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Indigo naturalis upregulates claudin-1 expression in human keratinocytes and psoriatic lesions
Journal of Ethnopharmacology 145 (2013) 614–620
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Indigo naturalis upregulates claudin-1 expression in human keratinocytes and psoriatic lesions Yin-Ku Lin a,b, Hsiao-Wen Chen b,c, Yann-Lii Leu d, Yueh-Lung Yang a,b, Yu Fang b, Tsong-Long Hwang d,e,n a
Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung 204, Taiwan. Department of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan. c Division of Urology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan. d Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan. e Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan 333, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 September 2012 Received in revised form 2 November 2012 Accepted 25 November 2012 Available online 7 December 2012
Ethnopharmacological relevance: Indigo naturalis is used in traditional Chinese medicine to treat various dermatoses. Our previous clinical studies showed that indigo naturalis is an effective treatment for psoriasis. Herein, the capabilities of indigo naturalis extract and its derivatives to increase claudin-1 expression and tight junction (TJ) function in human keratinocytes and psoriatic lesions were further studied. Materials and methods: Claudin-1 expression in psoriatic plaques with or without indigo naturalis treatment was analyzed by immunohistochemical methods. In primary human keratinocytes, the expression of claudin-1 was analyzed by fluorescent immunostaining, a real-time RT-PCR, and Western blot analysis. The effect of indigo naturalis on TJs was evaluated by measuring the transepithelial electrical resistance (TEER) and paracellular tracer flux. Results: The indigo naturalis extract upregulated mRNA and protein expressions of claudin-1 and function of TJs in primary human keratinocytes in concentration-dependent manners. Its main components, indirubin, indigo, and tryptanthrin, exerted synergistic effects on upregulating TJ functions in primary human keratinocytes. In addition, indigo naturalis increased the activity of protein kinase C (PKC), and a known potent PKC inhibitor, Ro318220, attenuated the indigo naturalisinduced claudin-1 expression. Significantly, restoration of claudin-1 was observed in healed psoriatic lesions after indigo naturalis treatment. Conclusions: Indigo naturalis upregulates claudin-1 expression and restores TJ function in keratinocytes. Our data also suggest that indirubin, indigo, and tryptanthrin have a synergistic effect on TJ function. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Claudin-1 Indigo naturalis Keratinocyte Psoriatic lesion Tight junction
1. Introduction Psoriasis is a chronic inflammatory skin disease characterized by erythematous papules and plaques with silvery scales that can appear on any surface of the body. Abnormalities in epidermal keratinocytes and dysfunction of the immune system are two major pathological mechanisms in the development of psoriasis (Lowes et al., 2007; Purnak and Purnak, 2012). In addition to hyperproliferation, aberrant epidermal differentiation of keratinocytes, and infiltration of inflammatory cells, a disturbance in
Abbreviations: TEER, transepithelial electrical resistance; PKC, protein kinase C; TJ, tight junction * Corresponding author. Graduate Institute of Natural Products, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan 333, Taoyuan, Taiwan. Tel./fax: þ 886 3 2118506. E-mail address: [email protected] (T.-L. Hwang). 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.11.044
the epidermal barrier function that accompanies alterations of epithelial junctional molecules such as tight junction (TJ) proteins, is associated with cutaneous epithelial inflammatory processes typified by psoriasis (Chung et al., 2005; Purnak and Purnak, 2012). Alteration of TJ proteins is an early event in psoriasis, and the impaired skin barrier function in psoriatic lesions may consequently contribute to the hyperproliferation of keratinocytes and cytokine activation of lymphocytes (Kirschner et al., 2009). Claudin-1 in the stratum granulosum is directly involved in the barrier function of mammalian skin and plays a critical role in human epidermal TJ function and keratinocyte proliferation (De Benedetto et al., 2011; Furuse et al., 2002). Significantly, the expression of claudin-1 is markedly lower in active plaque psoriasis than in healthy skin or uninvolved psoriatic skin lesions, indicating that low levels of claudin-1 expression are involved in the pathophysiology of psoriasis (Kirschner et al., 2010; Watson et al., 2007). Therefore, interest
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was generated about the potential of claudin-1 as a therapeutic target for developing new drugs to treat psoriasis. Indigo naturalis is a traditional Chinese herbal medicine widely used in clinical practice for treating various infectious and inflammatory skin diseases, such as carbuncles, furuncles, and eczema. Our previous clinical trials found that indigo naturalis ameliorates skin plaque psoriasis and psoriatic nails (Lin, 2011; Lin et al., 2007, 2008, 2011, 2012b). In in vitro studies, we also demonstrated that indigo naturalis inhibits proliferation, promotes differentiation, and reduces oxidative stress in epidermal keratinocytes, and inhibits the human neutrophil proinflammatory response and suppresses tumor necrosis factor-a-induced vascular cell adhesion molecule-1 expression in endothelial cells (Chang et al., 2010; Lin et al., 2009a, 2009b, 2012a). However, the detailed regulatory mechanisms of indigo naturalis have not been clarified and still need to be explored. In this study, we hypothesized that indigo naturalis ameliorates the severity of skin diseases by increasing claudin-1 expression and aiding barrier reestablishment. The effects of indigo naturalis and its three major components, indigo, indirubin, and tryptanthrin, on the function of TJs and expression of claudin-1 in primary human keratinocytes were investigated. Also, differences in claudin-1 expression were analyzed and compared between indigo naturalis-treated psoriatic plaques, vehicle-treated psoriatic plaques, and normal prelesional psoriatic skin.
2. Materials and methods The study protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taoyuan, Taiwan (CGMH IRB no. 96-1053C). All experiments adhered to principles of the Declaration of Helsinki. 2.1. Reagents Indigo naturalis powder prepared from Strobilanthes formosanus Moore (Acanthaceae) was purchased from Guang Sheng Trading (Taipei, Taiwan). The purity was ascertained by Dr. Rong-Chi Yang, the chief of the Chinese Herbal Pharmacy at Chang Gung Memorial Hospital. A voucher specimen (SF-1) was deposited in the herbarium of Chang Gung University. Indigo naturalis powder was dissolved in dimethyl sulfoxide (DMSO) at a ratio of 1:10 (w/v), sterilized by filtration (through a pore size of 0.2 mm), and was stored at 20 1C for subsequent bioassay testing. Fingerprints and quantity analyses of standard samples of indigo, indirubin, and tryptanthrin were established by Dr. Yann-Lii Leu. The Highperformance liquid chromatography (HPLC) analysis was performed on an Agilent 1100 series HPLC system (ECOM, Prague, Czech Republic) equipped with a quaternary pump, a vacuum degasser, an autosampler, a UV detector, and HP ChemStation software. The Indigo naturalis extract contained 0.7% indigo, 0.4% indirubin, and 0.04% tryptanthrin as determined by HPLC. Pure indigo was purchased from Fluka (Buchs, Switzerland), indirubin was obtained from Alexis (Lausen, Switzerland), and tryptanthrin was purchased from Wako (Osaka, Japan). Indigo naturalis, indigo, indirubin, and tryptanthrin dissolved in DMSO were prepared as stock solutions, diluted in culture media, and then used to treat epidermal keratinocytes. A rabbit anti-claudin-1 polyclonal antibody (pAb) RB-9209-P0 was purchased from Neomarkers (Fremont CA, USA) and a rabbit phospho-(Ser) PKC substrate antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Fluorescein isothiocyanatelabeled goat anti-rabbit immunoglobulin G was used as the secondary antibody for immunofluorescence microscopy.
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2.2. Cell culture Human neonatal foreskins obtained within 4 h of elective circumcision were used to culture primary human keratinocytes as previously described (Lin et al., 2009b). Keratinocytes were cultured in fresh keratinocyte serum-free medium (KSFM) (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 1 ng human recombinant epidermal growth factor/mL (Gibco) and 25 mg bovine pituitary extract/mL (Gibco). Primary human keratinocytes in the second to the fourth passages were used for the following experiments. 2.3. Cytotoxicity test Primary human keratinocytes were treated with various concentrations of indigo naturalis or DMSO alone for 24 h. Cytotoxicity was determined using a colorimetric MTT (Sigma, St. Louis, MO, USA) assay. Cell viability was evaluated at 570 nm with a 630-nm reference filter. Control values for DMSO alone were subtracted from all readings, and the mean optical densities were calculated. 2.4. Skin samples used in the study Skin samples consisted of paraffin-embedded punch biopsy samples that had been obtained from six patients (four men and two women, whose ages ranged 28–53 years, with a mean of 36.4710.1 years) with psoriasis who had completed a previous clinical trial at Chang Gung Memorial Hospital in Taoyuan or Taipei, and whose psoriatic plaques had cleared. In that trial, patients with psoriasis had been randomized to receive an 8-week course of topical indigo naturalis ointment or vehicle alone (Lin et al., 2007). 2.5. Immunofluorescence microscopy Primary human keratinocytes were grown on glass coverslips, treated with 0, 50, or 500 mg/ml indigo naturalis extract for 24 h at 37 1C. Cell were fixed in 10% formaldehyde for 15 min at room temperature, and then incubated in methanol at 20 1C for 10 min. After blocking with a solution containing 1% bovine serum albumin (BSA) and 1% goat serum in phosphate-buffered saline (PBS), an appropriate dilution of the primary monoclonal antibody: anti-claudin-1 was added, and samples were incubated for 1 h. Samples were then washed with PBS, and the secondary anti-rabbit antibody conjugated with FITC was added and allowed to react for 1 h. After washing with PBS, cells were stained with propidium iodide (PI), and coverslips were mounted with fluorescent mounting medium (Dakocytomation, Carointeria, CA, USA). Fluorescence signals were analyzed with a Nikon DXM1200 microscope and Nikon ACT-1 image analysis software. All images from the experiments were acquired and processed with the same settings, and representative areas were scanned. Paraffin sections of skin samples were deparaffinized and rehydrated. After blocking in solution containing 1% BSA and 1% goat serum in 1 PBS, appropriate dilution of primary monoclonal antibody: anti-claudin1 was added and incubated overnight. Subsequently, the samples were washed with 1 PBS and the secondary anti-rabbit antibody conjugated with FITC was added and incubated for 30 min. Then the procedures of immunofluorescence staining were the same as above described. 2.6. Quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis Total RNA from primary human keratinocytes was isolated, and a quantitative real-time RT-PCR was performed using an
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SYBR Green and MxPro-Mx3000P QPCR machine (Stratagene, La Jolla, CA, USA). Aliquots (20 ng) of complementary (c)DNA were used for each quantitative PCR, and each reaction was run in triplicate. The primer pair for claudin-1 (forward 50 -GTTGGGCTTCATTCTCGC-30 and reverse 50 -CTGCACCTCATCGTCTTCC-30 ) was provided by MDBio (Taipei, Taiwan). Differences in relative gene expressions between experimental groups were determined using MxPro software (Stratagene), and GAPDH was used as an internal control. All real-time RT-PCRs were performed in triplicate, and changes in gene expression were reported as multiples of increases relative to the untreated controls.
2.7. Western blot analysis Western blots were carried out as previously described (Lin et al., 2007). Briefly, whole-cell lysates were prepared, and protein concentrations were determined using a Pierce BSA protein assay (Rockford, IL, USA) with BSA as the standard. The same protein amounts were loaded on a sodium dodecylsulfate (SDS) polyacrylamide gel (10 or 12%) for electrophoresis. Gels were blotted overnight onto polyvinylidene difluoride (PVDF) membranes. The protein bands were confirmed using an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Little Chalfont, UK).
2.8. Measurement of transepithelial electrical resistance (TEER) and paracellular flux Keratinocytes were plated onto transwells with 6-mm-diameter inserts and a pore size of 0.4 mm (Millicell-PCF inserts, with an effective membrane area of 0.6 cm2, Millipore, Billerica, MA, USA). TEER measurements of individual monolayers were repeatedly performed with a Millicell-ERS epithelial voltohmmeter (Millipore) using different concentrations of indigo naturalis at 24 h with reproducibly placed electrodes. TEER values were calculated by subtracting the contribution of the bare filter and medium (TEER¼(R sample—R blank) effective membrane area; where R sample is the value of each sample, and R blank is the value of the bare filter and medium). The TEER of a keratinocyte sheet reflects the transepithelial permeability of water-soluble ions. A higher TEER value indicates lower ionic permeability.
Fig. 1. Effects of indigo naturalis on epidermal barrier function. The barrier function was monitored by measuring the transepithelial electrical resistance (TEER) and paracellular flux of 4-kDa FITC-dextran. (A) The TEER of keratinocytes was measured 24 h after incubation with different concentrations of indigo naturalis extract or 0.1% DMSO. (B) The TEER of keratinocytes cultured in 0.6-cm transwells was measured at 0, 12, 24, and 36 h after treatment with 500 mg/ml indigo naturalis. (C) Paracellular diffusion of 4-kDa FITC-dextran through keratinocytes cultured in a 0.6-cm transwell was measured at 24 h with either different concentrations of indigo naturalis extract or 0.1% DMSO. Data are presented as the mean7SEM from three experiments. npo0.05 indicates a statistically significant difference between indigo naturalis-treated and untreated cells.
Fig. 2. Indigo naturalis extract increased expression levels of claudin-1 mRNA and protein. (A) Cultured human keratinocytes were pretreated with either 0, 10, 50, 250, or 500 mg/ml indigo naturalis extract or 0.1% DMSO for 24 h. A quantitative real-time PCR analysis of claudin-1 in cultured human keratinocytes was performed using GAPDH as a reference gene. Results are presented as the mean 7SEM of three independent experiments. np o0.05 indicates a statistically significant difference between indigo naturalis-treated and untreated cells. (B) Protein expression of claudin-1 was analyzed by Western blotting. Tubulin was used as the internal control. Experiments were repeated at least three times with similar results.
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The fluorescent tracer permeability assay analyzes TJ integrity and paracellular permeability. Monolayers of keratinocytes were grown on a transwell filter and treated with different concentrations of drugs for 24 h. Before conducting the assay, 4-kDa FITCdextran (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 10 mg/ml of P buffer (10 mM HEPES (pH 7.4), 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl2, and 145 mM NaCl). Each part of the medium including the apical and basal compartments of the keratinocyte sheet was replaced with 250 ml of P buffer containing FITC-dextran or 750 ml of P buffer, respectively. After 2 h of incubation at 37 1C, the amount of FITC-dextran that had diffused from the apical to the basal side of the cellular sheet was measured with a VICTOR3 multilabel reader (PerkinElmer, Waltham, MA, USA). The flux of FITC-dextran can be used to determine the permeability of the solutes beyond the keratinocyte sheet. A lower flux of FITC-dextran means a lower permeability beyond the paracellular pathway. The experiment was repeated three times in triplicate. 2.9. Statistical analysis Data are expressed as the mean7standard error of the mean (SEM). Differences between groups were evaluated using Student’s t-test. A value of po0.05 was considered to indicate statistical significance.
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3. Results 3.1. Indigo naturalis increased TJ function in primary human keratinocytes TJ function in cell culture is generally measured using TEER or paracellular tracer flux (Harhaj and Antonetti, 2004). Indigo naturalis (10–500 mg/ml) did not produce a cytotoxic effect in primary human keratinocytes, as assayed by the MTT method (data not shown). These data agree with our previous results showing that 500 mg/ml of indigo naturalis is the threshold concentration for causing cytotoxic effects (Lin et al., 2009b). Therefore, 500 mg/ml of indigo naturalis was the maximum concentration used in this study. Indigo naturalis extract (10–500 mg/ml) produced a significant concentration- and timedependent increase in the TEER in cultured monolayer keratinocytes (Fig. 1A and B). However, this increase was followed by a slight decrease in the TEER over the next 36 h (Fig. 1B), which was likely because of detachment of keratinocytes from the insert surface. In addition, we observed a marked reduction in the flux of 4-kDa FITC-dextran from the apical to the basal side following incubation with different concentrations of indigo naturalis (10–500 mg/ml) for 24 h (Fig. 1C). There was no significant difference in flux between the DMSO-treated group and untreated control group.
Fig. 3. Indigo naturalis extract upregulated claudin-1 expression in cultured keratinocytes in a (A) concentration- and (B) time-dependent manner as analyzed by immunofluorescent staining. Expression levels of claudin-1 in keratinocytes were determined by immunofluorescent staining using monoclonal anti-claudin-1 antibodies and then with a FITC-conjugated secondary antibody (green fluorescence). Cell nuclei were stained with propidium iodide (orange fluorescence). Scale bar: 20 mm. Experiments were repeated at least three times with similar results. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.2. Indigo naturalis upregulated the messenger (m)RNA and protein expressions of claudin-1 in primary human keratinocytes Keratinocytes were treated with indigo naturalis (10–500 mg/ ml) for 24 h, and levels of claudin-1 mRNA and protein expressions were analyzed by a quantitative real-time RT-PCR and Western blot analysis. Results showed that mRNA and protein levels of claudin-1 in primary human keratinocytes increased in concentrationdependent manners after indigo naturalis treatment (Fig. 2A and B). There was no significant difference in the level of mRNA or protein between the untreated control and vehicle-treated groups.
3.3. Indigo naturalis enhanced the cytosolic and cell junctional expressions of claudin-1 in primary human keratinocytes To confirm whether indigo naturalis upregulates the expression of claudin-1 in epidermal keratinocytes, an immunofluorescent analysis of claudin-1 expression was performed. The immunofluorescent study revealed that treatment with the indigo naturalis extract led to increases in claudin-1 expression in a concentrationdependent manner in primary human keratinocytes (Fig. 3A). The fluorescence intensity of claudin-1 proteins was highest in large intracellular particles and the intercellular borders of indigo naturalis-treated keratinocytes. In addition, the relative abundance of claudin-1 also increased in a time-dependent manner (Fig. 3B).
3.4. Indigo, indirubin, and tryptanthrin had a synergistic effect in increasing TJ function in primary human keratinocytes Using high-performance liquid chromatography (HPLC), we determined that the concentrations of the three major components in 500 mg/ml indigo naturalis extract were 13.3 mM indirubin, 7.3 mM indigo, and 0.8 mM tryptanthrin (Lin et al., 2009a, 2009b). We found that none of the components individually exerted an effect comparable to that exerted by indigo naturalis, and that only when the three components were added together was there an increase in the TEER of keratinocytes similar to that with indigo naturalis (Fig. 4A). Although the three major components of indigo naturalis exerted a synergistic effect on upregulating TJ function, indirubin accounted for the majority of the effect (Fig. 4B). The results of Western blotting also demonstrated that indirubin upregulated the expression of claudin-1 in a concentration-dependent manner in cultured human keratinocytes (Fig. 4C).
3.5. Role of protein kinase C (PKC) in indigo naturalis-induced claudin-1 expression To further delineate the signaling pathways involved in indigo naturalis-induced claudin-1 expression, cells were co-incubated with a number of pharmacological inhibitors. Treatment with the PKC inhibitor, Ro318220 (3 mM), but not inhibitors of p38 mitogen-activated protein kinase (MAPK) (SB203580, 10 mM), ERK (PD98059, 10 mM), JNK (SP600125, 10 mM), or PI3K (LY294002, 10 mM), inhibited the expression of claudin-1 (Fig. 5A). These results suggested that PKC, but not MAPK or PI3K, mediates the expression of claudin-1 induced by indigo naturalis. To further examine whether indigo naturalis causes PKC activation, phosphorylation of the PKC substrate was assayed using a phospho-(Ser) PKC substrate antibody (Cell Signaling Technology) and determined by Western blotting. As shown in Fig. 5B, stimulation of cultured keratinocytes with indigo naturalis resulted in the rapid phosphorylation of PKC substrate at 130–55 kDa.
Fig. 4. The combination of indirubin, indigo, and tryptanthrin exerted a synergistic effect on enhancing the transepithelial electrical resistance (TEER) of keratinocytes. (A) The TEER was measured in keratinocytes after incubation with 500 mg/ml indigo naturalis, 13.3 mM indirubin, 7.3 mM indigo, or 0.8 mM tryptanthrin. The TEER in keratinocytes with the combined addition of 13.3 mM indirubin, 7.3 mM indigo, and 0.8 mM tryptanthrin was also measured. (B) The TEER of keratinocytes was measured 24 h after respective incubation with different concentrations of indigo, indirubin, and tryptanthrin. Data are presented as the mean 7 SEM from three experiments. npo 0.05 indicates a statistically significant difference between treated and untreated cells. (C) Cultured keratinocytes were pretreated with indirubin (5 and 20 mM) or 0.1% DMSO for 24 h. Protein expression of claudin-1 was analyzed by Western blotting. Tubulin was used as the internal control. Experiments were repeated at least three times with similar results.
3.6. Expression of claudin-1 in psoriatic lesions returned to normal after treatment with indigo naturalis In vehicle-treated psoriatic plaques, the expression of claudin1 was very weak and was only slightly detected in upper spinous layers (Fig. 6A). However, in tissue sections taken and prepared from healed psoriatic plaques after indigo naturalis topical therapy, the expression of claudin-1 had normalized and was similar to that found in normal perilesional psoriatic skin samples (Fig. 6B and C). The staining pattern of claudin-1 resembled that
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of ‘chicken-wire’ and was strongly detected in cellular junctions of the granular and spinosum layers, but weakly detected in the basal layer (Fig. 6B).
4. Discussion Clinical studies showed that topical indigo naturalis ointment is an effective treatment for psoriasis (Lin, 2011; Lin et al., 2007, 2008, 2011, 2012b). In this study, we showed that indigo naturalis extract upregulated claudin-1 expression and enhanced TJ function in cultured primary human keratinocytes. In addition, we found that the expression of claudin-1 returned to normal after treatment with indigo naturalis and was similar to that found in normal perilesional psoriatic skin. Our findings suggest that indigo naturalis can promote the restoration of damaged epidermal structures. TJs are intercellular structures that are required for epithelial barrier formation and which function in a wide variety of tissues
Fig. 5. Expression of claudin-1 induced by indigo naturalis was meditated by activation of the protein kinase C (PKC) pathway. (A) The expression of claudin1 was induced by 500 mg/ml indigo naturalis for 24 h. Pharmacological inhibitors were preincubated for 1 h before the addition of indigo naturalis. (B) Cultured human keratinocytes were pretreated with 250 mg/ml indigo naturalis or 0.1% DMSO for 0–60 min. Phosphorylation of the PKC substrate was analyzed by Western blotting. Tubulin was used as the internal control. Experiments were repeated at least three times with similar results.
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including the skin (Kirschner and Brandner, 2012). Studies showed that inflammatory skin disorders such as atopic dermatitis and psoriasis exhibit alterations in TJ expression and distribution (Brandner, 2009; Segre, 2006). Claudin-1 is involved in the skin barrier function and is critical for the structural and functional elements of the epithelial TJ barrier (Furuse et al., 2002). Previous in vitro studies showed that claudin-1 overexpression was correlated with an increased TEER and reduced flux of 4-kDa dextran in MDCK cells (Harhaj and Antonetti, 2004). The association between claudin-1 expression and the development of the epidermal barrier was also demonstrated in human keratinocyte monolayers. De Benedetto et al. (2011) found that selective downregulation of claudin-1 expression markedly increased the paracellular permeability, decreased resistance, and enhanced proliferation. In addition, loss of TJ function in keratinocytes was demonstrated in diseased skin (Ohnemus et al., 2008). Interestingly, in this study, we found that indigo naturalis and its active component, indirubin, significantly enhanced claudin-1 expression and that the increase in expression coincided with an increase in the TEER. These novel findings suggested that indigo naturalis helps restore the function of TJs in the epidermis. In recent years, some researchers suggested TJs may be a target for preventing and treating inflammatory skin diseases, especially with topical drug discovery and development (Kondoh et al., 2008). In TCM clinical practice, indigo naturalis is used topically to treat various infectious and inflammatory skin diseases. Besides its anti-inflammatory, antibacterial, and antiviral effects, restoring TJs may also be one of the mechanisms by which indigo naturalis ameliorates these skin disorders. It was noted that restoration of claudin-1 expression was observed in healed psoriatic lesions after indigo naturalis treatment. Results again support the upregulation of claudin-1 expression by indigo naturalis may ameliorate the severity of skin diseases. The intracellular signaling mechanisms responsible for claudin1 expression in human keratinocytes are very complex and not completely understood. We found that co-treatment with a PKC inhibitor, but not co-treatment with inhibitors of p38 MAPK, ERK, JNK, or PI3K, led to the inhibition of claudin-1 expression in indigo naturalis-treated cells. In addition, we found that treatment with indigo naturalis resulted in an increase in PKC activity in cultured keratinocytes, suggesting that indigo naturalis-induced claudin-1 expression is mediated, at least in part, by activating the PKC pathway. Similar findings were noted in primary human nasal and pancreatic duct epithelial cells (Koizumi et al., 2008; Yamaguchi et al., 2010). Recently, indigo naturalis and indirubin have also been shown to inhibit EGFR activation and CDC25B expression in cultured keratinocytes (Hsieh et al., 2012). It is notable that EGFR has an important negative regulatory role on the expression of
Fig. 6. Expression of claudin-1 was regained in psoriatic skin sections treated with indigo naturalis. Skin biopsies were taken from a 40 year-old male (with a duration of psoriasis of 10 years) after 8 weeks of treatment. The psoriatic lesions were located on his bilateral legs. (A) Vehicle-treated lesion. (B) Healed psoriasis after indigo naturalis treatment. (C) Normal perilesional skin. Representative results of the immunohistochemical staining analysis using anti-claudin-1antibodies are shown. Photos at high magnification are shown in the lower corner. Scale bar: 100 and 50 mm.
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epidermal TJs (Tran et al., 2012). Therefore, inhibition of EGFR activation by indigo naturalis is also a possible mechanism for increasing claudin-1 expression. Further study is needed to explore this hypothesis. Our finding that the expression of claudin-1 in active psoriatic plaques is lower than those in normal perilesional psoriatic skin and healthy skin is consistent with findings from a number of studies (Brandner et al., 2006; Kirschner and Brandner, 2012; Kirschner et al., 2009, 2010; Watson et al., 2007). However, other studies showed that the levels of expression of claudin-1 in psoriatic plaques do not significantly differ from levels seen in perilesional psoriatic skin and healthy skin (Itoh et al., 2005; Peltonen et al., 2007). Further studies are needed to elucidate the roles that claudin-1 alterations play in psoriasis. One limitation of this preliminary study was a lack of in vivo or in vitro investigations of the impact of indigo naturalis on the immune system, such as expression of cytokines and T cells, which might suppress expression of claudin-1 and the epidermal barrier function. It is not clear from the in vitro findings whether indigo naturalis has a similar restorative effect in vivo where immune system factors are present. In conclusion, indigo naturalis promotes claudin-1 expression and enhanced TJ function in primary human keratinocytes. Our data show that the PKC pathway is involved in indigo naturalis-induced claudin-1 expression. The three major components of indigo naturalis, indirubin, indigo, and tryptanthrin, exerted a synergistic effect on upregulating TJ function, with indirubin accounting for the majority of the effect. The results of this study provide additional evidence in support of the effectiveness of indigo naturalis as a treatment for psoriasis.
Acknowledgments This study was supported by grants from the Chang Gung Memorial Hospital (CMRPG260192) and National Science Council (NSC97-2320-B-182A-003), Taiwan. References Brandner, J.M., 2009. Tight junctions and tight junction proteins in mammalian epidermis. European Journal of Pharmaceutics and Biopharmaceutics 72, 289–294. Brandner, J.M., Kief, S., Wladykowski, E., Houdek, P., Moll, I., 2006. Tight junction proteins in the skin. Skin Pharmacology Physiology 19, 71–77. Chang, H.N., Pang, J.H., Yang, S.H., Hung, C.F., Chiang, C.H., Lin, T.Y., Lin, Y.K., 2010. Inhibitory effect of indigo naturalis on tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells. Molecules 15, 6423–6435. Chung, E., Cook, P.W., Parkos, C.A., Park, Y.K., Pittelkow, M.R., Coffey, R.J., 2005. Amphiregulin causes functional downregulation of adherens junctions in psoriasis. Journal of Investigative Dermatology 124, 1134–1140. De Benedetto, A., Rafaels, N.M., McGirt, L.Y., Ivanov, A.I., Georas, S.N., Cheadle, C., Berger, A.E., Zhang, K., Vidyasagar, S., Yoshida, T., Boguniewicz, M., Hata, T., Schneider, L.C., Hanifin, J.M., Gallo, R.L., Novak, N., Weidinger, S., Beaty, T.H., Leung, D.Y., Barnes, K.C., Beck, L.A., 2011. Tight junction defects in patients with atopic dermatitis. Journal of Allergy and Clinical Immunology 127, e771–e777. Furuse, M., Hata, M., Furuse, K., Yoshida, Y., Haratake, A., Sugitani, Y., Noda, T., Kubo, A., Tsukita, S., 2002. Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. Journal of Cell Biology 156, 1099–1111. Harhaj, N.S., Antonetti, D.A., 2004. Regulation of tight junctions and loss of barrier function in pathophysiology. International Journal of Biochemistry & Cell Biology 36, 1206–1237.
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Alteration of tight junction proteins is an early event in psoriasis: putative involvement of proinflammatory cytokines. American Journal of Pathology 175, 1095–1106. Koizumi, J., Kojima, T., Ogasawara, N., Kamekura, R., Kurose, M., Go, M., Harimaya, A., Murata, M., Osanai, M., Chiba, H., Himi, T., Sawada, N., 2008. Protein kinase C enhances tight junction barrier function of human nasal epithelial cells in primary culture by transcriptional regulation. Molecular Pharmacology 74, 432–442. Kondoh, M., Yoshida, T., Kakutani, H., Yagi, K., 2008. Targeting tight junction proteins-significance for drug development. Drug Discovery Today 13, 180–186. Lin, Y.K., 2011. Indigo naturalis oil extract drops in the treatment of moderate to severe nail psoriasis: a small case series. Archives of Dermatology 147, 627–629. Lin, Y.K., Chang, C.J., Chang, Y.C., Wong, W.R., Chang, S.C., Pang, J.H., 2008. Clinical assessment of patients with recalcitrant psoriasis in a randomized, observerblind, vehicle-controlled trial using indigo naturalis. Archives of Dermatology 144, 1457–1464. Lin, Y.K., Chen, H.W., Yang, S.H., Leu, Y.L., Huang, Y.H., Yen, H.C., 2012a. Protective effect of indigo naturalis extract against oxidative stress in cultured human keratinocytes. Journal of Ethnopharmacology 139, 893–896. Lin, Y.K., Leu, Y.L., Huang, T.H., Wu, Y.H., Chung, P.J., Su Pang, J.H., Hwang, T.L., 2009a. Anti-inflammatory effects of the extract of indigo naturalis in human neutrophils. Journal of Ethnopharmacology 125, 51–58. Lin, Y.K., Leu, Y.L., Yang, S.H., Chen, H.W., Wang, C.T., Pang, J.H., 2009b. Anti-psoriatic effects of indigo naturalis on the proliferation and differentiation of keratinocytes with indirubin as the active component. Journal of Dermatological Science 54, 168–174. Lin, Y.K., See, L.C., Chang, Y.C., Huang, Y.H., Chen, J.L., Tsou, T.C., Leu, Y.L., Shen, Y.M., 2011. Treatment of psoriatic nails with indigo naturalis oil extract: a noncontrolled pilot study. Dermatology 223, 239–243. Lin, Y.K., See, L.C., Huang, Y.H., Chang, Y.C., Tsou, T.C., Leu, Y.L., Shen, Y.M., 2012b. Comparison of refined and crude indigo naturalis ointment in treating psoriasis: randomized, observer-blind, controlled, intrapatient trial. Archives of Dermatology 148, 397–400. Lin, Y.K., Wong, W.R., Chang, Y.C., Chang, C.J., Tsay, P.K., Chang, S.C., Pang, J.H., 2007. The efficacy and safety of topically applied indigo naturalis ointment in patients with plaque-type psoriasis. Dermatology 214, 155–161. Lowes, M.A., Bowcock, A.M., Krueger, J.G., 2007. Pathogenesis and therapy of psoriasis. Nature 445, 866–873. Ohnemus, U., Kohrmeyer, K., Houdek, P., Rohde, H., Wladykowski, E., Vidal, S., Horstkotte, M.A., Aepfelbacher, M., Kirschner, N., Behne, M.J., Moll, I., Brandner, J.M., 2008. Regulation of epidermal tight-junctions (TJ) during infection with exfoliative toxin-negative Staphylococcus strains. Journal of Investigative Dermatology 128, 906–916. Peltonen, S., Riehokainen, J., Pummi, K., Peltonen, J., 2007. Tight junction components occludin, ZO-1, and claudin-1, -4 and -5 in active and healing psoriasis. British Journal of Dermatology 156, 466–472. Purnak, T., Purnak, S., 2012. The inflammatory markers in psoriasis. British Journal of Dermatology 167, 457–458. Segre, J.A., 2006. Epidermal barrier formation and recovery in skin disorders. Journal of Clinical Investigation 116, 1150–1158. Tran, Q.T., Kennedy, L.H., Leon Carrion, S., Bodreddigari, S., Goodwin, S.B., Sutter, C.H., Sutter, T.R., 2012. EGFR regulation of epidermal barrier function. Physiological Genomics 44, 455–469. Watson, R.E., Poddar, R., Walker, J.M., McGuill, I., Hoare, L.M., Griffiths, C.E., O’Neill, C.A., 2007. Altered claudin expression is a feature of chronic plaque psoriasis. Journal of Pathology 212, 450–458. Yamaguchi, H., Kojima, T., Ito, T., Kimura, Y., Imamura, M., Son, S., Koizumi, J., Murata, M., Nagayama, M., Nobuoka, T., Tanaka, S., Hirata, K., Sawada, N., 2010. Transcriptional control of tight junction proteins via a protein kinase C signal pathway in human telomerase reverse transcriptase-transfected human pancreatic duct epithelial cells. American Journal of Pathology 177, 698–712.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Indigo naturalis upregulates claudin-1 expression in human keratinocytes and psoriatic lesions Yin-Ku Lin a,b, Hsiao-Wen Chen b,c, Yann-Lii Leu d, Yueh-Lung Yang a,b, Yu Fang b, Tsong-Long Hwang d,e,n a
Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung 204, Taiwan. Department of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan. c Division of Urology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan. d Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan. e Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan 333, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 September 2012 Received in revised form 2 November 2012 Accepted 25 November 2012 Available online 7 December 2012
Ethnopharmacological relevance: Indigo naturalis is used in traditional Chinese medicine to treat various dermatoses. Our previous clinical studies showed that indigo naturalis is an effective treatment for psoriasis. Herein, the capabilities of indigo naturalis extract and its derivatives to increase claudin-1 expression and tight junction (TJ) function in human keratinocytes and psoriatic lesions were further studied. Materials and methods: Claudin-1 expression in psoriatic plaques with or without indigo naturalis treatment was analyzed by immunohistochemical methods. In primary human keratinocytes, the expression of claudin-1 was analyzed by fluorescent immunostaining, a real-time RT-PCR, and Western blot analysis. The effect of indigo naturalis on TJs was evaluated by measuring the transepithelial electrical resistance (TEER) and paracellular tracer flux. Results: The indigo naturalis extract upregulated mRNA and protein expressions of claudin-1 and function of TJs in primary human keratinocytes in concentration-dependent manners. Its main components, indirubin, indigo, and tryptanthrin, exerted synergistic effects on upregulating TJ functions in primary human keratinocytes. In addition, indigo naturalis increased the activity of protein kinase C (PKC), and a known potent PKC inhibitor, Ro318220, attenuated the indigo naturalisinduced claudin-1 expression. Significantly, restoration of claudin-1 was observed in healed psoriatic lesions after indigo naturalis treatment. Conclusions: Indigo naturalis upregulates claudin-1 expression and restores TJ function in keratinocytes. Our data also suggest that indirubin, indigo, and tryptanthrin have a synergistic effect on TJ function. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Claudin-1 Indigo naturalis Keratinocyte Psoriatic lesion Tight junction
1. Introduction Psoriasis is a chronic inflammatory skin disease characterized by erythematous papules and plaques with silvery scales that can appear on any surface of the body. Abnormalities in epidermal keratinocytes and dysfunction of the immune system are two major pathological mechanisms in the development of psoriasis (Lowes et al., 2007; Purnak and Purnak, 2012). In addition to hyperproliferation, aberrant epidermal differentiation of keratinocytes, and infiltration of inflammatory cells, a disturbance in
Abbreviations: TEER, transepithelial electrical resistance; PKC, protein kinase C; TJ, tight junction * Corresponding author. Graduate Institute of Natural Products, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan 333, Taoyuan, Taiwan. Tel./fax: þ 886 3 2118506. E-mail address: [email protected] (T.-L. Hwang). 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.11.044
the epidermal barrier function that accompanies alterations of epithelial junctional molecules such as tight junction (TJ) proteins, is associated with cutaneous epithelial inflammatory processes typified by psoriasis (Chung et al., 2005; Purnak and Purnak, 2012). Alteration of TJ proteins is an early event in psoriasis, and the impaired skin barrier function in psoriatic lesions may consequently contribute to the hyperproliferation of keratinocytes and cytokine activation of lymphocytes (Kirschner et al., 2009). Claudin-1 in the stratum granulosum is directly involved in the barrier function of mammalian skin and plays a critical role in human epidermal TJ function and keratinocyte proliferation (De Benedetto et al., 2011; Furuse et al., 2002). Significantly, the expression of claudin-1 is markedly lower in active plaque psoriasis than in healthy skin or uninvolved psoriatic skin lesions, indicating that low levels of claudin-1 expression are involved in the pathophysiology of psoriasis (Kirschner et al., 2010; Watson et al., 2007). Therefore, interest
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was generated about the potential of claudin-1 as a therapeutic target for developing new drugs to treat psoriasis. Indigo naturalis is a traditional Chinese herbal medicine widely used in clinical practice for treating various infectious and inflammatory skin diseases, such as carbuncles, furuncles, and eczema. Our previous clinical trials found that indigo naturalis ameliorates skin plaque psoriasis and psoriatic nails (Lin, 2011; Lin et al., 2007, 2008, 2011, 2012b). In in vitro studies, we also demonstrated that indigo naturalis inhibits proliferation, promotes differentiation, and reduces oxidative stress in epidermal keratinocytes, and inhibits the human neutrophil proinflammatory response and suppresses tumor necrosis factor-a-induced vascular cell adhesion molecule-1 expression in endothelial cells (Chang et al., 2010; Lin et al., 2009a, 2009b, 2012a). However, the detailed regulatory mechanisms of indigo naturalis have not been clarified and still need to be explored. In this study, we hypothesized that indigo naturalis ameliorates the severity of skin diseases by increasing claudin-1 expression and aiding barrier reestablishment. The effects of indigo naturalis and its three major components, indigo, indirubin, and tryptanthrin, on the function of TJs and expression of claudin-1 in primary human keratinocytes were investigated. Also, differences in claudin-1 expression were analyzed and compared between indigo naturalis-treated psoriatic plaques, vehicle-treated psoriatic plaques, and normal prelesional psoriatic skin.
2. Materials and methods The study protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taoyuan, Taiwan (CGMH IRB no. 96-1053C). All experiments adhered to principles of the Declaration of Helsinki. 2.1. Reagents Indigo naturalis powder prepared from Strobilanthes formosanus Moore (Acanthaceae) was purchased from Guang Sheng Trading (Taipei, Taiwan). The purity was ascertained by Dr. Rong-Chi Yang, the chief of the Chinese Herbal Pharmacy at Chang Gung Memorial Hospital. A voucher specimen (SF-1) was deposited in the herbarium of Chang Gung University. Indigo naturalis powder was dissolved in dimethyl sulfoxide (DMSO) at a ratio of 1:10 (w/v), sterilized by filtration (through a pore size of 0.2 mm), and was stored at 20 1C for subsequent bioassay testing. Fingerprints and quantity analyses of standard samples of indigo, indirubin, and tryptanthrin were established by Dr. Yann-Lii Leu. The Highperformance liquid chromatography (HPLC) analysis was performed on an Agilent 1100 series HPLC system (ECOM, Prague, Czech Republic) equipped with a quaternary pump, a vacuum degasser, an autosampler, a UV detector, and HP ChemStation software. The Indigo naturalis extract contained 0.7% indigo, 0.4% indirubin, and 0.04% tryptanthrin as determined by HPLC. Pure indigo was purchased from Fluka (Buchs, Switzerland), indirubin was obtained from Alexis (Lausen, Switzerland), and tryptanthrin was purchased from Wako (Osaka, Japan). Indigo naturalis, indigo, indirubin, and tryptanthrin dissolved in DMSO were prepared as stock solutions, diluted in culture media, and then used to treat epidermal keratinocytes. A rabbit anti-claudin-1 polyclonal antibody (pAb) RB-9209-P0 was purchased from Neomarkers (Fremont CA, USA) and a rabbit phospho-(Ser) PKC substrate antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Fluorescein isothiocyanatelabeled goat anti-rabbit immunoglobulin G was used as the secondary antibody for immunofluorescence microscopy.
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2.2. Cell culture Human neonatal foreskins obtained within 4 h of elective circumcision were used to culture primary human keratinocytes as previously described (Lin et al., 2009b). Keratinocytes were cultured in fresh keratinocyte serum-free medium (KSFM) (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 1 ng human recombinant epidermal growth factor/mL (Gibco) and 25 mg bovine pituitary extract/mL (Gibco). Primary human keratinocytes in the second to the fourth passages were used for the following experiments. 2.3. Cytotoxicity test Primary human keratinocytes were treated with various concentrations of indigo naturalis or DMSO alone for 24 h. Cytotoxicity was determined using a colorimetric MTT (Sigma, St. Louis, MO, USA) assay. Cell viability was evaluated at 570 nm with a 630-nm reference filter. Control values for DMSO alone were subtracted from all readings, and the mean optical densities were calculated. 2.4. Skin samples used in the study Skin samples consisted of paraffin-embedded punch biopsy samples that had been obtained from six patients (four men and two women, whose ages ranged 28–53 years, with a mean of 36.4710.1 years) with psoriasis who had completed a previous clinical trial at Chang Gung Memorial Hospital in Taoyuan or Taipei, and whose psoriatic plaques had cleared. In that trial, patients with psoriasis had been randomized to receive an 8-week course of topical indigo naturalis ointment or vehicle alone (Lin et al., 2007). 2.5. Immunofluorescence microscopy Primary human keratinocytes were grown on glass coverslips, treated with 0, 50, or 500 mg/ml indigo naturalis extract for 24 h at 37 1C. Cell were fixed in 10% formaldehyde for 15 min at room temperature, and then incubated in methanol at 20 1C for 10 min. After blocking with a solution containing 1% bovine serum albumin (BSA) and 1% goat serum in phosphate-buffered saline (PBS), an appropriate dilution of the primary monoclonal antibody: anti-claudin-1 was added, and samples were incubated for 1 h. Samples were then washed with PBS, and the secondary anti-rabbit antibody conjugated with FITC was added and allowed to react for 1 h. After washing with PBS, cells were stained with propidium iodide (PI), and coverslips were mounted with fluorescent mounting medium (Dakocytomation, Carointeria, CA, USA). Fluorescence signals were analyzed with a Nikon DXM1200 microscope and Nikon ACT-1 image analysis software. All images from the experiments were acquired and processed with the same settings, and representative areas were scanned. Paraffin sections of skin samples were deparaffinized and rehydrated. After blocking in solution containing 1% BSA and 1% goat serum in 1 PBS, appropriate dilution of primary monoclonal antibody: anti-claudin1 was added and incubated overnight. Subsequently, the samples were washed with 1 PBS and the secondary anti-rabbit antibody conjugated with FITC was added and incubated for 30 min. Then the procedures of immunofluorescence staining were the same as above described. 2.6. Quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis Total RNA from primary human keratinocytes was isolated, and a quantitative real-time RT-PCR was performed using an
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SYBR Green and MxPro-Mx3000P QPCR machine (Stratagene, La Jolla, CA, USA). Aliquots (20 ng) of complementary (c)DNA were used for each quantitative PCR, and each reaction was run in triplicate. The primer pair for claudin-1 (forward 50 -GTTGGGCTTCATTCTCGC-30 and reverse 50 -CTGCACCTCATCGTCTTCC-30 ) was provided by MDBio (Taipei, Taiwan). Differences in relative gene expressions between experimental groups were determined using MxPro software (Stratagene), and GAPDH was used as an internal control. All real-time RT-PCRs were performed in triplicate, and changes in gene expression were reported as multiples of increases relative to the untreated controls.
2.7. Western blot analysis Western blots were carried out as previously described (Lin et al., 2007). Briefly, whole-cell lysates were prepared, and protein concentrations were determined using a Pierce BSA protein assay (Rockford, IL, USA) with BSA as the standard. The same protein amounts were loaded on a sodium dodecylsulfate (SDS) polyacrylamide gel (10 or 12%) for electrophoresis. Gels were blotted overnight onto polyvinylidene difluoride (PVDF) membranes. The protein bands were confirmed using an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Little Chalfont, UK).
2.8. Measurement of transepithelial electrical resistance (TEER) and paracellular flux Keratinocytes were plated onto transwells with 6-mm-diameter inserts and a pore size of 0.4 mm (Millicell-PCF inserts, with an effective membrane area of 0.6 cm2, Millipore, Billerica, MA, USA). TEER measurements of individual monolayers were repeatedly performed with a Millicell-ERS epithelial voltohmmeter (Millipore) using different concentrations of indigo naturalis at 24 h with reproducibly placed electrodes. TEER values were calculated by subtracting the contribution of the bare filter and medium (TEER¼(R sample—R blank) effective membrane area; where R sample is the value of each sample, and R blank is the value of the bare filter and medium). The TEER of a keratinocyte sheet reflects the transepithelial permeability of water-soluble ions. A higher TEER value indicates lower ionic permeability.
Fig. 1. Effects of indigo naturalis on epidermal barrier function. The barrier function was monitored by measuring the transepithelial electrical resistance (TEER) and paracellular flux of 4-kDa FITC-dextran. (A) The TEER of keratinocytes was measured 24 h after incubation with different concentrations of indigo naturalis extract or 0.1% DMSO. (B) The TEER of keratinocytes cultured in 0.6-cm transwells was measured at 0, 12, 24, and 36 h after treatment with 500 mg/ml indigo naturalis. (C) Paracellular diffusion of 4-kDa FITC-dextran through keratinocytes cultured in a 0.6-cm transwell was measured at 24 h with either different concentrations of indigo naturalis extract or 0.1% DMSO. Data are presented as the mean7SEM from three experiments. npo0.05 indicates a statistically significant difference between indigo naturalis-treated and untreated cells.
Fig. 2. Indigo naturalis extract increased expression levels of claudin-1 mRNA and protein. (A) Cultured human keratinocytes were pretreated with either 0, 10, 50, 250, or 500 mg/ml indigo naturalis extract or 0.1% DMSO for 24 h. A quantitative real-time PCR analysis of claudin-1 in cultured human keratinocytes was performed using GAPDH as a reference gene. Results are presented as the mean 7SEM of three independent experiments. np o0.05 indicates a statistically significant difference between indigo naturalis-treated and untreated cells. (B) Protein expression of claudin-1 was analyzed by Western blotting. Tubulin was used as the internal control. Experiments were repeated at least three times with similar results.
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The fluorescent tracer permeability assay analyzes TJ integrity and paracellular permeability. Monolayers of keratinocytes were grown on a transwell filter and treated with different concentrations of drugs for 24 h. Before conducting the assay, 4-kDa FITCdextran (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 10 mg/ml of P buffer (10 mM HEPES (pH 7.4), 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl2, and 145 mM NaCl). Each part of the medium including the apical and basal compartments of the keratinocyte sheet was replaced with 250 ml of P buffer containing FITC-dextran or 750 ml of P buffer, respectively. After 2 h of incubation at 37 1C, the amount of FITC-dextran that had diffused from the apical to the basal side of the cellular sheet was measured with a VICTOR3 multilabel reader (PerkinElmer, Waltham, MA, USA). The flux of FITC-dextran can be used to determine the permeability of the solutes beyond the keratinocyte sheet. A lower flux of FITC-dextran means a lower permeability beyond the paracellular pathway. The experiment was repeated three times in triplicate. 2.9. Statistical analysis Data are expressed as the mean7standard error of the mean (SEM). Differences between groups were evaluated using Student’s t-test. A value of po0.05 was considered to indicate statistical significance.
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3. Results 3.1. Indigo naturalis increased TJ function in primary human keratinocytes TJ function in cell culture is generally measured using TEER or paracellular tracer flux (Harhaj and Antonetti, 2004). Indigo naturalis (10–500 mg/ml) did not produce a cytotoxic effect in primary human keratinocytes, as assayed by the MTT method (data not shown). These data agree with our previous results showing that 500 mg/ml of indigo naturalis is the threshold concentration for causing cytotoxic effects (Lin et al., 2009b). Therefore, 500 mg/ml of indigo naturalis was the maximum concentration used in this study. Indigo naturalis extract (10–500 mg/ml) produced a significant concentration- and timedependent increase in the TEER in cultured monolayer keratinocytes (Fig. 1A and B). However, this increase was followed by a slight decrease in the TEER over the next 36 h (Fig. 1B), which was likely because of detachment of keratinocytes from the insert surface. In addition, we observed a marked reduction in the flux of 4-kDa FITC-dextran from the apical to the basal side following incubation with different concentrations of indigo naturalis (10–500 mg/ml) for 24 h (Fig. 1C). There was no significant difference in flux between the DMSO-treated group and untreated control group.
Fig. 3. Indigo naturalis extract upregulated claudin-1 expression in cultured keratinocytes in a (A) concentration- and (B) time-dependent manner as analyzed by immunofluorescent staining. Expression levels of claudin-1 in keratinocytes were determined by immunofluorescent staining using monoclonal anti-claudin-1 antibodies and then with a FITC-conjugated secondary antibody (green fluorescence). Cell nuclei were stained with propidium iodide (orange fluorescence). Scale bar: 20 mm. Experiments were repeated at least three times with similar results. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.2. Indigo naturalis upregulated the messenger (m)RNA and protein expressions of claudin-1 in primary human keratinocytes Keratinocytes were treated with indigo naturalis (10–500 mg/ ml) for 24 h, and levels of claudin-1 mRNA and protein expressions were analyzed by a quantitative real-time RT-PCR and Western blot analysis. Results showed that mRNA and protein levels of claudin-1 in primary human keratinocytes increased in concentrationdependent manners after indigo naturalis treatment (Fig. 2A and B). There was no significant difference in the level of mRNA or protein between the untreated control and vehicle-treated groups.
3.3. Indigo naturalis enhanced the cytosolic and cell junctional expressions of claudin-1 in primary human keratinocytes To confirm whether indigo naturalis upregulates the expression of claudin-1 in epidermal keratinocytes, an immunofluorescent analysis of claudin-1 expression was performed. The immunofluorescent study revealed that treatment with the indigo naturalis extract led to increases in claudin-1 expression in a concentrationdependent manner in primary human keratinocytes (Fig. 3A). The fluorescence intensity of claudin-1 proteins was highest in large intracellular particles and the intercellular borders of indigo naturalis-treated keratinocytes. In addition, the relative abundance of claudin-1 also increased in a time-dependent manner (Fig. 3B).
3.4. Indigo, indirubin, and tryptanthrin had a synergistic effect in increasing TJ function in primary human keratinocytes Using high-performance liquid chromatography (HPLC), we determined that the concentrations of the three major components in 500 mg/ml indigo naturalis extract were 13.3 mM indirubin, 7.3 mM indigo, and 0.8 mM tryptanthrin (Lin et al., 2009a, 2009b). We found that none of the components individually exerted an effect comparable to that exerted by indigo naturalis, and that only when the three components were added together was there an increase in the TEER of keratinocytes similar to that with indigo naturalis (Fig. 4A). Although the three major components of indigo naturalis exerted a synergistic effect on upregulating TJ function, indirubin accounted for the majority of the effect (Fig. 4B). The results of Western blotting also demonstrated that indirubin upregulated the expression of claudin-1 in a concentration-dependent manner in cultured human keratinocytes (Fig. 4C).
3.5. Role of protein kinase C (PKC) in indigo naturalis-induced claudin-1 expression To further delineate the signaling pathways involved in indigo naturalis-induced claudin-1 expression, cells were co-incubated with a number of pharmacological inhibitors. Treatment with the PKC inhibitor, Ro318220 (3 mM), but not inhibitors of p38 mitogen-activated protein kinase (MAPK) (SB203580, 10 mM), ERK (PD98059, 10 mM), JNK (SP600125, 10 mM), or PI3K (LY294002, 10 mM), inhibited the expression of claudin-1 (Fig. 5A). These results suggested that PKC, but not MAPK or PI3K, mediates the expression of claudin-1 induced by indigo naturalis. To further examine whether indigo naturalis causes PKC activation, phosphorylation of the PKC substrate was assayed using a phospho-(Ser) PKC substrate antibody (Cell Signaling Technology) and determined by Western blotting. As shown in Fig. 5B, stimulation of cultured keratinocytes with indigo naturalis resulted in the rapid phosphorylation of PKC substrate at 130–55 kDa.
Fig. 4. The combination of indirubin, indigo, and tryptanthrin exerted a synergistic effect on enhancing the transepithelial electrical resistance (TEER) of keratinocytes. (A) The TEER was measured in keratinocytes after incubation with 500 mg/ml indigo naturalis, 13.3 mM indirubin, 7.3 mM indigo, or 0.8 mM tryptanthrin. The TEER in keratinocytes with the combined addition of 13.3 mM indirubin, 7.3 mM indigo, and 0.8 mM tryptanthrin was also measured. (B) The TEER of keratinocytes was measured 24 h after respective incubation with different concentrations of indigo, indirubin, and tryptanthrin. Data are presented as the mean 7 SEM from three experiments. npo 0.05 indicates a statistically significant difference between treated and untreated cells. (C) Cultured keratinocytes were pretreated with indirubin (5 and 20 mM) or 0.1% DMSO for 24 h. Protein expression of claudin-1 was analyzed by Western blotting. Tubulin was used as the internal control. Experiments were repeated at least three times with similar results.
3.6. Expression of claudin-1 in psoriatic lesions returned to normal after treatment with indigo naturalis In vehicle-treated psoriatic plaques, the expression of claudin1 was very weak and was only slightly detected in upper spinous layers (Fig. 6A). However, in tissue sections taken and prepared from healed psoriatic plaques after indigo naturalis topical therapy, the expression of claudin-1 had normalized and was similar to that found in normal perilesional psoriatic skin samples (Fig. 6B and C). The staining pattern of claudin-1 resembled that
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of ‘chicken-wire’ and was strongly detected in cellular junctions of the granular and spinosum layers, but weakly detected in the basal layer (Fig. 6B).
4. Discussion Clinical studies showed that topical indigo naturalis ointment is an effective treatment for psoriasis (Lin, 2011; Lin et al., 2007, 2008, 2011, 2012b). In this study, we showed that indigo naturalis extract upregulated claudin-1 expression and enhanced TJ function in cultured primary human keratinocytes. In addition, we found that the expression of claudin-1 returned to normal after treatment with indigo naturalis and was similar to that found in normal perilesional psoriatic skin. Our findings suggest that indigo naturalis can promote the restoration of damaged epidermal structures. TJs are intercellular structures that are required for epithelial barrier formation and which function in a wide variety of tissues
Fig. 5. Expression of claudin-1 induced by indigo naturalis was meditated by activation of the protein kinase C (PKC) pathway. (A) The expression of claudin1 was induced by 500 mg/ml indigo naturalis for 24 h. Pharmacological inhibitors were preincubated for 1 h before the addition of indigo naturalis. (B) Cultured human keratinocytes were pretreated with 250 mg/ml indigo naturalis or 0.1% DMSO for 0–60 min. Phosphorylation of the PKC substrate was analyzed by Western blotting. Tubulin was used as the internal control. Experiments were repeated at least three times with similar results.
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including the skin (Kirschner and Brandner, 2012). Studies showed that inflammatory skin disorders such as atopic dermatitis and psoriasis exhibit alterations in TJ expression and distribution (Brandner, 2009; Segre, 2006). Claudin-1 is involved in the skin barrier function and is critical for the structural and functional elements of the epithelial TJ barrier (Furuse et al., 2002). Previous in vitro studies showed that claudin-1 overexpression was correlated with an increased TEER and reduced flux of 4-kDa dextran in MDCK cells (Harhaj and Antonetti, 2004). The association between claudin-1 expression and the development of the epidermal barrier was also demonstrated in human keratinocyte monolayers. De Benedetto et al. (2011) found that selective downregulation of claudin-1 expression markedly increased the paracellular permeability, decreased resistance, and enhanced proliferation. In addition, loss of TJ function in keratinocytes was demonstrated in diseased skin (Ohnemus et al., 2008). Interestingly, in this study, we found that indigo naturalis and its active component, indirubin, significantly enhanced claudin-1 expression and that the increase in expression coincided with an increase in the TEER. These novel findings suggested that indigo naturalis helps restore the function of TJs in the epidermis. In recent years, some researchers suggested TJs may be a target for preventing and treating inflammatory skin diseases, especially with topical drug discovery and development (Kondoh et al., 2008). In TCM clinical practice, indigo naturalis is used topically to treat various infectious and inflammatory skin diseases. Besides its anti-inflammatory, antibacterial, and antiviral effects, restoring TJs may also be one of the mechanisms by which indigo naturalis ameliorates these skin disorders. It was noted that restoration of claudin-1 expression was observed in healed psoriatic lesions after indigo naturalis treatment. Results again support the upregulation of claudin-1 expression by indigo naturalis may ameliorate the severity of skin diseases. The intracellular signaling mechanisms responsible for claudin1 expression in human keratinocytes are very complex and not completely understood. We found that co-treatment with a PKC inhibitor, but not co-treatment with inhibitors of p38 MAPK, ERK, JNK, or PI3K, led to the inhibition of claudin-1 expression in indigo naturalis-treated cells. In addition, we found that treatment with indigo naturalis resulted in an increase in PKC activity in cultured keratinocytes, suggesting that indigo naturalis-induced claudin-1 expression is mediated, at least in part, by activating the PKC pathway. Similar findings were noted in primary human nasal and pancreatic duct epithelial cells (Koizumi et al., 2008; Yamaguchi et al., 2010). Recently, indigo naturalis and indirubin have also been shown to inhibit EGFR activation and CDC25B expression in cultured keratinocytes (Hsieh et al., 2012). It is notable that EGFR has an important negative regulatory role on the expression of
Fig. 6. Expression of claudin-1 was regained in psoriatic skin sections treated with indigo naturalis. Skin biopsies were taken from a 40 year-old male (with a duration of psoriasis of 10 years) after 8 weeks of treatment. The psoriatic lesions were located on his bilateral legs. (A) Vehicle-treated lesion. (B) Healed psoriasis after indigo naturalis treatment. (C) Normal perilesional skin. Representative results of the immunohistochemical staining analysis using anti-claudin-1antibodies are shown. Photos at high magnification are shown in the lower corner. Scale bar: 100 and 50 mm.
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epidermal TJs (Tran et al., 2012). Therefore, inhibition of EGFR activation by indigo naturalis is also a possible mechanism for increasing claudin-1 expression. Further study is needed to explore this hypothesis. Our finding that the expression of claudin-1 in active psoriatic plaques is lower than those in normal perilesional psoriatic skin and healthy skin is consistent with findings from a number of studies (Brandner et al., 2006; Kirschner and Brandner, 2012; Kirschner et al., 2009, 2010; Watson et al., 2007). However, other studies showed that the levels of expression of claudin-1 in psoriatic plaques do not significantly differ from levels seen in perilesional psoriatic skin and healthy skin (Itoh et al., 2005; Peltonen et al., 2007). Further studies are needed to elucidate the roles that claudin-1 alterations play in psoriasis. One limitation of this preliminary study was a lack of in vivo or in vitro investigations of the impact of indigo naturalis on the immune system, such as expression of cytokines and T cells, which might suppress expression of claudin-1 and the epidermal barrier function. It is not clear from the in vitro findings whether indigo naturalis has a similar restorative effect in vivo where immune system factors are present. In conclusion, indigo naturalis promotes claudin-1 expression and enhanced TJ function in primary human keratinocytes. Our data show that the PKC pathway is involved in indigo naturalis-induced claudin-1 expression. The three major components of indigo naturalis, indirubin, indigo, and tryptanthrin, exerted a synergistic effect on upregulating TJ function, with indirubin accounting for the majority of the effect. The results of this study provide additional evidence in support of the effectiveness of indigo naturalis as a treatment for psoriasis.
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