Putrescine Upregulates Melanogenesis Through Modulation of MITF Transcription Factor in B16F1 Mouse Melanoma Cells

SUMMARY Research background Ageing is a biochemical, metabolic and genetic physiological phenomenon. The suppression of melanin biosynthesis, evident in the greying of the hair, is a hallmark of ageing resulting from translation failure, reduced enzyme activity and cellular senescence. Putrescine, the smallest member of the polyamine family and an organic chemical, is present in living mammalian cells and plays a crucial role in regulating skin melanogenesis. Therefore, the purpose of this study is to explore the effect of putrescine on the signalling pathways of melanogenesis in melanoma cells. Experimental approach Melanin production capacity of putrescine was analysed using a tyrosinase activity assay. To assess the cell viability of B16F1 cells exposed to putrescine, a tetrazolium dye MTT assay was performed. The effect of putrescine on melanin synthesis in the presence of H2O2 was evaluated using various in vitro assays in B16F1 cells. The effect of putrescine on melanin production in B16F1 cells was determined using a specific melanin production assay. Gene expression was analysed using real-time polymerase chain reaction (RT-PCR). Furthermore, the effect of putrescine on the expression of proteins related to melanin production in the cells treated with H2O2 was analysed by immunofluorescence and Western blot analysis. Results and conclusions Putrescine increased tyrosinase activity and showed no cytotoxicity in B16F1 cells. In addition, putrescine effectively scavenged H2O2, as shown by the reduction of intracellular H2O2 amounts in 2',7'-dichlorofluorescin diacetate analysis, and promoted melanin production in living cells. The stimulation of melanogenesis by putrescine was attributed to the increased expression of Mitf, Tyr, Trp-1 and Trp-2 genes. Immunofluorescence assays revealed that putrescine enhanced the expression of proteins associated with melanogenesis and upregulated TYR, TRP-1 and TRP-2 via the microphthalmia-associated transcription factor (MITF) and increased the expression of methionine sulfoxide reductases A (MSRA) and B (MSRB) in the cells treated with H2O2, effectively promoting melanogenesis. These results suggest that putrescine can be used to stimulate melanin synthesis. Novelty and scientific contribution This is the first study to investigate the effect of putrescine on the signalling pathways of melanogenesis in B16F1 melanoma cells. The results confirm that putrescine can promote melanogenesis through the expression of TYR, TRP-1 and TRP-2 via the MITF in cells treated with H2O2. Putrescine can be used exclusively as a cosmetic product to prevent premature greying of hair.


INTRODUCTION
Melanin pigments constitute an integral component of the signaling system, originating from the amino acid L-tyrosine and representing the outcome of intricate metabolic processes governing the synthesis of skin, eye, and hair pigments (1).In melanocytes, the synthesis of melanin is stimulated by the interaction between the melanocyte surface receptor and alphamelanocyte-stimulating hormone (α-MSH) (2).Melanocytes produce melanin, a pigment transported to keratinocytes through vesicles called melanosomes at the epidermalepidermal junction of the skin (3).Recent research has unequivocally demonstrated that senescence constitutes a genuine constraint on cancer development, including melanoma (4).
Melanomas arise from the malignant proliferation of cutaneous melanocytes pigmentproducing cells located in the basal layer of the epidermis (5).Melanomagenesis describing the pathophysiology of melanoma, relies on sequential changes in specific genes and pathways regulating crucial cell processes and influencing metabolic or molecular mechanisms (6).The primary function of melanin is to shield DNA from UV radiation and serve as an antioxidant against reactive oxygen species (ROS), which induce cellular damage and Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.3 oxidative stress (7).However, alterations in genetic physiology and biochemical metabolism frequently occur during the aging process.Aging phenotypes are an inevitable aspect of human life.The accumulation of translational defects and cellular damage, coupled with reduced enzyme activity involved in cell metabolism, constitutes the major characteristics of cellular aging (8).Aging pigment decreases with age, leading to dysfunctional cells and disrupted homeostasis, leading to hair graying.Moreover, previous research has identified oxidative stress as a significant contributor to melanoma development (9).Tyrosinase (TYR), a copper-containing metalloprotein, along with tyrosinase-related protein 1 (TRP-1) and TRP-2, plays a crucial role in melanin synthesis (10).Additionally, the microphthalmia-associated transcription factor (MITF) is essential to melanocytes and melanogenesis differentiation by regulating multiple pigmentation-related genes, including TYR, TRP-1, and TRP-2 (11).However, an increase in hydrogen peroxide (H2O2) leads to a reduction in antioxidant enzymes including methionine sulfoxide reductase A and B (MSRA and MSRB).This disruption impairs the normal synthesis of melanogenesis and tyrosinase, resulting in depigmented diseases (12).
The polyamine, aliphatic small polycationic chemicals, plays a crucial function in the regulation of human skin melanogenesis (13).Putrescine (1,4-diaminobutane) is the smallest member of the polyamine family and is common in live mammalian cells (14).It is a polycationic chemical molecule produced from L-ornithine or regulated by ornithine decarboxylase (15).It can interact with negatively charged compounds inside the cell due to its natural substrate.Moreover, polyamines are essential for the regulation of melanogenesis in human skin.According to previous findings, putrescine can stimulate pigment production by tyrosinase in human skin explants and diminish the intracellular H2O2 of polyamine catabolism activities (13).However, few studies have been performed on the involvement of melanin synthesis activity and the mechanism of melanogenesis by putrescine.Moreover, it is unknown whether putrescine impacts the stimulation of melanin synthesis to postpone the beginning of premature greying of the hair.Therefore, the purpose of this study is to examine the impact of putrescine on the melanogenesis signaling pathway of B16F1 mouse melanoma cells.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.4 Putrescine dihydrochloride (1,4-butanediamine dihydrochloride, 1,4-diaminobutane dihydrochloride), diacetyldichlorofluorescein (DCF), H2O2, vitamin C, potassium ferricyanide, ferric chloride (FeCl3), trichloroacetic acid (TCA), tyrosinase, α-MSH, MTT (3-(4,5-dimethyl-2yl)-2,5-diphenyltetrazolium bromide) reagent, and RIPA lysis buffer were obtained from Sigma-Aldrich (Louis, MO, USA).The western blot antibodies were sourced from Santa Cruz Biotechnology (Santa Cruz, California, USA).TRIzol reagent was purchased from ThermoFisher Scientific (CA, USA).All of the reagents with analytical grades were used for the experiment.

Tyrosinase activity assay
Tyrosinase activity was tested to validate the initial stage of melanin synthesis.Initially, various concentrations of 3 µL of putrescine were added to a mixture of 237 µL of 0.1 M sodium phosphate (pH 6.5) and 40 µL of tyrosine (1.5 mM) in a microtube with continuous vortexing.Subsequently, 20 µL of 1,500 units/mL tyrosinase was introduced, followed by a 30 min incubation at 37 °C.Data were recorded with a spectrophotometer (SpectraMax M3, Molecular Devices, Sunnyvale, California, USA) at an absorbance value of A475 nm.The negative control included 0.1 % vitamin C. The blank group consisted of Na3PO4 and tyrosine.The control group comprised Na3PO4, tyrosine, and tyrosinase.The amount of tyrosinase activity (%) was determined by comparing the OD of the putrescine group to that of the control group using the formula: Tyrosinase activity=(A(putrescine treatment group)/A(control group))•100 /1/

Cell lines and cell cultures
ATCC (American Type Culture Collection, Manassas, Virginia, USA) supplied a mouse melanoma cancer cell line (B16F1).They were cultured individually as monolayers using DMEM containing FBS (10 %) with antibiotic reagents (penicillin at 10,000 U/mL and streptomycin/amphotericin at 10,000/2,500 g/mL) in a 5 % CO2 humidified environment at 37 °C.

MTT assay
The cell viability of B16F1 mouse melanoma cells was assessed with putrescine (0.25, 0.5, 1, and 2 mM) using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (16).B16F1 cells were seeded at a density of 1×10 6 cells/mL with 10 % DMEM-FBS in 24-well plates in an incubator at 37 °C, 5 % CO2 and a humidified environment.Following a Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
5 24 h treatment with varied doses of putrescine, MTT reagent (5 mg/mL, 400 µL) was applied and incubated for 4 h.The blank group consisted of DMEM supplement solution without putrescine treatment.The insoluble purple formazan was dissolved in dimethyl sulfoxide (DMSO), and absorbance (A) values were measured using a visible spectrophotometer (SpectraMax M3, Molecular Devices, Sunnyvale, California, USA) at a wavelength of A570 nm.

Diacetyldichlorofluorescein (DCF) fluorescence assay
DCF is a widely used fluorescent biomarker for ROS production (17).A 96-well plate was seeded with B16F1 cells at a density of 1×10

Melanin production assay
The melanin assay was conducted following the procedure outlined by Kang and Kim with modifications (22).B16F1 cells (1×10 6 cells/mL) were seeded in a 24-well plate with 10 % DMEM-FBS media and incubated for 24 h at 37 °C in a 5 % CO2 humidified atmosphere incubator.Various concentrations of putrescine and the positive control, α-MSH, at 6 μM were applied and incubated for 72 h.The blank group represents the group containing DMEM supplement solution without putrescine treatment.The control group represents the group containing H2O2 (250 μM) treatment.The concentration of H2O2 dissolved in 1X PBS is 250 μM.The production of melanin was examined under a microscope.Then, 400 μL of sodium hydroxide (1 N, NaOH) was used to dissolve the cell pellets.The cell lysates were immediately transferred at room temperature to a 96-well plate.The absorbance of the solution was measured at a wavelength of A490 nm using a visible spectrophotometer (SpectraMax M3, Molecular Devices, Sunnyvale, California, USA).

Reverse transcription polymerase chain reaction (RT-PCR) analysis
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing. 6 The cells at a density of 1×10 6 cells/mL were seeded and incubated in the presence of H2O2.Subsequently, the cells were treated with different concentrations of putrescine (0.25, 0.5, 1, and 2 mM) and incubated for 24 h.The blank group consisted of DMEM supplement solution without putrescine treatment.The control group included H2O2 at 250 μM treatment.
The cells were lysed with Trizol reagent.Total RNA was then purified using chloroform, isopropyl alcohol, and 75 % ethanol.RNA concentration was measured using a UV-VIS spectrophotometer at A260/A280 nm wavelength.RocketScript Reverse Transcriptase (Bioneer, Daejeon, South Korea) was employed with diluted RNA (concentration 0.2 µg/µL; volume 5µL) to synthesize cDNA.mRNA was amplified by PCR using Thermocycler T-Gradient Thermo Block (Biometra, Kent, UK).The PCR reaction was performed in the conditions of denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, and synthesis at 72 °C for 60 sec (30 cycles).Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as a reference gene.

Immunofluorescence assay
Jeon and Kim's protocol for immunofluorescence staining was followed (20).The cells (1×10 5 cells/mL) were seeded in an 8-well chamber.After 24 h of incubation in the presence of H2O2, putrescine (0.25, 0.5, 1, and 2 mM) was treated and incubated with 5 % CO2 at 37 °C for 24 h.The blank group consisted of DMEM supplement solution without putrescine Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

Western blot analysis
The standard Western blotting protocols were executed.The cells (1×10 6 cells/mL) were then treated with putrescine at concentrations of 0.25, 0.5, 1, and 2 mM and incubated for 24 was used to assess the protein expression levels (21).Calculating the relative protein expression was carried out by normalizing the level of the target protein band to the level of the control band.The formula used for the calculation was as follows: Relative expression (RE)=Intensity of the target protein of interest band/Intensity of the loading control band /2/ Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing. 8

Statistical analysis
The data were presented as the mean plus standard deviation (SD).All experiments were performed in triplicate (n=3).One-way analysis of variance (ANOVA) and Duncan's multiple range test were used to examine group differences.The SPSS program (Version 16; SPSS Inc., Illinois, USA) was used to conduct the statistical analysis (22).Statistically significant differences were indicated by a p-value of *, p<0.05, **, p<0.01, and ***, p<0.001.

Effect of putrescine on tyrosinase activity, cell cytotoxicity, and intracellular H2O2 production
Tyrosinase is a key enzyme in the process of melanogenesis and a critical diagnostic for identifying the enzyme in the melanin synthesis pathway (23).Commonly used to enhance skin tone and cure dermatoses, tyrosinase inhibitors may effectively suppress the formation of pigment (24).The purpose of the tyrosinase activity test was to assess the functioning of putrescine in melanin synthesis.Fig. 1a showed a reduction in tyrosinase activity in the vitamin C-treated group serving as a negative control.The putrescine-treated group increased tyrosinase activity from 0.5 to 2 mM (p<0.001), compared to the control group.Due to the direction of enzyme-oligoamine interactions, polyamines play a pivotal role in mammalian melanogenesis (25).A prior study reported that spermidine, a polyamine compound, represented tyrosinase activity (5.1 %) at 200 μM of the spermidine-treated group (26).In this research, the action of putrescine increased the activity of tyrosinase.These findings indicate that it may have the potential for putrescine to stimulate melanogenesis via antioxidant properties.Based on the ratio of live cells to dead cells, an MTT test for cell viability was performed.At these concentrations, putrescine did not cause cell death (Fig. 1b).This result suggests that putrescine is nontoxic on B16F1 cells.To further address the potential toxicity of putrescine, concentrations of putrescine ranging from 0.25 to 2 mM were used to assess its influence on melanogenesis, but no evidence of cell death was observed.However, putrescine above 10 mM displays the enhancement of cytotoxicity by considerably lowering cell viability on breast cancer BT474 cells, according to an earlier study (27).Decomposition of H2O2 may produce hydroxyl radicals, which may cause DNA damage and initiate lipid peroxidation (28).
We used the fluorescent indicator, DCFH-DA, which produces DCF and fluoresces green when it interacts with H2O2 in cells (29) to measure intracellular changes of H2O2 in living cells.
The effect of putrescine on the scavenging activity of intracellular H2O2 was examined in connection to the growing H2O2 concentration during aging and levels of reactive oxygen species (ROS) in B16F1 cells.Fig. 1c showed a negative control, vitamin C decreased H2O2 Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
by 79 %.Putrescine at the highest concentration (2 mM) reduced the quantity of H2O2 by 14 % (p<0.05).These data imply that putrescine may inhibit the production of H2O2 by eliminating the accumulated H2O2, leading to restoring melanin production.A prior study reported that while the concentration of spermidine at 4000 μM was increased, the intracellular H2O2 level was decreased by 83.2 % (26).These data imply that putrescine may inhibit the production of H2O2 by eliminating the accumulated H2O2, leading to restoring melanin production.While using melanoma cells provides valuable insights, especially in cancer research, it is important to recognize the following limitations when studying drug effects on melanogenesis, Melanoma cells have mutations and altered signaling pathways that are not present in normal melanocytes.These changes can significantly affect putrescine response, making it difficult to extrapolate findings to normal cells.In addition, melanoma cells often have higher proliferation rates and different growth characteristics compared to normal melanocytes.Therefore, the results obtained from melanoma cells may not be generalizable to other types of melanocytes.
Therefore, a further experiment is required to verify the positive effect on melanin production in normal human melanocytes instead of mouse melanoma cells that have been widely used to study melanin production but also human skin as a clinical study.Melanoma cells are cancer cells that proliferate more rapidly than normal melanocytes.They often exhibit genetic variations and mutations that are associated with the development of melanoma.Studying these cells could provide insights into the genetic factors influencing melanogenesis and melanoma progression.

Impact of putrescine on melanin synthesis in B16F1 cells
In general, the antioxidant activity of aging hair follicles decreases, leading to a buildup of H2O2.An increase in H2O2 may result in the death of melanocytes and a decrease in melanin production (30).Based on the identification of melanocytes among the basal cells of the epidermis, skin homeostasis is highly regulated by melanogenesis, which includes the generation and distribution of melanin (1).Throughout the aging process, melanin plays a function in neutralizing free radicals and reactive oxygen species.H2O2 accumulates in aging hair follicles due to a reduction in antioxidants, causing melanocyte apoptosis and inhibiting melanin synthesis (1,22).To evaluate the melanogenesis of B16F1 cells in the presence of H2O2, the effect of putrescine on melanin synthesis was studied.Fig. 2a and Fig. 2b displayed the quantity of intracellular melanin synthesis.α-MSH, a neuropeptide of the melanocortin family, is the most important activator and regulator bioactive peptide in melanogenesis.It binds to the melanocortin 1 (MC1R) receptor on the cell surface and stimulates cyclic Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
10 adenosine monophosphate (cAMP), which modulates pigmentation and acts as tyrosinase in melanocytes, resulting in melanin synthesis (31,32).It is advantageous to investigate the impact of putrescine on melanin formation.The treatment with α-MSH, a positive control, increased melanin synthesis by 50 % compared with the control group.The presence of putrescine raised melanin production in a dose-dependent manner (p<0.05).Putrescine treatment groups with 0.5, 1, and 2mM increased the melanin content by 21 %, 30 %, and 41 %, respectively.The melanin synthesis results demonstrated that putrescine promotes melanin formation.Similarly, putrescine increases tyrosinase concentrations (21).This study shows that putrescine might be used to restore melanin synthesis.Polyamine catabolism causes the activation of melanogenesis, according to prior research (21).ROS is a free radical that produces oxidative damage to lipids, DNA, and proteins (33).Constant exposure of cell molecules to oxidative damage from free radicals generated by the body results in a range of age-related ailments, including cancer and metabolic diseases such as hair graying (34,35).
This finding indicates that putrescine massively improved the formation of melanin in H2O2treated cells.

Effect of putrescine on the gene expression related to melanin production
The effect of putrescine on gene expressions (Mitf, Tyr, Trp-1, and Trp-2) was determined using RT-PCR analysis.As demonstrated in Fig. 3a and Fig. 3b, the Mitf and Trp-2 genes were more highly expressed in the α-MSH treatment group compared to the control group.
Specifically, the highest quantity of putrescine (2 mM) substantially increased the expression of the Mitf and Trp-2 genes (p<0.001).Compared to the control group, the expression levels of the Mitf and Trp-2 gene were increased by 10-fold and 2-fold, respectively.The levels of Mitf gene expression increased by 1,012 %.Moreover, the putrescine groups upregulated the expression level of Tyr with the increase in the dose (p<0.05) while the Trp-2 level increased in the α-MSH group (p<0.001).Putrescine improved the action of melanogenesis by elevating the Mitf and Tyr levels in B16F1 cells.These results were consistent with prior research on polyamines and putrescine (13,26).Putrescine, a polyamine, has been demonstrated by Sridharan et al. (13) to stimulate human epidermal melanogenesis.The upregulation in the RNA levels of Tyr and Trp-1, which are indispensable enzymes for melanogenesis, in putrescine-treated cells was observed, indicating that putrescine promotes melanogenesis by upregulating the essential enzymes needed for melanogenesis, which raises the amount of melanin in primary melanocytes (13).Furthermore, Kang and Kim (26) reported that spermidine, a polyamine molecule, has been shown to affect the expression of the Mitf gene.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing. 11 Spermidine enhanced expression in H2O2-treated compared to non-treated B16F1 cells, demonstrating that spermidine can regulate the process of melanin formation.These results suggest that putrescine may be effective for modulating the process of melanin production.

Effect of putrescine on melanogenesis-related protein expression in immunofluorescences analysis
An immunofluorescence staining study was conducted to evaluate whether putrescine affects the expression levels of TRP-1, TRP-2, MITF, TYR, MSRA, and MSRB in B16F1 cells treated with H2O2.By using fluorescent microscopy, the fluorescence signals of DAPI, CY3, and FITC were observed as blue, red, and green signal staining, respectively.In the expression of TYR, MITF, TRP-1, TRP-2, MSRA, and MSRB, the fluorescence intensity level of α-MSH, a positive control, was greater than that of the control group (Fig. 4).The expression levels of TYR, MITF, TRP-2, and MSRB were notably elevated in the 2 mM putrescine treatment group.Ko and Kim (9) demonstrated that H2O2 affects melanogenesis aging in human melanocytes with immunofluorescence analysis.H2O2-treated cells showed significantly higher expression levels of MSRA and MSRB compared to young cells, while TYR and TRP-1 showed significantly lower expression levels.These results suggest that H2O2 plays a crucial part in melanogenesis by regulating antioxidant enzyme expression.These findings suggest that putrescine may stimulate melanin synthesis by increasing melanogenesis-related protein expression.

Impact of putrescine on the protein expression associated with melanin production
Western blot analysis was used to investigate the effect of putrescine expression on melanogenesis-related protein expression in B16F1 cells treated with H2O2 using antibodies against TYR, TH, MITF, TRP-1, TRP-2, MSRA, MSRB, and catalase.α-MSH served as the positive control.In Fig. 5a, the putrescine groups above 0.5 mM upregulated TRP-1 expression levels in a dose-dependent manner (p<0.001).Moreover, putrescine substantially raised the expression of TRP-2 in a dose-dependent manner (p<0.01).Consistent with our findings, prior research found that putrescine increases the expression levels of TYR and TRP-1 in epidermal cells (21).The expression level of catalase increased in the presence of 2 mM putrescine (p<0.01) (Fig. 5b).Among these, MITF is a crucial regulator of melanocyte development and skin pigmentation by controlling the TYR expression, the initial enzyme, TRP-1, and TRP-2 (36).Catalase is one of the most important antioxidant enzymes, breaking down cellular H2O2 to generate oxygen and water, significantly reducing oxidative stress (37).
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
12 A previous study showed that the total cellular melanin level was directly associated with catalase-specific mRNA, protein, and enzymatic activity in human melanocytes (38).Jeon et al. reported that catalase in the lysosomal fraction was affected by reducing melanin color through antioxidant enzymes and H2O2 treatment, suggesting a potential connection between catalase and melanin production (39).Fig. 5c displayed the expression levels of TYR and tyrosine hydroxylase (TH).TH is a key enzyme in the early stages of melanogenesis, catalyzing the conversion of tyrosine to L-DOPA, which is a precursor for melanin synthesis (40).A prior study showed that melanin content and TH activity were markedly increased in B16-F10 melanoma cells (41).Contrary to our expectations, the putrescine-treated groups decreased TYR and TH expression levels compared to the control group.Nevertheless, the putrescine-treated group increased the expression levels of TYR and TH in a dose-dependent manner.Fig. 5d exhibited the MSRA and MSRB expression levels, proteins that catalyze the enzymatic conversion of methionine sulfoxide to methionine to restore the biological function of oxidatively-damaged, inactive proteins such as TYR, TRP-1, and TRP-2 (42).Compared with the control group, putrescine upregulated the expression of MSRA and MSRB by α-MSH to 234.23 % and 620.56 %, respectively.Furthermore, the MSRA expression level raised dose-dependently in the presence of putrescine concentrations greater than 0.5mM (p<0.001).
Particularly, the putrescine-treated group at 2 mM demonstrated a 346 % increase in MSRA expression.Furthermore, the MSRB expression level increased by 766 % in the presence of 2 mM putrescine.The previous study reported that the reduced expression of glutathione reductase, catalase, and MSRA, three antioxidant proteins, lowered melanin production and increased lipofuscin generation (43).Thus, our results imply that the increased expression of MSRA and MSRB by putrescine may stimulate melanin synthesis.As shown in Fig. 6, the accumulation of H2O2 is a key factor regulating the reduction of melanin synthesis in cells by inhibiting the MITF signaling pathway, leading to the aging of melanocytes and oxidizing enzymes.However, putrescine could be used as a potential activator of expression factors such as TYR, TRP-1, and TRP-2, to modulate melanin synthesis via the MITF signaling pathway.It also stimulates the development of MSRA and MSRB, acting as a promoter of melanin production, addressing the issue of graying hair, and suggesting the possibility of elucidating the aging mechanism.

CONCLUSIONS
Overall, it is demonstrated that putrescine from polyamines may be capable of inducing melanogenesis by enhancing tyrosinase activity.Putrescine exhibits low toxicity to B16F1 Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

23
and donkey anti-mouse conjugated CY3 antibody with a mouse polyclonal TYR, MSRA, and MSRB (red signal).The DAPI was used to stain a blue signal of cell nuclei.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

7 treatment.
The control group consisted of H2O2 at 250 μM treatment.The cells were treated with 10 % formaldehyde and fixed for 15 minutes.Subsequently, the cells were washed with 1X PBS containing 0.5 % Tween-20.The cells were rinsed with 1X PBS containing 0.1 % Tween-20 and the cells were blocked for 1 h with 5 % Donkey normal serum.The targeting antibodies (anti-MITF, anti-TYR, anti-TRP-1, anti-TRP-2, anti-MSRA, and anti-MSRB) and secondary antibodies (donkey anti-goat conjugated FITC and donkey anti-mouse conjugated CY3) were performed.The ratio of CY3 and FITC was 1:400 and 1:200, respectively.DAPI solution was applied to the chamber of the slide.The iRiS Digital Cell Imaging System (Logos Biosystems, Anyang, Gyeonggi-do, South Korea) was used to examine immunofluorescence cells.

13 mouse
melanoma cells and decreases H2O2 synthesis by lowering intracellular H2O2 expression levels.The activation of protein expression factors such as TYR, TRP-1, and TRP-2 modulates melanin synthesis via the MITF signaling pathway and stimulates the development of MSRA and MSRB.Putrescine has the potential to be developed as a new anti-aging ingredient and could easily be incorporated into cosmetics designed to prevent skin aging and premature graying of hair.Our experiments certainly warrant additional investments in this line of research.

Fig. 1 .*
Fig. 1.The effect of putrescine on tyrosinase activity, cell viability and H2O2 scavenging activity (a) The effect of putrescine on tyrosinase activity was examined using tyrosine as a substrate.The negative control was vitamin C at 0.1 %.The blank group consisted of sodium phosphate buffer and tyrosine, and the control group consisted of sodium phosphate buffer, tyrosine, and tyrosinase.(b) The effect of putrescine on cell viability was investigated by MTT assay.The blank group consisted of DMEM supplement solution without putrescine treatment.(c)The effect of putrescine on H2O2 scavenging activity was performed with DCFH-DA.The blank

Fig. 2 .* 21 Fig. 3 .
Fig. 2. The effect of putrescine on melanin production in H2O2-treated B16F1 cells.(a) The melanin production in the presence of putrescine in B16F1 cells was observed at a magnification of 200X (scale bar: 100 µm) using a microscope.(b) The total amount of melanin production in B16F1 cells was quantified using ImageJ.The blank group represents the group containing DMEM supplement solution without putrescine treatment.The control group represents the sodium phosphate (250 μM) treatment.α-MSH as a positive control stimulated melanin production.The control group and the sample groups differed statistically significantly, as represented by the asterisks (*, p<0.05) using one-way ANOVA analysis (Duncan's multiple range test).

Fig. 4 .
Fig. 4. The immunofluorescence images of TRP-1, TRP-2, MITF, TYR, MSRA, and MSRB under the aging process with the putrescine-treated group in B16F1 cells.The blank group consisted of DMEM supplement solution without putrescine treatment.The control group consisted of H2O2at 250 μM treatment.The cells were identified using a donkey anti-goat conjugated FITC antibody labeled with a polyclonal TRP-1, TRP-2, and MITF (green signal)

Fig. 5 .
Fig. 5.The effect of protein expression on the production of melanin by putrescine.Under H2O2-treated conditions, the TRP-1, TRP-2 (a), MITF, Catalase (b), TYR, TH (c), MSRA, and MSRB (d) levels in B16F1 cells treated with various concentrations of putrescine (0.25, 0.5, 1, and 2 mM) were examined.The blank group consisted of DMEM supplement solution without putrescine treatment.As a positive control, α-MSH (6 μM) was employed.The control group consisted of H2O2 (250 μM)-treated.The control group (β-actin) was used to normalize the relative levels of target protein expression.The control group and the sample groups differed statistically significantly, as represented by the asterisks (**, p<0.01 and ***, p<0.001) using one-way ANOVA analysis (Duncan's multiple range test).

Fig. 6 .
Fig. 6.The schematic diagram for the effect of putrescine on melanogenesis stimulated by α-MSH in the MITF signaling pathway.