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. 2016 Jun 3;17(6):879.
doi: 10.3390/ijms17060879.

Preventive Effects of Rhodiola rosea L. on Bleomycin-Induced Pulmonary Fibrosis in Rats

Ke Zhang  1 , Xiao-Ping Si  2 , Jian Huang  3 , Jian Han  4 , Xu Liang  5 , Xiao-Bo Xu  6 , Yi-Ting Wang  7 , Guo-Yu Li  8 , Hang-Yu Wang  9 , Jin-Hui Wang  10   11
Affiliations

Preventive Effects of Rhodiola rosea L. on Bleomycin-Induced Pulmonary Fibrosis in Rats

Ke Zhang et al. Int J Mol Sci. .

Abstract

Rhodiola rosea L. (RRL) possesses a wide range of pharmacological properties, including lung-protective activity, and has been utilized in folk medicine for several 100 years. However, the lung-protective mechanism remains unclear. This study investigated the possible lung-protective activity mechanism of RRL in a pulmonary fibrosis (PF) rat model. Lung fibrotic injury was induced in Sprague-Dawley rats by single intratracheal instillation of saline containing bleomycin (BLM; 5 mg/kg). The rats were administered 125, 250, or 500 mg/kg of a 95% ethanol extract of RRL for 28 days. The animals were killed to detect changes in body weight, serum levels of glutathione (GSH) and total superoxide dismutase (T-SOD), as well as lung tissue hydroxyproline (HYP) content. Tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), and interleukin 6 (IL-6) levels were measured in bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assay. Hematoxylin and eosin, Masson's trichrome, and immunohistochemical staining were performed to observe the histopathological changes in lung tissues. Additionally, target-related proteins were measured by Western blotting. RRL alleviated the loss of body weight induced by instilling BLM in PF rats, particularly at the 500 mg/kg per day dose. RRL reduced HYP (p < 0.01) and increased GSH and T-SOD contents. BALF levels of TNF-α, TGF-β1, and IL-6 decreased significantly in the RRL-treated groups. Expression levels of matrix metalloproteinase-9 (MMP-9) and α-smooth muscle actin decreased significantly in a dose-dependent manner in response to RRL. Moreover, the levels of TGF-β1 and tissue inhibitor of metalloproteinase-1 in lung tissues also decreased in the RRL-treated groups. RRL alleviated BLM-induced PF in rats. Our results reveal that the protective effects of RRL against fibrotic lung injury in rats are correlated with its anti-inflammatory, antioxidative, and anti-fibrotic properties. MMP-9 may play important roles in BLM-induced PF.

Keywords: MMP-9; Rhodiola rosea L.; TGF-β1; bleomycin; pulmonary fibrosis.

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Figures

Figure 1
Figure 1
Effects of RRL (Rhodiola rosea L.) on the levels of transforming growth TNF-α (tumor necrosis factor-α), TGF-β1 (transforming growth factor-β1) and IL-6 (interleukin 6) in the BALF (bronchoalveolar lavage fluid) of BLM (bleomycin)-treated rats. (** p < 0.01 vs. the normal control group; # p < 0.05 vs. the BLM-treated group; ## p < 0.01 vs. the BLM group). (A) normal group; (B) model group; (C) PAG (prednisone acetate) group; (D–F) are RRL group treated with 125, 250 and 500 mg/kg.
Figure 2
Figure 2
The H & E of the lung tissues in rats (Bar = 100 μm). (A) normal group; (B) model group; (C) PAG group; (D–F) are RRL groups treated with 125, 250 and 500 mg/kg, respectively.
Figure 3
Figure 3
The Masson examinations of the lung tissues in rats (Bar = 100 μm). (A) normal group; (B) model group; (C) PAG group; (D–F) are RRL groups treated with 125, 250 and 500 mg/kg, respectively.
Figure 4
Figure 4
(I) lung tissue HYP (hydroxyproline) level in rats; (II) serum T-SOD (Total superoxide dismutase) level in rats; (III) serum GSH (glutathione hormone) level in rats. (A) normal group; (B) model group; (C) PAG group; (D–F) are RRL groups treated with 125, 250 and 500 mg/kg, respectively. Compared with the Model group. ** p < 0.01. Compared with control group, # p < 0.05; ## p < 0.01.
Figure 5
Figure 5
Effects of RRL on the levels of α-smooth muscle actin (α-SMA) in the lung tissues after bleomycin (BLM)-induced in rats. (a) representative immunohistochemistry image (Bar = 50 μm). (A) normal group; (B) model group; (C) PAG group; (D–F) are RRL groups treated with 125, 250 and 500 mg/kg, respectively; (G) negative control of omitted first antibody; (H) negative control of omitted second antibody; (b) the quantitative analysis of α-SMA protein in lung tissues. Data represent the mean ± standard deviation (SD) (n = 3) (** p < 0.01 vs. normal group, # p < 0.05, ## p < 0.01, vs. model group).
Figure 5
Figure 5
Effects of RRL on the levels of α-smooth muscle actin (α-SMA) in the lung tissues after bleomycin (BLM)-induced in rats. (a) representative immunohistochemistry image (Bar = 50 μm). (A) normal group; (B) model group; (C) PAG group; (D–F) are RRL groups treated with 125, 250 and 500 mg/kg, respectively; (G) negative control of omitted first antibody; (H) negative control of omitted second antibody; (b) the quantitative analysis of α-SMA protein in lung tissues. Data represent the mean ± standard deviation (SD) (n = 3) (** p < 0.01 vs. normal group, # p < 0.05, ## p < 0.01, vs. model group).
Figure 6
Figure 6
Effects of RRL on the levels of matrix metalloproteinase-9 (MMP-9) in the lung tissues after bleomycin (BLM)-induced in rats. (a) representative immunohistochemistry image (Bar = 50 μm). (A) normal group; (B) model group; (C) PAG group; (D–F) are RRL groups treated with 125, 250 and 500 mg/kg, respectively; (G) negative control of omitted first antibody; (H) negative control of omitted second antibody; (b) the quantitative analysis of MMP-9 protein in lung tissues. Data represent the mean ± standard deviation (SD) (n = 3) (*** p < 0.001 vs. normal group, ### p < 0.001 vs. model group).
Figure 7
Figure 7
Effects of RRL on the levels of tissue inhibitors of metalloproteinase-1 (TIMP-1) in the lung tissues after bleomycin (BLM)-induced in rats. (a) representative immunohistochemistry image (Bar = 50 μm). (A) normal group; (B) model group; (C) PAG group; (D–F) are RRL groups treated with 125, 250 and 500 mg/kg, respectively; (G) negative control of omitted first antibody; (H) negative control of omitted second antibody; (b) the quantitative analysis of TIMP-1 protein in lung tissues. Data represent the mean ± standard deviation (SD) (n = 3) (*** p < 0.001 vs. normal group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. model group).
Figure 8
Figure 8
Expression level of TIMP-1, MMP-9 and α-SMA of the lung tissues in rats. (A) normal group; (B) model group; (C) PAG group; (D–F) are RRL groups treated with 125, 250 and 500 mg/kg, respectively.
Figure 9
Figure 9
Effects of RRL on the levels of TGF-β1 in the lung tissues after bleomycin (BLM)-induced in rats. (a) representative immunohistochemistry image (Bar = 50 μm). (A) normal group; (B) model group; (C) PAG group; (D–F) are RRL groups treated with 125, 250 and 500 mg/kg, respectively; (G) negative control of omitted first antibody; (H) negative control of omitted second antibody. Note the absence of the brown coloration; (b) the quantitative analysis of TGF-β1 protein in lung tissues. Data represent the mean ± standard deviation (SD) (n = 3) (*** p < 0.001 vs. normal group, # p < 0.05, ### p < 0.001 vs. model group).
Figure 10
Figure 10
The docking results of the seven compounds interacting with the target protein fibroblast activation protein-α (FAP-α). (A) 3,4,5-trihydroxybenzoic acid (2S-cis)-3,4-dihydro-5,7-dihydroxy -2-(3,4,5-trihydroxyphenyl)-2H-benzo[b]pyran-3-yl ester; (B) Herbacetin-3-O-β-d-glucopyranoside-7-O-α-l-rhamnoside; (C), Kaempferol-7-O-α-l-rhamnoside; (D) Rhodiocyanoside A; (E) Rosarin; (F) Rosavin; (G) Rhodionin.
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