responses. Overall, hesperidin pretreatment protected skeletal muscle against tourniquet-induced IR injury and was associated with enhanced Nrf2-mediated antioxidant defenses and attenuation of NF-κB-associated inflammation and NLRP3/caspase-1-associated responses. These findings support further investigation of hesperidin as a potential prophylactic intervention for reducing tourniquet-associated lower-limb IR injury.
PMID:42775776 | DOI:10.3791/72823