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

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), L‐glutamine and penicillin/streptomycin were obtained from GIBCO BRL/Invitrogen (Carlsbad, CA, USA). Flavokawain A (FKA) was purchased from LKT Laboratories, Inc. (St Paul, MN, USA). TGF‐β1 was purchased from Pepro TechInc. (Rocky Hill, NJ, USA). Antibodies against phospho‐Smad3, Smad3, phospho‐Smad2 and Smad2 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against fibronectin, MMP‐9, MMP‐2, TIMP‐1, F‐actin, Nrf2, NQO‐1, Keap‐1, histone and β‐actin were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). 4′,6‐Diamidino‐2‐phenylindole dihydrochloride (DAPI) was obtained from Calbiochem (La Jolla, CA, USA). 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), dihydrofluorescein‐diacetate (DCFH₂‐DA) and N‐acetyl‐cysteine (NAC) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Antibodies against HO‐1 and α‐SMA were purchased from Abcam (Cambridge, UK). All other chemicals were purchased from either Merck & Co., Inc. (Darmstadt, Germany) or Sigma‐Aldrich (St. Louis, MO, USA).

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% CO₂ in humidified conditions. Cultures were harvested and the cell number was determined by counting cell suspensions with a hemocytometer.

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.

Cell viability was determined by the MTT colorimetric assay. Cells (5 × 10⁴ 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 measured at 570 nm (A₅₇₀) using an ELISA microplate reader (μ‐Quant, Winoosky, VT, USA). The percentage (%) of cell viability was calculated as: (A₅₇₀ of treated cells/A₅₇₀ of untreated cells) × 100.

A7r5 cells were seeded in a 10‐cm dish at a density of 1 × 10⁶ 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.

A7r5 cells (2 × 10⁴ cells/well) were seeded onto an 8‐well glass Tek chamber and pre‐treated with FKA (2‐30 μM) for 2 h and then stimulated with or without TGF‐β1 (10 ng/mL) for 24 h. After treatment, cells were fixed in 2% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X‐100 for 10 min and then incubated for 1 h with anti‐F‐actin, anti‐Smad3 or anti‐Nrf‐2 primary antibodies in 1.5% FBS. The cells were then incubated with a FITC (fluorescein isothiocyanate)‐conjugated (488 nm) secondary antibody for an additional 1 h in 6% bovine serum albumin. Following this, cells were stained with 1 μg/mL DAPI for 5 min. The stained cells were washed with PBS and visualized using a fluorescence microscope at 200× magnification.

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 pre‐treated 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%).

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.

Invasion assay was performed using BD Matrigel invasion chambers (Bedford, MA, USA). For the assay, 10 μL Matrigel (25 mg/50 mL) was applied to 8‐μm polycarbonate membrane filters, and 1 × 10⁵ cells were seeded to the Matrigel‐coated filters in 200 μL of serum‐free medium containing FKA (2‐30 μM) and/or TGF‐β1 (10 ng/mL). The bottom chamber of the apparatus contained 750 μL of complete growth medium. Cells were allowed to migrate for 24 h at 37°C. After 24 h incubation, the non‐migrated cells on the top surface of the membrane were removed with a cotton swab. The migrated cells on the bottom side of the membrane were fixed in cold 75% methanol for 15 min and washed 3 times with PBS. The cells were stained with Giemsa stain solution and then de‐stained with PBS. Images were obtained using an optical microscope (200 × magnification), and invading cells were quantified by manual counting. The inhibition of invading cells was quantified and expressed on the basis of untreated control cells, which were set as 1‐fold.

The accumulation of intracellular ROS in A7r5 cells was quantified by a fluorescence spectrophotometer using DCFH₂‐DA as described previously.30 Briefly, A7r5 cells at a density of 4 × 10⁵ 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, DCFH₂‐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 DCFH₂. 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.

A7r5 cells were transfected with Nrf2 siRNA using Lipofectamine RNAiMAX (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. For the transfection, cells were grown in DMEM containing 10% FBS and plated in 6‐well plates to 60% confluence at the time of transfection. The next day, the culture medium was replaced with 50 μL of Opti‐MEM, and the cells were transfected using the RNAiMAX transfection reagent. For each transfection, 5 μL RNAiMAX was mixed with 250 μL of Opti‐MEM and incubated for 5 min at room temperature. In a separate tube, siRNA (100 pM, for a final concentration of 100 nM in 1 mL of Opti‐MEM) was added to 250 μL of Opti‐MEM, and the siRNA solution was added to the diluted RNAiMAX reagent. The resulting siRNA/RNAiMAX mixture (500 μL) was incubated for an additional 25 min at room temperature to allow complex formation. Subsequently, the solution was added to the cells in the 6‐well plates, for a final transfection volume of 1 mL. After incubation for 6 h, the transfection medium was replaced with 2 mL of standard growth medium, and the cells were cultured at 37°C. Cells were co‐incubated with FKA (7.5 μM) for 2 h in the presence or absence of TGF‐β1 (10 ng/mL) according for the indicated times, and changes in the protein expressions of Nrf2 (0.5 h), HO‐1 (8 h), NQO‐1 (8 h) and γ‐GCLC (8 h) were determined by Western blotting.

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.

FKA and its cytotoxicity on human SMC (A7r5) cells were determined by treating cells with increasing concentrations of FKA (0‐60 μM) for 24 h (Figure 1A). MTT assay results showed that the viability of A7r5 cells remains the same (100%) even after FKA treatment, which indicates that FKA up to 60 μM did not produce any cytotoxic effects (Figure 1B). We further evaluated the effects of FKA (0‐30 μM) on morphology, and cell viability of A7r5 cells was determined in the presence or absence of TGF‐β1 (10 ng/mL) induction. Images from optical microscopy showed no morphological changes following FKA treatment (Figure 1C). FKA treatment (0‐30 μM) significantly suppressed the TGF‐β1 (10 ng/mL)‐induced increased viability of A7r5 cells (Figure 1D). These results indicated that FKA is not cytotoxic for A7r5 cells, despite preventing the excessive growth induced by TGF‐β1.

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‐induced onset of fibrosis, the changes in α‐SMA and fibronectin protein levels were determined. Western blot data showed that the α‐SMA (Figure 2B) and fibronectin (Figure 2C) levels were significantly greater in A7r5 cells following TGF‐β1 treatment (10 ng/mL, 24 h). The fibronectin and α‐SMA levels with TGF‐β1 were increased ~2.5‐fold and ~5‐fold, respectively, when compared with control cells (Figure 2B and C). However, FKA pretreatment (0‐30 μM, 2 h) substantially suppressed both the fibronectin and α‐SMA elevation in a concentration‐dependent manner, with 30 μM FKA more effective than other concentrations.

TGF‐β1 increases the phosphorylation of various Smads, particularly Smad3, which forms a heterodimer complex with Smad4 and then translocates into the nucleus to induce fibrotic genes.13, 14 To 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 (pGL3‐SBE4) transcriptional activity. Consistent with the decreased p‐Smad3 levels, FKA (0‐30 μM) pretreatment significantly inhibited the Smad3 transcriptional activity in a dose‐dependent manner against TGF‐β1 stimulation (Figure 3B). Correspondingly, increased nuclear accumulation of p‐Smad3 and Smad3 with TGF‐β1 was substantially suppressed by FKA pretreatment (Figure 3C). FKA‐mediated inhibition of p‐Smad3/Smad3 nuclear localization simultaneously increased Smad3 levels in the cytosol. Nevertheless, increased Smad3 in the cytosol with FKA treatment was greater than the p‐Smad3 up‐regulation (Figure 3C). These results showed that FKA can inhibit the TGF‐β1‐activated Smad3 signaling to avert the onset of vascular fibrosis.

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 We hypothesized that anti‐fibrotic properties of FKA were possibly associated with decreased cell migration and invasion against TGF‐β1 stimulation. The anti‐migration effect of FKA against TGF‐β1 activation was determined by performing a classical wound‐healing assay. In this assay, we found that 24‐ hour TGF‐β1 treatment induced significant migration of A7r5 cells (>3‐fold), which was effectively inhibited by FKA treatment (2 h), particularily at 7.5 and 30 μM doses (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 and B show the imaged and quantified invading cells following treatments. Similar to migration, TGF‐β1 profoundly increased the invasion ability of A7r5 cells, by approximately 5‐fold. Interestingly, FKA treatment substantially restrained the TGF‐β1‐induced invasion ability in a dose‐dependent manner. Although FKA exhibited anti‐migration and anti‐invasion properties in the presence of TGF‐β1, FKA alone did not affect the endogenous migration and invasion potential of A7r5 cells.

MMPs represent a family of proteolytic enzymes that degrades ECM components and plays an important role in cell migration and invasion. TIMPs reduce excessive ECM degradation, and an imbalance between MMPs and TIMPs underlies the pathogenesis of vascular fibrosis.17 Based on the anti‐migration and anti‐invasion properties of FKA, we determined the effects of FKA on MMP‐9, MMP‐2 and TIMP‐1 expression levels in TGF‐β1‐stimulated SMCs. Western blot results showed that TGF‐β1 stimulation (10 ng/mL) for 24 h significantly up‐regulated MMP‐9 and MMP‐2 expressions, while it down‐regulated TIMP‐1 levels in A7r5 cells, which facilitated migration/invasion (Figure 6A‐C). Intriguingly, FKA pretreatment (0‐30 μM) remarkably suppressed the TGF‐β1‐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.

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.

To further explore the mechanistic role of ROS in the anti‐fibrotic effects of FKA, we determined the nuclear localization of p‐Smad3 in ROS‐quenched conditions (FKA/NAC pretreatment) following TGF‐β1 stimulation. A7r5 cells were treated with FKA (7.5 μM) or an ROS inhibitor (NAC, 2 mM) for 2 h prior to TGF‐β1 stimulation (10 ng/mL), and then immunofluorescence staining was performed to confirm the nuclear import of p‐Smad3. Consistent with Western blot data (Figure 3), images from immunofluorescence assay showed that the TGF‐β1‐induced nuclear accumulation of p‐Smad3 was obliterated by FKA as well as NAC pretreatment (Figure 8). Nuclear localization of p‐Smad3 in FKA treated cells was similar with that of NAC pre‐treated cells. However, Smad3 was restrained in the nucleus of control cells (Figure 8). These findings affirmed that the ROS‐quenching ability of FKA contributed to the inhibition of TGF‐β1/Smad3 signaling in human vascular smooth muscle cells.

It has been described that ROS signaling is involved in TGF‐β1‐mediated fibrosis through activation of proliferation, migration and/or ECM deposition.2 Owing to its potent ROS scavenging activity, we speculate that FKA may diminish ROS‐activated migration in TGF‐β1‐stimulated A7r5 cells. To validate the anti‐migration property of FKA, a typical wound‐healing assay was performed, and migration ability was compared with NAC pretreatment. For this assay, confluent monolayers of A7r5 cells were incubated with FKA (7.5 μM) or NAC (2 mM) for 2 h in the presence or absence of TGF‐β1 (10 ng/mL) stimulation for 24 h. We found that TGF‐β1‐stimulated migration (~4‐fold) was substantially inhibited by both FKA and NAC pretreatment. This anti‐migration effect was visualized by noting the widely opened wound areas in FKA or NAC pre‐treated cells, even after 24 h (Figure 9A and B). Since ROS signaling is required for TGF‐β1‐induced migration, suppression of ROS production either by FKA or NAC could contribute to the decreased migration ability of A7r5 cells with TGF‐β1 stimulation.

The ROS quenching ability of natural compounds is accompanied by enhanced antioxidant responses. Nrf2, a redox‐sensitive zipper protein, translocates into the nucleus in the presence of ROS, binds to ARE in the promoter region and transcribes a set of antioxidant genes.3 Since Nrf2 signaling is critically involved in fibrosis pathology, we investigated the effects of FKA on Nrf2 activation in vascular smooth muscle cells. First, we found that FKA treatment (0‐30 μM for 0‐4 h) dose‐ and time‐dependently augmented the total Nrf2 levels in A7r5 cells (Figure 10A). Next, we determined the Nrf2 levels in nuclear and cytosolic fractions of cells following FKA treatment (0‐30 μM for 0.5 h). Western blot results showed that FKA incubation dose‐dependently increased Nrf2 levels in both fractions, with a maximal elevation noted with 7.5 μM (Figure 10B). For further confirmation, we treated cells with FKA (7.5 μM, 0.5‐1 h), and 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).

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).

Next, to demonstrate the subsequent effects of FKA on endogenous antioxidant genes, we measured the changes in HO‐1, NQO‐1 and γ‐GCLC genes in FKA‐treated (7.5 μM, 0‐16 h) A7r5 cells. Western blot data showed that all antioxidant genes were time‐dependently 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.

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 Nrf2 activation for FKA‐induced antioxidant responses, we developed a Nrf2 gene knockdown model by transfecting siRNA into A7r5 cells. Changes in antioxidant gene expression were determined following FKA treatment (7.5 μM). Western blot results showed successful execution of Nrf2 knockdown, as we noted the abolishment of the Nrf2 protein band in siNrf2‐transfected cells even in the presence of FKA (Figure 11C). Consequently, up‐regulated HO‐1, NQO‐1 and γ‐GCLC proteins in control siRNA cells were not observed in Nrf2‐knockdown cells (Figure 11C). These results demonstrate that the FKA‐induced antioxidant response is mediated through Nrf2/ARE signaling in A7r5 cells.