Huperzine A Attenuates Oxidative Stress-Induced H9c2 Cardiomyoblast Injury Through AKT-Associated Survival Signaling

Oxidative stress is a major contributor to cardiomyoblast injury and cardiovascular disease progression. Huperzine A (HupA), a natural Lycopodium alkaloid, exhibits antioxidant and anti-inflammatory properties, but its cardioprotective mechanisms remain unclear. This study aimed to investigate the effects of HupA on oxidative stress-induced H9c2 cardiomyoblast injury and elucidate the underlying molecular mechanisms. Network pharmacology, molecular docking, molecular dynamics simulations, and cellular thermal shift assay (CETSA) were integrated to predict and investigate AKT1 involvement. An in vitro model of tert-butyl hydroperoxide (tBHP)-induced injury in H9c2 rat cardiomyoblasts was established. Oxidative stress status, mitochondrial function, and AKT-associated signaling were evaluated. The involvement of AKT signaling was examined using MK-2206. HupA significantly improved cell viability in a dose-dependent manner and markedly reduced lipid peroxidation and intracellular and mitochondrial ROS accumulation. In addition, HupA restored antioxidant defenses by increasing superoxide dismutase activity and glutathione levels. Mitochondrial dysfunction induced by tBHP was effectively alleviated, as evidenced by preserved mitochondrial membrane potential and reduced mitochondrial superoxide production. Furthermore, HupA enhanced AKT phosphorylation and MCL-1 expression while inhibiting extracellular HMGB1 release. Importantly, pharmacological inhibition of AKT largely abolished these protective effects, including antioxidant restoration, mitochondrial preservation, and anti-cell death activity. HupA protects H9c2 cardiomyoblasts against oxidative injury through AKT-associated survival signaling, thereby attenuating oxidative stress and preserving mitochondrial integrity. These findings suggest HupA as a promising therapeutic candidate in H9c2 cardiomyoblasts under oxidative stress conditions.