Ganoderma lucidum polysaccharide inhibits UVB-induced melanogenesis by antagonizing cAMP/PKA and ROS/MAPK signaling pathways

2.1 Culture of cells

In the presence of 5% CO₂ at 37°C, immortalized human melanocyte cell line PIG1 (a gift from Dr. Caroline Le Poole, Loyola University Chicago) was cultured in the 254 (#M-254-500; Gibco, CA) medium supplemented with 5% fetal bovine serum (Biological Industries, Israel), 1% Penicillin-Streptomycin (Gibco), and 1% human melanocyte growth supplement (HMGS) (#S0025; Gibco). Meanwhile, melanoma cells from kunming (KM) mice (B16F10 cells) were cultured in dulbecco's modified Eagle medium (DMEM) (Hyclone, UT) medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin (Gibco).

2.2 Determination of cell viability

After exposure to UVB irradiation, GLP (purchased from Shanxi Ciyuan Biotech Co., Ltd., Xi'an, China), or Malto (Yuanye Biotech Co., Ltd., Shanghai, China) for 24 hr, and the viability of B16F10 cells was determined with MTT assay. Specific procedures involved in methyl thiazolyl tetrazolium (MTT) essay can be referred to in our previously published paper (Zeng et al., 2017).

2.3 Determination of melanin content

B16F10 cells (2.5 × 10⁵ cells per 35 mm dish) were divided into four groups: control group, UVB group (20 mJ/cm² × 3d), GLP group (40 ug/ml), UVB+GLP group (20 mJ/cm² UVB×3d, 40 ug/ml GLP). The cells were digested, centrifuged, and then photographed. Thereafter, the cells were lysed with 100 μl 1 mol/L NaOH in a water bath at 70°C for 2 hr. Then, the absorbance of the cells was determined at 490 nm with a microplate reader (PerkinElmer EnVision xcite, UK).

2.4 Reverse transcription polymerase chain reaction experiments

Total RNA in B16F10 and PIG1 cells was extracted with TRizol reagent (Invitrogen, Carlsbad, CA), and RNA was transcribed reversely into complementary DNA with Toyobo RT kit (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's instructions. The reverse transcription polymerase chain reaction (RT-PCR) reaction was performed on a real-time fluorescence quantitative PCR instrument (LightCycler 480II, Mannheim, Germany) using a KOD SYBR® qPCR kit (Toyobo Co., Ltd.). All messenger RNA expression levels were normalized by GAPDH. Primer sequences are shown in Supporting Information Table 1.

2.5 Determination of intracellular ROS content

After B16F10 cells were treated with UVB irradiation, GLP, or UVB+GLP for 24 hr, the ROS content in the cells was determined by ROS detection kit (KeyGen Biotech. Co., Nanjing, China) in accordance with the instructions. The cells were observed under a fluorescence microscope (Olympus IX71, JPN). Fluorescence intensity was measured with a multifunction microplate reader (PerkinElmer EnVision xcite).

2.6 Western blotting

Cell proteins were extracted with lysis buffer and centrifuged at 12,000 rpm for 10 min at 4°C. The protein concentration was determined by the bicinchoninic acid (BCA) protein assay, and then the proteins were boiled for 5 min before the experiment. Twenty micrograms of protein sample was applied to each well, separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Well Biological Co., Ltd., Changsha, China) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). After 2 hr of blocking with 5% bovine serum albumin (BSA), the membranes were incubated overnight with antibodies to Myosin (#3402; CST), Rab27A (#69259; CST), MITF (#MABE78; Abcam), TYRP1 (#MABC592; Millipore), TYRP2 (NBP1-56058; Novusbio), TYR (ab180753; Abcam), cAMP response element-binding protein (CREB; #9197; CST), p-CREB (#9198; CST), p38 (#9926; CST), extracellular signal regulated kinase (ERK) (#9926; CST), c-Jun N-terminal kinase (JNK) (#9926; CST), P-p38 (#9910; CST), P-ERK (#9910; CST), P-JNK (#9910; CST) or GAPDH (ab181602; Abcam). The PVDF membrane was washed with phosphate buffered solution+Tween 20 (PBST) and then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) or anti-mouse IgG secondary antibody (CST) for 90 min at room temperature. The binding antibody was detected by electrochemiluminescence (ECL) chemiluminescence.

2.7 cAMP-Glo^TM assay

The cells were lysed using the cAMP-Glo™ Assay kit (Promega Corporation, Madison, WI) to release cAMP, followed by addition of test solution containing PKA and kinase substrate cAMP. The Kinase-Glo reagent was then added to terminate the PKA reaction, and ATP was detected by luciferase reaction. A multifunction microplate reader was used to determine cAMP concentration.

2.8 Immunofluorescence microscopy

B16F10 cells were grown on coverslips. After treatment with UVB, GLP or UVB+GLP, the cells were fixed with 4% paraformaldehyde for 30 min. Afterwards, they were incubated in PBS with 1% BSA for 20 min at 37°C. The cells were then incubated with the melanosome marker NKI-beteb antibody (NBP2-29407; Novusbio) for 45 min at 37°C, and the cells were rinsed three times with PBS and incubated with Alexa488-labeled goat anti-mouse IgG (#4408; CST) for 30 min. Finally, the nuclei were dyed blue with 4’,6-diamidino-2-phenylindole (DAPI). A laser confocal microscope (ZEISS LSM800, JPN) was used to take images.

2.9 Determination of mitochondrial membrane potential

JC-1 mitochondrial membrane potential was determined according to the instruction manual provided by the manufacturer (Beyotime, Shanghai, China). The cells were observed under a fluorescence microscope (Olympus IX71, JPN) and the fluorescence intensity was measured on a multifunction microplate reader.

2.10 In vivo experiments

On the first day after hatching, zebrafish in the normal control (NC) group were cultured with ordinary culture medium; zebrafish in the UVB group were exposed to 300 mJ/cm² UVB irradiation every day; zebrafish in the GLP group were cultured with GLP with a concentration of 4 mg/ml; zebrafish in the UVB+GLP group were irradiated with 300 mJ/cm² UVB every day, and at the same time cultured in GLP with a concentration of 4 mg/ml. The same treatment continued for 5 days. Changes in pigment content in zebrafish were observed. KM mice and guinea pig were epilated with a hair removal cream (Veet; HuBei, China) on the back and then divided into four groups. The mice in NC group were wet-applied with PBS on the back skin for 1 hr every day; the mice in UVB group were exposed with 300 mJ/cm² UVB irradiation on the back three times a week; the mice in GLP group were wet-applied with 40 mg/ml GLP solution on the back for 1 hr every day; the mice in UVB+GLP group was irradiated with 300 mJ/cm² UVB on the back three times a week, and, at the same time, wet-applied with 40 mg/ml GLP solution on the back for 1 hr every day. The experiment lasted for 8 weeks. Changes in the skin pigmentation of KM mice were observed by dermoscopy. After the KM mice were killed, the skin tissue on the back was taken, paraffin-embedded, sliced, and stained with silver stain. The guinea pig was divided into the same four groups. However, the UVB dosage was 500 mJ/cm². Twenty four hours after the treatment, changes in the guinea pig skin were observed.

2.11 Statistical analysis

The SPSS19.0 software was used for all statistical analysis. All experiments were performed in triplicate at the minimum. Data are presented as mean ± standard deviation. Significance tests were conducted on the data groups using analysis of variance followed by a comparison between the specific groups using Student's t test. p < 0.05 in all cases was considered statistically significant. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

3.1 GLP antagonizing UVB-induced melanogenesis

In this study, the toxic effects of GLP on B16F10 cells were first explored by MTT assay, and it was found that low concentrations of GLP (<160 ug/ml) had no obvious toxic effects on B16F10 cells. But when treated with whitening agent kojic acid (20 ug/ml) on a regular basis, B16F10 cells showed a significant decrease in viability (Figure 1a). MTT results showed that a low dose of UVB (≦60 mJ/cm²) had no significant influence on cell viability after treatment with different doses of UVB for B16F10 cells. When the UVB dosage was more than 60 mJ/cm², however, cell viability decreased significantly in a dose-dependent manner (Figure 1b). Because UVB at a dose of 60 mJ/cm² did not produce toxic effects on B16F10 cells, a total dose of 60 mJ/cm² UVB was chosen for the subsequent experiments (Figure 1b). After B16F10 cells were irradiated with 20 mJ/cm² UVB for three consecutive days (a total dose of 60 mJ/cm²), the cells were collected. Compared with those in the control group, the cells in UVB group were significantly darker. In comparison with the UVB group, the cells in the UVB + GLP group fade in color (Figure 1c). The pigment content of B16F10 cells were further determined by the NaOH method. The results showed that compared with the control group, the pigment content of the UVB group increased significantly; compared with the UVB group, the pigment content of the UVB + GLP group decreased (Figure 1d). After the melanosomes were stained with the immunofluorescence technique, there was an upward trend in the number of melanosomes in B16F10 cells which were irradiated with UVB, and they tended to be distributed to the cell membrane. Compared with the UVB group, melanosome in B16F10 cells in the UVB+GLP group decreased, and they were more likely to be distributed around the nucleus (Figure 1e). After the treatment of cells with only GLP, both the distribution of melanosomes and melanin content experienced no obvious change.

3.2 GLP antagonizing the expression of genes related to UVB-induced melanin synthesis, and transport

Because GLP can inhibit UVB-induced melanogenesis, the melanin synthesis and transport genes were further determined by RT-PCR and western blotting. It was found that, after B16F10 cells were exposed to repeated UVB irradiation, the expression of both synthesis-related genes such as MITF, TYR, TYRP1, and TYRP2, and transport-related genes like Myosin and Rab27A showed a remarkable upward trend. Compared with the UVB group, the expression of MITF, TYR, TYRP1, TYRP2, Myo5a, and Rab27A in the UVB+GLP group was downregulated. Meanwhile, treatment of cells with GLP only indicated no significant impact on the expression of melanin synthesis and transport genes (Figure 2a,b). To exclude the possibility of nonspecific physical blockage of UVB by GLP, we added Maltodextrin (Malto), whose molecular weight and chemical structure is similar to that of GLP, as another control substance. Malto is another kind of polysaccharide, which is formed during the degradation of starch or glycogen. We found that Malto had no significant effect on UVB-induced melanogenesis (Supporting Information Figure 2).

3.3 GLP inhibits UVB-induced melanogeneisis by antagonizing PKA and MAPK signaling pathways

It has been reported that UVB mediates melanogenesis by activating the PKA signaling pathway (Drira & Sakamoto, 2016; Ren et al., 2016). Consistent with previous studies, it was found in our study that CREB phosphorylation levels, key molecules of the PKA signal pathway, in UVB-stimulated B16F10 cells increased. Compared with the UVB group, the phosphorylation level of CREB in the UVB+GLP group dropped significantly. GLP alone had no effect on CREB phosphorylation level (Figure 3a). The MAPK signaling pathway also played a key role in melanogenesis. With UVB irradiation, the phosphorylation levels of ERK, JNK, P38 experienced a significant increase. Compared with the UVB group, ERK, JNK, P38 phosphorylation levels in the UVB+GLP group were significantly lowered. Treatment with GLP alone had no significant effect on MPAK family proteins (Figure 3a). The above results suggest that GLP can antagonize UVB-induced pigmentation by inhibiting the PKA and MAPK signaling pathways.

To further verify that GLP antagonized UVB-induced pigmentation through the PKA and MAPK signaling pathways, the cells were treated with PKA, ERK, and P38/JNK activators, respectively. WB results showed the activation of PKA, ERK, P38, and JNK pathways, respectively (Figure 3c). Compared with the UVB+GLP group, the color of B16F10 cells in each group deepened (Figure 3b). The RT-PCR results showed that all the activators could promote melanogenesis-related gene expression (Figure 3d).

3.4 GLP reduces UVB-induced ROS and cAMP

Our previous studies found that after exposure of fibroblasts to UVB irradiation, intracellular ROS levels increased significantly (Zeng et al., 2017). The current study found that B16F10 intracellular ROS levels experienced a significant increase as well after repeated UVB irradiation. Compared with the UVB group, the intracellular ROS levels in UVB+GLP group decreased, whereas GLP alone had no significant effect on ROS levels in B16F10 cells (Figure 4a,b). UVB irradiation could lead to mitochondrial damage, which may result in the generation of large amounts of ROS (Swalwell, Latimer, Haywood, & Birch-Machin, 2012). After exposure of B16F10 cells to UVB irradiation, a JC-fluorescent probe was used to determine mitochondrial membrane potential, the results of which showed that mitochondrial membrane potential decreased significantly, suggesting mitochondrial damage. Compared with the UVB group, the mitochondrial membrane potential of the UVB+GLP group increased (Figure 4c,d). Because cAMP is capable of activating the PKA signaling pathway, the level of cAMP was determined, which showed that repeated UVB irradiation of cells would lead to a significant increase in intracellular cAMP levels. Compared with the UVB group, the cAMP content in the UVB+GLP group dropped significantly, whereas GLP alone had no significant effect on the intracellular cAMP content (Figure 4e).

3.5 GLP inhibits UVB-induced melanogenesis by antagonizing PKA and MAPK signaling pathways in PIG1 cells

To further verify that GLP could antagonize UVB-induced melanogenesis, human melanocyte PIG1 cells were used for reconfirmation. After exposure of PIG1 cells to UVB irradiation, the expression of MITF, TYR, TYRP1, TYRP2, Myosin, and Rab27A were upregulated (Figure 5b), and the phosphorylation levels of CREB, ERK, JNK, and P38 increased significantly. In comparison, an opposite trend was found in the UVB+GLP group. GLP alone had no significant effect on the melanogenesis genes and phosphorylation levels of CREB, ERK, JNK, and P38 in PIG1 cells. These results suggest that GLP can antagonize UVB-induced melanogenesis in PIG1 cells by inhibiting the PKA and MAPK signaling pathways.

3.6 GLP antagonizing UVB-induced skin pigmentation in vivo

To explore whether GLP can antagonize UVB-induced pigmentation in vivo, a UVB-induced pigmentation model was constructed by treating zebrafish with UVB irradiation. After treatment of zebrafish juveniles with UVB irradiation for 5 days, increase in melanin content was observed. Compared with the UVB group, the melanin content in the UVB+GLP group experienced a downward trend (Figure 6a). To explore whether GLP was capable of sunscreen, depilated guinea pigs were irradiated with UVB, which resulted in obvious sunburn damage and erythema reaction. Compared with the UVB group, the guinea pigs in the UVB+GLP group suffered less sunburn damage and erythema reaction (Figure 6b). The results obtained in vivo confirmed GLP could antagonize UVB-induced pigmentation and sunburn reactions. At the same time, GLP could also antagonize UVB-induced pigmentation in KM mice (Supporting Information Figure 1).