AdipoRon Alleviates Free Fatty Acid-Induced Myocardial Cell Injury Via Suppressing Nlrp3 Inflammasome Activation

Figures

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  Figure 1. The effects of AdipoRon on PA-induced cell injury in H9c2 myocardial cells. H9c2 myocardial cells were incubated with PA (100μM) in the absence or presence of indicated concentrations of AdipoRon (20 μM) for 18 h. Specifically, in the combination group of the two, it was pretreated with AdipoRon half an hour and then incubated PA until to 18h. Cell viability was determined via MTT assay (A, B or C) and LDH release (D). Photomicrographs from phase-contrast microscopy (E). The values represent means ± SD from three separate experiments. *P<0.05, **P<0.01 vs Ctr treatment (DMSO or 0 group); #P<0.05, ##P<0.01 vs PA treatment. Bar = 200 µm. Abbreviations: Ctr, control; PA, palmitic acid; ADN, AdipoRon.

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  Figure 2. The effects of AdipoRon on PA-induced cell apoptosis in H9c2 myocardial cells. H9c2 myocardial cells were incubated with PA (100 μM) in the absence or presence of indicated concentrations of AdipoRon (20 μM) for 18 h. Specifically, in the combination group of the two, it was pretreated with AdipoRon half an hour and then incubated PA until to 18h. Apoptotic cells were defined by the flow cytometry (A). The values represent means ± SD from four separate experiments (B). **P<0.01, #P<0.05 vs PA treatment. Abbreviations: Ctr, control; PA, palmitic acid; ADN, AdipoRon.

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  Figure 3. The effects of AdipoRon on PA-induced the mitochondrial membrane potential and ROS production in H9c2 cells. After preincubation for 30 min with 20 μM AdipoRon, 100 μM PA was added to the medium for 18 h in H9c2 cells and followed by a 30 min incubation with JC-1 and H2O2-sensitive fluorescent probe DCF-AM (20 μM). Flow cytometry showed that the mitochondrial membrane potential with JC-1 probe reduced from red fluorescence and green fluorescence (A). Fluorescent images show the ROS level in control cells (left), H9c2 cells stimulated with PA (middle), in the presence of PA+ AdipoRon (right) and in the presence of AdipoRon (B). Fluorescence intensity of cells was measured with software. *P<0.05, **P<0.01, #P<0.05 vs PA treatment. Data show the mean ± SEM of 3 independent analyses. Bar = 50 µm. Abbreviations: Ctr, control; PA, palmitic acid; ADN, AdipoRon.

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  Figure 4. The effect of AdipoRon on ameliorating PA-induced expression of Nlrp3 inflammasome. H9c2 myocardial cells were incubated with PA (100 μM) in the absence and presence of indicated concentrations of AdipoRon (20 μM) for 18 h. Western blot analysis for protein levels of p-p65/p65 (A, B), Nlrp3, caspase-1 and ASC (A, C). The supernatant was detected according to manufacturer’s protocol by Elisa assay (D). Data are expressed as the mean ± SD. *P<0.05, #P < 0.05, vs PA group. Abbreviations: Ctr, control; PA, palmitic acid; ADN, AdipoRon.

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  Figure 5. The effect of AdipoRon on ameliorating PA-induced formation of Nlrp3 inflammasome. (A) Representative fluorescent microscopic images showing the colocalization of Nlrp3/caspase-1. (B) Summarized data showing PCC of Nlrp3/caspase-1 and ASC (n= 4). Data are expressed as the mean ± SD. **P < 0.01, #P < 0.05, vs PA group. Bar = 20 µm. Abbreviations: Ctr, control; PA, palmitic acid; ADN, AdipoRon.

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  Figure 6. The effect of AdipoRon on ameliorating PA-induced formation of Nlrp3 inflammasome. (A) Representative fluorescent microscopic images showing the colocalization of Nlrp3/ASC. (B) Summarized data showing PCC of Nlrp3/caspase-1 and ASC (n= 4). Data are expressed as the mean ± SD. **P < 0.01, ##P < 0.01 vs. PA group. Bar = 20 µm. Abbreviations: Ctr, control; PA, palmitic acid; ADN, AdipoRon.

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  Figure 7. Nlrp3 blockade ameliorated PA-induced cell apoptosis and ROS generation in H9c2 cells. H9c2 cells were cultured in 100 μM PA with or without pretreatment of Nlrp3 siRNA. Protein expression was detected with Nlrp3 siRNA incubation in 24 h and 48 h (A). Cell apoptotic rate was detected by flow cytometry (B). ROS was measured by fluorescent staining (C). Data were presented as the mean ± SD from three separate experiments. *P < 0.05 vs Ctr group; **P < 0.01; #P < 0.05 vs PA treatment; &P < 0.05, &&P < 0.01 vs PA +ADN treatment. Bar = 50 µm. Abbreviations: Ctr, control; PA, palmitic acid.

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  Figure 8. Caspase inhibitor ameliorated PA-induced cell apoptosis and ROS generation in H9c2 cells. H9c2 cells were cultured in 100 μM PA with or without pretreatment of pan-caspase inhibitor (z-vad-fmk, 100 μM). A. Cell viability was detected by the MTT assay (A). Cell apoptotic rate was detected by flow cytometry (B). ROS was measured by fluorescent staining (C). Data were presented as the mean ± SD from three separate experiments. *P<0.05, **P < 0.01; #P < 0.05, ##P < 0.01 vs PA treatment; &P < 0.05 vs PA +ADN treatment. Bar = 50 µm. Abbreviations: Ctr, control; PA, palmitic acid.

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  Figure 9. The proposed signal pathway with AdipoRon in H9c2 cells.