Puerarin Inhibits NLRP3-Caspase-1-GSDMD-Mediated Pyroptosis via P2X7 Receptor in Cardiomyocytes and Macrophages

Abstract

Diabetic cardiomyopathy (DCM) is a critical complication of long-term chronic diabetes mellitus, and it is characterized by myocardial fibrosis and myocardial hypertrophy. Previous studies have shown that the pyroptosis pathway was significantly activated in DCM and may be related to the P2X7 receptor. However, the role of the P2X7 receptor in the development of DCM with pyroptosis is still unclear. In this study, we aimed to explore the mechanism of puerarin and whether the P2X7 receptor can be used as a new target for puerarin in the treatment of DCM. We adopted systematic pharmacology and bioinformatic approaches to identify the potential targets of puerarin for treating DCM. Additionally, we employed D-glucose-induced H9C2 rat cardiomyocytes and lipopolysaccharide-treated RAW264.7 mouse mononuclear macrophages as the in vitro model on DCM research, which is close to the pathological conditions. The mRNA expression of cytokines in H9C2 cells and RAW264.7 macrophages was detected. The protein expressions of NLRP3, N-GSDMD, cleaved-caspase-1, and the P2X7 receptor were investigated with Western blot analysis. Furthermore, molecular docking of puerarin and the P2X7 receptor was conducted based on CDOCKER. A total of 348 puerarin targets and 4556 diabetic cardiomyopathy targets were detected, of which 218 were cross targets. We demonstrated that puerarin is effective in enhancing cardiomyocyte viability and improving mitochondrial function. In addition, puerarin is efficacious in blocking NLRP3-Caspase-1-GSDMD-mediated pyroptosis in H9C2 cells and RAW264.7 cells, alleviating cellular inflammation. On the other hand, similar experimental results were obtained by intervention with the P2X7 receptor antagonist A740003, suggesting that the protective effects of puerarin are related to the P2X7 receptor. The molecular docking results indicated key binding activity between the P2X7 receptor and puerarin. These findings indicate that puerarin effectively regulated the pyroptosis signaling pathway during DCM, and this regulation was associated with the P2X7 receptor.

Keywords: H9C2 cell; P2X7 receptor; RAW264.7 macrophages; diabetic cardiomyopathy; puerarin; pyroptosis.

Figure 1

Network pharmacology. (A) Venn diagram of puerarin (blue) and diabetic cardiomyopathy genes (red), (B) GO analysis, and (C) top ten KEGG pathways. The color scales indicate the different thresholds for the p-values, and the sizes of the dots represent the number of genes corresponding to each term.

Figure 2

Puerarin reverses D-glucose-induced cytotoxicity and mitochondrial dysfunction in H9C2 cells. (A) Effects of mannitol on cell viability in H9C2 cells for 24 h. (B) The difference between mannitol and glucose on cell viability in H9C2 cells for 24 h. (C) Effects of puerarin on H9C2 cells viability under normoxia conditions. (D) Effects of puerarin on cell viability in D-glucose-induced H9C2 cell injury. (E) Fluorescence image of JC-1 staining in H9C2 cells exposed to 25 mmol/L D-glucose for 24 h. Scale bar: 100 μm. (F) Quantitative analysis of the ratio of red fluorescence to green fluorescence in (E). (G) Effects of puerarin (30 μmol/L) on the mitochondrial complex of H9C2 cells induced by D-glucose (25 mmol/L) for 24 h. (H,I) The representative profile of O₂ concentration change and relative level of O₂ flux per volume of H9C2 cells in the high glucose model. (J) The summarized data of mitochondrial respiration in glucose and puerarin group, including routine, CI, and CI plus CII oxidative phosphorylation, CI and CII leak, as well as CI plus CII electron transfer system. (K) Representative profiles and statistical data of mitochondrial respiration of H9C2 cells treated with glucose (25 mmol/L, 24 h). (L,M) Effects of puerarin on routine respiration, maximum mitochondrial respiration (MMR), and residual respiration consumption (RRC) of H9C2 cells induced by D-glucose (25 mmol/L, 24 h). Data are shown as mean ± SEM (n = 3). ^# p < 0.05, ^## p < 0.01 vs. control, * p < 0.05, ** p < 0.01 vs. model.

Figure 3

Puerarin inhibited pyroptosis in H9C2 cells induced with D-glucose. Effects of puerarin on IL-1β (A), IL-18 (B), and TNF-α (C) mRNA expression of H9C2 induced with glucose (25 mM) for 24 h. Effects of puerarin (1–30 μmol/L) on the expression of (D) cleaved-caspase-1, (E) N-GSDMD, (F) NLRP3, and (G) the P2X7 receptor. (H) Puerarin decreases pyroptosis-related protein expression from H9C2 cells. Cells were pretreated with BzATP (100 μmol/L) and/or A740003 (50 μmol/L) for 2 h prior to puerarin, and with or without puerarin (30 μmol/L) for 24 h. LPS ± BzATP induced Caspase-1 and GSDMD expression that was inhibited by puerarin, similar to the effect of A740003. Relative density analysis of the protein bands was shown by the Western blot with β-actin as a control. Data are shown as mean ± SEM (n = 3). ^# p < 0.05, ^## p < 0.01 vs. control, * p < 0.05, ** p < 0.01 vs. model.

Figure 4

Puerarin attenuates LPS-induced inflammation in RAW264.7 cells. (A) Effects of puerarin on RAW264.7 cell viability under normoxia conditions. (B) Effects of puerarin on cell viability in RAW264.7 cells for 48 h. (C) Effects of puerarin on the NO production of RAW264.7 cells induced by LPS. Puerarin downregulates IL-1β (D), IL-18 (E), and NLRP3 (F) mRNA expression. Data are shown as mean ± SEM (n = 3). ^# p < 0.05, ^## p < 0.01 vs. control, * p < 0.05, ** p < 0.01 vs. model.

Figure 5

Puerarin decreased NO (A–D) production and mRNA expression (E–G) in RAW264.7 cells stimulated with LPS (100, 200 ng/mL), BzATP (10 μmol/L or 100 μmol/L), A740003 (50 μmol/L or 100 μmol/L), and with or without puerarin (3, 10, 30, 100 μmol/L) for 48 h. LPS ± BzATP induced NO production that was inhibited by puerarin, similar to the effect of A740003. Effects of puerarin (3–100 μmol/L) on the expression of (H) cleaved-caspase-1 and (I) N-GSDMD. Data are shown as mean ± SEM (n = 3). ^## p < 0.01 vs. control, * p < 0.05, ** p < 0.01 vs. model.

Figure 6

Molecular docking diagram. Molecular models of the binding of puerarin with the P2X7 receptor and the results are shown as 3D diagrams. (A) P2X7 receptor activate site 1–puerarin, (B) P2X7 receptor activate site 1–original ligand 1, (C) P2X7 receptor activate site 2 –puerarin, (D) P2X7 receptor activate site 2–original ligand 2, (E) P2X7 receptor activate site 3–puerarin, (F) P2X7 receptor activate site 3–original ligand 3, (G) P2X7 receptor activate site 4–puerarin, and (H) P2X7 receptor activate site 4–original ligand 4.

Figure 7

Schematic diagram of possible signal pathways of puerarin in D-glucose-induced H9C2 cells and LPS-induced RAW264.7 cells.