Gomisin N Inhibits Melanogenesis Los ntawm Regulating PI3K / Akt Thiab MAPK / ERK Txoj Kev Teeb Meem hauv Melanocytes

Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791

Abstract: Gomisin N, ib qho ntawm cov lignan tebchaw pom nyob rau hauv Schisandra Chinensis tau pom tias muaj cov tshuaj tiv thaiv oxidative, anti-tumorigenic, thiab anti-inflammatory kev ua ub no nyob rau hauv ntau yam kev tshawb fawb.Ntawm no peb qhia, thawj zaug, lub anti-melanogenic ua tau zoo ntawm Gomisin N nyob rau hauv mammalian. cellas thiab nyob rau hauv zebrafish embryo. Gomisin N ua rau txo qis melanin cov ntsiab lus tsis muaj cellular toxicity. Txawm hais tias nws tsis muaj peev xwm hloov kho cov kev ua haujlwm catalytic ntawm ncebtyrosinasehauv vitro,Gomisin Ndownregulated qhov qhia theem ntawm cov proteins tseem ceeb uas ua haujlwm hauvmelanogenesis. Gomisin N downregulated melanocortin 1 receptor (MC1R), adenylyl cyclase 2, microphthalmia-associated transcription factor (MITF), tyrosinase,tyrosinase-related protein-1(TRP-1), thiab tyrosinase-related protein-2 (TRP-2). Tsis tas li ntawd, Gomisin N-kho Melan-A cellexhibited nce p-Akt thiab p-ERK qib, uas txhais tau hais tias kev ua kom PI3K / Akt thiab MAPK / ERK txoj hauv kev tuaj yeem ua haujlwm rau inhibit.melanogenesis. Peb kuj tau lees paub tias Gomisin txo qis melanin ntau lawm los ntawm kev tawm tsam ntawm MITF,tyrosinase, TRP-1, thiab TRP-2 nyob rau hauv nas thiab tib neeg lub hlwb raws li nyob rau hauv tsim zebrafish embryos. Ua ke, peb xaus lusGomisin Ninhibits melanin synthesis los ntawm kev tawm tsam ntawm MITF thiab melanogenicenzymes, tej zaum los ntawm kev hloov kho PI3K / Akt thiab MAPK / ERK txoj hauv kev.

Ntsiab lus:Schisandra Chinensis;Gomisin N; lignan;melanogenesis; tawv nqaij dawb

cistanche muaj daim tawv nqaij ua haujlwm

1. Taw qhia

Melanin yog cov xim muaj nyob rau hauv cov tsiaj feem ntau xws li tawv nqaij, plaub hau, qhov muag, pob ntseg, pob txha, lub plawv, thiab lub hlwb [1,2].Melanogenesisyog ib tug complex txheej txheem uas muaj ntau yam signaling pathways koom. melanocortin 1 receptor (MC1R) yog ib qho tseem ceeb regulator hauvmelanogenesis, signaling los ntawm nws cov ligands xws li melanocyte-stimulating hormone (MSH) thiab adrenocorticotropic hormone (ACTH) [3]. Cov tawv nqaij melanin yog biosynthesized los ntawm melanocytes nyob rau hauv lub epidermis thiab ces pauv mus rau keratinocytes, qhov uas lawv ua si lub luag hauj lwm tseem ceeb nyob rau hauv kev tiv thaiv ntawm daim tawv nqaij los ntawm absorbing UV hluav taws xob los ntawm tshav ntuj thiab scavenging reactive dawb radicals [4,5]. Melanin synthesis thiab hloov mus rau hauv daim tawv nqaij thiab plaub hau follicles yog tswj los ntawm qhov muaj ntawm nws precursors [6]. L-tyrosine thiab L-dihydroxyphenylalanine (L-DOPA), cov substrates loj ntawm melanogenic enzymes, kuj ua haujlwm zoo li cov tshuaj hormones zoo li regulators hauv melanogenesis [7]. Ntawm qhov tod tes, overproduction ntawm melanin ua rau undesirable skincares xws li freckles thiab melasma [8,9]. Melanogenesis tuaj yeem cuam tshuam tus cwj pwm ntawm ib txwm thiab malignant melanocytes los ntawm kev hloov kho cov khoom elastic ntawm cov hlwb [10]. Txawm hais tias cov receptors rau serotonin thiab melatonin qhia nyob rau hauv cutaneous cells ua lub luag haujlwm tseem ceeb hauv kev tswj hwm cellularhomeostasis, ntau ntau ntawm melanin los ntawm kev hloov pauv hormonal tsis tuaj yeem ua rau muaj kab mob hauv daim tawv nqaij [11].

Yog li, tau muaj kev siv zog ntau los qhia txog cov txheej txheem molecular uas tswj melanogenesis yog thawj kauj ruam rau kev kho cov tawv nqaij hyperpigmentary. Tsis tas li ntawd, ntau yam ntawmtawv nqaij dawbCov neeg ua haujlwm uas inhibit melanin synthesis tau raug txheeb xyuas los ntawm cov nroj tsuag thiab cov nroj tsuag uas tsis yog cog thiab ua lag luam siv hauv cosmetics [12,13]. Cov tshuaj tua kab mob uas siv ntau tshaj plaws suav nrog hydroquinone, mequinol, arbutin, Kojic acid, ascorbic acid, thiab retinicacid [12,14]. Txawm li cas los xij, muaj ntau yam kev txwv ntawm lawv cov kev siv hauv kev kho mob hnyav lossis mob ntev ntawm hyperpigmentation hauv tib neeg. Piv txwv li, hydroquinone, txawm hais tias nws tau ntev tau siv fordepigmentation txij li xyoo 1960, tuaj yeem ua rau tawv nqaij khaus thiab tiv thaiv kab mob dermatitis [15,16]. Nws kuj ua rau DNA puas tsuaj los ntawm kev ua kom muaj cov pa oxygen reactive thiab kev loj hlob ntawm exogenous ochronosis nyob rau hauv mammalian hlwb [17,18]. Lwm yam paub zootyrosinaseinhibitors xws li Kojic acid thiab ascorbic acid tsis tsuas yog muaj cov tawv nqaij tsis zoo, kev ruaj ntseg, thiab kev ua kom dawb tab sis kuj tuaj yeem ua rau cytotoxicity, dermatitis, thiab erythema los ntawm kev siv mus sij hawm ntev [19,20]. Hauv qhov no, muaj kev xav tau ntau ntxiv los tsim kom muaj kev nyab xeeb thiab ua haujlwm zoo dua rau kev kho cov tawv nqaij hyperpigmentation. Cov tshuaj ntsuab siv nyob rau hauv cov tshuaj ib txwm siv yuav muab lwm qhov chaw rau kev txheeb xyuas cov tshuaj ntxuav hniav dawb tshiab uas tswj cov kauj ruam tseem ceeb hauvmelanogenesisnrog tsawg los yog tsis muaj phiv [21].

Schisandra chinensis, tseem hu ua Northern fine-flavor berry, yog ib txwm pom nyob rau sab qaum teb Tuam Tshoj, sab hnub tuaj Russia, Nyiv, thiab Kauslim [22]. Cov nroj tsuag no tau ntev tau siv rau kev kho mob hmo ntuj qhov muag tsis pom kev, tawv nqaij hlawv, aseptic o, thiab kab mob siab hauv cov tshuaj orientalmedicine [23,24]. S. chinensis txiv hmab txiv ntoo extract thiab nws cov lignan tebchaw tau pom tias muaj ntau yam teebmeem tshuaj nyob rau hauv murine cell kab. Piv txwv li, Schisandra A thiab C muaj cov tshuaj tiv thaiv oxidative, thaum Schisandra B muaj anti-fibrotic, anti-inflammatory, anti-oxidant, thiab anti-apoptoticactivities [25]. Lwm qhov sib xyaw lignanGomisin N(Daim duab 1A) tau tshaj tawm tias inhibit oxidative stress-induced apoptosis los ntawm inhibiting qhov tso tawm ntawm cytochrome C los ntawm mitochondria mus rau cytoplasm, cleavage ntawm caspase 3 thiab PARP, thiab Ca2 ntxiv -induced mitochondrial permeabilitytransition nyob rau hauv H9cyte rat [7,8]. Interestingly, ob peb txoj kev tshawb fawb siv nas qauv thiab tib neeg cov kab mob ntawm daim tawv nqaij ntawm tes tau nthuav tawm cov kev kho mob ntawm S. chinensis rau kev kho mob ntawm daim tawv nqaij.Lee li al. qhia tias methanol extract ntawm S. chinensis txiv hmab txiv ntoo alleviated hu dermatitis cov tsos mob los ntawm kev txo zus tau tej cov pro-inflammatory cytokines xws li TNF- thiab IFN- thaum topicallyapplied [8,24]. Kang et al. qhia tau tias Schisandra Fructus dej extract inhibited IκB ua kom, li nosuppressing zus tau tej cov TNF- , IL-6, thiab GM-CSF nyob rau hauv tib neeg mast cell kab HMC-1 [26].Cov kev tshawb pom no coj peb mus postulate tias S. chinensis lignans tuaj yeem cuam tshuam rau ntawm daim tawv nqaij ua haujlwm ntau dua.Gomisin N, ib qho ntawm cov lignancompounds loj hauv S. Chinensis, hauv kev tswj hwmmelanogenesis, yog li ntsuas nws cov peev txheej siv los ua tus neeg sawv cev acosmeceutical. Ntawm no, peb tshaj tawm tiasGomisin Ninhibits melanin biosynthesis yam tsis muaj cellular toxicity nyob rau hauv tib neeg thiab nas hlwb, nrog rau hauv zebrafish embryos.

2. Cov txiaj ntsig

2.1. Cov teebmeem ntawm Gomisin N ntawm Melanin Tsim thiab Cell Viability

Txhawm rau txheeb xyuas qhov cuam tshuam ntawm Gomisin N ntawmmelanogenesis, peb kho nas melanocytes nrog sib txawv concentrations ntawmGomisin Nrau 72 h, thiab tom qab ntawd ntsuas kev hloov pauv hauv cov ntsiab lus melanin. Gomisin Ntreatment txo cov ntsiab lus melanin ob qho tib si nyob rau hauv ib txwm melanocyte cell kab Melan-A thiab hauv B16F10 melanoma hlwb nyob rau hauv ib koob tshuaj raws li tsis muaj cellular toxicity (Daim duab 1B, C). Peb pom tias Gomisin N inhibited -MSH-induced melanin ntau lawm hauv B16F10 hlwb, uas yog piv nrog cov txiaj ntsig tau los ntawm kev kho PTU [27] (Daim duab 1C). Txhawm rau ntsuas seb qhov txo qis ntawm melanin li casGomisin NKev kho mob yog vim qhov txo qis ntawm tyrosinase, peb tau ua L-DOPAstaining assay hauv tib neeg epidermal melanocyte (NHEM) hlwb. Raws li pom nyob rau hauv daim duab 1E, NHEMcells kho nrog Gomisin N pom tau txo qis L-DOPA piv nrog cov hlwb tsis kho.Txawm li cas los xij, cov nyhuv inhibitory ntawm Gomisin N ntawm melanin ntau lawm tsis tseem ceeb hauv humanmelanoma MNT-1 hlwb (Daim duab 1D) .

2.2. Cov teebmeem ntawm Gomisin N ntawm Tyrosinase Activity

Mus xyuas sebGomisin NinhibitstyrosinaseKev ua hauv vitro, peb siv nceb tyrosinase thiab B16 melanoma cell lysates. Kojic acid, tus paub zoo tyrosinase inhibitor, tau siv los ua kev tswj xyuas qhov zoo. Thaum kho rau nceb tyrosinase, thaum Kojic acid txo qis qhov enzymaticactivity, Gomisin N tsis ua rau muaj kev hloov pauv ntawm L-DOPA rau dopachrome (Daim duab 2A). Txawm li cas los xij, thaum kho rau B16 melanoma hlwb, Gomisin N ua rau txo qis. hauvtyrosinasekev ua ntawm cov cell lysate nyob rau hauv ib koob tshuaj raws li (Daim duab 2B). Ntxiv mus, thaum kho nrog -MSH hauv B16 hlwb,Gomisin Nthim rov qab qhov nce hauv dopachrome tsim tawm los ntawm -MSH (Daim duab 3C).Qhov tseem ceeb, Gomisin N tau tshwm sim zoo dua li Kojic acid los tiv thaiv cellular.Kev ua haujlwm ntawm tyrosinasentawm B16 hlwb raws li -MSH stimulus. Cov kev tshawb pom no qhia tau hais tias qhov inhibitory nyhuv ntawm GomisinN ntawm melanin ntau lawm pom nyob rau hauv nas thiab tib neeg hlwb (Daim duab 1) tsis yog vim nws muaj nuj nqi mus ncaj qha inhibit lub catalytic kev ua si ntawm tyrosinase. Txawm li cas los xij, nws tseem muaj peev xwm hais tias Gomisin Nregulates qhov kev qhia ntawm tyrosinase lossis lwm cov proteins uas ua lub luag haujlwm tseem ceeb hauvmelanogenesis.

2.3. Cov teebmeem ntawm Gomisin N ntawm qhov tsis ua haujlwm ntawm MC1R Txoj Kev Qhia Tawm

Peb tau nrhiav kom paub meej cov txheej txheem hauv qab lub luag haujlwm rau inhibitory nyhuv ntawm GomisinN ntawm melanin ntau lawm. Peb xav li ntawdGomisin Ntuaj yeem tswj cov cim qhia cov protein uas koom nrogmelanogenesisthiab yog li inhibit melanin synthesis. Melanocortin 1 receptor (MC1R) yog amelanocytic G protein-coupled receptor uas ua haujlwm ua tus tswj hwm tseem ceeb hauv melanin synthesis. Kev ua haujlwm ntawm MC1R los ntawm nws cov ligand -MSH los yog adrenocorticotropic hormone (ACTH) ua rau muaj kev nce hauv adenylyl cyclase, uas nyob rau hauv lem upregulates intracellular cAMP theem [2,3]. Yog li ntawd, qib kev hloov pauv ntawm MITF tau nce los ntawm Protein kinase-C (PKA) / teb cov ntsiab lus-bindingprotein (CREB) txoj hauv kev [2,28]. Txhawm rau soj ntsuam cov txiaj ntsig kev tswj hwm ntawm Gomisin N ntawm MC1R signalingpathway, peb tau tshawb xyuas cov theem qhia ntawm MC1R thiab nws cov lus qhia qis qis tom qab kho Melan-A hlwb nrog Gomisin N. Peb pom tias Gomisin N txo qis cov protein ntau ntawm MC1R thiab adenylyl cyclase 2. raws li kev noj tshuaj raws li kev noj tshuaj (Daim duab 3A–C).Raws li xav tau,Gomisin N-kho cov hlwb kuj pom cov protein tsawg tsawg ntawm MITF thiab nws cov hom phiaj tyrosinase, TRP-1, thiab TRP-2 (Daim duab 3A, D-G). Cov kev tshawb pom no qhia tias Gomisin N inhibitsmelanin-tsim enzymes los ntawm inactivating MITF ntawm MC1R signaling txoj kev.

2.4. Cov teebmeem ntawm Gomisin N ntawm Phosphorylation ntawm Akt thiab ERK1/2 hauv Melan-A Cells

PI3K / Akt thiab MAPK / ERK txoj hauv kev tau paub tias muaj kev koom tes hauv melanogenesis bytranscriptionally lossis post-transcriptionally regulating MITF [29,30]. Txhawm rau ntsuas seb GomisinN puas cuam tshuam rau cov kev taw qhia no, peb tau soj ntsuam cov xwm txheej phosphorylation ntawm Akt thiab ERK1/2 los ntawm Western blot tsom xam. Raws li pom hauv daim duab 3H–J, kev kho mob siab (30 µM) ntawmGomisin NQhov tseem ceeb ntawm phosphorylation ntawm Akt thiab ERK. Cov ntaub ntawv no qhia tias cov nyhuv inhibitory ntawm Gomisin N raumelanogenesiszoo li yuav cuam tshuam nrog P13K / Akt thiab MAPK / ERK txoj hauv kev.

2.5. Gomisin N Inhibited Melanogenesis hauv Zebrafish Embryos

Peb tom ntej no tsom mus tshuaj xyuas seb Gomisin N puas muaj txiaj ntsig hauv kev tiv thaivmelanogenesishauv vivo. Txog qhov kawg no, peb tau kho zebrafish embryos nrog Gomisin N rau 72 h ntawm qhov ntau ntawm 1, 10, 20, thiab 30 µM, thiab tom qab ntawd ntsuas cov qib ntawm melanogenic proteins. Peb pom tias Gomisin N kev kho mob inhibited melanin tsim nyob rau hauv tsim zebrafish embryos.Gomisin N-kho embryos showeda txo melanin cov ntsiab lus nyob rau hauv ib tug concentration-dependent yam piv nrog untreatedcontrol (Daim duab 4). Peb kuj pom tias cov qib protein ntawmtyrosinase, MITF, TRP-1, thiab TRP-2 tau txo qis los ntawm Gomisin N kho (Daim duab 5). Cov txiaj ntsig no qhia tau tias Gomisin N inhibitsmelanogenesis hauv vivo los ntawm kev tswj hwm qhov hloov pauv MITF thiab nws cov hom phiaj.tyrosinase, TRP-1, thiab TRP-2.

2.6. Gomisin N thim rov qab Rapamycin-induced Melanogenesis hauv tib neeg MNT-1

Txawm hais tias Gomisin N cuam tshuam ntau heevmelanogenesisnyob rau hauv Melan-A thiab B16 hlwb thiab inzebrafish embryos, peb tsis tau soj ntsuam nws cov nyhuv rau tib neeg MNT-1 melanoma hlwb. Peb cia siab tias cov txiaj ntsig ntawm Gomisin N tuaj yeem kuaj pom hauv MNT-1 hlwb nyob rau hauv cov xwm txheej uas melanogenesis raug tswj los ntawm qhov tsim nyog stimulus. Rapamycin tau pom tias ua rau melanogenesis los ntawm kev nce tyrosinase kev ua haujlwm thiab cov protein ntau ntawm MITF, tyrosinase, TRP-1, thiab TRP-2 [31], ib nrab los ntawm kev ua kom autophagy [32]. Peb tau soj ntsuam cov theem ntawmtyrosinase, MITF,TRP-1, thiab TRP-2 hauv MNT-1 hlwb los ntawm Western blot tsom xam, tom qab kev kho nrog Gomisin N andrapamycin. Kev kho mob Rapamycin ua rau MITF, TRP-1, thiab TRP-2 qib tab sis tsis muaj txiaj ntsig rautyrosinasetheem (Daim duab 6). Txawm li cas los xij, Gomisin N kev kho mob tau thim rov qab cov txiaj ntsig ntawm rapamycin ntawm MITF, TRP-1, thiab TRP-2 nyob rau hauv ib qho concentration-dependent yam. Cov nyhuv rov qab ntawmGomisin Ntawm tsam rapamycin tau zoo dua ntawm qhov concentration ntawm 20 thiab 30 µM tshaj 10 µM.Cov txiaj ntsig no qhia tias Gomisin N inhibitsmelanogenesisnyob rau hauv tib neeg MNT-1 melanoma hlwb los ntawm kev tswj cov transcription factor MITF thiab nws cov hom phiaj TRP-1 thiab TRP-2.

3. Kev sib tham

Lub luag haujlwm tseem ceeb ntawm melanin yog los tiv thaiv cov hlwb ntawm daim tawv nqaij tiv thaiv UV hluav taws xob [33–35] .Hyperpigmentation, qhov tshwm sim ntawm kev tsim tawm ntau dhau ntawm melanin hauv daim tawv nqaij, ua rau tsis xav tau kev zoo nkauj thiab cuam tshuam nrog dermatitis thiab mob qog noj ntshav ntawm daim tawv nqaij. Ob peb tsab ntawv ceeb toom tau tawm tswv yimmelanogenesisraws li lub hom phiaj tseem ceeb rau kev kho mob metastatic melanoma [36,37]. Yog li, muaj kev xav tau ntau ntxiv los tsim cov tshuaj tiv thaiv melanogenic uas tswj cov melanogenesis yam tsis muaj cellulartoxicity [38]. Muaj ntau txoj hauv kev koom nrog hauv daim tawv nqaij melanogenesis [39,40]. Raws li ligand binding, MC1R txhim kho cov kev ua ntawm adenylyl cyclase, uas tom qab ntawd nce intracellular theem ntawm cAMP [41,42]. CAMP-dependent activation ntawm PKA / CREB txoj hauv kev tau tshaj tawm dav dav los txhim kho qib kev hloov pauv ntawm MITF, yog li txhim kho melanin synthesis [43]. MITFfunctions ua tus thawj tswj hwm ntawm peb qhov loj melanogenic enzymes tyrosinase, TRP-1, thiab TRP-2 hauv vertebrates [3,21,44]. Cov enzymes no yog transmembrane proteins nyob rau hauv melanosomalmembrane ntawm melanocytes. Tyrosinase tswj tus nqi txwv cov kauj ruam hauvmelanogenesisconvert L-tyrosinase rau L-DOPA [23]. TRP-1 thiab TRP-2 kuj tseem ua lub luag haujlwm tseem ceeb hauv kev tsim cov tshuaj melanin, txawm tias lawv txoj haujlwm tsis nkag siab tag nrho.

Nyob rau hauv txoj kev tshawb no, Gomisin N, ib tug lignan compound ntawm S. chinensis pom depigmenting kev ua si yam tsis muaj cellular toxicity. Gomisin N inhibited melanin synthesis nyob rau hauv kab lis kev cai mammalian cell kab raws li zoo raws li nyob rau hauv zebrafish embryos. Gomisin N zoo li muaj txiaj ntsig zoo dua li kev tswj hwm zoo PTU inhibiting melanin ntau lawm hauv Melan-A hlwb (Daim duab 1B). Gomisin N txo cov ntsiab lus melanin nyob rau hauv concentration-dependent yam. Piv nrog rau pawg tswj tsis tau kho, 10 µM ntawmGomisin Ntxo cov melanin cov ntsiab lus los ntawm 40 feem pua, tsis muaj cellular toxicity. Cov kev ua los tiv thaiv melanogenic ntawm 10-µM Gomisin N yog piv nrog rau ntawm 100-µM PTU hauv Melan-A hlwb. Ib yam li ntawd, Gomisin Nappeared muaj zog dua PTU hauv -MSH-activated B16F10 hlwb, qhov twg cov teebmeem ntawm 5- thiab 10-µM Gomisin N yog piv nrog cov ntawm 10- thiab {{12. }}µM PTU, ntsig txog (Daim duab 1C).NHEM hlwb kho nrog Gomisin N pom tau tias txo qis L-DOPA, uas qhia tias GomisinN inhibitstyrosinasekev ua haujlwm hauv cov kab lis kev cai (Daim duab 1E). Cov kev tshawb pom no tau coj peb mus rau kev tshawb fawb ntxiv txog cov txheej txheem hauv qab uas Gomisin N inhibits melanogenesis.

Peb tshuaj xyuas sebGomisin Nncaj qha modulates lub catalytic kev ua ntawmtyrosinaseNyob rau hauv vitro.Tsis zoo li Kojic acid, Gomisin N tsis qhia inhibitory cuam tshuam rau nceb tyrosinase kev ua (Daim duab 2A). Txawm li cas los xij, kev ua haujlwm ntawm cellular tyrosinase hauv B16 melanoma cell lysates tau txo qis los ntawm Gomisin N ob qho tib si nrog thiab tsis muaj -MSH kev kho mob (Daim duab 2B, C). Inhibition ofcellular tyrosinase kev ua ntawm Gomisin N raws li -MSH stimulus tau pom tias yog qhov tseem ceeb tshaj qhov kev tswj xyuas zoo Kojic acid (Daim duab 2C).

Peb tau tshaj tawm tias kev ua haujlwm los tiv thaiv melanogenic ntawm Gomisin N tuaj yeem tshwm sim los ntawm kev hloov pauv lossis kev hloov pauv tom qab kev tswj hwm ntawm tyrosinase thiab tyrosinase-related proteins (TRPs).Rau qhov ua tau zoo, peb ntsuas qhov qhia txog qib ntawm cov cim qhia hauv MC1R txoj hauv kev, ib qho tseem ceeb rau kev txiav txim siab rau qhov no. kom muaj nuj nqis thiab zoo ntawm melanin ntau lawm hauv melanocytes. Cia siab tias, peb pom tias Gomisin N txo qis ntawm MC1R thiab adenylyl cyclases 2 hauv Melan-A hlwb (Daim duab 3A–C). Tsis tas li ntawd,Gomisin Ntxo qis kev qhia ntawm MITF thiab nws lub hom phiaj cov proteins suav nrog tyrosinase, TRP-1, thiab TRP-2 (Daim duab 3A, D-G). Cov txiaj ntsig no qhia tias qhov txo qis ntawm cov ntsiab lus melanin thaum kho Gomisin N tshwm sim los ntawm kev tsis ua haujlwm ntawm MC1R txoj hauv kev.

Ntawm qhov tod tes, PI3K / Akt thiab MAPK / ERK txoj hauv kev tuaj yeem phosphorylate MITF, thiab yog li tuaj yeem hloov pauv hloov pauv nws cov haujlwm [45]. Txawm li cas los xij, tag nrho cov txiaj ntsig ntawm kev ua haujlwm ntawm PI3K / Akt thiab MAPK / ERK txoj hauv kevmelanogenesismuaj teeb meem. Ob txoj hauv kev PI3K / Akt thiab MAPK / ERK yog tsim los ua haujlwm hauv tib neeg melanomas vim muaj kev hloov pauv hloov pauv [46]. C2-ceramide-mediated depigmentation hauv Mel-Ab hlwb paub tias muaj tshwm sim los ntawm kev txo qis p-Akt qib [47]. Muaj ob peb lub ntuj sib txuas uas ua kom melanogenesis los ntawm kev tswj hwm p-ERK qib hauv B16 melanoma hlwb [28]. Hauv qhov sib piv, tseem muaj pov thawj tias nce p-ERK thiab p-Akt qib inhibit melanin synthesis [28,48]. Qhov nyuaj ntawm kev tswj hwm ntawm melanogenesis tuaj yeem piav qhia qee qhov los ntawm qhov tseeb tias phosphorylation txhim kho cov kev ua haujlwm ntawm MITF, tab sis ib txhij induces ubiquition-proteosome-dependentdegradation ntawm MITF [26,49-51]. Peb cov ntaub ntawv qhia tau hais tias ob qho tib si p-Akt thiab p-ERK qib tau upregulatedin Gomisin N-kho Melan-A hlwb (Daim duab 3H-J). Qhov no txhais tau hais tias PI3K / Akt thiab MAPK / ERKpathways tuaj yeem pab txhawb rau inhibition ntawm melanin ntau lawm.

Peb ntxiv validated lub anti-melanogenic kev ua si ntawm Gomisin N nyob rau hauv lub zebrafish nyob rau hauv vivo qauv.Gomisin N-kho zebrafish embryos pom ib tug tseem ceeb txo nyob rau hauv melanin pigmentation (Daim duab 4). Tsis tas li ntawd, Gomisin N tau txo qis cov qib ntawmtyrosinase, MITF, TRP-1, thiab TRP-2 hauv kev tsim cov embryos zebrafish. Cov kev tshawb pom no sib sau qhia tiasGomisin Ninduces depigmentation los ntawm downregulating cov kev qhia ntawm MITF thiab melanogenic enzymes nyob rau hauv vivo.Cov anti-melanogenic kev ua ntawm Gomisin N tau ntxiv paub tseeb hais tias nyob rau hauv tib neeg melanoma MNT -1 hlwb stimulated los ntawm rapamycin. Txawm hais tias Gomisin N ua rau tsuas yog hloov me me ntawm cov ntsiab lus melanin hauv MNT-1 hlwb (Daim duab 1D), nws muaj txiaj ntsig zoo rau kev thim rov qab rapamycin-induced upregulation ntawm MITF, TRP-1, thiab TRP-2 hauv ib tug concentration-dependent yam (Daim duab 6A, C–E). Coj ua ke, cov kev cai tswjfwm ntawmGomisin Nntawm MITF thiab melanogenic enzymes tau tsim dua tshiab nyob rau hauv nas thiab tib neeg lub hlwb nrog rau hauv zebrafish embryos.

Txhawm rau xaus, txoj haujlwm no qhia tias Gomisin N tuaj yeem muaj peev xwm ua tau zoo li qhov tshiabtawv nqaij dawbtus neeg sawv cev. Gomisin N zoo li inhibitmelanogenesislos ntawm kev tawm tsam qhov kev qhia ntawm MITF ntawm txoj kev MC1R, es tsis txhob ncaj qha hloov cov kev ua haujlwm catalytic ntawmtyrosinasethiab TRPs. Txawm hais tias cov txheej txheem ntxaws ntxaws tseem yuav tsum tau piav qhia, Gomisin N-induced depigmentation zoo li yuav cuam tshuam nrog kev ua haujlwm ntawm PI3K / Akt thiab MAPK / ERK txoj hauv kev (Daim duab 7).

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Khoom siv

RPMI1640 tau yuav los ntawm Gibco-BRL (Gaithersburg, MD, USA). Dulbecco's modifiedEagle's medium (DMEM), fetal bovine serum (FBS), thiab penicillin-streptomycin (PS) tau yuav los ntawm Hyclone (Carlsbad, CA, USA). Melanocyte kev loj hlob nruab nrab yog yuav los ntawm PromoCell (Heidelberg, Lub teb chaws Yelemees). Phenylmethylsulfonyl fluoride (PMSF), 12-O-tetradecanoylphorbol-13-acetate(TPA), Kojic acid, 1-phenyl-2-}thiourea (PTU), nceb tyrosinase, 3, {{ 9}}dihydroxy-l-phenylalanine (L-DOPA), -MSH, dimethyl sulfoxide (DMSO), thiab paraformaldehyde tau yuav los ntawm SigmaChemical Co. (St. Louis, MO, USA).Gomisin NQhov chaw yog muab los ntawm Chul Young Kim (Hanyang University, Ansan, Kauslim). Rapamycin tau yuav los ntawm Sigma-Aldrich (St. Louis, MO, USA).

4.2. Cell Culture

Cov nas melanoma cell kab B16F10 tau muab los ntawm Kauslim Cell Line Bank (Seoul, Kauslim).Murine melanocyte Melan-A hlwb [52] yog ib qho khoom plig pub dawb los ntawm Dr. Byeong Gon Lee (lub koom haum SkinResearch, Amore Pacific Co., Yongin -si, Korea). Tib neeg MNT-1 cov hlwb melanoma tau muab los ntawm Aeyeong Lee (Collage of Medicine hauv Dongguk University, Goyang-si, Kauslim). Lub hauv paus ib txwm tib neeg epidermal melanocytes (NHEM) tau yuav los ntawm PromoCell (Heidelberg, Lub teb chaws Yelemees). Melan-A hlwb tau loj hlob hauv RPMI 1640 nruab nrab (Gibco, Carlsbad, CA, USA) ntxiv nrog 10% FBS, 1% PS, thiab 200 nM TPA. DMEM ntxiv nrog 10 feem pua ​​​​FBS thiab 1 feem pua ​​PS tau siv los tswj Melan-A hlwb thiab NHEM hlwb. Tag nrho cov hlwb raug incubated ntawm 37 ◦C hauv 5 feem pua ​​CO2 incubator.

4.3. Kev ntsuas ntawm Melanin Cov ntsiab lus

Melan-A hlwb tau muab noob rau hauv 24- lub phaj zoo (1 × 105cells/well), kho nrogGomisin N, thiab tom qab ntawd incubated rau 72 h. Tom qab 72 h, cov ntsiab lus melanin tau ntsuas raws li tau piav qhia yav dhau los [53].Rov qab, tom qab tshem tawm cov kab lis kev cai, cov hlwb raug ntxuav peb zaug nrog PBS. Tom qab ntawd, sodium hydroxide tov (1 mL, 1 N) tau ntxiv rau txhua qhov dej kom yaj melanin. Lub absorbanceat 405 nm tau ntsuas siv tus nyeem ntawv microplate. Qhov kev ntsuam xyuas no tau rov ua dua nrog B16F10 hlwb (2 × 104cells/well) thiab MNT{10}} hlwb ua raws li tib txoj kev.

4.4. Western Blot Analysis

Melan-A cov noob tau cog rau hauv 100 hli lauj kaub tais diav (1 × 106cells / phaj) thiab kho nrog 1, 5, lossis 10 µMGomisin Nrau peb hnub ntawm 37 ◦C. Cells raug ntxuav nrog PBS thiab tom qab ntawd sau los ntawm scraper.Detached cells tau muab tso rau hauv 1 ml ntawm PBS thiab centrifuged ntawm 7500 rpm rau 5 min. Tom qab tshem tawm cov kev daws teeb meem saum toj kawg nkaus, cov cell pellets tau lysed nrog lysis buffer (50 mM Tris-HCl, pH 8.0, 0.1% SDS, 150 mM NaCl, 1% NP-40, 0.02% sodium azide, 0.5% sodium deoxycholate, 100 µg/mL PMSF, 1 g/mL aprotinin) rau 24 teev ntawm 4 ◦C. Tag nrho cov proteins tau muab rho tawm los ntawm kev siv ultracentrifuge ntawm 12, 000 rpm rau 30 min ntawm 4◦C. Cov ntsiab lus protein tau ntsuas los ntawm kev ntsuas Bradford. Cov Proteins (30 µg) tau muab sib cais siv a10 feem pua ​​​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel thiab xa mus rau anitrocellulose membrane. Lub membrane raug thaiv rau 1 h nrog 5 feem pua ​​​​skim mis nyuj nyob rau hauv Tris-buffered salinewith Tween -20 (TBST), thiab ces incubated rau 12 h ntawm 4 ◦C nrog thawj cov tshuaj tiv thaiv hom phiaj -tubulin (Santa Cruz, CA, USA ), MITF (Cell Signaling, Danvers, MA, USA), tyrosinase (Cell Signaling), ERK (Cell Signaling), phospho-ERK (Cell Signaling), AKT (Cell Signaling), phospho-AKT (Cell Signaling), MC1R ( Santa Cruz), adenylyl cyclases 2 (Santa Cruz), TRP-1 (Santa Cruz), thiab TRP-2 (Santa Cruz).Tom qab tshem cov thawj cov tshuaj tiv thaiv, cov membranes tau ntxuav peb zaug nrog TBST thiab incubated nrog lwm yam. Cov tshuaj tiv thaiv kab mob (Lavbit anti-tshis IgG-HRP; nas los tiv thaiv luav HRP, Santa Cruz) rau 1 h.Cov membranes tau kho nrog txhim kho chemiluminescence reagent siv ChemiDoc XRS ntxiv rau cov duab kos duab (Bio-Rad, Hercules, CA, USA). Densitometric tsom xam ntawm cov bands tau ua tiav siv Image MasterTM 2D Elite software (version 3.1, GE Healthcare, Chicago, IL, USA).

4.5. Tyrosinase Activity Assay

Txhawm rau kwv yees cov nyhuv inhibitory ntawm Gomisin N ntawm ncebtyrosinasekev ua haujlwm, tyrosinase tau tsim nrog 1, 5, lossis 10 µMGomisin Nlos yog qhov zoo tswj Kojic acid. Txhua tus qauv yog yaj methanol. L-DOPA (8.3 mM) thiab nceb tyrosinase (125 U) tau diluted hauv 80 mM phosphatebuffer (pH 6.8). 40 µL ntawm txhua tus qauv thiab 120 µL ntawm L-DOPA tau muab sib xyaw rau hauv 96- zoo phaj, tom qab ntawd ntxiv ntawm 40 µL ntawm diluted nceb tyrosinase. Cov phiajcim tau muab tso rau 15 feeb, thiab qhov nqus tau ntsuas ntawm 490 nm siv tus nyeem ntawv microplate.

TyrosinaseKev ua haujlwm hauv B16 melanoma cell lysates tau ntsuas nrog lossis tsis muaj -MSH kev kho mob, raws li yav dhau los tau piav qhia los ntawm Ohguchi li al. [54], nrog kev hloov kho me ntsis. Cell lysate tau npaj raws li tau piav qhia saum toj no hauv Western Blot tsom xam ib feem. Tag nrho cov proteins hauv supernatant tau ntsuas los ntawm kev soj ntsuam ntawm Bradford siv Bovine Serum Albumin raws li tus qauv [55]. Ib qho sib npaug ntawm cov proteins tau diluted thiab siv rau qhov kev ntsuam xyuas tyrosinase.

inhibit tyrosinase kev ua haujlwm

4.6. L-DOPA Staining hauv NHEM Cells

NHEM hlwb tau muab noob rau hauv {{{0}}} lub phaj zoo thiab incubated rau 72 teev nrog Gomisin N. Cells tau kho nrog 4 feem pua ​​paraformaldehyde rau 40 min, tom qab ntawd kho nrog 0.1 feem pua ​​Triton X{ {6}} rau 2 min.L-DOPA (0.1 feem pua) tau ntxiv rau txhua qhov dej, tom qab ntawd incubation rau 2 teev. Tom qab tshem tawm cov tshuaj, cov hlwb raug ntxuav ob zaug nrog PBS. Cov duab tau yees duab los ntawm microscope.

4.7. Zebrafish Experiments

Zebrafish embryo tau txais los ntawm Zebrafish Resource Bank (Daegu, Kauslim). Embryoswere kho nrog Gomisin N rau 72 teev. Cov nyhuv depigmenting ntawmGomisin Nntawm zebrafish embryoswas pom nyob rau hauv lub stereomicroscope. Rau Western blot tsom, Gomisin N-kho embryoswere lysed siv lysis tsis, los ntawm tag nrho cov proteins tau npaj raws li hais saum toj no.

5. Cov lus xaus

Peb qhov txiaj ntsig txhawb kev pom tias Gomisin N muaj peev xwm siv tau los ua cov khoom noj muaj txiaj ntsig thiabtawv nqaij dawbtus neeg sawv cev.Gomisin Nyog ib qho tseem ceeb ntawm lignan tebchaw hauv S. Chinensis. Qhov tseeb, S. Chinensis yog tshuaj ntsuab siv los kho ntau yam kab mob tib neeg. Txawm li cas los xij, kev tshawb fawb txuas ntxiv yog tsim nyog los ua pov thawj kev nyab xeeb ntawm Gomisin N ntawm daim tawv nqaij. Yog li ntawd, hauv vivo kev tshawb fawb thiab kev sim tshuaj ntsuam xyuas yuav muaj peev xwm ua kom pom tseeb dua qhov ua tau zoo ntawm GomisinN. Hauv kev xaus, qhov kev tshawb fawb no qhia tiasGomisin Ntej zaum yuav yog ib tug muaj peev xwm hypo-pigmentary neeg sawv cev thiabnaturaltawv nqaij dawbtus neeg sib tw rau kev lag luam tshuaj pleev ib ce.

cistanche txhim kho kom dawb

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