A Self‐Sustaining Antioxidant Strategy for Effective Treatment of Myocardial Infarction

Figures

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  Figure 1. Characterization of SSSe NPs. A) Schematic illustration of the therapeutic effects of SSSe NPs. B) TEM image of SSSe NPs; scale bar: 100 nm. Insert: enlarged image of SSSe NPs; scale bar: 2 nm. C) Zeta potential distribution intensity of SSSe NPs. D) XPS spectra of C1s and E) Se3d in SSSe NPs. F) O2 ·−, G) ·OH and H) H2O2 scavenging ability of different concentrations of SSSe NPs. I) Release degree of Se in SSSe NPs with or without H2O2.

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  Figure 2. Distribution of SSSe NPs in vivo A) Schematic illustration of the fate of the heart in mice with MI. B) A schematic showing SSSe NP treatment of mice with MI. C) Through leaky myocardial circulation, SSSe NPs entered the infarct tissue. D) FITC‐loaded SSSe NPs. E) Representative ex vivo fluorescence imaging of mouse hearts following tail vein injection of SSSe NP‐FITC into control and MI mice. F) Image of myocardial tissues showing FITC‐loaded SSSe NPs entering the MI site on day 3 after MI injury. G) Image of myocardial tissues showing FITC‐loaded SSSe NPs entering the MI site on day 7 after MI injury. H) Quantification of FITC‐labeled SSSe NPs in different organs on day 1 after MI surgery. I–J) TEM images showing SSSe NPs (yellow arrow) loaded on mitochondria in myocardial tissue. All data are presented as the mean ± S.D. (n = 3). Statistical significance was calculated by an unpaired two‐tailed Student's t‐test. ns P > 0.05, * P < 0.05.

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  Figure 3. SSSe NPs administration improves cardiac dysfunction after MI. A) HE staining showing a smaller infarcted area in the myocardium in the SSSe NP‐treated groups than in the NS/NAC groups on day 14 after acute MI. B) Quantification analysis of the infarct/healthy area. All data are presented as the mean ± S.D. (n = 6). C–E) Measurement of cardiac function by echocardiography, including LVEF, LVFS, and LVPWs on day 14 after MI. F) Representative M‐mode images of echocardiography in each treatment group on day 14 after MI. AW: anterior wall; PW: posterior wall. All data are presented as the mean ± S.D. (n = 9). G) Volcano plots showing differentially expressed genes (DEGs) with logFC > 2.15 and FDR P < 0.05 in MI tissue treated with saline or SSSe NPs. The blue points indicate downregulated DEGs. The red points indicate upregulated DEGs. H) Chord diagram showing overrepresented Gene Ontology (GO) terms for the genes that were differentially expressed during hyperopia induction. In the chord diagrams, overrepresented GO terms are shown on the right, and genes contributing to this overrepresentation are shown on the left. I) Heatmaps showing DEGs based on GO analysis of RNA‐seq data of…

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  Figure 4. SSSe NPs treatment alleviates stress‐induced mitochondrial injury in myocardial cells. A) Immunostaining of mitochondria (red MitoTracker) and nuclei (blue) in H9C2 cells treated with or without FITC‐labeled SSSe NPs (green). B) Measurement of mitochondrial membrane potential (MMP) following the treatment of PBS/SSSe NPs with H2O2 or erastin and JC‐1 staining. The JC‐1 monomer accumulates in mitochondria, indicating that the membrane potential decreased. C‐D) H9C2 cells were stained with MitoSOX Red, which is a mitochondrial superoxide indicator, and the fluorescence intensity of MitoSOX was quantified. The amount of superoxide generated in mitochondria increased during apoptosis (treatment with H2O2) and ferroptosis (treatment with erastin) but decreased in response to SSSe NPs treatment. E) Quantification of MMP of H9C2 cells in each group, and the ratio was decreased during apoptosis (treatment with H2O2) and ferroptosis (treatment with erastin) but increased in response to the administration of SSSe NPs. F) Quantitative real‐time PCR analysis of mitochondrial (mt) DNA: mtDNA content was normalized to nuclear DNA. G) ATP concentrations were measured using an ATP assay kit.…

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  Figure 5. SSSe NPs administration alleviates apoptosis and ferroptosis in the myocardial cells of MI mice. A‐B) The proposed pathway by which SSSe NPs reduces apoptosis and ferroptosis in myocardial cells through GPx4‐related ROS scavenging. C) Measurement of ROS levels in heart tissues 7 days after MI using a DCFH‐DA probe (green). D‐E) Western blot showing GPx4 protein levels in each group 3 days after MI, and the relative protein levels of GPx4 were normalized to β‐actin. MI resulted in a significant decrease in GPx4 protein expression, and this effect was reversed by SSSe NPs. F) Tissue glutathione peroxidase (GSH‐Px) activity was assessed using a GSH‐Px assay kit. (n = 6). (G) Western blot analysis of apoptosis‐related proteins (Bcl‐2, Bax, caspase‐8, caspase‐3, and Cyt‐c) in heart tissues on day 3 after MI. All data are presented as the mean ± S.D. (n = 3). H) Representative immunofluorescent images revealed TUNEL‐positive cells in heart tissues on day 3 after MI. The images in the middle panel are magnified in the same areas as in the upper panel (green: TUNEL, blue: DAPI). (I) Immunocytochemical analysis of redox‐active iron deposits in heart tissues, as measured by a modified…

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  Figure 6. SSSe NPs administration alleviates myocardial inflammation and fibrosis in MI mice. A) Immunofluorescence staining of CD68 (macrophage marker, red) with the mannose receptor (CD206, M2 marker, green) and (CD86, M1 marker, red) in heart tissues. The nuclei were stained with DAPI (blue). (B) RT‒qPCR analysis of the mRNA levels of the proinflammatory cytokines IL‐1β, IL‐6, and TNF‐α, as well as the anti‐inflammatory cytokine IL‐10. (C) Representative images of Masson trichrome staining of infarcted cardiac tissue showing fibrosis on day 14 after MI. D) Quantification of the area of fibrosis in the heart in each treatment group. All data are presented as the mean ± S.D. (n = 6). E–F) Western blot showing the expression of TGF‐β on day 3 after MI. The protein level was significantly decreased after treatment with SSSe NPs compared to saline. All data are presented as the mean ± S.D. (n = 3). Statistical significance was calculated via one‐way ANOVA followed by a post hoc Bonferroni test. Ns P > 0.05, * P < 0.05, **P < 0.01; *** P < 0.001.

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  Figure 7. SSSe NPs administration improves angiogenesis in MI hearts A) The proposed pathway by which SSSe NPs activate angiogenesis in endothelial cells by regulating ROS production. B) Quantification of the numbers of α‐SMA‐positive arterioles. All data are presented as the mean ± S.D. (n = 6) C) Representative immunofluorescent staining of α‐SMA (red) in the cardiac infarct junction region (bottom) on day 14 after MI, and the nuclei were stained with DAPI (blue). D–E) Representative Western blot showing VEGF A, p‐FAK, FAK, p‐ERK1/2, ERK1/2, p‐P38, and P38 expression in HUVECs treated with PBS or SSSe NPs under H2O2 injury conditions. All data are presented as the mean ± S.D. (n = 3). F) Typical images of the tube formation assay. G) Angiogenesis was measured by an in vitro tube formation assay, and the bar graph shows the percentage of branch lengths and points. All data are presented as the mean ± S.D. (n = 3). H) Migration was measured by in vitro scratch experiments; the bar graph shows the percentage of scratch closure. All data are presented as the mean ± S.D. (n = 3). Statistical significance was calculated via one‐way ANOVA followed by a post hoc Bonferroni test. Ns P > 0.05, * P…