Background

Melanin is synthesized in melanosomes of melanocytes from basal layers of the epidermis and is transported to epidermal keratinocytes through dendrites to provide skin pigmentation and protection from ultraviolet light and external stimuli (Fajuyigbe and Young 2016; Fitzpatrick et al. 1967). However, excessive melanogenesis and abnormal melanin distribution may lead to the formation of melasmas and freckles or may cause abnormal hyperpigmentation, such as spots during aging (Amaro-Ortiz et al. 2014). Melanogenesis is triggered by the conversion of tyrosine to dopaquinone by tyrosinase (Pillaiyar et al. 2017). Dopaquinone is then converted to dopachrome in the presence of thiol groups, and this metabolite is then converted by a tyrosinase-related protein (TRP)-2 into 5,6-dihydroxyindole-2-carboxylic acid, which is in turn converted by TRP-1 to indole-5,6-quinone-2-carboxylic acid to produce melanin (Tsukamoto et al. 1992; Boissy et al. 1998). The cyclic monophosphate/protein kinase A (cAMP/PKA) pathway is the main mode of signal transduction for melanin production, wherein cAMP, through PKA and cAMP-responsive element-binding protein 1 (CREB1), enhances the expression of microphthalmia-associated transcription factor (MITF) (D’Mello et al. 2016). MITF is a critical transcription factor for melanogenesis and is known to enhance the transcription of tyrosinase and its related proteins TRP-1 and TRP-2 (Buscà and Ballotti 2000; Saha et al. 2006). The cAMP-derived α-melanocyte-stimulating hormone (α-MSH) is a neuropeptide from proopiomelanocortin (POMC) that is produced in the hypophysis and in various other organs including the skin. In the skin, α-MSH is produced by melanocytes, Langerhans cells, fibroblasts, and endotheliocytes and, particularly, in keratinocytes following exposure to ultraviolet light (Chakraborty et al. 1996). In addition, α-MSH induces melanogenesis by increasing intracellular cAMP levels and membrane expression of the melanocortin-1 receptor (MC1R) (Videira et al. 2013). Therefore, modulation of intracellular signal transduction by α-MSH is recognized as an important target for the control of melanogenesis (Nasti and Timares 2015). Multiple studies report effective skin-whitening effects of treatments that modulate MITF expression, and although the melanogenic gene tyrosinases, TRP-1 and TRP-2, have been implicated, control of tyrosinase activity and expression did not affect melanogenesis in isolation.

Coptis chinensis (C. chinensis) is a perennial plant of the Ranunculaceae family. C. chinensis produces the active ingredient of berberine, which is an isoquinoline alkaloid, and other substances such as coptisine, epiberberlin, ferulic acid, magnoflorine, palmatine, and worenine. Berberine reportedly has excellent antibacterial, anti-inflammatory, haemostatic, hypotensive, and anticancer activities and has been used to inhibit the central nervous system activity to treat nephritis and induce bronchial smooth muscle expansion (Tang et al. 2016; Lee et al. 2003). Previous studies of the anti-inflammatory effects of C. chinensis extracts showed inhibition of lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α production in peritoneal macrophages, inhibition of apoptosis in pancreatic cells, TNF-α production in keratinocytes, and improvements in memory disorders (Kim et al. 2007; Enk et al. 2007; Wang et al. 2005). In the present study, the signalling mechanisms of the C. chinensis extracts were investigated to confirm skin-whitening effects.

Materials and methods

Cell line and culture

B16F10 cells were supplied by the Department of Dermatology of the College of Medicine at Seoul National University (Korea). Cells were subcultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene Inc., Korea) containing 10% fetal bovine serum (FBS) (v/v), 1 mM glutamine, 100 units/mL penicillin, and 50 μg/mL streptomycin in 100-mm cell culture dishes in a 37 °C incubator containing 5% CO2.

Extraction of C. chinensis

C. chinensis (100 g) plant samples were sonicated for 2 h in round flasks containing 1 L of 70% ethanol. The resulting extracts were decompressed and concentrated using a rotary evaporator (Eyela, Japan) and then dried using a freezing dryer for use in experiments.

Cytotoxicity

Cell growth inhibitory effects of C. chinensis extracts were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA) assays. In these experiments, cells were seeded into 96 wells at 2 × 103 cells/well and then cultured for 24 h at 37 °C in an incubator containing 5% CO2. Cells were then cultured with 0–120 μg/mL C. chinensis extracts for 24 h. Following treatments, cells were cultured with MTT for 4 h to allow formation of formazan crystals. The culture medium was then removed, the formazan crystals were dissolved by adding dimethyl sulphoxide (DMSO; Biopure, Canada), and cell survival rates were determined by measuring absorbance at 540 nm using a Microplate Reader (Bio-Rad, USA).

Assays of melanin contents

B16F10 cells were seeded into six-well plates at 5 × 104 cells/well and then cultured for 24 h at 37 °C in 5% CO2. Cells were then treated with 200 nM α-MSH (Sigma-Aldrich) for 48 h to induce melanogenesis. Subsequently, numbers of harvested cells were counted, and absorption was measured at 400 nm after lysing cells with 1 N NaOH.

qRT-PCR analysis

To determine changes in the expression of α-MSH mRNA following treatments with C. chinensis extracts, B16F10 cells were cultured in six-well plates at 5 × 104 cells/well. The cells were then treated with 200 nM α-MSH for 48 h to induce melanogenesis. The cells were then harvested, and extracted RNA was used to synthesize cDNA using Moloney murine leukaemia virus (M-MLV) reverse transcriptase (Enzynomics, Korea) at 37 °C for 1 h. Quantitative real-time PCR (qRT-PCR) was then performed using cDNA as a template, and gene expression levels in C. chinensis-treated cells were compared with those in untreated controls. Reaction mixes contained HOT FIREPol EvaGreen PCR Mix Plus (Solis BioDyne, Estonia), 1 pmol of forward primer and reverse primers (Table 1) and 10 ng of cDNA, and qRT-PCR was performed using a LineGene K (BioER, China) instrument with initial denaturation at 94 °C for 3 min, followed by 40 cycles of denaturation (94 °C, 30 s), annealing (58 °C, 30 s), and polymerization (72 °C, 30 s). PCRs were validated using melting curves, and gene expression levels were normalized to that of β-actin. The expression of miR-340 was determined using PCR according to the instructions of the miScript SYBR® Green PCR Kit (Qiagen, Germany) and a LineGene K instrument (BioER). Expression levels were normalized to that of U6 miRNA.

Table 1 Primers used in the qRT-PCR analyses
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miRNA transfection

To determine whether miR-340 regulates MITF expression in B16F10 cells following treatment with C. chinensis extracts, we transformed cells with anti-miR-340 (Bioneer, Korea) using RNAi max (Invitrogen, USA) and negative scramble miRNA as a control.

Western blotting analysis

B16F10 cells were seeded into six-well plates at 5 × 104 cells/well and then cultured for 24 h. Subsequently, the cells were treated with C. chinensis extracts and 200 nM α-MSH and then cultured for an additional 48 h. The cells were lysed and centrifuged in radioimmunoprecipitation assay (RIPA) buffer, and supernatants were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 10% gels. Proteins were then transferred to nitrocellulose membranes (Bio-Rad), blocked for 1 h in Tris-buffered saline/Tween 20 (TBS/T) buffer containing 5% skimmed milk and incubated with primary monoclonal antibodies against MITF, tyrosinase, and β-actin. Protein bands were detected using appropriate secondary antibody Immobilon Western Chemiluminescent HRP substrate (Thermo Scientific, USA), and images were developed using ChemiDoc (Bio-Rad).

Luciferase assays

B16F10 cells were transfected with reporter plasmid (pGL3-MITF-3′UTR, 1 μg) and normalization plasmid (pCMV-β-gal, 0.2 μg) using Lipofectamine 2000 (Invitrogen). The cells were cultured for 24 h under appropriate conditions and then treated with reagents for 24 h. The cells were then harvested in 100 μL of ×1 luciferase lysis buffer (Promega, USA) at 4 °C for 10 min and then centrifuged at 12,000 rpm for 10 min. Luciferase assays were then performed in the resulting supernatants using luciferase reagent (Promega) and a Luminometer (Veritas, USA). Luciferase activities were normalized to β-galactosidase, as determined using O-nitrophenyl-β-D-galactopyranoside assays.

Statistical analysis

All experiments were independently performed in triplicate. Data are expressed as means ± standard deviations. Statistical analyses were performed using Microsoft Excel, and differences were considered significant when p < 0.05.

Results

Regulation of melanogenesis by C. chinensis extracts

Cytotoxic activities of C. chinensis extracts in mouse melanoma cells were determined using MTT assays, and effective concentration ranges were calculated. B16F10 melanoma cell survival rates were 90% or higher in the presence of up to 15 μg/mL C. chinensis extracts (Fig. 1a). Melanin contents in the mouse melanoma cells were then measured to assess skin-whitening effects of C. chinensis extracts. Under these conditions, α-MSH-mediated melanogenesis was inhibited by treatments with C. chinensis extracts (Fig. 1b).

Fig. 1
figure 1

C. chinensis extracts decreased α-MSH-mediated melanogenesis in B16F10 cells. a Cytotoxicity of C. chinensis extracts. b Melanin contents in C. chinensis-treated B16F10 mouse melanoma cells. Statistically significant differences are indicated with an asterisk for p < 0.05

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C. chinensis extracts also inhibited tyrosinase activities by more than 50% at 15 μg/mL (Fig. 2a), which was related to concomitant decreases in tyrosinase expression (Fig. 2b, d). In subsequent qRT-PCR and Western blotting analyses, decreases in melanogenesis were correlated with tyrosinase mRNA and protein expression levels and with MITF protein expression (Fig. 2c, d).

Fig. 2
figure 2

C. chinensis extracts decrease the expression and activity of melanogenesis-related genes. a C. chinensis-mediated changes in tyrosinase activity. b Tyrosinase mRNA expression. c MITF mRNA expression. d Melanogenesis-related protein expression. Statistically significant differences are indicated with an asterisk for p < 0.05

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Regulation of miR-340 expression by C. chinensis extracts

Previous studies showed that MITF translation is regulated by miR-340. Accordingly, treatments of the present B16F10 melanoma cells with C. chinensis extracts at 0, 7.5, and 15 μg/mL increased miR-340 expression in a concentration-dependent manner (Fig. 3).

Fig. 3
figure 3

Regulation of miR-340 expression by C. chinensis extracts. C. chinensis extracts increased miR-340 expression in a concentration-dependent manner. Statistically significant differences are indicated with an asterisk for p < 0.05

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Regulation of MITF by C. chinensis extracts is mediated by miR-340

To confirm the roles of the miR-340–MITF axis in the regulation of melanogenesis by C. chinensis extracts, we performed reporter assays of the miR-340 target on the MITF promoter. In these experiments (Fig. 4), luciferase activity was decreased following treatments with C. chinensis extracts but was recovered following transfection with anti-miR-340.

Fig. 4
figure 4

C. chinensis extracts regulated melanogenesis via the miR-340–MITF axis. a C. chinensis-mediated changes in luciferase activity of the miR-340 target sequence. b miR-340-mediated blockade of C. chinensis-induced luciferase activity of the miR-340 target sequence. Statistically significant differences are indicated with an asterisk for p < 0.05

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Discussion

During melanogenesis, melanin is produced from L-tyrosine by the key enzyme tyrosinase (Pillaiyar et al. 2017; Kwon et al. 2014; D’Mello et al. 2016), and various skin-whitening raw materials have been developed to inhibit the expression and activity of tyrosinases (Gunia-Krzyżak et al. 2016; Lee et al. 2016; Choi et al. 1998). The present study shows that C. chinensis extracts inhibit melanogenesis by decreasing tyrosinase activity (Fig. 2a) and expression (Fig. 2b, d). Moreover, we investigated the roles of α-MSH and MITF in tyrosinase-mediated melanogenesis and showed that MITF protein but not mRNA expression was decreased in the presence of C. chinensis, suggesting regulation by post-transcriptional mechanisms. Among potential regulators, miRNAs of 16 to 29 nucleotides are known to regulate protein expression by inhibiting translation (Bartel 2004; Murchison and Hannon 2004). Previous studies showed that miR-340 regulates melanogenesis by directly inhibiting MITF (Goswami et al. 2015). In agreement, miR-340 expression was increased and luciferase activity of the MITF reporter sequence was decreased following the present treatments with C. chinensis extracts (Fig. 4). Moreover, transfection with anti-miR-340 restored MITF protein levels under these conditions, confirming that miR-340 inhibits translation of MITF in the presence of C. chinensis extracts. Taken together, the present data show that C. chinensis extracts inhibit MITF translation by increasing miR-340 expression, leading to decreased tyrosinase protein and mRNA expression and reduced melanogenesis.

Conclusions

To investigate the potential of C. chinensis extracts as a raw material for skin-whitening cosmetics, we initially determined changes in tyrosinase activities in B16F10 melanoma cells and showed that C. chinensis extracts inhibit melanogenesis in a concentration-dependent manner. Subsequent Western blotting experiments showed that tyrosinase and MITF protein expression levels were decreased by C. chinensis extracts, whereas only tyrosinase mRNA levels were affected under these conditions, indicating that MITF is regulated at the protein level. Accordingly, the known MITF translation regulator miR-340 was induced following treatments with C. chinensis extracts, and the resulting suppression of MITF was mitigated by an anti-miR-340 antibody. The results of the present study confirm the skin-whitening effects of C. chinensis extracts and demonstrate that miR-340-mediated inhibition of MITF translation leads to decreased tyrosinase expression and melanin synthesis.