Journal: Journal of Innate Immunity

Article Title: Potential Role of Aerobic Exercise in Attenuating Diabetic Cardiomyopathy via Modulation of P2X4-Mediated NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1159/000548603

Figure Lengend Snippet: Exercise training suppressed PANX1/P2X4/NLRP3 signaling and reduced myocardial oxidative stress. a A PPI network of NLRP3-related protein and purinergic signaling-related protein. b qRT-PCR analysis of PANX1 mRNA levels among different groups, n = 9. c qRT-PCR analysis of relative mRNA expression of P2X1–7 to GAPDH (housekeeping) in the heart of normal mice, n = 4. d qRT-PCR analysis of P2X1, P2X4, P2X5, P2X7 mRNA levels among different groups, n = 6–9. e Representative WB images of PANX1, P2X4, P2X7, TXNIP, NLRP3 protein, n = 9. f , g qRT-PCR analysis of TXNIP and NFE2L2 mRNA levels among different groups, n = 6–9. h The MDA levels among different groups, n = 4. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM. See also online supplementary Figure S1. SED, sedentary; EX, exercise; DM, diabetes mellitus; qRT-PCR, quantitative real-time-PCR; PPI, protein-protein interaction.

Article Snippet: The membranes were then blocked in QuickBlockTM Blocking Buffer for Western Blot (Beyotime, P0252, China) for 2 h at room temperature and then incubated with primary antibodies directed against PANX1 (12595-1-AP, Proteintech, China), IL-1β (sc-12742, Santa Cruz, USA), P2X7 (A10511, ABclonal, China), caspase-1 (A0964, ABclonal, China), GSDMD (BS-14287R, Bioss, China), P2X4 (66416-1-Ig, Proteintech, China), NLRP3 (DF7438, affinity, China), TXNIP (sc-166234, Santa, USA), NF-κb p65 (WL01980, Wanleibio, China), p-NF-κb (WL02169, Wanleibio, China), β-tubulin (AF7011, affinity, China), HSP90 (ab13492, Abcam, USA), and GAPDH (sc-47724, Santa, USA) and horseradish peroxidase-conjugated secondary antibodies (115-035-003, 111-035-003, Jackson Lab, USA).

Techniques: Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Innate Immunity

Article Title: Potential Role of Aerobic Exercise in Attenuating Diabetic Cardiomyopathy via Modulation of P2X4-Mediated NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1159/000548603

Figure Lengend Snippet: Hyperlipid-induced P2X4 upregulation and NLRP3 inflammasome activation. a The viability of H9C2 cells treated with high Glu/high PA; experiments were performed in 4 biological replicates with 3 technical replicates. b LDH content in the supernatant of H9C2 cells treated with high Glu/high PA. Experiments were performed in 4 biological replicates with 3 technical replicates. c Representative WB images of PANX1, NLRP3, P2X7, P2X4 protein, n = 4–6. d Caspase-1 activity of H9C2 cells treated with high Glu/high PA; all experiments were performed in 4 biological replicates. e The viability of H9C2 cells treated with high Glu/high PA, MCC950, KCl, and CaCl 2 ; experiments were performed in 3 biological replicates with 3 technical replicates. f The viability of H9C2 cells treated with KCl; experiments were performed in 3 biological replicates with 3 technical replicates. g Representative images of H9C2 stimulated with high Glu/high PA and stained with ROS dye. White arrowheads indicate membrane rupture and ballooning (pyroptosis), while black arrowheads indicate cell shrinkage and apoptotic body formation (apoptosis). Scale bar, 100 μm, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the CON group (5.5 m m glucose) or comparison as indicated. Data are represented as mean ± SEM. See also online supplementary Figure S2. Glu, glucose; PA, palmitic acid; LDH, lactate dehydrogenase; blank, no treatment; Con, mannitol and BSA treatment as control; CON, normal diet mice; HFD, high-fat diet mice; HE, HFD and exercise mice.

Article Snippet: The membranes were then blocked in QuickBlockTM Blocking Buffer for Western Blot (Beyotime, P0252, China) for 2 h at room temperature and then incubated with primary antibodies directed against PANX1 (12595-1-AP, Proteintech, China), IL-1β (sc-12742, Santa Cruz, USA), P2X7 (A10511, ABclonal, China), caspase-1 (A0964, ABclonal, China), GSDMD (BS-14287R, Bioss, China), P2X4 (66416-1-Ig, Proteintech, China), NLRP3 (DF7438, affinity, China), TXNIP (sc-166234, Santa, USA), NF-κb p65 (WL01980, Wanleibio, China), p-NF-κb (WL02169, Wanleibio, China), β-tubulin (AF7011, affinity, China), HSP90 (ab13492, Abcam, USA), and GAPDH (sc-47724, Santa, USA) and horseradish peroxidase-conjugated secondary antibodies (115-035-003, 111-035-003, Jackson Lab, USA).

Techniques: Activation Assay, Activity Assay, Staining, Membrane, Comparison, Control

Journal: Journal of Innate Immunity

Article Title: Potential Role of Aerobic Exercise in Attenuating Diabetic Cardiomyopathy via Modulation of P2X4-Mediated NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1159/000548603

Figure Lengend Snippet: P2X4 and P2X7 were upregulated in the hearts of HFD mice and restrained by exercise intervention. a Representative M-mode echocardiograms of the LVs are shown. b–f Quantification of corrected LV mass ( b ), LVEF ( c ), LVFS values ( d ), LVIDs ( e ), and LVIDd ( f ) among different groups, n = 5. g Representative WB images of PANX1, P2X7, P2X4, NLRP3, caspase-1 protein, n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM. See also online supplementary Figures S3 and S4. CON, normal diet mice; HFD, high-fat diet mice; HE, HFD and exercise mice.

Article Snippet: The membranes were then blocked in QuickBlockTM Blocking Buffer for Western Blot (Beyotime, P0252, China) for 2 h at room temperature and then incubated with primary antibodies directed against PANX1 (12595-1-AP, Proteintech, China), IL-1β (sc-12742, Santa Cruz, USA), P2X7 (A10511, ABclonal, China), caspase-1 (A0964, ABclonal, China), GSDMD (BS-14287R, Bioss, China), P2X4 (66416-1-Ig, Proteintech, China), NLRP3 (DF7438, affinity, China), TXNIP (sc-166234, Santa, USA), NF-κb p65 (WL01980, Wanleibio, China), p-NF-κb (WL02169, Wanleibio, China), β-tubulin (AF7011, affinity, China), HSP90 (ab13492, Abcam, USA), and GAPDH (sc-47724, Santa, USA) and horseradish peroxidase-conjugated secondary antibodies (115-035-003, 111-035-003, Jackson Lab, USA).

Techniques: