Qhov Inhibitory Effect Ntawm Curcumin Derivative J147 Ntawm Melanogenesis Thiab Melanosome Thauj Los Ntawm Kev Ua Haujlwm ERK-Mediated MITF Degradation Part 1

Kev siv tshuaj curcumin thiab tshuaj hloov kho curcumin (CMC) rau suppressing melanogenesis thiab tyrosinase kev ua haujlwm tau raug lees paub. J147 yog hloov kho version ntawm curcumin nrog superior bioavailability thiab stability. Txawm li cas los xij, tsis muaj ntawv tshaj tawm txog cov teebmeem ntawm J147 ntawm pigmentation hauv vitro thiab hauv vivo. Hauv peb cov kev tshawb fawb, peb tau tshawb xyuas cov teebmeem hypo pigmentary ntawm J147 kev kho mob ntawm melanocytes thiab tshawb nrhiav cov txheej txheem hauv qab. Cov kev tshawb fawb tam sim no tau qhia tias J147 suppressed ob leeg basal thiab -MSH-induced melanogenesis, nrog rau txo melanocyte dendrite extension thiab melanosome thauj. J147 tau ua lub luag haujlwm no feem ntau los ntawm kev ua kom lub cev muaj zog ntxiv-txheej txheem protein kinase (ERK) txoj hauv kev. Thaum ua haujlwm, nws ua rau MITF degradation thiab txo qis-tswj cov lus qhia ntawm tyrosinase, TRP-1, TRP-2, Myosin Va, Rab27a, thiab Cdc42, thaum kawg inhibiting melanin synthesis thiab melanosome thauj. Tsis tas li ntawd, cov teebmeem hypo pigmentary ntawm J147 tau pom nyob rau hauv vivo hauv tus qauv zebrafish thiab UVB-induced hyperpigmentation qauv hauv cov xim av guinea npua. Peb qhov kev tshawb pom kuj tau qhia tias J147 tsis muaj cytotoxicity hauv vitro thiab hauv vivo. Ua ke, cov ntaub ntawv no tau lees paub tias J147 tuaj yeem ua pov thawj muaj txiaj ntsig zoo raws li kev nyab xeeb ntawm daim tawv nqaij dawb.

Cistanchekuj muaj cov haujlwm ntawmtxhawb kev tsim collagen, uas tuaj yeem nce elasticity thiab luster ntawm daim tawv nqaij thiab pab kho cov tawv nqaij puas. CistanchePhenylethanol Glycosidesmuaj ib tug tseem ceeb down-regulating nyhuv rautyrosinasekev ua haujlwm, thiab cov txiaj ntsig ntawm tyrosinase tau pom tias muaj kev sib tw thiab thim rov qab inhibition, uas tuaj yeem muab lub hauv paus kev tshawb fawb los tsim thiab siv covcov khoom xyaw ua kom dawbhauv Cistanche. Yog li ntawd, cistanche muaj lub luag haujlwm tseem ceeb hauv kev ua kom tawv nqaij. Nws ua tauinhibit melanin ntau lawmkom txo tau cov xim thiab dullness; thiab txhawb kev tsim collagen rautxhim kho daim tawv nqaij elasticitythiab radiance. Vim qhov kev lees paub dav dav ntawm cov teebmeem ntawm cistanche, ntautawv nqaij dawbCov khoom lag luam tau pib infuse cov khoom xyaw tshuaj ntsuab xws li Cistanche kom tau raws li cov neeg siv khoom xav tau, yog li ua lag luam tus nqi ntawm Cistanche hauv cov khoom siv tawv nqaij dawb. Hauv cov ntsiab lus, lub luag haujlwm ntawm cistanche hauv daim tawv nqaij dawb yog qhov tseem ceeb. Nws cov nyhuv antioxidant thiab collagen-tsim cov nyhuv tuaj yeem txo cov xim thiab dullness, txhim kho daim tawv nqaij elasticity thiab luster, thiab yog li ua tiav cov nyhuv whitening. Tsis tas li ntawd, daim ntawv thov dav ntawm Cistanche hauv cov khoom siv tawv nqaij ua kom pom tseeb tias nws lub luag haujlwm hauv kev lag luam tsis tuaj yeem kwv yees.

Keywords: J147, hypo pigmentary teebmeem, melanosome thauj, ERK txoj kev, MITF degradation

Nyem rau ntawm Cistanche Tubulosa rau Whitening

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Taw qhia

Cov tawv nqaij pigmentation nyob ntawm ob qho tib si melanin synthesis thiab kev faib tawm hauv txheej txheej epidermis. Melanin yog tsim nyob ib ncig ntawm lub nucleus ntawm melanocytes thiab khaws cia hauv melanosomes (Tian li al., 2021). Tom qab maturing, melanosomes migrated raws microtubules thiab actin filaments mus rau dendrite cov lus qhia ntawm lub hlwb thiab thaum kawg mus rau cov nyob sib ze keratinocytes kom tiav cov txheej txheem tis (Beaumont li al., 2011; Ohbayashi thiab Fukuda, 2012). Raws li qhov xwm txheej ntawm lub cev, melanin tiv thaiv tib neeg cov tawv nqaij los ntawm kev puas tsuaj ntawm ultraviolet (UV), tshuaj lom neeg, thiab lwm yam ib puag ncig (Slominski li al., 2004; Yousaf et al., 2020). Txawm li cas los xij, kev tsim khoom ntau dhau thiab sib xyaw ua rau muaj qhov ua rau hyperpigmentation thiab cuam tshuam nrog cov kab mob ntawm daim tawv nqaij xws li post-inflammatory melanoderma, melasma, thiab hnub ci lentigines, ua rau muaj kev puas siab puas ntsws zoo heev (Pillaiyar li al., 2017). Yog li ntawd, nws yog ib qho tsim nyog los tsim cov tshuaj tua kab mob kom zoo thiab nyab xeeb.

Nyob rau hauv xyoo tas los no, melanin cell biology tau dhau los ua qhov kev tshawb fawb dav dav thiab ntau cov proteins tseem ceeb uas ua rau melanogenesis thiab melanosome thauj tau raug elucidated, them txoj hauv kev los txheeb xyuas melanin synthesis inhibitors. Tyrosinase tsuas yog tsim nyog rau melanogenesis thiab inhibition ntawm tyrosinase catalytic kev txiav txim yog feem ntau txoj kev los txo melanin ntau lawm (D'Mello li al., 2016). Ntau lub npe hu ua tyrosinase inhibitors, suav nrog arbutin thiab kojic acid, twb tau tsim los ua cov tshuaj pleev ib ce (Ding li al., 2020). KIF5b thiab Rab27A-Melanophilin-Myosin Va complex pab txhawb rau kev thauj mus los ntawm melanosome (Ohbayashi thiab Fukuda, 2012; Noguchi li al., 2014). Cdc42 tswj dendrite elongation, uas yog qhov tseem ceeb rau kev hloov melanosome (Luo, 2000). Qhov inhibition ntawm cov proteins saum toj no tuaj yeem txo qis kev thauj mus los ntawm melanosome, uas yog ib qho tseem ceeb rau kev tsim cov tawv nqaij dawb. Microphthalmia-associated transcription factor (MITF) yog ib qho kev hloov pauv zoo hauv melanogenesis. Tsis tas li ntawd, MITF tseem tswj hwm kev thauj mus los ntawm melanosome los ntawm inducing kev qhia ntawm Rab27a thiab Cdc42 (Noguchi li al., 2016). Ntau qhov kev taw qhia txoj hauv kev koom nrog pigmentation los ntawm kev tswj hwm qib qhia ntawm MITF. Kev ua kom muaj cAMP protein kinase A (PKA) txhawb nqa pigmentation los ntawm cAMP cov lus teb cov ntsiab lus khi cov protein (CREB) nyob ntawm kev tswj hwm ntawm MITF qhia (Rodríguez thiab Setaluri, 2014). Hloov pauv, kev ua kom muaj zog ntawm cov teeb liab tswj hwm cov protein kinase (ERK) inhibits melanogenesis los ntawm kev ua kom MITF degradation (Lv li al., 2020a). Ntau cov tshuaj tiv thaiv melanogenic tau tsim los ua kom lub hom phiaj tyrosinase kev ua, kev hloov pauv melanosome, lossis cov kev qhia txog melanogenic. Txawm li cas los xij, ob peb tus neeg inhibitors tau kawm hauv vivo thiab pom cov txiaj ntsig zoo (Pillaiyar li al., 2017).

Curcumin yog diarylheptanoid compound cais los ntawm rhizome ntawm Curcuma longa (Zingiberaceae) thiab siv los ua daj tsw los yog xim hauv cov khoom noj (Zheng J. et al., 2018). Cov kev tshawb fawb tau qhia nws ntau yam kev ua haujlwm ntawm lub cev, suav nrog kev tiv thaiv kab mob, tiv thaiv oxidative, anti-amyloid, thiab kev tiv thaiv qog nqaij hlav (Liu li al., 2016; Zheng Y. li al., 2018). Sib nrug los ntawm cov no, curcumin inhibits tyrosinase kev ua haujlwm thiab suppresses melanogenesis hauv melanocytes (Lee et al., 2010; Tu et al., 2012). Tab sis, qhov tsis zoo bioavailability ntawm curcumin txwv nws daim ntawv thov (Karthikeyan li al., 2020). Txhawm rau daws qhov teeb meem no, J147 yog tsim los ua ib qho chaw muaj zog ntawm curcumin derivative nrog ntau dua stability thiab bioavailability (Li et al., 2020). J147 muaj cov nyhuv neuroprotective thiab tam sim no nyob rau hauv theem kuv kev kuaj mob rau Alzheimer's kab mob. Txawm li cas los xij, tseem tsis tau tshaj tawm txog qhov cuam tshuam ntawm J147 ntawm pigmentation hauv vitro thiab hauv vivo.

Cov khoom siv thiab cov txheej txheem

Reagents

J147 (J302241), -MSH (M118985), thiab tyrosinase los ntawm nceb (T128536) tau txais los ntawm Aladdin (Shanghai, Suav). Peb tau txais cov tshuaj tiv thaiv kab mob tiv thaiv Myosin Va (sc-365986), KIF5b (sc-133184), GP100 (sc-393094), Cdc42 (sc-8401), Rab27a (sc{{ 13}}), p-JNK (sc-6254), JNK (sc-7345), p-p38 MAPK (sc-166182) thiab p38 MAPK (sc{21}}) los ntawm Santa Cruz (CA, USA). Cov tshuaj tiv thaiv tiv thaiv MITF (97800), p-MEK (2338), MEK (4694), p-ERK (4370), thiab ERK (4695) tau txais los ntawm Cell Signaling Technology (MA, USA). Cov tshuaj tiv thaiv tiv thaiv tyrosinase (ab180753), TRP-1 (ab235447), TRP-2 (ab221144), cytokeratin (ab7753), thiab S100 (ab133519) tau txais los ntawm Abcam (Cambridge, UK). p38 inhibitor SB203580 (S1863), ERK inhibitor PD98059 (S1805), BCA protein assay kit (P0012), cell lysis buffer (P0013), thiab -actin (AF5001) tau txais los ntawm Beyotime (Shanghai, Suav). RT-qPCR cov khoom siv (RR036A) tau yuav los ntawm Takara Biomedical Technology (Beijing, Suav).

Cell Culture

MTT Assay

Cell viability raug tshuaj xyuas los ntawm MTT assay (Yun li al., 2020). Luv luv, cov hlwb tau muab noob rau hauv 96- cov phiaj zoo thiab kho nrog J147 (1–8 μM) rau 48 teev. Tom qab ntawd, cov hlwb raug ntxuav nrog PBS thiab hloov nrog MTT tov (20 μL). Tom qab incubation rau ntxiv 4 h, cov tshuaj supernatant raug tshem tawm thiab DMSO (200 μL) tau ntxiv rau txhua qhov dej. Thaum kawg, qhov kho qhov muag absorbance ntawm 570 nm tau txiav txim siab.

Kev ntsuas ntawm Melanin Cov ntsiab lus

Cells nrog qhov ceev ntawm 2 × 105 hlwb / mL tau muab noob rau hauv 6- cov kab lis kev cai zoo. Tom qab 24 h incubation, hlwb tau coj los sib txawv koob tshuaj J147 (1, 2, 4 μM) thiab nrog los yog tsis muaj -MSH (60 nM) stimulation. Tom qab 48 teev kev kho mob, cov hlwb raug sau thiab tag nrho cov melanin hauv cov cell pellet tau yaj hauv 100 μL ntawm NaOH ua haujlwm daws (1 mol / L, 10 feem pua ​​​​DMSO) ntawm 80 degree C rau 1 h, thiab qhov nqus tau ntsuas ntawm 405. nm (Lv et al., 2015; Lv et al., 2019).

Tyrosinase Activity Assay

Cellular tyrosinase kev ua haujlwm tau raug tshuaj xyuas raws li tau piav qhia yav dhau los (Liao li al., 2017; Lv li al., 2020a). Hauv luv luv, cov hlwb tau lysed los ntawm cell lysis tsis tom qab ntxuav peb zaug, thiab tom qab ntawd tus supernatant rau tyrosinase kev ua ub no tau txais los ntawm centrifuging cov lysates. 100 μL PBS (0.1M, pH 6.5) contenting 10 ug proteins tov nrog 100 μL 0.1 feem pua ​​L-DOPA. Lub phaj yog incubated ntawm 37 degree rau 1 h, thiab tom qab ntawd kho qhov muag absorbance ntawm 475 nm.

Qhov cuam tshuam ncaj qha ntawm J147 ntawm tyrosinase kev ua haujlwm tau sim los ntawm lub xov tooj ntawm tes tsis pub dawb raws li tau piav qhia dhau los (Lv li al., 2020a). Nyob rau hauv luv luv, cov tshuaj tiv thaiv rau kev txiav txim siab ntawm nceb tyrosinase kev ua si tau ua nyob rau hauv ib tug 96- zoo phaj thiab cov tshuaj tiv thaiv sib tov muaj nceb tyrosinase (10 units), L-tyrosine (0.03 feem pua, 50 μL), thiab 100 μL PBS. (0.1 M, pH 6.5) ntxiv nrog ntau qhov sib txawv ntawm J147. Tom qab incubation ntawm 37 degree rau 10 min, absorbance ntawm 475 nm tau ntsuas siv lub microplate spectrophotometer.

Masson-Fontana Ammoniacal Silver Staining

Txhawm rau txheeb xyuas cov xim melanin, daim tawv nqaij, thiab melanocytes tau kho nyob rau hauv formalin thiab stained raws li tus qauv raws tu qauv (Gu li al., 2018; Lv li al., 2020b). Nyob rau hauv luv luv, slides raug ntxuav 3 zaug nrog dej deionized thiab ces incubated nyob rau hauv ammoniacal silver tov nyob rau hauv chav tsev kub rau 12 teev. Tom qab rinsing zoo nyob rau hauv dej deionized, slides tau incubated nyob rau hauv hypo daws rau 5 min. Tom qab ntawd, cov swb tau muab yaug dua thiab rov ua dua nrog cov xim liab nruab nrab rau lwm 5 feeb. Thaum kawg, tom qab ntxuav kom huv si, cov swb tau pom nyob hauv Nikon-Eclipse-Ti microscope.

Immunofluorescence rau Melanosome Transfer

Cov txheej txheem coculture ntawm B16F10 thiab HaCaT hlwb tau tsim los ntawm cov tais diav, raws li tau piav qhia dhau los (Lee li al., 2015; Lv li al., 2020c). Tom qab kev kho J147, cov hlwb tau txhaj tshuaj tiv thaiv GP100 thiab anti-Cytokeratin raws li tus qauv txheej txheem. Cov duab tau coj los ntawm Nikon-Eclipse-Ti microscope.

Immunohistochemistry rau S-100

Immunohistochemistry rau S-100 tau ua raws li tau piav qhia yav dhau los (Lee et al., 2013; Lv et al., 2020b). Cov lus piav qhia luv luv yog raws li nram no: swb tau thaiv nrog 5 feem pua ​​BSA ntawm 25 degree C rau 1 h thiab tom qab ntawd incubated nrog anti-S-100 thawj cov tshuaj tiv thaiv ntawm 4 degree C thaum hmo ntuj. Hnub tom qab, cov slides raug ntxuav 3 zaug nrog TBST tov thiab incubated nrog cov tshuaj tiv thaiv theem nrab. Tom qab ntawd, cov slides raug kho nrog aminoethyl carbazole los tsim cov ntu thiab tau pom nyob rau hauv lub tshuab tsom.

Rov qab Transcription-PCR

Cellular tag nrho RNA tau muab rho tawm los ntawm TRIzol reagent thiab quantified spectrophotometrically. Tom qab ntawd, SuperScript II Reverse Transcriptase tau siv los ua ke ib leeg-strandedcDNA ua raws li cov lus qhia tsim khoom.Oligonucleotide primers tau yuav los ntawm GenScript(Nanjing, Suav teb). Cov sequences ntawmMITFgene primers yog 5′- AGAGCAGGGCAGAGAGAGTGAGTG -3′, 5′-AACTGATTCCAGGCTGATGATGTC -3′. Cov sequences ntawmGAPDHnoobprimers yog 5′- AGGTCGGTGTGAACGGATTTG-3′, 5′- TSIAGACCATGTAGTTGAGGTCA -3′. PCR cov khoom yogcais los ntawm electrophoresis ntawm 1 feem pua ​​agarose gels thiab kuaj pomnyob rau hauv lub teeb ultraviolet (Li et al., 2013).

Western Blotting

Proteins (40 ug) tau sib cais los ntawm SDS-PAGE gels thiab xa mus rau nitrocellulose lim (NC) daim nyias nyias los ntawm kev hloov hluav taws xob electrophoretic (Bio-Rad). NC daim nyias nyias tau thaiv nrog 3 feem pua ​​​​BSA hauv TBST tov ntawm chav tsev kub rau 1.5 h. Tom qab ntawd cov ntaub so ntswg tau incubated nrog thawj cov tshuaj tiv thaiv ntawm 4 degree thaum hmo ntuj. Hnub tom qab, cov blots tau ntxuav 3 zaug nrog cov tshuaj TBST thiab tom qab ntawd incubated nrog peroxidase-conjugated thib ob cov tshuaj tiv thaiv ntawm 25 degree rau 1 h, thiab pom los ntawm kev siv cov tshuaj kho kom zoo (Lv li al., 2020d).

Kev txiav txim siab ntawm Melanin Cov ntsiab lus hauv Zebrafish Model

Luv luv, synchronized embryos tau sau thiab arrayed los ntawm pipette (peb mus rau plaub embryos ib qhov dej nrog 200 μL embryo nruab nrab hauv 96-zoo daim hlau). Tom qab ntawd, J147 thiab PTU tau yaj hauv 0.1 feem pua ​​​​DMSO thiab tau ntxiv rau qhov nruab nrab embryo ntawm 35 txog 60 h (25 h raug). Kev cuam tshuam ntawm J147 ntawm melanogenesis ntawm zebrafish tau pom nyob rau hauv stereomicroscope (Zheng J. li al., 2018).

Phenotype-raws li kev ntsuam xyuas thiab UVB-Induced Hyperpigmentation Guinea Pig Model

8 guinea pigs xim av (6 lub lis piam, kwv yees li 25{23}}-300 g) tau yuav los ntawm Lub Tsev Haujlwm Saib Xyuas Tsiaj Tsiaj Science (Beijing, Suav). Cov npua guinea no tau khaws cia nyob ib leeg nyob rau hauv ib qho chaw kub thiab av noo nyob rau hauv ib tug 12-h lub teeb / tsaus voj voog. Cov chaw sib cais (1 cm diametrical voj voog) ntawm sab nraub qaum ntawm txhua tus tsiaj tau raug 500 mJ / cm2 UVB (Sigma SH-4, Shanghai, Tuam Tshoj) ib hnub ib zaug rau 1 lub lis piam los tsim cov qauv hyperpigmentation. Tom qab ntawd lub tsheb (PEG400/EtOH=7:3) thiab J147 (1 feem pua) tau muab rau cov cheeb tsam hyperpigmented (20 μL tov ib lub voj voog) ob zaug hauv ib hnub rau 3 lub lis piam. Lub degree ntawm pigmentation tau soj ntsuam los ntawm kev xam qhov ΔL-tus nqi raws li L-tus nqi ntsuas los ntawm spectrophotometer (YS3010, 3nh, Shenzhen, Tuam Tshoj), raws li hauv qab no: ΔL=L (ntawm txhua hnub ntsuas)-L (hnub 0) (Lee et al., 2013; Lv et al., 2020b). Tag nrho cov txheej txheem tsiaj hauv txoj kev tshawb no tau pom zoo los ntawm pawg saib xyuas tsiaj thiab siv ntawm Changzhou University.

Kev txheeb cais

TSEEM CEEB

J147 Txo Melanogenesis thiab tus naj npawb ntawm Dendrites hauv B16F10 Cells

Tus qauv ntawm J147 yog qhia hauv daim duab 1A. Ua ntej, qhov kev sim ntawm tes tau ua los tshawb xyuas yog tias J147 yog cytotoxic rau B16F10 hlwb. Raws li pom nyob rau hauv daim duab 1B, tsis muaj cov teebmeem cytotoxic ntawm J147 tau pom nyob rau ntawm qhov ntau ntawm 1-8 μM tom qab 48 teev. Tom qab ntawd, peb tshuaj xyuas qhov cuam tshuam ntawm J147 ntawm melanin synthesis. Raws li pom hauv daim duab 1C, J147 inhibited basal melanin synthesis. -MSH txhawb nqa melanogenesis hauv melanocytes. Qhov nce hauv melanogenesis tshwm sim los ntawm -MSH tau thim rov qab tom qab kev kho J147. Raws li txoj cai, Masson-Fontana ammoniacal silver staining pom tias J147 zoo kawg nkaus txo cov ntsiab lus melanin hauv B16F10 hlwb nrog lossis tsis muaj -MSH (Daim duab 1D). Tsis tas li ntawd, tus naj npawb thiab qhov ntev ntawm dendrites thiab melanin concentration hauv dendrites kuj tau txo qis thaum piv nrog cov hlwb tsis kho (Daim duab 1D). Cov txiaj ntsig tau pom tias J147 txo qis melanogenesis thiab dendrite tsim yam tsis muaj teebmeem cytotoxic.

J147 Inhibited Cellular Tyrosinase Kev Ua Haujlwm thiab Kev Qhia Txog Tyrosinase, TRP-1, TRP-2

Cov tsev neeg tyrosinase ntawm cov protein (tyrosinase, TRP-1, thiab TRP-2) tau koom nrog hauv melanogenesis. Ntawm cov no, tyrosinases yog cov enzymes tseem ceeb tswj txoj hauv kev melanin biosynthetic (D'Mello li al., 2016). Txhawm rau txiav txim siab seb J147 cuam tshuam txog kev ua haujlwm tyrosinase, peb thawj zaug siv L-DOPA oxidation txoj kev los txiav txim siab txog cov teebmeem ntawm J147 ntawm kev ua haujlwm ntawm tes ntawm tyrosinase. J147 (1–4 μM) tau pom tias ua rau muaj kev cuam tshuam loj heev ntawm kev ua haujlwm ntawm cellular tyrosinase nyob rau hauv ib koob tshuaj nyob rau hauv B16F10 hlwb (Daim duab 2A). Tom ntej no, kev soj ntsuam tyrosinase kev ua ub no tau ua los txiav txim siab qhov cuam tshuam ncaj qha ntawm J147 ntawm kev ua tyrosinase. Raws li pom nyob rau hauv daim duab 2B, tsis muaj kev cuam tshuam inhibitory tau pom, qhia tias J147 tsis cuam tshuam rau cov haujlwm enzymatic ntawm nceb tyrosinase (Daim duab 2B). Raws li peb paub, melanogenesis yog tswj los ntawm cov dej num thiab cov nqi ntawm cov tyrosinases. Txhawm rau tshawb xyuas yog tias cov tshuaj tiv thaiv melanogenic ntawm J147 muaj feem cuam tshuam nrog kev qhia ntawm tyrosinase, western blot tsom xam. Raws li pom hauv daim duab 2C, cov theem ntawm tyrosinase qhia tau txo qis tom qab J147 kev kho mob nrog lossis tsis muaj -MSH, thiab cov txiaj ntsig tib yam tau pom hauv TRP-1 thiab TRP{23}} qhia. Cov kev soj ntsuam no tau qhia tias J147 inhibited cellular tyrosinase kev ua haujlwm thiab melanogenesis los ntawm kev txo qis cov theem ntawm tyrosinase, TRP-1, thiab TRP-2.

J147 Inhibited Melanosome Thauj los ntawm Kev Tswj Xyuas Kev Qhia ntawm Myosin Va, Rab27a thiab Cdc42

Tib neeg daim tawv nqaij pigmentation yog txiav txim los ntawm melanin synthesis nrog rau kev faib ntawm melanin. Nyob rau hauv mammalian melanocytes, melanin yog tsim nyob rau hauv lub cell lub cev thiabmigrate nrog actin filamentsthiab microtubules rau dendrites, thiab thaum kawgmus rau cov neeg nyob sib ze keratinocytes kom tiavkev faib tawmtxheej txheem (Ohbayashi thiab Fukuda, 2012). Raws li qhia hauvDaim duab 1D, J147 markedly txo cov dendrite tsim.Tsis tas li ntawd, melanin concentration hauv dendrites kuj yogtxo qis raws li cov xim melanin tau sib sau ua ke hauv perinuclearcheeb tsam. Tom ntej no, peb co-cultured B16F10 thiab HaCaT hlwb rautshawb xyuas yog J147 influencesmelanosome hloov mus raukeratinocytes los ntawm confocal microscopy. Kev faib tawm ntawmmelanin tau pom nyob rau hauv HaCaT hlwb hauv kev sib koom ua keqauv (Daim duab 3A). Nyob rau hauv sib piv, thaum lub co-culture qauv yogkho nrog J147 rau 48 h, melanosome granular signals hauvHaCaT hlwb tau loj heevtxo (Daim duab 3A).

Txhawm rau kom paub meej ntxiv txog lub hauv paus txheej txheem ntawm inhibition cuam tshuam ntawm melanosome hloov los ntawm J147, peb tau tshuaj xyuas ntau yam tseem ceeb hauv kev thauj mus los ntawm melanosome. KIF5b kho kom haum rau sab nrauv melanosome thauj nrog microtubules, thiab Rab27a Melanophilin-Myosin Va complex pab txhawb nqa melanosome thauj nrog actin filaments hauv melanocytes (Reiner li al., 2020). Tsis tas li ntawd, Cdc42 stimulates dendrites tsim nyob rau hauv melanocytes, uas kuj plays lub luag hauj lwm tseem ceeb nyob rau hauv melanosome thauj. Raws li tau pom hauv daim duab 3B, qhov kev qhia ntawm Cdc42, Myosin Va, thiab Rab27a, tab sis tsis yog KIF5b tau poob qis heev tom qab J147 kev kho mob nyob rau hauv cov xwm txheej nrog lossis tsis muaj -MSH. Peb qhov kev tshawb pom tau qhia tias J147 inhibited melanosome thauj los ntawm kev txo qis ntawm Myosin Va, Rab27a, thiab Cdc42.

J147 Accelerated MITF Degradation

MITF yog ib qho ntawm cov tswj hwm tseem ceeb rau melanogenesis thiab nws tswj cov noob hloov ntawm tyrosinase, TRP-1, TRP-2, Cdc42, thiab Rab27a (Noguchi et al., 2016; Kim et al., 2019 ). Peb thawj zaug tshuaj xyuas qhov cuam tshuam ntawm J147 ntawm MITF cov ntawv sau tseg. Raws li pom nyob rau hauv daim duab 4A, -MSH zoo kawg li nce MITF transcription, txawm li cas los xij, tsis muaj kev hloov pauv tau pom tom qab kev kho J147, qhia tias J147 tsis txo qis MITF qhia. Tom ntej no, peb tshuaj xyuas yog J147 cuam tshuam rau kev txhais ntawm MITF. Raws li pom nyob rau hauv daim duab 4B, tom qab -MSH kev kho mob, MITF protein ntau nce, peaked ntawm 4 h, thiab pib poob ntawm 8 h (Daim duab 4B). Qhov sib txawv, cov protein ntau ntawm MITF tsis txo qis ntawm 4 h tom qab J147 kev kho mob, tab sis ntawm 8 h cov MITF protein ntau tau poob qis mus rau qhov tsis paub meej, qhia tias J147 tsis cuam tshuam rau kev txhais ntawm MITF tab sis remarkedly accelerates MITF protein degradation.

J147 Txhaum Melanogenesis Los ntawm MEK / ERK Signaling Pathway

Mitogen-activated protein kinases (MAPK) kev taw qhia txoj hauv kev, suav nrog cov xov tooj hluav taws xob ntxiv-tswj cov protein kinase (ERK), p38, thiab c-jun N-terminal kinase (JNK), ua lub luag haujlwm tseem ceeb hauv pigmentation (Lee li al., 2013). ERK phosphorylation yog paub los pab txhawb kev degradation ntawm MITF thiab nws thiaj li dissipate lub melanogenic stimuli, uas sawv cev rau ib tug loj tsis zoo tswv yim mechanism (Kwon li al., 2017). Txawm li cas los xij, kev ua haujlwm ntawm p38 thiab JNK txoj hauv kev tseem muaj teeb meem. Li no, peb tau tshuaj xyuas cov txiaj ntsig ntawm J147 ntawm MAPK kev taw qhia txoj hauv kev. Raws li pom hauv daim duab 5A, J147 tau qhib MEK / ERK thiab p38, thaum tsis muaj kev cuam tshuam nyob rau hauv phosphorylation ntawm JNK signaling txoj kev.

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