Baicalin Down-Regulates IL-1β-Stimulated Extracellular Matrix Production in Nasal Fibroblasts

Human recombinant IL-1β was obtained from Biovision (Milpitas, CA). Baicalin was provided by Sigma (St. Louis, MO). Inhibitors of extracellular signal-regulated kinase (ERK; U0126), p38 (SB203580), and c-Jun N-terminal kinase (JNK; SP600125) were purchased from Calbiochem (Billerica, MA). Inhibitors of Akt (LY294002) and NF-κB (BAY-117082) were provided by Sigma. Antibodies against phospho-ERK (p-ERK), total-ERK (t-ERK), p-p38, t-p38, p-JNK, t-JNK, p-Akt, t-Akt, p-p50, p50, p-p65, p65, p-IκBα, IκBα, fibronectin, α-smooth muscle actin (α-SMA), and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Patients were recruited from the Department of Otorhinolaryngology, Korea University Medical Center, Korea. Written informed consent was obtained from each patient, and the study was approved by the Korea University Medical Center Institutional Review Board. All patients provided their written informed consent to participate in this study and had no history of allergy, asthma, or aspirin sensitivity. Inferior turbinate-derived fibroblasts were isolated from six patients' (three males and three females; mean age, 32.8 ± 7.9 years) surgical tissues by enzymatic digestion with collagenase (500 U/ml, Sigma), hyaluronidase (30 U/ml, Sigma), and DNase (10 U/ml, Sigma). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen^TM, Carlsbad, CA), 1% (v/v) 10,000 units/ml penicillin, and 10,000 μg/ml streptomycin (Invitrogen^TM). According to our previous study, the purity of obtained cells was confirmed by characteristic spindle-shaped cell morphology and flow cytometry [3]. Cells were used in experiments from the fourth to seventh cell passages.

To determine the cytotoxic effects of baicalin in nasal fibroblasts, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma) assay was used. Fibroblasts were treated with baicalin (0–400 μM) for 72hours. And then, incubated with MTT for 4 hours, and the reaction was interrupted by adding dimethyl sulfoxide. A fluorescence microplate reader (F2000; Hitachi, Ltd., Tokyo, Japan) was used at 570 nm to measure the results.

Nasal fibroblasts were pre-treated with baicalin (0–50 μM) for 1 hour, and then stimulated with IL-1β (10 ng/ml) for 24 hours. Total RNA was isolated with the Trizol reagent (Invitrogen^TM). Two micrograms of RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen^TM). Polymerase chain reaction (PCR) was performed with the following primers: α-SMA (sense 5′ - GGT GCT GTC TCT CTA GCC TCT GGA—3′, anti-sense 5′ - CCC ATC AGG CAA CTC GAT ACT CTT C—3′, 322 bp), fibronectin (sense 5'—GGA TGC TCC TGC TGT CAC - 3', anti-sense 5'- CTG TTT GAT CTG GAC CTG CAG—3', 386 bp), collagen type I (sense 5′- CAT CAC CTA CCA CTG CAA GAA C—3′, anti-sense 5′- ACG TCG AAG CCG AAT TCC—3′, 278 bp), and GAPDH (sense 5'- GTG GAT ATT GTT GCC ATC AAT GAC C—3', anti-sense 5'- GCC CCA GCC TTC TTC ATG GTG GT—3', 271 bp). Amplification reactions were performed as follows: an initial 5-minute denaturation step at 94°C; followed by 30 cycles at 94°C for 45 seconds, 55–65°C for 45 seconds, and 72°C for 45 seconds; and a final extension step at 74°C for 5 minutes. All reactions were performed in a 20 μl volume. Products were electrophoresed on a 1.5% agarose gel and visualized by staining with ethidium bromide. The gels were captured and visualized with a Molecular Imager ChemiDoc XRS+ (Bio-Rad, Hercules, CA).

For detection of α-SMA, fibronectin and β-actin, nasal fibroblasts were pre-treated for 1 hour with the following baicalin (50 μM) and specific inhibitors: U0126 (ERK inhibitor, 10 μM), SB203580 (p38 inhibitor, 10 μM), SP600125 (JNK inhibitor, 10 μM), LY294002 (AKT inhibitor, 10 μM), and BAY-117082 (NF-κB inhibitor, 1 μM). After then, cells were stimulated IL-1β (10 ng/ml) for 72 hours. For detection of p-ERK, p-p38, p-JNK, p-Akt and p-IκBα, nasal fibroblasts were pre-treated for 1 hour with the following baicalin and specific inhibitors: U0126, SB203580, SP600125, LY294002, BAY-117082. And then, cells were stimulated IL-1β for various times (0–120 minutes). Cells were lysed in PRO-PREP^TM protein extraction solution (iNtRON Biotechnology, Seongnam, Korea). Lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinyl difluoride membranes (Millipore Inc., Billerica, MA). Membranes were blocked with 5% skim milk solution and incubated with the following antibodies: α-SMA, fibronectin, p-ERK, t-ERK, p-p38, t-p38, p-JNK, t-JNK, p-Akt, t-Akt, p-p50, p50, p-p65, p65, p-IκBα, IκBα and β-actin. Blots were visualized with HRP-conjugated secondary antibodies and an ECL system (Pierce, Rockford, IL).

Nasal fibroblasts on coverslips were pre-treated for 1 hour with the baicalin (50 μM), and then, stimulated with IL-1β (10 ng/ml) for 72 hours. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in 1% bovine serum albumin for 10 minutes, blocked with 5% bovine serum albumin for 1 hour at room temperature, and incubated overnight at 4°C with monoclonal anti-α-SMA or polyclonal anti-fibronectin. Fibroblasts were then incubated with anti-mouse Alexa Fluor 488 (Invitrogen^TM) or anti-rabbit Alexa Fluor 555 (Invitrogen^TM) secondary antibodies. Finally, coverslips were counterstained with 4′-6-diamidino-2-phenylindole. Stained fibroblasts were captured and visualized under a confocal laser scanning microscope (LSM700, Zeiss, Oberkochen, Germany).

Nasal fibroblasts were plated and grown to confluence in 6-well tissue culture dishes. A straight scratch was made in the cells using a pipette tip. Scratched cells were immediately rinsed with phosphate buffered saline and DMEM medium containing 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen^TM), 1,000 unit/ml penicillin, and 1,000 μg/ml streptomycin (Invitrogen^TM) was added. Cells were incubated with IL-1β (10 ng/ml) alone or in conjunction with baicalin (50 μM) up to 48 hours. Images were obtained with a microscope (Olympus BX51; Olympus, Tokyo, Japan. The cell migration rate was expressed as the ratio of the migration distance in control cells (100%).

Fibroblasts were seeded in the upper chamber of transwell chambers (Corning Life Sciences, MA). Then, DMEM containing 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen^TM), 1,000 unit/ml penicillin, and 1,000 μg/ml streptomycin (Invitrogen^TM) was added with IL-1β (10 ng/ml) alone or in conjunction with baicalin (50 μM) to the lower chamber up to 48 hours. The cells on the upper surface of the membrane were removed with cotton swabs. Cells on the lower surface of the membrane were stained with Diff-Quik stain (Sysmex, Kobe, Japan). Images of the stained cells from five selected views were captured with a microscope at 200x magnification at 0, 48 hours.

Nasal fibroblasts were pre-treated for 1 hour with the following baicalin (50 μM) and specific inhibitors: U0126 (ERK inhibitor, 10 μM), SB203580 (p38 inhibitor, 10 μM), SP600125 (JNK inhibitor, 10 μM), LY294002 (AKT inhibitor, 10 μM), and BAY-117082 (NF-κB inhibitor, 1 μM). After then, cells were stimulated with IL-1β (10 ng/ml) for 72 hours. As previously reported [13], total soluble collagen in cell culture supernatants was quantified using the Sircol collagen assay (Biocolor, Belfast, UK). One milliliter of Sirius red dye, an anionic dye that reacts specifically with basic side chain groups on collagen under the assay conditions, was added to 400 μl of supernatant and incubated with gentle rotation for 30 minutes at room temperature. The samples were centrifuged at 12,000 rpm for 10 minutes, and the collagen–dye complex precipitate was collected and re-solubilized in 0.5 M sodium hydroxide. The dye concentration was estimated by spectrophotometry at 540 nm (Beckman Coulter, Fullerton, CA). The absorbance was directly proportional to the amount of newly formed soluble collagen in the cell culture supernatant.

Rat-tail tendon collagen type I was purchased from BD Biosciences (Bedford, MA). Collagen gels were prepared as previously described by mixing rat-tail tendon collagen type I, serum-free DMEM, and cells. Fibroblasts were then mixed with the neutralized collagen solution (pH 7.4) so that the final cell density in the collagen solution was 3 x 10⁵ cells/ml, and the final collagen concentration was 0.75 mg/ml. Aliquots (0.5 ml/well) of the mixture of cells in collagen were cast into each well of 24-well tissue culture plates and allowed to polymerize at room temperature, generally completed in 20 minutes. After polymerization, the gels were gently released from the 24-well tissue culture plates and transferred to 6-well culture plates containing 1.5 ml serum free-DMEM with IL-1β, baicalin, or both. The gels were then incubated at 37°C in a 5% CO₂ atmosphere for 3 days. The area of each gel was measured with Image J analyzer (National Institutes of Health, Bethesda, MD). Data are expressed as a percentage of the initial gel area.

Nasal fibroblasts were pre-treated for 1 hour with the baicalin (50 μM), and then, stimulated with IL-1β (10 ng/ml) for 72 hours. Aliquots of medium conditioned (10 μl) by cells were analyzed using collagen zymography for matrix-metalloproteinase (MMP)-1 in 0.4 mg/mL collagen-10% polyacrylamide gels. Following electrophoresis, the gels were washed twice with 2.5% Triton X-100 for 30 minutes while shaking to remove sodium dodecyl sulfate-polyacrylamide and to renature the MMP-1 in the gels. Renaturated gels were incubated in developing buffer containing 100 mM Tris–HCl, 5 mM CaCl2, 0.005% Brij-35, and 0.001% NaN3 (pH 8.0), overnight at 37°C. Gels were stained with 0.25% Coomassie brilliant blue G-250 (50% methanol, 10% acetic acid) and destained using destaining solution (50% methanol, 10% acetic acid). Proteinase activity was observed as cleared (unstained) regions. Finally, the gels were dried for 2 hours using a gel dryer (Bio-Rad Labs).

Results were obtained from at least three independent experiments. Statistically significant differences between control and experimental data were analyzed with unpaired t test or one-way analysis of variance followed by Tukey's test (GraphPad Prism, version 5, Graph Pad Software, San Diego, CA). Significance was established at the 95% confidence level. P values less than 0.05 were accepted as statistically significant.

To determine the cytotoxic effects of baicalin on nasal fibroblasts, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma) assay was used. Baicalin was not cytotoxic for 72 hours at concentrations up to 50 μM (S1 Fig). To examine the inhibitory effects of baicalin on α-SMA, fibronectin and collagen accumulation in nasal fibroblasts, cells were treated with IL-1β with or without baicalin at different concentrations (0–50 μM). Baicalin significantly down-regulated expression of IL-1β-induced α-SMA, fibronectin and collagen type I mRNA, as assessed by RT—PCR (p < 0.05, Fig 1A). IL-1β-induced expression of α-SMA and fibronectin protein was suppressed by baicalin (Fig 1B). Expression levels of collagen protein were determined with a Sircol soluble collagen assay. IL-1β-stimulated expression of collagen protein was significantly down-regulated by baicalin treatment (p < 0.05, Fig 1C). Localization of α-SMA and fibronectin was determined by immunocytochemical staining (Fig 1D). Expressions of α-SMA and fibronectin protein were markedly down-regulated by baicalin in a dose-dependent manner. These results indicate that IL-1β-stimulated myofibroblast differentiation and ECM production were significantly inhibited by baicalin.

To evaluate the effects of baicalin on IL-1β-stimulated migration of nasal fibroblasts, scratch and transwell migration assays of cell migration were performed. Nasal fibroblasts were treated with mitomycin C to inhibit cell proliferation. Then, fibroblasts were treated with IL-1β with or without baicalin. The migration distances were significantly higher in cells treated with IL-1β alone compared with cells treated with IL-1β and baicalin (p < 0.05, Fig 2A and 2B). In addition, baicalin also significantly suppressed IL-1β-stimulated migration in a transwell migration assay (p < 0.05, Fig 2C and 2D). Taken together, these results suggest that baicalin inhibits IL-1β-stimulated migration of nasal fibroblasts.

To investigate the effect of baicalin on collagen contraction and activity of MMP-1, fibroblasts were treated with IL-1β with or without baicalin. Baicalin significantly inhibited IL-1β-stimulated contraction as assessed by a collagen gel contraction assay (p < 0.05, Fig 3A). MMP-1 enzymatic activity was analyzed by zymography. IL-1β-stimulated expression of MMP-1 was significantly down-regulated by baicalin treatment (p < 0.05, Fig 3B). These results indicate that baicalin inhibits IL-1β-stimulated collagen contraction and activity of MMP-1 in nasal fibroblasts.

To verify whether the MAPK (ERK, p38, and JNK) and Akt signaling pathway is involved in α-SMA, fibronectin, and collagen expressions, fibroblasts were treated with IL-1β with or without baicalin and MAPK or Akt inhibitors. We examined the activation of MAPK and Akt signal proteins by western blot and determined that IL-1β activated all three MAPKs (ERK, p38, and JNK) and Akt. Baicalin markedly down-regulated the phosphorylation of the three MAPKs and Akt (Fig 4A). In addition, IL-1β-stimulated expression of α-SMA, fibronectin, and collagen were significantly down-regulated by all three MAPK inhibitors and the Akt inhibitor (p < 0.05, Fig 4B and 4C). These results indicate that baicalin regulates IL-1β-stimulated myofibroblast differentiation and ECM production via MAPK and Akt signaling pathways in nasal fibroblasts.

To determine the role of NF-κB signaling in the expression of α-SMA, fibronectin and collagen, fibroblasts were treated with IL-1β, with or without baicalin or an NF-κB inhibitor (BAY-117082). Expressions of p-p50 and p-p65 protein, the active forms of NF-κB subunits, were up-regulated by IL-1β, and then markedly inhibited by baicalin treatment (Fig 5A). Moreover, the treatment of IL-1β showed significantly greater phosphorylation of IκBα (NF-κB inhibitor) than IL-1β-untreated nasal fibroblasts, while baicalin significantly down-regulated (Fig 5B). Further, the NF-κB inhibitor (BAY-117082) significantly down-regulated IL-1β-stimulated production of α-SMA, fibronectin, and collagen (p < 0.05, Fig 5C and 5D). These results suggest that the effect of baicalin on IL-1β-stimulated myofibroblast differentiation and ECM production may be mediated by NF-κB pathways in nasal fibroblasts.