Introduction

Doxorubicin (DOX) is a chemotherapeutic agent widely used for treating solid and hematologic tumors1. However, its clinical use is hindered by serious side effects, especially cardiotoxicity2. DOX can cause gradual dilation of the ventricles, ultimately leading to heart failure3. Oxidative stress, iron metabolism, inflammation, and calcium overload may be involved in DOX-induced cardiotoxicity; however, the exact mechanism remains unclear4.

Pyroptosis is a recently discovered, novel type of programmed cell death accompanied by inflammation. It differs from apoptosis and necrosis in terms of morphology and pathophysiological mechanisms5. In previous studies, we demonstrated that pyroptosis is involved in DOX-induced cardiotoxicity. Formation of the Nod-like receptor (NLR) family pyrin ___domain-containing 3(NLRP3) inflammasome is an important step in pyroptosis 6. NLRP3 inflammasomes can drive the activation of caspase-1 or caspase-4/5/11, which convert interleukin (IL)-1β, IL-18, and gasdermin (GSDMD) into their active forms 5, induce cell membrane perforation and then cause cell death. We previously reported that the inhibition of NLRP3 inflammation can alleviate DOX-induced pyroptosis 6.

Mesenchymal stem cells (MSCs) are pluripotent stem cells found in the bone marrow, subcutaneous adipose tissue, lungs, and dental pulp 7. They can differentiate into a variety of cell types, including bone, cartilage, cardiac muscle, skeletal muscle, vascular endothelial cells, and vascular smooth muscle cells 8. For example, bone marrow mesenchymal stem cells (BMSCs) can differentiate into osteoblasts and chondrocytes under induced culture conditions, participating in the regeneration and repair of bone and cartilage tissues. This is of significant importance for the treatment of fractures, bone defects, and diseases such as osteoarthritis. However, with increasing age, the ability of BMSCs to differentiate into osteoblasts decreases while their ability to differentiate into adipocytes increases, resulting in age-related bone loss 9,10,11. BMSCs have the capacity to differentiate into multiple cell types, including osteoblasts and adipocytes 12,13,14,15. It has been reported that during the aging process, the ability of BMSCs to differentiate into osteoblasts decreases while their ability to differentiate into adipocytes increases, leading to age-related bone loss 16,17,18. MSCs have protective activities against acute myocardial infarction, ischemic cardiomyopathy, diabetic cardiomyopathy, and other cardiac diseases 19. The earliest study on the cardiovascular protective effect of MSCs was to protect ischemic cardiomyocytes by the intravenous injection of MSCs. Later studies reported that the supernatant of the MSC culture medium had the same effect as MSCs of improving cardiac function after myocardial infarction. Follow-up studies confirmed that exosomes in the supernatant of MSCs play a cardioprotective role. Exosomes are 40–160 nm in diameter and contain diverse biomolecules including lipids, proteins, and nucleic acids 20. Exosomes, lipid nanoparticles (LNPs), PLGA nanoparticles, and vesicles are all nanoscale materials used in drug delivery and biological research. They share some similarities, such as their ability to encapsulate and deliver drugs or bioactive substances. However, they exhibit differences in terms of structure, origin, composition, and applications. Exosomes, naturally produced by the body, are nanosized particles with specific biological functions. Lipid nanoparticles and PLGA nanoparticles are artificially prepared carriers using lipids and polymers as materials, respectively. Vesicles can refer to cellular structures or artificially prepared nanoscale particles. Therefore, exosomes have unique advantages in terms of biocompatibility. Exosomes play vital roles in intercellular communication and material exchange through intercellular transfer of nucleic acids and specific proteins 21. The protective effects of bone marrow stem cell–derived exosomes (BMSC-Exos) on the cardiovascular system have been the focus of recent studies. Lee et al. reported that BMSC-Exos inhibited the progression of hypoxia-induced pulmonary hypertension 22. Lai et al. reported that BMSC-Exos can reduce infarct size in a myocardial ischemia–reperfusion injury model 23. In the same period, studies have reported that exosomes secreted by endothelial progenitor cells (EPC-Exo) differentiated from BMSCs can promote endothelial function, protect cardiac microcirculation, and inhibit cardiac fibrosis 24,25. However, whether BMSC-Exos or EPC-Exos can alleviate DOX-induced pyroptosis remains unclear.

In this study, BMSC-Exos and EPC-Exos were added to DOX-treated cells and rats. The in vitro and in vivo results showed that BMSC-Exos could inhibit Dox-induced pyroptosis, whereas EPC-Exos could not. To investigate the molecular mechanisms, both exosomes were sequenced. Sequencing revealed higher expression of long non-coding RNA human gastric cancer with higher expression of transcript 1 (lncRNA GHET1) in BMSC-Exo. One study demonstrated that lncRNA GHET1 could bind to insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to regulate the stability of downstream mRNA 26. Our recently published study showed that IGF2BP1 could bind to NLRP3 mRNA to enhance its stability of NLRP3 mRNA, thereby increasing NLRP3 expression and leading to cardiomyocyte pyroptosis. We found that lncGHET1 decreased NLRP3 mRNA stability by binding to insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), contributing to decreased NLRP3 expression and improving cardiomyocyte pyroptosis. Thus, our study provides a potentially effective therapeutic strategy for DOX-induced cardiotoxicity.

Methods and materials

Culture and characterization of BMSCs and EPCs

Three-week-old male Sprague–Dawley rats were euthanized with sevoflurane and tissues were collected from the tibia and femur. The bone marrow was rinsed in a culture flask containing the prepared medium. BMSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, USA) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 20% fetal bovine serum. EPCs were cultured in an EPC induction medium (EGM-2; Lonza, USA). The cells were cultured at 37 °C in a 5% CO2 atmosphere for 24 h. Nonadherent cells were discarded, and the adherent cells were cultured for approximately 3 days until they reached approximately 90% confluency. BMSCs and EPCs at passage three were used for the exosome collection.

Alizarin red staining

After aspirating the culture medium and gently washing the cells three times with PBS, the cells were fixed with a paraformaldehyde solution. Subsequently, 2 mL of Alizarin Red working solution was added, and the cells were stained at room temperature for 5–10 min. Following the removal of the Alizarin Red staining solution, cells were gently washed three times with PBS to thoroughly eliminate excess dye. Additional three washes with PBS were performed before placing the culture dish under a microscope for observation of the osteogenic staining effect.

Isolation and characterization of exosomes

BMSCs and EPCs were incubated for 48 h in a complete medium and cultured for 24 h in 10% exosome-free fetal bovine serum. The culture supernatant was collected in centrifuge tubes to remove nonadherent cells, dead cells, and cellular debris. The supernatant was centrifuged for 20 min at 2000 × g at 4 °C, and the supernatant was pipetted off and transferred to polyallomer tubes or polycarbonate bottles appropriate for the ultracentrifugation rotor. One side of each ultracentrifuge tube was marked with a waterproof marker, and the tube was oriented in the rotor with the mark facing up and centrifuged for 30 min at 10,000 × g at 4 °C. The supernatant was transferred to a fresh tube, centrifuged for 70 min at 100,000 × g at 4 °C, and removed to collect the exosome fraction. The pellet was resuspended in phosphate buffered saline (PBS) and centrifuged twice at 100,000 × g for 60 min at 4 °C twice. Protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime, China). Transmission electron microscopy was used to examine the samples after they were fixed with 1% glutaraldehyde and applied to a carbon-coated copper grid. The size distribution and concentration of the particles were evaluated by nanoparticle tracking analysis (NTA) using a ZetaView PMX 110 (Particle Metrix, Germany). Surface markers were verified by western blotting with anti-CD63 and CD9. The exosomes were stored at − 80 °C.

RNA extraction and sequencing

Total RNA was extracted from exosome using PureLink RNA Mini Kit (Massachusetts, Thermo Fisher Scientific). Some samples were concentrated to reach the desired concentration of 1 mg/ml using a vacuum centrifuge. The presence of small RNA was verified using a Bioanalyzer 2100 (Agilent, CA, USA), ensuring no signs of degradation, as indicated by the OD ratio of 260/280. The initial RNA amount was 10 mg, and the preparation protocol strictly adhered to the manufacturer's recommendations. Small RNA was isolated from the total RNA using a 15% Novex TBE-Urea PAGE gel. The region corresponding to small RNA molecules with sizes ranging from 18 to 30 nucleotides (nt) was excised, fragmented, and the RNA was eluted in 0.3 M NaCl. A purification step was performed using a Spin X column. Subsequently, a 5'-adapter was ligated at 20 °C for 6 h. Small RNA molecules with the 5'-adapter were separated on a 15% Novex TBE-Urea PAGE gel (Invitrogen, CA, USA), and the band with sizes between 40 and 60 nt was excised, fragmented, and eluted in 0.3 M NaCl, followed by purification on a Spin X column. A 3'-adapter was ligated at 20 °C for 6 h to the small RNAs with the 5'-adapter. These RNA molecules were isolated on a 10% Novex TBE-Urea PAGE gel, and the band with sizes between 70 and 90 nt was excised, fragmented, and eluted in 0.3 M NaCl. A final purification step was performed using a Spin X column. GlycoBlue and ethanol were added, followed by precipitation at – 80 °C for 30 min and centrifugation at 14,000 rpm for 25 min. The RNA pellet was reconstituted in 4.5 ml of RNase-free water. Reverse transcription and amplification were carried out, and the resulting cDNA was separated on a 6% Novex TBE PAGE gel. The amplified cDNA band was excised, fragmented, and the RNA was eluted in Gel Elution Buffer and purified on a Spin X column. Glycogen and ethanol were added for precipitation, followed by centrifugation at 14,000 rpm and 4 °C for 20 min. The cDNA pellet was dissolved in 10 ml of Resuspension Buffer. The quality of the generated cDNA library was assessed through quantitative real-time PCR to ensure its quality and the proper addition of adapters. High-throughput sequencing of the cDNA was performed using an Illumina Genome Analyzer IIx (Illumina, CA, USA) in a 36 bp single-read run. Image analysis and base calling were carried out using Illumina GA pipeline software version 1.5.1. Sequences with a chastity score of less than 0.6 on two or more bases within the first 25 bases were excluded.

Exosome uptake assay

H9C2 cells were seeded at a density of 5 × 104 cells per well in a six-well plate and incubated overnight. Fresh culture medium containing 10 μg of PKH26 (Sigma, USA)-labeled exosomes (BMSC-Exo/EPC-Exo) was added, and the cells were incubated for 24 h. The cells were washed with Hank's Balanced Salt Solution (HBSS) containing 0.01% sucrose and fixed with 2% paraformaldehyde. Subsequently, the cells were washed with HBSS, and images were acquired using fluorescence microscopy.

Animal experiments

Six-week-old Sprague–Dawley rats (24 males; body weight, 90–100 g) were purchased from the Experimental Animal Center of Basic Medicine, Zhejiang Chinese Medical University (Zhejiang, China). According to the predefined inclusion criteria, all rats had a left ventricular ejection fraction greater than 60% and exhibited normal vitality. The rats were randomly divided into four groups: control (CON), DOX, DOX + BMSC-Exo, and DOX + EPC-Exo, each group consisted of 6 rats, all experimenters were aware of the group assignments. To simulate a normal lifestyle, rats were housed in a dark room with a 12-h light/dark cycle and fed ad libitum. Animals in the DOX group were injected with 3 mg/kg DOX via the tail vein once a week for 5 weeks (total dose, 15 mg/kg) to induce cardiotoxicity. Using an insulin syringe, inject 50 µg of BMSC-Exo or EPC-Exo into the rats in the DOX + BMSC-Exo and DOX + EPC-Exo groups via the tail vein in a single injection. Following this, administer DOX injection as described above 27. After 5 weeks, since all rats survived, no exclusions were necessary, and all could proceed to subsequent testing, all rats were sacrificed by sevoflurane inhalation. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Shaoxing Hospital of Zhejiang University (Shaoxing City, China), We confirm the study was reported in accordance with ARRIVE guidelines, and all methods were performed in accordance with the relevant guidelines and regulations.

Cardiomyocyte culture and regents

The H9C2 cell line, obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China), was cultured in DMEM supplemented with 10% fetal bovine serum and 5 mg/mL penicillin/streptomycin in a humidified incubator with 5% CO2 at 37 °C. DOX (MCE, USA) at a concentration of 2 µM was used.

Vector construction and cell transduction

Small interfering RNAs was designed and synthesized by GenePharma (Shanghai, China) to target the lncRNAs GHTB1 and IGF2BP1. The small interfering RNAs were transfected into H9C2 cells using Lipofectamine 3000 transfection reagent (Invitrogen, USA) according to the manufacturer’s instructions. After 48 h of transfection, the cells were collected and the knockout efficiency was detected via quantitative reverse transcription polymerase chain reaction.

Echocardiography

Cardiac function was evaluated using an iE33 system (Philips Medical, Best, Netherlands) equipped with an s5-1probe (12–12 MHz) through the performance of two-dimensional M-mode echocardiography. Fraction shortening (FS) and left ventricular ejection fraction (LVEF) were measured during five consecutive cardiac cycles.

Transmission electron microscopy

Left ventricular tissues from rats were fixed in 2.5% glutaraldehyde overnight at 4 °C, post-fixed with 1% buffered osmium tetroxide, dehydrated in a graded acetone series, and embedded in epoxy resin. Ultrathin Sects. (70 nm) were obtained, stained with uranyl acetate/lead citrate, and observed using TEM.

Calcein-AM staining assay

Cells cultured in chamber slides were washed twice with PBS. The cells were then incubated with calcein-AM solution (Abmole USA) for 30 min. After the incubation, the medium was replaced with fresh preheated medium at 37 °C and then incubated in the dark for 30 min at 37 °C. The culture medium was washed twice with PBS and serum-free cell culture medium was added. Nuclear staining was performed using Hoechst. Images were captured using a fluorescence microscope (Nikon).

Immunohistochemistry staining analyses

Immunohistochemistry was used to visualize pyroptosis using the following antibodies: anti-NLRP3 (1:100; ab214185; Abcam, Cambridge, MA, USA), anti-cleaved caspase-1 (1:100, ab2302; Abcam), and tissue sections were routinely dewaxed and fixed with 3% H2O2 for 15 min at room temperature to quench endogenous peroxidases. The sections were incubated with the primary antibody at 4 °C overnight and then incubated with the secondary antibody for 30 min, followed by the substrate (3,3-diaminobenzidine) for 5 min at 37 °C. Images were obtained using a Olympus FSX100 microscope.

RNA fluorescence in situ hybridization

The lncGHTB1 probe was purchased from RiboBio (Guangzhou, China). According to the manufacturer's instructions, a commercial kit (RiboBio) was used to conduct fluorescence in situ hybridization. Specifically, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton, and incubated overnight with a specific probe. Imaging was performed with a Nikon A1Si laser scanning confocal microscope.

RNA pull-down assay

In vitro transcription of the lncRNA GHTB1 was performed using Ambion RNA polymerase (Ambio Life), followed by purification with an RNeasy Plus Mini Kit (Qiagen) and treatment with DNase I (Qiagen). To prepare the cell lysates, a ProteaPrep Zwitterionic Cell Lysis Kit was used, along with mass spectrometry grade anti-RNase (Protea®), a protease/phosphatase inhibitor cocktail, panobinostat, and methylstat supplemented lysis buffer. The proteins pulled down using GHTB1 were detected by western blotting.

RNA immunoprecipitation protocol assay

RNA immunoprecipitation protocol (RIP) assays were conducted using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions. An anti-IGF2BP1 antibody (ab184305; Abcam) was used in the RIP assays.

Real-time reverse-transcription PCR

Total RNA was extracted from heart tissues using TRIzol reagent (Invitrogen, California) according to the manufacturer's instructions. The RNA was then reverse-transcribed using oligo (dT) primers. Real-time PCR was performed on an ABI 7300 RT-PCR Detection System (Applied Biosystems, Foster) with gene-specific primers and SYBR Premix Ex Taq (TaKaRa, Dalian). Each RT-qPCR reaction was conducted in triplicate. The relative expression levels of the target gene were calculated using the 2−△△CT method. Primer sequences are provided in the Supplementary Materials.

Western blotting procedure and antibodies

To prepare samples for analysis, myocardial tissues and H9C2 cells were lysed using RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails. Following protein separation by SDS-PAGE, PVDF membranes were used for protein transfer. After blocking with skimmed milk at room temperature for 1 h, the membranes were incubated with specific antibodies against β-actin (sc58673; Santa Cruz), NLRP3 (ab214185; Abcam), caspase-1 (ab2302; Abcam), IL-18 (ab18672; Abcam), IL-1β (ab2105; Abcam), and GSDMD-N (Bioss, Beijing, China, catalog bs-14287R) overnight at 4 °C (the blots were cut prior to hybridisation with antibodies during blotting). The next day, the membranes were treated with a secondary antibody for 1 h and visualized using an enhanced chemiluminescence (ECL) detection kit (Massachusetts; Thermo Fisher Scientific). Protein bands were analyzed using Quantity One software.

Statistical analyses

Statistical analyses were performed using GraphPad Prism software version 6.0 and SPSS13.0 software. All data are presented as mean ± standard deviation (SD). Statistical differences were analyzed using one-way ANOVA followed by Tukey’s post-hoc test. Statistical significance was set at p < 0.05.

Ethics statement

The animal study was reviewed and approved by Experimental Animal Ethics Committee of Shaoxing People’s Hospital. All methods were performed in accordance with the relevant guidelines and regulations.

Results

Characterization and identification of exosomes

BMSC and EPC were isolated and cultured until the 3rd generation. Supernatants were collected and exosomes were extracted. Microscopy showed that the cell morphology of BMSCs was irregular, and that of EPCs was polygonal or spindle-shaped, both BMSCs and EPCs were observed to adhere and grow along the culture dish (Fig. 1A). Immunofluorescence showed CD90 and CD105 expression in BMSCs, and CD34 and VEGFR2 expression in EPCs (Fig. 1B). The Alizarin Red staining results revealed the presence of multiple calcium nodules in the BMSCs culture dish after osteogenic induction, indicating the osteogenic differentiation capability of our BMSCs (Fig. 1C). As shown in Fig. 1D, electron microscopy revealed the lipid bilayer membrane-encapsulated structure of exosomes. NTA showed that the peak diameters of BMSC-Exo and EPC-Exo were 153 and 136.1 nm (Fig. 1F). Western blot analysis revealed that the exosome marker proteins CD63 and CD9 were positively expressed in BMSC-Exos and EPC-exos (Fig. 1E). Using PKH26 to trace exosomes in H9C2 cells, it was found that exosomes were evenly distributed in the cells (Fig. 1G). These results showed that BMSC and EPC secrete exosomes with common exosomal features.

Figure 1
figure 1

Characterization and functional validation of exosomes derived from BMSCs and EPCs. (A) Micrographs of microscopy of BMSCs and EPCs. (B) Immunofluorescence of marker protein of BMSCs and EPCs. (C) Alizarin Red staining of BMSCs and EPCs after osteogenic induction. (D) Micrographs of scanning electron microscopy of MSC-Exos. (E) Western blot analysis of CD63 and CD9 expressions of BMSC-Exos and EPC-Exo. (F) The particle size and particleconcentration of Exo analyzed by nanoparticle tracking analysis. (G) PKH26-labeled exosomes enter cells through endocytosis in H9C2 cells.

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BMSC-Exo exerted better protection against Dox-induced pyroptosis than EPC-Exo in vitro

To verify whether BMSC-Exos and EPC-Exos can inhibit DOX-induced cardiotoxicity, BMSC-Exos and EPC-Exos were added to the preparations of cardiomyocytes treated with DOX. DOX-treated cardiomyocytes exhibited swelling and cell membrane rupture (Fig. 2A). This damage was reduced in BMSC-Exo, and the EPC-Exo group had the same result as the DOX group (Fig. 2A). Calcein-AM staining revealed decreased cell death in the BMSC-Exo group compared with that in the EPC-Exo group (Fig. 2B). The effects of the two exosome preparations on DOX-induced cardiomyocyte pyroptosis were further investigated. Western blot analysis demonstrated that cells treated with BMSC-Exo group had greater reduction in the expressions of NLRP3, cleaved caspase-1, and GSDMD-N compared to Dox and EPC-Exo group (Fig. 2C). These data suggest that BMSC-Exos exert protective effects against DOX-induced pyroptosis in vitro.

Figure 2
figure 2

BMSCs-Exo improve Dox-induced cardiomyocyte pyroptosis. (A) Micrographs of microscope of cardiomyocyte. (B) cardiomyocyte viability showed by Calcein-AM (green) staining. (C) Western bolt analyse showed expression of cardiomyocyte pyroptosis. n = 3 per group. *p < 0.05.

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BMSC-Exo exerted better protection against Dox-induced pyroptosis than EPC-Exo in vivo.

A rat model of Dox-induced pyroptosis, achieved by intravenous injection of Dox, was used to determine the protective effects of BMSC-Exos and EPC-Exos in vivo. The rat groups included control, DOX, DOX + BMSC-Exo, and DOX + EPC-Exo groups. After 4 weeks, echocardiography showed that, compared with the control group, the LVEF and FS of the DOX treatment group decreased. And LVEF and FS were significantly improved in the DOX + BMSC-Exo group compared to those in the DOX group, and were rarely improved in the DOX + EPC-Exo group compared to those in the DOX group (Fig. 3A). After conducting hematoxylin and eosin staining of rat cardiac tissue, it was observed that DOX administration resulted in significant injury to the cardiomyocytes, which was effectively reduced by treatment with BMSC-Exo, but only marginally reduced by treatment with EPC-Exo (Fig. 3B). Additionally, results from Masson staining indicated a significant increase in myocardial fibrosis in the DOX and DOX + EPC-Exo groups compared to the control, whereas it was comparable between the control and DOX + BMSC-Exo groups. (Fig. 3C,D). TEM showed that cardiomyocyte damage was relieved in the DOX + BMSC-Exo group (Fig. 3E). TEM of cardiomyocyte ultrastructure revealed extensive cardiomyocyte damage in the DOX and DOX + EPC-Exo groups compared to that in control hearts, including increased inter-mitochondrial distance and disconnected cardiac myofibers. The DOX + BMSC-Exo group showed improvement compared to the DOX and DOX + EPC-Exo groups (Fig. 3E). Immunohistochemistry was performed to confirm the occurrence of cardiomyocyte pyroptosis induced by DOX, by examining the expression of pyroptosis markers. As anticipated, rats that received DOX and DOX + EPC-Exo exhibited significantly higher expression levels of NLRP3, cleaved caspase-1, and GSDMD-N, in comparison to the saline control group (Fig. 3F). Pyroptosis was significantly relieved in the DOX + BMSC Exos group (Fig. 3G). Collectively, these results indicate that BMSC-Exos exerted better protection against Dox-induced pyroptosis than EPC-Exos in vivo.

Figure 3
figure 3

BMSCs-Exo improve cardiac function and cardiomyocyte pyroptosis after Dox. (A) Morphological changes and quantitative analysis of LVEF and FS in rat hearts detected by echocardiography after treatment with DOX, BMSC-Exo or EPCs-Exo. (B) H&E staining for assessment of myocardial injury after treatment with DOX and/or DHM (Scale bar = 100 μm, n = 6). (C) Masson staining for assessment of myocardial fibrosis after treatment with DOX and/or DHM (Scale bars = 100 μm, n = 6). (D) Quantitative analysis of fibrotic area. (E) TEM of cardiomyocyte ultrastructure. (F) The expression levels of pyroptosis-related proteins in myocardial tissues from CON, DOX, DOX + BMSCs-Exo, EPCs-Exo group were determined by IHC analysis. (G) The expression levels of pyroptosis-related proteins in myocardial tissues from CON, DOX, DOX + BMSCs-Exo, EPCs-Exo group were determined western bolt. n = 6 per group, *p < 0.05.

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LncRNA GHET1 was a key component of BMSC-Exo to inhibit Dox-induced pyroptosis

To clarify the mechanism by which BMSCs alleviate DOX-induced pyroptosis, we performed RNA sequencing analysis of BMSC-Exo and EPC-Exo. The results showed that the expression of lncRNA GHET1 was higher in BMSC-Exos than in EPC-Exos (Fig. 4A). PCR results also showed that lncRNA GHET1 was more highly expressed in BMSC-Exos than in EPC-Exos (Fig. 4B). To determine whether lncRNA GHET1 plays a protective role in DOX-induced myocardial toxicity and pyroptosis, we constructed lncRNA GHET1 overexpression plasmids and transfected them into H9C2 cardiomyocytes using vector transfection. Under the intervention of doxorubicin, we found that overexpression of lncRNA GHET1 could reduce the swelling and membrane rupture of DOX-treated cardiomyocytes (Fig. 4C). Calcein-am staining indicated that overexpression of lncRNA GHET1 reduced the cell mortality induced by doxorubicin (Fig. 4D). WB was used to detect pyroptosis-related proteins, and it was found that the protein expression of NLRP3, Casepase-1 and GSDMD-N significantly decreased in the DOX + P-lncrNA GHET1 group (Fig. 4E). These findings suggest that the lncRNA GHET1 plays a protective role in cardiomyocyte pyroptosis induced by DOX.

Figure 4
figure 4

Screening of differential genes of BMSCs-Exo and EPCs-exo. (A) RNA sequencing analysis showed lncGHET1 has higher expression in BMSCs-Exo than in EPCs-Exo. (B) PCR proved lncGHET1 has higher expression in BMSCs-Exo than in EPCs-Exo. (C) Micrographs of microscope of cardiomyocyte. (D) cardiomyocyte viability showed by Calcein-AM (green) staining. (E) Western bolt analyse showed expression of cardiomyocyte pyroptosis. n = 3 per group, *p < 0.05.

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LncGHET1 reduces DOX-mediated pyroptosis, which is regulated by histone acetylation at the lncRNA GHET1 gene promoter

To investigate the mechanism underlying the high expression of lncRNA GHET1 in BMSCs, we assessed whether there were any epigenetic modification sites in the promoter region of lncRNA GHET1. By searching the gene analysis website (http://genome.ucsc.edu/), lncRNA GHET1 was found to be a feasible H3K27ac binding site in the promoter region (Fig. 5A). Treatment with the C646 acetyltransferase inhibitor suppressed lncRNA GHET1 expression (Fig. 5B).

Figure 5
figure 5

LncGHET1 is regulated by acetylization. (A) The potential binding area by H3K27ac at the promoter region of lncGHET1 were predicted by using an online prediction software (http://genome.ucsc.edu/). (B) The expression of lncGHET1 is inhibited by C646 acetyltransferase inhibitor. n = 3 per group, *p < 0.05.

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LncRNA GHET1 binds IGF2BP1 to regulate cardiomyocyte pyroptosis

Based on the minimum free energy and partition function, we predicted that nucleotides 351–570 in the GHET1 lncRNA transcript could form a stem-loop structure, which is crucial for its physical loading with targeted proteins (Fig. 6A). To determine the subcellular localization of GHET1 in cardiomyocytes, we conducted various experimental assays, including RNA fluorescence in situ hybridization, which indicated that GHET1 was mainly present in the cytoplasm (Fig. 6B). This finding suggests that GHET1 may regulate downstream signals posttranscriptionally. Yang et al. study show GHET1 could bind IGFBP1 to regulate mRNA expression in gastric cancer cells. The GHET1 may bind IGF2BP1 to affect the pyroptosis of cardiomyocyte. Firstly Immunofluorescence analysis was used to investigate the cellular ___location of IGF2BP1. The results showed that the IGF2BP1 protein was primarily located in the cytoplasm of cardiomyocytes, consistent with GHET1's ___location (Fig. 6C). And to confirm the direct binding between GHET1 and IGF2BP1, we conducted RIP and RNA pull-down assays, which revealed that GHET1 was enriched with IGF2BP1 (Fig. 6D,E). Knockdown of GHET1 did not alter the expression of IGF2BP1, indicating that GHET1 may exert its functional role by binding to IGF2BP1. Furthermore, overexpression of IGF-2BP1 upregulated NLRP3 mRNA expression in cardiomyocytes, while inhibition of IGF2BP1 had the opposite effect (Fig. 6F,G). Most importantly, the overexpression of GHET1 reversed the IGF2BP1-induced transcriptional upregulation of NLRP3 (Fig. 6H).

Figure 6
figure 6

LncGHET1 bind with IGF2BP1 to play critical role in pyroptosis. (A) Prediction of 351–370 nt lncGHET1 structure was based on minimum free energy (MFE) and partition function (http://rna.tbi.univie.ac.at/). Oval area showed the core area binding with IGF2BP1. (B) RNA Fluorescent were performed to verify the sub-cellular distribution of lncRNA GHET1 in H9c2 cells. (C) The subcellular distribution of IGF2BP1 was visualized by immunofluorescence in H9C2 cells. (D) RNA pulldown assay was performed to identify the direct interaction between GHET1 and IGF2BP1 in cardiomyocytes, and western blot was used to show the bands pull down by lncGHET1. (E) RIP assay was done by using specific antibodies: anti-IGF2BP1 and anti-IgG. qRT-PCR was then used to examine the expression lncGHET1 by IGF2BP1 in cardiomyocytes. (F) Expression of NLRP3 after overexpression of IGF2BP1. (G) Expression of NLRP3 after low expression of IGF2BP1. (H) Expression of NLRP3 after over-expression of IGF2BP1 or simultaneous over-expression of IGF2BP1 and GHET1. n = 3 per group, *p < 0.05.

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LncRNA GHET1 reduces mRNA stability of NLRP3 by recruiting IGF2BP1

Given the potential role of IGF2BP1 in mRNA stability modulation, we hypothesized that the lncRNA GHET1 may guide IGF2BP1 to affect the stabilization of NLRP3 mRNA, thus mitigating DOX-induced pyroptosis. To investigate this possibility, we created a GHET1 and IGF2BP1 overexpression vectors and observed that the overexpression of IGF2BP1 counteracted the GHET1-induced decrease in NLRP3 expression (Fig. 7A). Furthermore, we used RIP detection to demonstrate that NLRP3 mRNA was enriched in the IGF2BP1 precipitate, indicating that IGF2BP1 can directly interact with NLRP3 (Fig. 7B). Overall, these findings suggest that the combination of lncRNA GHET1 and IGF2BP1 can impede NLRP3 mRNA stability, which result in a reduction of cell pyroptosis.

Figure 7
figure 7

LncGHET1 regulates pyroptosis via decrease the mRNA stability of NLRP3. (A) NLRP3 expression was determined in cardiomyocytes treated with p-lncGHET1 and (or) si-IGF2BP1. (B) RIP assay was done by using specific antibodies: anti-IGF2BP1 and anti-IgG. qRT-PCR was then used to examine the expression NLRP3 pull down by IGF2BP1 in cardiomyocytes. n = 3 per group, *p < 0.05.

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Discussion

In this study, exosomes from BMSCs were found to protect against DOX-induced cardiotoxicity. Mechanistically, lncRNA GHET1 from BMSC-Exos decreased the expression of NLRP3 by combining with IGF2BP1 to inhibit its mRNA, in turn inhibiting the pyroptosis pathway (Fig. 8).

Figure 8
figure 8

A scheme of the proposed mechanisms. The expression of lncRNA GHET1 in BMSCs is regulated by the acetylation of its upstream promoter sequence H3K27. Exosome released by BMSCs containing lncGHET1 enters cardiomyocytes. lncGHET1 downregulates NLRP3 expression through decreasing mRNA stability via bind with IGF2BP1, resulting in the inhibition of pyroptosis.

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Pyroptosis differs from apoptosis and necrosis in terms of morphology and pathophysiological mechanisms 28. It is an important innate inflammatory immune response and is widely involved in the development of infectious diseases, cardiovascular diseases, and tumors 5. We reported for the first time that pyroptosis is an important mechanism of DOX-induced cardiotoxicity and that inhibition of pyroptosis can reduce this cardiotoxicity 6. The activation of pyroptosis is mainly regulated by inflammasomes 29. Inflammatory bodies are large-molecular-weight multiprotein complexes located in cells. Studies have focused on NOD-like receptor protein 1 (NLRP1), melanoma deficiency, deficiency factor (Absent in Melanoma 2, AIM-2), NLRP3, NLRC4, and other inflammatory bodies. NLRP3 is the most widely studied and important member of the Nod-like receptor family 30. It is composed of an NLRP3 protein scaffold, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 precursor. The amount of NLRP3 directly affects the formation of NLRP3 inflammasomes and the final activation of pyroptosis 31. We previously reported that DOX can increase the expression of NLRP3, thereby activating inflammasomes in cardiomyocytes and inducing pyroptosis in cardiomyocytes 6.

BMSCs protect against DOX-induced cardiotoxicity 19,32. In 2017, Allah et al. reported that BMSCs derived from human umbilical cord blood could slow DOX-induced cardiotoxicity 33. Zaki et al. subsequently found that BMSCs derived from rat bone marrow could also inhibit DOX cardiotoxicity 34. However, these studies did not explore the specific mechanisms underlying the protective effects of BMSCs against DOX-induced cardiotoxicity. Subsequent studies confirmed that exosomes in the supernatant of BMSCs play a cardioprotective role 35. Exosomes are rich in proteins and RNA species (including mRNA, microRNAs, lncRNAs, and competing endogenous RNA) and mediate material exchange and signal transmission between cells. Lee et al. found that BMSC-Exos inhibited the progression of hypoxia-induced pulmonary hypertension 22. Huang et al. showed that atorvastatin can increase H19 expression to promote the protective effect of BMSC-Exos on acute myocardial infarction 36. Moreover, other studies demonstrated that exosomes secreted by EPCs differentiated from BMSCs promote endothelial function, protect the cardiac microcirculation, and inhibit heart fibrosis 24,25.

In this study, BMSC-Exos and EPC-Exos interfered with DOX cardiotoxicity in cells and in vivo in rats. BMSC-Exos reduced DOX-induced heart damage caused by DOX, whereas EPC-Exos did not. BMSC-Exos also impede DOX-induced pyroptosis. To further explore the inhibition of DOX-induced cardiomyocyte pyroptosis by BMSC-Exos, we performed high-throughput sequencing of BMSC-Exos and EPC-Exos. Expression of lncRNA GHET1 was observed in BMSC-Exos but not in EPC-Exos. Furthermore, lncRNA GHET1 knockout and overexpression in vitro showed that overexpression of lncRNA GHET1 alleviated DOX-induced pyroptosis.

The lncRNA GHET1 contains 1913 nucleotides and is located on chromosome 7 of the genome. It was originally found early in gastric cancer tissue, the basis of its designation 26. LncRNA GHET1 was highly expressed in BMSCs. In contrast, the expression was low in EPCs differentiated from BMSCs and cardiomyocytes. During this process, the activity of the upstream promoter of lncRNA GHET1 is related to events that include DNA methylation and histone acetylation modifications near the promoter. Epigenetic factors regulate gene expression. Werber et al. found that lncRNA Fendr interacts with the TrxG/MLL complex to cause H3K27 methylation, which leads to a decrease in the expression of target genes in the heart and ultimately causes cardiac dysplasia 37. We previously reported that lncRNA TINCR expression is regulated by the acetylation of its upstream promoter region, H3K27. To explore the reasons for the high expression of lncRNA GHET1 in BMSCs but low expression in EPCs, we searched a gene database (http://genome.ucsc.edu/) for the lncRNA GHET1 promoter. The presence of many H3K27ac modification markers in the region suggests that lncRNA GHET1 expression may be regulated by H3K27 acetylation. BMSCs were treated with an acetyltransferase inhibitor, C646, and the results of quantitative reverse transcription polymerase chain reaction showed that C646 could inhibit lncRNA GHET1expression in BMSCs. The results of the present study suggest that the high expression of lncRNA GHET1 in BMSCs is regulated by acetylation of its upstream promoter sequence H3K27.

Research on lncRNA GHET1 has mainly focused on tumors. LncRNA GHET1 can promote the proliferation, invasion, and epithelial-mesenchymal transition of tumor cells through the expression of downstream target proteins 38,39. Yang et al. found that lncRNA GHET1 increases c-Myc by binding to IGF2BP1 in gastric cancer cells to stabilize mRNA, thereby increasing the expression of c-Myc protein and promoting the proliferation of gastric cancer cells 26. IGF2BP1 is a member of the IGF2 mRNA-binding protein family. The encoded protein contained four Kelvin homology domains and two RNA recognition motifs. As a posttranscriptional regulatory protein, the importance of IGF2BP1 has been affirmed by recent studies. IGF2BP1 binds to a variety of mRNAs, regulates mRNA stability, and affects the expression of target genes 40,41. Our recent study showed that IGF2BP1 is essential for maintaining the stability of NLRP3 mRNA. IGF2BP1 binds to NLRP3 mRNA and enhances its stability, thereby increasing NLRP3 expression and leading to cardiomyocyte pyroptosis 6. In this study, the minimum free energy, partition function, and database search (http://rna.tbi.univie.ac.at/) allowed us to predict that for lncRNA GHET1, the 351–570 bp nucleotides in the transcript form a stem-loop structure that binds to IGF2BP1. The direct interactions were verified through the use of RIP and RNA pull-down assays. Additionally, the functional analysis revealed that the overexpression of IGF2BP1 counteracted the GHET1-induced cardiomyocyte pyroptosis triggered by DOX. These findings suggest that GHET1 modulates the NLRP3 mRNAs related to cardiomyocyte pyroptosis by interacting with IGF2BP1.

Our study had several limitations. First, only three BMSC-Exos and EPC-Exos samples were RNA sequenced. Secondly, the effects of lncGHET1 on DOX-induced pyroptosis and the tracer after exosome uptake were only verified in cell experiments and not in vivo, and we cannot determine the duration of biomolecule release following exosome injection into the rat's body. Thirdly, this study focuses on investigating the role of LncGHET1 in counteracting doxorubicin-induced cardiotoxicity through combating myocardial pyroptosis. However, other LncRNAs have indeed shown differential expression, and it remains unknown whether they are also involved in counteracting doxorubicin-induced cardiotoxicity and through what mechanisms. This is an area of investigation that we aim to explore in our future studies. Fourth, we observed that BMSC-Exo is slightly larger in size compared to EPC-Exo. While previous studies have reported the sizes of these vesicles, with BMSC Exo ranging from 100 to 150 nm 42 and EPC Exo measuring approximately 100 nm 43, the underlying cause of this phenomenon remains unexplored. This intriguing question represents a valuable avenue for future investigation. Fifth, although we identified BMSCs using immunofluorescence and alizarin red staining, there are still some identification markers that have not been tested. In our future work, we will further examine additional characterization markers.

In conclusion, exosomes from BMSCs improve DOX-induced myocardial toxicity and pyroptosis by lncGHET1 binding with IGF2BP1 to influence the stability of NLRP3 mRNA. These findings revealed a specific function of the GHET1/IGF2BP1/NLRP3 pathway in cardiomyocyte pyroptosis induced by DOX and may aid in the development of a treatment for DOX myocardial toxicity. These findings will inform studies intended to lessen or eliminate cardiotoxicity after chemotherapy.