The in vitro and in vivo depigmentation activity of coenzyme Q0, a major quinone derivative from Antrodia camphorata, through autophagy induction in human melanocytes and keratinocytes

Background Coenzyme Q0 (CoQ0), a novel quinone derivative of Antrodia camphorata, has been utilized as a therapeutic agent (including antioxidant, anti-inflammatory, antiangiogenic, antiatherosclerotic, and anticancer agents); however, its depigmenting efficiency has yet to be studied. Methods We resolved the depigmenting efficiency of CoQ0 through autophagy induction in melanoma (B16F10) and melanin-feeding keratinocyte (HaCaT) cells and in vivo Zebrafish model. Then, MPLC/HPLC analysis, MTT assay, Western blotting, immunofluorescence staining, LC3 transfection, melanin formation, GFP-LC3 puncta, AVO formation, tyrosinase activity, and TEM were used. Results CoQ0-induced autophagy in B16F10 cells was shown by enhanced LC3-II accumulation, ATG7 expression, autophagosome GFP-LC3 puncta, and AVOs formation, and ATG4B downregulation, and Beclin-1/Bcl-2 dysregulation. In α‐MSH-stimulated B16F10 cells, CoQ0 induced antimelanogenesis by suppressing CREB-MITF pathway, tyrosinase expression/activity, and melanin formation via autophagy. TEM data disclosed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in α‐MSH-stimulated B16F10 cells. CoQ0-inhibited melanogenesis in α‐MSH-stimulated B16F10 cells was reversed by pretreatment with the autophagy inhibitor 3-MA or silencing of LC3. Additionally, CoQ0-induced autophagy in HaCaT cells was revealed by enhanced LC3-II accumulation, autophagosome GFP-LC3 puncta and AVO formation, ATG4B downregulation, ATG5/ATG7 expression, and Beclin-1/Bcl-2 dysregulation. In melanin-feeding HaCaT cells, CoQ0 induced melanin degradation by suppressing melanosome gp100 and melanin formation via autophagy. TEM confirmed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in melanin-feeding HaCaT cells. Treatment with 3-MA reversed CoQ0-mediated melanin degradation in melanin-feeding HaCaT cells. In vivo study showed that CoQ0 suppressed endogenous body pigmentation by antimelanogenesis and melanin degradation through autophagy induction in a zebrafish model. Conclusions Our results showed that CoQ0 exerted antimelanogenesis and melanin degradation by inducing autophagy. CoQ0 could be used in skin-whitening formulations as a topical cosmetic application. Supplementary Information The online version contains supplementary material available at 10.1186/s12964-024-01537-6.


Background
Melanogenesis is the synthesis of melanin, a darkly pigmented biopolymer produced by lysosome-like organelles called melanosomes present in melanocytes.Melanogenesis is considered a complex physiological process that involves both melanocytes and keratinocytes.Functionally, melanin is produced from melanocytes and transferred to keratinocytes that surround the melanocytes.The ratio between melanocytes and keratinocytes is approximately 1:36.Therefore, the metabolism (formation and degradation) of melanosomes and melanin determines the skin tone [1].In addition to melanin metabolism, intrinsic (skin types and genetic background) and extrinsic factors (sunlight exposure levels and pollution) also influence skin color.The most important positive regulator of melanogenesis is melanocortin 1 receptor (MC1R), which is expressed on melanocytes and stimulated by keratinocyte-secreted α-melanocyte stimulating hormone (α-MSH) [2].Activated MC1R subsequently activates cAMP response element-binding protein (CREB) and microphthalmiaassociated transcription factor (MITF), a principal factor regulating melanogenesis [2].The MITF protein controls the transcription and translation of pigmentary tyrosinase and tyrosinase-related protein-1/-2 (TRP-1/-2) catalytic enzymes, leading to melanin production [3].Melanin aids in shielding the skin from risky environmental influences (toxic drugs, chemicals, and UV radiation).The most common target in inhibiting pigmentation and melanogenesis pathway is direct inhibition of tyrosinase catalytic activity.Several skin whitening compounds have been used through tyrosinase inhibition [4,5].Therefore, most of the commercial skin-whitening products are categorized as tyrosinase inhibitors to lessen hyperpigmentation and preventing cytotoxicity to normal/healthy melanocytes.
Previous studies have documented novel mechanisms and the role of autophagy in depigmenting skin [6].Defective autophagy mechanisms are associated with various skin pigmentation disorders [7].Our previous studies confirmed that Vitamin C derivatives (3-O-ethyl ascorbic acid), pterostilbene, and ellagic acid have skin depigmentation efficacy through autophagy induction in melanocytes or keratinocytes [6,8,9].In the cellular microenvironment, autophagy functions as a housekeeping process.Autophagy removes misfolded or aggregated proteins and clears damaged/unnecessary organelles in the cell through a lysosome-dependent mechanism [10].The key autophagic microtubule-associated protein light chain 3 (LC3) is soluble and widely present in mammalian cells.LC3 exists in cytoplasmic LC3-I and membrane-bound LC3-II (transformed from LC3-I) forms.The conversion of LC3-I to LC3-II is necessary for autophagy.A stable interaction occurs between LC3-II and the autophagosome membrane during autophagy.In the process of autophagy, autophagosomes fuse with lysosomes to form autolysosomes, and intraautophagosomal components are degraded by lysosome hydrolases.Thus, the development of autophagy inducers confers extensive clinical benefit for the treatment of diseases as well as for the regulation of human skin pigmentation by melanocytes and keratinocytes [11].
CoQ 0 , a novel quinone derivative of Antrodia camphorata, is the major molecule of CoQ without an isoprenoid side chain.Chemically, CoQ 0 is known as 2,3-dimethoxy-5-methyl-1,4-benzoquinone [14].CoQ 0 biological pharmacology has been reported in vivo and in vitro and in several studies.CoQ 0 exerts multiple actions including stimulating insulin production in the islets of Langerhans, anti-angiogenic and anti-oxidative defenses, and pharmacological actions against various cancers [14].However, researchers have not assessed the ability of CoQ 0 to reduce skin pigmentation.To the best of our knowledge, this investigation is the first to delineate the autophagy mediated depigmenting effects of CoQ 0 in in vitro (B16F10 and HaCaT cells) and in vivo (zebrafish model).

Antrodia camphorata fermented broth preparation from submerged culture
Antrodia camphorata was collected from Nantou County, Taiwan.All AC specimens used were saved in the CMU repository and named "CMU-AC010".Dr. Shy-Yuan Hwang from 'The Endemic Species Research Institute' in Nantou, Taiwan, characterized the fermented broth prepared from Antrodia camphorata.The samples were prepared as previously described [15].All powdered samples were rendered in DMEM containing 1% FBS (pH 7.4) and were saved at -20 °C.Approximately 2 to 4 batches of fermented Antrodia camphorata culture were involved in our experiments.To create a concentrated cultured broth, it was vacuum-sealed and then frozen.From 5 liters, this process produced 46.8 g of dry matter.

Cell culturing and CoQ 0 stock preparation
Murine melanoma B16F10 cells (Cat # CRL-6475) were purchased from the American Type Culture Collection (ATCC, VA, USA).Human skin keratinocyte HaCaT cells (Cat # 300493) were obtained from Cell Line Services (CLS, Eppelheim, Germany).Complete DMEM supplemented with FBS and antibiotics was used to culture HaCaT and B16F10 cells, as described previously [9].A 100 mM CoQ 0 stock concentration was prepared in DMSO and stored at -20 °C until further use.

MTT (cell viability) assay
Cultured cells (HaCaT or B16F10) were treated with varying concentrations of CoQ 0 for the given conditions.After treatments, the cells were washed with PBS, and MTT was added to each well (0.5 mg/mL for 2 h).Following the incubation period, solubilization of cytoplasmic formazan crystals was performed using DMSO solution (0.8 mL), and the color intensity that developed was quantified using an ELISA plate reader.The wavelength was set to 570 nm (λ 570 ) [6].

Immunoblotting assay
The immunoblotting assay was conducted to determine the expression of various proteins present in the cells (HaCaT/B16F10) exposed to varying concentrations of CoQ 0 for different durations.The protocols for harvesting protein samples and the immunoblotting assay were described previously [16].Densitometry analyses were conducted to measure the protein expression levels, and commercial software (AlphaEase, Genetic Technology Inc., Miami, FL, USA) was used for data acquisition.β-Actin, histone H3, and GAPDH served as loading control proteins.

Immunofluorescence staining
Immunofluorescence staining was performed as described previously [16].Briefly, 3-MA (1 mM, 1 h)pretreated or nontreated HaCaT cells were subsequently exposed to various concentrations of CoQ 0 (0-5 μM for 24 h).After incubation, the cells were subjected to staining using different primary (anti-LC3B, anti-gp100, anti-tyrosinase, and anti-MITF) and fluorescein isothiocyanate (FITC)-conjugated (488 nm) secondary antibodies.DAPI was used for nuclear (counter)staining.After staining, the distribution of antibodies in the stained cells was examined under a fluorescence microscope.

LC3 transfection studies
Cells (B16F10/HaCaT) were grown in 6-well plates and used for transfection when they reached 50% confluence.A commercial transfection kit (Lipofectamine RNAiMAX from Invitrogen, Carlsbad, CA, USA) was used for transfection.The procedure used to transfect LC3 plasmids was described in a previous study [6].

Estimation of the melanin content
Cells (B16F10/HaCaT) were cultured in a 10-cm dish to 50% confluence.Cells were collected and rinsed with PBS.At the end of the treatments, the cells were rinsed with PBS again and harvested to measure the melanin content.This procedure was explained in detail in a previous study [17].

GFP-LC3 puncta formation
The GFP-LC3 fusion protein was used to detect autophagosomes (cellular GFP-LC3 puncta) in the cells.The Premo ™ Autophagy Sensor LC3B-GFP Kit from Thermo Fisher Scientific (Waltham, MA, USA) was used according to the manufacturer's protocol [18].After incubation, the culture medium was replaced with fresh medium, and the cells were subjected to CoQ 0 treatment for the indicated durations.After treatments, the cells were washed with PBS, and cellular GFP-LC3 puncta were observed under a laser scanning confocal microscope.

Detection of acidic vesicular organelles (AVOs) using acridine orange staining
Acridine orange (AO) is a fluorescence-based cationic dye that is also cell membrane-permeable and serves as a marker dye to detect AVOs that accumulate within cells.
Based on this proportionality principle, the level of AVOs present in the cells was categorized as high (red), intermediate (yellow), and low (green).The protocol for AO staining was followed as described in a previous study [6].After treatments, 1 μg/mL AO stain prepared in PBS + 5% FBS was applied for approximately 15 min, and the cells were observed under a fluorescence microscope to detect AVOs.For the quantification of fluorescence, Olympus Softimage Solution software (Olympus Imaging America, Inc., PA, USA) was applied.

Tyrosinase activity
Tyrosinase activity was measured in B16F10 cells.The method used to determine tyrosinase activity was described in a recent study [9].The following formula was applied to measure cellular tyrosinase activity:

Melanin-feeding HaCaT cells
The Sigma-Aldrich Chemical Company supplied synthetic melanin (St.Louis, USA).Melanin (25 ng/mL) was fed to HaCaT cells for 24 h before they were given CoQ 0 for the indicated time [16].

Transmission electron microscopy (TEM)
TEM was used to observe the key cellular events in B16F10 and melanin-feeding HaCaT cells.After the treatments were applied, cells were fixed and prepared for TEM observation (Tecnai 12, FEI, Hillsboro, Oregon, USA) as described previously [6].

In vivo zebrafish studies
The depigmenting effect of CoQ 0 in vivo was assessed in the zebrafish model.These in vivo experiments were approved by the China Medical University Institutional Animal Care and Use Committee (IACUC).The protocol used to measure this parameter was described in a previous report [6].Changes in zebrafish viability due to exposure to CoQ 0 , the heartbeat rate, and endogenous pigmentation were monitored through a Z16 stereomicroscope (Leica Microsystems, Ernst-Leitz-Strasse, Germany).Image-Pro Plus software (Media Cybernetics, Inc., Rockville, USA) was used to analyze the data.The methodology used to measure melanin production in zebrafish was described previously [6].

Analyses of LC3B and tyrosinase protein expression in zebrafish embryos
Zebrafish embryos (9 hpf ) were treated with CoQ 0 (0-10 μM) or vehicle (0.1% DMSO) for up to 72 hpf.After incubation, proteins were extracted from the embryos using Tyrosinase activity(%) = 475 of sample 475 of control × 100 a previously reported method [6].The extracted proteins were subjected to Western blotting to measure LC3B and tyrosinase protein expression.The immunoblotting procedure is described earlier in this section.

Statistical analysis
Analysis of variance (ANOVA) and Dunnett's test were performed for pairwise comparisons among the control and test groups.SigmaPlot 10.0 statistical software was employed for the analyses.The data are reported as fold or 100% changes.

Isolation and analysis of CoQ 0 derived from Antrodia camphorata by MPLC and HPLC analysis
MPLC profiling was performed on the fermented culture broths of Antrodia camphorata, which were separated into ten fractions (AC-1 to AC-10, Fig. 1A).The CoQ 0 concentrations of the AC-1 to AC-10 fractions derived from Antrodia camphorata are shown in Fig. 1B.HPLC analysis revealed that the highest CoQ 0 concentration was 86.6 mg/g in the AC-6 fraction (Fig. 1B).The effect of autophagy on the antimelanogenesis of AC-1 to AC-10 fractions (0-20 μg/mL, Fig. 2A-J) was subsequently assessed using immunoblotting in melanoma B16F10 cells.Interestingly, the AC-6 fraction remarkably upregulated the expression of the autophagy marker LC3-II and remarkably downregulated the expression of key melanogenesis-related tyrosinase in B16F10 cells (Fig. 2F and K).
The results suggested that CoQ 0 derived from Antrodia camphorata might be an effective compound for use in depigmenting cosmetics.Consequently, we aimed to delineate the depigmenting mechanisms of CoQ 0 , a novel quinone derivative of Antrodia camphorata, by inducing autophagy in melanoma B16F10 and keratinocyte HaCaT cells.

CoQ 0 suppressed melanogenesis in unstimulated or α-MSH-stimulated B16F10 cells
We determined the cytotoxic concentration of CoQ 0 (0-7.5 μM for 24, 48, or 72 h) in B16F10 cells.MTT results demonstrated that compared to the untreated control, B16F10 cell viability was significantly decreased after treatment with 5 μM CoQ 0 , and this repression was also aggravated with increasing CoQ 0 incubation time (Fig. 3A).Therefore, ≤ 5 μM CoQ 0 was considered a nontoxic or subcytotoxic CoQ 0 concentration for further in vitro experiments in this investigation (Fig. 3A).In B16F10 cells, we first examined the efficacy of CoQ 0 with respect to the protein expression involved in melanogenesis.The immunoblotting data revealed that CoQ 0 dosedependently decreased the levels of the MC1R, p-CREB, CREB, p-MITF, MITF, and tyrosinase proteins (Fig. 3B).

CoQ 0 inhibited MITF nuclear translocation through the ERK, JNK and PI 3 K/AKT pathways in B16F10 cells
We next investigated the roles of different signaling pathways in CoQ 0 -mediated inhibition of MITF nuclear translocation in B16F10 cells [3].Various pharmacological inhibitors were used for this experiment.The immunoblot results showed that the ERK (PD98059), JNK (SP600125), or PI3K/AKT (LY294002) inhibitors remarkably reversed the CoQ 0 (5 μM)-induced decrease in p-MITF and nuclear MITF protein levels, suggesting that the ERK, JNK, and PI3K/AKT pathways play crucial roles (Fig. 4A and B).Moreover, CoQ 0 (5 μM) increased p-ERK, p-JNK, and p-PI3K/AKT in a time-and dose-dependent manner (Fig. 4C and D).This result confirmed that CoQ 0 suppressed MITF nuclear translocation in B16F10 cells via the ERK, JNK, and PI3K/AKT pathways.In B16F10 cells, the differential expression patterns of an autophagy marker (LC3-I/II) and its associated proteins were determined after treatment with CoQ 0 .The immunoblotting data suggested that in the presence of CoQ 0 (0-5 μM for 24 h), LC3-I/II expression was significantly upregulated in B16F10 cells (Fig. 5A  and B).Autophagy-related 4B cysteine peptidase (ATG4B) negatively regulates autophagy and represses autophagy by eliminating lipid conjugates from LC3 (LC3-II).Therefore, ATG4B serves as a negative regulator of autophagy [21].Hence, the results confirmed that ATG4B expression was significantly downregulated by CoQ 0 treatment in B16F10 cells (Fig. 5A and  B) and that ATG7 functions as an E-1 enzyme at the beginning of autophagy for ubiquitin-like proteins, including ATG12 and ATG8 [22].During the initiation of autophagy, ATG7 acts as a critical regulator of autophagosome assembly [22].ATG7 expression was significantly upregulated by CoQ 0 treatment in B16F10 cells (Fig. 5A and B).These results suggested that CoQ 0 enhanced autophagy in B16F10 cells.
CoQ 0 increased autophagosome GFP-LC3 puncta in B16F10 cells GFP-LC3 was transiently transfected into B16F10 cells to further determine whether CoQ 0 stimulated the development of autophagosomes using confocal microscopy.The data confirmed that CoQ 0 increased autophagosome formation, as inferred by the increased number of green LC3 puncta in cytoplasmic B16F10 cells (Fig. 5C and D).Interestingly, this result was reversed (significantly downregulated) by the autophagy inhibitor 3-MA, suggesting that autophagosome GFP-LC3 formation was facilitated in CoQ 0 -treated B16F10 cells (Fig. 5C and D).

CoQ 0 increased AVO formation in B16F10 cells
The autophagy-inducing efficacy of CoQ 0 (0-5 μM for 24 h) was further analyzed by measuring AVO formation.AVO levels were measured by performing AO staining.AVOs significantly accumulated in CoQ 0 -treated B16F10 cells (Fig. 6A and B).Moreover, 3-MA (an early autophagy inhibitor) and CQ (a late autophagy inhibitor) were used to evaluate the interconnection with respect to CoQ 0 -induced AVO accumulation.Pretreatment with 3-MA (1 mM) suppressed this AVO accumulation; however, pretreatment with CQ (1 μM) enhanced the effect in CoQ 0 -treated B16F10 cells (Fig. 6A and B).Furthermore, B16F10 cells treated with 3-MA or CQ alone displayed a significant decrease or increase in AVO formation, respectively (Fig. 6A and B).

CoQ 0 repressed p-MITF and tyrosinase expression through autophagy in B16F10 cells
The effect of autophagy on the antimelanogenesis effect of CoQ 0 was subsequently assessed.For this experiment, 3-MA was administered to determine the key melanogenesis-associated tyrosinase and p-MITF levels using immunofluorescence staining in B16F10 cells.CoQ 0 (0-5 μM for 24 h) mediated a decrease in p-MITF expression, and this effect was also reversed by 3-MA (Fig. 7A and B).Furthermore, CoQ 0 significantly reduced tyrosinase levels, whereas LC3B levels were significantly upregulated in B16F10 cells (Fig. 7C,  D and E).Notably, CoQ 0 -induced inhibition of tyrosinase expression was reversed by pretreatment with 3-MA (Fig. 7C, D and E).The data demonstrated that autophagy plays a crucial role in the antimelanogenesis effect of CoQ 0 in B16F10 cells.The effects of CoQ 0 and/or 3-MA on tyrosinase activity/expression and melanin production were measured in α-MSH-stimulated B16F10 cells.Increased tyrosinase expression/activity (Fig. 8A and B) and melanin levels (Fig. 8C) were observed in α-MSH-stimulated B16F10 cells.However, CoQ 0 (0-5 μM for 24 h) treatment substantially reduced tyrosinase expression/activity and melanin levels in α-MSH-stimulated B16F10 cells (Fig. 8A, B and C).Moreover, in the presence of 3-MA (1 mM), the attenuating effects of CoQ 0 were reversed in α-MSH-stimulated B16F10 cells (Fig. 8A, B and C), confirming that autophagy was directed to inhibiting melanogenesis in α-MSHstimulated B16F10 cells.

CoQ 0 modulated autophagy-associated proteins in favor of autophagy in HaCaT cells
We examined the cell viability of HaCaT cells treated with CoQ 0 .MTT assay showed that CoQ 0 reduced HaCaT cell viability, with a maximum decrease in viability observed after 7.5 and 10 μM CoQ 0 treatment (Fig. 10A).The results implied that CoQ 0 concentration ≤ 5 μM was noncytotoxic for HaCaT cells.This dose was used for subsequent studies.The effect of CoQ 0 (0-5 μM) on autophagy-associated proteins, such as LC3-I/II, ATG4B, ATG5, ATG7, Beclin-1, and Bcl-2, was tested in HaCaT cells.The immunoblotting data indicated that CoQ 0 stimulation dose-dependently upregulated autophagic LC3-I/II protein expression (Fig. 10B).In addition, a decrease in the anti-autophagic  10B).ATG5 is activated by ATG7 and combines with ATG12 and ATG16L1 to form a complex, and this complex is required for the conjugation of phosphatidylethanolamine to LC3-I to produce LC3-II [24].Immunofluorescence staining confirmed that CoQ 0 significantly increased LC3B expression, and 3-MA reversed CoQ 0 -mediated LC3B accumulation in HaCaT cells (Fig. 10C and D).Furthermore, CoQ 0 treatment dose-dependently upregulated Beclin-1 expression but downregulated Bcl-2 expression (Fig. 10E and F).The Beclin-1/Bcl-2 ratio was significantly increased, leading to a shift in the cellular environment to promote autophagy in CoQ 0 -treated HaCaT cells (Fig. 10E and F).These data suggest that CoQ 0 induces autophagy in HaCaT cells.

CoQ 0 provoked antimelanogenesis in zebrafish embryos
The in vivo antimelanogenesis effect of CoQ 0 was further examined using zebrafish embryos as an experimental model.The effects of CoQ 0 (0-15 μM at 9 hpf ) on the viability and heart rate of zebrafish embryos at 72 hpf were first determined.Viability and heartbeat data measured through a stereomicroscope indicated that increasing concentrations of CoQ 0 (up to ≤10 μM) did not exert significant effects on the viability and heart rate of zebrafish at up to 72 hpf, suggesting that testing ≦10 μM CoQ 0 in the context of in vivo experimentation is safe (Fig. 13A and B).Later, CoQ 0 (0-10 μM at 72 hpf )-mediated endogenous body pigmentation and melanin formation were measured in zebrafish.Stereomicroscopic and melanin assay data showed that CoQ 0 dose-dependently decreased endogenous body pigmentation and melanin formation in 72 hpf zebrafish (Fig. 13C and D).Interestingly, preexposure to 3-MA (1 mM 3-MA+10 μM CoQ 0 ) significantly reversed CoQ 0 -mediated antimelanogenesis in zebrafish (Fig. 13C and D).Moreover, the proteins derived from zebrafish (72 hpf ) were subjected to immunoblotting, and the results suggested that CoQ 0 dose-dependently increased LC3B (favoring autophagy) and decreased tyrosinase (indicating antimelanogenesis) expression (Fig. 13E).PTU (4.4 μM) was used as a positive control.The results confirmed that autophagy plays a critical role in CoQ 0 -induced antimelanogenesis.

Discussion
Cosmetic compounds with depigmenting potential are in high demand for skin beautification and lightening.Therefore, the identification of new depigmenting compounds that possess antimelanogenic properties is urgently needed [26].However, due to insufficient scientific evidence regarding the risks (carcinogenicity, allergies, and other side effects), many consumers have serious concerns about their usage.These compounds may be derived from natural sources for safety.CoQ 0 , a major quinone derivative from Antrodia camphorata, is a redox-active ubiquinone compound often found as a component of the mitochondrial respiratory chain [14].In this study, we elucidated the autophagy mediated depigmentation activity of CoQ 0 in in vitro (B16F10 and melanin-feeding keratinocyte HaCaT cells) and in vivo (zebrafish model) (Fig. 14).
The use of cosmetic products or ingredients in animal studies is forbidden.In Taiwan, Europe, and other countries, animal tests create ethical concerns.Zebrafish embryos are a common vertebrate model system in biochemical investigations because they exhibit many physiological and genetic similarities to mammals [27].In depigmentation studies, the effects of cosmetic ingredients on the skin/surface of a zebrafish are easy to observe.In the present study, CoQ 0 decreased endogenous body pigmentation and melanin formation in zebrafish by inducing autophagy.Moreover, CoQ 0 (up to ≤10 μM) did not affect the viability and heartbeat of zebrafish, indicating that there is no toxicity to CoQ 0 -treated zebrafish.Consistent with the in vitro results, the in vivo data derived from 3-MA application confirmed that autophagy plays a pivotal role in CoQ 0 -triggered antimelanogenesis and melanin degradation.As a result, the screening of zebrafish embryos for CoQ 0 incited antimelanogenesis and melanin degradation replaced animal trials for the use of CoQ 0 in cosmetic products or ingredients.
In melanocytes, the mechanism of α-MSH-stimulated melanogenesis is well defined.A number of transcription factors, including CREB and MITF, are involved in melanogenesis [2].Tyrosinase and TRP-1/-2 are crucial melanogenic enzymes.Enzyme transcription is closely controlled by MITF, and MITF is thus viewed as a master regulator of melanogenesis [3].Our results showed that CoQ 0 triggered antimelanogenic effect was regulated by the MC1R-cAMP-CREB-MITF pathways.In unstimulated or α-MSH-stimulated B16F10 cells, CoQ 0 repressed MC1R/CREB/MITF, tyrosinase, and TRP-1/-2 expression and melanin production, suggesting that CoQ 0 exerted strong antimelanogenesis effects on melanocytes.Interestingly, CoQ 0 suppressed MITF nuclear translocation via the ERK, JNK, and PI3K/AKT pathways, suggesting the complexity of the melanogenesis process.ERK activation enhances the antimelanogenic effect in α-MSH-stimulated melanocytes [2].MITF instability and degradation are caused by activated ERK phosphorylating MITF at Ser73.JNK inhibits MITF production through CREB pathways [28].As has been previously discovered, JNK activation inhibits the CREB-MITF signaling pathway which could be enhanced pharmacologically to suppress melanogenesis.PI3K/AKT signaling is an important antimelanogenic pathway and is frequently activated in melanoma cells [29].PI3K/AKT activation and subsequent GSK3β inhibition provoke antimelanogenesis through dephosphorylation of MITF at Ser298 which inhibits binding to tyrosinase and TRP-1/-2 promoters and decrease melanin formation [30].Our findings were consistent with previous studies implicating the important roles of ERK, JNK, and PI3K/AKT activation in the inhibition of MITF nuclear translocation, and therefore anti-melanogenesis.
The development of autophagy-inducing agents offers latent clinical benefits for the treatment of diseases and for the inhibition of abnormal skin pigmentation by melanocytes and keratinocytes [31].Autophagy is a conserved self-catabolic process that helps protect the cellular microenvironment from different stresses; thus, basal autophagy is necessary for cellular homeostasis.The contribution of autophagy to melanocyte and keratinocyte biology has already been documented [7].Moreover, earlier studies suggested that autophagy regulators and melanosome biogenesis-associated regulators have overlapping molecular mechanisms [31].In the current study, we verified the efficacy of CoQ 0 in inducing autophagy mediated antimelanogenesis in B16F10 and HaCaT cells.CoQ 0 -induced autophagy in B16F10 and HaCaT cells was shown by enhanced LC3-II accumulation, ATG4B downregulation, autophagosome GFP-LC3 puncta and AVO formation, and Beclin-1/Bcl-2 dysregulation.However, pretreatment with 3-MA reversed these activities, suggesting that antimelanogenic effect of CoQ 0 is mediated via autophagy in B16F10 and HaCaT cells.
Melanocytes and keratinocytes interact during melanogenesis and melanin formation [31].Our results revealed that CoQ 0 treatment did not markedly alter melanocyte and keratinocyte viability, indicating that CoQ 0 is safe for those skin cells.Autophagy-mediated antimelanogenesis was reported to affect physiological skin color [31].In α-MSH-stimulated B16F10 cells, CoQ 0 triggered antimelanogenesis by inhibiting p-MITF, tyrosinase expression/activity, and melanin formation via autophagy.TEM provided evidence of melanosome engulfment by autophagosomes, and autolysosomes formation, following CoQ 0 treatment in α-MSH-stimulated B16F10 cells.Notably, these CoQ 0 -mediated antimelanogenesis effects were reversed in 3-MA-pretreated cells.LC3 silencing also significantly suppressed the expression of early-and late-stage melanosome marker proteins and melanin levels in LC3-silenced cells.These results cumulatively indicated that autophagy induction by CoQ 0 negatively affects the melanogenesis and hyperpigmentation in skin cells.
Melanosomes, which are lysosome-related organelles, are where melanin is kept after being produced by melanocytes and transferred to nearby keratinocytes.Physiological skin color is influenced by the balance between the quantity of melanosomes and the level of phagocytic activity of keratinocytes that feed on melanin [16].Melanosomes express the transmembrane antigen protein gp100, and gp100 is involved in melanosome maturation [25].Initial data showed that in melanin-feeding HaCaT cells, CoQ 0 inhibited melanosome associated gp100 expression.Notably, CoQ 0 inhibited melanin formation in melanin-feeding HaCaT cells.Further analyses using TEM revealed that CoQ 0 enabled autophagosomes to engulf melanosomes and autolysosome formation in melanin-feeding HaCaT cells.However, CoQ 0 mediated melanin degradation, which was restored in 3-MA pretreated cells.The results confirmed that CoQ 0 promoted autophagic flux, leading to melanin degradation in melanin-feeding HaCaT cells.From our results, the primary target of CoQ 0 appears to be cell surface expression of MC1R which exhibited significantly decreased levels by CoQ 0 Fig. 12 CoQ 0 suppressed melanosome associated gp100 expression and triggered melanin degradation by inducing autophagy in melanin-feeding HaCaT cells.Melanin-treated HaCaT cells were pretreated with or without 3-MA (1 mM, 1 h), followed by treatment with CoQ 0 (0-5 μM, 24 or 72 h).A The gp100 and LC3B expressions (24 h) were determined using immunoblotting.B, C The gp100 expression (24 h) as assessed by immunofluorescence staining.D The intracellular melanin levels (72 h) were estimated using the procedures described in the methodology section.The results are the mean ± SD (n=3).**p < 0.01; ***p < 0.001 compared with untreated cells.## p< 0.01; ### p < 0.001 compared with CoQ 0 -treated cells.E TEM was used to analyze the CoQ 0 promoted formation of melanosome-engulfing autophagosomes, and autolysosomes.Melanin-treated HaCaT cells were pretreated with or without 3-MA (1 mM, 1 h) followed by CoQ 0 (0 or 5 μM, 24 h).M = mitochondria.The blue, yellow, and red arrows indicate melanin/melanosomes, autophagosomes containing melanin/melanosomes, and autolysosomes, respectively treatment (0-5 μM) in B16F10 cells.There were concomitant reduced expressions of MC1R downstream signaling mediators including p-CREB, CREB, MITF, and tyrosinase.However, previous researches have suggested that MC1R expression and melanogenesis is also impacted by environmental signals (UV-R, cellular stresses) and various growth factor signaling (ET-1, FGF etc.,) [32,33].Therefore, further studies are required to ascertain the precise molecular mechanisms of CoQ 0 in anti-melanogenesis/melanin degradation.

Conclusions
This investigation is the first to demonstrate that CoQ 0 , a major quinone derivative from Antrodia camphorata, exerts autophagy mediated depigmenting effects on keratinocytes and melanocytes.In B16F10 Fig. 13 CoQ 0 triggered antimelanogenesis and melanin degradation in zebrafish embryos.A-E Zebrafish embryos, 9 hpf, were treated with or without 3-MA (1 mM), followed by vehicle (0.1% DMSO) or CoQ 0 (0-15 μM) up to 72 hpf.A, B The viability (%) and heart rate (beats/min) of zebrafish were measured using a stereomicroscope.C, D CoQ 0 suppressed melanogenesis in zebrafish.PTU (4.4 μM) served as a positive control.E At the end of treatments, proteins were extracted, and an immunoblotting assay was conducted to measure LC3-II and tyrosinase expression.F, G CoQ 0 triggered melanin degradation in zebrafish.Zebrafish at 72 hpf were treated with or without 3-MA (1 mM) followed by CoQ 0 (0 or 10 μM) for 24 h (72+24 hpf ) with or without α-MSH (1 μM) stimulation.The change in endogenous body pigmentation (melanin levels) in zebrafish (lateral views are shown) was measured as described in the methodology section.The results are the mean ± SD (n=3).*p < 0.05; **p < 0.01; ***p < 0.001 compared with control zebrafish embryos.## p < 0.01; ### p < 0.001 compared with CoQ 0 -treated zebrafish and melanin-feeding HaCaT cells, CoQ 0 exerted antimelanogenesis and melanin degradation effects by inducing autophagy.The in vivo results confirmed that CoQ 0 inhibited endogenous body pigmentation in zebrafish embryos by instigating autophagy.Altogether, our findings suggested that CoQ 0 might be used as a depigmenting ingredient in the skin care formulations.

Fig. 1
Fig.1CoQ 0 isolation from Antrodia camphorata by MPLC and HPLC analysis.A Antrodia camphorata was eluted by a mobile phase consisting of water and methanol by using an RP-18 MPLC glass column.B Ten fractions (AC-1 to AC-10) were ascertained by using an RP-C18 HPLC column.A series of standard solutions (CoQ 0 ) were created by diluting the stock solution, and they were utilized to calculate the CoQ 0 concentration of the AC-1 to AC-10 fractions.The final CoQ 0 product was dissolved in 0.1% DMSO