The effect of silymarin (Silybum marianum) on human skin fibroblasts in an in vitro wound healing model

Abstract

Context: Silymarin, a flavonolignan from Silybum marianum (L.) Gaertn. (Asteraceae), has been reported to have antioxidant and anti-inflammatory properties. Therefore, it may be worthwhile to study the effect of silymarin on wound healing.

Objective: To evaluate the effect of silymarin on human fibroblast cells in an in vitro model of wound healing.

Materials and methods: Human fibroblast cells were treated with different concentrations (4.5, 9, 18, 36 µg/mL) of silymarin. The effects of silymarin on cell viability, proliferation, collagen synthesis, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-bromo-2′-deoxy-uridine, hydroxyproline analysis and real-time PCR, respectively. The effect of silymarin on cellular antioxidant status was determined by protection against hydrogen peroxide (H₂O₂)-induced cell injury and free radical scavenging activity (ABTS assay) of the cells.

Results: Results of the present study indicate that pretreatment of fibroblast cells with silymarin significantly protected cells against H₂O₂-induced injury (p < 0.05). After an 18 h treatment of cells with 36 µg/mL silymarin, total antioxidant capacity of cells significantly increased (p < 0.05). Furthermore, pretreatment of human fibroblast cells with silymarin significantly inhibited lipopolysaccharide (LPS)-induced COX-2 mRNA expression (p < 0.001). There was no significant difference in fibroblast proliferation and collagen synthesis between treatment and control groups (p > 0.05).

Discussion and conclusion: Silymarin may be useful as a therapeutic agent for the treatment of cutaneous wounds through its antioxidation and anti-inflammation effects.

Keywords::

• Antioxidant capacity
• collagen synthesis
• cyclooxygenase-2

Introduction

Wound healing is a complex process characterized by inflammation, cell proliferation and remodeling of the extracellular matrix (Diegelmann & Evans, 2004). Fibroblast cells play an important role in wound healing by initiating the proliferative phase of repair. These cells secrete the collagens and the glycosaminoglycans of the new granulation tissue and subsequently affect the remodeling of the granulation tissue into mature dermis (Singer & Clark, 1999).

Impairment in each of wound healing processes can lead to healing delay or even the inability for dermal wound healing. Reactive oxygen species (ROS) that are produced in response to cutaneous injury may impair the healing process (Gupta et al., 2002). Furthermore, the prolonged inflammation phase may lead to cell destruction and altered composition of extracellular matrix with subsequent failure in epithelization (Dovi et al., 2003; Gutiérrez-Fernández et al., 2007). Hence, if a compound has antioxidant and anti-inflammation potential, it can be a good therapeutic agent for accelerating the wound healing process (Houghton et al., 2005). Among the inflammatory pathways, nitric oxide synthase (NOS) and cyclooxygenase (COX) and their products play an important role in wound healing (Stallmeyer et al., 1999; Abd-El-Aleem et al., 2001). Human fibroblasts express two NOS isoforms, the constitutively expressed endothelial NOS (eNOS) and the inducible NOS (iNOS) form. iNOS is involved in provoking and maintaining inflammatory phase (Soneja et al., 2005). COX enzymes control the initial steps of the inflammation process by converting arachidonic acid to prostaglandins (PGs) and thromboxane. Like NOS, COX has two isoforms. COX-1, a constitutive enzyme, is expressed at a constant level and does not fluctuate in response to various stimuli. COX-2, an inducible enzyme, is up-regulated in the early inflammatory response and produce a large amount of proinflammatory PGs at the inflammatory site (Futagami et al., 2002).

Silymarin, a mixture of bioactive flavonolignans obtained from Silybum marianum (L.) Gaertn. (Asteraceae), possesses strong antioxidant, anti-inflammatory and anticancer activity (Katiyar et al., 1997, 2002; Meeran et al., 2006; Gu et al., 2007). It also modulates many molecular changes caused by xenobiotics and ultraviolet radiation to protect the skin (Zhao et al., 2000; Katiyar, 2002; Meeran et al., 2006; Svobodova et al., 2006, 2007).

Although silymarin has antioxidant and anti-inflammation effects, its effect on wound healing has not been investigated to date. The aim of the present study was to investigate the effect of silymarin on human fibroblast cells in an in vitro model of wound healing.

Materials and methods

Materials

Human neonatal foreskin fibroblast cells were obtained from Shaheed Beheshti Skin Research Center. Silymarin, lipopolysaccaride (LPS; Escherichia coli 0111:B4), tumor necrosis factor-α (TNF-α), phorbol 12-myristate 13-acetate, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (Seelze, Germany). Cell culture reagents were purchased from GIBCO Life Technologies (Paisley, UK) and all the other reagents were obtained from Sigma.

Cell culture and treatment

Human fibroblast cells were grown in Dulbecco modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 g/mL streptomycin sulfate and l-glutamine, at 37°C in a humidified 5% CO₂ atmosphere. Cells were treated with serial concentrations (4.5, 9, 18, 36 µg/mL) of silymarin that was dissolved in 100% DMSO.

Fibroblast proliferation and viability assay

Assessment of cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, human fibroblast cells (2 × 10⁴ well) were seeded in 96-well plates and treated with different concentrations of silymarin for 24 and 48 h. Cells were incubated for an additional 3 h with MTT, followed by treatment with 100 μL of DMSO for 1 h to solubilize formazan violet crystals in the cells. The absorbance of each well was determined by spectrophotometer at dual wavelengths of 540 and 620 nm with a microplate reader.

MTT tetrazolium assay may lead to false positive results when testing natural compounds with intrinsic reductive potential (Peng et al., 2005). Therefore, cells were washed several times before adding MTT.

Cell proliferation was also measured by determining the extent of 5-bromo-2′-deoxy-uridine (BrdU) incorporation into DNA of human fibroblast cells (ELISA kit, Roche, Germany). The amounts of BrdU incorporated into the fibroblast cells were assayed according to the manufacturer’s instructions.

Protection against damage to fibroblasts by hydrogen peroxide

The method used for the hydrogen peroxide (H₂O₂) assay was described by Annan and Houghton (2008). Fibroblast cells were seeded at 4 × 10³ cells/well in 96-well plates and incubated at 37°C until near confluence. The concentration of H₂O₂ inducing about 50% inhibition of the MTT response in fibroblasts was determined to be 2.5 × 10⁻³ M H₂O₂ and was used for all experiments.

To assess the protective effect of silymarin on cells, three different types of experiments were performed. In the first experiment, the cells were pretreated with silymarin overnight and then were treated with H₂O₂ for 3 h. In the second experiment, the cells were pretreated with silymarin overnight and then were treated with silymarin and H₂O₂ simultaneously for 3 h. In the third experiment, cells were treated with silymarin and H₂O₂ simultaneously for 3 h.

Catalase (250 IU/mL), a scavenger of H₂O₂, was used as the positive control in all experiments. After the incubation period, the MTT assay was used to quantify the degree of protection of fibroblast cells by silymarin against H₂O₂ damage.

Determination of total antioxidant capacity

Fibroblast cells (2 × 10⁶) were seeded in 25 cm² flask and treated with different concentrations of silymarin for 3 and 18 h. After treatment, cells were scraped, pelleted and re-suspended in 1 mL of 5 mM phosphate buffer, pH: 7.4. Cell membranes were disrupted by sonication on ice and the cell lysates centrifuged at 4°C, 10,000g, for 15 min. The supernatant was separated from cell debris and used for total antioxidant assay measurement. The results were normalized to the protein content of the cell lysates.

Cellular antioxidant levels were determined by measuring the ability of antioxidants in the sample to inhibit the oxidation of ABTS (2,2′-azino-di-[3-ethylbenzthiazoline sulphonate]) to the ABTS⁺ radical by H₂O₂ with the Total Antioxidant Assay Kit (Cayman, USA). According to manufacturer’s instructions, the amount of ABTS⁺ radical was monitored by reading the absorbance at 405 nm. Torolox concentrations from 0 to 0.35 mM were used to make a standard curve. The capacity of the antioxidants in the samples to prevent ABTS⁺ oxidation were determined according to the standard curve (Miller et al., 1993).

Measurement of collagen synthesis by hydroxyproline analyses

The assay was performed following the protocol published by Stipcevic et al. (2006) with small modification. Briefly, cells were seeded at a density of 2 × 10⁶ in 75 cm² flask and treated with different concentrations of silymain for 12 days. Ascorbic acid solution was added every day in concentration of 50 µg/mL to the medium. After 12 days, cells were harvested by rubber policeman in 1 mL of 10 mM PBS. Cell membranes were disrupted by sonicator and total protein amount was quantified using Bradford (1976). For quantifying hydroxyproline, 0.5 mL of sonicated samples was mixed with 0.5 mL of 12 N HCl and incubated in 115°C oven for 18 h. The residue was re-suspended in 250 μL deionized water and 50 μL was mixed with 450 μL of deionized water to perform assay. The incubation of samples were as follows: 20 min incubation with 250 μL of 0.05 M chloramine T solution [chloramine T solution = 1.41 g chloramine T (Sigma–Aldrich,) dissolved in 20 mL dH₂O, 30 mL methyl cellosolve (2-methoxyethanol) and 50 mL citric acid buffer]; 5 min incubation with 250 μL of 3.15 M perchloric acid; 20 min incubation with 250 μL of 20% p-dimethylbenzaldehyde in 60°C water bath dissolved in methyl cellosolve to a final volume of 50 mL and heated to 60°C to dissolve the reagent. Absorbance was measured at 540 nm. Hydroxyproline (Sigma–Aldrich) concentrations from 0 to 10 µg/mL were used to make a standard curve.

COX-2 and iNOS mRNA expression analysis

Human fibroblast cells (5 × 10⁵ cells/well) were seeded in 6-well plates and allowed to grow for 24 h in a growth medium at 37°C and 5% CO₂. Cells were then washed twice with PBS and then pretreated with silymarin at various concentrations for 18 h. After pretreatment, LPS (10 µg/mL) was added, and the cells were incubated at 37°C and 5% CO₂ for 24 h. For iNOS induction, LPS (1–10 µg/mL) plus TNF-α (20–50 ng/mL) or LPS (1–10 µg/mL) plus phorbol 12-myristate 13-acetate (PMA) (20–50 ng/mL) were used.

Total cellular RNA was extracted from cells using RNeasy protect cell mini kit (Qiagen, Hilden, Germany) and its quality was controlled. Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using QuantiTect Rev. Transcription kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Real-time PCR was performed using SYBR premix Ex Taq (Qiagen, Hilden, Germany) and predesigned primers for COX-2, iNOS, and GAPDH genes (QuantiTect Primer Assay, Qiagen, MD, USA). Each cycle consisted of denaturation at 95°C for 10 s, and combined annealing and extension at 60°C for 30 s. A standard curve was generated from dilution series constructed from pooled cDNA. Relative changes in gene expression were quantified using ΔΔC_T method.

Statistical analysis

Analysis of variance was used to compare means in different groups. Values of less than 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001). Data were expressed as mean ± SE.

Results

Effect of silymarin on the viability and proliferation of human fibroblast cells

The effects of silymarin on the viability and proliferation of human fibroblast cells were examined by the MTT assay and by measuring the amounts of BrdU incorporated into cellular DNA in the presence of serial concentrations of silymarin for 24 and 48 h are shown in Figure 1.

Figure 1.  Effect of silymarin on cellular viability and proliferation of human fibroblast cells. Cells were treated with different concentrations of silymarin for 24 and 48 h. (A) Cell viability was determined by the MTT assay. (B) Cell proliferation was determined by DNA (BrdU) incorporation assay. The data are expressed as mean ± SE from at least three separate experiments.

Results showed that silymarin does not have any cytotoxic and proliferative effect on fibroblast cells in the studied concentration range (4.5–36 µg/mL) and the data obtained by the cell proliferation ELISA assay are well correlated with those obtained by MTT assay.

Effect of silymarin on fibroblast injury by H₂O₂

As shown in Figure 2A and 2B, when the cells were pretreated with silymarin overnight before the application of the H₂O₂, the MTT assay showed that the cells were protected. However, when cells were incubated with different concentrations of silymarin and H₂O₂ simultaneously for 3 h at 37°C, it was observed that fibroblast cells were not protected against H₂O₂ damage by various concentrations of the silymarin (Figure 2C).

Figure 2.  Effect of silymarin on hydrogen peroxide-induced damage in fibroblasts using different treatment regimens. (A) Pretreatment only. Fibroblasts were incubated with various concentrations of silymarin overnight before exposure to hydrogen peroxide without silymarin for 3 h. (B) Fibroblasts were pretreated with silymarin overnight and then exposed to hydrogen peroxide and silymarin for 3 h. (C) Fibroblasts were incubated with silymarin and hydrogen peroxide simultaneously for 3 h. The data are expressed as mean ± SE of three separate experiments. Treated cells were compared with cells exposed only to hydrogen peroxide. *p < 0.0.05, **p < 0.01, ***p < 0.001.

Effect of silymarin on fibroblast total antioxidant capacity

The antioxidant capacity of silymarin was evaluated according to the ABTS decoloration. As shown in Figure 3, incubation of silymarin led to increase in total antioxidant capacity of cell extract (reaching statistical significant after 18 h incubation).

Figure 3.  Total antioxidant capacity of cell extracts after 3 and 18 h incubation of the cells with various concentrations of silymarin. Total antioxidant capacity was measured by monitoring of ABTS solution decolorization in supernatants of cell homogenates after incubation with silymarin for various times. Results represent mean ± SE, *p < 0.05 compared to the control group.

Effect of silymarin on fibroblast collagen synthesis

Collagen synthesis was determined by measuring hydroxyproline levels after fibroblast treatment with different concentrations of silymarin is shown in Figure 4. The results demonstrated that silymarin has no effect on collagen synthesis compared to the control group (p > 0.05).

Figure 4.  The effect of silymarin on in vitro collagen synthesis was measured by hydroxyproline assay. Data are present as mean ± SE. No significant difference was seen between groups.

Effect of silymarin on LPS-induced COX-2 expression

Incubation with LPS significantly increased the expression of COX-2 mRNA in fibroblast cells. Through this study, iNOS mRNA was not induced in human fibroblast cells regardless of inflammatory stimuli. As it is evident in Figure 5, pretreatment with silymarin significantly inhibits LPS-induced mRNA expression of COX-2 (p < 0.001).

Figure 5.  Real-time RT-PCR measured effect of silymarin on COX-2 mRNA expression of human fibroblast cells. Cells were incubated first with indicated concentrations of silymarin for 18 h and then in the absence or presence of LPS (10 µg/mL) for 24 h. The COX-2 mRNA level was normalized by GAPDH RNA level. Data are mean ± SE of triplicates from an experiment repeated three times. ***p < 0.001 compared with cells treated only with LPS.

Discussion

Wound healing is categorized into three phases: inflammatory phase (including homeostasis and inflammation), proliferative phase (including granulation, contraction and epithelialization) and the remodeling phase which ultimately determines the appearance of the healed tissue and its strength (Diegelmann & Evans, 2004).

Regulation of inflammation and oxidation are important in the process of wound healing. Production of free radicals in response to cutaneous injury, may delay the healing process through the destruction of lipids, proteins and extracellular matrix elements (Russo et al., 2002). Antioxidant activity of silymarin was previously demonstrated in vitro and in vivo. Most of these reported antioxidant activities were based on cell-free biochemical tests (Psotová et al., 2001; Asghar & Masood, 2008; Turgut et al., 2008; Köksal et al., 2009).

In the present study, we have found that silymarin has a protective effect on cells against H₂O₂. The fibroblast cells that were pretreated with silymarin were well protected against H₂O₂ while cells treated with silymarin and H₂O₂ simultaneously were not protected. These results suggest that the protection is not due to direct interaction of the silymarin and H₂O₂. Therefore, the protective effect of silymarin could be attributed to an indirect effect by altering the cell membrane and limiting the damage induced by H₂O₂. Also in this study, we used a cell-based in vitro assay for testing the antioxidant activities of silymarin. Similarly, total antioxidant capacity was significantly increased in cells that were treated with silymarin for 18 h. These findings further support that silymarin has an indirect effect on ROS condition. It was demonstrated that a number of endogenous antioxidants, including GSH, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), gluthatione reductase, and glucose 6-phosphate dehydrogenase can be induced by silymarin (Ramakrishnan et al., 2006). Oral administration of silymarin has been shown to reduce lipid peroxidation of brain tissues in acetaminophen-induced toxicity by enhancing antioxidant enzymes (Nencini et al., 2007). Silymarin also inhibited tetradecanoyl phorbol-13-acetate (TPA)-induced lipid peroxidation and depletion of antioxidant enzymes such as SOD, catalase and GPx in mouse skin (Lahiri-Chatterjee et al., 1999; Zhao et al., 1999). Furthermore, Toklu et al. (2007) showed that administration of silymarin was effective against burn-induced oxidative damage and depressed TNF-α and reversed malondialdehyde and glutathione levels in rats.

On the other hand, a prolonged inflammatory phase may lead to cell destruction and altered composition of the extracellular matrix with subsequence of epithelization (Gutiérrez-Fernández et al., 2007). One of the early responses to inflammatory stimuli in cells which is involved in the repair processes is the induction of COX-2 (Futagami et al., 2002). Upregulation of COX-2 is involved in the persistent inflammation in chronic wounds. Studies show that high COX-2 expression causes a delay in healing and production of a scar (Wilgus et al., 2003, 2004). The present investigation has clearly shown that silymarin pretreatment attenuated the LPS-induced COX-2 mRNA expression in skin fibroblasts. Previously, it was found that silymarin down-regulates COX-2 induction in macrophage cell line J774A.1 (Raso et al., 2001). Furthermore, it has been shown that silymarin is a potent and specific inhibitor of nuclear transcription factor κB, and also modulates immune response, by augmenting synthesis of anti-inflammatory cytokines, such as IL-10, IL-12 (Schümann et al., 2003; Meeran et al., 2006). Silymarin also inhibits chemically induced expression of TNF-α and interleukin-1α (IL-1α) and reduces TPA-induced of myeloperoxidase, lipoxygenase, and COX-2 activities in mouse skin (Zhao et al., 1999; Zi et al., 1997).

Fibroblast viability and proliferation are important in the granulation formation in wound healing. Fibroblasts secrete collagens and subsequently affect the remodeling of granulation tissue into mature dermis (Lau et al., 2007). In this study, results of BrdU incorporation and MTT assays revealed that silymarin had no effect on fibroblast proliferation in the studied concentration range.

In addition, by measuring hydroxyproline content, we found that silymarin did not exhibit a significant effect on collagen content in fibroblast cell culture. It was shown previously that silibinin, the major component of silymarin, has no effect on type I collagen synthesis in vitro (Abul Hashem et al., 2008).

Conclusion

In conclusion, this study showed that silymarin has antioxidant and anti-inflammatory effects in human fibroblast cells, but does not have any effect on total collagen and cell proliferation. Therefore, silymarin may be useful as a therapeutic agent for the treatment of cutaneous wounds through antioxidation and anti-inflammation effects.

Declaration of interest

This article is a part of the PhD dissertation which was financially supported by Tehran University of Medical Sciences (Grant No. 88-1-30-8602) and Food and Drug Control Laboratory and Research Center. The authors report no conflict of interest.