A Chinese Herbal Formula Ameliorates Pulmonary Fibrosis by Inhibiting Oxidative Stress via Upregulating Nrf2

Abstract

This study aimed to explore the protective effects of a Chinese herbal formula, Jinshui Huanxian formula (JHF), on experimental pulmonary fibrosis and its underlying mechanisms. After being treated with single dose of bleomycin (5 mg/kg) intratracheally, rats were orally administered with JHF and pirfenidone from day 1 to 42, then sacrificed at 7, 14, 28, or 42 days post-bleomycin instillation. JHF ameliorated bleomycin-induced pathological changes, collagen deposition in the rat lung and recovered pulmonary function at different days post-bleomycin instillation. In lungs of JHF-treated rats, the levels of total superoxide dismutase, catalase and glutathione were higher, and myeloperoxidase and methane dicarboxylic aldehyde were lower than those in vehicle-treated rats, respectively. Additionally, JHF inhibited the expression of NADPH oxidase 4 (NOX4) and increased the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) in lung tissues. In vitro, JHF and ruscogenin, a compound of Ophiopogonis Radix contained in JHF, significantly inhibited transforming growth factor β1 (TGF-β1)-induced differentiation of fibroblasts. Furthermore, JHF markedly decreased the level of reactive oxygen species in TGF-β1-induced fibroblast. In line with this, upregulation of NAD(P)H: quinone oxidoreductase 1 and heme oxygenase 1, and downregulation of NOX4 were found in JHF-treated fibroblast induced by TGF-β1. While on the other hand, Nrf2 siRNA could suppress the JHF-mediated inhibition effect on alpha-smooth muscle actin (α-SMA), and FN1 expression induced by TGF-β₁ in fibroblasts. These results indicated that JHF performed remarkably therapeutic and long-term effects on pulmonary fibrosis in rat and suppressed the differentiation of fibroblast into myofibroblast through reducing the oxidative response by upregulating Nrf2 signaling. It might provide a new potential natural drug for the treatment of pulmonary fibrosis.

Keywords: Jinshui Huanxian formula; Nrf2; fibroblast; oxidative stress; pulmonary fibrosis.

FIGURE 1

Jinshui Huanxian formula (JHF) inhibited bleomycin-induced the decline of FVC, increase of Lung coefficient and up-expression of TGF-β1. (A) FVC. (B) Lung coefficient. (C) Immunohistochemical staining of TGF-β1 in rat lung tissue, scale bars: 200 μm. Values represented as mean ± SEM. ^##P < 0.01, versus control group. ^∗∗P < 0.01, ^∗P < 0.05, versus model group. ^{Δ Δ} P < 0.01, ^Δ P < 0.05, versus matched time point model group.

FIGURE 2

The therapeutic effect of Jinshui Huanxian formula (JHF) on bleomycin-induced PF rats. (A) Hematoxylin and eosin stained, scale bars: 100 μm. (B) Masson trichrome stained, scale bars: 100 μm. (C) Ashcroft score, (D) Hydroxyproline. Values represented as mean ± SEM.^##P < 0.01, ^#P < 0.05, versus control group. ^∗∗P < 0.01, ^∗P < 0.05 versus model group. ^{Δ Δ} P < 0.01, ^Δ P < 0.05, versus matched time point model group.

FIGURE 3

Jinshui Huanxian formula (JHF) suppressed α-SMA, COL-I, and COL-III expression in bleomycin-induced PF rats. Immunohistochemical analysis of α-SMA (A), COL-I (B), and COL-III (C), scale bars: 200 μm. Values represented as mean ± SEM. ^##P < 0.01, versus control group. ^∗∗P < 0.01, ^∗P < 0.05, versus model group. ^{Δ Δ} P < 0.01, ^Δ P < 0.05, ANOVA versus matched time point model group.

FIGURE 4

Jinshui Huanxian formula (JHF) ameliorated bleomycin-induced oxidative stress in rat lung tissue and serum. (A,B) T-SOD activity, (C,D) the level of GSH content, (E,F) CAT activity. (G,H) MDA level, (I,J) MPO activity. Values represented as mean ± SEM. ^##P < 0.01, ^#P < 0.05, versus control group. ^∗∗P < 0.01, ^∗P < 0.05, vs. model group.

FIGURE 5

Jinshui Huanxian formula (JHF) inhibited expression of NOX4 and increased expression of Nrf2 mRNA and protein on PF rats. (A,B) The expressions of NOX4 and Nrf2 mRNA in lung tissue. (C–E) The expression of NOX4 and Nrf2 protein in lung tissue. Values represented as mean ± SEM. ^##P < 0.01, ^#P < 0.05, versus control group. ^∗∗P < 0.01, ^∗P < 0.05, versus model group.

FIGURE 6

Jinshui Huanxian formula (JHF) inhibited TGF-β1-induced differentiation of MRC-5 cells. (A) Immunofluorescence analysis of α-SMA and FN1 expression in TGF-β1-treated, α-SMA in green, FN1 in red and DAPI in blue, scale bars: 20 μm. Values represented as mean ± SEM. ^∗∗P < 0.01, ^∗P < 0.05, versus control group. (B) The protein level of α-SMA in TGF-β-treated fibroblasts. Values represented as mean ± SEM. ^##P < 0.01, versus control group. ^∗∗P < 0.01, ^∗P < 0.05, versus model group. (C–G) The effect of JHF on mRNA and protein levels of α-SMA and FN1 in TGF-β1-induced fibroblasts. Values represented as mean ± SEM. ^##P < 0.01, versus control group. ^∗∗P < 0.01, versus model group.

FIGURE 7

Total ion chromatogram of Jinshui Huanxian formula (JHF)-containing serum, and the effect of ruscogenin on the differentiation of fibroblast. (A) The 20 compounds were identified in JHF-containing serum: 1, Catalpol; 2, geniposide; 3, leonuride; 4, bilobalide; 5, Peimine; 6, hesperidin; 7, Peiminine; 8, Ginsenoside Re; 9, Ginkgolide B; 10, Ginkgolide A; 11, Anemoside B4; 12, Icraiin; 13, Ophiopogonin D; 14, Ginsenoside Rb1; 15, Ginsenoside Rc; 16, Nobiletin; 17, Methylophiopogonanone B; 18, Paeonol; 19, Ruscogenin; 20, 3,29-Dibenzoyl rarounitriol. (B) The effect of ruscogenin on NIH-3T3 cell viability was detected by MTT analysis. (C,D) The NIH-3T3 cells were treated with TGF-β1 (7.5 ng/ml) and different concentrations of ruscogenin (2.5, 5, 10 μg/ml) for 24 h. Then the protein levels of FN and COL-I in the supernatant of NIH-3T3 cells were detected. Values represented as mean ± SEM. ^##P < 0.01, versus control group. ^∗∗P < 0.01, ^∗P < 0.05, versus model group.

FIGURE 8

Jinshui Huanxian formula (JHF) reduced the level of ROS in TGF-β1-induced MRC-5 cells. (A) After being treated with TGF-β1 JHF and PFD for 24 h, MRC-5 cells were incubated with CM-H2DCFDA (10 μM) for 30 min, fluorescence of DCF was measured by flow cytometry. (B) ROS levels in MRC-5 cells were quantified and presented. Values represented as mean ± SEM. ^#P < 0.05, versus control group. ^∗P < 0.05, versus model group.

FIGURE 9

The effect of Jinshui Huanxian formula (JHF) on mRNA and protein levels of Nrf2, HO1, NQO-1, and NOX4 in TGF-β1-induced fibroblasts. (A–D) RT-PCR analysis of Nrf2, HO1, NQO-1, and NOX4 mRNA expression in TGF-β1-induced fibroblasts. Reported values are means ± SEM. of the relative fold inductions calculated with the Δ ΔCt method considering control values as the calibrator, i.e., expression level = 1. (E–H) Representative Western blot analysis of Nrf2, HO1, NQO-1, and NOX4 protein expression and quantitation with mean values ± SEM. ^##P < 0.01, ^#P < 0.05, versus control group. ^∗∗P < 0.01, ^∗P < 0.05, versus model group.

FIGURE 10

Nrf2 siRNA prevented the anti-fibrosis effect of Jinshui Huanxian formula (JHF) on TGF-β1-induced MRC-5 cells. (A) MRC-5 cells were transfected with Cy3-labeled siRNA (red), scale bars: 300 μm. (B) The effect of JHF on the mRNA level of Nrf2 in TGF-β1-treated fibroblasts transfected with Nrf2 siRNA. (C) The effect of JHF on the mRNA levels of α-SMA, FN1 in TGF-β1-treated fibroblasts transfected with Nrf2 siRNA. Values represented as mean ± SEM. ^aaP < 0.01, versus NC control group. ^bbP < 0.01, ^bP < 0.05, versus NC model group. ^ccP < 0.01, versus Nrf2 siRNA control group.