Cardiomyocyte pyroptosis inhibited by dental pulp-derived mesenchymal stem cells via the miR-19a-3p/IRF-8/MAPK pathway in ischemia–reperfusion

Figure 1. Cardioprotective effects of DP-MSCs in heart I/R. (A) TTC staining of hearts from I/R model mice at different times after I/R (n = 3); (B) Quantification of the infarct volumes in different groups at different times after I/R (n = 3); (C) Masson’s trichrome staining of hearts from I/R model mice at different times after I/R (n = 3), scar bar: 1250 µm; (D) Quantification of the area of myocardial fibrosis in different groups at different times after I/R (n = 3); (E) Systolic function and ventricular wall thickness of hearts from I/R model mice on the 1st week and 4th week after I/R (n = 5); (F) Quantification of systolic function and ventricular wall thickness on the 1st week and 4th week after I/R (n = 5). All the data are presented as the mean ± standard deviation. DP-MSC: Dental pulp-derived mesenchymal stem cell; EF: Ejection fraction; FS: Fractional shortening; I/R: Ischemia–reperfusion; TTC: 2,3,5-Triphenyltetrazolium chloride staining; LVAWd: Left ventricular anterior wall thickness in diastole; LVAWs: Left ventricular anterior wall thickness in systole; LVIDd: Left ventricular internal diameter in diastole; LVIDs: Left ventricular internal diameter in systole. *P <0.001,…

Figure 2. Cardiomyocyte pyroptosis is inhibited by DP-MSC transplantation after I/R injury. (A) Detection and quantification of TUNEL staining of heart tissues from I/R model mice in different groups on the 2nd hour, 1st day, 3rd day 7th day, 14th day, and 28th day after I/R (n = 3), scar bar: 50 µm; (B) The relative expression level of GSDMD-N, Caspase-1, and Caspase-11 in heart tissue from I/R model mice (n = 3) on the 2nd hour, 1st day, 3rd day, 7th day, 14th day, and 28th day after I/R were analyzed by Western blotting (B) and reverse transcription-quantitative polymerase chain reaction (C). All the data are presented as the mean ± standard deviations. DP-MSC: Dental pulp-derived mesenchymal stem cell; GSDMD-N: Gasdermin D-N terminal; I/R: Ischemia–reperfusion; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick-end labeling. *P <0.001, †P <0.01, ‡P <0.05.

Figure 3. miR-19a-3p inhibited I/R-induced myocardial pyroptosis by the inhibition of the IRF-8/MAPK pathway. (A) Bioinformatics analysis revealed that miR-19a-3p was able to bind to the 3′-untranslated region of IRF-8. Gene Set Enrichment Analysis revealed that high expression of IRF-8 might promote MAPK activation and pyroptosis; The relative expression level of IRF-8, P-P38, P-ERK1/2, and P-JNK in I/R-exposed cardiomyocytes (n = 3) were analyzed by Western blotting (B) and RT-qPCR (C); The relative expression level of GSDMD-N, Caspase-1, and Caspase-11 in I/R-exposed cardiomyocytes (n = 3) were analyzed by Western blotting (D) and RT-qPCR (E); (F) TUNEL staining of the hearts of I/R model mice in the different groups after I/R (n = 3), scar bar: 50 µm. All the data are presented as mean ± standard deviation. ES: Enrichment score; FDR: False discovery rate; GSDMD-N: Gasdermin D-N terminal; I/R: Ischemia–reperfusion; IRF-8: Interferon regulatory factor 8; MAPK: Mitogen-activated protein kinase; RT-qPCR: Reverse transcription-quantitative polymerase chain reaction; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick-end labeling. *P <0.001, †P <0.01.

Figure 4. DP-MSCs downregulated IRF-8/MAPK signaling and inhibited pyroptosis in I/R cardiomyocytes via the overexpression of miR-19a-3p. (A) Relative mRNA expression levels of miR-19a-3p in I/R-exposed cardiomyocytes (n = 3). The relative expression level of IRF-8, P-ERK1/2, P-P38, and P-JNK in I/R-exposed cardiomyocytes (n = 3) were analyzed by Western blotting (B) and RT-qPCR (C); (D) TUNEL staining of I/R cardiomyocytes from different groups after I/R (n = 3), scar bar: 100 µm; The relative expression level of GSDMD-N, Caspase-1, and Caspase-11 in I/R-exposed cardiomyocytes (n = 3) were analyzed by Western blotting (E) and RT-qPCR (F). All the data are presented as the mean ± standard deviations. GSDMD-N: Gasdermin D-N terminal; I/R: Ischemia–reperfusion; IRF-8: Interferon regulatory factor 8; MAPK: Mitogen-activated protein kinase; MSC: Mesenchymal stem cell; NC: Negative control; RT-qPCR: Reverse transcription-quantitative polymerase chain reaction; OE: Overexpression; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick-end labeling. *P <0.001, †P <0.01.