Hydroxytyrosol attenuates obesity-induced myocardial fibrosis by inhibiting NLRP3/CYLD/NOX4 mediated oxidative stress

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  Fig. 1. Hydroxytyrosol (HT) improved myocardial injury, oxidative stress and myocardial remodeling in obese mice. A Represents the flow chart of the mouse intervention. B Indicates the trajectory of body weight (n = 6), * compared with the control group (CON), # compared with the high-fat diet (HFD) + HT group. C Reflects the levels of heart weight (n = 6). D Represents the gross appearance of the hearts at 12 and 16 weeks of dietary intervention. E–H Show fasting blood glucose (FBG) (n = 6), serum creatine kinase-myocardial band (CK-MB) (n = 6), serum lactate dehydrogenase (LDH) (n = 6), and cardiac malondialdehyde (MDA) (n = 6) in mice after 16 weeks of dietary intervention, respectively. I Represents Western blot of NADPH oxidase (NOX4) in heart tissue after 16 weeks of dietary intervention (n = 3). J Represents Masson staining and hematoxylin and eosin (H&E) staining of the hearts at 16 weeks of dietary intervention. In Masson staining, the left images show a ×40 field of view, while the right images display a magnified section of a specific area. In H&E staining, the left images depict transverse sections of myocardial tissue, while the right images show longitudinal sections. Taken…

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  Fig. 2. Pyroptosis and CYLD activation were involved in the initiation of high-fat diet (HFD)-induced myocardial fibrosis, which was inhibited by hydroxytyrosol (HT). A Represents the Western blot of pyroptosis-related proteins and CYLD in the hearts of mice (n = 3). B Indicates the serum level of interleukin (IL)-1β and IL-18 (n = 6). C Shows the epresentative images of cardiac mitochondrial ultrastructure. The green arrow indicates normal mitochondria, the red arrow indicates the disruption of mitochondrial membrane integrity, and the yellow arrow indicates shrinkage and rounded mitochondria as well as the scarcity or absence of mitochondrial cristae, taken at ×8.0 k magnification, scale bars: 2.0 μm (n = 3). D Denotes the immunofluorescence co-localization of NLRP3, F4/80 (the macrophage marker) and cTnT (the cardiomyocyte marker) in myocardial tissue at the 16th week of HFD treatment, taken at ×20 magnification, scale bars: 50 μm (n = 3). *P < 0.05, **P < 0.01.

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  Fig. 3. Palmitic acid (PA) stimulation of H9C2 cells for 24 hours led to the activation of rat cardiac fibroblasts (RCFs). A Represents the flowchart of the co-culture of H9C2 cells and RCFs. B–D Show RCFs activation-related proteins detected by immunofluorescence after co-culturing with H9C2 cells stimulated by PA for 12 h and 24 h, taken at ×20 magnification, scale bars: 50 μm (n = 3). E Conveys the flowchart of the intervention of RCFs with the medium from H9C2 cells stimulated by PA. F Reflects the levels of RCFs activation-related proteins after intervention with the medium from H9C2 cells stimulated by PA for 12 h and 24 h, as detected by Western blot (n = 3). *P < 0.05, **P < 0.01.

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  Fig. 4. Hydroxytyrosol (HT) ameliorated palmitic acid (PA)-induced H9C2 cells injury, oxidative stress and rat cardiac fibroblasts (RCFs) activation. A, B Represent the cell viability of H9C2 cells detected by the CCK-8 assay under different concentrations of HT treatment and co-treatment with different concentrations of HT and PA (n = 3). C Shows the intracellular reactive oxygen species (ROS) levels in H9C2 cells after 24 h of treatment with PA or PA + HT, taken at ×20 magnification, scale bars: 50 μm (n = 3). D Denotes Western blot of NADPH oxidase (NOX4) in H9C2 cells (n = 3). E Shows the mitochondrial ROS levels in H9C2 cells after 24 h of treatment with PA or PA + HT, taken at ×10 magnification, scale bars: 100 μm (n = 3). F, G Imply the malondialdehyde (MDA) levels in H9C2 cells (n = 6) and the lactate dehydrogenase (LDH) levels in the medium of H9C2 cells (n = 6), respectively. H–J Indicate the detection of RCFs activation-associated proteins by immunofluorescence after co-culture with H9C2 cells that interferenced with PA and PA + HT for 24 h, taken at ×20 magnification, scale bars: 50 μm (n = 3). K Shows the detection of RCF activation-related proteins by Western blot in RCFs…

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  Fig. 5. HT alleviated PA-induced cardiomyocytes pyroptosis and associated increase in CYLD protein. A Represents the Western blot of pyroptosis-related proteins and CYLD in H9C2 cells treated with PA and PA + HT for 24 h (n = 3). B Denotes the levels of interleukin (IL)-18 (n = 6) and IL-1β (n = 5) in the medium of H9C2 cells treated with PA and PA + HT for 24 h. *P < 0.05, **P < 0.01.

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  Fig. 6. Downregulation of NLRP3 could abate PA-induced pyroptosis, CYLD activation and oxidative stress in cardiomyocytes, as well as cardiac fibroblast activation. A Shows the knockdown efficiency of NLRP3 in H9C2 cells treated with small interfering RNA (siRNA, n = 3). B–G Respectively represent the Western blot of pyroptosis-related proteins and CYLD in H9C2 cells, the levels of interleukin (IL)-1β (n = 5) and IL-18 (n = 6) in the culture medium of H9C2 cells, the level of reactive oxygen species (ROS) in H9C2 cells (×20 magnification, scale bars: 50 μm, n = 3), the level of mitochondrial ROS in H9C2 cells (×10 magnification, scale bars: 100 μm, n = 3), the Western blot of NADPH oxidase (NOX4) in H9C2 cells (n = 3), and the levels of lactate dehydrogenase (LDH) in the medium of H9C2 cells (n = 6) after transfection with si-NC or si-NLRP3 for 24 h before PA intervention. H Denotes the flowchart of treating RCFs with the medium of H9C2 cells after si-RNA and PA intervention. I conveys the Western blot of RCFs activation-related proteins after treating RCFs with the culture medium of H9C2 cells intervened with si-RNA and PA (n = 3). J Shows the flowchart of co-culture of RCFs and H9C2…

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  Fig. 7. Knockdown of CYLD alleviated PA-induced H9C2 cells damage, oxidative stress and RCFs activation. A Conveys the knockdown efficiency of CYLD in H9C2 cells treated with small interfering RNA (siRNA, n = 3). B–F Represent the Western blot of key pyroptosis proteins and CYLD (n = 3), the cellular reactive oxygen species (ROS) detected by DCFH probe (×20 magnification, scale bar: 50 μm, n = 3), the mitochondrial ROS level detected by MitoSOX red probe (×10 magnification, scale bar: 100 μm, n = 3), the Western blot of NADPH oxidase (NOX4) (n = 3), and lactate dehydrogenase (LDH) level in the culture medium (n = 6), respectively, after H9C2 cells were transfected with si-NC or si-CYLD for 24 h followed by PA intervention. G Indicates the Western blot of RCFs activation-related proteins after treating RCFs with the culture medium from H9C2 cells after si-RNA and PA intervention (n = 3). H–J Shows the immunofluorescence of RCFs activation-related proteins after co-culturing RCFs and H9C2 cells intervened with si-RNA and PA intervention, taken at ×20 magnification, scale bars: 50 μm (n = 3). *P < 0.05, **P < 0.01.

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  Fig. 8. Knockdown of NOX4 inhibited RCFs activation indirectly induced by PA-stimulated H9C2 cells injury. A Conveys the knockdown efficiency of NOX4 in H9C2 cells treated with small interfering RNA (siRNA, n = 3). B Indicates the Western blot of RCFs activation-related proteins in RCFs after treated with the culture medium from H9C2 cells intervened with si-RNA and PA (n = 3). C Shows the immunofluorescence of α-SMA in RCFs after co-culturing RCFs and H9C2 cells intervened with si-RNA and PA, taken at ×20 magnification, scale bars: 50 μm (n = 3). *P < 0.05, **P < 0.01.

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  Fig. 9. Hydroxytyrosol (HT) ameliorated myocardial fibrosis in obese mice through restraining oxidative stress induced by the activation of NLRP3/CYLD/NOX4. A Represents the deubiquitination level of NOX4 by CYLD in the cardiac tissue after 16 weeks of dietary intervention, detected by co-immunoprecipitation (Co-IP, n = 3). B Indicates the deubiquitination level of NOX4 by CYLD in H9C2 cells after 24 h of intervention with PA and PA + HT, detected by Co-IP (n = 3). C Implies the Western blot of NOX4 in H9C2 cells after pre-treatment with MG132 (10 μM) before PA and/or HT intervention (n = 3). D Shows the overexpression efficacy of NLRP3 in H9C2 cells treated with a plasmid overexpressing NLRP3 (n = 3). E Implies the Western blot of NLRP3, CYLD, and NOX4 in H9C2 cells after transfection with the plasmid for 24 h before PA and/or HT intervention (n = 3). F Represents the overexpression efficacy of CYLD in H9C2 cells treated with a plasmid overexpressing CYLD (n = 3). G Indicate the Western blot of NOX4 and NLRP3 in H9C2 cells after transfection with the plasmid for 24 h before PA and/or HT intervention (n = 3). H Shows the molecular docking of NLRP3 and HT (AutoDock Tool was used to…