Leonurine inhibits cardiomyocyte pyroptosis to attenuate cardiac fibrosis via the TGF-β/Smad2 signalling pathway

Figures

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  Fig 1. Leonurine enhanced cardiac function in rats with ISO-induced heart failure by improving haemodynamic variables. Cardiac function is responsive to haemodynamic variables, including LVSP, LVEDP, -dP/dt max, and +dP/dt max. Leonurine increased (A) LVSP level, and decreased levels of (B) LVEDP, (C) -dP/dt max, and (D) +dP/dt max compared with the model group. ##P<0.01, control group vs. model group; **P<0.01, *P<0.05, leonurine groups vs. model group. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; -dP/dt max, the falling rate of LVEDP; +dP/dt max, the rising rate of LVEDP.

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  Fig 2. Leonurine reduced myocardial injury in rats with ISO-induced heart failure. Myocardial injury is responsive to LDH and CK. Leonurine decreased levels of (A) CK and (B) LDH in plasma in heart failure rats induced by ISO. #P<0.05, ##P<0.01, model group vs. control group; *P<0.05, **P<0.01, ***P<0.001, leonurine groups vs. model group. CK, creatine kinase; LDH, lactic dehydrogenase.

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  Fig 3. Leonurine reduced the area of cardiac fibrosis in rats with ISO-induced heart failure. (A) Masson staining on the heart, blue = fibrous collagen. (B) H&E staining on the heart, pink = fibrous collagen. (C) PSR staining on the heart, red = fibrous collagen (bar, 1 mm; magnification, ×4, ×40). H&E, haematoxylin and eosin; PSR, Sirius red.

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  Fig 4. Leonurine reduced the expression of proteins related to fibrosis in heart tissues from rats with ISO-induced heart failure. (A) Compared to ISO-induced rats, leonurine decreased the expression of proteins related to fibrosis including (B) FN1, (C) α-SMA, and (D) COL3A1. ##P<0.01, ###P<0.001, model group vs. control group; **P<0.01, leonurine groups vs. model group.

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  Fig 5. Leonurine reduced the expression of proteins related to pyroptosis in heart tissues from rats with ISO-induced heart failure. (A) Compared to ISO-induced rats, leonurine decreased expressions of pyroptosis proteins including (B) GSDMD, (C) cleaved GSDMD, (D) Caspase 1, (E) cleaved Caspase 1, and the level of (F) IL-1β in plasma. #P<0.05, ##P<0.01, ###P<0.001, model group vs. control group; *P<0.05, **P<0.01, ***P<0.001, leonurine groups vs. model group.

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  Fig 6. Leonurine reduced the expression of proteins related to fibrosis in H9c2 cells stimulated by TGF-β. (A) Compared to H9c2 cells induced by TGF-β for 48 hours, leonurine decreased the expression of proteins related to fibrosis including (B) FN1, (C) α-SMA, and (D) COL3A1. #P<0.01, ###P<0.001, model group vs. control group; *P<0.05, **P<0.01, ***P<0.001, 20 μM leonurine group vs. model group.

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  Fig 7. Leonurine reduced the expression of proteins related to pyroptosis in H9c2 cells stimulated by TGF-β. (A) Compared to H9c2 cells induced by TGF-β for 48 hours, leonurine decreased the expression of proteins related to pyroptosis including (B) GSDMD, (C) cleaved GSDMD, (D) Caspase 1, (E) cleaved Caspase 1, and the level of (F) IL-1β. #P<0.05, model group vs. control group; *P<0.05, 20 μM leonurine group vs. model group.

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  Fig 8. Leonurine decreased ROS production by flow cytometric detection in H9c2 cells stimulated by TGF-β. DCFH-DA was used to evaluate the intracellular ROS level. DCFH-DA is a nonfluorescent ester of the dye fluorescent product in the presence of ROS. (A) The peak value of the model group shifted to the right, and the peak value of the leonurine group shifted to the left. (B) ROS production increased with TGF-β exposure, and pretreatment with leonurine inhibited the ROS increase after TGF-β stimulation. ##P<0.01, model group vs. control group; **P<0.01, 20 μM leonurine group vs. model group. DCFH-DA, 2,7-dichlorofluorescein diacetate.

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  Fig 9. Leonurine inhibited pyroptosis by regulating TGF-β/Smad2 signalling pathway in H9c2 cells stimulated by TGF-β. (A) Compared to H9c2 cells induced by TGF-β for thirty minutes, leonurine decreased the expressions of Smad2 phosphorylation and cleaved Caspase 1 (B) The ratio of Smad2 phosphorylation to Smad2 expression and the ratio of cleaved Caspase 1 to Caspase 1 expression. (C) The fluorescence of Smad2 protein was detected under a confocal laser scanning microscope. Compare to TGF-β induced model group, the intensity fluorescence of Smad2 protein was weakened after leonurine treatment. #P<0.05, ##P<0.01, model group vs. control group; *P<0.05, ***P<0.001, 20 μM leonurine group vs. model group.

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  Fig 10. Diagram of the potential mechanism of the effects of leonurine attenuating cardiac fibrosis in heart failure. Leonurine inhibited the activation of Smad2 phosphorylation and regulated the TGF-β/Smad2 signalling pathway, thereby the expression of proteins related to pyroptosis including Caspase 1 and GSDMD. The release of a large number of inflammatory factors like IL-1β caused cardiac fibrosis and promoted the pathological process of heart failure. TGF-β, transforming growth factor-β; TGF R1, transforming growth factor receptor 1; TGF R2, transforming growth factor receptor 2; Smad2, small mothers against decapentaplegic 2; P-Smad2, phospho-Smad2; IL-1β, Interleukin 1 beta; Caspase 1, cysteinyl aspartate specific proteinase 1; GSDMD, gasdermin-D.