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. 2016 Jun;37(6):794-804.
doi: 10.1038/aps.2016.36. Epub 2016 May 2.

Paeoniflorin suppresses TGF-β mediated epithelial-mesenchymal transition in pulmonary fibrosis through a Smad-dependent pathway

Yu Ji  1 , Yan-Nong Dou  1 , Qian-Wen Zhao  1 , Ji-Zhou Zhang  1 , Yan Yang  1 , Ting Wang  1 , Yu-Feng Xia  2 , Yue Dai  1 , Zhi-Feng Wei  1
Affiliations

Paeoniflorin suppresses TGF-β mediated epithelial-mesenchymal transition in pulmonary fibrosis through a Smad-dependent pathway

Yu Ji et al. Acta Pharmacol Sin. 2016 Jun.

Abstract

Aim: Paeoniflorin has shown to attenuate bleomycin-induced pulmonary fibrosis (PF) in mice. Because the epithelial-mesenchymal transition (EMT) in type 2 lung endothelial cells contributes to excessive fibroblasts and myofibroblasts during multiple fibrosis of tissues, we investigated the effects of paeoniflorin on TGF-β mediated pulmonary EMT in bleomycin-induced PF mice.

Methods: PF was induced in mice by intratracheal instillation of bleomycin (5 mg/kg). The mice were orally treated with paeoniflorin or prednisone for 21 d. After the mice were sacrificed, lung tissues were collected for analysis. An in vitro EMT model was established in alveolar epithelial cells (A549 cells) incubated with TGF-β1 (2 ng/mL). EMT identification and the expression of related proteins were performed using immunohistochemistry, transwell assay, ELISA, Western blot and RT-qPCR.

Results: In PF mice, paeoniflorin (50, 100 mg·kg(-1)·d(-1)) or prednisone (6 mg·kg(-1)·d(-1)) significantly decreased the expression of FSP-1 and α-SMA, and increased the expression of E-cadherin in lung tissues. In A549 cells, TGF-β1 stimulation induced EMT, as shown by the changes in cell morphology, the increased cell migration, and the increased vimentin and α-SMA expression as well as type I and type III collagen levels, and by the decreased E-cadherin expression. In contrast, effects of paeoniflorin on EMT disappeared when the A549 cells were pretreated with TGF-β1 for 24 h. TGF-β1 stimulation markedly increased the expression of Snail and activated Smad2/3, Akt, ERK, JNK and p38 MAPK in A549 cells. Co-incubation with paeoniflorin (1-30 μmol/L) dose-dependently attenuated TGF-β1-induced expression of Snail and activation of Smad2/3, but slightly affected TGF-β1-induced activation of Akt, ERK, JNK and p38 MAPK. Moreover, paeoniflorin markedly increased Smad7 level, and decreased ALK5 level in A549 cells.

Conclusion: Paeoniflorin suppresses the early stages of TGF-β mediated EMT in alveolar epithelial cells, likely by decreasing the expression of the transcription factors Snail via a Smad-dependent pathway involving the up-regulation of Smad7.

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Figures

Figure 1
Figure 1
The chemical structure of paeoniflorin.
Figure 2
Figure 2
The effects of paeoniflorin (Pae) and prednisone (Pred) on the expression levels of FSP-1, α-SMA and E-cadherin on bleomycin-induced fibrosis in mouse lung tissues. On d 21 after BLM instillation, mice were sacrificed using an excess of chloral hydrate hydrochloride anesthesia, and their lung tissues were isolated. The expression and localization of FSP-1, α-SMA and E-cadherin protein in the lower left sides of lung tissues were examined using immunohistochemistry. n=6. ##P<0.01 vs normal. *P<0.05, **P<0.01 vs model.
Figure 3
Figure 3
The effects of paeoniflorin (Pae) on the cell viability in A549 cells. Cell viability was detected using MTT assays. A549 cells were seeded into 96-well plates and then incubated with serum-free RPMI-1640 for 2 h. Then, cells were incubated with Pae (1, 3, 10 and 30 μmol/L) in the presence or absence of TGF-β1 (2 ng/mL) for 20 h, and their viability was detected using MTT assays as described in the Methods section. (A) Cell viability was analyzed in A549 cells treated with Pae. (B) Cell viability was analyzed in A549 cells treated with Pae and TGF-β1. Data are expressed as the mean±SD. n=3.
Figure 4
Figure 4
The effects of different concentrations of paeoniflorin (Pae) on TGF-β1-induced epithelial-mesenchymal transition in A549 cells. A549 cells were seeded into plates and incubated with serum-free RPMI 1640 for 2 h. Then, the cells were incubated with TGF-β1 (2 ng/mL) or TGF-β1 (2 ng/mL) plus Pae (1, 3, 10 and 30 μmol/L) for the indicated intervals. (A) The expression levels of E-cadherin, Vimentin and α-SMA were detected using Western blot analysis. (B) The amounts of type I and type III collagen in the cell supernatants were detected using ELISA and Real-Time Quantitative PCR assays. Data are expressed as the means±SD. n=3. ##P<0.01 vs normal. *P<0.05, **P<0.01 vs TGF-β1.
Figure 5
Figure 5
Time course of the effect of paeoniflorin (Pae) on TGF-β1-induced epithelial-mesenchymal transition in A549 cells. A549 cells were seeded into plates and incubated with serum-free RPMI-1640 for 2 h. Then, the cells were incubated with TGF-β1 (2 ng/mL) or TGF-β1 (2 ng/mL) plus Pae (1, 3, 10 and 30 μmol/L) for the indicated intervals. (A) Cell morphology was recorded using an inverted microscope (magnification 200×). (B) The expression levels of E-cadherin, Vimentin and α-SMA were detected using Western blot analysis, as described in the Methods section. GAPDH was used as the internal control. (C) The protein levels of E-cadherin, Vimentin and α-SMA were determined using Real-Time Quantitative PCR, as described in the Methods section. (D) The cell supernatants were collected, and the amount of type I collagen was detected using ELISA, as described in the Methods section. The data are expressed as the mean±SD. n=3. ##P<0.01 vs normal. *P<0.05, **P<0.01 vs TGF-β1.
Figure 6
Figure 6
The effects of paeoniflorin (Pae) on the expression levels of transcription factors in TGF-β1-stimulated A549 cells. A549 cells were seeded into plates and incubated with serum-free RPMI-1640 for 2 h. Then, the cells were incubated with Pae (1, 3, 10 and 30 μmol/L) in the presence or absence of TGF-β1 (2 ng/mL) for the indicated time interval. The expression levels of Snail, Slug, ZEB1, ZEB2 and Twist mRNAs were measured using Real-Time Quantitative PCR, as described in the Methods section. (A) Time course showing Snail, Slug, ZEB1, ZEB2 and Twist mRNA expression. (B) The effects of Pae (1, 3, 10, and 30 μmol/L) on the expression levels of Snail, Slug, ZEB1, ZEB2 and Twist mRNAs. The data are expressed as the mean±SD, n=3. *P<0.05, **P<0.01 vs normal (in Figure 6A); #P<0.05, ##P<0.01 vs normal. *P<0.05, **P<0.01 vs TGF-β1 (in Figure 6B).
Figure 7
Figure 7
The effects of paeoniflorin (Pae) on the activation of the Smad-dependent pathway in A549 cells stimulated using TGF-β1. A549 cells were seeded into plates and incubated with serum-free RPMI-1640 for 2 h. Then, the cells were incubated with Pae (1, 3, 10 and 30 μmol/L) and stimulated using TGF-β1 (2 ng/mL) for the indicated time interval. The expression levels of p-Smad2 and p-Smad3 were detected using Western blot analysis. GAPDH was used as the internal control. (A) Time course showing p-Smad2 and p-Smad3 expression. (B) The effects of Pae (1, 3, 10, and 30 μmol/L) on p-Smad2 and p-Smad3 expression levels. The data are expressed as the mean±SD, n=3. #P<0.05, ##P<0.01 vs normal. *P<0.05, **P<0.01 vs TGF-β1.
Figure 8
Figure 8
The effects of paeoniflorin (Pae) on the activation of the Smad-independent pathway and the levels of Smad 7 and ALK5 in A549 cells stimulated by TGF-β1. A549 cells were seeded into plates and incubated with serum-free RPMI-1640 for 2 h. Then, the cells were incubated with Pae (1, 3, 10 and 30 μmol/L) and stimulated using TGF-β1 (2 ng/mL) for the indicated time interval. The expression levels of p-Akt/Akt, p-p38/p38, p-ERK/ERK and p-JNK/JNK were detected using Western blot analysis. GAPDH was used as the internal control. (A) Time course showing p-Akt/Akt, p-ERK/ERK, p-JNK/JNK and p-p38/p38 protein expression levels. (B) The effects of Pae (1, 3, 10, and 30 μmol/L) on p-Akt/Akt, p-Erk/Erk, p-JNK/JNK and p-p38/p38 protein expression levels. (C) The effects of Pae (1, 3, 10, and 30 μmol/L) on Smad7 and ALK5 levels. The data are expressed as the mean±SD, n=3. ##P<0.01 vs Normal, *P<0.05 vs TGF-β1.
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