Allicin protects against LPS-induced cardiomyocyte injury by activating Nrf2-HO-1 and inhibiting NLRP3 pathways

Figures

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  Fig. 1. Allicin increases the viability of rat cardiomyocytes treated with LPS. H9c2 cells were treated with LPS (10 µg/mL) for 12, 24, and 36 h. (A) Cell viability was assessed by CCK-8 assay. (B) Cell injury was detected by LDH release assay. H9c2 cells were incubated with Allicin (25, 50, and 100 µM) for 24 h, (C) viability and (D) LDH content were measured. H9c2 cells were pretreated with Allicin (0, 25, 50, and 100 µM) for 2 h, followed by incubation with LPS (10 µg/mL) for a further 24 h. (E) CCK-8 assay and (F) LDH release assay was performed. ELISA assay was carried out to measure the concentrations of IL-1β (G), IL-6 (H), and TNF-α (I) in culture media of H9c2 cells. ANOVA was performed to analyze the data. ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. LPS group

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  Fig. 2. Effect of Allicin on cell apoptosis and oxidative stress in cardiomyocytes. H9c2 cells were pretreated with Allicin (0, 25, 50, and 100 µM) for 2 h, followed by incubation with LPS (10 µg/mL) for a further 24 h. (A) Representative TUNEL and DAPI‑stained images. Magnification, x200. (B) Quantitative analysis of TUNEL staining. (C) Cell apoptosis was determined by measurement of caspase-3 activity. The MDA level (D) and SOD activity (E) were measured by respective commercial kits. ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. LPS group

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  Fig. 3. Allicin activated Nrf2-HO-1 signaling pathway in LPS-induced cardiomyocytes. H9c2 cells were pretreated with 50 µM Allicin, followed by incubation with LPS (10 µg/mL) for 24 h. RT-qPCR showed that Allicin did not change mRNA expression of Nrf2 (A) but enhanced mRNA expression of HO-1 (B) in H9c2 cells with LPS. (C) Representative western blot images investigating Nrf2 and HO-1. Allicin did not change Nrf2 protein (D) but enhanced HO-1 protein (E). (F) Representative western blot images investigating nuclear Nrf2 protein. (G) Quantitative results of western blot analysis on nuclear Nrf2. **P < 0.01 vs. control group; ##P < 0.01, ###P < 0.001 vs. LPS group

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  Fig. 4. Allicin suppresses NLRP3 inflammasome activation by LPS in cardiomyocytes. H9c2 cells were pretreated with 50 µM Allicin, followed by incubation with LPS (10 µg/mL) for 24 h. (A) Representative western blot bands investigating NLRP3 pathway proteins. Quantitative results of western blot analysis on expressions of NLRP3 (B), pro-caspase-1 (C), cleaved caspase-1 (p10) protein (D), pro-IL-1β (E), and pro-IL-1β (F). ***P < 0.001 vs. control group; ###P < 0.001 vs. LPS group

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  Fig. 5. Nrf2 mediates the protective effects of Allicin on LPS-induced injury and NLRP3 inhibition. H9c2 cells were transfected with Nrf2 siRNA, and the silencing efficiency was verified by western blot at 48 h after transfection (A, B). (C) Cell viability of H9c2 cells with Nrf2 silencing. (D) Representative western blot images of HO-1 and NLRP3. (E-F) Quantitative results show that Allicin increased HO-1 protein and decreased NLRP3 protein, which was reversed by silencing of Nrf2 (siNrf2). ***P < 0.001 vs. control group; ###P < 0.001 vs. LPS group; $$$P < 0.001 vs. Allicin group