Kaempferol promotes melanogenesis and reduces oxidative stress in PIG1 normal human skin melanocytes

Abstract Vitiligo is an autoimmune disease characterized by depigmentation. Kaempferol is a flavonoid compound with broad anti‐inflammatory and antioxidant properties. The purpose of this study was to investigate the effect of kaempferol on melanogenesis in PIG1 normal human skin melanocytes and its response to oxidative stress. The effect of kaempferol on melanin synthesis in PIG1 normal human skin melanocytes was explored by measuring tyrosinase activity, melanin content, mRNA and protein expression of key enzymes and expression of related pathway proteins. The effects of kaempferol pretreatment on cell viability, apoptosis, ROS level and HO‐1 protein level under H2O2 stimulation were explored. When treated with kaempferol, the tyrosinase activity and melanin content of PIG1 cells increased, the mRNA and protein expressions of TYR, TRP1, TRP2 and MITF increased, and the phosphorylation level of ERK1/2 increased. Upon the stimulation of H2O2, kaempferol reduced the production of ROS, decreased apoptosis and increased the protein expression of HO‐1 in PIG1 cells. In addition, kaempferol inhibited oxidative stress‐induced melanin reduction and promoted melanin synthesis in PIG1 cells and protected against H2O2‐induced oxidative stress damage.

Regulated cell death ensures that the homeostasis of the organism is maintained. The endoplasmic reticulum is a key organelle involved in protein homeostasis, and kaempferol as a flavonoid may regulate endoplasmic reticulum stress and autophagy and have protective effects on dysfunctional cells. 9 By analysing the relationship between the chemical structure of flavonoids and promoting melanogenesis, existing studies infer that phenyl group play an important role in stimulating melanogenesis, so we infer that KF has a role in promoting melanin synthesis. 10 Photochemotherapy using 8-methoxypsoralen (8-Mop) and long-wave ultraviolet radiation is commonly used to treat vitiligo. 11 The main potential side effects of topical photochemotherapy are uncontrolled light reactions manifested by severe sunburn, blisters and abnormally dark pigmentation. 12 Animal experiments and longterm follow-up studies of photochemotherapy patients have shown that photochemotherapy has a certain carcinogenic potential, and the mutagenicity of photochemotherapy in microorganisms and cell cultures has also been studied. 13 The exploration of vitiligo treatment has never stopped. Our research explored the effect of KF on PIG1 normal human skin melanocytes, providing more research basis and new directions for drug development for KF in the treatment of depigmented skin diseases such as vitiligo.

| Cell culture and treatment
PIG1 normal human skin melanocytes (Otwo Biotech, Shenzhen, China) were cultured in high glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37°C in 5% carbon dioxide. KF (Aladin) was dissolved in DMSO, and the final concentration of DMSO in the medium was one-thousandth.

| Cell viability
Cell viability was determined using the Cell Counting Kit-8 (NovaBio). PIG1 was inoculated in a 96-well plate at a density of 5000/well, treated with KF after adhering, and CCK-8 solution added according to the instructions. After incubation for 1 h, the microplate reader measured the absorbance at 450 nm.

| Tyrosinase activity measurements
After KF treatment, the culture medium was discarded, the cells washed three times with PBS, cell lysate added, lysed on ice for 30 min and then it was scraped off with a cell scraper and then put into an EP tube at 12000 rpm for 5 min at 4°C; then, the supernatant was taken and 500 μl L-DOPA (1 mM) added; 200 μl/well was added to the 96-well plate, and after incubation at 37°C for 3 h, the absorbance was measured at 475 nm with a microplate reader.

| NaOH assay of melanin content
After KF treatment, the cells were collected, and 1 mL of 1 M NaOH was added and then the solution was heated at 80°C for 1 h, and 200 μl/well was added to a 96-well plate. The microplate reader measured absorbance at 405 nm.

| Quantitative polymerase chain reaction (qPCR)
After the cells were treated with KF, trizol was added to lyse the cells, shaken at room temperature for 15 s, kept undisturbed for 3 min, and centrifuged at 12000 rpm, 4°C for 15 min. The upper colourless water sample layer was taken, an equal volume of isopropanol was added and mixed well; the mixture was kept undisturbed at room temperature for 10 min, centrifuge at 12,000 rpm for 10 min at 4°C and 1 mL of 75% ethanol was added to wash and then centrifuged at 17,108 g for 5 min at 4°C and the supernatant discarded, and finallyair dried for 3 min, DEPC100UL added and stored at −80°C. RNA was reverse transcribed into cDNA using a reverse transcription kit (NovaBio R202) according to the manufacturer's instructions. Target gene expression was assayed using SYBR qPCR mix (NoviaBio Q204) according to the manufacturer's instructions.

| Protein extraction and western blotting
Cells were cultured and processed in 10-mm dishes. The cells were discarded from the culture medium, washed three times with PBS, added with 500 μl of cell lysis solution (RIPA: PMSF = 100:1), lysed on ice for 30 minutes, scraped off with a cell scraper and placed in an EP tube, centrifuged at 4°C and 12,000 rpm for 5 min, and the supernatant was taken to a new EP tube, added with Ladding buffer

| Immunofluorescence (IF)
Cells were plated in six-well plates, and after treatment, they were washed three times with PBS, fixed with 4% formaldehyde for 20 min and then washed three times with PBS. Cells were stained with antibodies and washed three times with PBS. Nuclei were stained with DAPI fluorescent dye and then washed three times with PBS and observed under an inverted fluorescence microscope.

| Cell apoptosis measurement
After the cells were treated with KF for 24 h, they were stimulated with H 2 O 2 for 1 h. Cells were trypsinized without EDTA and collected in tubes, then cells were washed twice with PBS and resuspend in 100 μL of PBS. According to the kit instructions, cells were stained with Annexin V-FITC/PI apoptosis kit and detected by flow cytometry.

| Intracellular ROS measurement
After 24 h of KF treatment, cells were stimulated with H 2 O 2 for 1 h.
We washed the cells twice with PBS to remove all medium and FBS and then diluted the DCFH-DA probe to 1:1000 with medium and added it to each well. We incubated cells at 37°C for 30 min and then washed them three times with a serum-free medium. Fluorescence was observed and recorded under an inverted fluorescence microscope and then the fluorescence intensity was measured using ImageJ. Cells were trypsinized without EDTA and collected in tubes for detection by flow cytometry, and FlowJo software was used to analyse cell mean fluorescence intensity.

| Statistical analysis
Data in this work are presented as mean ± standard deviation (SD), and statistical analysis was performed using GraphPad Prism (version 6.0) Student's t-test or one-way analysis of variance (anova) for multiple group comparisons. Values of p < 0.05 were considered significant. All experiments were repeated at least three times.

| KF promoted melanogenesis in PIG1 cells
At the outset of the experiment, we investigated the non-toxic extent of KF to PIG1 cells using the CCK8 assay. PIG1 cells were

| KF activated the MAPK (ERK1/2) signalling pathway in PIG1 cells
To explore the mechanism by which KF promotes melanogenesis, we compared the phosphorylation and total levels of ERK1/2 in MAPK signalling pathways and AKT after treating PIG1 cells with different concentrations (0, 1, 10 μM) of KF for 24 h. The results showed that the total level of ERK1/2 was consistent after KF treatment, and the level of p-ERK increased in a concentration-dependent manner; the protein levels of AKT and PAKT did not change ( Figure 2H,I). From this, we concluded that KF activated the MAPK (ERK1/2) pathway in PIG1 cells, but did not affect the AKT pathway.

| KF reduced H 2 O 2 -induced apoptosis
We used H 2 O 2 to simulate the oxidative stress state of cells and explored the role of KF in cellular oxidative stress. We pretreated   KF enhanced cell viability under oxidative stress ( Figure 3A). The measurement of cell apoptosis by flow cytometry showed that cell apoptosis was significantly increased after hydrogen peroxide treatment, the apoptosis rate in the KF pretreatment group was significantly lower than that in the pure H 2 O 2 group, and KF had a protective effect on apoptosis caused by oxidative stress ( Figure 3B,C).

| KF reduced H 2 O 2 -induced intracellular ROS production by upregulating HO-1
Reactive oxygen species generation is highly correlated with hy- showed the same result ( Figure 4C).
To explore the mechanism of KF scavenging ROS, we detected the level of HO-1 protein in PIG1 cells. We found that there was no significant difference between the KF group and the control group.
Under the stimulation of H 2 O 2 , the expression of the HO-1 protein was significantly increased, and the expression of HO-1 protein in the cells of the KF pretreatment group was significantly higher than that of the pure H 2 O 2 stimulation group ( Figure 4D,E).

| KF protects melanin reduction caused by H 2 O 2
Existing studies have shown that excess H 2 O 2 and ROS impair biological processes, including the key melanin synthase, thereby reducing melanin synthesis. 14 Figure 5C,D). Existing studies have shown that kaempferol increases melanin content and dendritic length in melanoma cells and promotes cell migration. 16 The effect of KF on normal human skin melanocytes has never been reported, and our study fills the gap of KF's role in promoting melanogenesis and inhibiting oxidative stress in normal human skin melanocytes.

F I G U R E 4 KF reduced ROS generation in PIG1 cells stimulated by
Whether it is topical drug application, phototherapy or systemic treatment, vitiligo treatment is mainly to promote melanin   production and reduce the loss of melanocytes 17,18 ; KF works in both ways and can be used as a new option for clinical drug development. As a natural flavonol, kaempferol has the characteristics of poor water solubility but strong lipophilicity, which is beneficial to the percutaneous absorption of drugs for local treatment and improves the utilization rate of drugs. 19 At the same time, KF is a good metal ion chelating ligand, and the effect of KF can be enhanced by metal chelation. 20 MAPK/ERK (mitogen-activated protein kinases/extracellular signal-regulated kinases) signalling is essential for the proliferation and differentiation of melanocytes. 21 The kinase ERK in the MAPK signalling pathway is involved in the activation of the melanocyte receptor through ligand binding to its extracellular domain. 22 By binding to its receptor, ligand activation leads to the upregulation of MITF. 23 The PI3K/Akt (phosphatidylinositol 3′-kinase/Akt) signalling pathway regulates the cell cycle through GSK-3 and the protein cyclin D1 and plays a key role in melanocyte proliferation and apoptosis. 24 ROS include oxygen radicals such as superoxide anion radicals and hydroxyl radicals and nonradical oxidants such as hydrogen peroxide and singlet oxygen. 25 Under physiological conditions, the balance between ROS generation and ROS scavenging is highly controlled, and unregulated oxidative and reductive stress can lead to severe cellular damage and even unnecessary cell death, leading to whole organ and organismal failure. 26 Oxidative stress is considered one of the possible pathogenic events responsible for melanocyte loss. The poor circulation of tetrahydrobiopterin in the epidermis of vitiligo patients may be highly correlated with the production of intracellular H 2 O 2 . 27 Increased levels of ROS in melanocytes may lead to defective apoptosis leading to the release of abnormal proteins that can act as self-antigens leading to autoimmunity. 28 HO-1 is considered a major protein in diseases caused by oxidative and inflammatory damage. It has become an important target protein that can be upregulated against various stressful events by regulating intracellular levels of pro-oxidative haeme and other benefits conferred with by-products such as carbon monoxide and biliverdin. 29,30 HO-1 acts as a cytoprotective molecule against various forms of cell death, including apoptosis, necrosis and regulated cell death programmes (necroptosis, pyroptosis and ferroptosis). 31 As an effective cytoprotective agent, the regulation of its expression level has potential therapeutic value in the treatment of vitiligo. 32

ACK N O WLE D G E M ENTS
This work was supported by the National Natural Science Foundation of China (grant number 81573031).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.