Inhibition of the Sp1/PI3K/AKT signaling pathway exacerbates doxorubicin-induced cardiomyopathy

Abstract

Objective

This study aimed to investigate the interaction and underlying mechanisms between specificity protein 1 (Sp1) and the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway in the context of doxorubicin-induced cardiomyopathy (DIC).

Methods

A rat model of DIC was established by intraperitoneal injection of doxorubicin (1 mg/kg) twice a week for eight weeks. Cardiac function was evaluated using echocardiography, and myocardial histopathology was assessed by hematoxylin-eosin (HE) staining. In vitro, H9c2 cardiomyocytes were treated with doxorubicin (2 μmol/L) to induce cardiotoxicity, followed by co-treatment with the Sp1 inhibitor plicamycin or the PI3K/AKT inhibitor LY294002. Cell viability was measured by the CCK-8 assay. Oxidative stress markers, including reactive oxygen species (ROS) and lactate dehydrogenase (LDH), were quantified using flow cytometry and colorimetric assays. Apoptosis was detected via TUNEL staining, and protein expression of Sp1, PI3K, AKT, and Caspase-3 was analyzed by Western blotting.

Results

Doxorubicin treatment significantly impaired cardiac function in rats, as evidenced by an increase in both left ventricular internal diameters during diastole (LVIDd) and systole (LVIDs), along with decreased ejection fraction (EF) and fractional shortening (FS) (p < 0.01). Myocardial HE staining in doxorubicin-treated rats revealed disorganized cardiomyocyte structures, edema, and cellular necrosis. In vitro, doxorubicin exposure led to reduced H9c2 cell viability, elevated ROS and LDH levels, and increased apoptosis rates (p < 0.01). Western blotting demonstrated that doxorubicin significantly downregulated the expression of Sp1, PI3K, and AKT while upregulating Caspase-3. Inhibition of Sp1 or PI3K/AKT exacerbated these effects, resulting in further cardiac dysfunction, oxidative stress, and apoptosis. Moreover, Sp1 inhibition led to decreased PI3K/AKT pathway activation, while PI3K/AKT inhibition reciprocally suppressed Sp1 expression, indicating a bidirectional regulatory relationship.

Conclusion

Doxorubicin induces cardiotoxicity by promoting oxidative stress and apoptosis through the downregulation of the Sp1/PI3K/AKT signaling pathway. Inhibition of this pathway exacerbates cardiac injury, suggesting that targeting Sp1 and PI3K/AKT may offer novel therapeutic strategies for the prevention and treatment of DIC.