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. 2019 Mar 5:10:151.
doi: 10.3389/fphar.2019.00151. eCollection 2019.

Wedelolactone Attenuates Pulmonary Fibrosis Partly Through Activating AMPK and Regulating Raf-MAPKs Signaling Pathway

Affiliations

Wedelolactone Attenuates Pulmonary Fibrosis Partly Through Activating AMPK and Regulating Raf-MAPKs Signaling Pathway

Jin-Yu Yang et al. Front Pharmacol. .

Abstract

Pulmonary fibrosis is common in a variety of inflammatory lung diseases, there is currently no effective clinical drug treatment. It has been reported that the ethanol extract of Eclipta prostrata L. can improve the lung collagen deposition and fibrosis pathology induced by bleomycin (BLM) in mice. In the present study, we studied whether wedelolactone (WEL), a major coumarin ingredient of E. prostrata, provided protection against BLM-induced pulmonary fibrosis. ICR or C57/BL6 strain mice were treated with BLM to establish lung fibrosis model. WEL (2 or 10 mg/kg) was given daily via intragastric administration for 2 weeks starting at 7-day after intratracheal instillation. WEL at 10 mg/kg significantly reduced BLM-induced inflammatory cells infiltration, pro-inflammatory factors expression, and collagen deposition in lung tissues. Additionally, treatment with WEL also impaired BLM-induced increases in fibrotic marker expression (collagen I and α-SMA) and decrease in an anti-fibrotic marker (E-cadherin). Treatment with WEL significantly prevented BLM-induced increase in TGF-β1 and Smad2/3 phosphorylation in the lungs. WEL administration (10 mg/kg) also significantly promoted AMPK activation compared to model group in BLM-treated mice. Further investigation indicated that activation of AMPK by WEL can suppressed the transdifferentiation of primary lung fibroblasts and the epithelial mesenchymal transition (EMT) of alveolar epithelial cells, the inhibitive effects of WEL was significantly blocked by an AMPK inhibitor (compound C) in vitro. Together, these results suggest that activation of AMPK by WEL followed by reduction in TGFβ1/Raf-MAPK signaling pathways may have a therapeutic potential in pulmonary fibrosis.

Keywords: AMPK; Eclipta prostrata; bleomycin; pulmonary fibrosis; wedelolactone.

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Figures

FIGURE 1
FIGURE 1
WEL attenuated bleomycin (BLM)-induced pulmonary fibrosis in ICR mice. One week after 5 mg/kg bleomycin (BLM) treatment, mice were orally administered with two doses of WEL-L (2 mg/kg/day) and WEL-H (10 mg/kg/day) and prednisone (PNS, 6 mg/kg) once a day for 7 or 21 days. (A) The chemical structure of WEL, changes of body weight (B), survival rate (C), and pulmonary index (D) were shown in different groups. (E) The HYP contents in lung tissues were determined by an assay kit. (F) The levels of pro-inflammatory cytokines (IL-1β, TNF-α, and TGF-β1) in lung tissue from different groups at day 14 were detected by ELISA assay. Data are shown as mean ± SD (n = 10). #p < 0.05, ##p < 0.01 vs. the control group; p < 0.05, ∗∗p < 0.01 vs. the BLM group.
FIGURE 2
FIGURE 2
WEL protects against bleomycin-induced pathological changes of lungs in ICR mice. One week after BLM treatment (5 mg/kg), mice were orally administered with WEL-L (2 mg/kg) or WEL-H (10 mg/kg) and prednisone (6 mg/kg, positive drug) once a day for 7 or 21 days. Representative pictures (×200) of HE-stained (A) and Masson’s trichrome-stained (B) lung sections from mice on day 14 or day 28 were shown. Bar = 100 μm. The inflammation (C) and fibrosis (D) score numbers of 0–3, corresponding to the grades of –, +, ++, and +++, were evaluated by experienced pathologists in a blinded fashion. Data are presented as the mean ± SD (n = 10). ##p < 0.01 vs. the control group; p < 0.05, ∗∗p < 0.01 vs. the BLM alone group.
FIGURE 3
FIGURE 3
WEL ameliorated bleomycin (BLM)-induced pulmonary fibrosis in C57/BL6 mice. One week after 5 mg/kg BLM treatment, mice were orally administered with WEL (10 mg/kg) once a day for 14 days. (A) Body weight, (B) survival rate, and (C) pulmonary index of BLM mice and BLM mice that received WEL were determinated on day 21 (n = 6). (D) Representative pictures (×200) of HE-stained and Masson’s trichrome-stained lung sections from mice on day 21 were shown. Bar = 100 μm. The inflammation (E) and fibrosis (F) score numbers of 0–3, corresponding to the grades of –, +, ++, and +++, were evaluated by experienced pathologists in a blinded fashion. (G) HYP contents in lung tissues were determined by a assay kit. The protein expressions (H) of α-SMA and collagen I (Col I) in lung tissues were determined by Western blotting. The mRNA levels (I) of α-SMA and collagen I (Col I) in lung tissues were determined by PCR analysis. Data are presented as mean ± SD (n = 9). p < 0.05, ∗∗p < 0.01.
FIGURE 4
FIGURE 4
WEL regulated TGF-β/Smad signaling pathway and AMPK activation in lung tissue in bleomycin-induced PF in C57/BL6 mice. One week after 5 mg/kg BLM treatment, mice were orally administered with WEL (10 mg/kg) once a day for 14 days. The protein expression of TGF-β1 (A) and the phosphorylation levels of Smad2/3 (C) in lung tissues were determined by Western blotting. (B) The mRNA levels of TGF-β1 in lung tissues were determined by PCR analysis. (D) The protein phosphorylation levels of AMPK in lung tissues were determined by Western blotting. Data are presented as mean ± SD (n = 9). p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
FIGURE 5
FIGURE 5
WEL ameliorated TGF-β-induced myofibroblast differentiation partly through Raf1-MAPK signaling pathway and AMPK activation in primary mouse lung fibroblasts (PLFs). The cells were pretreated with compound C (50 μM) or solvent for 1.5 h and subsequently incubated with/without TGF-β1 (10 ng/ml), WEL or solvent for 48 h. (A) The effect of WEL (0.1–100 μM) on PLFs proliferation cells were measured by the MTT assays. (B) The expression of α-SMA in PLFs treated with/without TGF-β1 was detected by Western blotting. (C) The inhibition of WEL or compound C on PLFs proliferation cells were measured by the MTT assays. (D) Expression of p-AMPK/AMPK in PLFs treated with/without TGF-β1 or compound C were determined by Western blotting. (E) The expression of α-SMA in PLFs treated with/without TGF-β1 or compound C were determined by Western blotting. (F) Protein expressions of Raf1, JNK/p-JNK, p38/p-p38, and ERK1/2/p-ERK1/2 in PLFs treated with/without TGF-β1 were detected by Western blotting. Data are presented as mean ± SD (n = 5). p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. NS, non-significant.
FIGURE 6
FIGURE 6
Regulation of WEL on the EMT of alveolar epithelial cells partly and its inflammation in TGF-β1 mediated MLE-12 cells. The cells were pretreated with compound C (50 μM) or solvent for 1.5 h and subsequently incubated with/without TGF-β1 (TGF, 10 ng/ml), WEL or solvent for 48 h. (A,B) Effects of WEL (0.1–100 μM) on proliferation cells were measured by the MTT assays. (C–J) Protein expressions and mRNA levels of α-SMA, Vimentin, Col I, and E-cadherin in MLE-12 cells treated with/without TGF-β1 were detected by Western blotting and PCR analysis. (K) Effect of compound C (0.1–100 μM) on proliferation cells were measured by the MTT assays. (L) The expression of α-SMA in MLE-12 cells treated with/without TGF-β1 or compound C were determined by Western blotting analysis. (M) Protein expressions of Raf1, JNK/p-JNK, p38/p-p38, and ERK1/2/p-ERK1/2 in MLE-12 treated with/without TGF-β1 were detected by Western blotting analysis. Data are presented as mean ± SD (n = 5). p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. NS, non-significant.

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