Introduction

Cardiac fibrosis is a biological process characterized by the uncontrolled accumulation of extracellular matrix (ECM) in response to ischemia, hypoxia, or pressure overload1. This condition is a common pathophysiological feature in nearly all types of heart disease, leading to increased left ventricular stiffness, cardiomyocyte hypertrophy, arrhythmias, and congestive heart failure (HF), which in turn raises mortality rates2,3,4. Cardiac fibrosis is responsible for approximately 800,000 fibrosis-related deaths annually worldwide5. The extent of cardiac fibrosis is considered a critical indicator of mortality and adverse cardiac events5.

The exact mechanisms driving cardiac fibrosis are not well understood, and the absence of evidence-based effective therapies makes managing this condition particularly challenging6. Currently, no treatment has been proven to effectively repair injured myocardium and stimulate myocardial regeneration. Consequently, addressing cardiac fibrosis remains a significant medical issue. Recent studies have shown that extracellular vesicles (EVs), especially exosomes, can enhance heart repair, promote myocardial regeneration, and reduce cardiac fibrosis7,8,9. These beneficial effects occur through anti-inflammatory, anti-apoptotic, pro-angiogenic and anti-fibrotic mechanisms9,10. Exosomes derived from bovine milk are safe for both animals and humans and can be produced in large quantities11,12. Additionally, when administered orally, bovine milk exosomes are highly stable in the gastrointestinal tract and easily bioavailable13. Our previous research found that orally administered bovine milk exosomes reduced cardiac fibrosis and improved cardiac function impaired by the condition14. However, the precise mechanisms by which bovine milk exosomes affect cardiac fibrosis remain unclear.

Long non-coding RNAs (lncRNAs) are a novel class of endogenous non-coding RNAs with transcripts longer than 200 nucleotides, playing crucial roles in various cardiovascular disease (CVD) pathophysiological processes6. Although some studies have documented the role of lncRNAs in cardiac fibrosis15,16, their specific contributions to disease progression remains less understood. To address this gap, we performed transcriptome sequencing analysis of the left ventricle in rats with cardiac fibrosis treated with oral bovine milk exosomes. Functional analysis of differentially expressed lncRNAs (DELs) and differentially expressed mRNAs (DEMs) was conducted to provide new insights into potential therapeutic strategies for cardiac fibrosis.

Results

Bovine milk exosomes alleviated cardiac fibrosis and enhanced cardiac function in isoproterenol (ISO)-induced rats

As shown in Fig. 1a, ECM deposition in the left ventricle of ISO-induced cardiac fibrosis rat was markedly reduced following oral administration of bovine milk exosomes, as assessed by Hematoxylin and Eosin (H&E) and Masson’s trichrome staining. Quantitative analysis confirmed that rats treated with bovine milk exosomes exhibited fewer cellsand a lowerpercentage of collagen area compared with PBS- treated rats (Fig. 1b, c). Echocardiographic measurements demonstrated improved cardiac function in the bovine milk exosomes-treated group. Specifically, end-systole volume (ESV), end-diastole volume (EDV), and left ventricular internal dimension at end-systole (LVIDs) and end-diastole (LVIDd), as well as end-systole volume (ESV) and end- diastole volume (EDV), were altered in a manner consistent with improved function. Furthermore, left ventricular posterior wall thickness at end-systole (LVPWs) and end-diastole (LVPWd), cardiac output (CO), ejection fraction (EF) and left ventricular fractional shortening (FS) were significantly enhanced compared with PBS control (Fig. 1d, e). These data indicate that bovine milk exosomesattenuate cardiac fibrosis and promotol functional recoveryin ISO-induced rats.

Fig. 1
figure 1

The cardiac fibrosis levels and cardiac function in ISO-induced cardiac fibrosis rats treated orally with bovine milk exosomes or PBS. (a) ECM deposition in the left ventricle assessed by H&E stain and Masson’s trichrome staining (n = 3, Scale bar:50 μm). (b) Quantitative analysis of cell counts in the left ventricle(H&E staining). (c) Quantitative analysis of collagen area in the left ventricle(Masson’s trichrome staining). (d) Representative echocardiographic analysis of cardiac function(n = 3). (e) Quantitative analysis of cardiac function parameters of (n = 3). LVIDd: left ventricular internal dimension at end-diastole; LVIDs: left ventricular internal dimension at end-systole; LVPWs: left ventricular posterior wall thickness at end-systole; LVPWd: left ventricular posterior wall thickness at end-diastole; SV: stroke volume; ESV: end-systole volume; EDV: end-diastole volume; CO: cardiac output; EF: ejection fraction; FS: fractional shortening; *: p < 0.05; #: p < 0.005; ##: p < 0.001.

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Transcriptomic high-throughput sequencing revealed changes in expression profiles

Using the Illumina HiSeq X-ten platform, 676,993,654 raw reads were obtained, fo which 641,870,791 mapped to the rat Rnor 6.0 reference genome, with an average mapping rate of 97.13%. The raw and processed data have been submitted in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information’s Gene, with accession number SRP300824.

In each sample, transcripts were distributed across all chromosomes in a nearly uniform manner (Fig. 2a). Pearson’s correlation coefficient of global transcript expression levels were calculated and visualized in heatmaps, which revealed marked differences between bovine milk exosomes-treated and PBS-treated rats, but only slight differences within each group (Fig. 2b, c). In total,  22,601 mRNAs and 10,068 lncRNAs were detected. Differential expression analysis, with thresholds of P < 0.05 and absolute log2fold change |log2FC|> 1, identified 116 differentially expressed mRNAs (DEMs; 62 upregulated and 54 downregulated) and 141 differentially expressed lncRNAs (DELs; 72 upregulated and 69 downregulated; Fig. 2d). The distributions of DEMs and DELs were visualized using volcano plots (Fig. 2e, f). Hierarchical clustering further distinguished expression patterns betweenthe exosomes- and PBS-treated groups (Fig. 2g, h). Detailed information for the top 20 upregulated and top 20 downregulated DEMs and DELs is provided in Tables 1 and 2.Collectively, these findings demonstrated that orally administered bovine milk exosomes significantly altered lncRNA and mRNA expression patterns in the left ventricle of ISO-induced cardiac fibrosis rats.

Fig. 2
figure 2

Bovine milk exosomes altered mRNA and lncRNA expression profile in ISO-induced cardiac fibrosis rats. (a) Circos plot showing genome-wide transcript distribution across all chromosomes. From inside to outside: PBS1, PBS2, PBS3, Exosomes1, Exosomes2, Exosomes3. (b, c) Heatmap of inter-sample correlation base on Pearson’s correlation coefficients. High intra-group correlation indicates consistency, while low intergroup correlation indicates divergence. (d) Summary of overall and differently expressedtranscripts (e, f) Volcano plot of DEMs and DELs. Vertical lines represent twofold change cufoffs. The horizontal line indicates P = 0.05; red dots denote upregulated genes and blue dots denote downregulated genes. (g, h) Hierarchical clustering of DEMs and DELs. The color scale log10FPKM, with green indicating downregulation and red indicating upregulation.

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Table 1 Detailed informationon the top 20 upregulated and top 20 down-regulated mRNAs.
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Table 2 Detailed information on the top 20 upregulated and top 20 downregulated lncRNAs.
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Validation of DELs and DEMs expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR)

To confirm the reliability of sequencing results, 10 DEMs and 10 DELs were selected for RT-qPCR analysis based on biological relevance, expression level, and potential roles in pathways associated with cardiac fibrosis. All transcripts were successfully amplified, and melting curves analysis showed single peaks, indicating primer specificity. As shown in Fig. 3, the RT-qPCR results were consistent with the RNA sequencing data. Among the DEMs Ciart, Cd151, Per2, Per3, H3f3c, Dbp,Tnc weresignificantly upregulated, while the remain genes were downregulated. Similarly, six DELs (XR 001841620.1, TCONS 00025336, TCONS 00002367, TCONS 00027989, TCONS 00029872, TCONS 00036358) were significantly upregulated, while the other four were downregulated (Fig. 3).

Fig. 3
figure 3

RT-qPCR validation of selected DEMs and DELs in cardiac tissue, confirming concordance with RNA sequencing. *: P < 0.05; **: P < 0.01; #: P < 0.005; ##: P < 0.001.

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Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of DEMs and DELs

GO enrichment analysis revealed that DEMs were primarily associated with biological process (BP): chromosome segregation, response to light stimulus, circadian regulation of gene expression and cellular response to calcium ions; cellular component (CC): ECM, proteinaceous extracellular matrix, synapse and endoplasmic reticulum; and molecular function (MF): pyridoxal phosphate binding, voltage-gated potassium channel activity, protein heterodimerization activity and sequence-specific RNA polymerase II transcription factor activity (Fig. 4a). Potential target genes of DELs were enriched in BP: regulation of complement activation, complement activation, the lectin pathway, complement activation, ubiquitin-dependent protein catabolic process and apoptotic mitochondrial changes; CC: endomembrane system, microtubule organizing center, cytoskeleton and nuclear envelope; and MF: peptidase activity, serine-type endopeptidase activity, scaffold protein binding, and calcium—dependent protein binding (Fig. 4b).

Fig. 4
figure 4

GO and KEGG enrichment analysis for DEMs and DELs. (a, b) GO enrichment of DEMs and DELs showing the top 30 terms across biological process (green), cellular component (blue) and molecular function (red). (c, d) KEGG enrichment analysis for DEMs and DEL showing the top 30 pathways. (e, f) Enrichment scatteplots of DEMs and DEL in KEGG pathways.

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Similarly, KEGG analysis showed that, after filtering out non-relevant pathways, DEMs were primarily enriched in neomycin, kanamycin and gentamicin biosynthesis, taurine and hypotaurine metabolism, fatty acid biosynthesis (Fig. 4c, e). In contrast, the potential target genes of DELs were mainly enriched in alpha-Linolenic acid metabolism, galactose metabolism, staphylococcus aureus infection (Fig. 4d, f). The overlap between the GO enrichment and KEGG analysis of DEMs and DELs suggested potential crosstalk between these two transctipt classes in ISO-induced cardiac fibrosis treat with bovine milk exosomes.

Protein–protein interaction (PPI) network Meanwhile,PPI network of DEMs and their functional enrichment analysis were shown in Fig. 5, illustrating potential interactions and biologcal roles of these genes.

Fig. 5
figure 5

Construction of the protein–protein interaction network of DEMs.

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Co-expression analysis of DELs and DEMs

As illustrat described in Material and methods, a co-expression network built using all DELs and all DEMs was too large to visualize, even with stringent criteria (correlation coefficient ≥ 0.8, P < 0.05). Therefore, a refined co-expression network was constructed based on DELs and DEMs which was atheir potential target DEMs (Fig. S1). Most of DELs and their trans-regulated DEMs were highly interconnected, with XR 593247.1, XR 001836784.1, XR 001836203.1, TCONS 00011576, TCONS 00025336, and TCONS_00022095 serving as hub lncRNAs, and Fos, Gypa Myom3, Rhox5, Slc17a9, Tmem171, and Tonsl as hub mRNAs (Fig. 6).

Fig. 6
figure 6

Co-expression network of DELs and their potential target mRNAs. (a) Circos plot showing chromosomal distribution of DEMs and DELs. The outer circle represents autosomes of the rat;  the second and third circles show DEMs, with red indicating upregulation and green indicating downregulation; column height represents the number of differentially expressed genes in that interval. The fourth and fifth circles show DELs with the same color coding. The inner circle indicates the top 500 co-expressing DEM–DEL pairs. (b) Co-expression network of DEL regulating mRNA in cis. (c) Co-expression network of DEL regulating mRNA in trans (red circles: DELs, green triangles: DEMs. (d) Transcription factor-target gene analyses network (red circle: DELs, green triangle: DEMs, yellow square: key transcription factors).

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Competing endogenous RNA (ceRNA) network of DELs, miRNAs and DEMs

Because lncRNA an act by sponging miRNA, and thereby regulate mRNA translation, a ceRNA network was constructed based on sequence complementarity, maximum binding free energy, and expression consistency. The analysis identified several core miRNAs, including miR-136-5p, miR-539-5p, miR-144-3p, miR-124-3p, miR-203b-3p, which may play central roles in DEL-mediated ceRNA regulation mechanisms (Fig. S2).

Discussion

Using high-throughput transcriptome sequencing, we identified significant alterations in lncRNA and mRNA expression in cardiac fibrosis rats treated with orally administration of bovine milk exosomes compared with PBS controls. Bioinformatic investigations revealed that these differentially expressed genes are involved in multiple biological processes and pathways,with DELs potentially regulating DEMs both directly and indirectly.

Previous studies on cardiac fibrosis have mainly focused on functional gene changes, providing extensive into disease pathogenesis. In contrast, our study emphasizes the therapeutic impact of exosomes, aiming to elucidate the involved pathways and gene regulatory mechanisms involved8,13. Our earlier work demonstrated that intragastrically administered bovine milk exosomes exert protective effects on cardiac fibrosis14. Given the regulatory roles of lncRNAs in gene expression, they may contribute significantly to fibrosis progression.

RNA sequencing enable comprehensive assessment of gene expression changes, simultaneous capturing mRNA, lncRNA, and circRNA profiles. Although circRNAs detection rates were relatively low, a functional analysis of DEMs and DELs highlighted involvement in circadian rhythms, ECM remodeling, endomembrane system, and immune responses.

Circadian rhythms play a critical role in cardiovascular physiology, and disruption of circadian genes has been associated with ventricular remodeling and HF17,18,19. The expression of circadian rhythm–related genes such as aryl hydrocarbon receptor nuclear translocator like (ARNTL), period 1–2 (PER1-PER2), and CIART were significantly upregulated following exosome treatment. Immune-related pathways, including complement activation, were also prominent, suggesting that immune modulation contributes to the therapeutic effects of bovine milk exosomes20,21.

LncRNAs, as emergingregulators of cardiac fibrosis, may influence gene expression through various mechanisms, including scaffolding and miRNA sponging15,16. Co-expression and ceRNA network analyses supported this regulatory potential. However, further experimental validation is required to confirm these findings.

Exosomes, including those derived from bovine milk, carry proteins, lipids, and nucleic acids, regulating diverse biological processes and offering cardioprotective effects22,23,24,25,26. Challenges remain regarding effective dosing, targeted delivery, and potential off-target effects22,23. Nevertheless, bovine milk exosomes are safe for oral administration, making them promising candidates for therapeutic intervention.

Our study reveals that bovine milk exosomes significantly modulate lncRNA and mRNA expression in cardiac fibrosis,  particularly affecting circadian rhythms, immune modulation, and ECM remodeling pathways. Limitations include the absence of in vivo functional assays, limited mechanistic validation, and challenges in quantifying exosome uptake in cardiac tissue. Future studies should employ precise tracking techniques, lineage tracing, and explore RNA markers in biofluid-derived exosomes as potential non-invasive diagnostics, as well as their translational potential in human cardiac fibrosis.

Materials and methods

Cardiac fibrosis model establishment and animal treatment

All animal experimental methods were approved by the Committee on Ethics at Xiangya Hospital, Central South University (No. 202103510), and conducted in accordance with the standards of the International Association for the Study of Pain (IASP). This study is also reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

Healthy adult male Sprague Dawley rats were obtained from the Central South University experimental animal center in Changsha, China. Rats were housed in clear plastic cages with wood chip bedding under specific pathogen-free conditions, maintained at 22 ± 2 °C, with a 12-h light/dark cycle, as specified in the Guide for the Care and Use of Laboratory Animals27.

The cardiac fibrosis model was established by subcutaneous injection of ISO (10 mg/kg/day) for 14 consecutive days, following the protocol of Hu et al.8. Rats received ISO (10 mg/kg/day) subcutaneously for 14 consecutive days. After fibrosis induction, rats were divided into two groups (n = 6): Exosomes group: received 600 μg bovine milk exosomes (1 μg/μl) orally via an orogastric tube. PBS group:  received an equivalent volume of PBS14. Bovine milk exosomes were obtained from Umibio Co. Ltd. (Shanghai, China), and identified by transmission electron microscopy, nanoparticle tracking analysis, and exosome marker proteins, as previously described14. Both treatments were administered for 7 consecutive days following the establishment of cardiac fibrosis. In vivo untake and dosing effectiveness were also confirmed in earlier work14. Following fibrosis induction, both treatments were administered once daily for 7 consecutive days.

H&E staining and Masson’s trichrome staining

H&E and Masson’s trichrome staining were utilized to detect pathological changes, particularly ECM deposition. Fourteen days after treatment, thoracotomy was performed under ketamine (50 mg/kg) anesthesia. Rats were euthanized by exsanguination under anesthesia, and hearts were harvested, fixed in 4% paraformaldehyde for 24 h, dehydrated, and embedded in paraffin. Cross-sectional of the left ventricle were cut into 10 slices (4 μm thick) for staining and histological evaluation. Slices were dewaxed and stained according to standard protocols28. Cell counts (H&E) and collagen area percentage(Masson) were quantitatively analyzed.

Transthoracic echocardiography

Echocardiography was to assess cardiac structure and function29. Rats were anesthetized intraperitoneally with ketamine (50 mg/kg), and echocardiogram data were collected using the Vivid E7 system (GE Vingmed, Horten, Norway). Measured parameters included LVIDd, LVIDs, LVPWs, and LVPWd. Function indices included stroke volume (SV),  ESV, EDV, CO, EF, and left ventricular FS.

Tissue collection and RNA extraction

Fourteen days post-treatment, rats were deeply anesthetized intraperitoneally with ketamine (50 mg/kg). Hearts were collected by thoracotomy and perfused with heparinized saline to remove blood. Left ventricles were snap-frozen and preserved in liquid nitrogen. Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion, USA) following the manufacturer’s instructions. RNA Integrity Number (RIN) was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and samples with RIN >7 were used for further analysis.

Three left ventricles in each group were selected for RNA sequencing and RT-qPCR to ensure biological replicates.

Construction of complementary DNA (cDNA) libraries and sequencing

The TruSeq® Stranded Total RNA with Ribo-Zero Gold kit (Cat. no. RS-122-2301, Illumina, USA) was used to create strand-specific libraries. Ribosomal RNA was removed from total RNA using the Ribo-Zero rRNA removal kit (Illumina, USA). First-strand cDNA was synthesized using fragmented RNA as the template, with six-base random hexamers, followed by second-strand cDNA synthesis incorporating dUTP for strand marking. Adenylate 3′ ends and ligate adapters were added to the double-stranded cDNA. After PCR amplification and cDNA fragment purification, library insert size and concentration were measured using a Qubit® 2.0 Quantometer (Invitrogen, USA) and an Agilent 2100 bioanalyzer (Agilent Technologies, USA). Libraries were sequenced with PE150 on the Illumina sequencing platform (Illumina, HiSeq X Ten, USA) according to the manufacturer’s protocol. Library construction, sequencing, and data analysis were conducted by OE Biotech Co. Ltd. (Shanghai, China).

Quantification of Gene expression and differential expression analysis

Sequencing raw reads were filtered to obtain high-quality clean reads: ribosomal RNAs were removed using SortMeRNA30, and low-quality bases or reads and adapters were filtered out using Trimmomatic software31. Clean reads were aligned to the rat Rnor 6.0 reference genome using Hisat2 (V.2.0.4)32. Genomic and gene alignment were assessed for all samples. The Fragments Per Kilobase of exon model per Million mapped reads (FPKMs) with reference annotation were assessed using Stringtie (V.1.3.0)33,34. Novel candidate lncRNA transcripts were predicted using Guffcompare (V.0.9.8) software35. Transcripts with coding potential were screened out using CPC36, CNCI37, Pfam38 and PLEK39 to obtain lncRNA predicted sequences. The estimateSizeFactors function of the DESeq (2012) R package40 was selected to normalize the counts, and the p-value and FC values were calculated for the difference comparison using nbinomTest. Differentially expressed mRNAs (DEMs) and differentially expressed lncRNAs (DELs) were identified using edgeR28, with filter criteria set at p-value < 0.05 and |log2FC|> 1.

RT-q PCR

Total RNA was extracted from the left ventricle of cardiac fibrosis rats using the mirVana miRNA Isolation Kit (Ambion, USA). RNA yield was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and RNA integrity was assessed via agarose gel electrophoresis stained with ethidium bromide. Quantification was performed with a two-step process: RT and PCR. Each RT reaction consisted of 0.5 μg RNA, 2 μl of 5 × TransScript All-in-one SuperMix for qPCR, and 0.5 μl of gDNA Remover, in a total volume of 10 μl. RT reactions were conducted in a GeneAmp® PCR System 9700 (Applied Biosystems, USA) for 15 min at 42 °C and 5 s at 85 °C. The RT reaction mix was then diluted × 10 in nuclease-free water and stored at − 20 °C. Real-time PCR was performed using the LightCycler® 480 Ⅱ Real-time PCR Instrument (Roche, Swiss) with a 10 μl PCR reaction mixture including 1 μl of cDNA, 5 μl of 2 × PerfectStartTM Green qPCR SuperMix, 0.2 μl of forward primer, 0.2 μl of reverse primer, and 3.6 μl of nuclease-free water. Reactions were incubated in a 384-well optical plate (Roche, Swiss) at 94 °C for 30 s, followed by 45 cycles of 94 °C for 5 s and 60 °C for 30 s. Each sample was run in triplicate for analysis. A melting curve analysis was performed at the end of the PCR cycles to validate the specific generation of the expected PCR product. Primer sequences were designed in the laboratory and synthesized by TsingKe Biotech based on mRNA sequences obtained from the NCBI database (Table S1). mRNA expression levels were normalized to GAPDH and calculated using the 2−ΔΔCt method41.

Target prediction of DELs

LncRNAs can regulate gene expression in cis and trans manners42. The target prediction of DELs included both cis and trans predictions. Potential trans target coding genes were identified by sequence complementary analysis using the Basic Local Alignment Search Tool (BLAST), with target genes determined based on complementary energy evaluated by RNAplex43; Potential cis-target genes were those transcribed within 10 kb upstream or downstream of the lncRNA genomic location43. DELs and their corresponding target DEMs in cis and trans were then identified.

GO and KEGG enrichment analysis

GO and KEGG enrichment analyses were conducted to evaluate the functions of identified DEMs and DELs and to analyze potential pathways involved in cardiac fibrosis in rats treated with bovine milk exosomes or PBS. GO analysis for DEMs and DEL targets was performed using DAVID Bioinformatics Resources 6.8, with p-values and false discovery rates used to assess analysis reliability44. GO analysis included biological processes, cellular components, and molecular functions. KEGG pathway analysis provided further understanding of the functions and interactions among DEMs and DELs45.

PPI network

The PPI network was constructed using the STRING database and visualized with Cytoscape software 3.9.046. A minimum confidence score of 0.4 was used after filtering unconnected nodes. The confidence score was derived from neighborhoods on the chromosome, gene fusion, phylogenetic co-occurrence, homology, co-expression, experimentally determined interaction, database annotations, and automated text-mining.

Co-expression analysis of DELs and DEMs

The co-expression network of DELs and DEMs was constructed based on correlations between DELs and DEMs. The Pearson correlation coefficient (PCC) for each DEL and DEM pair was calculated. Significant correlation pairs with |PCC|> 0.99 and p < 0.05 were used to build the co-expression network, visualized with Cytoscape software 3.9.046.

Transcription factor (TF) prediction of DELs

The Match-1.0 Public transcription factor prediction tool was used to predict potential TFs for DELs47. Gene-TF regulatory interaction networks were constructed and visualized. TFs binding to the upstream 2000 bp region and the downstream 500 bp region of the DEL starting site were predicted based on the co-expression network.

CeRNA analysis

The ceRNA analysis was performed to predict possible target bindings of lncRNA/mRNA and miRNA. The ceRNA network was constructed using miRanda with a maximum binding free energy of less than − 2048. For visualization, the threshold of |PCC| of relative expression value of DELs and DEMs was set at more than 0.98. The network was constructed and visualized using Cytoscape software 3.9.046.

Statistical analysis

Statistical analysis was performed using SPSS20.0, with all data presented as mean ± standard deviation (SD). The Student’s t-test was used to determine differences between groups, with p < 0.05 considered statistically significant. Graphs were generated with GraphPad Prism v8.0 (GraphPad, USA).

Conclusion

In summary, this study demonstrates that oral administration of bovine milk exosomes induces significant alterations in the lncRNA and mRNA expression profiles of rats with cardiac fibrosis. The differentially expressed genes were mainly enriched in pathways related to circadian rhythms, immune regulation, and angiogenesis, underscoring their potential roles in cardiac remodeling. These findings not only advance understanding of the molecular mechanisms underlying the therapeutic effects of bovine milk exosomes but also highlight potential targets for future translational research in the treatment of cardiac fibrosis.