Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

Figures

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  Fig. 1. Bufalin ameliorated cardiac dysfunction, reduced infarct size and attenuated apoptosis during I/R injury. Mice were subjected to 45 min of ischemia and 24 h of reperfusion with or without bufalin treatment. Echocardiography was performed before and after I/R injury. A-C LVEF and FS measurements were used to evaluate cardiac function (n = 5). D-E TTC/EB staining was performed to determine the area at risk and myocardial infarct size in the different groups (n = 5). F-G Caspase 3, Bcl-2 and Bax expression levels were measured by immunoblotting. All the bands were normalized to the β-actin band and the relative expressions were calculated as fold changes compared to the Sham + PBS group (control group, n = 3). H TUNEL staining analysis was used to detect apoptosis in the myocardium. TUNEL-positive cell ratios were calculated by comparing the TUNEL-positive area with the DAPI-stained area (n = 5). TTC (Triphenyl tetrazolium chloride), EB (Evans Blue). Data are shown as the mean ± SEM. All P values were determined by two-way ANOVA with Tukey’s multiple comparisons test

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  Fig. 2. Bufalin attenuated macrophage pyroptosis and reduced IL-1β release into the myocardium and serum during I/R injury. A-C The RNA sequencing data were extracted from GSE 126772. The mRNA levels of Nlrp3, Pycard (ASC) and Il-1b in macrophages isolated from heart after MI were shown as fold change compared to the Pre group (control group, n = 2,3). D Macrophages were identified and isolated from the heart by flow cytometry. E–F Immunoblotting was performed to assess the expression levels of NLRP3, GSDMD and IL-1β in macrophages after I/R injury with or without bufalin treatment. Each target band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Sham + PBS group (control group, n = 3). G Representative IL-1β expression and localization in murine hearts are shown by IHC analysis in longitudinal sections of whole hearts. For each mouse, 5 representative zones in the left ventricle were chosen randomly to determine the percentage of IL-1β-positive ratio by ImageJ (n = 3). H IL-1β levels in the peripheral blood of mice that were subjected to I/R injury and were treated with or without bufalin were measured by ELISA (n = 6).…

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  Fig. 3. Bufalin attenuated H/R-treated macrophage pyroptosis in vitro. Macrophages were subjected to 1 h of hypoxia and 24 h of reoxygenation treated with PBS or bufalin. A-B Proteins were extracted from cells and immunoblotting analysis was performed. The expressions of NLRP3, GSDMD, and IL-1β were normalized to that of β-actin and then compared with the Sham + PBS group (control group, n = 3). C PI staining was used to examine the plasma membrane integrity of macrophages. Positive cells were counted and normalized to the total number of cells (n = 5). D Cell viability was assessed by measuring the quantity of ATP in macrophages subjected to different treatments (n = 3, 6). E IL-1β levels in the culture medium of macrophages were measured by ELISA (n = 3). Data are presented as the mean ± SEM. P values were calculated by two-way ANOVA with Tukey’s multiple comparisons test (B, C, and E) or Šídák’s multiple comparisons test (D)

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  Fig. 4. Bufalin suppressed macrophage pyroptosis by restoring autophagic flux and reducing ROS. A-B Immunoblotting was used to measure LC3 and P62 protein expression levels in macrophages that were subjected to H/R for different times. All the target bands were normalized to the β-actin band, and then, expression was compared with the H/R 0 h group (control group, n = 3). C p62 mRNA was extracted from cells. The levels of p62 in different groups were measured by qRT‒PCR. The Cq results were normalized to Mus-18 s and compared with the 0 h group (control group, n = 6). D DCFH-DA staining emitted green fluorescence and represented the ROS levels in macrophages that were subjected to H/R for different times (n = 5). E–F Immunoblotting was performed to measure LC3 and p62 protein expression in macrophages that were subjected to H/R with or without Bufalin treatment. Each protein band was normalized to the β-actin band and the expression was calculated by comparison with the Con + PBS group (control group, n = 3). G DCFH-DA staining emitted green fluorescence and represented the ROS levels in macrophages (n = 5). H-I Mice were subjected to 45 min of ischemia and 24 h of reperfusion with or…

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  Fig. 5. Bufalin restored autophagic flux by suppressing P62 pathway in H/R-induced macrophages. Macrophages were transfected with the p62-overexpression plasmid for 24 h and then subjected to H/R with or without bufalin. A-B The protein levels of P62 were measured by immunoblotting. The bands were normalized to the β-actin band, and the relative expressions were calculated as the fold changes compared to the control group (n = 3). C The mRNA levels of p62 were measured by qRT‒PCR. The Cq results were normalized to Mus-18 s and then compared with the control group (n = 3). D-E Proteins were extracted from H/R-treated macrophages that were infected with vector or OE-p62 plasmid in advance and treated with bufalin or PBS. Immunoblotting was performed to measure the expressions of NLRP3, GSDMD and IL-1β. Each band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Con + PBS group (control group, n = 3). F PI staining was used to assess pyroptosis in macrophages. Positive ratios were calculated as the percentage of positive cells compared with total number of cells (n = 5). G IL-1β levels in the culture medium of macrophages were…

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  Fig. 6. Bufalin reduced cardiomyocytes apoptosis by attenuating macrophage pyroptosis. Cardiomyocytes were cultured with or without the culture medium of H/R-induced macrophages. A-B Caspase 3, cleaved-Caspase 3, Bcl-2 and Bax levels in cardiomyocytes were assessed. After immunoblotting, the target band was normalized to the β-actin band, and relative expression was calculated as the fold change compared to the control group (n = 3). C TUNEL staining was used to identify DNA breaks in cardiomyocytes. The TUNEL-positive ratio was normalized to the number of total cells (n = 5). D-E Cardiomyocytes were cultured with the culture medium of H/R-induced macrophages which were treated with or without bufalin. Immunoblotting analysis was performed to measure the Caspase 3, cleaved-Caspase 3, Bcl-2 and Bax levels in cardiomyocytes. Each protein band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Normoxia + PBS group (control group, n = 3). F TUNEL staining was performed to assess the apoptosis of cardiomyocytes. The TUNEL-positive cells were normalized to the total cells (n = 4). Data are presented as the mean ± SEM. P values were…