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

Introduction

As a widely used anthracycline drug, doxorubicin (Dox) is applied in several cancer treatments.¹ However, the application of Dox in clinical is limited because of Dox induced cardiotoxicity (DIC) caused by its cumulative doses.² The DIC usually leads to irreversible myocardial injury or congestive heart failure, as well as is related with several mechanisms, such as calcium overload, inflammation, mitochondrial injury, oxidative stress, apoptosis and autophagy.^3,4 Considering the powerful anti-tumor effect of Dox, it is extremely necessary to seek out the protective drugs against DIC. Up to now, dexrazoxane is the only drug for DIC treatment approved by FDA, but its application is limited due to the several deficiencies.⁵ Hence, the development of an effective and safe drug to overcome DIC at present is very urgent.

The Na⁺/H⁺ exchangers (NHEs) are plasma membrane proteins and maintain the intracellular pH and sodium homeostasis through exchanging the intracellular H⁺ and extracellular Na⁺.⁶ It was reported that there were about nine NHE exchanger isoforms and the NHE1 isoform was mainly expressed in cardiac tissues, which played an essential role in the cardiac function.⁷ Previous researches reported that the up-regulated NHE1 activity was participated in calcium overload, inflammation, oxidative stress, apoptosis, autophagy and so on.^8–10 In addition, hyperactivity of NHE1 was associated with several cardiovascular diseases, specially cardiac hypertrophy and heart failure.^11,12 It was reported that the increased NHE1 activity could induce the over-expression of gene, which was the cause of cardiac hypertrophy.¹³ While, the knock-down of NHE1 gene expression could alleviate cardiac hypertrophy in rats.¹⁴ It was reported that the NHE1 activation led to the intracellular Na⁺ accumulation, which induced the intracellular calcium overload through the Na⁺/Ca²⁺ exchanger (NCX) and contributed to cardiac injury.¹⁵ Up to now, there were several reports showed that the NHE1 activity was up-regulated in the DIC model, and inhibition of NHE1 activity could alleviate DIC by oxidative stress and apoptosis inhibition.^16–18 As a typical NHE1 inhibitor, cariporide exerted protection against DIC in rats by suppressing oxidative stress, inflammation and apoptosis through the Akt/glycogen synthase kinase 3 beta (GSK-3β) and sirtuin 1 (Sirt1) pathways.¹⁹ What is more, previous reports also exhibited that inhibition of NHE1 activity led to reactive oxygen species (ROS) generation, inflammation and apoptosis inhibition.¹⁹ Considering the effectiveness above, the NHE1 inhibitor has an important value in the treatment of DIC. Unfortunately, the NHE1 inhibitors such as cariporide, eniporide and zoniporide frequently used in clinical did not achieve the expected action in the acute myocardial infarction reperfusion.²⁰ Previous researches showed that the NHE1 inhibitors above had a certain therapeutic effect on the patients in the early stage of severe ischemic cardiomyopathy, however, there was no intervention to avoid infarction.²⁰ Therefore, the development of new NHE1 inhibitors is particularly urgent.

Our previous research reported that N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide (GBVAM), a new efficient NHE1 inhibitor, had a super NHE1 activity inhibition than cariporide, as well as exerted protection on the Dox-induced H9c2 cell injury.²¹ However, whether GBVAM has the protective effect on DIC mice as well as the related mechanism is unknown. In this research, we assumed that GBVAM targeted NHE1 to exert the protection against DIC, such as apoptosis, calcium overload, inflammation, mitochondrial injury, oxidative stress and autophagy in vivo and in vitro. As an NHE1 activator, LiCl was applied to verify the effect of NHE1 in the protection of GBVAM against DIC.²² Therefore, we used Dox to induce the H9c2 cell injury and DIC mice model to investigate the protection of GBVAM and the relevant mechanisms.

Materials and Methods

Reagents

GBVAM was synthesized and reported by our lab previously.²¹ The GBVAM, cariporide (HY-19693, MedChemExpress), LY294002 (T2008, TargetMol), Dox (RH456534, RHAWN) were all diluted with DMSO. The LiCl (HY-Y0649, MedChemExpress) was diluted with distilled water. All the store solutions were stored at −20 °C for further application.

Animals

The male C57BL/6J mice (7 weeks old) were obtained from Changsheng Biotechnology [SCXK (Liao) 2020–0001] and housed in the specific pathogen-free environment with the temperature set at 24~26 °C. The experimental protocol was approved by the Animal Care and Use Committee, Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College (IRM/2-IACUC-2511-011) in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. The mice were randomly divided into five groups using a computer-generated random sequence, with six mice per group. The researchers for drug treatment, sample collection and data analysis were blinded to the group allocation. The sample size was determined according to previous reports about DIC treatment, our pre-experiment results and the subsequent experiment. The mice in different groups were treated as follows.

1. Control group: the mice were treated with the saline at the same time.
2. Dox group: the mice were treated with Dox (4mg/kg/day) by intraperitoneal injection once every 4 day for 3 weeks with the cumulative total amount was 16 mg/kg.
3. Dox+low dose GBVAM group (Dox+L-GBVAM): the mice were treated with Dox (4 mg/kg/day) by intraperitoneal injection once every 4 day and GBVAM (5 mg/kg/day) daily by intragastric administration for 3 weeks.
4. Dox+high dose GBVAM group (Dox+H-GBVAM): the mice were treated with Dox (4 mg/kg/day) by intraperitoneal injection once every 4 day and GBVAM (10 mg/kg/day) daily by intragastric administration for 3 weeks.
5. Dox+low dose GBVAM+LiCl group (Dox+L-GBVAM+LiCl): the mice were treated with Dox (4 mg/kg/day) by intraperitoneal injection once every 4 days, while treated with GBVAM (5 mg/kg/day) and LiCl (2 mg/kg/day) daily by intragastric administration for 3 weeks.

After received the treatment above, the mice were proceeded the following experiment. Finally, all the mice were sacrificed with intraperitoneal injection of tribromoethanol for the blood and cardiac tissue obtained.²²

Echocardiography and Electrocardiogram Experiments

For the echocardiography and electrocardiogram experiments, the mice were anesthetized with isoflurane (3%–4% for induction, 1.5%–2% for maintenance) via inhalation. The echocardiography experiment was performed by Vevo 2100 (FUJIFILM, Tokyo, Japan) on the mice. The cardiac function parameters were recorded as reported.²³ The electrocardiogram was further applied to estimate the cardiac function and the related parameters were recorded.²⁴

Serum Biochemical Assays

The blood samples of mice were collected for the biochemical assays. The lactate dehydrogenase (LDH, A020-1, NanJing JianCheng), atrial natriuretic peptide (ANP, ml105994, mlbio), N-terminal pro-B-type natriuretic peptide (NT-ProBNP, ml003242B, mlbio), cardiac troponin T (cTnT, ml231456, mlbio), superoxide dismutase (SOD, A001-3, NanJing JianCheng), malondialdehyde (MDA, A003-2, NanJing JianCheng) and creatine kinase myocardial band (CK-MB, ml107303, mlbio) were detected following the instructions.

Histological Analysis

The obtained cardiac tissues were fixed with 4% paraformaldehyde and dehydrated by ethanol, which were embedded in paraffin at last. Then, the tissues were sliced into 4 μm sections and stained with hematoxylin and eosin stain (HE) and Masson’s trichrome stain (Masson) for histological analysis.

Cell Culture and Treatment

The rat cardiomyocytes H9c2 cells, human breast carcinoma MCF-7 cells and mouse breast carcinoma 4T1 cells (ATCC, America) were cultured with DMEM or RPMI 1640 (C11995500BT or C11875500BT, Gibco) added with 10% FBS (FSP500, ExCell bio) and 1% penicillin-streptomycin (PB180120, Solarbio). The H9c2 cells in the following groups were treated as follows.

1. Control group: the H9c2 cells were treated with normal culture medium.
2. Dox-treated group: the H9c2 cells were treated with 2 μM Dox for 24h.
3. Dox+GBVAM-treated groups: the H9c2 cells were treated with 2 μM Dox and GBVAM (1 μM or 10 μM) for 24h.
4. Dox+GBVAM+LiCl-treated group: the H9c2 cells were treated with 2 μM Dox, 1 μM GBVAM and 5 mM LiCl for 24h.
5. Dox+GBVAM+LY294002-treated group: the H9c2 cells were treated with 2 μM Dox, 1 μM GBVAM and 10 μM LY294002 for 24h.

Cell Viability Measurement

To investigate the cytotoxicity of GBVAM on the H9c2 cells, the cells were treated with culture medium and different concentrations of GBVAM (0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM and 20 μM). In order to select the Dox concentration to induce the cytotoxicity on the H9c2 cells, the cells were divided into the control group and Dox treated groups, which were treated with culture medium and different concentrations of Dox (0.25 μM, 0.5 μM, 1 μM, 2 μM, 5 μM and 10 μM). In order to investigate the protective effect of GBVAM on the H9c2 cells, the cells were divided into the control group, Dox treated group as well as Dox+GBVAM co-treated groups, which were treated with culture medium, 2 μM Dox, 2 μM Dox and GBVAM (0.05 μM, 0.1 μM, 0.5 μM, 1 μM and 10 μM). In order to investigate whether GBVAM had the anti-tumor effect, the 4T1 cells and MCF-7 cells were divided into the control group, GBVAM treated groups, Dox treated groups and Dox+GBVAM co-treated groups, which were treated with culture medium, GBVAM (0.5 μM, 1 μM and 10 μM), Dox (2 μM), Dox (2 μM) and GBVAM (0.05 μM, 1 μM and 10 μM). After the treatment, the cell viability was calculated as previously reported through MTT.

LDH and the Oxidative Stress Indicators Measurement

The LDH, SOD and MDA levels of cells was recorded by the instructions. The ROS was measured with DCFH-DA fluorescent probe (ID3130, Solarbio, ex/em=488 nm/525 nm) and the mean fluorescence intensity (MFI) was analyzed by ImageJ.

NHE1 Activity Measurement

In order to explore the NHE1 activity inhibition effect at the physiological condition, the H9c2 cells were divided into control, NH₄Cl and various concentrations of GBVAM (0.05~20 μM) groups. Moreover, the control, NH₄Cl, Dox, Dox+various concentrations of GBVAM (0.05 μM, 1 μM and 10 μM) groups were further prepared to investigate the NHE1 inhibition of GBVAM in the presence of Dox. The effect of GBVAM on the NHE1 activity was measured by BCECF AM fluorescence (S1006, Beyotime). We also investigated the long-term effect of GBVAM on the NHE1 activity by fluorescence microscope. After treated with the corresponding drug for 24 h, the H9c2 cells were incubated BCECF AM working solution (5 μM). Ultimately, the images were observed by fluorescence microscope (ex/em=488 nm/535 nm) and MFI was analyzed by ImageJ.^25,26

Actin-Tracker Green, Mito-Tracker Red CMXRos and TUNEL Staining

The H9c2 cells were stained with the Actin-Tracker Green solution (C1033, Beyotime, ex/em=495 nm/518 nm), Mito-Tracker Red CMXRos solution (C1035, Beyotime, ex/em=579 nm/599 nm) and TUNEL working solution (KTA2011, Abbkine, Atlanta, ex/em=555 nm/565 nm) to explore the cell size, membrane potential of mitochondria and TUNEL positive cells. The MFI was analyzed by ImageJ. The apoptosis ratio was expressed as follows: apoptosis ratio=apoptosis cells/total cells×100%.

Transcriptomic Analysis

Total RNA of H9c2 cells was extracted by TRIZOL (10606ES60, YEASEN, Shanghai, China). The RNA isolation, quality control, library construction and sequencing were carried out by Guangdong Magigene Biotechnology Co.Ltd. (Guangdong, China, https://cloud.magigene.com/login). Then, the sequencing was performed on the Illumina HiSeq 2500 high-throughput sequencing platform. The DEGs were analyzed by the DESeq2 with the cut-off set at a 2-fold change and significant threshold set at P<0.05. Finally, functional annotation and functional enrichment analysis of the original test sequence were carried out by Gene Ontology (GO,https://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes(KEGG,http://www.kegg.jp/)analysis. The GO enrichment analysis of DEGs included biological processes(BP), cellular components(CC) and molecular functions(MF), as well as KEGG pathway analysis were performed by clusterProfiler package.^27,28

Molecular Docking and Dynamics Simulation

The crystal structure of NHE1 (PDB: 7DSX) was obtained from the Protein Data Bank (PDB, https://www.rcsb.org/) and uniprot database (https://www.uniprot.org/). The three-dimensional structure of GBVAM was obtained by Chemdraw and the molecular docking was conducted with AutoDock Tools (version 1.5.7). The molecular visualization of the NHE1-GBVAM complex was obtained using PyMOL 3.1, as well as the interactions were obtained using LigPlot (version 2.2.8). The docking analysis of NHE1-mTOR complex and NHE1-p53 complex were used the HDOCK server (http://hdock.phys.hust.edu.cn/). The 3D structures of NHE1 (PDB: 7DSX), mTOR (PDB:1FAP) and p53 (PDB:1AIE) were obtained from the Uniprot database and PDB database. In order to evaluate the structural stability of NHE1-GBVAM complex, we performed the molecular dynamics simulation within 100 ns using the GROMACS software package (version 2020.6). The AMBER99SB and GAFF force field were selected for NHE1 and GBVAM ligand, respectively. The system was solvated in the Single Point Charge Extended (SPC/E) three-site water model. Subsequently, the system underwent energy minimization, constant volume and constant pressure. Then, the trajectory data included root mean square deviation (RMSD), radius of gyration (Rg), root mean square fluctuation (RMSF) and solvent accessible surface area (SASA) were recorded every 1 ps. The data analysis and visualization were performed using Visual Studio Code (version 1.102.1) and Python (version 3.13.7).²⁹

Cellular Thermal Shift Assay (CETSA)

After the cells lysed and centrifuged, the protein was obtained by collecting the supernatant. The protein was incubated with DMSO and GBVAM (5 μM) at 37 °C for 30 min. The two groups protein were further divided into seven tubes, which were boiled for 5 min at different temperatures (37 °C, 42 °C, 47 °C, 52 °C, 57 °C, 62 °C, 67 °C). Finally, the supernatant was collected for following Western blot.

Western Blot Analysis

After electrophoresed by SDS-PAGE and transferred to PVDF membranes, the proteins were incubated the primary antibody at 4°C overnight (Table S1). The next day, the corresponding secondary antibody (ABclonal, AS014) was incubated at room temperature for 1 h. Ultimately, the blots were visualized with the Ultra high-sensitivity ECL kit (GK10008, GlpBio) and the band intensity was calculated with ImageJ.

Statistical Analysis

All data were expressed as mean ± SEM. For multiple comparisons, one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc tests was used. When P < 0.05, the difference was considered statistically significant.

Results

GBVAM Inhibited the NHE1 Activity and Protein Expression

GBVAM presented in Figure 1A was a novel inhibitor of NHE1 activity previously reported.²¹ In the following experiment, the MTT assay was applied under the different concentrations of GBVAM, with the results presented in Figure 1B illustrating that GBVAM at the concentrations from 0.05 μM to 20 μM did not affect H9c2 cell viability. We further confirmed the inhibitory effect of GBVAM on the NHE1 activity at different concentrations (0.05 μM~20 μM). The results in Figure 1C indicated that the incubation of GBVAM (0.05 μM~20 μM) for 30 min could inhibit NHE1 activity obviously in short term. What is more, the incubation of GBVAM (0.05 μM~20 μM) for 24 h could also inhibit the NHE1 activity measured with the fluorescence microscope (Figure 1D).

Figure 1 The effect of GBVAM on cell viability and NHE1 activity in H9c2 cells. (A) Chemical structure of GBVAM. (B) Cell viability at different doses of GBVAM (0 μM~20 μM), n=6. (C) MFI of BCECF AM under GBVAM (0.05 μM~20 μM), n=6. (D) Fluorescence intensity images of BCECF AM. *P < 0.05, ***P < 0.001.

The Results of Molecular Docking and Dynamics Analysis

We found that GBVAM played the action on NHE1 with a high affinity (−7.31±0.035 kcal/mol) and located at ASP267, GLU346, ASP159, ASP59 and HIS275 (Figure 2A and Figure s1). The CETSA results in Figure 2B illustrated that cells treated with GBVAM exhibited a higher NHE1 protein expression compared with the DMSO treated cells at the same temperature, which showed that GBVAM acted directly on NHE1. The binding stability of the NHE1-GBVAM complex was further evaluated with molecular dynamics analysis under 100 ns simulation. The RMSD, RMSF, Rg and SASA (Figure 2C–F) exhibited that the binding between NHE1 and GBVAM was relatively stable.

Figure 2 The molecular docking and dynamics stimulation analysis. (A) Molecular docking results of GBVAM and NHE1, n=5. (B) Cellular thermal shift assay, n=5. (C) The RMSD of GBVAM, NHE1, and NHE1-GBVAM complex. (D) The RMSF of NHE1-GBVAM complex. (E) The Rg of NHE1-GBVAM complex. (F) The SASA of NHE1-GBVAM complex. **P < 0.01, ***P < 0.001.

GBVAM Inhibited the Increased Activity and Protein Expression of NHE1 Caused by Dox

Therefore, we firstly tested whether GBVAM could attenuate the H9c2 cell injury induced by Dox via NHE1 inhibition. The 2 μM Dox was selected to establish the cell injury model (Figure s2). GBVAM restored the decreased H9c2 cell viability in Dox group to different extents (Figure 3A and B), as well as the increased LDH level caused by Dox (Figure 3C). We also found that GBVAM inhibited the 4T1 and MCF-7 viability and had a synergistic inhibitory effect on the 4T1 and MCF-7 viability with Dox (Figure s3 and 4). The results indicated that GBVAM alleviated the H9c2 cell injury via NHE1 inhibition without affecting the anti-tumor effect of Dox. It was reported the increased NHE1 activity is one of the factors causing DIC.^17,18 Therefore, we explored whether GBVAM could reduce the increased NHE1 activity caused by Dox. The results in Figure 3D and E illustrated that GBVAM (1 µM and 10 µM) could notably inhibit the increased NHE1 activity caused by Dox. What is more, GBVAM exerted a better inhibitory effect on the NHE1 activity than cariporide (Figure s5). In addition, 1 µM and 10 µM GBVAM could inhibit the elevated NHE1 protein expression caused by Dox (Figure 3F).

Figure 3 The effect of GBVAM on NHE1 activity and expression co-treated with Dox. (A) Morphological changes of H9c2 cells at different concentrations of GBVAM (0 μM~10 μM) co-treated with 2 μM Dox. (B) Cell viability at different concentrations of GBVAM (0 μM~10 μM) co-treated with 2 μM Dox, n=6. (C) Quantification of LDH release, n=5. (D) Representative BCECF AM fluorescence intensity images. (E) MFI of BCECF AM, n=6. (F) Representative Western blotting images and quantitative analysis of NHE1 protein, n=5. *P < 0.05, **P < 0.01, ***P < 0.001.

Effects of GBVAM on Transcriptomic in the Presence of Dox

The samples of cells were collected for RNA-seq analysis. As shown in Figure 4A, there were 1379 DEGs identified between Dox treated group, and Dox+GBVAM co-treated group, among which 186 DEGs were up-regulated (red) and 1193 DEGs were down-regulated (green). What is more, there were 378 DEGs identified by comparing control vs Dox, and Dox vs Dox+GBVAM co-treated groups (Figure 4B). The cluster analysis between control, Dox and Dox+GBVAM co-treated groups indicated that the DEGs in Dox and Dox+GBVAM co-treated groups were significant (Figure 4C).

Figure 4 Transcriptomics analysis of Dox injury protection by GBVAM in H9c2 cells. (A) Volcano plots of DEGs. (B) Venn diagrams with 378 DEGs. (C) Heatmap of DEGs. (D–F) Bubble chart of the top 15 of the enriched BP (D), CC (E) and MF (F). (G) Bar chart of the top 15 of KEGG pathway analysis. (H and I) The molecular docking of NHE1 and p53 (H), NHE1 and mTOR (I). The genes marked with red highlighted box represented the ones we interested in and be further studied in detail.

Subsequently, the GO enrichment stated the targets associated with BP included nucleobase-containing compound metabolic process, cellular response to stress, regulation of cellular process, intrinsic apoptotic signaling pathway and autophagy, etc. (Figure 4D). The targets associated with CC included nucleus, intracellular membrane-bounded organelle, chromosome, protein-containing complex and cytosol, etc. (Figure 4E). The targets associated with MF were mainly involved in nucleic acid binding, ubiquitin-like protein peptidase activity, ATP-dependent activity, acting on DNA, protein kinase activity and phosphatidylinositol 3-kinase activity, etc. (Figure 4F).

The KEGG enrichment analysis displayed that the targets were significantly associated with p53, mTOR and autophagy, etc. (Figure 4G and Figure s6). We further applied HDOCK (http://hdock.phys.hust.edu.cn/) to predict the combination between NHE1 and p53 or mTOR with the results presented in Figure 4H and I. The docking scores for p53 and mTOR with NHE1 were −283.25 and −287.09. Therefore, the results above indicated that the inhibition of GBVAM on NHE1 might be associated with mTOR and p53 signaling pathways.

GBVAM Alleviated DIC of Mice via NHE1 Inhibition

The protection of GBVAM on mice cardiac injury via NHE1 inhibition was further investigated in this section. The animal experiment design was presented in Figure 5A. At first, GBVAM could significantly decrease the heart weight/body weight (HW/BW) ratio in the Dox group, which was reversed partially by LiCl (Figure 5B and C). The serum LDH, CK-MB, ANP, NT-ProBNP and cTnT levels in the Dox group were all down-regulated by GBVAM (Figure 5D–H). While LiCl could reverse the effect of GBVAM above (Figure 5D–H). In Figure 5I, HE and Masson staining revealed that GBVAM attenuated the cardiac injury. The results above suggested that NHE1 inhibition was involved in the protection of GBVAM on the cardiac injury induced by Dox.

Figure 5 The effect of GBVAM on the DIC via NHE1 inhibition in mice. (A) Schematic diagram of the animal experiment design. (B) Images of heart. (C) Heart weight/body weight (HW/BW) of mice, n=6. (D–H) Cardiotoxicity indicators of serum, such as LDH (D), CK-MB (E), ANP (F), NT-ProBNP (G) and cTnT (H) in the indicated groups, n=6. (I) Representative images of HE staining and Masson staining of cardiac tissue.**P < 0.01, ***P < 0.001.

GBVAM Improved Cardiac Function of DIC Mice via NHE1 Inhibition

The echocardiography and electrocardiogram were applied to estimate the cardiac function. The HR, RR interval, QT interval, QRS interval and QTc interval were all used as parameters to further estimate the cardiac function. As Figure 6A and B shown, GBVAM could elevate HR level in Dox treated mice, as well as decline the prolonged RR, QT, QRS and QTc in the Dox-treated mice. Moreover, the results showed that GBVAM down-regulated left ventricular internal dimension at end-diastole (LVIDd) and left ventricular internal dimension at end-systole (LVIDs), as well as up-regulated left ventricular posterior wall thickness at end-diastole (LVPWd), left ventricular posterior wall thickness at end-systole (LVPWs), ejection fraction (EF) and fractional shortening (FS) levels in Dox group (Figure 6C and D). The protection of GBVAM on cardiac function was all reversed by the LiCl treatment.

Figure 6 The effect of GBVAM on the cardiac function caused by Dox via NHE1 inhibition in mice. (A) Representative images of electrocardiogram. (B) The parameters of electrocardiogram, such as HR, QRS, QT, QTc and RR, n=6. (C) Representative images of echocardiography. (D) The parameters of echocardiography, such as LVIDd, LVIDs, LVPWd, LVPWs, EF and FS, n=6. *P < 0.05, **P < 0.01, ***P < 0.001.

GBVAM Attenuated Apoptosis and Autophagy of DIC Mice via NHE1 Inhibition

As Figure 7A and B shown, for the Dox-treated mice, GBVAM increased SOD and decreased MDA, which illustrated that GBVAM could inhibit the oxidative stress response by NHE1 inhibition. Figure 7C and D results indicated that NHE1 protein expression was inhibited by GBVAM in vivo. As transcriptomics results indicated that p53 and mTOR were all involved in the effect of GBVAM, which were associated with apoptosis and autophagy.^30,31 Therefore, we investigated whether GBVAM had an effect on apoptosis and autophagy induced by Dox. The results in Figure 7C and D showed that GBVAM alleviated the cardiomyocyte apoptosis caused by Dox through decreasing Bax/Bcl-2 ratio and p53 levels. Meanwhile, GBVAM also regulated the autophagy induced by Dox through decreasing the Beclin1 and LC3-II expression, and increasing p62 (Figure 7E and F).

Figure 7 The effect of GBVAM on apoptosis and autophagy in DIC mice via NHE1 inhibition. (A) Serum SOD activity, n=6. (B) Serum MDA content, n=5. (C) Representative Western blotting images of NHE1, p53, Bax and Bcl-2 protein. (D) Quantitative analysis of Western blots for the NHE1, p53, Bax and Bcl-2 protein expression, n=5. (E) Representative Western blotting images of Beclin 1, p62 and LC3-II protein. (F) Quantitative analysis of Beclin 1 protein, p62 protein and LC3-II protein expression, n=5. **P < 0.01, ***P < 0.001.

GBVAM Inhibited Cardiotoxicity Caused by Dox via NHE1 Inhibition in H9c2 Cells

We further verified whether GBVAM played the protective role on the Dox induced myocardial injury through NHE1 inhibition in vitro. At first, GBVAM reduced the increase of cell size caused by Dox significantly (Figure 8A and B), which was abolished by the LiCl addition. As shown in Figure 8C–F, GBVAM could reduce the oxidative stress in H9c2 cells caused by Dox including down-regulating ROS and MDA, and up-regulating SOD level. What is more, LiCl also reversed the effect of GBVAM on the membrane potential of mitochondria in the Dox group (Figure 8G and H). The effect of GBVAM on the oxidative stress and the membrane potential of mitochondria were all reversed by LiCl addition (Figure 8), which indicated that the protective effects of GBVAM above were achieved by NHE1 inhibition in vitro.

Figure 8 The effect of GBVAM on the cardiotoxicity via NHE1 inhibition. (A) Representative images of Actin-Tracker fluorescence staining. (B) Quantitative analysis of cell surface size, n=8. (C) SOD activity, n=6. (D) Representative images of DCFH-DA fluorescence staining. (E) MFI of DCFH-DA, n=5. (F) MDA content, n=7. (G) Representative images of Mito-Tracker fluorescence. (H) MFI of Mito-Tracker, n=8. *P < 0.05, **P < 0.01, ***P < 0.001.

GBVAM Participated in Apoptosis Inhibition and Autophagy Regulation Induced by Dox in H9c2 Cells via NHE1 Inhibition

In Figure 9A and B, the increased apoptosis cells were decreased by GBVAM treatment significantly. Furthermore, GBVAM also alleviated the Bax/Bcl-2 ratio, c-caspase-3 and p53 expressions in the Dox group, which indicated that GBVAM could attenuate the apoptosis induced by Dox via NHE1 inhibition (Figure 9C and D). The results in Figure 9E and F indicated that GBVAM could regulate the autophagy induced by Dox by decreasing the Beclin 1 and LC3-II expression, as well as increasing p62 expression via NHE1 inhibition.

Figure 9 The effect of GBVAM on the apoptosis and autophagy via NHE1 inhibition. (A) Representative fluorescence images of TUNEL staining. (B) Quantitative analysis of TUNEL positive cells, n=7. (C) Representative Western blotting images of p53, c-caspase-3, Bax and Bcl-2 protein. (D) Quantitative analysis of Bax/Bcl-2 protein ratio, p53 protein and c-caspase-3 protein expression, n=5. (E) Representative Western blotting images of p62, LC3-II and Beclin 1 protein. (F) Quantitative analysis of p62 protein, LC3-II protein and Beclin 1 protein expression, n=5. **P < 0.01, ***P < 0.001.

GBVAM Participated in the Apoptosis Inhibition and Autophagy Regulation Induced by Dox in H9c2 Cells via PI3K/Akt/mTOR Pathway

The previous researches reported that PI3K/Akt pathway was participated in apoptosis and autophagy regulation, which was further verified by the KEGG analysis in our research.^32,33 What is more, mTOR was associated with apoptosis and autophagy regulation, which was also the downstream of PI3K/Akt pathway.^34–36 Figure 10A and B results manifested that GBVAM up-regulated the p-PI3K, p-Akt and p-mTOR expressions in Dox group, which was alleviated by LiCl significantly. Therefore, we expected the anti-apoptosis and autophagy regulation of GBVAM were achieved by PI3K/Akt/mTOR activation. The results in Figure 10C–F indicated that PI3K inhibitor, LY294002 could alleviate the effect of GBVAM on the cell apoptosis. Furthermore, LY294002 also alleviated the effect of GBVAM on the cell autophagy regulation (Figure 10G and H). Therefore, we got the conclusion that the anti-apoptosis and autophagy regulation of GBVAM were achieved by PI3K/Akt/mTOR activation.

Figure 10 The effect of GBVAM on apoptosis and autophagy caused by Dox in H9c2 cells via PI3K/Akt/mTOR pathway. (A) Representative Western blotting images of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR protein. (B) Quantitative analysis of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR protein expression, n=5. (C) Representative fluorescence images of TUNEL positive cells staining. (D) Quantitative analysis of the TUNEL positive cells staining, n=5. (E) Representative Western blotting images of Bax, Bcl-2 and p53 protein. (F) Quantitative analysis for Bax, Bcl-2 and p53 protein expression, n=5. (G) Representative Western blotting images of Beclin 1, LC3-II and p62 protein. (H) Quantitative analysis for Beclin 1, LC3-II and p62 protein expression, n=5. **P < 0.01, ***P < 0.001.

Discussion

Up to now, many researchers devote themselves to reducing DIC, which limited the application of Dox for the cancers. But there are many obstacles due to the numerous and complex mechanisms of DIC.^37–39 Currently, dexrazoxane is the only therapeutic drug for DIC, although it has several side effects.^5,40 Therefore, exploring valid drugs against DIC to expand its clinical application is urgent. GBVAM was a recently synthesized NHE1 inhibitor in our lab, and we explored protection of GBVAM on DIC in this research.²¹ In our previous research, we reported that GBVAM had a super inhibition on NHE1 activity than cariporide, a classical NHE1 inhibitor.²¹ Moreover, GBVAM exhibited a considerable protective effect on the Dox-induced H9c2 cells injury.²¹ But the related mechanism above is still unknown. Therefore, we investigated the role of NHE1 on the protection of GBVAM on DIC and the mechanisms.

NHE1 is the main isoform of NHE in the cardiac tissue, which is contributed to the acid-base, electrolyte and volume homeostasis in the body.^6,41 The NHE1 activation could lead to the intracellular Na⁺ accumulation, which led to the intracellular Ca²⁺ overload through the NCX.⁴² It was reported that the intracellular Ca²⁺ overload had several negative effects on the cardiac, such as cardiac hypertrophy, cardiomyocyte fibrosis, apoptosis, cardiac systolic dysfunction as well as arrhythmias occurrence, which were all the causes of HF.^17,18 It was reported that several compounds played protection on DIC by alleviating oxidative stress, apoptosis and inflammation through NHE1 inhibition.^16–18 Therefore, we firstly verified the effective concentration of GBVAM on the decreased H9c2 cell viability and increased NHE1 activity caused by Dox. Then, we investigated the NHE1 protein expression under the GBVAM treatment. It was reported that the NHE1 activity could affect the NHE1 protein expression,¹³ and our results indicated that GBVAM could inhibit the increased NHE1 protein expression caused by Dox in a long term effect. Furthermore, the molecular docking, molecular dynamics and CETSA experiment results indicated that GBVAM had a high affinity for NHE1. The KEGG pathway enrichment analysis indicated that the DEGs was enriched in p53, mTOR signaling pathway and autophagy, mainly. Therefore, in vivo and in vitro experiment, we explored the protection of GBVAM. There were several mechanisms involved in the DIC development, such as apoptosis, inflammation, oxidative stress, autophagy and ferroptosis.^37–39 Our results indicated that GBVAM weakened the oxidative stress in vivo and in vitro via NHE1 inhibition. Moreover, GBVAM also alleviated the apoptosis and autophagy caused by Dox through NHE1 inhibition. Our results also indicated that GBVAM could protect the DIC on mice through improving the cardiac function and morphology deficiency. Considering the importance of PI3K/Akt pathway in apoptosis and autophagy, we further researched the role of p53 and mTOR in the effect of GBVAM.^33,43,44 Our present results showed that PI3K/Akt pathway was activated by GBVAM in the Dox group, which was reversed by LiCl, an NHE1 activator. What is more, our results also indicated that the PI3K inhibitor LY294002 could reverse the protection of GBVAM through bating anti-apoptosis and autophagy regulation. Therefore, we assumed that GBVAM played the protective effect on the DIC in vivo and in vitro by NHE1 inhibition, which might be associated with the PI3K/Akt pathway. Although we had confirmed the protection of GBVAM on DIC; however, the limitations also existed. In the future research, we will further investigate the correlation between the PI3K/Akt pathway and NHE1 inhibition, particularly in the cardiomyopathy.

Conclusion

In conclusion, our results indicated that GBVAM as a NHE1 inhibitor played the protective effect on DIC mice by alleviating the oxidative stress, apoptosis and autophagy by NHE1 inhibition. In addition, the vitro experiment results indicated that GBVAM played the protective effect on Dox-induced H9c2 cell injury against apoptosis and autophagy via the inhibition of NHE1 and PI3K/Akt/mTOR pathway. Therefore, the protection of GBVAM achieved by NHE1 inhibition was via PI3K/Akt/mTOR pathway properly, which still needs to be confirmed in the future research.