Ntu 2: Kev Tiv Thaiv Kab Mob, Antioxidant, Moisturizing, Thiab Antimelanogenesis Los Ntawm Quercetin 3-O- -D-Glucuronide hauv Tib Neeg Keratinocytes Thiab Melanoma Cells Ntawm Kev Ua Kom NF-κB Thiab AP -1 Pathways

Yog xav paub ntxiv, hu rautina.xiang@wecistanche.com

Nyem qhov link mus kawm part1:https://www.xjcistanche.com/news/part-1-anti-inflammatory-antioxidant-moistu-55118257.html

3. Kev sib tham

Hauv kev tshawb fawb yav dhau los, Q-3-G tau siv los tiv thaiv pob ntseg edema, peritoneal permeability, thiab pulmonary edema [50], ua lub zogantioxidantNyob rau hauv cov ntshav plasma nrog tsawg-density lipoprotein [42], txo qis peroxynitrite-induced oxidative modification [51], exerting ananti-inflammatorycuam ​​tshuam los ntawm inhibiting JNK thiab ERK signaling txoj kev nyob rau hauv LPS kev sib tw [52] thiab muaj anticancer zog tiv thaiv lub mis mob hlwb [56]. Txawm li cas los xij, tsis zoo li nws cov niam txiv tau kawm dav dav,quercetin, cov txiaj ntsig tshuaj, thiab cov txheej txheem ntawm Q-3-G tseem yuav tau tshawb xyuas. Rau qhov zoo tshaj plaws ntawm peb txoj kev paub, lub luag haujlwm ntawm Q-3-G ntawm kev tiv thaiv daim tawv nqaij tsis tau qhia meej meej. Hauv txoj kev tshawb no, siv ntau yam kev ntsuam xyuas hauv vitro, peb tau tsom mus rau tus yam ntxwv ntawm kev tiv thaiv ntawm daim tawv nqaij ntawm Q-3-G tiv thaiv UVBor H2O2,-induced oxidative stress thiab o, tawv nqaij hydration hauv HaCaT (ib tug tib neeg keratinocyte cell kab), thiabantimelanogenesiskev ua haujlwm hauv B16F10 (ib kab murine melanoma cell kab).

Kev ua tau zoo ntawm HaCaT, B16F10, thiab HEK293T hlwb tau txiav txim siab tom qab kev kho mob nrog Q-3-G dhau ntawm ntau yam tsis-cytotoxic concentrations. Cov txiaj ntsig tau pom tias tsis muaj qhov cuam tshuam tseem ceeb ntawm cell viability nyob rau hauv HaCaT, B16F10, thiab HEK293T hlwb tshaj qhov ntau ntawm Q-3-Gup rau 20 μM (Figures 2a,3a, thiab 4f). Lub cell viability nyob rau hauv HaCaT, B16F10, thiab HEK293T hlwb ntawm qhov ntau ntawm O-3-G mus txog 20 μM tau pom tias qis dua qhov concentration ntawm quercetin thaum nws siv los tshawb xyuas cov teebmeem cytotoxic hauv SCC{{18} } thiab HSC{19}} hlwb (50 μM) 【57】 los yog phev viability (50-100μM) 【58】. Txawm li cas los xij, Q-3-G tau pom tias muaj tshuaj lom tsawg dua piv rau nws cov aglycone [59]. Qhov tsis muaj pab pawg OH dawb ntawm peb txoj haujlwm tuaj yeem ua rau muaj kev phom sij tsawg dua ntawm Q-3-G [29].

UVB rays nrog lub wavelength ntawm 280-315 nm nkag mus rau sab sauv txheej ntawm daim tawv nqaij thiab zoo dua li UVA thiab UVC[60]. UVB rays yog vim li cas rau feem ntau cov qog nqaij hlav ntawm daim tawv nqaij thiab cell tuag. Tsis tas li ntawd, kev puas hlwb hauv HaCaT hlwb tau pom tias raug ntxias los ntawm UVB irradiation [61]. Yog li ntawd, peb tau tshawb xyuas lub cell muaj peev xwm ntawm HaCaT hlwb tom qab UVB irradiation piv nrog Q-3-G kho. Raws li tau pom nyob rau hauv cov ntaub ntawv dhau los, ib qho ntawm cov ntsiab lus tseem ceeb uas ua rau cov hlwb puas, cov ntaub so ntswg, thiab lub cev yog UVB raug [62,63]. Q-3-G rov qab kho qhov txo qis ntawm cov cell viability tshwm sim los ntawm UVB irradiation (Daim duab 2b-d). Cov txiaj ntsig tau pom tias zoo ib yam li cov kev tshawb fawb dhau los ntawm cov nyhuv thiab cov txheej txheem ntawm UVB-induced cytotoxicity hauv HaCaT ntawm quercetin [64].

Nyem rau nws kom tau txais cov khoom lag luam ntau cov ntaub ntawv

Ntau qhov kev tshawb fawb tau piav qhia tias UVB tuaj yeem txhawb cov lus teb mob hnyav, ua rau muaj teeb meem ntawm daim tawv nqaij [1,2,4,27].UVB ua kom cov enzymes tseem ceeb xws li COX-2 thiab cytokines xws li TNF- . COX-2 yog ib qho enzyme uas muaj feem xyuam nrog kev mob uas tshwm sim los ntawm cov cytokines pro-inflammatory cytokines thiab cov neeg nruab nrab [65], uas txhawb cov inflammatory teb. Vim tias COX-2 tsim cov inflammatory mediator PGE-2, nws induces ib tug inflammatory teb [66,67], thiab TNF- yog qhov loj pro-inflammatory cytokine [68] nyob rau hauv cov lus teb inflammatory. Cov lus teb inflammatory no ua rau cov tawv nqaij puas, uas ua rau cov tawv nqaij laus [69]. mRNA qhia theem ntawm cov inflammatory enzyme COX-2 thiab cov pro-inflammatory cytokines TNF- tau inhibited los ntawm Q-3-G (Daim duab 2e,). Raws li cov txiaj ntsig tau tshaj tawm yav dhau los, quercetin kuj txo qis cov noob qhia thiab kev tsim tawm ntawm TNF- [70]. Hais txog cov khoom antioxidant, DPPH thiab ABTS radical scavenging assays yog ceev, txhim khu kev qha, thiab rov tsim dua txoj hauv kev rau kev ntsuam xyuas hauv vitro.kev ua haujlwm antioxidantntawm cov ntshiab tebchaw thiab cov nroj tsuag rho tawm [54] thiab tau siv dav hauv ntau txoj hauv kev los tshawb xyuas cov kev ua ub no ntawm cov tebchaw ntuj lossis cov extracts [1,46,71,72]. Q-3-G tau pom los txo cov reactivity ntawm radicals nyob rau hauv ob qho tib si kev soj ntsuam thiab ntws cytometry nyob rau hauv concentration-dependent yam ntxwv. Cov txiaj ntsig no yog ua raws li kev tshawb fawb yav dhau los, uas Q-3-G cuam tshuam qhov tsim ntawm ROS hauv pheochromocytoma PC-12 hlwb [34]. Dhau li ntawm kev ua tshuaj lom neeg nrog cov nyhuv antioxidant, peb tau pom tias Q-3-G nce qib ntawm Nrf2, yog ib qho tseem ceeb ntawm kev tswj hwm ntawm oxidative kev nyuaj siab hauv hlwb. Ob peb lub tebchaw lossis cov khoom siv ntuj tsim tau raug tshaj tawm tias muaj cov khoom tiv thaiv antioxidant los ntawm Nrf2- nyob ntawm ARE signaling [46,73,74]. Ua ke, cov kev tshawb pom no qhia tias Q-3-G muaj cov tshuaj tiv thaiv antioxidant (Daim duab 2g-j). Melanosome los ntawm melanin synthesis tshwm sim hauv intracellular organelles [75-77]. Kev tsim tawm ntau dhau ntawm melanin tuaj yeem cuam tshuam rau cov tawv nqaij tshwm sim thiab cov kab mob ntawm daim tawv nqaij hyperpigmentation. Peb pom tias kev kho nrog O-3-G hauv c -MSH-induced B16F10 hlwb inhibited melanin secretion. Ib yam li hauv kev ua haujlwm dhau los, intracellular melanogenesis tseem tswj tau los ntawm quercetin metabolites thiab qee cov quercetin-3-O- -D-glucopyranosides [24,78,79].

Kev tuav cov tawv nqaij noj qab haus huv yuav tsum muaj cov tawv nqaij txaus, thiab NMFs thiab HA muaj lub luag haujlwm tseem ceeb hauv cov txheej txheem no [12]. FLG yog ib feem ntawm daim tawv nqaij barrier, yog li qhov nce qib ntawm FLG qhia yuav cuam tshuam nrog moisturizing [80]. Hauv peb txoj kev tshawb fawb, Q-3-G tau pom tias cuam tshuam rau ntawm daim tawv nqaij kom cov dej noo los ntawm kev nce FLG thiab TGM-1 (Daim duab 3c,d). Cov kev qhia theem ntawm FLG thiab TGM-1 raug txwv los ntawm JNK, IKK , thiab IkBainhibitors (SP600125 thiab Bay11-7082, raws li). Peb tsom mus rau MAPK-txog enzymes vim tias peb cov kev tshawb fawb tsom los txiav txim siab cov txheej txheem molecular uas Q-3-Gexerts nws cov teebmeem tiv thaiv tawv nqaij. JNK plays ib qho tseem ceeb hauv oxidative-stress-induced keratinocyte apoptosis [81]. Lub phosphorylation ntawm c-Jun, TAK1, MKK4, thiab INK tau nce los ntawm Q-3-G. Tsis tas li ntawd, retinol tsis tau nce phosphorylation ntawm c-Jun, TAK1, MKK4, lossis JNK, qhia tias muaj qee qhov sib txawv ntawm cov yam ntxwv inhibitory ntawm Q-3-G thiab retinol hauv AP-1 kev taw qhia txoj kev. Raws li hauv cov txiaj ntsig tau tshaj tawm yav dhau los, JNK-c-Jun/AP-1 txoj hauv kev yog cuam tshuam nrog cov nyhuv anti-apoptotic ntawmquercetin[82]. Piv nrog rau txoj kev tshawb fawb dhau los [83], los ntawm kev tswj hwm NF-kB thiab AP -1 cov kev taw qhia, cov kev no kuj tau pom tias muaj feem cuam tshuam rau hauv lub hauv paus txheej txheem ntawm quercetin. Nyob rau hauv txoj kab nrog cov txheej txheem ntawm nws cov niam txiv sib xyaw, peb cov txiaj ntsig tau pom tias txoj hauv kev NF-kB kuj tseem suav nrog kev tiv thaiv tawv nqaij ntawm Q-3-Gaby qhov ua kom p50, p65, IKK, IkB, Akt, thiab Src tom qab kho nrog Q-3-G hauv HaCaT hlwb. Tsis tas li ntawd, Q-3-G upregulated AP-1- mediated Luc thiab NF-B-Luc kev ua (Daim duab 4). Cov kev tshawb pom no qhia tias MAPK-mediated AP-1 thiab NF-kB signaling pab txhawb Q-3-G-mediated daim tawv nqaij tiv thaiv cov khoom.

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Khoom siv

Q-3-Gwas tau los ntawm Sigma Aldrich Co. (St.Louis, MO, USA).HaCaT, HEK293T, thiab B16F10 hlwb tau yuav los ntawm American Type Culture Collection (Rockville, MD, USA).ABTS diammonium ntsev, DPPH, ascorbic acid (AA), ABTS, thiab retinol tau yuav los ntawm Sigma Chemical Co.(St.Louis, MO, USA). Fetal bovine serum(FBS), Dulbecco's Modified Eagle Medium (DMEM), thiab phosphate-buffered saline (PBS) tau yuav los ntawm Gibco (Grand Island, NY, USA).3-(4-5-Dimethylthiazol{{ 7}}yl)-25-diphenyltetra-sodium bromide (MTT) tau yuav los ntawm Amresco (Brisbane, Australia). TRILzol thiab PCR premix tau yuav los ntawm Bio-D Inc. (Seoul, Kauslim). Cov khoom siv cDNA synthesis tau yuav los ntawm Thermo Fisher Scientific (Waltham, MA, USA). Forward thiab thim rov qab primers rau PCR (polymerase saw cov tshuaj tiv thaiv) tau tsim los ntawm Macrogen, Inc. (Seoul, Kauslim). Tag nrho cov tshuaj tiv thaiv kab mob ntsig txog cov phosphorylated lossis tag nrho cov ntaub ntawv ntawm lub hom phiaj protein tau yuav los ntawm Cell Signaling Technology (Beverly, MA, USA).

4.2.Cell Cultures

HaCaT thiab B16F10 hlwb tau coj mus kuaj hauv DMEM nrog 10 feem pua ​​​​FBS thiab 1 feem pua ​​​​cov tshuaj tua kab mob (streptomycin thiab penicillin). HEK293T hlwb tau coj mus kuaj hauv DMEM nrog 5% FBS thiab 1% tshuaj tua kab mob. Cov kab ntawm tes no tau tsim nyob rau hauv 5 feem pua ​​​​CO, ntawm 37 degree .

4.3. Cell Viability Tests

B16F10 hlwb tau cog rau ntawm 2 × 10 degree hlwb ntawm ib qhov dej hauv 96- cov phiaj zoo rau 24 h, qhov sib txawv ntawm qhov sib txawv ntawm Q-3-G(0-20 μM) tau ntxiv rau lawv, thiab ces lawv tau incubated rau 48 teev. HaCaT hlwb tau plated nyob rau hauv 96- zoo daim hlau, thiab cov hlwb tau noob ntawm 6 × 10 degree cell / mL. Tom qab incubation ib hmos, cov hlwb tau incubated nrog txawv concentrations ntawm Q-3-G ({14}} μM) rau 24 teev. HEK293T hlwb tau cog rau ntawm 6 × 10 degree hlwb / mL hauv 96- cov phaj zoo rau 24 teev thiab tom qab ntawd kho nrog Q-3-G (0-20 μM). Cov hlwb incubated tau kho nrog 10μL / qhov dej ntawm MTT tov, thiab tom qab 3 teev tau kho nrog 100 μL ntawm MTT Stopping Solution. Siv cov qauv MTT kev ntsuas rau kev ntsuas cell viability [84], qhov nqus ntawm 570 nm tau ntsuas siv tus nyeem ntawv (BioTek Instruments, Winooski, VT, USA).

4.4.mRNA Kev Ntsuam Xyuas los ntawm Semi-Quantitative RT-PCR thiab Quantitative Real-Time PCR

HaCaT hlwb raug kho nrog Q-3-G (5-30 μM) lossis retinol (10ug/mL) rau 12 h.RNA tau ua los ntawm TRI reagent raws li tau tshaj tawm yav dhau los [85]. Cov khoom siv cDNA synthesis tau siv los ua kom cov DNA ntxiv. RT-PCR tau ua tiav siv cov kev thim rov qab thiab rau pem hauv ntej primers. Primers rau RT-PCR thiab PCR real-time muaj npe nyob rau hauv Table 1.

4.5. DPPH Assay

Kev ntsuam xyuas DPPH tau siv los ntsuas qhov muaj peev xwm rub tawm ntawm Q-3-G. Methanol tau siv los ua kom yaj DPPH rau qhov kawg ntawm 250 μM. Tom qab ntawd, 475 mL ntawm DPPHwas

reacted nrog 5 mL ntawm Q-3-G (0-40 μM) los yog AA (500 mM) ntawm 37 degree rau 30 min. Kev tswj zoo yog AA. Qhov kev txiav txim siab ntawm DPPH scavenging effect thiab xam tau ua raws li tau piav ua ntej [86].

4.6.ABTS Assay

Kev ntsuam xyuas ABTS tau ua raws li tau tshaj tawm yav dhau los [72]. Luv luv, sib npaug ntawm 7.4 mM ABTS tov thiab 2.4 mM poov tshuaj persulfate tov tau tov thiab khaws cia nyob rau hauv chav tsev kub thaum hmo ntuj los tsim ABTS radical cations. ABTS cov kev daws teeb meem tau ntxiv rau txhua lub qhov dej ntawm 96- cov phaj zoo, nrog rau qhov sib ntxiv ntawm Q-3-G (0-40 μM) lossis AA (500 mM) ib qhov dej. Cov khoom sib xyaw tau muab tso rau ntawm 37 degree rau 30 min, thiab tom qab ntawd lawv qhov nqus ntawm 730 nm tau ntsuas.

4.7. Cellular ROS Assay los ntawm Flow Cytometry

Txhawm rau txheeb xyuas qhov tsim ntawm intracellular ROS, oxidation-sensitive dye, 2',7'-dichlorodihydrofluorescein diacetate (DCFDA), tau siv. HaCaT hlwb raug UV, thiab tom qab ntawd cov hlwb raug sau thiab rov ua dua nrog 300 ul PBS nrog 20 μM DCFDA rau 30 min ntawm 37 degree hauv qhov tsaus ntuj. Cov hlwb raug ntxuav nrog PBS thiab cov fluorescence tau ntsuas ntawm 485/535 nm los ntawm Beckman CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA).

4.8.Western Blot Analysis

HaCaT hlwb raug noob ntawm qhov ntom ntawm 6 × 10 degree cell / mL. Tom qab incubation thaum hmo ntuj, qhov sib txawv ntawm Q-3-G (0-10 μM) los yog retinol (10 ug/mL) tau ntxiv thiab incubated ntxiv rau 24 teev. Protein npaj thiab tag nrho-cell lysates tau ua raws li tau piav qhia yav dhau los [87]. Cov tshuaj tiv thaiv tshwj xeeb tau siv los txheeb xyuas tag nrho daim ntawv thiab phosphorylated daim ntawv ntawm Jun, JNK, MKK4, TAK1, p50, p65, IKK, IkB, Src, thiab Akt, uas tau pom nrog chemiluminescence reagents [88].

4.9. Melanin Cov ntsiab lus thiab kev zais cia

B16F10 hlwb tau muab noob ntawm qhov ntom ntawm 2 × 10 degree cell / mL rau hauv 12- cov phiaj zoo thiab incubated thaum hmo ntuj. Cov kab lis kev cai nruab nrab tau hloov pauv rau qhov nruab nrab tshiab ntxiv nrog Q-3-G (10-20 μM) lossis arbutin (1 mM). Melanin ntau lawm tau txhawb nqa nrog a-MSH (100 nM) rau 48 teev. Rau kev ntsuam xyuas melanin secretion, qhov optical ntom ntawm 475 nm tau ntsuas. Tom qab ntawd, cov hlwb raug sau los txiav txim siab txog lub cev melanin con-tent, siv lub lysis tsis rau lyse lub hlwb thiab pelleting lawv los ntawm centrifugation (12, 000 rpm, 5 min). Cov concentrated cell pellets tau yaj nyob rau hauv 100 mL dissolving buffer (1 M NaOH, 10 feem pua ​​DMSO) thiab yaj ntawm 55 degree. Lub absorbance tau ntsuas ntawm 405 nm siv tus nyeem ntawv qhov muag pom [89].

4.10.UVB Irradiation

HaCaT hlwb tau muab noob rau ntawm qhov ceev ntawm 1 × 105 hlwb / mL rau hauv 6- cov phaj zoo siv MEM tsis muaj ntshav qab zib thiab raug rau 24 teev ntawm kev tshaib plab. HaCaT hlwb raug kho ua ntej nrog G-3-Q rau 30 feeb thiab tom qab ntawd UVB irradiation. Tom qab ntawd, HaCaT hlwb raug ntxuav nrog PBS thiab raug UVB irradiation (UVBLamp BLX-312, Vilber Lourmat, Fabkis). Lub zog ntawm UVB irradiation yog 30 mJ / cm2² 【90】.

4.11.H2O2 Kev kho mob

Cov hlwb HaCaT tau muab noob rau hauv 6- cov phiaj zoo thiab raug rau 24 teev ntawm kev tshaib kev nqhis peb-hauv cov ntshav tsis muaj MEM. Cov hlwb raug pretreated nrog ntau qhov sib txawv ntawm Q-3-G(5-10 μM thiab tom qab 30 min tau incubated nrog H2O2 (500 μM) rau 12 teev.

4.12. Luciferase Reporter Gene Assay

HEK293T hlwb tau muab noob ntawm qhov ntom ntawm 3 × 10 degree cell / mL rau hauv 24- cov phiaj zoo. Cov hlwb HEK293T tau pauv nrog 2 ug ntawm plasmids uas muaj AP-1-Luc lossis NF-kB-Luc thiab b-galactosidase, ua haujlwm polyethylenimine[91]. Tom qab 24 teev ntawm incubation, cov hlwb raug kho nrog Q-3-G (5-10 μM) lossis retinol (10 ug/mL) rau 24 teev ntxiv. Qhov kev ntsuam xyuas luciferase tau ua los ntawm lub luminometer ntawm qhov nqus ntawm txhua tus qauv tau ntsuas ntawm 475 nm siv Spectramax 250 microplate nyeem ntawv (Molecular Devices, San Jose, CA, USA), raws li tau tshaj tawm yav dhau los [92].

4.13. Cell Morphology Shooting

HaCaT hlwb tau muab noob rau hauv 6- cov phiaj zoo ntawm lub cell ceev ntawm 1 × 10 degree cell/mL. Cov hlwb HaCaT raug kho nrog G-3-Q rau 30 feeb thiab tom qab ntawd UVB irradiation. Tom qab 24 teev, 4 ×, 10 ×, thiab 20 × lub hom phiaj tsom iav duab tau raug coj los (Olympus, Tokyo, Nyiv).

4.14 ib. Kev txheeb cais

Tag nrho cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab ± tus qauv sib txawv ntawm tsawg kawg peb qhov kev sim ywj pheej. Qhov kev xeem Mann-Whitney thiab Tub Ntxhais Kawm T-test tau siv los sib piv qhov sib txawv ntawm cov pawg. Ib p-value<0.05 was="" regarded="" as="" statistically="" significant.="" all="" statistical="" tests="" were="" carried="" out="" using="" spss(ver.25,="" spss="" inc.,="" chicago,="" il,="">

5. Cov lus xaus

Peb cov kev tshawb fawb pom tau tias Q-3-Gexhibited anti-inflammatory thiab antioxidant kev ua ub no tiv thaiv tej yam ntawm oxidative kev nyuaj siab thiab o nyob rau hauv UVB irradiation thiab HO, raug. Peb kuj tau pom tias Q-3-G muaj peev xwm moisturizer-stimulating hauv HaCaT hlwb. Ntxiv nrog rau cov nyhuv antimelanogenesis ntawm Q-3-G, nws tau pom tias inhibit melanin secretion hauv -MSH-induced B16F10. Thaum kawg, cov txiaj ntsig ntawm Q-3-G tau kho los ntawm kev ua kom AP-1 thiab NF-kB teeb liab txoj hauv kev. Kev tiv thaiv ntawm daim tawv nqaij ntawm Q-3-G hauv HaCaT tib neeg keratinocytes hlwb thiab B16F10 murine melanoma hlwb tau sau tseg hauv daim duab 5. Txoj kev tshawb no tau tsim tias Q-3-G tuaj yeem ua tau zoo vim nws cov tshuaj tiv thaiv, antioxidant, moisturizing, thiab antimelanogenesis zog hauv tib neeg keratinocytes thiab melanoma hlwb los ntawm kev ua kom NF-kB thiab AP -1 txoj hauv kev. Hauv kev xaus, cov txiaj ntsig tau qhia meej tias qhov sib txuas metabolite no muaj peev xwm tiv thaiv cov hlwb ntawm daim tawv nqaij. Kev tshawb nrhiav ntxiv yog xav tau los ua pov thawj cov txiaj ntsig ntawm Q-3-G nyob rau hauv ntau yam hauv vivo qauv, tshwj xeeb tshaj yog hais txog cov qauv tiv thaiv tawv nqaij.

Cov ntaub ntawv

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7. Che, DN; Xie, GH; Cho, BO; Shin, JY; Kang, HJ; Jang, SI Tiv thaiv cov teebmeem ntawm txiv hmab txiv ntoo extract tiv thaiv UVB-induced kev puas tsuaj hauv C57BL nas ntawm daim tawv nqaij. J. Phom. Photobiol. B 2017, 173, 551–559. [CrossRef]

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