Mesenchymal stem cell-derived exosomal microRNA-182-5p alleviates myocardial ischemia/reperfusion injury by targeting GSDMD in mice

Figures

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  Fig. 1. I/R induces myocardial cell injury and pyroptosis in the mouse myocardial tissues. A Representative images of NLRP3 protein bands in myocardial tissues of mice; B Western blot analysis of NLRP3 protein in myocardial tissues of mice, normalized to GAPDH; C The concentration of IL-1β and IL-18 in myocardial tissue homogenate supernatant of mice determined by ELISA; D LDH release in myocardial tissues of mice detected by LDH kit. E ROS content in myocardial tissues of mice evaluated by DCFH-DA; F The cell viability in myocardial tissues of mice measured by Calcein-AM/PI double staining; G The TUNEL-positive apoptotic cells (red) in myocardial tissues of mice; H The cell survival rate in myocardial tissues of mice measured by trypan blue staining; I Representative images of ASC, pro-caspase-1, and caspase-1 protein bands in myocardial tissues of mice; J Western blot analysis of ASC, pro-caspase-1, and caspase-1 proteins in myocardial tissues of mice, normalized to GAPDH; N = 10 for mice in each group. Measurement data were expressed as mean ± standard deviation. Data comparison between two groups was conducted by unpaired t-test. *P < 0.05 vs. sham-operated mice.

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  Fig. 2. GSDMD is robustly expressed in myocardial tissues of I/R mice. A mRNA expression of GSDMD in myocardial tissues of mice with I/R detected by RT-qPCR, normalized to GAPDH; B Representative images of GSDMD and GSDMD N protein bands in myocardial tissues of mice with I/R. C Western blot analysis of GSDMD protein and GSDMD N in myocardial tissues of mice with I/R, normalized to GAPDH; D Immunohistochemistry staining of GSDMD protein in myocardial tissues of mice with I/R. N = 10 for mice in each group. Measurement data were expressed as mean ± standard deviation. Data comparison between two groups was conducted by unpaired t-test. *p < 0.05 vs. sham-operated mice.

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  Fig. 3. GSDMD induces H/R-induced myocardial cell pyroptosis and injury. A The relative mRNA expression of GSDMD, NLRP3, IL-1β, and IL-18 in H/R-exposed myocardial cells detected by RT-qPCR; B Western blot analysis of GSDMD, NLRP3, IL-1β, IL-18, and GSDMD N proteins in H/R-exposed myocardial cells, normalized to GAPDH; C The viability of H/R-exposed myocardial cells assessed by Calcein-AM/PI double staining. D The H/R-exposed myocardial cell survival rate measured by trypan blue staining; E LDH release in H/R-exposed myocardial cells detected by LDH kit; F ROS production in H/R-exposed myocardial cells stained with DCFH-DA; G Western blot analysis of ASC, pro-caspase-1, and caspase-1 proteins as well as a cleaved caspase-1/pro-caspase-1 ratio in H/R-exposed myocardial cells, normalized to GAPDH. Measurement data were expressed as mean ± standard deviation. The comparison among multiple groups was conducted with one-way ANOVA, followed by Tukey’s post hoc test. Each cell experiment was repeated three times independently. *P < 0.05 vs. untreated myocardial cells, #P < 0.05 vs. H/R-exposed myocardial cells, &P < 0.05 vs. H/R-exposed myocardial cells transfected with oe-NC, @ P < 0.05 vs.…

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  Fig. 4. miR-182-5p targets GSDMD. A miR-182-5p putative binding sites in GSDMD 3′UTR predicted by starBase; B The relative expression of miR-182-5p in I/R-injured myocardial tissues detected by RT-qPCR, normalized to U6; C The correlation of miR-182-5p expression with GSDMD expression in I/R-injured myocardial tissues; D The miR-182-5p expression in myocardial cells determined by RT-qPCR; E The binding of miR-182-5p to GSDMD in HEK-293T cells confirmed by dual-luciferase reporter assay; F The miR-182-5p expression and relative mRNA expression of GSDMD in myocardial cells detected by RT-qPCR; G Representative images of GSDMD protein bands; H Western blot analysis of GSDMD protein in myocardial cells, normalized to GAPDH. N = 10 for mice in each group. Measurement data were expressed as mean ± standard deviation. The comparison between two groups was conducted by unpaired t-test. Each cell experiment was repeated three times independently. *P < 0.05 vs. sham-operated mice or HEK-293T cells or myocardial cells transfected with mimic-NC, #P < 0.05 vs. myocardial cells transfected with inhibitor-NC.

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  Fig. 5. Exosomal miR-182-5p from MSCs reduces GSDMD expression in myocardial cells. A The uptake of exosomes by myocardial cells observed under an inverted microscope (× 200); B The relative expression of miR-182-5p in MSCs and MSC-derived exosomes determined by RT-qPCR, normalized to U6; *P < 0.05 vs. the MSCs transfected with mimic-NC or their derived exosomes; #P < 0.05 vs. the MSCs transfected with inhibitor-NC or their derived exosomes. C miR-182-5p expression and GSDMD mRNA level in myocardial cells co-cultured with miR-182-5p mimic/inhibitor-transfected MSCs detected by RT-qPCR, normalized to U6 and GAPDH, respectively; D Western blot analysis of GSDMD protein in myocardial cells co-cultured with miR-182-5p mimic/inhibitor-transfected MSCs, normalized to GAPDH. Measurement data were expressed as mean ± standard deviation. The comparison between two groups was performed using an unpaired t-test and comparison among multiple groups was assessed with one-way ANOVA, followed by Tukey’s post hoc test. Each cell experiment was repeated three times independently. *P < 0.05 vs. the blank myocardial cells, #P < 0.05 vs. myocardial cells co-cultured with non-transfected MSCs, &P < 0.05 vs.…

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  Fig. 6. Exosomal miR-182-5p from MSCs inhibits inflammation and pyroptosis of H/R-exposed myocardial cells via inhibition of GSDMD. H/R-exposed myocardial cells were co-cultured with MSC-derived exosomes or not. GSDMD expression was silenced in H/R-exposed myocardial cells while miR-182-5p expression was altered in MSC-derived exosomes using mimics or inhibitors before co-culture. A miR-182-5p expression and GSDMD mRNA level in H/R-exposed myocardial cells detected by RT-qPCR, normalized to U6 and GAPDH, respectively; B Western blot analysis of GSDMD protein and GSDMD N in H/R-exposed myocardial cells, normalized to GAPDH; C The levels of IL-1β and IL-18 in the supernatant of H/R-exposed myocardial cells determined by ELISA; D The viability of H/R-exposed myocardial cells assessed by Calcein-AM/PI double staining; E The survival rate of H/R-exposed myocardial cells measured by trypan blue staining; F LDH release in H/R-exposed myocardial cells detected by LDH kit; G ROS production in H/R-exposed myocardial cells examined by DCFH-DA; H Western blot analysis of ASC, pro-caspase-1, and caspase-1 proteins as well as the cleaved caspase-1/pro-caspase-1 ratio in H/R-exposed myocardial cells,…

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  Fig. 7. Exosomal miR-182-5p from MSCs diminishes I/R-induced myocardial cell pyroptosis and delays I/R injury in vivo. I/R-treated mice were injected with exo-miR-182-5p mimic (exosomes derived from miR-182-5p mimic-transfected MSCs) or exo-mimic-NC (exosomes derived from mimic-NC-transfected MSCs). A The echocardiography of cardiac functions of I/R mice; B MI size diagram calculated by the cross-sectional imaging; C Determination of LVEF of I/R mice; D Determination of LVFS of I/R mice; E Determination of LVESD of I/R mice; F Determination of LVEDD of I/R mice; G The expression of miR-182-5p and GSDMD mRNA level in myocardial tissues of I/R mice detected by RT-qPCR, normalized to U6 and GAPDH respectively; H The levels of IL-1β and IL-18 in myocardial tissue homogenate supernatant of I/R mice determined by ELISA; I LDH release in myocardial tissues of I/R mice detected by LDH kit; J ROS production in myocardial tissues of I/R mice measured by DCFH-DA; K The cell viability in myocardial tissues measured by Calcein-AM/PI double staining; L The cell survival rate in myocardial tissues of I/R mice measured by trypan blue staining; M Representative images of ASC, pro-caspase-1, and caspase-1…

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  Fig. 8. A schematic diagram showing potential molecular mechanisms of exosomal miR-182-5p targeting GSDMD in myocardial I/R injury. miR-182-5p shuttled by exosomes from MSCs can be internalized by myocardial cells, and then targets GSDMD and reduces its expression, suppressing myocardial cell injury and pyroptosis, and delaying the myocardial I/R injury in mice.