GBVAM as a Novel NHE1 Inhibitor Alleviates Doxorubicin-Induced Cardiotoxicity via PI3K/Akt/mTOR Pathway

Background: The Na + /H + exchanger isoform 1 (NHE1) inhibitor is an effective agent applied to prevent doxorubicin (Dox) induced cardiotoxicity (DIC). This research investigated the protective effect of N -(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide (GBVAM), a novel NHE1 inhibitor synthesized by our lab on the DIC model. Methods: We established the DIC mice by Dox intraperitoneal injection (16 mg/kg) for 21 days and divided the mice into Dox, Dox+GBVAM (5 mg/kg/day or 10 mg/kg/day) and Dox+GBVAM (5 mg/kg/day)+LiCl (2 mg/kg/day) groups. The H9c2 cells were divided into control, Dox (2 μM), Dox (2 μM)+GBVAM (1 μM or 10 μM), Dox (2 μM)+GBVAM (1 μM)+LiCl (5 mM), and Dox (2 μM)+GBVAM (1 μM)+LY294002 (10 μM) groups. We applied LiCl and LY294002 as NHE1 activator and phosphatidylinositol 3-kinase (PI3K) inhibitor to estimate the effect of NHE1 and PI3K pathway in the protection of GBVAM. In vivo, we examined the myocardial enzymes, cardiac function and oxidative stress indicators. In vitro, we also investigated the cardiomyocytes apoptosis and autophagy indicators as well as the PI3K/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathways. Results: Our results indicated that GBVAM played the protective effect on DIC in vivo by reversal on the myocardial enzymes, cardiac function and oxidative stress. The transcriptomic analysis data indicated that the differentially expressed genes (DEGs) associated with the protective effect of GBVAM included autophagy, mTOR and p53. Moreover, GBVAM also had an inhibition on the cardiomyocytes apoptosis and autophagy caused by Dox, which were all attenuated by LiCl treatment in vivo. The vitro experiment showed that GBVAM alleviated the oxidative stress, apoptosis and autophagy via inhibition of NHE1 and PI3K/Akt/mTOR signaling pathways. In addition, the inhibition of PI3K/Akt/mTOR signaling pathways caused by DOX could be activated by GBVAM, which were attenuated by LiCl addition. Conclusion: Our results indicated that GBVAM inhibited apoptosis and autophagy in DIC model by NHE1 inhibition, which might be via PI3K/Akt/mTOR signaling pathway. The diagram illustrates a biochemical pathway involving NHE1, a sodium-hydrogen exchanger, in a cellular membrane. Doxorubicin (Dox) and GBVAM influence NHE1 activity, affecting sodium and hydrogen ion exchange. This regulation impacts the PI3K, Akt and mTOR pathways, shown with phosphorylation and directional arrows. In the mitochondrion, Bcl-2, Bax, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) are involved, with arrows indicating changes. The lysosome is associated with LC3 and Beclin 1. In the nucleus, p62 and p53 are shown with arrows indicating regulation. The diagram highlights interactions between these cellular components and pathways.Potential mechanisms of the protective effect of GBVAM on DIC. The yellow arrow indicates the direction of action. The upward red arrow indicates the up-regulated action. The downward red arrow indicates the down-regulated action. The brown arrow indicates the ion exchange. The blue arrow indicates the inhibited action. Keywords: NHE1 inhibitor, doxorubicin, cardiotoxicity, apoptosis, autophagy, PI3K/Akt/mTOR