Magnesium Ion-Doped Mesoporous Bioactive Glasses Loaded with Gallic Acid Against Myocardial Ischemia/Reperfusion Injury by Affecting the Biological Functions of Multiple Cells

Figures

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  Scheme 1. Scheme of the preparation and evaluation of the MgNPs/GA.

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  Figure 1. Characterisation of MgNPs at different concentrations. (A) Scanning electron microscope images of MgNPs at different concentrations, scale bar: 50nm. (B) TEM-EDS images at different concentrations, scale bar: 50nm. (C–E) N2 adsorption-desorption analysis and pore size distributions of 2%MgNPs and 6%MgNPs. (F) FTIR spectra of 6%MgNPs. (G) XPS survey scan and peak fitting of MgNPs at different concentrations. (H) Particle size distribution. (I) Gallic acid release profile of 2%MgNPs and 6%MgNPs in vitro. Results are presented as mean ± SD (N = 3).

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  Figure 2. Biocompatibility and haemocompatibility of MgNPs/GA. (A) Cell viability at 24h and 72h after treatment of HL-1 and HUVECs in different samples. (B) Live-dead staining of samples from each group after treatment with HL-1, scale bar:40µm. (C) CLSM images of the morphology of HL-1 after 24 h treatment with each group, scale bar: 50µm. (D) fluorescence microscope images of the JC-1 staining after 24 h treatment with each group, scale bar: 50µm. (E) Uptake of MgNPs by HL-1, scale bar: 20µm. (F) Bio-Scanning Electron Microscope Images of HL-1 after 24 h treatment with MgNPs. (G) Thromboresistance test of each group, Results are presented as mean ± SD (N = 3), NS = not significant (P > 0.05), *P < 0.05, **P < 0.01.

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  Figure 3. Association of MgNPs/GA Treatment with Inflammatory Signaling Pathways. (A) Displays the differential gene expression (log2 FoldChange) and q-values (-log10 adjusted P-values) from DESeq2 differential analysis between the I/R group (N = 3) and the MgNPs/GA group (N = 3). Each point represents a gene, with significantly differentially expressed genes (q < 0.05) color-coded. (B) Heatmap illustrates the expression of inflammation-related genes in I/R mice treated with MgNPs/GA. Inflammation-related genes were obtained from the Molecular Signatures Database (MSigDB) gene sets. Gene set enrichment analysis (GSEA) results show (C) pathway-normalized enrichment scores based on significance levels and (D) corresponding enrichment plots for the inflammation response signaling pathway.

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  Figure 4. In vitro antioxidant effects of MgNPs/GA. (A) Fluorescent probe DCFH-DA staining images of HL-1 after 24 h treatment with each groups, scale bar: 50µm. (B) Detection of intracellular DCFH-DA fluorescent probe by flow cytometry. (C) Percentage of apoptosis detected by flow cytometry. (D) Quantification of DCFH-DA staining with fluorescent probes. (E) Quantification of the mean fluorescence intensity after flow cytometry. (F) Quantification of the percentage of HL-1 apoptosis after flow cytometry detection. (G–I) MDA, SOD and GPx levels were measured by ELISA kits. Results are presented as mean ± SD (N = 3), NS = not significant (P > 0.05), *P < 0.05, **P < 0.01, ****P < 0.001.

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  Figure 5. In vitro assessment of angiogenesis. (A and B) Cell migration assay and quantitative analysis, scale bar: 50µm. (C) Branch point quantitative analysis of tube formation. (D) Mean tube length quantitative analysis of tube formation. (E) Images of tube formation of HUVECs after 12 h treatment with each groups, scale bar: 50µm. Results are presented as mean ± SD (N = 3), **P < 0.01.

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  Figure 6. Anti-inflammatory effects of MgNPs/GA in vitro. (A and B) CD206/iNOS immunofluorescence images and quantitative analysis of fluorescence intensity, scale bar: 50µm. (C) PCR analysis of the expression of M1-related genes TNF-α, IL-8 and M2-related genes ARG1 and CD163. (D–F) Detection of inflammatory factors TNF-α, IL-1β and IL-6. Results are presented as mean ± SD (N = 3), *P < 0.05, **P < 0.01, ****P < 0.001.

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  Figure 7. Immunofluorescence staining of histopathological sections after I/R animal model. (A) Schematic diagram of the establishment of the I/R mouse model and protocol for evaluating therapeutic effects. (B) Detection of reactive oxygen species in the region of injured myocardium 24h after I/R in mice, scale bar: 100µm. (C) Detection of Angiogenesis in Areas of Injured Myocardium 14 Days After I/R in mice, scale bar: 100µm. (D) Quantitative analysis of ROS fluorescent probe staining of histopathologic sections. (E) Quantitative analysis of CD31 immunofluorescence staining of histopathological sections. (F–H) The number of M2 macrophages in the region of injured myocardium was detected and quantitatively analyzed 14 days after I/R in mice, scale bar: 100µm. Results are presented as mean ± SD (N = 6), **P < 0.01, ****P < 0.001.

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  Figure 8. MgNPs/GA Enhances Cardiac Function in I/R Mice. (A) Survival curves for each group of mice over 28 days. (B) Changes in body weight over 28 days in each group of mice. (C and D) TTC staining to assess infarct size in I/R mice. (E–I) Cardiac function indexes were detected by cardiac ultrasound after 28 days in I/R mice. Results are presented as mean ± SD (N = 6), *P < 0.05, **P < 0.01, ****P < 0.001.