Puerarin attenuates myocardial ischemia‑reperfusion injury by inhibiting myocardium pyroptosis via the NRF2/HO‑1 signaling pathway

Abstract

Myocardial ischemia‑reperfusion injury (MIRI) can trigger inflammatory responses and cause pyroptosis. Puerarin (Pue), as a traditional medicine, exhibits potential value in cardiac protection. However, the mechanism by which Pue regulates pyroptosis in MIRI remains to be fully elucidated. The present study aimed to explore the cardioprotective effects of Pue against MIRI and reveal the underlying mechanisms of these effects. Sprague‑Dawley rats were used to establish in vivo models of MIRI, while H9C2 rat embryonic cardiomyocytes were employed as in vitro models. Echocardiography was performed to measure cardiac function. Triphenyltetrazolium chloride/Evans blue staining, hematoxylin‑eosin staining, Masson's trichrome staining and immunohistochemistry were employed to assess the pharmacodynamic effects of Pue. The expression of molecules related to pyroptosis, such as nuclear factor E2‑related factor 2 (NRF2) and heme oxygenase‑1 (HO‑1) were detected by immunofluorescence, Hoechst 33342/PI staining, reverse transcription‑quantitative PCR and western blot analyses. The results of the present study showed that Pue pretreatment reduced the area of myocardial infarction and decreased the expression of pyroptosis‑related molecules. Additionally, Pue was shown to reverse H2O2‑induced mitochondrial dysfunction in cardiomyocytes and inhibit nucleotide‑binding oligomerization domain‑like receptor family pyrin domain‑containing 3 (NLRP3)/caspase‑1/gasdermin D (GSDMD)‑mediated pyroptosis. Pue was also shown to stimulate the nuclear translocation of NRF2 and increase the expression of HO‑1. Furthermore, Pue further demonstrated its anti‑pyroptotic effects by activating the NRF2/HO‑1 pathway. The present study revealed that Pue can protect injured myocardium after MIRI by inhibiting NLRP3/caspase‑1/GSDMD‑mediated pyroptosis. The mechanism of action for these cardioprotective effects relied upon downregulation of the NRF2/HO‑1 signaling pathway. The findings of the present study provided a novel strategy for the clinical application of puerarin in the treatment of MIRI.

Keywords: heme oxygenase‑1; inflammation; myocardial ischemia‑reperfusion injury; nuclear factor E2‑related factor 2; puerarin; pyroptosis.

Figure 1.

Pue displays cardioprotective effects against myocardial ischemia-reperfusion injury in vivo. (A) Timeline of Pue administration and schematic of the rat I/R injury model. (B) Changes in cardiac function (LVIDd, LVIDs, LVEF) and heart rate were observed in each group at 2 h after myocardial I/R. (C) Triphenyltetrazolium chloride staining of myocardial tissues. (D) Quantitative comparison of infarction areas among each group (n=8). (E) Masson's trichrome staining of myocardial tissues (scale bar, 100 µm). **P<0.01, ***P<0.001 and ****P<0.0001 vs. sham group; ^##P<0.01, ^###P<0.001 and ^####P<0.0001 vs. I/R group. Pue, puerarin; LVIDd, left ventricular internal dimension at end-diastole; LVIDs, left ventricular internal dimension at end-systole; LVEF, left ventricular ejection fraction; I/R, ischemia/reperfusion.

Figure 2.

Pue inhibits the key proteins of the inflammasome and pyroptosis in myocardial ischemia-reperfusion injury. (A) Hematoxylin-eosin staining showed the inflammatory condition of myocardial tissue (n=8; scale bar, 100 µm). (B) Immunohistochemistry detected the positive expression of inflammasome markers in myocardial tissues of each group (scale bar, 100 µm). (C) The AOD values of inflammasome markers obtained by immunohistochemical staining. (D) Expression of pyroptotic proteins and HO-1. (E) Quantification of relative mRNA levels of key pyroptotic markers. (F) Semi-quantification of relative expression levels of pyroptotic proteins and HO-1. (G) Protein expression of nuclear NRF2 was assessed by western blotting. (H) Semi-quantification of relative expression levels of NRF2 obtained by western blotting. **P<0.01, ***P<0.001 and ****P<0.0001 vs. sham group; ^####P<0.0001 vs. I/R group.. AOD, average optical density; HO-1, heme oxygenase-1; ASC, apoptosis-associated speck-like protein; NLRP3, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; GSDMD, gasdermin D; Pue, puerarin; I/R, ischemia/reperfusion.

Figure 3.

Pue inhibits pyroptosis in cardiomyocytes. (A) JC-1 was used to detect the mitochondrial membrane potential of cardiomyocytes (scale bar, 100 µm). (B) Quantitative analysis of the ratio of red to green fluorescence. (C) Western blot analysis showed the expression levels of key proteins involved in pyroptosis in cardiomyocytes. (D) Representative morphology of mitochondria obtained by transmission electron microscopy (scale bar, 1 µm). The green arrow showed normal mitochondria, while the red arrow indicated organelle vacuolation. (E) Semi-quantification of the relative expression levels of key pyroptotic proteins in cardiomyocytes from western blotting results. ****P<0.0001 vs. control group; ^##P<0.01 and ^####P<0.0001 vs. H₂O₂ group. Pue, puerarin; ASC, apoptosis-associated speck-like protein; GSDMD, gasdermin D; NLRP3, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3.

Figure 4.

Pue activates the NRF2 pathway to alleviate cardiomyocyte pyroptosis. (A) Western blot assays were used to detect NRF2 expression in cardiomyocytes of the OE-NC group and the OE-NRF2 group (n=3). (B) Semi-quantification of NRF2 protein levels detected in western blotting. (C) Immunofluorescence was used to detect nuclear translocation of NRF2 in each group of cells (n=3). (D) HO-1 expression was detected by immunofluorescence staining in each group of cells (n=3). (E) Quantitative analysis of the fluorescence intensities of NRF2 and HO-1 immunofluorescence staining. (F) Activity of LDH in each group. **P<0.01 and ****P<0.0001 vs. OE-NC group; ^###P<0.001 and ^####P<0.0001 vs. H₂O₂ + OE-NC group. OE, overexpression; NC, negative control; NRF2, nuclear factor E2-related factor 2; Pue, puerarin; HO-1, heme oxygenase-1; LDH, lactate dehydrogenase.

Figure 5.

Pue alleviates cardiomyocyte pyroptosis via the NRF2/HO-1 signaling pathway. (A) Hoechst 33342/PI staining detected the apoptotic state of H9C2 cells (magnification, ×400) (n=3). (B) Quantitative analysis of PI-positive cells. (C) Western blotting of nuclear NRF2 expression in each group (n=3). (D) Western blotting images of key pyroptotic proteins and HO-1 in each treatment group (n=3). (E) Semi-quantification of the expression of pyroptotic proteins and HO-1 in each group of cells. (F) Semi-quantification of NRF2 protein expression. ****P<0.0001 vs. OE-NC group; ^###P<0.001 and ^####P<0.0001 vs. H₂O₂ + OE-NC group. Pue, puerarin; OE, overexpression; NC, negative control; NRF2, nuclear factor E2-related factor 2; ASC, apoptosis-associated speck-like protein; NLRP3, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; GSDMD, gasdermin D; HO-1, heme oxygenase-1.

Figure 6.

Schematic diagram of the molecular mechanism of Pue-mediated pyroptosis inhibition. NRF2, nuclear factor E2-related factor 2; HO-1, heme oxygenase-1; ARE, antioxidant response element; NLRP3, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; ASC, apoptosis-associated speck-like protein; GSDMD, gasdermin D.