Cardioprotective Effects of Puerarin-V on Isoproterenol-Induced Myocardial Infarction Mice Is Associated with Regulation of PPAR-Υ/NF-κB Pathway

Abstract

Puerarin is a well-known traditional Chinese medicine which has been used for the treatment of cardiovascular diseases. Recently, a new advantageous crystal form of puerarin, puerarin-V, has been developed. However, the cardioprotective effects of puerarin-V on myocardial infarction (MI) heart failure are still unclear. In this research, we aim to evaluate the cardioprotective effects of puerarin-V on the isoproterenol (ISO)-induced MI mice and elucidate the underlying mechanisms. To induce MI in C57BL/6 mice, ISO was administered at 40 mg/kg subcutaneously every 12 h for three times in total. The mice were randomly divided into nine groups: (1) control; (2) ISO; (3) ISO + puerarin injection; (4⁻9) ISO + puerarin-V at different doses and timings. After treatment, cardiac function was evaluated by electrocardiogram (ECG), biochemical and histochemical analysis. In vitro inflammatory responses and apoptosis were evaluated in human coronary artery endothelial cells (HCAECs) challenged by lipopolysaccharide (LPS). LPS-induced PPAR-Υ/NF-κB and subsequently activation of cytokines were assessed by the western blot and real-time polymerase chain reaction (PCR). Administration of puerarin-V significantly inhibits the typical ST segment depression compared with that in MI mice. Further, puerarin-V treatment significantly improves ventricular wall infarction, decreases the incidence of mortality, and inhibits the levels of myocardial injury markers. Moreover, puerarin-V treatment reduces the inflammatory milieu in the heart of MI mice, thereby blocking the upregulation of proinflammatory cytokines (TNF-α, IL-1β and IL-6). The beneficial effects of puerarin-V might be associated with the normalization in gene expression of PPAR-Υ and PPAR-Υ/NF-κB /ΙκB-α/ΙΚΚα/β phosphorylation. In the in vitro experiment, treatment with puerarin-V (0.3, 1 and 3 μM) significantly reduces cell death and suppresses the inflammation cytokines expression. Likewise, puerarin-V exhibits similar mechanisms. The cardioprotective effects of puerarin-V treatment on MI mice in the pre + post-ISO group seem to be more prominent compared to those in the post-ISO group. Puerarin-V exerts cardioprotective effects against ISO-induced MI in mice, which may be related to the activation of PPAR-γ and the inhibition of NF-κB signaling in vivo and in vitro. Taken together, our research provides a new therapeutic option for the treatment of MI in clinic.

Keywords: NF-κB; PPAR-Υ; anti-apoptosis; anti-inflammation; myocardial infarction; puerarin-V.

Figure 1

Structure of puerarin-V and differences in crystal characterization between puerarin-V and puerarin. (A) The chemical structure of puerarin-V. (B) Representative images of powder X-ray diffraction (PXRD) of puerarin-V (a) and puerarin (b); representative images of differential scanning calorimetry (DSC) of puerarin-V (c) and puerarin (d).

Figure 2

Cardioprotective effects of puerarin-V in the MI mice. (A) Representative images of ECG. (a)–(i) correspond to groups: (a) control group; (b) model group at day 5 after the MI induction; (c) puer i.v.-40 mg/kg group at day 5 after the MI induction; (d), (e) and (f): post-puerarin-V groups (10, 30, and 100 mg/kg, respectively) at day 5 after the MI induction; (g), (h) and (i): pre + post-puerarin-V groups (10, 30, and 100 mg/kg, respectively) at day 5 after the MI induction. (B) Effects of puerarin-V on ST segment deviation (mV) of MI mice. (C) TTC-stained heart slices arranged in order and representative images of heart samples from the indicated groups of mice are shown in the photos. The white area represents infarcted tissue. (D) The heart wet weight/body weight ratio in each group. (E,F) The survival rate of MI mice and normal mice in each group. All mice were observed for mortality until day 6. Data are shown as mean ± SEM (n = 8–10). ^## p < 0.01 vs. control group. * p < 0.05, and ** p < 0.01 vs. model group.

Figure 3

Puerarin-V treatment suppressed myocardial inflammation and reduced myocardial cell damage in the MI mice. (A) Representative images of H&E staining of right ventricular cardiomyocytes in different groups. Small images were 100×, and the big images were 200×. Scale bar = 100 μm. (B–D) The serum levels of AST, LDH and cTn-T. Data are shown as mean ± SEM (n = 6). ^### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. model group.

Figure 4

Puerarin-V reduced myocardial inflammation in the heart of MI mice. (A) Immunohistochemical analysis of macrophage marker CD68 in heart cross sections. The original magnification of the images was 200×. The scale bar is 100 μm. (B) The percent of CD68⁺ cells (n = 4). (C–E) The mRNA levels of IL-6, TNF-α, and IL-1β (n = 6). (F–H) The quantitative analyses of IL-6, TNF-α, and IL-1β by ELISA (n = 6). Data are shown as mean ± SEM. ^### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. model group.

Figure 5

Puerarin-V inhibited ISO-induced apoptosis in the heart of MI mice. (A) The TUNEL-positive cells were shown by the immunohistochemistry method. The original magnification of the images was 200×. The scale bar is 100 μm. (B) The percent of TUNEL-positive cells (n = 4). (C,D) Western blot analyses for Bax and Bcl-2 expression, and normalization of these two expression by the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (n = 6). Data are shown as mean ± SEM. ^## p < 0.01, and ^### p < 0.001 vs. control group. ** p < 0.01, and ***p < 0.001 vs. model group.

Figure 6

Puerarin-V attenuated ISO-induced inflammation in the MI mice associated with the PPAR-γ/NF-κB pathway: (A) PPAR-γ; (B) p-NF-κB/NF-κB; (C) p-IκB-α/IκB-α; and (D) p-IKKα/β/IKKα/β. Relative density analysis of the protein bands was shown by the western blot with GAPDH as control. Data are shown as mean ± SEM (n = 6). ^### p < 0.001 vs. control group. * p < 0.05, and ** p < 0.01 vs. model group.

Figure 7

Puerarin-V attenuated LPS-induced inflammation in HCAECs associated with the PPAR-γ/NF-κB pathway. (A,B) The mRNA levels of TNF-α and IL-1β. (C) Confluent cultures of HCAEC were preincubated for 2 h with puerarin-V (3 μM). Thereafter, different concentrations of LPS were added and cells were cultivated for 24 h. The black bars show the cultures without puerarin-V treatment; the blue bars show the puerarin-V treated cultures. (D) Confluent cultures of HCAEC were preincubated for 2 h with different amounts of puerarin-V (μM). Thereafter, LPS (100 ng/ml) was added and cells were cultivated for 24 h. (E) PPAR-γ expression. (F) p-NF-κB/NF-κB expression. (G) p-IκB-α/IκB-α expression. (H) p-IKKα/β/IKKα/β expression. Relative density analysis of the protein bands was shown by the western blot with GAPDH as control. Data are shown as mean ± SEM (n = 4–6). ^## p < 0.01, and ^### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. model group.

Figure 8

Effects of puerarin-V on cell viability and apoptosis-related protein expression in HCAECs exposed to LPS. (A) Effects of puerarin-V on cell viability under normaxia conditions. (B) Effects of puerarin-V on cell viability in LPS-induced cell injury. (C) Cleaved caspase 3 expression. (D) Bcl-2 expression. (E) Bax expression. Relative density analysis of the protein bands was shown by the western blot with GAPDH as control. Data are shown as mean ± SEM (n = 4). ^## p < 0.01, and ^### p < 0.001 vs. control group. * p < 0.05, and ** p < 0.01 vs. model group.

Figure 9

Schematic representation of animal experimental design.