Andrographolide attenuates epithelial‐mesenchymal transition induced by TGF‐β1 in alveolar epithelial cells

Abstract Andrographolide (Andro), a component from Chinese medicinal herb Andrographis paniculata, could alleviate pulmonary fibrosis in rodents. Yet, whether and how Andro mitigates epithelial‐mesenchymal transition (EMT) induced by TGF‐β1 remain unknown. This study aimed to explore the effect of Andro on TGF‐β1‐induced EMT in human alveolar epithelial cells (AECs) and the mechanisms involved. We illustrated that Andro inhibited TGF‐β1‐induced EMT and EMT‐related transcription factors in alveolar epithelial A549 cells. Andro also reduced TGF‐β1‐induced cell migration and synthesis of pro‐fibrotic factors (ie CCN‐2, TGF‐β1), matrix metalloproteinases (ie MMP‐2, MMP‐9) and extracellular matrix (ECM) components (ie collagen 1), implying the inhibiting effect of Andro on TGF‐β1‐induced EMT‐like cell behaviours. Mechanistically, Andro treatment not only repressed TGF‐β1‐induced Smad2/3 phosphorylation and Smad4 nuclear translocation, but also suppressed TGF‐β1‐induced Erk1/2 phosphorylation and nuclear translocation in A549 cells. And treatment with ALK5 inhibitor (SB431542) or Erk1/2 inhibitors (SCH772984 and PD98059) remarkably reduced EMT evoked by TGF‐β1. In addition, Andro also reduced TGF‐β1‐induced intracellular ROS generation and NOX4 expression, and elevated antioxidant superoxide dismutase 2 (SOD2) expression, demonstrating the inhibiting effect of Andro on TGF‐β1‐induced oxidative stress, which is closely linked to EMT. Furthermore, Andro remarkably attenuated TGF‐β1‐induced down‐regulation of sirtuin1 (Sirt1) and forkhead box O3 (FOXO3), implying that Andro protects AECs from EMT partially by activating Sirt1/FOXO3‐mediated anti‐oxidative stress pathway. In conclusion, Andro represses TGF‐β1‐induced EMT in AECs by suppressing Smad2/3 and Erk1/2 signalling pathways and is also closely linked to the activation of sirt1/FOXO3‐mediated anti‐oxidative stress pathway.


| INTRODUC TI ON
Pulmonary fibrosis (PF) is one of chronic maladies, which is characterized by an extravagant accumulation of extracellular matrix (ECM), causing scarring and damage of lung architecture, thereby limiting gas exchange. 1 Eventually, most of the patients with PF die of irreversible deterioration of pulmonary function. 2 Now, the occurrence of PF around the world is gradually increasing and the median survival is probably 3-5 years. 3 Hence, the treatment of PF remains a major issue to be addressed.
Epithelial-mesenchymal transition (EMT) is a critical pathological process in PF. 4,5 It is characterized by numerous molecular and pathophysiological alterations influencing cell phenotype and behaviour, during which epithelial cells gradually lose the original characteristics and have mesenchymal properties. 6 EMT can be induced within alveolar epithelial cells (AECs) by hypoxia, reactive oxygen species (ROS) and transforming growth factor (TGF)-β1. 7,8 Among these factors, TGF-β1, mainly secreted by epithelial cells, fibroblasts and macrophages, is markedly up-regulated in the lung tissues of PF patients and is the most latent inductor of EMT during PF progression. Both TGF-β1-treated primary AECs and alveolar epithelial A549 cells were observed a transition from epithelial cell morphology to a mesenchymal cell phenotype. 5,9 TGF-β1-induced EMT is largely regulated by Smad-dependent or Smad-independent pathways. 10,11 Hence, inhibition of TGF-β1-dependent EMT process may be a promising treatment strategy for PF.
Andrographolide (Andro) ( Figure 1A) is a diterpenoid derived from Andrographis paniculata, which has been shown to have numerous bioactivities such as anti-inflammatory, antioxidant and anti-EMT properties. [12][13][14] Recently, Andro was found to inhibit bleomycin (BLM)-induced PF in rodents by inhibiting inflammatory response, oxidative stress, fibroblast proliferation and differentiation. [15][16][17] Andro was also found to mitigate silica-induced PF by inhibiting inflammation, oxidative stress and EMT phenotype. 18 Nevertheless, the direct effects of Andro on the TGF-β1-induced EMT within AECs remain unknown.
The goal of this study was to explore the direct effects of Andro on the TGF-β1-induced EMT in AECs and explore the feasible molecular mechanisms. We illustrated that Andro ameliorated TGF-β1-induced EMT in AECs through suppressing Smad2/3 and Erk1/2 signalling pathways. Andro also activated Sirt1/FOXO3-mediated anti-oxidative stress pathway to suppress EMT in AECs.

| Chemicals and reagents
Andro was purchased from Chengdu Herbpurify Co., Ltd

| Cell culture
Human A549 cells were bought from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and grew in DMEM (Gibco, NY, USA) with 10% FBS (Biochrom, Berlin, Germany), 100 KU/L penicillin and 100 mg/L streptomycin at 37°C in 5% CO 2 .

| Morphological analysis
A549 cells were seeded into a six-well plate and incubated with 2, 5 and 10 μM of Andro in the presence and absence of TGF-β1 (5 ng/mL; Novoprotein) for 24 h. Cell morphology was randomly captured with an inverted microscope (Leica, Wetzlar, Germany).

| Immunofluorescence staining
Immunofluorescence staining was performed as described previously. 17

| Wound healing assay
A549 cells were cultured in a six-well plate and treated with different concentrations of Andro (2, 5 or 10 μM) in the presence and absence of TGF-β1 as indicated. The wound healing assays were performed as described by Yang et al 19 The wounds were observed using bright field microscopy (Leica, Weztlar, Germany), and multiple images were obtained at areas flanking the intersections of the wound and the marker lines after the scratch at 0, 6, 12 and 24 hours. Images were obtained for analysis using ImageJ software. After incubation for 20 minutes in the dark, the cells were washed three times with PBS, and the ROS level was examined with a fluorescence microscope.

| Real-time qPCR
Real-time qPCR was performed as described previously. 17 Briefly, total RNA was extracted from A549 cells using TRIzol reagent

| Western blot
Western blot was performed as described previously. 17 Briefly, the samples of A549 cells were homogenized in radioimmune precipita-

| Data and statistical analysis
Data were expressed as means ± SEM. Statistical analysis was performed with one-way ANOVA. Differences between means were accept as significant at P < 0.05.

| Effect of Andro on the viability of alveolar epithelial A549 cells
To investigate the cytotoxicity of Andro in AECs, human alveolar epithelial A549 cells were incubated with varying concentrations of Andro (0, 1, 2, 5, 10 and 20 μM) for 24 hours. CCK-8 assay showed that Andro induced no significant cytotoxicity at the concentrations of 1, 2, 5 and 10 μM ( Figure 1B), indicating that at the maximum concentration of 10 μM Andro did not affect A549 cell viability. Therefore, in the follow-up experiments, the highest concentration of Andro we used was 10 μM.

| Andro represses TGF-β1-induced EMT and EMT-related transcription factors in A549 cells
We assessed whether Andro repressed TGF-β1-induced EMT in A549 cells. TGF-β1-induced EMT was confirmed by the surviving cell's phenotypic changes from an epithelial shape to a fibroblast-like shape  Figure 2F,G,J,K). Additionally, we found that membrane-bound E-cadherin ( Figure 2C) was significantly elevated, whereas nucleus E-cadherin and β-catenin were down-regulated by Andro treatment ( Figure 2D). Collectively, these data illustrated that Andro has a critical impact on inhibiting TGF-β1-induced EMT and on preserving the epithelial phenotype.

| Andro regulates TGF-β1-induced EMT-like cell behaviours in A549 cells
Apart from phenotypic alterations, AECs also produce various profibrotic cytokines/growth factors such as TGF-β1 and connective tis-
Taken together, these data suggest that the inhibition of Smad2/3 by Andro is involved in the attenuation of EMT in epithelial A549 cells.
These data indicate that Andro repressed TGF-β1-induced activation of Erk1/2 signalling to inhibit EMT in AECs.

| Andro reduces TGF-β1-induced ROS generation and modulates the oxidant/antioxidant imbalance in A549 cells
Since ROS can mediate many cellular functions including EMT, we investigated whether Andro affects ROS synthesis during TGF-β1induced EMT. TGF-β1 dramatically increased ROS levels in A549 cells, which was reduced by Andro intervention ( Figure 6A). Similarly, fluorescence microscopy revealed that TGF-β1 significantly enhanced DCF intensity, and Andro repressed this effect to near basal levels ( Figure 6B). ROS can be induced by the imbalanced oxidant/antioxidant enzymes, such as enhanced NADPH oxidase 4 (NOX4) activity and reduced SOD2 levels. We, therefore, investigated whether the inhibition of ROS production by Andro is due to the regulation of the oxidant/ antioxidant imbalance. Western blot analysis showed that TGF-β1 significantly enhanced NOX4 protein expression and decreased SOD2 protein levels; however, these changes were ameliorated by Andro treatment ( Figure

β1-induced EMT in A549 cells
Sirt1/FOXO3 signalling can regulate oxidative stress and EMT. 10, 22 We, therefore, determined Sirt1 and FOXO3 expressions in the absence or presence of Andro in response to TGF-β1 stimulation. The protein expressions of Sirt1 and FOXO3 were significantly decreased in TGF-β1-treated A549 cells. Expression levels of Sirt1 and FOXO3 were elevated to near baseline by Andro treatment (Figure 7). These data suggest that Andro activated Sirt1/FOXO3-mediated anti-oxidative stress pathway to suppress TGF-β1-induced EMT in A549 cells.

| D ISCUSS I ON
Epithelial-mesenchymal transition has been cumulatively considered as a mechanism for fibrotic diseases, and it plays a critical role in the development of PF. 8 Because TGF-β1 is a primary inducer of PF, Pulmonary fibrosis is characterized by extravagant accumulation of fibroblasts/myofibroblasts and ECM in the lung that destroys its architecture and function. 23 Although the pathogenesis of PF is largely unknown, epithelial cells, in particular type II AECs, have been identified as one of the critical players in this disease. 23 Primary human type II AECs undergo a TGF-β1-dependent EMT and thus acquire the ability to produce collagens, promoting PF. 8,24 Hence, TGF-β1 was widely used as a stimulator to trigger EMT in human type II AECs. 25

Alveolar epithelial cells, for example A549 cells, in response to
TGF-β1, can also undergo that EMT and differentiate into fibroblasts and myofibroblasts. 11 Recent studies have shown that Andro attenuated silica-induced EMT in mice, 18 and antagonized cigarette smoke-induced EMT and pulmonary dysfunction through inhibiting HOTAIR. 26 Nevertheless, the direct effects of Andro on TGF-β1-induced EMT in AECs remain uncertain. Here, we found that Andro treatment β-catenin to mediate cell adhesion. 33 Hence, we speculated that the overall enhancement in E-cadherin level by Andro attributes to the up-regulation of membrane-bound E-cadherin. In accordance with our hypothesis, Andro remarkably decreased E-cadherin expression in cytosolic and nuclear fractions whereas elevated its expression in the cell membrane fraction. Our speculation was also confirmed by the results that Andro-treated A549 cells also had an increase in the E-cadherin-binding protein, β-catenin in the membrane, and decreased β-catenin level in the nuclear.
The mechanism by which Andro inhibits TGF-β1-induced EMT while Smad3 silencing decreased Nox4 expression and prevented oxidative stress-induced damage. 47 Additionally, inhibition of Smad3 expression using SB431542 reversed TGF-β1-induced decrease in FOXO3 in A549 cells. 10 Similarly, Erk1/2 pathway, another specific SIRT1 target, is also involved in the regulation of FOXO3, and thus reduced oxidative stress and EMT. 48 These findings indicate that the down-regulation of Smad2/3 and Erk1/2 by Andro might be also involved in the regulation of FOXO3 and the alleviation of oxidative stress in A549 cells. In return, the reduction of oxidative stress by Andro may contribute to the inactivation of Smad2/3 and Erk1/2 signalling pathways as well, since inhibition of ROS production by NAC could inhibit the phosphorylation of Smad2/3 and Erk1/2.
In summary, we demonstrate firstly that Andro attenuates TGF-β1-induced EMT in alveolar epithelial A549 cells by suppressing TGF-β1-activated Smad2/3 and Erk1/2 signalling pathways. Andro also decreases ROS generation in TGF-β1-induced EMT process. We propose that Andro is a promising pharmacological tool for the treatment of PF.

ACK N OWLED G EM ENT
This research was supported by National Natural Science Foundation of China (grant no. 81703792).

CO N FLI C T S O F I NTE R E S T
There are no conflicts of interest to declare.