What question did this study set out to answer?

This study aims to explore the cardioprotective effects of hesperidin against isoproterenol-induced oxidative and apoptotic damage in H9c2 cardiomyocytes.

March 22, 2026

Investigating the Protective Effects of Hesperidin on Isoproterenol-Induced Injury in H9c2 Cardiomyocytes via the Akt/GSK3β Pathway

Key Result

Hesperidin dose-dependently protects H9c2 cardiomyocytes from isoproterenol-induced oxidative stress and apoptosis via the Akt/GSK3β pathway.

Key Points

This study aims to explore the cardioprotective effects of hesperidin against isoproterenol-induced oxidative and apoptotic damage in H9c2 cardiomyocytes.
Established isoproterenol injury model in H9c2 rat embryonic cardiomyocytes.
Applied varying doses of hesperidin and assessed cell viability using MTT and EdU assays.
Measured oxidative stress markers and apoptosis using multiple assays, including TUNEL and Western blotting.
Analyzed Akt/GSK3β pathway activation through quantitative real-time PCR and phospho-specific Western blotting.
Included co-treatment with an Akt-specific inhibitor to confirm the role of Akt.
Hesperidin enhanced cell viability and proliferation, reduced injury markers, and boosted antioxidant levels.
Hesperidin significantly decreased apoptosis and preserved mitochondrial membrane potential.
Western blot analysis showed increased levels of anti-apoptotic proteins and downregulated pro-apoptotic proteins.
Hesperidin treatment activated the Akt/GSK3β pathway, effects negated by the Akt inhibitor.

Structured PICO

Population

H9c2 rat embryonic cardiomyocytes treated with isoproterenol (ISO)

Intervention

Hesperidin (HES) at doses of 5, 10, and 20 μM

Comparator

Isoproterenol (ISO) alone and untreated control

Outcome

Cell viability and proliferative capacity, oxidative stress markers, apoptosis, and mitochondrial functionsurrogate

Hesperidin exerts cardioprotective effects against isoproterenol-induced injury in H9c2 cardiomyocytes by activating the Akt/GSK3β signaling pathway.

Main Result

Absolute Event Rate: 0% vs 0%

Abstract

Background: Isoproterenol (ISO) induces oxidative and apoptotic damage in cardiomyocytes. This study aimed to investigate whether hesperidin (HES) confers a protective effect against ISO-induced injury in H9c2 cardiomyocytes and to determine whether this effect modulates the Protein Kinase B (Akt)/Glycogen Synthase Kinase-3 Beta (GSK3β) signaling pathway. Methods: H9c2 rat embryonic cardiomyocytes were treated with ISO to establish the optimal injury concentration and with varying HES doses (5, 10, 20 μM) to define a safe and effective range (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT assay). The cells were divided into five groups: Control, ISO alone, and ISO plus HES at each concentration. The viability and proliferative capacity of the cells were assessed using MTT and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Markers of injury and oxidative stress—Lactate dehydrogenase (LDH) release, glutathione (GSH), catalase (CAT), and malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and intracellular reactive oxygen species (ROS)—were measured. Apoptosis and mitochondrial function were assessed using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC‑1) dye for membrane potential, and Western blotting for B-cell lymphoma 2 (Bcl‑2), Bcl-2-associated X protein (Bax), Cysteine-aspartic acid protease-3 (caspase‑3), PTEN-induced putative kinase 1 (PINK1), Parkin and cytochrome C. Akt/GSK3β pathway activation was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and phospho‑specific Western blotting. The role of Akt was confirmed by co-treatment with the Akt-specific inhibitor VIII. Results: Compared to ISO alone, HES dose-dependently enhanced cell viability and proliferation, significantly reduced LDH release, MDA, and ROS levels, while increasing GSH and CAT levels, as well as SOD activity (p p p p < 0.05), indicating activation of this signaling cascade. These protective effects were abolished in the presence of Akt inhibitor VIII, confirming reliance on Akt/GSK3β signaling. Conclusion: Hesperidin exerts significant cardioprotective effects by alleviating ISO-induced oxidative stress, mitochondrial impairment, and apoptotic injury in H9c2 cardiomyocytes, an effect largely attributed to its activation of the Akt/GSK3β signaling pathway, thereby highlighting its therapeutic potential in cardiac protection.

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