Molecular Mechanisms Ntawm Anti-Melanogenic Gedunin Muab los ntawm Neem Tsob Ntoo
Mar 28, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
Hwang-Ju Jeon 1, Kyeongnam Kim 1, Chaeeun Kim 2, Myoung-Jin Kim 1, Tae-Oh Kim 3,4,5,† andSung-Eun Lee 1,2,*,†
Abstract:Melanogenesis sawv cev rau ntau cov txheej txheem uas tsim melanin, tiv thaiv tawv nqaij (tawm tsam ultraviolet rays), thiab txiav txim siab tib neeg cov tawv nqaij xim. Cov tshuaj txo qis melaninproduction yeej ib txwm muaj nyob rau hauv kev thov nyob rau hauv lub lag luam kom hniav zoo nkauj vim hais tias ntawm kev tu tawv nqaij nyiam, xws li whitening. Lub ntsiab mechanism rau inhibiting melanin ntau lawm yog inhibition ntawm tyrosinase (TYR), ib tug tseem ceeb enzyme rau melanogenesis. Ntawm no, peb ntsuas qhov tseeb (Ged), tus neeg sawv cev limonoid, rau nws cov tshuaj tiv thaiv melanogenesis. Melanin ntau lawm hauv vitro tau txhawb nqa los ntawm alpha-melanocyte-stimulating hormone (-MSH) hauv B16F10 nas melanoma hlwb. Ged txo -MSH-stimulatedmelanin ntau lawm, inhibiting TYR kev ua thiab cov protein ntau. Peb tau lees paub qhov tshwm sim hauv vivo ina zebrafish qauv rau melanogenesis. Tsis muaj cov cim qhia txog kev toxicity thiab malformation ntawm zebrafishembryos thaum lub sij hawm kev loj hlob nyob rau hauv tag nrho cov kho concentrations. Ged txo tus naj npawb ntawm tsim zebrafish embryo pigment dots thiab melanin ntsiab lus ntawm embryos. Lub siab ua haujlwm siab ntawm Ged (100 µM) qis dua qhov kev tswj zoo, kojic acid (8 mM). Yog li ntawd, Ged tuaj yeem ua tus neeg sib tw nyiam rau cov tshuaj tiv thaiv melanogenesis.
Cov ntsiab lus: MITF; microphthalmia cuam tshuam nrog kev hloov pauv; TYR; tyrosinase; DQ; dopaquinone; TRP-1; tyrosinase-related protein 1; TRP-2; tyrosinase-related protein 2; MC1R; melanocortin 1 receptors; - MSH; - melanocyte-stimulating hormone; ACTH; adrenocorticotropic tshuaj hormones; ASP; agoutisignaling cov protein; cAMP; cyclic adenosine monophosphate; CREB; cAMP cov ntsiab lus teb
1. Taw qhia
Melanin yog synthesized nyob rau hauv melanosome, lysosome zoo li organelles ntawm melanocyte.Nyob rau hauv lub melanosome, melanin yog tsim nyob rau hauv ob pigments, eumelanin, thiab pheomelanin, sawv cev rau xim av-dub thiab liab-daj, raws li [1]. Lub synthesis ntawm cov melaninpigments pib los ntawm precursor, L-tyrosine, nrog oxidation cov tshuaj tiv thaiv ntawm L-tyrosine rau hauv acompound hu ua dopaquinone (DQ), thiab L-DOPA [2]. Hauv cov kauj ruam oxidation no, tyrosinase (TYR) ua raws li tus nqi txwv enzyme ntawm tag nrho cov melanin biosynthesis txoj kev [2,3]. TRP-1thiab TRP-2, ze ze rau cov proteins melanogenesis, catalyze eumelanin biosynthesis txoj hauv kev thiab stabilize thiab induce TYR kev ua [1]. Cov txheej txheem ntawm kev tsim melaninpigments (hu ua melanogenesis) tshwm sim nyob rau hauv melanosome ntawm melanocytes, ib qhov chaw formelanin synthesis thiab cia [4]. Nyob rau hauv lub melanogenesis signaling pathway, melanocortin1 receptor (MC1R) yog ib tug tswv cuab ntawm G-protein-coupled receptors. MCIR yog qhov pib taw qhia rau melanogenesis signaling thiab yog qhib los ntawm MC1R agonists zoo li ib tug -melanocyte-stimulatinghormone (-MSH), adrenocorticotropic hormone (ACTH), thiab agouti signaling protein (ASP) [1,2]. Kev ua kom muaj zog ntawm -MSH-MC1R kev taw qhia txoj hauv kev nce cyclic adenosinemonophosphate (cAMP) concentration thiab phosphorylates lub cAMP cov ntsiab lus teb (CREB). Phosphorylated CREB induces protein theem ntawm microphthalmia-associatedtranscription factor (MITF), ib tug transcription factor tswj melanin-synthesis-relatedgenes, TYR, tyrosinase-related protein-1 (TRP-1), thiab tyrosinase-related protein. -2 (TRP-2) los ntawm kev khi M-box ntawm cov noob no [1,5,6]. Ntawm tsib melanocortin receptors, MC1R muaj ntau tshaj melanocytes. Nws feem ntau tswj hwm eumelanin (dub-xim av xim) ntau lawm los ntawm kev ua kom MC1R thaum cov receptor no agonists, xws li a-MSH thiab ACTH, hloov mus rau cov receptor no [2,4,7].
Txawm hais tias muaj qhov sib txawv ntawm cov tsiaj nyeg thiab ntses, zebrafish muaj cov ntawv luam tawm ntawm MC5R thiab MC3R, uas cov ntses pufferfish tsis muaj [7], thiab txoj kev taw qhia no tseem muaj nyob rau hauv zebrafish thiab ua haujlwm raws nraim li nws ua hauv cov tsiaj melanocytes [7].Ntxiv mus, Tus naj npawb ntawm cov kev tshawb fawb ntawm inhibition ntawm melanin pigment tsim tau nce vim nws qhov kev tshawb fawb zoo, xws li kev loj hlob sai hauv cov embryos thaum ntxov thiab yooj yim ntawm kev soj ntsuam [8-11]. Hauv cov xwm txheej no, ntau qhov kev tshawb fawb yav dhau los tau pom tias qhov inhibition ntawm txoj kev taw qhia no txo qis melanin ntau lawm thiab pom muaj ntau yam inhibitors, suav nrog bisabolol-Angelone, 4-hydroxy-3-methoxy cinnamaldehyde, anddiphenyl-methylene-hydrazine carbo thiamine [12 — 14].
Gedunin (Ged), lub npe hu ua cua sov-shock protein 90 inhibitor, yog limonoid, pom nyob rau hauv Meliaceae tsob nroj genera. Cov limonoid no muaj ntau nyob rau hauv epicarp ntawm cov tub ntxhais hluas Indian neem ntoo (Azadirachta indica) txiv hmab txiv ntoo thiab muaj ntau yam kev ua ub no, xws li tshuaj tua kab mob, tshuaj tua kab mob, tshuaj tiv thaiv kab mob, tshuaj tua kab mob, tshuaj tua kab mob, tshuaj tua kab, tshuaj tua kab mob, thiab tshuaj tua kab mob [15–18]. Tsis muaj ntawv tshaj tawm txog cov txheej txheem ntawm kev tiv thaiv melanogeniceffect ntawm Ged.
Raws li cov tshuaj siv tshuaj los yog tus neeg sawv cev tshuaj pleev ib ce los kho cov kab mob melanin orskin hloov xim, Ged tej zaum yuav muaj qhov zoo vim tias nws muaj kev nyab xeeb, ib txwm muaj cov khoom cog [19]. Cov nyhuv anti-melanogenic ntawm Ged tau soj ntsuam hauv vitro thiab hauv vivo siv B16F10 nas melanoma hlwb thiab zebrafish embryos, ntsig txog, txhawm rau nrhiav tus neeg sawv cev zoo nkauj, yog ib qho ntawm cov khoom lag luam loj tshaj plaws ntawm kev lag luam tshuaj pleev ib ce.Simultaneously, los ntawm zebrafish, ib qho mob toxicity. kev sim kuj tau ua los qhia tias Ged tuaj yeem cuam tshuam rau tsiaj li cas.
2. Cov khoom siv thiab cov txheej txheem
2.1. Tshuaj thiab Reagents
Ged tau yuav los ntawm Microsource Discovery Systems, Inc. (Gayloardville, CT, USA).Melanogenesis stimulator, -MSH, anti-melanogenic compound, thiab kojic acid tau yuav los ntawm Sigma-Aldrich (St. Louis, MO, USA). Tag nrho lwm cov reagents tau siab dua qib molecular biology.
2.2. Cell Culture
Cov nas melanoma-carcinoma cell kab, B16F10, tau txais los ntawm American TypeCulture Collection (ATCC, Manassas, VA, USA) thiab kab lis kev cai nrog 10 feem pua fetal bovine serum(v/v), thiab penicillin muab Dulbecco-modified Eagle's media (GE Kev Noj Qab Haus Huv, Chicago, IL, USA) ntawm qhov chaw noo nrog 5 feem pua CO2. Cells tau coj mus kuaj kom txog thaum confluency ncav cuag 80 feem pua thiab muab faib peb zaug hauv ib lub lis piam nrog kev faib piv ntawm 1: 8. Cells tau soj ntsuam txhua txhua 12 teev, thiab kev hloov morphological raug kuaj xyuas. Tus lej ntawm cov hlwb siv tau khaws cia me me.
2.3. Cell Viability Assay
Txhawm rau soj ntsuam cov txiaj ntsig kev loj hlob ntawm Ged ntawm B16F10 nas melanoma cell kab, MTS kev soj ntsuam tau ua raws li cov txheej txheem qhia yav dhau los [20] siv CellTiter96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA). Luv luv, 1 × 103 hlwb tau noob nyob rau hauv txhua lub qhov dej ntawm 96-zoo phaj thiab incubated rau 24 h kom rov zoo. Tom qab incubation, Ged tau kho nrog ntau yam concentrations: 0, 3.12, 6.25,12.5 25, 50, thiab 75 µM. Tom qab 48 teev, 20 µL ntawm MTS kev tshuaj ntsuam xyuas tau ntxiv rau txhua qhov muaj 100-µL media nrog Ged thiab tsis muaj Ged. Tom qab ib qho ntxiv incubation rau 4 h, absorbance tau ntsuas ntawm 490 nm siv Multiskan GO spectrophotometer (ThermoScientific, Waltham, MA, USA). Cov ntaub ntawv absorbance tau normalized nrog kev tswj thiab sawv cev raws li qhov feem pua inhibition piv.
2.4. Kev txiav txim siab ntawm Melanin Cov ntsiab lus
Extracellular thiab intracellular melanin cov ntsiab lus tau txiav txim siab raws li kev hloov kho yav dhau los qhia [21]. -MSH (200 nM) pretreated melanoma hlwb raug incubated rau 72 h nrog thiab tsis muaj kojic acid (200 µM) thiab Ged (25 thiab 50 µM). Tom qab incubation, phenol-liab dawb kab lis kev cai xov xwm raug sau, thiab kab lis kev cai hlwb raug sau nrog RIPA lysis tsis. Cov noob qoob loo tau centrifuged ntawm 4 ◦C, 13, 000 rpm rau 15 min thiab pellets tau yaj hauv 1 M NaOH, 10 feem pua DMSO tov ntawm 95 ◦C rau 2 h. Lub absorbance ntawm cov ntaub ntawv sau thiab yaj melanin tau ntsuas ntawm 470 nm siv lub spectrophotometer. Tag nrho cov qauv raug normalized nrog lawv cov protein concentration txiav txim raws li BCA txoj kev.
2.5. Intracellular Tyrosinase Activity Assay
Cellular tyrosinase kev ua haujlwm tau txiav txim siab los ntawm kev ntsuas oxidation tus nqi ntawm L-dihydroxyphenylalanine (L-DOPA) raws li tau hais dhau los, nrog kev hloov kho me ntsis [22] .B16F10 nas melanoma hlwb raug pretreated nrog 200 nM ntawm -MSH rau 1 h ua raws li kev kho mob nrog 200 µM. kojic acid, nrog thiab tsis muaj Ged (25 thiab 50 µM), thiab incubated rau 72 h. Tom qab incubation, hlwb raug sau nrog lysis tsis muaj proteinaseinhibitor, thiab cov qauv yog centrifuged ntawm 13, 000 × g, 4 ◦C rau 15 min. Lub supernatant twb pauv mus rau lub raj tshiab. Txhua tus qauv (20 µL) thiab 80 µL ntawm 40 mM L-DOPA tau muab sib xyaw rau hauv 96- lub phaj zoo thiab incubated ntawm 37 ◦C rau 2 h. Absorbance ntawm 475 nm los ntawm oxidatedL-DOPA tau txiav txim siab siv lub microplate nyeem ntawv. Tus nqi absorbance yog normalized nrog cov protein concentration ntawm txhua tus qauv.
Cistanche extract txiaj ntsig: inhibits tyrosinase qhia.
2.6. RNA Isolation thiab qRT-PCR
Tag nrho RNA kev rho tawm thiab qRT-PCR tau ua tiav raws li ib txoj hauv kev tau piav qhia yav dhau los los ntawm [18]. Tag nrho RNA tau luv luv los ntawm txhua tus qauv siv Trizol reagent (Ambion, Austin, TX, USA). Extracted tag nrho RNA concentration tau txiav txim los ntawm absorbance ntawm 260 nm, thiab qhov zoo ntawm tag nrho RNA tau soj ntsuam nrog ib tug absorbance piv ntawm 260:280 thiab 260:230 nm siv Multiskan GO microplate nyeem ntawv (Thermo Scientific). TotalRNA (5 µg) tau muab tso rau hauv cDNA nrog Maxima thawj-strand cDNA synthesis kit (Thermo Scientific) thiab synthesized cDNA. mRNA qhia theem ntawm Mitf, Tyr, Trp-1, thiab Trp-2 tau txiav txim siab siv Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) thiab QuantStudio 3 Real-Time PCR instrument ( Applied Biosystems, Foster City, CA, USA) raws li tus kws qhia ntawv raws tu qauv. Kev qhia theem ntawm cov noob tau raug kho nrog glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
2.7. Immunoblot Analysis
Immunoblot tsom xam tau ua raws li cov txheej txheem yav dhau los tau piav qhia [23] .Proteins ntawm B16f10 hlwb tau sau los ntawm Ceti lysis buffer (Translab, Daejeon, Kauslim), thiab cov concentration tau txiav txim siab nrog Pierce BCA protein assay kit (Thermo Scientific). Ib qho sib npaug ntawm cov protein (30 µg) ntawm txhua tus qauv raug thauj mus rau sodiumdodecyl sulfate-polyacrylamide gel thiab electrophoresed. Tom qab sib cais, cov proteins raug xa mus rau ib daim nyias nyias ntawm nitrocellulose, thiab daim nyias nyias raug thaiv hauv 10 feem pua cov kua mis uas tsis yog-fatskim rau 2 teev. Blocked membranes tau incubated nrog thawj antibody nyob rau hmo ntuj ntawm 4 ◦C, tom qab los ntawm lwm incubation nrog lwm yam antibody ntawm 26 ◦C rau 2 h. Thawj thiab theem nrab cov tshuaj tiv thaiv tau diluted hauv qhov piv ntawm 1: 1000 thiab 1: 3000, feem. Thaum kawg, blotted daim nyias nyias tau sau tseg siv ChemiDoc (Bio-Rad, Hercules, CA, USA) nrog ECL reagent (SuperSignal, Thermo Scientific). Thawj thiab theem nrab antibodies tau yuav los ntawm Santa Cruz Biotechnology (Dallas, TX, USA).
2.8. Zebrafish Embryo Test
Wild-type zebrafish were thankfully obtained from Professor Tae-Lin Huh, the School of Life Science and Biotechnology, Kyungpook National University, Daegu, Republic of Korea. The strain was kept for more than eight generations in a laboratory condition of 26 ◦C ± 1 ◦C, and a day-to-night ratio of 8:16. General fish care and breeding conditions, as previously described, were followed [24]. Ten groups of zebrafish were mated to obtain embryos with a mating ratio (male: female, 1:2), and among obtained embryos, healthy embryos had >80 feem pua fertilization tus nqi thiab raug xaiv thiab siv rau kev sim. Kaum ob embryos noj qab nyob zoo nyob rau hauv txhua pab pawg neeg kho tau nyob rau hauv txhua lub qhov dej ntawm ib tug 96- zoo phaj nrog thiab tsis muaj Ged (25, 50, 75, thiab 100 µM) thiab kojic acid (8 mM) nyob rau hauv E3 media ntawm lub shield theem. 24 teev tom qab fertilization (h / pf), embryos tau decorionated, thiab qhov txawv txav tau kuaj xyuas txhua 24 teev txog 72 h / pf. Lub embryos 'phenotype tau yees duab nyob rau sab nraud thiab dorsalview los sib piv qhov sib txawv ntawm cov pab pawg siv BX53 upright microscope nrog DP80CCD (Olympus Life Science Solutions, Waltham, MA, USA). Tom qab picturing, embryos tau homogenized nrog Ceti lysis buffer (TransLab), thiab kev txiav txim siab ntawm melanin concentration tau ua, uas yog sib npaug rau B16F10 cell melanin kev txiav txim siab cov ntsiab lus.
2.9. Kev txheeb cais
Txhua qhov kev txheeb xyuas txheeb cais tau nthuav tawm hauv txoj kev tshawb no tau ua tiav siv GraphPad Prismv.8.0 (GraphPad Software, San Diego, CA, USA). Ib txoj kev ANOVA nrog Tukey qhov kev sim tau siv rau ntau qhov sib piv. Tag nrho cov ntaub ntawv yog sawv cev raws li qhov nruab nrab ± tus qauv sib txawv. Qhov sib txawv tseem ceeb tau suav tias yog qhov tseem ceeb ntawm p <>
3. Cov txiaj ntsig
3.1. Cytotoxic nyhuv ntawm Piperlongumine hauv B16F10 Cells
Ua ntej tshawb xyuas cov nyhuv anti-melanogenic ntawm Ged, peb tau ua qhov kev kuaj pom muaj zog hauv B16F10 kab xov tooj ntawm tes los teeb tsa cov koob tshuaj hauv qhov kev tshawb fawb no. Nyob rau hauv lub concentration ranges ntawm 3.12 mus rau 50 µM, hlwb tsis qhia ib tug inhibitory nyhuv ntawm kev loj hlob, thiab lawv cov txheeb ze proliferation yog los ntawm 91.0 feem pua rau 118.7 feem pua (Daim duab 1). Hauv qhov sib piv, txawm hais tias tsis muaj qhov tseem ceeb, 75 µM-kho pab pawg muaj lawv txoj kev loj hlob me ntsis inhibited (89.3 feem pua ; Daim duab 1). Yog li ntawd, qhov siab tshaj plaws concentration tau teem rau 50 µM nyob rau hauv seem ntawm tes-lineexperiments.
Daim duab 1. Cytotoxicity ntawm gedunin (Ged) hauv B16F10 hlwb
3.2. Gedunin Inhibits Melanin Production thiab Intracellular Tyrosinase Kev Ua Haujlwm hauv B16F10 MouseMelanoma Cells
Peb tau soj ntsuam cov nyhuv anti-melanogenic ntawm Ged hauv B16F10 nas melanoma hlwb. TheMC1R agonist, -MSH, zoo induced intracellular thiab extracellular melanin ntau lawm (Daim duab 2a–c). Cov xim ntawm cov ntaub ntawv sau tau tsaus nrog -MSH (Daim duab 2a). Cov xim tau ploj mus nrog qhov sib ntxiv ntawm Ged, thiab cov nyhuv yog nyob ntawm koob tshuaj (Daim duab 2a). Nyob rau hauv cov ntsiab lus ntawm tag nrho cov ntsiab lus melanin, -MSH nce melanin ntau lawm hauv melanocytes kwv yees li xya-fold siab dua li pawg tswj hwm thiab txhawb nqa melanin txo qis los ntawm kev ntxiv kojic acid, paub zoo tswj hauv melanogenesis (Daim duab 2d). Ged kuj txo qis stimuli-inducing melanin ntau lawm hauv melanocytes ntawm tag nrho cov concentrations testedin txoj kev tshawb no (Daim duab 2d). Ntxiv mus, hauv kev sim tyrosinase kev ua ub no, Ged tau pom tias muaj zog tiv thaiv kev cuam tshuam ntawm tus nqi txwv enzyme, tyrosinase, hauv melanogenesis (Daim duab 2e). Kev ua haujlwm ntawm tyrosinase hauv 50 µM Ged kho pab pawg tau txo qis los ntawm 20 feem pua thiab 12.24 feem pua piv rau cov tswj thiab a-MSH-stimulated pab pawg, feem.
Daim duab 2. Anti-melanogenic nyhuv ntawm tiag (Ged) ntawm alpha-melanocyte-stimulating hormone (-MSH)-stimulated melanin ntau lawm hauv B16F10 hlwb.
3.3. Gedunin Txo -MSH Induced Melanogenesis-Related Gene Expression
-MSH (200nM) induced mRNA qib ntawm Mitf; melanogenesis transcription factor; thiab lub hom phiaj genes Tyr, Trp-1, thiab Trp-2 (Daim duab 3). Kev kho mob ntawm Ged txo qis mRNA qib ntawm tag nrho cov noob nyob rau hauv ib koob tshuaj raws li qhov concentration ntawm 25 thiab 50 µM (Daim duab 3).Piv rau qhov zoo tswj, kojic acid (200 µM), Ged tau zoo dua los txo cov mRNA. theem ntawm melanogenesis ntsig txog cov noob hauv B16F10 hlwb. Tsis tas li ntawd, theem ntawm Mitfand Tyr poob los ntawm 0.10- thiab 0.26-fold qis dua -MSH induced cells, raws li, ntawm qhov concentration ntawm 50 µM (Daim duab 3a,b). Down-regulated mRNA theem ntawm cov noob no ua rau theem ntawm Tyr thiab txo cov TYR ntau lawm ntawm melanin tsawg dua li pawg tswj.
Daim duab 3. Qhov cuam tshuam ntawm kev txo qis ntawm melanogenesis ntsig txog cov noob.
3.4. Gedunin Txo TYR thiab TYR-1 Tus Nqi
TYR plays lub luag haujlwm tseem ceeb hauv melanogenesis, thiab qib protein ntawm cov enzyme no cuam tshuam rau melanogenesis ncaj qha. Peb tau sim lees paub qhov kev hloov pauv ntawm cov protein, TYR, ua rau txo qis mRNA qib los ntawm kev tshuaj ntsuam immunoblot. TYR tau pom zoo txo qis piv nrog kev tswj hwm, thiab -MSH tau kho cov koob tshuaj raws li qhov xav tau (Daim duab 4a). Tsis tas li ntawd, TRP-1, qhov tseem ceeb ntawm kev hloov pauv ntawm TYR, tau txo qis me ntsis (Daim duab 4a). Cov txiaj ntsig tau pom tseeb dua thaum txhua cov protein cov qhab yog normalized los ntawm lub tsev khaws cov protein, GAPDH (Daim duab 4b, c).
Daim duab 4. Western blotting ntawm alpha-melanocyte-stimulating hormone (-MSH) induced B16F10 hlwb nrog txoj kev kho.
3.5. Toxicity ntawm Gedunin tawm tsam Zebrafish hauv Early-Stage Development
Kev soj ntsuam toxicological ntawm Ged ntawm zebrafish thaum ntxov-theem kev loj hlob tau ua ua ntej ntsuas kev ntsuas tshuaj tiv thaiv melanogenic ntawm Ged hauv vivo. Morphological abnormality ntawmzebrafish embryos tsis tau pom nyob rau hauv ib qho kev kho concentration ntawm Ged (Daim duab 5a). Kev kuaj ntshav siab ntawm Ged, 25, 50, 75, thiab 100 µM, Ged-kho pab pawg muaj sia nyob tsis muaj qhov sib txawv tseem ceeb ntawm 72 h ntawm lub sijhawm kho tshuaj (Daim duab 5b).
Daim duab 5. Toxicity ntawm tiag (Ged) ntawm zebrafish thaum ntxov-theem kev loj hlob.
3.6. Gedunin Txo Melanin Cov ntsiab lus hauv Zebrafish Embryos
Vim hais tias Ged tsis muaj kev loj hlob toxicity nyob rau hauv zebrafish embryos, peb tau soj ntsuam theanti-melanogenic nyhuv ntawm ib tug series ntawm concentrations ntawm Ged, 25, 50, 75, thiab 100 µM. Hauv kev soj ntsuam theoptical, peb tshawb xyuas cov dots ntawm pigment zebrafish; lub overhead sab saib ntawmzebrafish embryos yog qhia nyob rau hauv daim duab 5a. Kojic acid muaj cov nyhuv inhibition ntawm pigmentproduction ntawm zebrafish embryo. Ged-kho zebrafish embryos kuj muaj tsawg dua pigmentdots ntawm lub taub hau (Daim duab 6a). Tsis tas li ntawd, tag nrho cov ntsiab lus melanin muab rho tawm los ntawm tag nrho lub cev ntawm zebrafish embryos txo qis tom qab Ged raug rau 72 h hauv kojic acid, apositive tswj, thiab Ged-kho pab pawg (Daim duab 6b, c)
Daim duab 6. Cov nyhuv ntawm qhov tseeb ntawm melanin ntau lawm hauv vivo.
4. Kev sib tham
Hauv txoj kev tshawb no, inhibition zog ntawm Ged tiv thaiv melanin ntau lawm raug soj ntsuam.Raws li peb cov kev tshawb pom, qhov inhibition muaj peev xwm ntawm Ged yog kwv yees li 11. 50 µM) qis dua li ntawm kojic acid (200 µM), kev tswj zoo. Nws tau txuam nrog kev ua haujlwm tsis muaj zog ntawm intracellulartyrosinase, ib qho enzyme uas hloov L-Dopa rau DQ, thawj tus thawj coj ntawm eumelanin thiab pheomelanin. Kev ua haujlwm Tyr txo qis ua rau qeeb ntawm tag nrho cov txheej txheem tsim khoom melanin thiab qhov tsis txaus ntawm ob qho khoom kawg, eumelanin thiab pheomelanin [1-3]. Cov ntawv sau tseg thiab TYR qib tau tswj hwm los ntawm ntau cov ntawv sau npe: Mitf, Trp-1, thiab Trp-2 [1,2]. Qhov txo qis ntawm cov protein thiab mRNA qib txhais tau hais tias qhib MC1R ntawm -MSH-MC1R-PKA-CREB signaling axis los ntawm -MSH raug downregulated nrog kev kho mob ntawm Ged ntawm ib tug concentration ntawm 25 thiab 50 µM, koob tshuaj-dependently nyob rau hauv ob quantitative PCR thiab Western blotting. Raws li tau piav qhia dhau los, kev txo qis ntawm MITF ua rau muaj qhov tsis txaus ntawm Tyr, Trp-1, thiab Trp-2 los ntawm kev cuam tshuam tsawg dua rau thaj tsam txhawb nqa ntawm cov noob no [25–27]. Txo cov kev hloov pauv no ua rau TYR mus rau theem qis thiab inhibits melanin pigment ntau lawm hauv B16F10 hlwb. Tsis tas li ntawd, cov txiaj ntsig tam sim no qhia tau tias TYR inhibitory nyhuv ntawm Ged, lwm qhov tseem ceeb ntawm melanin ntau lawm hauv melanocyte. Ged kuj pom tias muaj kev cuam tshuam rau pigmentproduction thaum lub sij hawm zebrafish thaum ntxov-theem kev loj hlob nyob rau hauv lub vitro thiab nyob rau hauv vivo qauv.Our cov kev tshwm sim pom tau hais tias kev kho mob nrog Ged rau 72 h markedly txo zebrafish embryopigmentation. Tag nrho cov ntsiab lus melanin thiab mRNA qib ntawm cov noob muaj feem cuam tshuam nrog kev muaj Ged, thiab qhov nyiam ntawm cov txiaj ntsig tau txhawb nqa cov cuab yeej vitroanti-melanogenesis ntawm Ged.
Ntxiv mus, lub peev xwm inhibition ntawm Ged muaj zog ntau dua kojic acid, ib qho ntawm lawv feem ntau paub cov tebchaw ua cov tshuaj pleev xim dawb hauv kev lag luam kom hniav zoo nkauj [6,28].Qhov concentration ntawm uas Ged ua tau qis dua kojic acid. Tsis tas li ntawd, ib daim ntawv tshaj tawm yav dhau los tau qhia tias lwm cov tshuaj ntxuav hniav dawb, arbutin, muaj cov tshuaj tiv thaiv melanogenic zoo ib yam li kojic acid [2]. Yog li ntawd, Ged yuav tsum raug suav hais tias yog ib qho tshuaj dawb hauv kev hloov pauv ntawm cov tam sim no siv arbutin lossis kojic acid, thiab ua ib qho tshuaj tiv thaiv melanomadrug txog nws cov khoom cog lus.
Anti-melanogenic cov khoom ntawm Ged tau hais ua ntej [29,30], tab sis cov kev tshawb fawb no tsis tau tsom mus rau cov txheej txheem tshwj xeeb ntawm cov nyhuv, thiab lawv tsuas pom kev hloov pauv ntawm cov teebmeem cytotoxic thiab melanin ntau lawm nyob rau hauv vitro, thaum muaj kev tiv thaiv kev loj hlob thiab inhibitory cuam tshuam rau. HSP 90 qhia tau hais txog [31–33]. Qhov ua tau ntawm Ged raws li tus neeg sawv cev tiv thaiv melanogenetic tau raug tshaj tawm; Txawm li cas los xij, cov kev tshawb fawb no tsis tau tsom mus rau cov txheej txheem tshwj xeeb ntawm cov nyhuv, thiab lawv tsuas pom kev hloov pauv ntawm cov teebmeem cytotoxic thiab melanin ntau lawm hauv vitro. Peb sim txiav txim siab txog qib cellular mechanism los ntawm kev lees paub cov qib mRNA thiab cov protein ntau ntawm cov noob qoob loo uas cuam tshuam txog melanin. Ua ke nrog cov ntaub ntawv dhau los, txoj kev tshawb fawb no tau pom qhov kev xav tshiab ntawm Gedas ib feem ntawm cov khoom xyaw kom zoo nkauj rau ob qho txiaj ntsig, zoo heev anticancer thiab anti-melanogenesis, uas yuav siv tau rau ob qho tib si UV-induced melanoma thiab tsub zuj zuj ntawm pigment, tshwj xeeb tshaj yog nyob rau tib lub sij hawm.
cistanche extract hmoov: ntshiab dawb radicals
5. Cov lus xaus
Hauv kev xaus, tag nrho cov txiaj ntsig no tau pom qhov sib xyaw tshiab, Ged, tuaj yeem siv los ua cov tshuaj tua kab mob rau melanin biosynthesis, uas txo qis qis qis qis proteinsTYR, TRP-1, thiab TRP-2 los ntawm tus nqi txo piv rau tus tswv. Kev tswj hwm cov protein, MITF, hauv ob qho tib si hauv vitro thiab hauv vivo systems ntawm zebrafish qauv.
cistanche ntsiav tshuaj
