Capsaicin alleviates doxorubicin-induced acute myocardial injury by regulating iron homeostasis and PI3K-Akt signaling pathway

CAP alleviated DOX-induced myocardial injury

The survival rate of mice pretreated with CAP (Figure 1A) was higher than that of the DOX group during the observed 20 days. To investigate the gross changes in the mouse heart, we performed echocardiography on four groups of mice (Figure 1B) and found that DOX worsened HR FS EF in mice, but CAP prevented the deterioration of the hearts of mice in the DOX group, resulting in a corresponding improvement in the short-axis shortening ratio of the left ventricular (LV) (Figure 1C left) and the Ejection Fraction (EF) (Figure 1C right). Upon dissecting the mouse hearts, we found that the hearts of mice in the DOX group were larger than those in the control(CTL) group, while the hearts of mice in the CAP+DOX group were smaller than those in the DOX group (Figure 1D left), and when considering the mice’s weight (Figure 1D right), the compensatory increase in heart volume caused by the CAP+DOX group was reduced, thereby reducing the compensation of the heart due to the decreased blood supply caused by DOX, which was sufficient to support the weight of the mice. Tissue analysis of the mouse hearts using HE staining showed that the arrangement of myocardial cells in the DOX group was more disorderly than that in the other three groups, and mice in the CAP+DOX group was apparently better than those in the DOX group (Figure 2A). Using Masson staining to observe the content of collagen fibers (Figure 2B), the hearts of mice in the CAP+DOX group contained significantly less collagen fibers than those in the DOX group (Figure 2C). Measurement of heart failure indicators showed that BNP and MYH7 in the heart muscle of mice in the CAP+DOX group were notably lower than those in the DOX group (Figure 2D). The decrease in these two genes, which serve as indicators of heart failure, suggests that CAP is effective in reducing DOX-induced heart failure [25, 26]. Additionally, we found that the group treated with CAP alone also demonstrated better performance than the CTL group, and we believe that CAP did play a key role in protecting cardiovascular function, as previously reported in the literature. KEGG disease enrichment was used to analyze the CAP+DOX group and DOX group using RNA-seq methodology, revealing differences in disease between the CAP+DOX and DOX groups, with heart failure ranking second (Figure 2E). In conclusion, CAP not only protected the heart under normal circumstances but also protected against DOX-induced myocardial disease.

Figure 1. CAP alleviated DOX-induced myocardial injury. (A) Survival rate of mice injected with DOX or CAP+DOX (n = 20). (B) Echocardiographic images of CTL group, CAP group, DOX and DOX + CAP groups in at day 4 after injection. (C) Shortening fraction (left) and ejection fraction of mice at day 4 after injection (right) (n = 6). (D) Weight of mice (left) and anatomical appearance of mice heart (right) (n = 5). Data are shown as the mean ± SEM. Statistical significance was determined using two-tailed student’s t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 2. CAP alleviated DOX-induced myocardial injury. (A) Cross-sectional staining of mouse heart, HE staining. (B) Masson staining of Cross-sectional staining of mouse heart. Scale: 50 µM (C) Percentage analysis of collagen fibers (n = 6). (D) mRNA expression levels ofMYH7 (left) and BNP (right) in mouse heart tissue on day 4 after injection (n = 6). (E) KEGG disease enrichment map of mice in DOX and CAP+DOX groups (n = 3). Data are shown as the mean ± SEM. Statistical significance was determined using two-tailed student’s t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

CAP mitigated DOX-induced ferroptosis in mice

Ferroptosis has been reported to play an important role in doxorubicin-induced cardiomyopathy (DIC) [27, 28]. Our research showed that CAP inhibited DOX-induced cardiac ferroptosis. Firstly, the content of 4-HNE in the heart muscle of mice in the four groups was determined by immunohistochemistry, and it was found that the content of 4-HNE in the heart tissue of mice in the CAP+DOX group was apparently lower than that in the DOX group (Figure 3A). As a product of lipid peroxidation, the decreased level of 4-HNE indicated that CAP reduced lipid peroxidation in cardiomyocytes. To further demonstrate that lipid peroxidation was inhibited, MDA, another product of this process, was detected, and as expected, MDA content in CAP+DOX group was also significantly lower than that in DOX group (Figure 3B, left). Furthermore, our results provide evidence that CAP reduces lipid peroxidation induced by DOX. In order to further confirm that CAP inhibits DOX-induced ferroptosis, and that accumulation of iron ions and depletion of GSH are key to ferroptosis, we detected non-heme iron content and GSH/GSSH content in mouse myocardium (Figure 3B, middle), and found that CAP did inhibit the occurrence of DOX-induced ferroptosis. Meanwhile, we also found that the cardiac iron in CAP+DOX group lower than DOX (Figure 3B, right). We found through RNA-seq that CAP had a important effect on DOX-induced ferroptosis, and we expressed the genes with notably differences related to ferroptosis using cluster heat maps (Figure 3C). Later, we verified the related genes in the figure. Firstly, mRNA of GPX4, the core factor of ferroptosis, and PTGS2, the product of ferroptosis, were performed. CAP could indeed apparently inhibit DOX-induced ferroptosis (Figure 3D, left). GPX4, as the core factor of ferroptosis, was significantly up-regulated in CAP+DOX (Figure 3D, right), and PTGS2, as a result of ferroptosis, was also decreased. We then performed protein-related verification and found that changes in GPX4 and PTGS2 were consistent with mRNA, and SLC7A11 upstream of GPX4 was also elevated (Figure 4A, 4B). In addition, FTH1, as an important protein for the treatment of ferrous ions, was also up-regulated, and the expression of FTH1 in CAP+DOX group was significantly higher than that in DOX group (Figure 4A, 4B).

Figure 3. CAP mitigated DOX-induced ferroptosis in mice. (A) The cardiac tissues of mice from each group were stained with anti - 4-HNE (left) and quantitative analysis was performed (right). Scale: 50 µM (B) Mouse myocardial MDA (left), myocardial glutathione to reduced glutathione ratio (middle), myocardial iron non-heme iron (right) (n − 5). (C) Cluster heat maps of genes associated with ferroptosis in dox and CAP+DOX groups (n = 3). (D) mRNA expression levels of GPX4 (left) and PTGS2 (right) in mouse myocardium (n = 6). Data are shown as the mean ± SEM. Statistical significance was determined using two-tailed student’s t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 4. CAP mitigated DOX-induced ferroptosis in mice. (A) Immunoprotein gel of COX-2, SLC7All, FTHI, GPX4 in mice (n = 3). (B) Relative expressions of COX-2, SLC7All, FTHI, GPX4 in mice. (C) Immunoprotein gel of Trf and SLC40A1 in mice (n = 3). (D) Relative expression levels of Trf and SLC40A1 in mice (n = 3). Data are shown as the mean ± SEM. Statistical significance was determined using two-tailed student’s t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Through RNA-seq analysis, we have discovered that CAP regulated two key components of intracellular iron content, SLC40A1 and Transferrin (TRF). SLC40A1, also known as FPN1, encodes the iron transport protein that exports iron out of cells, while TRF allows iron to be taken up by cells by binding to transferrin receptors (TFR) [29, 30]. Our experimental results have shown that CAP is capable of upregulating or downregulating SLC40A1 and TRF, thereby regulating intracellular iron content and inhibiting DOX-induced ferroptosis (Figure 4C, 4D). These findings indicated that CAP modulated ferroptosis by adjusting intracellular iron content.

CAP inhibited DOX-induced ferroptosis in H9C2 cells

To demonstrate that CAP exerted the same effect in vitro, we performed an in vitro experiment using H9C2 cells [31]. Methods described in previous literature were used to treat H9C2 with DOX and CAP. Firstly, we conducted a CCK-8 cell viability assay and found that CAP significantly increased cell viability in the DOX group (Figure 5A). We then performed a lactate dehydrogenase (LDH) cytotoxicity assay (Figure 5B) and found that CAP also reduced DOX-induced cell toxicity. In order to detect the occurrence of ferroptosis, we first measured the ratio of reduced glutathione to oxidized glutathione in cells, and found that CAP up-regulated this ratio (Figure 5C). We then tested for intracellular reactive oxygen species and cell death, and found that the CAP+DOX group was lower than the DOX group as evidenced by ROS and PI staining (Figure 5D). Finally, we measured the mRNA and protein levels of ferroptosis-related genes. The CAP+DOX group was elevated in the expression of GPX4 mRNA compared to the DOX group, while PTGS2, which is induced by DOX, was significantly reduced by CAP (Figure 6A). Testing for GPX4, COX-2, and FTH1 protein levels revealed that GPX4 and FTH1 were higher in the CAP+DOX group than in the DOX group, whereas COX-2 decreased in the CAP+DOX group (Figure 6B, 6C). Meanwhile, we validated the effects of TRF and SLC40A1 on cellular iron intake by modulating their expression levels in vitro (Figure 6B, 6C). Our results indicate that CAP regulated intracellular iron pool, iron metabolism to inhibit dox-induced ferroptosis by up- or down-regulating the expression of TRF and SLC40A1.

Figure 5. CAP inhibited DOX-induced ferroptosis in H9C2 cells. (A) H9C2 cells were treated with 2.5 µM DMSO or DOX or varying concentrations of CAP+ 2.5 µM DOX for 6 hours, followed by measurement of cell viability using cck-8 assay (n = 5). (B) H9C2 cells were treated with 2.5 µM DMSO or DOX or 0.4 µM CAP+ 2.5 µM DOX for 6 hours, followed by measurement of cell toxicity using LOH assay (n = 5). (C) H9C2 cells were treated as described previously for 6 hours, followed by measurement of intracellular GSH/GSSG levels (n = 5). (D) H9C2 cells were treated with 2.5 µM DMSO or DOX or 0.4 µM CAP + 2.5 µM DOX for 24 hours, and then observed under bright field (BF) and fluorescence modes for measurement of intracellular ROS levels (top) and cell death(bottom) (n = 3). Scale: 20 µM Data are shown as the mean ± SEM. Statistical significance was determined using two-tailed student’s t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0001.

Figure 6. CAP inhibited DOX-induced ferroptosis in H9C2 cells. (A) H9C2 cells were treated with 2.5 µM DMSO or DOX or 0.4 µM CAP+ 2.5 µM DOX for 6 hours, and then, qPCR was used for quantitative analysis of PTGS2 (right) and GPX4 (left) mRNA expression (n = 5). (B) Relative expressions of COX-2, SLC7All, FTHl, GPX4, TRF, SLC40Al in H9C2 (n = 3). (C) H9C2 cells were treated with 2.5 µM DMSO or DOX or 0.4 µM CAP+ 2.5 µM DOX for 8 or 12 hours, followed by extraction of proteins for Western blot analysis (n = 3). Data are shown as the mean ± SEM. Statistical significance was determined using two-tailed student’s t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

CAP relieved DOX-induced apoptosis of cardiomyocytes by regulating PI3K-Akt signaling pathway

As mentioned earlier, ferroptosis plays an important role in DOX-induced myocardial disease; however, apoptosis, as a common form of cell death, also plays a key role in DOX-induced myocardial disease [29]. DOX induced cardiomyocyte apoptosis by suppressing the PI3K-Akt pathway and regulating BCL2 and BAX [10]. Through KEGG pathway enrichment analysis, we discovered that the calcium ion channel and PI3K-Akt pathway were significantly enriched (Figure 7A). Therefore, to investigate the effect of CAP on DOX-induced apoptosis, we measured the mRNA and protein levels of apoptosis-related factors and found that BCL2, which was inhibited by DOX, was elevated in the presence of CAP, while DOX-induced BAX was decreased (Figure 7B). The ratio of BCL2 to BAX could be used to determine the degree of myocardial apoptosis [32]; we found that CAP inhibited DOX-induced apoptosis (Figure 7D top). We then validated the phosphorylation levels of PI3K and Akt upstream and found that DOX reduced the phosphorylation levels of PI3K and Akt, whereas CAP+DOX restored the phosphorylation levels of PI3K and Akt. Therefore, we concluded that CAP alleviated DOX-induced apoptosis by upregulating the phosphorylation levels of PI3K and Akt. CAP inhibited DOX-induced myocardial apoptosis in mice, so we replicated similar experiments at the cellular level. Through our experiments, we found that CAP suppressed DOX-induced apoptosis in H9C2 cells. We also performed PI3K-Akt pathway validation in our cellular experiments and obtained similar results (Figure 7C). We then analyzed the relevant apoptosis-related proteins and found that BCL2, as an anti-apoptotic protein [33], was significantly upregulated by CAP when DOX inhibits this gene; on the other hand, BAX, as a pro-apoptotic protein [34], was inhibited by CAP (Figure 7C). And the ratio of BCL2 and BAX decreased, so we could tell the apoptosis was reduced by CAP (Figure 7D bottom). We have determined that CAP activated the PI3K-Akt pathway and reduced DOX-induced cellular apoptosis.

Figure 7. CAP relieved DOX-induced apoptosis of cardiomyocytes by regulating PI3K-Akt signaling pathway. (A) KEGG pathway enrichment plots for DOX group and CAP+DOX group 24 hours after injection of DOX (n = 3). (B) Quantitative analysis of heart tissue collected from four groups of mice after injection of saline or CAP or DOX or CAP pretreatment followed by DOX treatment, using immunoblotting (n = 3). (C) Quantitative and analytical immunoblotting for H9C2 cells treated with 2.5 µM DMSO, DOX or 0.4 µM CAP+2.5 µM DOX for 12 hours (n = 3). (D) Ratio of the BCL2 and BAX proteins in mouse myocardial tissue (top) and in H9C2 cells (bottom), used to indicate risk of apoptosis (n = 3). Data are shown as the mean ± SEM. Statistical significance was determined using two-tailed student’s t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Mice and treatment

All protocols were approved by the Institutional Animal Care and Use Committee of the Huazhong Agricultural University (HZAUMO-2023-0128). Male C57BL/6J mice were housed in a temperature- and humidity-controlled room with normal illumination, fed a commercial diet (Animal Center of Huazhong Agricultural University), and given free access to water. Adult (6 to 8 weeks old) male animals were used in this study. The mice in capsaicin (CAP) + doxorubicin (DOX) group were subjected to one time intraperitoneal injection of CAP (HY-10448, MedchemExpress) in every 24 h for 3 days. The mice in CAP group were subjected to a single intraperitoneal injection of saline. The mice in DOX group were subjected to a single intraperitoneal injection of DOX (HY-15142, MedchemExpress, 20 mg/kg, body weight) for 1 or 4 days. The mice in CTL group were subjected to intraperitoneal injection of saline for the same time.

Cell culture

H9C2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) and 1% antibiotics penicillin and streptomycin (P/S) at 37°C and 5% CO2 in the incubator. Cells were treated with DOX or DMSO with or without CAP, and the concentration and cells were determined according to the specific experimental requirements.

Histopathological evaluation

Hearts of mice were fixed in 4% paraformaldehyde (pH 7.4) for several hours, embedded in paraffin, and serially sectioned at 5-µM thickness. The sections were stained with Hematoxylin and Eosin (H&E) for routine histological examination with a light microscope. To measure collagen deposits, select sections were stained with Masson. For each mouse, three adjacent sections were quantified using ImageJ software.

Immunofluorescence and immunohistochemistry

Tissues were treated as previously described. Anti-4-hydroxynonenal antibody (ab216880, Bioss) was used to detect protein expression in the heart tissue. Images were captured at 50× with an Olympus immunofluorescence microscope. Quantification of the relative intensity of protein staining was performed by automated image analysis in five randomly chosen 200× fields for each sample.

RT-qPCR

Total RNA was isolated from tissues or cells using Trizol (Vazyme), and RNA concentration and purity were measured using a spectrophotometry. RNA was reverse-transcribed using the PrimeScript RT reagent Kit (Vazyme) in accordance with the manufacturer’s instructions. Quantitative PCR was performed using lightcycle96 Real-Time System (Roche) in accordance with the manufacturer’s instructions. The fold difference in gene expression was calculated using the 2^−ΔΔCt method and is presented relative to GAPDH mRNA. All reactions were performed in triplicate. The primer sequences are in Supplementary Table 1.

RNA sequencing and data analysis

Whole-genome gene expression analysis was performed using the heart tissues from DOX- and DOX+CAP-treated mice (n = 3 per group) at 24 h. The total RNA was extracted using Trizol (Vazyme), and cDNA samples were sequenced using a sequencing system (illumina novaseq 6000). The reference Mus musculus genome and gene information were downloaded from the National Center for Biotechnology Information database. Raw reads were filtered to produce high-quality clean data. All the subsequent analyses were performed with the clean data. All the differentially expressed genes were used for heat map analysis and KEGG ontology enrichment analyses. For KEGG enrichment analysis, a P-value < 0.05 was used as the threshold to determine significant enrichment of the gene sets.

Biochemical analysis

Serum and cardiac malondialdehyde (MDA) levels were measured using a kit (S0131, Beyotime) in accordance with the manufacturer’s instructions. GSH/GSSG ratio was measured using a glutathione assay kit (No. S0052, Beyotime) according to the product instructions. The LDH kit was bought from Nanjing Jiancheng and was used to detect cytotoxicity according to the instructions.

Cell viability assay

Cell viability assay was performed using the CellTiter-Glo^® Luminescent CCK-8 Cell Counting Kit (A311, Vazyme). Cells were seeded at 5 × 10³ cells per well in a 96-well opaque plate. After the indicated treatment, the luminescent signal was measured using GloMax-Multi Detection System (Promega) in accordance with the manufacturer’s instructions. Based on the recorded luminescence, the percentage of cell viability was then calculated accordingly.

Measurement of ROS

H9C2 cells were seeded in a 24-well plate. Then the intracellular reactive oxygen species content was detected using Beyotime Reactive Oxygen kit (S0033S, Beyotime) according to the instructions.

Cell death measurement

H9C2 cells were seeded in a 24-well plate. After the treatment, cell death was determined by propidium iodide (PI) staining (10 ng/mL) coupled with microscopy.

Western blot analysis

Total proteins were extracted from the tissues by homogenizing in RIPA buffer containing protease inhibitors. The homogenate was cleared by centrifugation at 4°C for 30 min at 12,000 rpm, and the protein was collected. Protein concentration was measured using the BCA Protein Assay Kit (Vazyme). A total of 20 or 30 mg protein per sample was resolved in a 10–12% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.2% Tween-20, then incubated with primary antibodies at 4°C overnight. The following antibodies were used: anti-COX-2 (A1253, Abclonal), anti-SLC7A11 (CSB-PA145393, CUSABIO) anti-FTH1 (WL05360, Wanleibio), anti-GPX4 (A11243, Abclonal), anti-GAPDH (A1253, Abclonal), anti-Transferrin (CSB-PA00250E0Rb, CUSABIO), anti-SLC40A1 (PHJ61901, AntibodySystem), anti-Pi3k (4292S, Cellsignaling), anti-Akt (WLP001a, Wanleibio), anti-Phospho Pi3k (17366, Cellsignaling), anti-Phospho Akt (WL0003b, Wanleibio), anti-BCL2 (CSB-RA564360A0HU, CUSABIO), anti-BAX (CSB-MA484077, CUSABIO). The membrane was then trimmed according to the size of the target protein. The membranes were then washed and probed with the appropriate horseradish peroxidase-conjugated secondary antibodies (As104, Abclonal) and detected using the ECL System.

Statistical analysis

Data were analyzed and graphed using GraphPad Prism software, and all summary data are presented as mean ± s.e.m. Groups were compared using the student’s t-test. For the Kaplan-Meier survival plots, statistical significance was measured using the log-rank (Mantel-Cox) test. Compared with the control group, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Data availability

All study data are included in the Article and/or SI Appendix.