Sodium Alginate Hydrogel Infusion of Bone Marrow Mesenchymal Stem Cell‐Derived Extracellular Vesicles and p38α Antagonistic Peptides in Myocardial Infarction Fibrosis Mitigation

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  Figure 1. Influence of hypoxia induction and silencing or overexpression of the Mapk14 gene on CFs. A, RT‐qPCR detection of changes in Mapk14 and Acta2 mRNA expression in CFs after hypoxia induction; B, Western blot detection of p38α, p‐p38α, α‐SMA, and collagen I protein expression in CFs after hypoxia induction; C, ELISA detection of collagen I expression in the cell culture medium; D, RT‐qPCR detection of Mapk14 mRNA expression in CFs after Mapk14 gene silencing or overexpression; E, Western blot detection of p38α and p‐p38α protein expression in CFs after Mapk14 gene silencing or overexpression. Quantitative data in the figure are presented as mean±SD, with each cell experiment repeated 3 times. * indicates P<0.05 compared with the normal or sh‐NC group; # indicates P<0.05 compared with the oe‐NC group; F, CCK‐8 assay to detect the effect of Mapk14 silencing or overexpression on CF viability under hypoxic conditions; G, EDU assay to detect the effect of Mapk14 silencing or overexpression on CF proliferation under hypoxic conditions (scale bar: 25 μm); H, Transwell assay to detect the effect of Mapk14 silencing or overexpression on CF migration (scale bar: 25 μm); I, Annexin V/PI double…

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  Figure 2. Characterization and uptake observation of EVs‐PAP and EVs‐PAP@SA. A, Amino acid sequence of p38α antagonistic peptide; B, Zeta potential analysis of EVs‐PAP nanoparticles; C, Particle size distribution of EVs‐PAP nanoparticles; D, Expression of CD63, CD9, and TGS101 in EVs and EVs‐PAP detected by Western blot; E, Morphology of EVs‐PAP@SA and SA observed by scanning electron microscopy (white arrows indicate EVs‐PAP, scale bar: 500 nm); F, Characterization of SA and EVs‐PAP@SA by Fourier‐transform infrared spectroscopy; G, Drug release experiment of EVs‐PAP@SA; H, Uptake of PKH‐67‐labeled EVs‐PAP@SA in CFs detected by immunofluorescence staining (scale bar: 25 μm), blue represents DAPI nuclear staining, and green represents PKH‐67‐labeled EVs‐PAP@SA. The comparison between the 2 groups was conducted using a Mann–Whitney U test, and multiple groups were analyzed using Kruskal–Wallis test. The bar graphs represent the data as mean±SD, and each cell experiment was repeated 3 times (n=3). EV indicates extracellular vesicles; EVs‐PAP@SA, sodium alginate hydrogel loaded with extracellular vesicles and p38α antagonistic peptides; PAP, p38α antagonistic peptide; PDI, polymer dispersity…

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  Figure 3. Effects of EVs‐PAP@SA on CFs fibrosis. A, The effect of different concentrations of EVs‐PAP@SA on the viability of CFs cells under hypoxic conditions was assessed using the CCK‐8 assay; B, Expression changes of proinflammatory factors TNF‐α and IL‐1β in the culture supernatant of CFs after different treatments for 24 hours measured by ELISA; C, Changes in cell viability of CFs after different treatments for 24 hours measured by the CCK‐8 assay; D, Changes in proliferation capacity of CFs after different treatments for 24 hours measured by the EDU assay (scale bar: 25 μm); E, Changes in migration ability of CFs after different treatments for 24 hours measured by the Transwell assay (scale bar: 50 μm); F, Changes in apoptosis of CFs after different treatments for 24 hours detected by Annexin V/PI double staining flow cytometry. The column chart represents the apoptosis rate of cells in the Q2 and Q3 regions; G, Expression changes of α‐SMA protein in CFs after different treatments for 24 hours detected by immunofluorescence staining. Green represents α‐SMA protein, and blue represents DAPI nuclear staining (scale bar: 25 μm); H, Expression changes of collagen I protein in CFs after…

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  Figure 4. Influence of EVs‐PAP@SA on the cardiac function of MI mice. A, Changes in the baseline RR, PR, QRS intervals, and corrected QT interval (QTc) after tail vein injection of different drugs for 28 days; B, Changes in the echocardiogram of mice in each group after tail vein injection of different drugs for 28 days; C, Changes in the baseline LVEF, LVFS, LVIDd/TL, and LVIDs/TL of mice in each group after tail vein injection of different drugs for 28 days. D, Triphenyl tetrazolium chloride staining to detect MI in mice after tail vein injection of different drugs for 28 days, quantified by the ratio of the white stained area (infarct area) to the total area (scale bar: 1 mm); E, H&E staining to detect myocardial necrosis in mice after 28 days of injection with different drugs (scale bar: 1 mm/100 μm); F, Masson staining to detect interstitial fibrosis deposition in the ventricular muscle of mice from each group after 28 days of injection (myocardial cells appear red, interstitium collagen appears blue‐green), collagen volume fraction represents the ratio of blue staining area to the total atrial area (scale bar: 1 mm); G, Sirius Red staining to detect fibrosis area in mice from each…

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  Figure 5. Based on transcriptome sequencing analysis, possible mechanisms of EVs‐PAP@SA in improving myocardial fibrosis in mice. A, Heat map of differentially expressed genes between the PBS group (4 samples) and the EVs‐PAP@SA group (4 samples) in mouse heart tissue. Orange indicates upregulated genes, and blue indicates downregulated genes; B, Volcano plot of differentially expressed genes between the PBS group (4 samples) and the EVs‐PAP@SA group (4 samples). Orange dots represent upregulated genes, blue dots represent downregulated genes, and black dots represent nondifferentially expressed genes; C, PPI network diagram of Mapk14, with color grades from pink to red indicating increasing degree values of genes; D, KEGG pathway enrichment analysis results of differentially expressed genes; E, GO function enrichment analysis results of differentially expressed genes. BP indicates Biological Process; CC, Cellular Component; EV, extracellular vesicles; EVs‐PAP@SA, sodium alginate hydrogel loaded with extracellular vesicles and p38α antagonistic peptides; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen‐activated protein kinase; MF, Molecular Function; PAP,…

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  Figure 6. Influence of EVs‐PAP@SA on the TGF‐β1/SMAD and TRPC6/NFAT pathways in the fibrosis of CFs. A, RT‐qPCR detection of changes in Mapk14, Tgfb1, Smad2, Smad3, and Acta2 mRNA expression in CFs after treatment with different reagents for 24 hours; B, Western blot detection of changes in p38α, p‐p38α, α‐SMA, and collagen I protein expression in CFs after treatment with different reagents for 24 hours; C, ELISA to detect the expression of collagen I in the cell culture medium; D, Western blot detection of changes in TGF‐β1, SMAD2, p‐SMAD2, SMAD3, and p‐SMAD3 protein expression in CFs after treatment with different reagents for 24 hours; E, RT‐qPCR detection of changes in Srf, Nfat1, and Trpc6 mRNA expression in CFs after treatment with different reagents for 24 hours; F, Western blot detection of changes in SRF, NFAT1, TRPC6, and whether PAP could modulate protein expression in CFs after treatment with different reagents for 24 hours; G, Changes in intracellular Ca2+ influx in CFs after treatment with different reagents for 24 hours; H, Immunofluorescence staining to detect changes in SRF and TRPC6 protein expression in CFs after treatment with different reagents for 24 hours, with green…

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  Figure 7. Influence of EVs‐PAP@SA on the TGF‐β1/SMAD and TRPC6/NFAT signaling pathways in CFs fibrosis. A, EDU assay to detect changes in the proliferative capacity of CFs after treatment with different reagents for 24 hours (scale bar: 25 μm); B, Transwell assay to detect changes in the migration capacity of CFs after treatment with different reagents for 24 hours (scale bar: 50 μm); C, AnnexinV/PI double‐staining flow cytometry to detect changes in apoptosis of CFs after treatment with different reagents for 24 hours, with the column chart representing the percentage of cells in the Q2 and Q3 regions as the apoptotic rate; D, Western blot detection of changes in SMAD2, p‐SMAD2, SMAD3, and p‐SMAD3 protein expression in CFs after treatment with different reagents for 24 hours; E, Western blot detection of changes in SRF, NFAT1, CaN, α‐SMA, and collagen I protein expression in CFs after treatment with different reagents for 24 hours. CFs in the normal group were cultured in a 5% CO2, 37 °C incubator, whereas CFs in the other treatment groups (PBS group, PAP group, EVs@SA group, and EVs‐PAP@SA group) were cultured in a 1% O2, 5% CO2, 37 °C incubator to induce myocardial fibrosis. F, ELISA to…

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  Figure 8. Effects of EVs‐PAP@SA on regulating the TRPC6/NFAT pathway in myocardial fibrosis in mice. A, Immunofluorescent staining showing changes in the expression of α‐SMA and collagen I proteins (green fluorescence represents α‐SMA protein, red fluorescence represents collagen I protein) as well as SRF and TRPC6 proteins (green fluorescence represents SRF protein, red fluorescence represents TRPC6 protein) in the myocardial tissues of mice from different treatment groups after 28 days of injection therapy, with DAPI nuclear staining shown in blue (scale bar: 50 μm). B, Western blot analysis showed changes in the expression of SRF, NFAT1, TRPC6, and proteins in the myocardial tissues of mice from different treatment groups after 28 days of injection therapy. C, ELISA analysis shows changes in the expression of inflammatory factors TNF‐α and IL‐1β in the serum of mice from different treatment groups after 28 days of injection therapy. The normal group of mice underwent thoracotomy without ligation, whereas the other treatment groups (PBS group, PAP group, EVs@SA group, and EVs‐PAP@SA group) were subjected to heart ligation to induce MI. The multiple groups were analyzed using 1‐way ANOVA.…