Dexmedetomidine attenuates myocardial ischemia-reperfusion injury in vitro by inhibiting NLRP3 Inflammasome activation

Figures

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  Fig. 1. Experimental protocol and four time points for detecting the beating rate of cardiomyocytes (CMs). a Experimental protocols. Cardiac fibroblasts (CFs), cardiomyocytes (CMs) and cocultured CMs and CFs (CMs + CFs) were randomly assigned to one of five groups: 1) C group, in which the cells were incubated under normal conditions in a CO2 incubator; 2) M group, in which the cells were exposed to hypoxia/reoxygenation as we described above; 3) D group, in which the cells were pretreated with 1 μg/ml dexmedetomidine (DEX) two hours before hypoxia/reoxygenation; 4) 950 group, in which the cells were treated with 1 μM MCC950 during hypoxia/reoxygenation and two hours before it; and 5) D + 950 group, in which the cells were treated with 1 μM MCC950 during DEX preconditioning and hypoxia/reoxygenation,. b Four time points for detecting the myocardial cell beat frequency of cocultured CMs and CFs (CMs + CFs). We detected the beat frequency of CMs at these four time points as indicated by the arrow. 1–2 (group M): 1, normal; 2, after hypoxia/reoxygenation. 3–4 (group D): 3, normal; 4, after hypoxia/reoxygenation. DEX, dexmedetomidine; CMs, cardiomyocytes; CFs, cardiac fibroblasts; CMs + CFs,…

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  Fig. 2. Comparison of the effect of DEX pretreatment on CM beating rates. a The beating rate of CMs in group M and group D of the cocultured CMs and CFs at normal times and after hypoxia/reoxygenation (H/R). b After H/R, the rate of decline in the beating rate of CMs pretreated with DEX was significantly lower than that of the other groups of cells. Decrease rate of beating frequency = (beating rate at the normal time - beating rate after H/R)/ beating rate at the normal time. Group M: cocultured CMs and CFs were subjected to H/R as described above. Group D: cocultured CMs and CFs were treated with 1 μg/ml DEX two hours before H/R. bpm, beats per minute. *P < 0.05 vs. the M group; data are expressed as the means ± SD (n = 5) and analyzed by One-way ANOVA analysis of variance

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  Fig. 3. Effects of DEX on cell viability reduced by H/R injury. Cell viability was detected by the CCK-8 assay. *P < 0.05 vs. the C group; #P < 0.05 vs. the M group; data are expressed as the means ± SD (n = 5) and analyzed by One-way ANOVA analysis of variance. CMs, cardiomyocytes; CFs, cardiac fibroblasts; CMs + CFs, cocultured CMs and CFs

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  Fig. 4. Localization and expression of NLRP3 in cocultured CFs and CMs. The protein expression of NLRP3, sarcomeric alpha actin and vimentin was observed in the C, M, and D groups (magnification, × 400) using a laser scanning confocal microscope. Sarcomeric alpha actin is a marker of CMs. Vimentin is a marker of CFs. Blue fluorescence indicates nuclear staining, green fluorescence indicates NLRP3 staining, purple fluorescence indicates sarcomeric alpha actinin staining (white arrow), and red fluorescence indicates vimentin staining (yellow arrow). CMs, cardiomyocytes; CFs, Cardiac fibroblasts

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  Fig. 5. Effects of DEX on inflammation and the NLRP3 inflammasome in H/R injury. a The levels of TNF-α were determined in CF supernatant by ELISA. b The levels of IL-18 were determined in CF supernatant by ELISA. c The mRNA levels of IL-1β, IL-18, TNF-α, NLRP3, Caspase1, and ASC in CFs treated with DEX, MCC950 and DEX + MCC950 examined by RT-qPCR. d The densitometric value of NLRP3, Caspase1, cleaved caspase-1, ASC and IL-1β protein bands in CFs treated with DEX, MCC950 and DEX + MCC950 examined by western blot assay. Full-length blots are presented in Supplementary Fig. 1–6). e The protein levels of NLRP3, Caspase1, cleaved caspase-1, ASC and IL-1β in CFs treated with DEX, MCC950 and DEX + MCC950 examined by western blot analysis. *P < 0.05 vs. the C group; #P < 0.05 vs. the M group; data are expressed as the means ± SD (n = 5) and analyzed by One-way ANOVA analysis of variance

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  Fig. 6. Localization and expression of NLRP3, cleaved caspase-1 and ASC in CFs. The protein expression of NLRP3, cleaved caspase1 and ASC was observed in the C, M, D, 950, and D + 950 groups (magnification, × 400). Blue fluorescence indicates nuclear staining, and green fluorescence indicates NLRP3, cleaved caspase1 or ASC staining

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  Fig. 7. Effects of DEX on cell apoptosis induced by H/R injury. The protein levels of Bcl2 and Bax in CFs, CMs, and CMs + CFs were determined by western blot assay. Full-length blots are presented in Supplementary Fig. 7–15. *P < 0.05 vs. the C group; #P < 0.05 vs. the M group; data are expressed as the means ± SD (n = 5) and analyzed by One-way ANOVA analysis of variance. CM, cardiomyocytes; CF, cardiac fibroblasts; CMs + CFs, cocultured CMs and CFs