Nc886, Tsis yog-Coding RNA, Yog Ib Tug Tshiab Biomarker Thiab Epigenetic Mediator Ntawm Cellular Senescence hauv Fibroblasts
Apr 14, 2023
Abstract:
Cov kev tshawb fawb ua haujlwm ntawm cov kab mob thiab tib neeg cov qauv tau qhia tias kev hloov pauv ntawm epigenetic tuaj yeem cuam tshuam rau cov txheej txheem ntawm kev laus. Tsis yog-coding RNA (ncRNA), yog ib qho ntawm cov neeg tswj hwm epigenetic, ua lub luag haujlwm tseem ceeb hauv kev hloov kho cov kev qhia ntawm mRNAs thiab lawv cov proteins. Nws tuaj yeem kho cov phenotype ntawm cov hlwb. Nws tau raug tshaj tawm tias nc886 (=vtRNA2-1 lossis pre-miR-886), ncRNA ntev, tuaj yeem cuam tshuam cov qog tsim thiab cov duab-kev puas tsuaj ntawm keratinocytes tshwm sim los ntawm UVB. Txoj kev tshawb no tsom los txiav txim siab lub luag haujlwm ntawm nc886 hauv kev rov ua dua tshiab ntawm fibroblasts thiab txiav txim siab seb cov khoom muaj peev xwm tswj tau nc886 kev qhia tuaj yeem tswj cov cellular senescence.
Hauv kev sib piv senescence fibroblasts, nc886 qhia tau txo qis thaum methylated nc886 tau nce. Muaj kev hloov pauv hauv cov cim biomarkers suav nrog SA- -kev ua haujlwm gal thiab kev qhia ntawm p16INK4A thiab p21Waf1/Cip1 hauv senescent hlwb. Cov kev tshawb pom no qhia tau hais tias qhov txo qis ntawm nc886 cuam tshuam nrog kev laus yog cuam tshuam nrog cellular senescence ntawm fibroblasts thiab hais tias nce nc886 qhia muaj peev xwm los suppress cellular senescence. AbsoluTea Concentrate 2. Cov txiaj ntsig no qhia tau tias nc886 muaj peev xwm ua lub hom phiaj tshiab rau kev tiv thaiv kev laus thiab tias ATC tuaj yeem yog cov khoom muaj zog epigenetic tiv thaiv kev laus.
Cellular senescence yog ib lo lus dav uas npog cov xwm txheej xws li kev ua haujlwm ntawm tes tsis muaj zog, txo qis kev muaj peev xwm, thiab DNA puas tsuaj los ntawm ntau yam. Cellular senescence tsis yog ib qho txheej txheem, kev kwv yees, tab sis kev sib xyaw ua ke ntawm ntau yam sib txawv ntawm lub cev, caj ces, thiab ib puag ncig. Hauv kev tshawb fawb, nws tau pom tias Cistanche tuaj yeem tiv thaiv kev laus vim tias txoj cai tiv thaiv kev laus ntawm Cistanche feem ntau cuam tshuam nrog ntau yam khoom xyaw uas nws muaj. Cistanche yog nplua nuj nyob rau hauv ntau yam khoom lom lom, xws li polysaccharides, flavonoids, acetophenones, thiab lwm yam. Ntawm lawv, cov polysaccharide yog ib qho ntawm cov khoom xyaw tseem ceeb ntawm Cistanche, uas muaj ntau yam kev noj qab haus huv xws li kev tiv thaiv kab mob, tiv thaiv oxidation, tswj cov metabolism, thiab lwm yam, thiab tuaj yeem txo qhov kev laus ntawm lub cev.
Nyem cistanche tubulosa cov txiaj ntsig
Ntsiab lus:
fibroblasts; replicate senescence; kev cai ntawm epigenetic; nc886 ;. ntsuab tshuaj yej extract.
1. Taw qhia
Cellular senescence nyob rau hauv lub xeev irreversible tom qab cell proliferation ntes tau tshwm sim raws li ib tug tseem ceeb contributor rau cov ntaub so ntswg tsis ua hauj lwm thiab kev laus ntawm cov kab mob [1]. Senescence yog tus cwj pwm los ntawm kev ua haujlwm siab ntawm senescence-associated beta-galactosidase (SA- -gal), nce kev qhia ntawm senescence biomarkers xws li p16INK4A thiab p21Waf1/Cip1, thiab txo qis ntawm LaminB1 [2]. Muaj ob hom kab mob cellular senescence: replicaative senescence thiab stress-induced premature senescence. Replicative senescence txhais tau tias yog qhov tshwm sim thaum lub cev ib txwm tsis sib faib tom qab ncav cuag cov lej ntawm kev faib. Nws muaj feem xyuam rau kev laus laus. Kev puas tsuaj tas mus li tuaj yeem ua rau muaj kev cuam tshuam tsis zoo ntawm cov fibroblasts, ua rau lub peev xwm ploj mus hloov kho thiab teeb tsa lub extracellular matrix (ECM). Cov yam ntxwv tseem ceeb ntawm cov laus dermis yog txo qis ntawm collagen fibers thiab nce kev tsim tawm ntawm matrix metalloproteinases (MMPs), uas ua rau nyias nyias thiab tsis sib haum xeeb ntawm dermis [3,4].
Epigenetic regulatory mechanisms yog cov neeg kho kom haum xeeb ntawm cov txheej txheem kev laus, hloov mus rau cov kev ntxhov siab thiab cov hnub nyoog hloov pauv hauv genomic thiab molecular ib puag ncig. Cov kev hloov pauv ntawm epigenetic no tshwm sim nyob rau ntau theem, suav nrog kev txo qis histone loj, deformation ntawm histone los ntawm kev hloov pauv tom qab kev hloov pauv thiab DNA methylation, hloov cov canonical histones nrog histone variants, thiab hloov pauv kev qhia ntawm noncoding RNA (ncRNA) thaum lub sij hawm cev xeeb tub thiab replicativ senescence [5 ,6] ib. nc886 yog 101 nucleotides ntev ncRNA uas tuaj yeem khi rau lub hom phiaj protein, yog li kho cov haujlwm ntawm cov protein thiab tswj cov noob qhia [7]. nc886 kuj tau raug pom zoo los ua cov qog nqaij hlav loj heev los ntawm nws cov qauv qhia thiab nws qhov chaw nyob ntawm tib neeg chromosome 5q31, qhov chaw ntawm cov qog suppressor gene. Ib qho tshwj xeeb ntawm nc886 kev qhia yog qhov tsis txaus ntseeg ntawm malignancies los ntawm CpG DNA methylation hauv cheeb tsam txhawb nqa [8]. Raws li cov kev tshawb fawb yav dhau los, peb tau pom tias qhov txo qis ntawm nc886 qhia los ntawm UVB irradiation yog txuam nrog nce COX-2 thiab MMP-9 los ntawm protein kinase RNA-activated (PKR) txoj hauv kev hauv keratinocytes thiab qhov ntawd. kev txhawb nqa ntawm nc886 qhia tuaj yeem yog lub tswv yim tseem ceeb los tsim cov khoom siv tiv thaiv UVB [9,10].
Tshuaj yej ntsuab tau kawm raws li kev kho mob rau ntau yam kab mob dermatological, xws li pob txuv, rosacea, psoriasis, kab mob warts, thiab txawm tias mob qog noj ntshav. Phenolic tebchaw nrog rau gallic acid (GC) thiab epigallocatechin gallate (EGCG) yog cov paub zoo active compounds nyob rau hauv ntsuab tshuaj yej. (-)-Epigallocatechin-3-(3"-O-methyl) gallate (3" Me-EGCG) yog O-methylated daim ntawv ntawm EGCG thiab muaj nyob rau hauv oolong tshuaj yej thiab tshuaj yej ntsuab. Tshwj xeeb, Jangwon No. 3 (Amorepacific ntau hom tshuaj yej ntsuab) muaj ntau dua 3 "Me-EGCG dua li lwm hom tshuaj yej ntsuab [11]. . Hauv txoj kev tshawb no, peb tau tshawb xyuas lub luag haujlwm ntawm nc886 hauv kev rov ua dua tshiab ntawm fibroblasts thiab txiav txim siab seb cov tshuaj yej ntsuab 3" Me-EGCG npaj (AbsoluTea Concentrate 2.0) uas tuaj yeem nce nc886 qhia tuaj yeem txhim kho cellular senescence.
2. Cov txiaj ntsig
2.1. Chemical Profile of AbsoluTea Concentrate 2.0 (ATC)
Cov tshuaj profile ntawm AbsoluTea Concentrate 2.0 (ATC) yog qhia hauv daim duab 1B. Cov txiaj ntsig tau txais los ntawm kev siv HPLC rau ATC npaj raws li tau piav qhia hauv Tshooj Txoj Cai. ATC muaj 30 feem pua lossis ntau dua 3 "Me-EGCG (Daim duab 1B)). Ob leeg hluas thiab senescent fibroblasts kho nrog ATC pom ntau ntxiv hauv nc886 qhia ntau dua li cov neeg kho nrog 70 feem pua ethanolic extract ntawm ntsuab tshuaj yej (Cov duab ntxiv S1A). Tsis tas li ntawd, SA- -gal qhia theem hauv senescent fibroblasts kho nrog ATC tau txo qis ntau dua li cov tshuaj yej ntsuab extract (Cov duab ntxiv S1B).
2.2. nc886 Regulates Cellular Senescence of Fibroblasts
Txhawm rau soj ntsuam lub luag haujlwm ntawm nc886 nyob rau hauv cellular senescence ntawm fibroblasts, peb siv tus qauv replicaative senescent los ntawm kev loj hlob thiab subculturing. Raws li pom nyob rau hauv Ntxiv daim duab S2, qhov kev ua ntawm SA- -gal thiab qhia theem ntawm senescent markers p16INK4A thiab p21Waf1/Cip1 tau nce thiab Lamin B1 qhia tau txo qis hauv senescent fibroblasts piv rau cov hluas hlwb. Ntau qhov kev tshawb fawb yav dhau los tau tshaj tawm tias kev laus yog tshwm sim los ntawm oxidative kev nyuaj siab. Txhawm rau kom paub meej tias qhov kev ntxhov siab ntawm oxidative no cuam tshuam nrog cellular senescence, ROS qib hauv cov hluas (p3) thiab senescent fibroblasts (p30) tau ntsuas. Cov txiaj ntsig tau pom tias qib ROS tau nce ntxiv nrog kev laus (Cov Duab Ntxiv S2D).
Txhawm rau soj ntsuam qhov muaj feem cuam tshuam ntawm nc886 ntawm cellular senescence hauv fibroblasts, nc886 gene qhia theem hauv txhua nqe lus tau txiav txim. Cov theem ntawm nc886 qhia tau txo qis nrog kev nce qib (Daim duab 2A). Nws tau raug tshaj tawm tias nc886 qhia tau txo qis nrog cov methylation ntawm CpG Islands tuaj. Txhawm rau txiav txim siab seb nc886 downregulation hauv senescent fibroblasts tau kho los ntawm methylation ntawm nc886, ib qho methylation-specific PCR tau ua. Raws li pom nyob rau hauv daim duab 2A, methylation ntawm nc886 tau nce nrog ib qho kev nce ntxiv (Daim duab 2A). Qhov tshwm sim no qhia tau hais tias qhov txo qis ntawm nc886 nyob rau hauv cov kab mob siab ntawm fibroblasts yog vim qhov nce hauv nc886 DNA methylation. Txhawm rau txheeb xyuas lub luag haujlwm ntawm nc886 hauv fibroblast senescence, kev hloov pauv ntawm cov cim senescent hauv nc886 overexpression thiab knock-down qauv tau txiav txim siab. Cov txiaj ntsig tau tshaj tawm tias cov pab pawg ntawm senescent fibroblasts nrog nc886 overexpression pom tau tias txo qis SA-beta-gal kev ua, txo qis kev qhia qib ntawm p16INK4A thiab p21Waf1 / Cip1, thiab nce qib ntawm laminB1.
Tsis tas li ntawd, qhov nce ntawm ROS hauv senescent fibroblasts tau txo qis los ntawm nc886 overexpression (Daim duab 2B). Conversely, nyob rau hauv cov pab pawg neeg ntawm cov hluas fibroblasts nrog nc886 knock-down, cellular senescence tau ceev thiab qhia theem ntawm cellular senescent markers xws li p16INK4A thiab p21Waf1/Cip1 tau nce (Daim duab 2C). Cov txiaj ntsig no qhia tias nc886 tuaj yeem tswj cov cellular senescence ntawm fibroblasts los ntawm kev tswj cov kev qhia ntawm senescence markers (Daim duab 2B, C).
2.3. ATC Mitigates Cellular Senescence los ntawm Regulating nc886 Qhia
Cov txiaj ntsig tau los saum toj no tau pom tias nce nc886 qhia tuaj yeem txhim kho cellular senescence los ntawm kev sib kho cov cim senescent. Txhawm rau soj ntsuam cov txiaj ntsig ntawm ATC ntawm nc886 qhia, qhia theem, thiab methylation ntawm nc886 hauv senescent fibroblasts raug soj ntsuam tom qab kev kho mob nrog ATC ntawm qhov tsis muaj cytotoxic concentration (Daim duab 3A). Raws li pom nyob rau hauv daim duab 3B, thiab C, ATC nce nc886 qhia thiab txo methylation ntawm nc886 nyob rau hauv ib tug concentration-dependent yam. Qhov txiaj ntsig no tau pom tias qhov nce ntawm nc886 qhia los ntawm ATC tau kho los ntawm kev cuam tshuam ntawm nc886 methylation. Tsis tas li ntawd, peb tau pom tias ATC inhibit SA{11}}kev ua haujlwm gal thiab txo qis kev qhia qib ntawm cov cim senescent p16INK4A thiab p21Waf1/Cip1 hauv senescent fibroblasts (Daim duab 3D, E). Txawm li cas los xij, qhov kev qhia ntawm LaminB1 paub tias yuav txo qis nrog kev laus tau nce hauv senescent fibroblasts kho nrog ATC (Daim duab 3E). Cov txiaj ntsig no qhia tau tias ATC tuaj yeem txo cov cellular senescence los ntawm kev nthuav tawm nc886.
Daim duab 2. nc886 tswj cellular senescence ntawm fibroblasts. (A) Kev nthuav qhia ntawm nc886 thiab methylated nc886 raws li txoj cai tau lees paub los ntawm PCR lub sijhawm tiag tiag thiab normalized nrog 18s rRNA. (B) Cov nc886 fragment amplified thiab purified los ntawm tib neeg genomic DNA tau kis mus rau senescent hlwb nrog Lipofectamine 3000. Overexpression tau lees paub los ntawm kev soj ntsuam qhov qhia ntawm nc886 los ntawm lub sijhawm PCR thiab SA{10}}gal qhia los ntawm fluorescence microscopy thiab flow cytometry. Kev nthuav qhia ntawm senescence marker protein thiab intracellular ROS tiam tau pom nyob rau hauv nc886 overexpression qauv. (C) Cells tau kis nrog anti-oligos (si-ctrl thiab si-nc886) ntawm ib tug concentration ntawm 250 ppm siv lipofectaminTM RNAiMAX reagent. nc886 knock-down tau lees paub los ntawm kev soj ntsuam qhov qhia ntawm nc886 los ntawm lub sijhawm PCR thiab SA- -gal qhia los ntawm fluorescence microscopy thiab flow cytometry. Kev qhia ntawm cov cim senescence tau lees paub nrog Western blot. Scale bars: 200 µm. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ± SD ntawm peb qhov kev soj ntsuam ywj pheej. p <0.05; **, p <0.01 piv rau kev tswj.
2.4. ATC tswj cov hnub nyoog ntsig txog kev hloov pauv ntawm ECM thiab SASP
Ib tug yam ntxwv ntawm fibroblast senescence yog qhov kev ua ntawm MMP -1, ib feem uas degrades collagen, yog nce, whereas collagen synthesis yog txo [15,16]. Qhov nce ntawm MMP-1 thiab qhov txo qis ntawm collagen tau lees paub hauv senescent fibroblasts nrog cov lej siab (Daim duab 4A).
Txawm li cas los xij, ATC inhibited qhov kev qhia ntawm MMP-1 thiab nce collagen synthesis hauv senescent fibroblasts. Cov txiaj ntsig no qhia tau tias ATC tuaj yeem txhim kho hnub nyoog hloov pauv ntawm ECM los ntawm kev tswj hwm ntawm MMP-1 kev qhia thiab collagen synthesis hauv senescent fibroblasts (Daim duab 4B). Ib qho senescence-associated secretory phenotype (SASP) nyob rau hauv uas senescent hlwb secrete tshwj xeeb pro-inflammatory yam, kev loj hlob yam, thiab proteolytic enzymes tau raug tshaj tawm los pab txhawb rau qhov pib thiab kev loj hlob ntawm cov kab mob uas muaj feem rau kev laus [17-19]. Peb tau pom cov txiaj ntsig ntawm ATC ntawm SASP tshwj xeeb tshaj yog cov cytokines xws li IL-1 , IL_6, thiab IL-8. Raws li pom nyob rau hauv daim duab 4C, ATC txo qis kev qhia theem ntawm senescence-induced IL-1 , IL-6, thiab IL-8. Qhov no qhia tias ATC tuaj yeem ua rau muaj kev tiv thaiv kev laus los ntawm kev tawm tsam ntawm SASP.
3. Kev sib tham
Senescent hlwb raug cov yam ntxwv morphological hloov pauv uas muaj qhov loj thiab feem ntau tsis xwm yeem nuclear thiab chromatin reorganization. Thaum muaj kev puas tsuaj los ntawm DNA puas, rov ua dua tshiab, lossis kev qhia txog oncogene, Lamin B1 feem ntau ploj. Ntau yam kev ntxhov siab sab hauv lossis sab nraud tuaj yeem ua rau DNA puas tsuaj teb (DDR) txoj hauv kev los qhib txoj hauv kev p53 thiab / lossis p16INK4A. p16INK4A tuaj yeem ua tsis tau Cdk4/6, ua rau muaj cov phosphorylated pRb, cuam tshuam cov kev cai ntawm E2F transcription factor, thiab induce cell voj voog ntes los yog senescence [20,21]. Kev tsim cov pa oxygen ntau ntxiv (ROS) feem ntau ua los ntawm kev ua haujlwm tsis zoo ntawm mitochondria tuaj yeem ua rau cellular senescence los ntawm DNA puas thiab transactivation ntawm senescence signaling pathways, xws li p53, p16INK4A, thiab p21Waf1/Cip1 [22].
Hauv txoj kev tshawb no, peb pom tias nc886 kev qhia tau txo qis nrog cov lej nce ntxiv thiab qhov kev txhim kho ntawm nc886 qhia tau txo qis cellular senescence los ntawm kev sib kho ntawm LaminB1, p16INK4A, p21Waf1 / Cip1, thiab ROS ntau lawm. nc886 yog transcribed los ntawm RNA polymerase III (Pol III) thiab silenced los ntawm CpG DNA hypermethylation nyob rau hauv ntau malignancies [8]. Peb pom tias qhov kev qhia ntawm nc886 tau txo qis nrog methylation ntawm CpG Islands nyob rau hauv senescent fibroblasts. Qhov txiaj ntsig no qhia tau hais tias hypermethylation ntawm CpG Islands tuaj cuam tshuam kev ua haujlwm ntawm nc886 raws li kev tawm tsam ntawm cellular senescence.
Cov kev qhia tshwj xeeb ntawm noob caj noob ces cuam tshuam los ntawm nc886 tsis meej. Lawv yuav suav nrog yav dhau los qhia rau lwm cov ncRNAs. nc886 tuaj yeem ua lub luag haujlwm tseem ceeb hauv cellular senescence los ntawm kev ncaj qha lossis tsis ncaj qha kev sib kho ntawm qhov kev taw qhia ntawm lub cev nyob rau theem ntawm cov qauv chromatin, kev ua haujlwm ntawm kev hloov pauv, post-transcriptional, thiab posttranslational gene regulation [23]. Piv txwv li, nc886 tuaj yeem kho qhov senescent txoj kev ntawm lub voj voog ntawm tes.
Nws tau raug tshaj tawm tias AK156230 raws li ncRNA cuam tshuam nrog kev cuam tshuam ntawm kev rov ua dua tshiab ntawm fibroblasts los ntawm nws lub luag haujlwm hauv autophagy thiab cell cycle. Downregulation ntawm AK156230 induced hlwb los qhia cov lus teb senescence los ntawm activating p53 thiab p21 thaum txo cyclin-dependent kinase 1 (CDK1) [24]. Senescence-associated ncRNA1 (SAL-RNA1), MALAT1, thiab MIAT yog lub npe hu ua cov tswj tsis zoo ntawm cellular senescence. Lawv raug txo qis hauv senescent fibroblasts. Kev txo qis ntawm cov noob no tuaj yeem ua rau muaj kev nce ntxiv hauv cov cim npe xws li SA-gal, p16, p21, thiab p53 [25]. Cov nyhuv ntawm MALAT1 ntawm cellular senescence tau ua tiav los ntawm kev poob ntawm oncogenic transcription factor b-Myb/Mybl2.
Txawm hais tias qhov kev nthuav qhia ntawm ntau yam ncRNAs cuam tshuam thaum lub sij hawm senescence, tsuas yog ob peb tus ua haujlwm koom nrog hauv senescence. Vim hais tias qhov chaw ntawm ncRNAs yog qhov tseem ceeb rau kev nkag siab txog cov txheej txheem koom nrog hauv kev ua haujlwm, nuclear thiab cytoplasmic ncRNAs tau sib tham sib cais thaum piav qhia txog cov txheej txheem molecular cuam tshuam rau senescence [26]. ncRNA nyob rau hauv lub cytoplasm yuav ua hauj lwm raws li ib tug neeg txhais lus regulator los ntawm lub hauv paus khub nrog lub hom phiaj mRNA [27]. ncRNA tuaj yeem cuam tshuam txog qib protein ntau los ntawm kev nce lossis txo mRNA stability [28].
Piv txwv li, thaum lub sij hawm RAS-induced senescence, cytoplasmic UCA1 pab txhawb rau p16 mRNA stabilization los ntawm sequestering hnRNPA1 [29]. nc886 muaj cov protein kinase RNA-activated (PKR) khi qhov chaw. PKR-nc886 khi tuaj yeem cuam tshuam PKR kev ua haujlwm. PKR, ib qho cytoplasmic receptor rau ob-stranded RNA (dsRNA), yog tswj los ntawm serine / threonine protein kinase uas yog qhib los ntawm kab mob, cytokines, oxidative kev nyuaj siab, thiab irradiation (xws li UV), ua rau tom qab induction ntawm o thiab apoptosis [20 ,21] [30,31] ib. nc886 khi rau PKR nrog kev sib raug zoo piv rau dsRNA thiab tiv thaiv PKR los ntawm kev ua haujlwm. nc886 knockdown tshwm sim hauv phosphorylation ntawm eukaryotic pib qhov tseem ceeb 2 subunit (eIF2) ntawm txoj kev PKR, uas ua rau cell tuag thiab inhibits ntiaj teb cellular protein synthesis. Nws tau raug tshaj tawm tias kev tsim cov COX-2, IL-8, thiab MMPs los ntawm TNF- yog kho los ntawm PKR txoj hauv kev hauv tib neeg chondrocytes [32].
Hauv kev tshawb fawb yav dhau los, peb kuj tau pom tias UVB-induced o tuaj yeem tswj tau los ntawm nc886-PKR txoj hauv kev hauv keratinocytes [10]. Ntxiv mus, PKR muaj feem xyuam rau oxidative stress-induced raug mob hauv neonatal cardiac myocytes. Inhibition ntawm PKR tiv thaiv H2O2-induced raug mob los ntawm attenuating apoptosis thiab o [33]. Kev mob o thiab oxidative kev nyuaj siab yog qhov tseem ceeb uas ua rau cov txheej txheem kev laus. Yog li, txoj kev PKR tuaj yeem kho cov cellular senescence ntawm fibroblasts induced los ntawm nc886 depletion. Txhawm rau nthuav tawm cov ncauj lus kom ntxaws txog kev tswj hwm kev koom tes hauv cov txiaj ntsig ntawm nc886 ntawm cov cellular senescence ntawm fibroblasts, xav tau kev tshawb fawb ntxiv.
Cov teebmeem antiaging ntawm ntsuab tshuaj yej extract tau dav tshaj tawm hauv cov kev tshawb fawb yav dhau los. Cov tshuaj tua kab mob antioxidant thiab tshuaj tiv thaiv kab mob ntawm cov tshuaj yej ntsuab yog cov txheej txheem paub zoo uas ua rau nws cov nyhuv tiv thaiv kev laus. Hauv kev sim ua ntej, peb pom tias ATC nce nc886 qhia tau ntau dua los ntawm ntau dua 70 feem pua ethanolic extract ntawm ntsuab tshuaj yej (Supplement Figure S1A). Cov txiaj ntsig no qhia tau tias cov ntsiab lus catechins suav nrog 3 "Me-EGCG tuaj yeem ua rau muaj kev txhawb nqa ntawm nc886 qhia. Txhawm rau txiav txim siab seb nc886 stimulating tus neeg saib xyuas tuaj yeem tiv thaiv cellular senescence, peb tau pom cov txiaj ntsig ntawm ATC ntawm senescent fibroblasts. ATC nce qhov kev qhia ntawm nc886 los ntawm inhibiting theem ntawm methylation ntawm nc886 noob nyob rau hauv senescent fibroblasts (Daim duab 3B, C). ATC kuj relieves cellular senescence los ntawm mediating senescent biomarkers xws li SA- -gal kev ua si, LaminB1, p16INK4A, thiab p21Waf1/CipE (Daim duab 3 ). Tsis tas li ntawd, nce MMP-1 thiab txo qis collagen tsim los ntawm cellular senescence tau rov qab los ntawm ATC (Daim duab 4B). SASP yam txawv me ntsis nyob ntawm seb hom cell thiab hom aging-induced stress, lawv feem ntau paub tias yog hom noob caj noob ces rau NF-κB uas tswj cov cytokines pro-inflammatory xws li IL-6 thiab IL{27}}.
Hauv txoj kev tshawb no, peb tau lees paub tias qhov kev nthuav qhia ntau ntxiv ntawm IL-1 , IL_6, thiab IL-8 hauv cov hlwb raug txwv los ntawm ATC (Daim duab 4C). Peb tsis tuaj yeem tshem tawm qhov ua tau tias qhov kev txhim kho ntawm cov tshuaj yej ntsuab ntawm cellular senescence tau ua tiav los ntawm ntau lub tswv yim, suav nrog nc886 kev tswj hwm, tiv thaiv oxidation, thiab tiv thaiv kab mob. Txawm li cas los xij, peb cov txiaj ntsig tau qhia tias nce nc886 los ntawm ATC ua lub luag haujlwm tseem ceeb hauv kev tawm tsam ntawm cellular senescence, tom qab ntawd cuam tshuam rau ECM kev tsim cov fibroblasts.
4. Cov ntaub ntawv thiab cov txheej txheem
4.1. Kev npaj ntawm ATC
Cov nplooj ntsuab qhuav qhuav (Camellia sinensis var. sinensis cv. Jangwon No. 3) siv rau txoj kev kawm tam sim no tau txais los ntawm cov tshuaj yej tsob ntoo cog hauv Dolsongi tshuaj yej vaj (33◦16023.900 N 126◦28058.300 E), Jeju, Kaus Lim Qab Teb. Cov nplooj qhuav qhuav (170 g) tau muab rho tawm nrog 70 feem pua EtOH (v/v) ntawm chav tsev kub rau 16 teev. Cov extract yog ces hmoov siv lyophilizer (TF10D, TEFIC BIOTECH, Xian, Tuam Tshoj). Txhawm rau npaj ATC, EtOH extract tau concentrated kaum-fold nrog lub evaporator teb thiab thauj mus rau ib kem ntim nrog AB-8 resin (puab txoj kab uas hla 5 cm, ntev 45 cm). Qhov hnyav elution ntawm kem tau ua nrog 4 zaug txaj ntim (BV) ntawm 20 feem pua, 30 feem pua, thiab 100 feem pua EtOH (v/v). Hauv txoj kev tshawb no, cov kuab tshuaj seem nyob hauv kem raug tshem tawm nrog lub tshuab cua compressor ua ntej elution. 30 feem pua EtOH eluate tau concentrated nees nkaum-tsib thiab siv rau ib kem polyamide (puab txoj kab uas hla 2.5 cm, ntev 40 cm). Cov kab polyamide tau eluted 5 zaug BV ntawm 10 feem pua thiab 100 feem pua EtOH. 100% EtOH eluate tau ua tib zoo mloog zoo thiab lyophilized.
4.2. Cell Culture thiab Cell Treatment nrog ATC
Tib neeg dermal fibroblasts (HDFs) tau yuav los ntawm ATCC (Manassas, VA, USA). Cell kab lis kev cai tau ua raws li kev tswj xyuas ntawm 37 ◦C nrog 5 feem pua CO2 siv Dulbecco's modified Eagle's nruab nrab (WELGENE, Daegu, Kauslim) ntxiv nrog 10 feem pua fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) thiab 1 feem pua ntawm cov tshuaj penicillin -streptomycin (WELGENE, Daegu, Kauslim). Cov noob tau cog rau hauv 6- cov phiaj zoo ntawm qhov ntom ntawm 2 × 105 hlwb / phaj. Tom qab ncav cuag 60 feem pua ntawm cov confluence, lawv tau kho nrog qhov qhia pom ntawm ATC rau 72 teev.
4.3. SA- -Gal Staining thiab Flow Cytometry Analysis
Cells raug stained siv SPiDER- -Gal (Dojindo, Kumamoto, Nyiv) raws li cov chaw tsim khoom cov lus qhia. Cells tau txheeb xyuas nrog EVOS® FL Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) thiab BD FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
4.4. Intracellular ROS ntsuas
Kev tsim tawm ntawm intracellular ROS tau saib xyuas siv C20, 70 -dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes), qhov taw qhia fluorescent ROS. Cells raug incubated nrog H2DCFDA (5 µM) rau 30 min ntawm 37 ◦C. Cell-associated fluorescence tau kuaj pom nrog BD FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
4.5. Real-Time PCR thiab Methylation-Specific PCR
Tag Nrho RNA tau muab rho tawm siv TRIzol (Thermo Fisher Scientific, Waltham, MA, USA). Kev ntsuas ntau ntawm RNA tau ua los ntawm Epoch microplate spectrophotometer (BioTek, Winooski, VT, USA). cDNA tau tsim los siv am-fiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Barker, TX, USA). Genomic DNA tau muab rho tawm siv AccuPrep® Genomic DNA Extraction Kit (BIONEER, Daejeon, Kauslim). Kev hloov pauv bisulfite tau ua los ntawm EZ DNA methylation cov khoom siv (Zymo Research, CA, USA). Txhawm rau ua qRT-PCR, AMPIGENE® cDNA Synthesis Kit (Enzo Life Sciences Inc., Farming-dale, NY, USA) thiab ABI7500 real-time PCR system (Ambion Inc, Austin, TX, USA) tau siv. Primer sequences siv nyob rau hauv txoj kev tshawb no yog piav nyob rau hauv Table 1.
4.6. Lactate Dehydrogenase Assay
Kev soj ntsuam LDH tau siv los txiav txim siab cytotoxicity los ntawm kev ntsuas lactate dehydrogenase (LDH) kev ua haujlwm tso tawm los ntawm cov hlwb puas. Qhov kev ntsuam xyuas tau ua tiav siv LDH Cytotoxicity WST Assay cov khoom (Enzo life sciences, Farmingdale, NY, USA) raws li cov chaw tsim khoom cov lus qhia.
4.7. DNA Transfection rau nc886 Overexpression thiab siRNA Transfection rau nc886 Knockdown
Kom tau txais DNA rau kev tshaj tawm ntawm nc886 hauv hlwb, DNA tau nthuav dav nrog AccuPower® PCR PreMix (BIONEER, Daejeon, Kauslim) siv genomic DNA ntawm HDFs ua tus qauv thiab cov primers hauv qab no tau piav qhia hauv Table 2.
Amplified DNA tau huv si siv QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). Cov DNA huv huv tau hloov pauv nrog LipofectamineTM 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). Txhawm rau txhawm rau nc886, cov hlwb tau hloov pauv nrog cov tshuaj tiv thaiv oligos (si-ctrl thiab si-nc886) ntawm qhov siab ntawm 250pM siv Lipofectamine™ RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA, USA).
4.8. Western Blotting
Cell lysates tau npaj nrog PRO-PREP ™ Protein Extraction Solution (iNtRON Biotechnology, Gyeonggi ua, Kauslim). Tag nrho cov proteins raug cais nrog NuPAGE electrophoresis system (Thermo Fisher Scientific, Waltham, MA, USA) thiab xa mus rau polyvinylidene difluoride (PVDF) daim nyias nyias. Immunoblotting tau ua tiav siv thawj cov tshuaj tiv thaiv kab mob tiv thaiv p16INK4A, p21Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA), MMP1, Col1A2, thiab -actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Scanning densitometric qhov tseem ceeb ntawm bands tau txheeb xyuas siv ImageJ, software version 1.52a (National Institutes of Health, Bethesda, MD, USA).
4.9. Enzyme-Linked Immunosorbent Assay
Tom qab qhia incubation, collagen (Procollagen Type I C-Peptide EIA Kit, Takara, Shiga, Nyiv) thiab MMP-1 (R&D Systems, Minneapolis, MN, USA) nyob rau hauv kab lis kev cai supernatant raug ntsuas siv cov enzyme-txuas immunosorbent assay (ELISA) cov khoom siv ua raws li cov chaw tsim khoom cov lus qhia.
4.10. Kev txheeb cais
Tag nrho cov txiaj ntsig tau nthuav tawm raws li qhov txhais tau tias ± tus qauv sib txawv (SD). Qhov sib txawv ntawm ob pawg tau soj ntsuam los ntawm t-test los yog tsom xam ntawm qhov sib txawv (ANOVA) siv GraphPad Prism. Kev txheeb cais tseem ceeb tau qhia tias yog p < 0 05 lossis p < 0.01.
5. Cov lus xaus
Hauv kev xaus, kev tswj hwm nc886 kev qhia tuaj yeem yog lub hom phiaj ntawm cellular senescence hauv fibroblasts, thiab ATC tuaj yeem yog cov khoom muaj zog epigenetic tiv thaiv kev laus.
Tus sau kev koom tes:
Conceptualization, YK, KH, and EJ; methodology YK, HJ, N.-HP, and K.-SL; cov ntaub ntawv curation, YK, HJ thiab EC; kev tshawb nrhiav, YK, HJ, EC, K.-SL, N.-HP, thiab EJ; project tswj, WP, DP, thiab EJ; cov peev txheej, WP thiab DP; saib xyuas, DP, thiab EJ; sau ntawv- thawj daim ntawv, YK; sau-saib xyuas & kho, N.-HP, KH, WP, DP, thiab EJ Txhua tus kws sau ntawv tau nyeem thiab pom zoo rau cov ntawv luam tawm ntawm cov ntawv sau.
Nyiaj txiag:
Qhov kev tshawb fawb no tsis tau txais nyiaj txiag sab nraud.
Institutional Review Board Statement:
Tsis siv tau.
Cov Lus Qhia Txog Kev Pom Zoo:
Tsis siv tau.
Cov ntaub ntawv muaj nyob:
Cov ntaub ntawv yuav muaj nyob rau ntawm kev thov.
Kev tsis sib haum xeeb ntawm kev txaus siab:
Cov kws sau ntawv tshaj tawm tsis muaj kev sib cav txog kev txaus siab.
Cov ntaub ntawv
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Yuna Kim 1, Hyanggi Ji 1, Eunae Cho 1, Nok-Hyun Park 2, Kyeonghwan Hwang 2, Wonseok Park 2, Kwang-Soo Lee 1, Deokhoon Park 1 thiab Eunsun Jung 1,*.
1 Biospectrum Life Science Institute, A-1805, U-TOWER, Yongin-si 16827, Kauslim; bioyn@biospectrum.com (YK); biocr@biospectrum.com (HJ); biozr@biospectrum.com (EC); bioyc@biospectrum.com (K.-SL); pdh@biospectrum.com (DP)
2 Basic Research thiab Innovation Division, Amorepacific Corporation R&D Center, Youngin-si 17074, Kauslim; aquareve@amorepacific.com (N.-HP); khhwang@amorepacific.com (KH); wspark@amorepacific.com (WP)
* Correspondence: bioso@biospectrum.com
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