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Changes Of Melanosomal Lumen PH And Ultrastructure Affect Melanogenesis And Antioxidative Capacity Of The Skin

Posted on:2020-02-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:F MiaoFull Text:PDF
GTID:1484305882490384Subject:Dermatology and Venereology
Abstract/Summary:PDF Full Text Request
Backgrounds and ObjectivesMelasma is a common,therapeutically challenging,and universally relapsing disorder of hyperpigmentation,which is most often observed in individuals with Fitzpatrick Skin TypesⅣthroughⅥ.It is also recognized to be more common among women living in Asians and African-Americans.At present,combined therapeutic interventions for melasma include laser,drug,microneedle and so on.But the curative effect is different and associated with universal relapses.Melanin is the most important pigment that determines the color of the skin.It is synthesized and stored by melanosomes in melanocytes(MCs)through a series of complex and fine enzymatic biochemical reactions.MS is the site for synthesis and storage of melanin,which is originated from lysosomal vesicles and encapsulated by monolayer plasma membrane.Melanosomal p H is approximately 4-5.Tyrosinase(TYR,EC1.14.18.1)catalyzes the rate-limiting reaction of melanogenesis and its optimal p H is 6.8.When the intraluminal p H of melanosomes is less than 5.5,TYR is almost completely inactivated.Vitamin C(vitamin C,VC)is also known as ascorbic acid.Many clinical studies have confirmed that VC and its derivatives have therapeutic effects on facial chloasma and other pigmentation diseases.We speculated whether VC and its derivatives could inhibit epidermal melanogenesis by affecting the p H value of melanosomes.Most skin lightening agents reduce melanogenesis by inhibiting the activity of TYR.However,it has been reported that tyrosinase inhibitors in sunscreens and cosmetics could enhance UV carcinogenicity.It may be the decline of melanin which protects from gene mutation and oxidative stress damage caused by UVR.Besides,the integrity of melanosomal ultrastructure is also critical to the photoprotection of keratinocytes.Degraded or broken melanosomes produce a large number of ROS under ultraviolet irradiation to weaken the photoprotective capacity of skin.Hydroquinone(HQ)is a representative tyrosinase-inhibited skin lightening agent,which is currently recognized as one of the effective drugs for the treatment of melasma.But it is unstable and it has been tested that HQ could lead to exogenous ochronosis,irreversible skin depigmentation,and murine hepatic adenomas,renal adenomas,and leukemia with large doses over extended time periods.The US Food and Drug Administration proposed a ban on over-the-counter HQ in 2006.Therefore,it is urgent to explore a safer and more effective ingredient to replace HQ.Arbutin(Arb)is a naturally derived p-hydroxybenzene-β-D-glucopyranoside.Deoxy-arbutin(D-Arb)is derived from the dehydroxylation of the glucose side chain of Arb.They are glucoside derivatives of HQ with stable chemical properties and low side effects.We compared the effects of HQ,Arb and D-Arb on the ultrastructure of melanosomes in order to elucidate the biological safety in the process of skin lightening.This study includes three parts:1.To study whether VC and its derivatives,magnesium ascorbyl phosphate(MAP)and 3-o-ethyl-L-ascorbic acid(AAE),could modulate the p H in melanocytes.Meanwhile,to determine the expression of sodium-dependent vitamin C transporter(SVCT)in melanocytes and the antioxidative effects of VC.2.To examine the melanin content and distribution in hyperpigmented guinea pig skins using Fontana-Masson staining,which were induced by 308 nm monochromatic excimer light and treated with HQ,Arb and D-Arb.Besides,the ultrastructural changes of melanosomes were also observed using transmission electron microscope(TEM).3.To compare the decolorization efficiency of lignin peroxidase(Li P),manganese peroxidase(Mn P)and laccase on eumelanin and pheomelanin,and to investigate the skin lightening effect in vivo through topical administration of Li P solution on hyperpigmented guinea pigs.Methods and Results1.Effects of various concentrations of VC,MAP and AAE(0.1,0.3,1,3,10mmol/L)on proliferation of MCs were examined using cell counting kit-8(CCK-8)reagent to determine the optimal drug concentration.The melanin content of MCs treated with 2μmol/Lα-MSH,3μmol/L HQ,1 mmol/L VC,1 mmol/L MAP and 1mmol/L AAE was investigated using sodium hydroxide pyrolysis assay after 48 hours.TYR activity of each group was determined using a dot blot apparatus and protein levels of TYR,Pmel17 and microphthalmia-associated transcription factor(MITF)were examined by western blot.The results showed that VC and its derivatives could inhibit melanogenesis more or less,which was related with the decrease of TYR activity but actually independent of the protein contents of TYR,Pmel17 and MITF.In addition,the effects of VC and its derivatives on the intracellular p H of MCs were observed using acridine orange(AO)and Lyso Sensor Green DND-189 staining.The results indicated that VC and its derivatives might acidify lysosomes,melanosomes and other lysosome-related organelles.Besides,concanamycin A(Con A)and ammonia chloride(NH4Cl)could eliminate the acidification and restore TYR activity and melanogenesis,which was irrelevant to the protein expression of TYR,Pmel17and MITF.DCFH-DA labeling was used to detect the ROS level of MCs treated with1 mmol/L VC and/or 3 J/cm2 UVA irradiation.The intracellular ROS level significantly increased in MCs exposed to UVA irradiation,whereas the UVA-induced oxidative stress could be attenuated by VC.The expression of SVCTs was determined by western blot and q PCR and the level of SVCT-2 m RNA was detectable in MCs.Phloretin,a kind of SVCT inhibitor,attenuated the acidification of MCs induced by VC.But the inhibition of phloretin is non-specific.The expression of SVCT-2 was significantly decreased through SVCT-2 si RNA transfection of MCs by western blot and q PCR.Besides,the acidification of MCs induced by VC was weakened.2.308 nm monochromatic excimer light(500 m J/cm2,every other day for a total of four exposures)was applied to the guinea pigs to induce hyperpigmentation and the dorsal skin was divided into six groups:sham-irradiated control,vehicle control,3%H2O2,5%HQ,10%Arb and 10%D-Arb.All chemicals were applied topically to the dorsal skin once a day for 10 days.The L*value was measured by a CR-10 Minolta chromameter before and 10 days after the treatment.The results showed that 5%HQ,10%Arb and 10%D-Arb could significantly attenuate the pigmentation induced by UVB irradiation.The melanin distribution in skin and the surface corneocytes was observed by Fontana-Masson silver staining.Statistical analyses of gray value were calculated by Image-J software.The results indicated that the melanin granules in basal and spinous layers of epidermis decreased significantly and the thickness of epidermis increased remarkably in 5%HQ group.Besides,10%Arb and 10%D-Arb also significantly decreased the content of melanin granules in epidermis but had no effect on the thickness of epidermis.The ultrastructural changes of melanosomes in epidermis were detected by TEM before and after treatment.After topical application of 5%HQ,marked ruptures in melanosomal membranes were found and some melanosomes occasionally manifested a bulb-like structure in the outer membranes.Meanwhile,discernible damage on melanosomal membrane was barely observed in the skin treated with 10%Arb and 10%D-Arb.3.Pheomelanin-enriched specimens were prepared from human hair and cutaneous melanoma tissue using alkaline lysis method,synthetic eumelanin was purchased from a commercial supplier.The same amount(0.02%)of melanin was incubated with the equal enzyme activity(0.2 U/ml)of Li P,Mn P and laccase for 3 h respectively.The absorbance at 475-nm(A475)in the enzyme-catalyzed solution was measured using ELISA microplate reader.The results showed that all three ligninolytic enzymes exhibited various degree of eumelanin and pheomelanin decolorization activity.The decolorization activity by Li P,Mn P and laccase was40%~70%,22%~42%and 9%~21%respectively.The experimental hyperpigmentation model was established in the dorsal skin of brownish guinea pigs using 308 nm excimer light radiation.Li P and heat-inactivated Li P solution were topically applied at each site.Meanwhile,3%HQ and vehicle cream were used as control.The results showed that similar lightening was seen in the skin treated with Li P solution and 3%HQ.The skin color(L*value)was recorded using a CR-10Minolta chromameter.Corneocytes were collected using adhesive taping method.The amount and distribution of melanin in the corneocytes and skin tissues was visualized by Fontana-Masson staining.The results showed that the amount of melanin granules in the corneocytes was 199.1±11.1 by Li P,which was fewer than untreated control(922.6±12.2)and heat-inactive control(988.7±13.2).The amount of melanin was decreased in the whole epidermis treated with HQ and the epidermis thickness was increased as well.In contrast,melanin of Li P group was decreased only in the superficial epidermis,the epidermis thickness seemed to be normal.Conclusions1.VC and its derivatives enter MCs through SVCT-2,and play the role of skin lightening by acidifying the lysosome-related organelles to inhibit the activity of tyrosinase reversibly.2.HQ may destroy the outer membranes of melanosomes,while Arb and D-Arb have little effect on the ultrastructure of melanosomes when they exhibit skin lightening capability.3.Li P exerts a potent decolorization activity for melanin extracted from hair and skin.It only affects the superficial layer of epidermis and did not interfere with the melanin synthesis of MCs in basal layer in vivo.
Keywords/Search Tags:Melanocyte, Melanosome, Photoprotection, Skin depigmenting agents, Vitamin C, Hydroquinone, Ligninolytic enzymes
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