Chalcone flavokawain A attenuates TGF‐β1‐induced fibrotic pathology via inhibition of ROS/Smad3 signaling pathways and induction of Nrf2/ARE‐mediated antioxidant genes in vascular smooth muscle cells

Abstract TGF‐β1 plays a crucial role in the pathogenesis of vascular fibrotic diseases. Chalcones are reportedly cancer chemo‐preventive food components that are rich in fruits and vegetables. In this study, flavokawain A (FKA, 2‐30 μM), a naturally occurring chalcone in kava extracts, was evaluated for its anti‐fibrotic and antioxidant properties in TGF‐β1‐stimulated vascular smooth muscle (A7r5) cells, as well as its underlying molecular mechanism of action. Immunofluorescence data showed down‐regulated F‐actin expression with FKA treatment in TGF‐β1‐stimulated A7r5 cells. Western blotting demonstrated that FKA treatment suppressed the expression of α‐SMA and fibronectin proteins under TGF‐β1 stimulation. Findings from wound‐healing and invasion experiments showed that FKA inhibits TGF‐β1‐mediated migration and invasion. Western blotting demonstrated that treatment with FKA down‐regulated MMP‐9 and MMP‐2 and up‐regulated TIMP‐1 expression. Further evidence showed that FKA decreased TGF‐β1‐mediated phosphorylation and the transcriptional activity of Smad3. TGF‐β1‐induced excessive ROS production was remarkably reversed by FKA treatment in A7r5 cells, and inhibition by FKA or N‐acetylcysteine (NAC) substantially diminished TGF‐β1‐induced p‐Smad3 activation and wound‐healing migration. Interestingly, FKA‐mediated antioxidant properties were associated with increased nuclear translocation of Nrf2 and elevated antioxidant response element (ARE) luciferase activity. Activation of Nrf2/ARE signaling was accompanied by the induction of HO‐1, NQO‐1 and γ‐GCLC genes in FKA‐treated A7r5 cells. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished the FKA‐mediated antioxidant effects, indicating that FKA may inhibit TGF‐β1‐induced fibrosis through suppressing ROS generation in A7r5 cells. Our results suggested that anti‐fibrotic and antioxidant activities of the chalcone flavokawain A may contribute to the development of food‐based chemo‐preventive drugs for fibrotic diseases.


| INTRODUCTION
Fibrosis can be described as a non-physiological scarring process in response to chronic diseases, wherein excessive extracellular matrix (ECM) deposition contributes to irreversible tissue damage and the malfunction of vital organs, including the liver, heart, lung, kidney and skin. 1,2 Various chemokines and growth factors are primarily involved in the onset and progression of fibrotic pathology; the cytokine transforming growth factor-β (TGF-β) is a crucial regulator of all types of fibrosis. 3 TGF-β family members are multifunctional proteins that regulate diverse cellular functions, including cell division, migration, invasion, adhesion, promotion of ECM production, tissue homeostasis and embryogenesis. [4][5][6] Of the 3 isoforms (TGF-β1, 2 and 3), TGF-β1 is the most important isoform for the cardiovascular system and is present in vascular smooth muscle cells, endothelial cells, myofibroblasts, macrophages and other hematopoietic cells. 6 Perturbation of TGF-β1 signaling pathways has been implicated in diverse human diseases, including fibrotic, autoimmune and cardiovascular diseases. 4,5 In particular, TGF-β1 exerts pleiotropic effects on cardiovascular cells and promotes vascular fibrotic diseases, including hypertension, atherosclerosis, cardiac hypertrophy and heart failure. 4,6 TGF-β1-induced vascular fibrosis is characterized by cytoskeletal rearrangements and alterations in F-actin assembly. 7,8 The increased ED-A form of fibronectin (ECM proteins) by TGF-β1 is required for the enhancement of α-smooth muscle actin (α-SMA) and collagen during fibrotic changes and wound healing. 9,10 TGF-β1 is known to activate several ECM components, including fibronectin, an essential protein for enhancement of α-SMA involved in fibrosis. 9 Myofibroblasts, which express α-smooth muscle actin (α-SMA) and show an enormous capacity for producing ECM and collagen (types I and III), also inhibit the activity of matrix metalloproteinases (MMPs) and are known to be the main effector cells responsible for cardiac fibrosis. 11,12 At the molecular level, TGF-β predominantly transmits signals through cytoplasmic proteins called Smads, which play a key role in vascular fibrosis. In VSMCs, TGF-β1 increases the phosphorylation of receptor-associated Smads (Smad2 and Smad3), which form heterodimers with Smad4. This complex translocates into the nucleus and triggers the transcription of genes, including fibronectin and type 1 collagen, that are involved in vascular fibrosis. 13,14 TGF-β1 is extensively involved in the development of fibrosis in various organs by disturbing the homeostatic microenvironment and promoting cell migration, invasion or hyperplastic changes. 15,16 MMPs, a family of proteolytic enzymes, degrade the ECM components and play an important role in cell migration and invasion. TIMP (tissue inhibitor of MMP) reduces excessive ECM degradation, and an imbalance in the MMPs to TIMPs ratio underlies the pathogenesis of vascular fibrosis. 17 The fibrotic events associated with TGF-β1 are coincident with the induction of ROS-producing enzymes and/or reduction of ROS scavenging enzymes. 18,19 In these circumstances, the redox-sensitive protein nuclear factor E2-related factor 2 (Nrf2) is reported to be involved in the dynamics of fibrogenesis. 18 In the presence of ROS, Nrf2 disassociates from its bound form (Nrf2-Keap1 complex), translocates to the nucleus and binds to a small Maf protein/ARE to induce the expression of a battery of antioxidant genes, including γ-GCLC, HO-1 and NQO-1. [20][21][22] Supplementation of antioxidants appears to modulate the degree of plasticity and severity of fibrosis. 18 Therefore, inhibition of TGF-β1 signaling or TGF-β1mediated ROS/Smad3 signaling might be a potential therapeutic strategy to treat vascular fibrosis. 18 Flavokawain A (FKA) is a naturally occurring chalcone, extracted from Piper methysticum Forst, commonly known as kava-kava. It has been reported that the age-standardized incidence of cancer in three kava-drinking Pacific counties (Fiji, Vanuatu and Samoa) is significantly lower than in their neighboring counties (Australia and New Zealand). 23 The therapeutic actions of kava extracts have been attributed to the presence of various secondary metabolites, including chalcones (flavokawains), alkaloids and several unique lactones (kavalactones). 24 To date, three types of flavokawains (FKA, FKB and FKC) have been identified, and FKA is the predominant chalcone, constituting up to 0.46% of kava extracts. 25 Consumption of kava extracts (kavalactones, FKB and contaminant hepatotoxins) has been reported to produce hepatotoxicity in humans and animals, but convincing evidence of kava-induced hepatotoxicity has not yet been established. 26 Previous studies have demonstrated that FKA preferably inhibits the growth of various cancer cells with no or minimal effect on the growth of several normal and cancer cells. 25,27,28 Dietary feeding of FKA to mice resulted in no adverse effects on major organ functions, instead inducing phase II antioxidant enzymes in liver, lung, prostate and bladder tissues. 29 Nevertheless, the effects of FKA on vascular muscle fibrosis and signaling molecules involved in fibrogenesis have not yet been investigated. We hypothesized that FKA could alleviate TGF-β1-mediated ROS/Smad3 signaling, thereby preventing fibrotic pathology in vascular smooth muscle cells. We determined the key molecular proteins demonstrating antifibrotic effects in FKA and revealed the underlying induction of antioxidant Nrf2/ARE signaling pathways.

| Cell culture
A rat aortic smooth muscle cell line (A7r5) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in DMEM medium supplemented with 10% fetal calf serum, 100 μg/mL penicillin and 1 μg/mL streptomycin, at 37°C with 5% CO 2 in humidified conditions. Cultures were harvested and the cell number was determined by counting cell suspensions with a hemocytometer.

| Sample treatment
For all TGF-β1 stimulated experiments, the supernatant was removed after FKA supplementation for 2 h, the cells were washed with phosphate-buffered saline (PBS) and the culture medium was replaced with new medium and then stimulated with or without TGF-β1 (10 ng/mL) for 24 h.

| MTT assay
Cell viability was determined by the MTT colorimetric assay. Cells (5 × 10 4 cells/well in 24-well plates) were treated with the indicated concentration of FKA alone for 24 h, or pre-treated with FKA for 2 h and then stimulated with or without TGF-β1 (10 ng/mL) for 24 h. MTT (0.5 mg/mL) in PBS was added to each well. After incubation at 37°C for 4 h, an equal volume of DMSO (400 μL) was added to dissolve the MTT formazan crystals and the absorbance was mea-

| Protein isolation and Western blot analysis
A7r5 cells were seeded in a 10-cm dish at a density of 1 × 10 6 cells/ dish. Next, the cells were incubated with the indicated concentrations of FKA for 2 h, then stimulated with or without TGF-β1 (10 ng/mL) for 24 h. Cells were detached and washed once in ice-cold PBS and then re-suspended in 100 μL lysis buffer containing 10 mM Tris-HCl [pH 8], 0.32 M sucrose, 1% Triton X-100, 5 mM EDTA, 2 mM dithiothreitol and 1 mM phenylmethyl sulfonyl fluoride.
The suspension was kept on ice for 20 min and then centrifuged at 15,000 × g for 30 min at 4°C. Total protein content was determined using the Bio-Rad protein assay reagent, with bovine serum albumin as a standard. Protein extracts were reconstituted in sample buffer (0.062 M Tris-HCl, 2% SDS, 10% glycerol and 5% β-mercaptoethanol), and the mixture was boiled for 5 min. Equal amounts (50 μg) of the denatured proteins were loaded onto each lane, separated on 8%-15% SDS polyacrylamide gels, followed by transfer of the proteins to polyvinylidene difluoride membranes overnight. Membranes were blocked with 0.1% Tween-20 in PBS containing 5% non-fat dried milk for 20 min at room temperature, and the membranes were reacted with primary antibodies overnight. The membranes were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody for 2 h. The blots were detected using an ImageQuant ™ LAS 4000 mini (Fujifilm, Tokyo, Japan) with an Enhanced Chemiluminescence substrate (Millipore, Billerica, MA). Densitometry analyses were performed using commercially available quantitative software (AlphaEase, Genetic Technology Inc. Miami, FL), with the control set as 1-fold, as shown below.

| Luciferase activity assay of Smad3 and ARE
The Smad3 and ARE transcriptional activity was measured using a dual-luciferase reporter assay system (Promega, Madison, WI). A7r5 cells were cultured in 24-well plates that had reached 70%-80% confluence and incubated for 5 h with serum-free DMEM that did not contain antibiotics. The cells were then transfected with either a pcDNA vector or a Smad3 (pGL3-SBE4-Luc reporter vector) plasmid/ ARE plasmid with β-galactosidase using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After plasmid transfection, cells were pretreated with FKA 7.5 μM for 0.5 to 4 h and then stimulated with or without TGF-β1 (10 ng/mL) for 24 h. Following treatment, the cells were lysed, and their luciferase activity was measured using a luminometer (Bio-Tek instruments Inc, Winooski, VA). The luciferase activity was normalized to the β-galactosidase activity in cell lysate, which was considered the basal level (100%).

| In vitro wound-healing repair assay
To assess the cell migration, A7r5 cells were seeded into a 12-well culture dish and grown in DMEM containing 10% FBS to a nearly confluent cell monolayer. The cells were re-suspended in DMEM medium containing 1% FBS, and a "wound gap" in the monolayers was carefully scratched using a culture insert. Cellular debris was removed by washing with PBS. Then, the cells were incubated with a non-cytotoxic concentration of FKA (2-30 μM) for 2 h and stimulated with or without TGF-β1 (10 ng/mL) for 24 h. The migrated cells were imaged (200× magnification) at 0 and 24 h to monitor the migration of cells into the wounded area, and the closure of the wounded area was calculated.

| Cell invasion assay
Invasion assay was performed using BD Matrigel invasion chambers

| Measurement of intracellular ROS generation
The accumulation of intracellular ROS in A7r5 cells was quantified by a fluorescence spectrophotometer using DCFH 2 -DA as described previously. 30 Briefly, A7r5 cells at a density of 4 × 10 5 cells/well in 12-well plates were pre-treated with FKA (7.5 μM) for 2 h in the presence or absence of TGF-β1 (10 ng/mL) stimulation for 30 min.
Then, the non-fluorescent probe, DCFH 2 -DA (10 μM), was added to the culture medium, and the cells were incubated at 37°C for another 30 min. After incubation, the cells were washed with warm PBS, and the ROS production was measured by changes in fluorescence due to the intracellular production of DCF caused by the oxidation of DCFH 2 . The DCF fluorescence was measured via fluorescence microscopy (200 × magnification) (Olympus, Center Valley, PA, USA). The fold-increase in ROS generation was compared with the vehicle-treated cells, which were arbitrarily set as 1.

| Transient transfection of siRNA targeting Nrf2
A7r5 cells were transfected with Nrf2 siRNA using Lipofectamine were determined by Western blotting.

| Statistical analyses
The results are presented as the mean ± standard deviation (mean ± SD). The obtained values in this study were analysed using analysis of variance followed by Dunnett's test for pair-wise comparison. The results are significant at *P < 0.05, **P < 0.01 and ***P < 0.001 compared to control cells and significant at # P < 0.05, ## P < 0.01 and ### P < 0.001 compared to TGF-β1-treated cells.  Figure 1C). FKA treatment (0-30 μM) significantly suppressed the TGF-β1 (10 ng/mL)-induced increased viability of A7r5 cells ( Figure 1D).

| Effects of FKA on the viability of SMC (A7r5) cells with or without TGF-β1-stimulation
These results indicated that FKA is not cytotoxic for A7r5 cells, despite preventing the excessive growth induced by TGF-β1.

| FKA suppresses TGF-β1-induced fibrosis via inhibition of F-actin, α-SMA and fibronectin in A7r5 cells
TGF-β1-induced vascular fibrosis is characterized by cytoskeletal rearrangements and alterations in F-actin assembly. 7,8 To determine TGF-β1-induced fibroblastic-type cytoskeletal rearrangements, we detected the F-actin structure and appearance in A7r5 cells by immunofluorescence staining. Cells following TGF-β1 treatment (10 ng/mL, 24 h) showed much thicker central stress fibers that were mostly oriented in parallel to the long axis of the cells (Figure 2A). Interestingly, the abnormal F-actin structure (stress fiber disruption) was not seen in FKA pre-treated (7.5 μM) cells (Figure 2A). Control cells without TGF-β1 stimulation showed randomly oriented cytoplasmic fibers.
TGF-β1 is known to activate several ECM components, including fibronectin, an essential protein for enhancement of α-SMA that is involved in fibrosis. 9 To define the effects of FKA on TGF-β1- The results are presented as the mean ± SD of three assays. Significant at ***P < 0.001 compared to control cells; significant at #P < 0.05 and ###P < 0.001 compared to TGF-β1-treated cells HSEU ET AL.
| 779 provide molecular evidence for the anti-fibrotic properties of FKA, we evaluated the Smad3 signaling in TGF-β1-stimulated A7r5 cells.
Cells were treated with FKA (0-30 μM, 2 h) prior to TGF-β1 stimulation (10 ng/mL), and then, changes in phosphorylated Smad3 and total Smad3 levels were determined. We found that TGF-β1 induced an enormous increase in p-Smad3 levels (>5-fold) in whole cell lysates, which was dose-dependently abolished by FKA pretreatment ( Figure 3A). Next, we assayed the Smad3 transcriptional activity with a luciferase reporter construct that was stably transfected into A7r5 cells. The results from the luciferase reporter assay revealed that TGF-β1 (10 ng/mL) stimulation profoundly increased the Smad3  Figure 4A and B). We found that TGF-β1-induced substantial increase in A7r5 cell migration (>3-fold), which was significantly inhibited by FKA pretreatment, particularly at 7.5 and 30 μM doses ( Figure 4A and B).
We further determined, via transwell invasion assay, the ability of cells to pass through a layer of ECM on a Matrigel-coated filter in FKA pre-treated (0-30 μM) cells following TGF-β1 stimulation (10 ng/ mL). Figure 5A  (p-Smad3) and total Smad3 protein levels were evaluated by Western blotting. B, Transcriptional activity of Smad3 (pGL3-SBE4-Luc) was monitored by luciferase reporter assay. Following plasmid transfection, cells were pre-treated with FKA and then stimulated with or without TGF-β1. Luciferase activity was determined and normalized to β-gal activity and shown as relative luciferase activity. C, p-Smad3 and total Smad3 protein levels were determined in nuclear and cytosolic fractions of cells. Histone and β-actin were used as controls. Relative changes in protein intensities were quantified using AlphaEaseFc 4.0 software and presented as a histogram, with a control set to onefold. Significant at **P < 0.01 and ***P < 0.001 compared to control cells; significant at ##P < 0.01 and ###P < 0.001 compared to TGF-β1-treated cells induced MMP-9 and MMP-2 elevation ( Figure 6A and B). Furthermore, inhibition of MMP activation with FKA was accompanied by a significant restoration of TIMP-1 levels in TGF-β1-treated cells ( Figure 6C).
These results demonstrate that FKA is able to inhibit TGF-β1-induced vascular cell migration and invasion, possibly through the inhibition of MMP-9/-2 activation and restoration of TIMP-1 degradation.

ROS production in A7r5 cells
It has been well-documented that increased ROS production is interlinked with TGF-β1 production and signaling, which may synergistically participate in the onset of fibrotic events. 2 Since ROS are key instigators in the pathophysiology of fibrosis, we determined whether FKA is able to inhibit the TGF-β1-induced ROS production in A7r5 cells. We found that TGF-β1 treatment (10 ng/mL) alone enormously increased (~6-fold) the intracellular ROS production, which was represented by increased DCF fluorescence ( Figure 7A and B). Nevertheless, FKA pretreatment (7.5 μM) resulted in decreased fluorescence intensity in A7r5 cells, indicating diminished TGF-β1-induced ROS production ( Figure 7A). Decreased ROS production with FKA was similar to the ROS levels observed in NAC pre-treated TGF-β1-stimulated cells. This evidence shows that FKA potently suppressed ROS production in a similar fashion to NAC.
The ROS scavenging activity of FKA may be involved in the inhibition of TGF-β1-induced ROS-mediated fibrotic pathology.

| Inhibition of ROS production by FKA obliterates nuclear localization of Smad3 in TGF-β1activated A7r5 cells
To

| Inhibition of ROS production by FKA diminishes TGF-β1-induced wound healing migration in A7r5 cells
It has been described that ROS signaling is involved in TGF-β1mediated fibrosis through activation of proliferation, migration and/

B
F I G U R E 7 FKA suppresses TGF-β1-induced intracellular ROS production in A7r5 cells. A-B, Cells were preincubated with or without FKA (7.5 μM) or NAC (2 mM) for 2 h and then stimulated with TGF-β1 (10 ng/mL) for 30 min. A, Intracellular ROS levels were indicated by DCF fluorescence and measured by fluorescence microscopy (200× magnification). B, The percentage of fluorescence intensity of DCF-stained cells was quantified by Olympus Soft Image Solution software. The percentage of fluorescence intensity (ROS) was compared with that of control cells, which were arbitrarily assigned a value of one. The results are presented as the mean ± SD of three assays. Significant at ***P < 0.001 compared to control; significant at ###P < 0.001 compared to TGF-β1-treated cells nuclear localization of Nrf2 was visualized using confocal microscopy. Immunofluorescence images confirmed that FKA treatment enhanced the nuclear accumulation of Nrf2, as indicated by strong Nrf2-staining in FKA-treated cells ( Figure 10C).

| FKA triggers ARE promoter activity and upregulates HO-1, NQO-1 and γ-GCLC expression levels in A7r5 cells
ARE, a cis-acting element, together with Nrf2 induces many antioxidant genes in response to chemical stress. The Nrf2-ARE transcriptional pathway plays an essential role in the regulation of antioxidant genes and elimination of ROS. 20 Being a potent Nrf2 activator, we hypothesized that FKA could augment ARE promoter activity and subsequently up-regulate antioxidant genes in vascular smooth muscle cells. ARE promoter activity was assayed in luciferase reporter co-transfected cells following FKA treatment (7.5 μM) for 0-4 h.
The results revealed that FKA treatment of A7r5 cells significantly augmented the luciferase activity derived from the ARE promoter ( Figure 11A).
Western blot data showed that all antioxidant genes were timedependently up-regulated by FKA treatment. In particular, HO-1 and γ-GCLC expression levels were gradually increased until 12 h, whereas NQO-1 appeared to be up-regulated until 16 h following treatment ( Figure 11B). Based on these findings, we demonstrated that FKA stimulates Nrf2/ARE transcriptional activation to promote antioxidant gene expression in human A7r5 cells. TGF-1 (10 ng/mL) F I G U R E 8 FKA impairs nuclear localization of Smad3 in TGF-β1-stimulated A7r5 cells. Cells were pre-treated with FKA (7.5 μM) or NAC (2 mM) for 2 h and then stimulated with or without TGF-β1 (10 ng/mL) for 24 h. Immunofluorescence staining was performed to detect the nuclear localization of Smad3. Following incubation with primary antibody (anti-Smad3) and conjugated secondary antibody, cells were stained with DAPI (1 μg/mL) for 5 min. The sub-cellular localization of Smad3 in all conditions was visualized under fluorescence microscopy (200 × magnification). The results are presented as the mean ± SD of three assays F I G U R E 9 Inhibition of ROS production by FKA diminishes TGF-β1-induced wound healing migration in A7r5 cells. Cells were pre-incubated with or without FKA (7.5 μM) or NAC (2 mM) for 2 h and then stimulated with TGF-β1 (10 ng/mL) for 24 h. A, Cells that migrated to the lower membrane were photographed (200× magnification). B, The percentage of migrated cells was quantified and expressed relative to untreated cells (control), which were set at onefold. To quantify migration, cells were counted in three microscopic fields per sample. The results are presented as the mean ± SD of three assays. Significant at *P < 0.05 and **P < 0.01 compared to control cells; significant at ###P < 0.001 compared to TGF-β1-treated cells

| Nrf2 knockdown diminishes FKA-induced antioxidant responses in A7r5 cells
Nrf2 signaling is critically involved in the ROS-mediated regulation of fibrosis; indeed, a lack of Nrf2 expression amplifies oxidative stress and exacerbates fibrotic pathology. 3 To emphasize the importance of

| DISCUSSION
Kava has been used to treat mild to moderate anxiety, insomnia and muscle fatigue in Western countries, which has led to its emergence as one of the ten best-selling herbal preparations. Several reports of severe hepatotoxicity in kava consumers led the U.S. Food and Drug Administration and authorities in Europe to restrict the sales of kava-containing products. 31 The maximum tolerant doses of some chalcones in rodents were shown to be more than 1-3 g/kg body weight. 29 FKA exhibited in vivo anti-tumor activity in a bladder cancer xenograft model and in the UPII-SV40T transgenic bladder cancer mouse model. 32 A recent study showed that FKA significantly inhibited LPS-induced activation of NF-kB, AP-1 and JNK/p38 MAPK signaling pathways. 33 Notably, FKA is more effective than FKB in promoting Nrf2 activation, antioxidant genes (HO-1 and γ-GCLC) and GSH levels. 24 Here, we hypothesized that FKA could alleviate TGF-β1-mediated ROS/Smad3 signaling, thereby preventing fibrotic pathology in vascular smooth muscle cells.
TGF-β1 plays a vital role in tissue remodeling in injured tissues, regulating cell growth and fibrosis. However, aberrant regulation of TGF-β1 leads to pathologic fibrosis via increased cell proliferation and excessive accumulation of ECM proteins. [34][35][36] The TGF-β1 sig- Relative changes in protein intensities were quantified using AlphaEaseFc 4.0 software and presented as a histogram, with the control set at onefold. C, Immunofluorescence staining to detect the Nrf2 nuclear translocation. Cells were exposed to (7.5 μM) for 0.5 or 1 h, fixed and permeabilized. Cells were incubated with anti-Nrf2 antibody followed by FITC-labeled secondary antibody and stained with DAPI (1 μg/mL) for 5 min. Then, the sub-cellular localization of Nrf2 was visualized using a confocal microscope (630× magnification). All results are presented as the mean ± SD of three assays. Significant at ***P < 0.001 compared to control cells and Smad4 acts as a common mediator of TGF-β1. 37 It has also been well documented that TGF-β1 increases ROS levels, which mediate the profibrogenic effects of TGF-β1 via a Smad pathway. 38  In addition, fibronectin impacts the tensile strength in smooth muscle cells 42 and can increase cytoskeletal organization and mechanical tension generation by cells. 43 In leiomyomas, mechanotransduction appears to regulate Smad3 activity 44 ; indeed, in other smooth muscle cells, Smad3 activation correlated with fibronectin polymerization. These data suggest that fibronectin cannot only impact ECM formation but can also regulate intracellular function within the leiomyocyte. 44 In further support of the role fibronectin plays in the interrelationship between the intracellular structure and ECM, disruption of cytoskeletal actin also disrupts fibronectin matrix organization, 45 while fibronectin can regulate smooth muscle cell entry into the cell cycle. 46 Here, we found that the FKA-induced suppression of fibronectin expression was mediated by inactivation of the Smad3 pathways in fibrosis development. Regulation of fibronectin by FKA provides a mechanism whereby FKA can impact fibrosis, ECM production and intracellular regulation.
Vascular muscle fibrosis is due to both excessive collagen synthesis and abnormal collagen turnover by matrix degrading enzymes, such as matrix metalloproteinases (MMPs). 47  F I G U R E 1 1 FKA up-regulates HO-1, NQO-1 and γ-GCLC genes following Nrf2/ARE activation, which is diminished with Nrf2 knockdown in A7r5 cells. A, The luciferase activity of ARE was measured after FKA treatment (7.5 μM) for 0.5-4 h. Luciferase activity was determined, normalized by β-gal activity and shown as relative luciferase activity. B, Cells were incubated with FKA (7.5 μM) for 1-16 h, and total cell lysate was subjected to Western blotting to monitor the changes in HO-1, NQO-1 and γ-GCLC proteins using specific antibodies. C, Cells were transfected with a specific siRNA against Nrf2 or a non-silencing control. Following transfection (24 h), the cells were incubated with or without FKA (7.5 μM) for the indicated time. Total Nrf-2 (0.5 h), HO-1 (8 h), NQO-1 (8 h) and γ-GCLC (8 h) were evaluated by Western blotting. The results are presented as the mean ± SD of three assays. Significant at **P < 0.01 and ***P < 0.001 compared to control cells degradation of ECM at wound sites, and the imbalance of MMP and TIMP results in scar formation. Other studies have indicated that TGF-β1 activates ECM in human glioma. 50,51 However, the effects of FKA on TGF-β1-induced vascular muscle cells are unknown. Here, we report that FKA attenuates migration and invasion in TGF-β1treated vascular cells. More interestingly, cells treated with FKA had reductions in TGF-β1 induced MMP-9/-2 activity via down-regulation of Smad3. As an early biomarker of fibrosis, TGF-β1 actuates synthesis of ECM components, such as MMPs. Early examination has shown that chalcone inhibited TGF-β1-induced fibrosis on diabetic nephropathy. 52,53 Here, we found that the chalcone FKA decreased the expression of fibronectin and down-regulated MMP-9/-2 and upregulated TIMP-1 expression in TGF-β1-stimulated A7r5 cells, which indicated that FKA might target both TGF-β1 and its downstream pro-fibrotic proteins.
TGF-β1-induced Smad signaling in ROS is associated with fibrosis progress and development, with differential involvement of Smad3, depending on the cellular context. 54 In smooth muscle cells, Smad3 signaling has been shown to play a key role in the induction of TGF-β1-mediated tissue damage. 55  indicating Nrf2-mediated inhibition of TGF-β/Smad signaling. 57 Several studies have shown that Nrf2 activators can potentially inhibit fibrosis, and Nrf2-null mice were more susceptible to fibrosis than wild-type mice, which signify that Nrf2 is a potential target for treatment of fibrosis. [58][59][60][61] Interestingly, many studies have revealed that FKA exhibited anti-inflammatory and antioxidant activities by activating Nrf2 pathways. 24,31 In addition, recent reports have also shown that FKA could activate a Nrf2 reporter gene and induce the expression of HO-1 and NQO-1. [62][63][64] However, whether FKA activates Nrf2-ARE signaling to limit fibrosis has not been previously studied. 65 Our result suggests that Nrf2 translocated to the nucleus activating ARE reporter gene and inturn inducing the expression of HO-1, NQO-1 and γ-GCLC.
To summarize, we demonstrated that FKA inhibited TGF-β1 signaling and phosphorylation of Smad3 via upregulation of Nrf2, in turn leading to the activation of antioxidant genes in combating ROS. We elucidate the components of FKA-mediated inhibition of vascular tissue fibrosis, as well as different underlying sub-molecular mechanisms that might be investigated in further studies. Unquestionably, elucidating the relative roles of every unique pathway involved in the general effects of FKA in vascular muscle cell protection remains a challenging and important area of study.

ACKNOWLEDG EMENTS
We would like to thank the Editor Prof. Stefan N.

DISCLOSURES
All authors declare that they have no conflicts of interest.