Induction of HO-1 by 5, 8-Dihydroxy-4',7-Dimethoxyflavone via Activation of ROS/p38 MAPK/Nrf2 Attenuates Thrombin-Induced Connective Tissue Growth Factor Expression in Human Cardiac Fibroblasts

Abstract

Heme oxygenase-1 (HO-1) has been shown to exert as an antioxidant and anti-inflammatory enzyme in cardiovascular inflammatory diseases. Flavonoids have been demonstrated to display anti-inflammatory and antioxidant effects through the induction of HO-1. 5,8-Dihydroxy-4',7-dimethoxyflavone (DDF), one of the flavonoid compounds, is isolated from Reevesia formosana. Whether DDF induced HO-1 expression on human cardiac fibroblasts (HCFs) remained unknown. Here, we found that DDF time- and concentration-dependently induced HO-1 protein and mRNA expression, which was attenuated by pretreatment with reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC) in HCFs. DDF-enhanced ROS generation was attenuated by NAC, but not by either diphenyleneiodonium chloride (DPI, Nox inhibitor) or MitoTempol (mitochondrial ROS scavenger). Interestingly, pretreatment with glutathione (GSH) inhibited DDF-induced HO-1 expression. The ratio of GSH/GSSG was time-dependently decreased in DDF-treated HCFs. DDF-induced HO-1 expression was attenuated by an inhibitor of p38 MAPK (p38i VIII) or siRNA, but not by MEK1/2 (PD98059) or JNK1/2 (SP600125). DDF-stimulated p38 MAPK phosphorylation was inhibited by GSH or p38i VIII. Moreover, DDF-induced HO-1 expression was mediated through Nrf2 phosphorylation and translocation into the nucleus which was attenuated by NAC or p38 siRNA. DDF also stimulated antioxidant response element (ARE) promoter activity which was inhibited by NAC, GSH, or p38i VIII. Interaction between Nrf2 and the ARE-binding sites on the HO-1 promoter was revealed by chromatin immunoprecipitation assay, which was attenuated by NAC, GSH, or p38i VIII. We further evaluated the functional effect of HO-1 expression on the thrombin-induced fibrotic responses. Our result indicated that the induction of HO-1 by DDF can attenuate the thrombin-induced connective tissue growth factor expression. These results suggested that DDF-induced HO-1 expression is, at least, mediated through the activation of the ROS-dependent p38 MAPK/Nrf2 signaling pathway in HCFs. Thus, the upregulation of HO-1 by DDF could be a candidate for the treatment of heart fibrosis.

Figure 1

Chemical structure of 5, 8-Dihydroxy-4′,7-dimethoxyflavone (DDF) isolated from Reevesia formosana.

Figure 2

DDF induced HO-1 protein and mRNA expression in HCFs. (a) HCFs were incubated with various concentrations of DDF (3, 10, and 30 μM) for the indicated time intervals (0, 2, 4, 8, 16, and 24 h). The levels of HO-1 and β-actin protein expression were examined by Western blot analysis. (b) Cells were incubated with various concentrations of DDF (1, 3, 10, 20, 30, and 50 μM) for 16 h. The cell viability was performed by using a CCK-8 kit. (c) Cells were incubated with 10 μM DDF for the indicated time intervals (0, 1, 2, 4, 6, and 8 h). The levels of HO-1 and GAPDH mRNA were analyzed by real-time PCR. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with control.

Figure 3

DDF-induced HO-1 expression requires ongoing transcription and translation in HCFs. (a, b) HCFs were pretreated with various concentrations of either (a) CHI or (b) Act. D for 1 h and then incubated with 10 μM DDF for 16 h. The levels of HO-1 and β-actin protein expression were examined by Western blot analysis. (c) Cells were pretreated with 30 nM Act. D for 1 h and then incubated with 10 μM DDF for 6 h. The levels of HO-1 and GAPDH mRNA were analyzed by real-time PCR. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with DDF alone.

Figure 4

Involvement of ROS generation in DDF-induced HO-1 expression. (a) HCFs were pretreated with various concentrations of NAC for 1 h and then incubated with 10 μM DDF for 16 h. The levels of HO-1 and β-actin protein expression were examined by Western blot analysis. (b) Cells were pretreated with 10 mM NAC for 1 h and then incubated with 10 μM DDF for 6 h. The levels of HO-1 and GAPDH mRNA were analyzed by real-time PCR. (c, d) The levels of ROS production were measured. Cells were treated with 10 μM DDF for the indicated time intervals (c). Cells were pretreated without or with 10 mM NAC, 10 μM DPI or 1 μM MitoTempol for 1 h, and then incubated with 10 μM DDF for 30 or 60 min (d). (e) Cells were pretreated with 10 mM NAC for 1 h and then incubated with 10 μM DDF for 1 h, and labeled with DCFDA or DHE. The fluorescence intensities of ROS accumulation were observed using a fluorescence microscope (magnification = 400×). (f) Cells were pretreated with either GSH or Trolox for 1 h and then incubated with 10 μM DDF for 16 h. The levels of HO-1 and β-actin protein expression were examined by Western blot analysis. (g) Cells were incubated with 10 μM DDF for the indicated time intervals, and then the ratio of GSG/GSSG was determined by a glutathione detection kit. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with control.

Figure 5

Involvement of p38 MAPK in DDF-induced HO-1 expression but not JNK and Erk. (a) HCFs were pretreated with various concentrations of p38 inhibitor VIII, PD98059, and SP600125 for 1 h, respectively, and then incubated with DDF (10 μM) for 16 h. The protein expression of HO-1 was examined by Western blot analysis. (b) Cells were pretreated with p38 inhibitor VIII for 1 h and then incubated with DDF (10 μM) for 6 h. The levels of HO-1 mRNA were analyzed by real-time PCR. (c) Cells were transfected with scrambled or p38α siRNA, and then incubated with DDF (10 μM) 16 h. The protein levels of p38, HO-1, and β-actin were determined by Western blot analysis. (d) Cells were treated with DDF (10 μM) for the indicated time intervals with or without preincubation with GSH (1 mM) and p38 inhibitor VIII (0.1 μM), respectively, or transfection with scramble or p38 siRNA. The levels of phospho- and total-p38 MAPK and GAPDH were determined by Western blot. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with DDF alone.

Figure 6

Involvement of Nrf2 in DDF induced HO-1 expression. (a) HCFs were transfected with scrambled or Nrf2 siRNA, and then incubated with DDF (10 U/ml) for 16 h. The protein levels of Nrf2, HO-1, and β-actin were determined by Western blot. (b) Cells were transfected with scrambled or Nrf2 siRNA then incubated with DDF for 6 h. The HO-1 mRNA expression was analyzed by real-time PCR. (c) Cells were pretreated with or without NAC (10 mM) or transfected with scrambled siRNA, p38 siRNA, or Nrf2 siRNA, individually, and then incubated with DDF (10 μM) for the indicated time intervals. The levels of phospho- and total-Nrf2, total p38 MAPK, and GAPDH were determined by Western blot. (d) Cells were treated with DDF (10 μM) for the indicated time intervals. Nuclear and cytosolic extracts were prepared, and the protein levels of Nrf2, p-Nrf2, Lamin A, and GAPDH were determined by Western blot. (e) Cells were pretreated with or without NAC (10 mM), GSH (1 mM), or p38i VIII (0.1 μM), then incubated with DDF for 2 h. Phosphorylation of Nrf2 nuclear translocation was labeled with FITC and observed by immunofluorescence microscopy. Scale bars, 50 μm. #P < 0.01, as compared with DDF alone.

Figure 7

DDF-mediated Nrf2 activates the HO-1 promoter via binding with the ARE site in HCFs. (a, b) HCFs were cotransfected with ARE promoter-Luc and β-galactosidase and then incubated with DDF (10 μM) for the indicated time intervals (1, 2, 3, 4, and 5 h) (a); pretreated with or without NAC (10 mM), GSH (1 mM), or p38i VIII (0.1 μM), then incubated with DDF (10 μM) for 3 h (b). The cell lysates were determined in ARE promoter luciferase activity. (c, d) The transcriptional activity of Nrf2 was determined by ChIP assay. Cells were treated with DDF (10 μM) for the indicated time intervals (1, 2, and 4 h) (c). Cells were pretreated with or without NAC (10 mM), GSH (1 mM), or p38i VIII (0.1 μM), then incubated with DDF for 2 h (d). #P < 0.01, as compared with DDF alone.

Figure 8

Upregulation HO-1 by DDF inhibits the thrombin-induced CTGF expression. (a) HCFs were incubated with various concentrations of thrombin (1, 3, and 10 U/ml) for the indicated time intervals (0, 2, 4, 8, 16, and 24 h). The levels of CTGF and β-actin protein expression were examined by western blot. (b) HCFs were pretreated without or with 10 μM DDF for 6 h and then challenged with 10 U/ml thrombin for 16 h. The levels of CTGF and β-actin protein expression were examined by western blot. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with DDF alone.

Figure 9

The schematic signaling pathways involved in DDF-induced HO-1 expression inhibited thrombin-stimulated CTGF induction in HCFs. DDF-induced HO-1 expression was, at least in part, mediated through the activation of the ROS-dependent p38 MAPK/Nrf2 pathway, which attenuated the thrombin-stimulated CTGF induction. “⟶” means “activated,” and “⊥” means “inhibited.”