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.