Apoptosis Resistance Ntawm Senescent Cells yog Ib qho Kev Tiv Thaiv Intrinsic Rau Senolysis Induced Los Ntawm Cardiac Glycosides

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

AbstractLub hom phiaj tshem tawm cov hlwb senescent, tsom xam, yog ib qho ntawm cov qauv tseem ceeb hauv kev kho kev laus. Lub plawv glycosides tsis ntev los no tau ua pov thawj tias yog dav-spectrum senolytics.Ntawm no peb tau sim cov khoom senolytic ntawm lub plawv glycosides rau tib neeg mesenchymal qia hlwb (hMSCs). Cardiac glycosides tsis muaj peev xwm senolytic rau senescent hMSCs ntawm ntau lub hauv paus chiv keeb. Siv cov txheej txheem lom neeg thiab bioinformatic peb piv kev txhim kho senescence hauv 'cardiac glycosides-sensitive'A549 thiab '-insensitive'hMSCs. Qhov tsis muaj kev tshuaj xyuas tau pom tias yuav tsum tau kho los ntawm kev siv cov poov tshuaj zoo thiab nce apoptosis tsis kam hauv senescent hMSCs. Tsis muaj zog "antiapoptotic tiv thaiv" predisposes hMSCs rau kev tsom xam. Peb tau nthuav tawm tias apoptosis tsis kam, yav tas los lees paub tias yog ib qho ntawm cov yam ntxwv ntawm senescence, qhov tseeb, tsis yog ib qho ntawm cov senescent hlwb. Ntxiv mus, tsuas yog apoptosis-proline senescent hlwb rhiab rau lub plawv glycoside-induced tsom xam. Yog li, peb tuaj yeem kwv yees tias qhov ua tau zoo ntawm kev tshuaj xyuas yuav nyob ntawm seb cov hlwb puas tau ua apoptosis-resistant piv rau lawv cov neeg sib tw loj hlob.

Ntsiab lusStem cells. Senolysis · Senescence · Apoptosis · Stress resistance

Thov nias ntawm no kom paub ntxiv

Taw qhia

Niaj hnub no cellular senescence yog suav hais tias yog ib qho kev tshwm sim ntawm yuav luag txhua hom kev loj hlob ntawm cov hlwb, nrog rau cov qog nqaij hlav thiab cov qia, nrog rau qee lub hlwb tom qab mitotic rau ntau yam kev ntxhov siab [1-3]. Cov nram qab no hom ntawm cellular senescence tuaj yeem txawv raws li lub hauv paus chiv keeb ntawm inducing yam: replicaative (vim DNA puas nyob rau hauv lub shortened telomere), oncogene-induced (nyob rau hauv teb rau aberrant activation ntawm oncogenic signaling), stress-induced ( kho los ntawm DNA puas tsuaj los ntawm oxidative kev nyuaj siab, kub poob siab, UV thiab y hluav taws xob, thiab lwm yam) thiab chemotherapy-induced (activated nyob rau hauv cov qog nqaij hlav cancer nyob rau hauv teb rau chemotherapeutic tshuaj)[4-7]. Muaj heterogeneity nyob rau hauv cov cim qhia los ntawm senescent hlwb nyob ntawm ob hom cell thiab ib qho kev thuam siv los ntxias senescence. Txawm li cas los xij, muaj ntau yam zoo sib xws rau feem ntau hom senescent hlwb. Cov yam ntxwv tseem ceeb ntawm senescence rau txhua hom kev faib tawm ntawm tes yog qhov tsis tuaj yeem hloov pauv tsis tau [8].cistanche tubulosa extractLub irreversibility ntawm lub voj voog ntawm tes yog tswj los ntawm cyclin-dependent kinase (CDK) inhibitors pl6 thiab p21 thiab feem ntau tswj los ntawm cov qog suppressor protein p53 [8,9]. Lwm qhov tseem ceeb ntawm cov hlwb senescent yog qhov ua kom muaj kev cuam tshuam DNA tsis tu ncua; cell hypertrophy, uas feem ntau tshwm sim los ntawm impaired ribosomal biogenesis thiab protein synthesis; cuam ​​​​tshuam ntawm lysosomal degradation thiab tsis ua haujlwm ntawm tus so degradation systems; nce kev ua haujlwm ntawm lysosomal enzyme senescence-sociated- -galactosidase; ntau yam kev hloov mitochondrial; tau txais cov senescence-associated secretory phenotype (SASP), muaj xws li pro-inflammatory yam, matrix-degrading enzymes, reactive oxygen hom, thiab lwm yam.; Kev hloov pauv ntawm epigenetic thiab chromatin toj roob hauv pes, suav nrog kev tsim cov senescence-sociated heterochromatic foci thiab senescence-associated distension ntawm satellites [8-10].Ib lo lus, senescent hlwb khaws cov metabolic kev ua ub no thiab vitality, tab sis lawv cov hauj lwm yog ho hloov pauv piv rau. lub hauv paus hlwb.

Cistanche tuaj yeem tiv thaiv kev laus

Nws yog tam sim no paub tseeb tias cov hlwb muaj peev xwm hloov kho qhov ib puag ncig microenvironment cuam tshuam rau ob lub hlwb nyob sib ze thiab cellular niches, uas tuaj yeem ua rau cov ntaub so ntswg ua haujlwm tsis zoo thiab yog li ntawd yuav cuam tshuam rau kev loj hlob ntawm cov laus thiab cov kab mob muaj hnub nyoog [11,12]. Ua raws li qhov xav tau, niaj hnub no kev saib xyuas ntau dua yog tsom rau cov tswv yim rau kev tsom "tua" ntawm cov hlwb senescent [13-15]. Txog rau qhov kawg no, cov chav kawm tshiab ntawm cov tshuaj hu ua senolytics yog nquag tsim. Senolytics lub hom phiaj qhia txoj hauv kev uas ua rau muaj kev tawm tsam ntawm cov hlwb ntawm apoptosis, yog li ua rau apoptosis nyiam hauv cov hlwb senescent [16]. Daim ntawv teev npe ntawm senolytics yog pheej rov ntxiv nrog cov neeg ua haujlwm tshiab. Lub npe nrov tshaj plaws nrog cov kev ua senolytic tau teev tseg yog navitoclax (Bcl -2 tsev neeg inhibitor), kev sib xyaw ntawm dasatinib (ib qho inhibitor ntawm ntau tyrosine kinases) thiab quercetin (natural flavonol), Hsp90 inhibitors, MDM2 inhibitors, FOXO{ {8}}p53 cuam tshuam peptide, BET tsev neeg protein degrader, uPAR-specific CAR-T, galacto-conjugated navitoclax thiab ntau yam senolytic natural compounds[16-24]. Tsis ntev los no, siv cov yeeb tshuaj los ntawm kev tshuaj ntsuam xyuas ob pawg kev tshawb fawb ywj pheej tau txheeb xyuas lub plawv glycosides, tshwj xeeb tshaj yog ouabain, digoxin, thiab bufalin, raws li broad-spectrum senolytics [25,26].cistanche tubulosa tshuaj xyuasNws yog tsim nyog hais tias yuav luag tag nrho cov lus tshaj tawm senolytics muaj kev txwv, xws li cov kev mob tshwm sim tsis zoo lossis tsis muaj txiaj ntsig rau qee hom cell. Mesenchymal qia hlwb (MSCs), pom zoo nyob rau hauv tag nrho cov kab mob tom qab yug me nyuam / cov ntaub so ntswg, yog tus cwj pwm los ntawm lub peev xwm los txuas ntxiv nws tus kheej (symmetric divisions) thiab sib txawv, yog li pab txhawb rau kev saib xyuas thiab rov tsim dua tshiab ntawm cov ntaub so ntswg [27]. Vim muaj cov khoom tshwj xeeb no, nrog rau cov muaj zog tiv thaiv kab mob thiab kev tiv thaiv kab mob, MSCs tau siv dav hauv kev kho cell hloov kho rau ntau yam kab mob, suav nrog ntshav qab zib mellitus, ntau yam sclerosis, myocardial infarction, thiab lwm yam [28]. Txawm li cas los xij, zoo ib yam li lawv cov progenies sib txawv thiab lwm cov hlwb tsis muaj zog, MSCs tuaj yeem ua rau muaj kev rov ua dua lossis ntxov ntxov hauv cov lus teb rau oncogenes 'kev ua kom muaj zog thiab kev ntxhov siab [29, 30].MSCs 'senescence muaj ntau qhov tsis xav tau tom qab, ntawm cov uas yog cov qaug zog. ntawm lub pas dej ntawm cov qia hlwb, txo cov ntaub so ntswg tu thiab rov tsim dua tshiab, tsis muaj peev xwm sib txawv, SASP-mediated senescence kis, txo cov khoom angiogenic, hloov kho cov qia cell niche [29,31-33]. Muab rau hauv tus account qhov tseem ceeb ntawm kev ua haujlwm zoo ntawm MSCs, senescent MSCs yuav raug suav hais tias yog lub hom phiaj tseem ceeb rau kev tshuaj xyuas. Raws li cov lus pom zoo no, ntau qhov kev tshuaj xyuas qhia txog qhov ua tau zoo ntawm kev tshem tawm MSCs tau tshaj tawm xyoo dhau los [29, 31,34,35]. Txawm li cas los xij, tsuas muaj qee qhov kev sim cov ntaub ntawv hais txog qhov teeb meem no [36-40].

Hauv qhov kev tshawb fawb tam sim no, peb pom thawj zaug uas mob plawv glycosides, uas yog ouabain thiab bufalin, tsis ua haujlwm hemolytic rau tib neeg MSCs (hMSCs) muab los ntawm endometrium (END-MSCs), cov ntaub so ntswg adipose (AD-MSCs), Kev kho hniav pulp (DP-MSCs) thiab Warton jelly (WJ-MSCs). Tsis tas li ntawd, peb paub tseeb tias ob lub plawv glycosides muaj peev xwm ua rau apoptosis nyiam nyob rau hauv Senes-cent A549 thiab SK-HEP-1, raws li tau piav qhia yav dhau los hauv kev tshawb fawb pilot [25,26]. Los ntawm kev ntsuam xyuas kev hloov pauv hauv ionic homeostasis tshwm sim los ntawm Nat / K ntxiv -ATPase thaiv thiab qhia qib ntawm cov noob muaj feem cuam tshuam peb qhia tias qhov tsis muaj ouabain-induced tsom xam yuav tau kho los ntawm kev txhim kho kev ua tau zoo ntawm cov nyiaj them rov qab K ntxiv rau hauv senescent END- MSCs piv rau senescent A549. Tsis tas li ntawd, siv cov ntaub ntawv bioinformatics siab heev peb pom tau hais tias qhov tsis zoo ntawm END-MSCs, resistant rau ouabain-induced tsom xam, yog nrog los ntawm kev nrhiav tau ntawm apoptosis-resistant phenotype, thaum senescence ntawm ouabain-sensitive A549 tsis yog. Qhov tseem ceeb, thaiv kev ua haujlwm ntawm antiapoptotic protein MCL-1 ua ntej kev kho mob plawv glycoside ua rau kev soj ntsuam ntawm apoptosis-resistant senescent END-MSCs. Yog li ntawd, peb muab pov thawj tseeb hais tias apoptosis-tiv thaiv-nce tsis yog ib qho tseem ceeb ntawm senescent hlwb. Raws li cov ntaub ntawv tau txais peb txiav txim siab tias qhov ua tau zoo ntawm kev tshuaj ntsuam xyuas yuav nyob ntawm seb cov hlwb puas tau los ua apoptosis-resistant thaum lub sij hawm kev loj hlob ntawm senescence.

Cov ntaub ntawv thiab cov txheej txheem

Cells

END-MSCs, WJ-MSCs, DP-MSCs, AD-MSCs, A549, thiab SK-Help tau txais los ntawm Lavxias Sau Npe ntawm Cell Cultures (Institute of Cytology, Saint-Petersburg, Russia). A549 thiab SK-Help kab tau txheeb xyuas los ntawm karyology, tumorigenicity, isoenzyme (LDH thiab G6PD) kev ntsuam xyuas, thiab STR tsom xam. Cells raug coj los ua tiav hauv nruab nrab DMEM F12 (Gibco BRL) ntxiv nrog 10 feem pua ​​​​FBS (HyClone), 1 feem pua ​​penicillin-streptomycin (Gibco BRL) thiab 1 feem pua ​​glutamic (Gibco BRL).Tag nrho cov hlwb raug kuaj xyuas cov kab mob mycoplasma.

Senescence- thiab stress-inducing tej yam kev mob

Rau oxidative stress-induced senescence, END-MSCs/WJ-MSCs raug kho nrog 200 uM / 100 uM HO (Sigma) rau 1 h. Rau doxorubicin-induced senescence, DP-MSCs / A549 tau kho nrog 1 μM ntawm doxorubicin (Veropharm) rau 3 hnub. Nyob rau hauv txhua rooj plaub, cov hlwb raug suav hais tias tsis yog ntxov tshaj 14 hnub tom qab kev kho mob. Rau replicaative senescence AD-MSCs ua ntej tshaj li 4th nqe lus raug txheeb xyuas raws li kev tswj cov hlwb thiab tom qab 10th raws li senescent sawv daws yuav. Rau etoposide-induced senescence A549 / END-MSCs / SK-Help tau kho nrog 2 uM / 5 uM / 3 uM etoposide (Veropharm) rau 3 hnub thiab tshuaj xyuas tsis pub dhau 7 hnub tom qab senescence induction. Nyob rau hauv cov ntaub ntawv no, kev kho mob tsim tag nrho coincided nrog cov piav nyob rau hauv txoj kev tshawb no los ntawm cov ntaub ntawv RNA-seq originated (Wang li al.,2017). Ob lub plawv glycosides peb tau siv los ua senolytic compounds - Ouabain (Sigma) thiab Bufalin (Calbiochem).

Txhawm rau sib piv cov kev ntxhov siab ntawm kev tswj hwm thiab senescent END-MSCs lossis A549, cov hlwb raug kho nrog 400/800 uM H O2 (Sigma) rau 1 h lossis nrog 0.3/1 uM staurosporine (Sigma). Cell viability raug soj ntsuam 48 teev tom qab kev ntxhov siab induction.

Txhawm rau thaiv MCL-1 kev ua END-MSCs tau ua ntej nrog 10 uM ntawm A-1210477(Sigma) rau 3 hnub.

Flow cytometry tsom xam

Kev ntsuas ntawm cell viability, proliferation, cell loj, auto-fluorescence, apoptosis tus nqi, caspase 3/7 cleavage, mitochondrial membrane muaj peev xwm, thiab membrane depolarization tau ua los ntawm flow cytometry. Flow cytometry tau ua tiav siv CytoFLEX (Beckman Coulter) thiab cov ntaub ntawv tau txais raug tshuaj xyuas siv CytExpert software version 2.0. Cov hlwb Adherent tau yaug ob zaug nrog PBS thiab sau los ntawm trypsinization. Detached hlwb tau sib sau ua ke thiab rov ua dua tshiab hauv nruab nrab thiab tom qab ntawd suav thiab ana-lyzed rau autofluorescence. Txhawm rau nkag mus rau cell viability, 50ug / ml propidium iodide (Life Technologies) tau ntxiv rau txhua tus qauv ua ntej kev tshuaj xyuas. Lub cell loj tau soj ntsuam los ntawm cytometric rau pem hauv ntej lub teeb tawg ntawm PI-tsis zoo hlwb. Apoptosis induction tau txheeb xyuas siv Annexin-V-APC (Invitrogen) thiab DAPI (Sigma) co-staining raws li cov lus qhia ntawm cov chaw tsim khoom. Kev ua haujlwm Caspase raug soj ntsuam siv CellEventTM Caspase-3/7 Green Flow Cytometry Assay Kit (Invitrogen) tom qab tsim cov txheej txheem. Kev poob ntawm mitochondrial daim nyias nyias muaj peev xwm raug soj ntsuam siv qhov ratiometric dye JC-1 (Invitrogen). Cov txheej txheem staining tau ua raws li cov neeg tsim khoom raws tu qauv. Rau daim nyias nyias depolarization, peb siv DiBAC4 (3) fluorescent sojntsuam (Invitrogen).

Senescence-associated -galactosidase staining

Senescence-associated -Galactosidase (SA- -Gal) staining tau ua tiav siv cov khoom siv senescence -galactosidase staining (Cell Signaling Technology) raws li cov chaw tsim khoom cov lus qhia. Kev txheeb xyuas ntau ntawm cov duab tau tsim nrog daim ntawv thov ntawm pob MatLab, raws li cov algorithm tau piav qhia yav dhau los [41]. Rau txhua qhov kev sim taw tes, tsis pub tsawg dua 50 lub hlwb raug xaiv los ntawm kev txheeb xyuas.

Western blotting

Western blotting tau ua raws li tau piav qhia yav dhau los [41]. SDS-PAGE electrophoresis, hloov mus rau nitrocellulose membrane, thiab immunoblotting nrog ECL (Thermo Scientific) kuaj tau ua raws li tus qauv tsim cov txheej txheem (Bio-Rad Laboratories). Antibodies tiv thaiv cov proteins hauv qab no tau siv: glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(clone 14C10), phospho-p53(Ser15), p21(clone 12D1), phospho-Rb (Ser807/811) , HMGB1 (clone D3E5), as well as horseradish peroxidase-conjugated tshis antirabbit IgG. Dilution tus nqi yog 1: 1000 rau tag nrho cov thawj cov tshuaj tiv thaiv thiab 1: 7000 rau cov theem nrab. Tag nrho cov tshuaj tiv thaiv tau yuav los ntawm Cell Signaling. Scion Image 4.0 (Scion Corporation) tau siv los xaiv thiab txiav txim siab keeb kwm yav dhau los rho tawm qhov ntom ntom ntawm cov bands hauv tag nrho cov gels thiab blots.

Transscriptomic tsom xam

Cov qauv los ntawm peb cov Gene Expression Omnibus (GEO)RNA-seq datasets tau siv hauv kev tshuaj ntsuam: GSE102639(GSM2742113-GSM2742114 thiab GSM2742121-GSM2742122forcontrol thiab senescentA549cells, raws li); GSE122081(GSM3454482-GSM3454484 thiab GSM{10}}GSM3454502 rau kev tswj thiab senescent IMR-90 hlwb, raws li); thiab peb cov ntaub ntawv teev npe GSE160702 (GSM{14}}GSM4877898 thiab GSM4877907-GSM4877910 rau kev tswj thiab senescent END-MSCs, feem). Cov ntaub ntawv rau tag nrho cov datasets tau ua tiav tib txoj kev.

Raw nyeem cov ntaub ntawv underwent zoo lim thiab adapter trimming ntawm FilterByTile thiab BBDuk scripts los ntawm BBtools pob (version 38.75) siv lub neej ntawd xaiv. Cov ntawv nyeem uas tseem tshuav tau ntxiv tau lim thiab txiav nrog kev siv cov ntawv trimester los ntawm FastqPuri pob-hnub (version 1.0.7).cistanche UKKev ua haujlwm trimming tau siv rau ob qhov kawg ntawm kev nyeem yog tias lawv muaj N's lossis lawv qhov zoo qis dua qhov zoo tshaj plaws tau teem rau 27, txhua qhov kev nyeem luv dua 25 lub hauv paus raug muab pov tseg. Kev tswj xyuas zoo ntawm trimming tau tuav nrog FastQC software (version 0.11.7) thiab FastqPuri scripts. Cov ntawv nyeem, dhau tag nrho cov haujlwm, suav nrog tsis pub tsawg dua 90 feem pua ​​​​ntawm cov ntaub ntawv thawj zaug.

Cov ntawv sau ntau ntxiv tau kwv yees siv Salmon lightweight mapping (version 1.1.0) khiav hauv hom kev xaiv xaiv. Cov npe ntawm cov decoys tau tsim los ntawm Gencode tib neeg siv genome GRCh38.p13 (tso tawm 33) thiab siv ntxiv rau kev tsim qhov Performance index ntawm concatenated transcriptome thiab genome Gencode siv cov ntaub ntawv (tso 33) siv lub kmer loj ntawm 21. Kev ua haujlwm tau ua haujlwm. nrog cov chij ntxiv —numBootstraps 30 — sequins — gcBias — validate mappings. Cov txiaj ntsig tau ua tiav yog nyob ib ncig ntawm 70 feem pua.

Kev ua cov ntaub ntawv ntxiv tau ua tiav siv R version 3.6.3 nrog Tidyverse sau cov pob (version 1.3.0). Kwv yees cov noob suav, metadata, thiab cov ntawv sau sau tau raug thauj mus rau hauv R thiab suav nrog rau qib gene siv tximeta (version 1.4.5). Cov txiaj ntsig suav matrix tau lim kom muaj cov kab uas muaj tsawg kawg yog 5 kwv yees suav thoob plaws txhua qhov qauv, qhov tshwm sim matrix muaj 20,400 noob.

Rau PCA thiab heatmap sawv cev, cov kev suav nyeem tau normalized siv log transformation los ntawm DESeq2 pob (version 1.26.0).cistanche wrkungHeatmaps tau tsim nrog kev siv cov noob lim (version 1.38.0) thiab heatmap(version 1.0.12)R pob. Kev lees paub ntawm kev faib cov qauv los ntawm qhov sib txawv ntawm qhov sib txawv tau ua los ntawm kev hloov pauv cov lej nyeem ib txwm los ntawm cov noob ntsig txog Gene Ontology lo lus "Cellular senescence"(GO 0090398). Kev txheeb xyuas qhov sib txawv thiab kev ntsuas kev hloov pauv tau suav nrog DESeq2 rau qhov hloov pauv kawg hauv cov qauv tsim qauv tswj-ling rau cov cell senescence xwm txheej, ouabain kev kho cov tshuaj tiv thaiv, thiab kev sib cuam tshuam ntawm cov ntsiab lus qhia. Txhawm rau ntxiv dag zog rau kev ntsuas qhov sib txawv, kev hloov pauv hloov kho siv kev sib xyaw ua ke ntawm kev hloov pauv kev kwv yees los ntawm lub xibtes pob (version 1.8.0) thiab qhia txog qhov kev hloov pauv ntxiv ntawm qhov sib npaug ntawm 0.667 tau siv. Qhov kev kwv yees qhov tshwm sim tau raug siv ntxiv rau kev ntsuas cov noob thiab khiav Gene Set Enrichment Analysis siv pawg profile (version 3.14.3) thiab fuse (version 1.12.0)R pob khoom nrog p tus nqi kho rau ntau qhov sib piv raws li Benjamin-Hochberg txoj kev. Affymetrix microarray cov qauv rau kev tswj thiab senes-cent AD-MSCs tau txais los ntawm GEO database (GSE66236) thiab ua tiav nrog Phantasus web-application nrog log2 thiab quantile suav normalization. Gene Set Enrichment Analysis tau ua nrog kev siv lub fuse (version 1.12.0)R pob.

RNA rho tawm, thim rov qab, thiab PCR lub sijhawm

Kev rho tawm RNA, thim rov qab, thiab PCR lub sijhawm tiag tiag tau ua raws li tau piav qhia hauv peb txoj kev tshawb fawb dhau los [32]. Reagents rau RNA rho tawm (ExtractRNA reagent), thim rov qab (MMLV RT cov khoom siv), thiab lub sijhawm PCR (HS SYBR cov khoom siv) tau txais los ntawm Evrogen. Gene qhia theem tau raug soj ntsuam siv lub sijhawm tiag tiag PCR BioRad CFX-96 amplifier (BioRad) nrog cov haujlwm hauv qab no rau txhua qhov kev tshawb nrhiav cov noob: 40 cycles ntawm melting rau 10 s ntawm 95 degree, annealing rau 15 s ntawm 57.5 degree , thiab synthesis rau 15 s ntawm 72 degree . Melting curves tsom xam tau siv los tswj qhov tshwj xeeb ntawm cov tshuaj tiv thaiv. Kev soj ntsuam hauv qab no ntawm cov ntaub ntawv tau txais tau ua tiav siv Bio-Rad CFX Manager software (BioRad) nrog tus qauv 2deltaCt kom muaj nuj nqis nrog kev siv GAPDH qhia raws li kev siv. Primer sequences yog teev nyob rau hauv Table 1.

Kev txheeb cais

Txhawm rau kom tau txais qhov tseem ceeb ntawm qhov sib txawv ntawm ob pawg Tub Ntxhais Kawm t-test lossis Welch's t-test raug siv. Rau ntau qhov sib piv ntawm pawg, ANOVA nrog Tukey's HSD tau siv. Tshwj tsis yog qhia lwm yam, tag nrho cov ntaub ntawv ntau tau qhia tias txhais tau tias ± SD thiab cov hnub qub qhia qhov sib txawv tseem ceeb raws li hauv qab no: ns, tsis tseem ceeb, * p<0.05,><><0.001. statistical="" analysis="" was="" performed="" using="" r="">

Cov txiaj ntsig

H2O2-kho END-MSCs nkag mus rau qhov ntxov ntxov senescence

Nyob rau hauv txoj kev tshawb no, peb siv tib neeg mesenchymal qia hlwb cais los ntawm desquamated endometrium (END-MSCs), uas ua tau raws li qhov tsawg kawg nkaus cov qauv qhia los ntawm ISCT rau kev txhais hMSCs [41]. Yav dhau los, peb tau tsim cov qauv kev sim uas muaj kev ntseeg siab los kawm txog ntau yam ntawm qhov ntxov ntxov ntxov ntawm END-MSCs [30]. Namely, peb tau pom tias END-MSCs raug rau sublethal oxidative kev nyuaj siab maj mam tau txais tag nrho cov yam ntxwv ntawm senescent hlwb, nrog rau cov DNA dam-hnub foci thiab active DNA kev puas tsuaj teb, irreversible cell voj voog ntes kho los ntawm classical p53/p21/Rb. txoj hauv kev, kev loj hlob poob, cell hypertrophy, zoo li SA- -Gal staining thiab kev loj hlob ntawm senescence-associated secretory phenotype [30, 32, 42]. Ntawm no peb siv tus qauv tsim los kawm txog cov nyhuv senolytic ntawm ouabain thiab qhia txog qhov kev hloov pauv hauv lub cev hauv ion homeostasis. Thaum xub thawj, los ntawm kev kwv yees qhov tsis zoo tshaj plaws peb tau lees paub tias kev txhaj tshuaj ib zaug (1 h, 200 μM) HO kev kho mob tau txaus los ua kom tsis muaj zog hauv END-MSCs. Qhov tseem ceeb, kev ntxhov siab END-MSCs raug suav tias yog senescent ob lub lis piam tom qab oxidative kev nyuaj siab; Yog li ntawd, tag nrho cov cim senescence tau soj ntsuam tsis pub dhau 14 hnub tom qab kev kho H-Oz. Tseeb tiag, H-Oz kev kho mob ntawm END-MSCs coj mus rau proliferation block, cell hypertrophy raws li qhia los ntawm kev loj hlob ntawm tes, tsub zuj zuj ntawm lipofuscin kuaj pom los ntawm qhov nce ntawm autofluorescence, tsos ntawm SA- -Gal, ua kom cov p21/Rb txoj hauv kev thiab kev poob ntawm HMGB1 ua ke txhawb kev tsim tsa lub cev nyob rau hauv cov kev sim xaiv (Fig. ac, e, f).

Qhov teeb meem loj tshaj plaws ntawm cov hlwb senescent ntawm cov ntaub so ntswg thiab cov kab mob hauv lub cev tau ntseeg tias yog qhov tshwm sim ntawm lawv qhov kev loj hlob ntev. Raws li cov ntsiab lus no, HO?-kho END-MSCs khaws cia muaj txiaj ntsig zoo txawm tias nyob rau theem lig ntawm kev txhim kho senescence (kev muaj peev xwm ntawm senescent END-MSCs raug soj ntsuam 17 hnub (14 hnub＋3 hnub) tom qab pib oxidative stress)(Fig.1d ). Txhawm rau ua kom tiav, sublethal HO2-kev kho mob ntawm END-MSCs yog tus qauv tsim nyog ntawm qhov ntxov ntxov senescence thiab muaj feem cuam tshuam los soj ntsuam cov teebmeem ntawm cov tshuaj senolytic ntawm END-MSCs.

Cardiac glycoside ouabain tsis muaj kev ua haujlwm senolytic ntawm senescent END-MSCs nyob rau hauv ntau qhov ntau.

Cov pov thawj tsis ntev los no qhia tau hais tias lub plawv glycosides, suav nrog ouabain, digoxin, thiab bufalin, sawv cev rau tsev neeg ntawm cov khoom sib txuas nrog hemolytic kev ua haujlwm [25,26]. Txawm hais tias niaj hnub no cardiac glycosides suav hais tias yog qhov dav-spectrum senolytics, cov ntaub ntawv hais txog lawv cov teebmeem ntawm senescent hMSCs tsis muaj. Yog li no, ntawm no peb tau sim seb ouabain puas muaj peev xwm ua rau muaj kev tuag xaiv hauv senescent END-MSCs. Yuav kom ua tau li ntawd, peb tau soj ntsuam qhov muaj peev xwm ntawm kev tswj hwm thiab senes-cent END-MSCs tom qab kev kho mob nrog ouabain ntawm qhov dav dav (ntawm 10-' txog 10 ~ M). Interestingly, tsis muaj kev xav tau coj mus rau qhov pom kev txo qis hauv kev muaj peev xwm ntawm ob qho tib si tswj thiab senescent hlwb nyob rau thawj hnub tom qab daim ntawv thov ouabain (Fig.2 thiab Supplemental Fig.S1).

Txawm li cas los xij, nyob rau hnub thib peb tom qab kev kho mob ouabain, peb tau nthuav tawm qhov kev txo qis ntawm kev siv tshuaj ntau ntxiv hauv kev muaj peev xwm ntawm kev tswj hwm END-MSCs (Fig. 2a thiab Supplemental Fig. S1). Tsis tau xav txog, cov hlwb senescent tau tawm tsam ntau dua rau ouabain ntawm txhua qhov kev sim siab. Raws li cov txiaj ntsig no, 10-M ouabain ua rau muaj kev tuag ntau dua apoptotic hauv cov hlwb tswj (20.75 feem pua ​​​​Ntxiv / PI-thiab 36.47 feem pua ​​​​Ntxiv / PI ntxiv ) piv rau cov neeg laus (15.01 feem pua ​​​​An＋/PI -thiab 12.15 feem pua ​​Ib qho ntxiv /PI ntxiv )(Fig.2b). Tau txais cov txiaj ntsig tau pom meej meej tias hauv cov ntsiab lus ntawm senescent END-MSCs ouabain tsis muaj kev ua haujlwm senolytic.

Txhawm rau tshem tawm cov teebmeem tshwm sim cuam tshuam nrog kev hloov pauv ntawm senescence-inducing stimuli ntawm senolytic kev txiav txim ntawm ouabain, peb tau ua ib qho kev sim ntxiv. Namely, peb kho END-MSCs nrog etoposide es tsis txhob oxidative kev nyuaj siab rau induce ntxov ntxov senescence.Citrus bioflavonoidsEtoposide-kho END-MSCs tso tawm tag nrho cov yam ntxwv zoo rau cov hlwb senescent (Fig.3a-e).

Qhov tseem ceeb, ouabain tsis muaj hemolytic ua rau etoposide-kho senescent END-MSCs (Fig. 3f thiab Supple-mental Fig. S2). Cov ntaub ntawv no muab kev pom zoo ntxiv rau cov txiaj ntsig saum toj no thiab cov pov thawj tias qhov tsis muaj ouabain-induced tsom xam tsis yog qhov tshwm sim ntawm cov pob zeb senescence uas siv los ua kom muaj senescence.

Fig.1 Validation of oxidative stress-induced END-MSCs premature senescence model. Senescent END-MSCs ib tug xoob proliferation, b undergo hypertrophy, c tau txais autofluorescence siab, khaws cia siab cell viability d thiab e zaub SA- -Gal kev ua ub no piv rau cov tswj. f Phosphorylation theem ntawm p53 thiab Rb thiab qhia theem ntawm p21 thiab HMGBI proteins nyob rau hauv kev tswj thiab senescent END-Ouabain tsis muaj hemolytic ua rau tib neeg mesenchymal qia hlwb ntawm ntau lub hauv paus chiv keeb Los nthuav peb cov kev soj ntsuam txog qhov tsis muaj ouabain-induced tsom xam nyob rau hauv END-MSCs , peb tau txheeb xyuas cov teebmeem ouabain ntawm hMSCs cais los ntawm lwm qhov chaw xws li cov ntaub so ntswg adipose, cov ntaub so ntswg, thiab Wharton's jelly. Txhawm rau ntxiv dag zog rau peb cov ntaub ntawv, peb siv cov qauv sib txawv ntawm senescence-replicative senescence rau AD-MSCs, doxorubicin-induced senescence rau DP-MSCs, thiab oxidative stress-induced senescence rau WJ-MSCs (Fig.4a-f).

Raws li pom nyob rau hauv Fig.4g, ntau hom hMSCs txawv ntawm qhov muaj peev xwm ua tau raws li kev kho mob ntawm ouabain, piv txwv li, ob qho tib si tswj thiab senescent DP-MSCs tau tiv taus ntau dua rau ouabain kev txiav txim dua li WJ-MSCs (Fig.4g thiab Supplemental Fig. S3b, c). Txawm li cas los xij, ouabain tsis tuaj yeem ua rau muaj kev cuam tshuam senolysis hauv ob hom senescent hMSCs (Fig. 4g thiab Supplemental Fig. S3). Ua ke, cov ntaub ntawv tau qhia pom tias tsis muaj kev tsom xam ntawm ouabain-induced yog ib qho zoo rau ntau hom hMSCs. MSCs. Cov txiaj ntsig tau nthuav tawm yog txhais tau tias ± SD. Rau ntau pab pawg sib piv ntawm a thiab d, ib-txoj kev ANOVA tau siv, n=3, ns tsis tseem ceeb, ***p<0.001. for="" pair="" comparisons="" at="" b,c="" and="" e="" welch's="" t-test="" was="" used,n="3" for="" b="" and="" c,n="50" for=""><0.001. scale="" bars="" for="" images="" are="" 500="">

GAPDH tau siv los tswj kev thauj khoom

Cardiac glycoside bufalin tsis tua senescent END-MSCS

Txhawm rau txheeb xyuas qhov tsis muaj senolytic kev txiav txim ntawm lub plawv glycosides ntawm hMSCs, peb tau thov bufalin, lwm qhov sib xyaw nrog cov kev ua haujlwm senolytic uas tau koom nrog hauv plawv glycosides tsev neeg [25]. Bufalin yuav luag tsis muaj kev cuam tshuam rau kev muaj peev xwm ntawm kev tswj hwm thiab H2O2- kho senes-cent END-MSCs nyob rau hauv ntau qhov concentration ntau (ntawm 10-7 mus rau 10-5 M) (Fig. 5a thiab Supplemental). Fig. S4a).

Zoo ib yam li peb tau pom rau kev kho mob ouabain, qhov tsis muaj kev tshuaj xyuas raws li bufalin yog ywj siab ntawm senescence-inducing stimuli, txij li thaum lub peev xwm ntawm ESCs uas senesced txawm nyob rau hauv teb rau oxidative kev nyuaj siab los yog rau etoposide yog unaffected (Fig. 5a, b, Supplemental Fig. . S4a, b). Cov txiaj ntsig tau piav qhia saum toj no tau lees paub tias lub plawv glycosides tau dhau los ua tsis tau zoo rau lub hom phiaj tuag induction hauv senescent END-MSCs. MSCs. Cov txiaj ntsig tau nthuav tawm yog txhais tau tias ± SD. Rau ntau pab pawg sib piv ntawm a thiab d, ib txoj kev ANOVA tau siv, n=3, ns tsis tseem ceeb, ***p<0.001. for="" pair="" comparisons="" at="" b,c="" and="" e="" welch's="" t-test="" was="" used,n="3" for="" b="" and="" c,n="50" for=""><0.001. scale="" bars="" for="" images="" are="" 500="" um.="" gapdh="" was="" used="" as="" a="" loading="">

Cardiac glycoside bufalin tsis tua senescent END-MSCS

Txhawm rau txheeb xyuas qhov tsis muaj senolytic kev txiav txim ntawm lub plawv glycosides ntawm hMSCs, peb tau thov bufalin, lwm qhov sib xyaw nrog cov kev ua haujlwm senolytic uas tau koom nrog hauv plawv glycosides tsev neeg [25]. Bufalin yuav luag tsis muaj kev cuam tshuam rau kev muaj peev xwm ntawm kev tswj hwm thiab H2O2- kho senes-cent END-MSCs nyob rau hauv ntau qhov concentration ntau (ntawm 10-7 mus rau 10-5 M) (Fig. 5a thiab Supplemental). Fig. S4a).

Zoo ib yam li peb tau pom rau kev kho mob ouabain, qhov tsis muaj kev tshuaj xyuas raws li bufalin yog ywj siab ntawm senescence-inducing stimuli, txij li thaum lub peev xwm ntawm ESCs uas senesced txawm nyob rau hauv teb rau oxidative kev nyuaj siab los yog rau etoposide yog unaffected (Fig. 5a, b, Supplemental Fig. . S4a, b). Cov txiaj ntsig tau piav qhia saum toj no tau lees paub tias lub plawv glycosides tau dhau los ua tsis tau zoo rau lub hom phiaj tuag induction hauv senescent END-MSCs.

Fig.2 Ouabain tsis muaj kev ua haujlwm hemolytic rau HO2- kho senes-cent END-MSCs nyob rau hauv ntau qhov ntau. Kev txheeb ze ntawm tes muaj peev xwm (feem pua) ntawm kev tswj hwm thiab senescent END-MSCs hauv 3 hnub tom qab kev kho mob nrog 1077,10 ~ 6, 10-5 M ouabain. b Apoptosis induction nyob rau hauv END-MSCs raws li 10-6 M ouabain soj ntsuam los ntawm Annexin V/DAPI ob chav staining. n=3 kev sim ywj pheej. Tag nrho cov ntaub ntawv sib raug rau qhov nruab nrab ± SD. Kev txheeb xyuas qhov tseem ceeb tau raug ntsuas los ntawm Welch's t-test: ns tsis tseem ceeb, *** p<>

Ob lub plawv glycosides ouabain thiab bufalin muaj peev xwm ua kom cov cell tuag xaiv hauv senescent A549 thiab SK-Hep1 hlwb.

Muab rau hauv tus account qhov tseeb tias peb cov txiaj ntsig tsis sib haum nrog cov ntaub ntawv pov thawj tsis ntev los no hais txog qhov dav-spectrum senolytic kev txiav txim siab ntawm lub plawv glycosides, peb txiav txim siab los tsim cov nyhuv no siv cov qauv ntawm tes piav qhia hauv cov kev tshawb fawb cuam tshuam [25,26]. Yog li, peb tau ua ntau qhov kev sim siv kev tswj hwm thiab senescent A549 lub ntsws carcinoma hlwb. Senescence hauv A549 hlwb raug ntxias los ntawm kev kho etoposide. Etoposide-induced senescence ntawm A549 hlwb yog ib qho nquag siv thiab yog li tus qauv zoo ntawm kev kho mob vim kev ntxhov siab [4]. Tsis tas li ntawd, ouabain tau pom tias xaiv tua etoposide-kho senescent A549 hlwb[25].Yuav ua pov thawj senescence hauv A549, peb tau soj ntsuam tus nqi proliferation, cell loj, tsub zuj zuj ntawm lipo-fine, SA- -Gal staining, qhov ua kom muaj xwm txheej ntawm txoj kev p53/p21/Rb, thiab qhia theem ntawm HMGB1 (Fig. 6a-e).

Txhawm rau txheeb xyuas cov haujlwm hemolytic ntawm ouabain rau cov qog nqaij hlav qog noj ntshav, peb thawj zaug kwv yees li ntawm cov koob tshuaj ntawm cov cell viability. Raws li peb cov txiaj ntsig tau piav qhia saum toj no, peb tsis tuaj yeem kuaj pom qhov poob qis ntawm tus lej ntawm kev tswj tau zoo lossis senescent A549 hlwb hauv 24 teev tom qab daim ntawv thov ouabain (Supplemental Fig. S5a). Txawm li cas los xij, hauv 3 hnub tom qab kev kho mob ouabain txo qis qhov muaj peev xwm ntawm senescent A549 hlwb hauv kev siv tshuaj raws li kev siv tshuaj, thaum tus naj npawb ntawm cov tswj A549 hlwb txo qis rau qhov tsawg dua (Fig.7a thiab Supplemental Fig. S5a). Namely, kwv yees li 90 feem pua ​​​​ntawm cov hlwb tswj tau khaws cia ntawm 10- M ouabain piv rau 50 feem pua ​​​​ntawm senescent A549 hlwb kho nrog tib koob tshuaj (Fig.7a). Ntxiv mus, senescent A549 hlwb tau ua rau bufalin-induced cell tuag ntau dua li lawv cov kev tswj hwm (Fig.7b)(Fig.5b thiab Supplemental Fig.S5b).

Raws li cov ntaub ntawv luam tawm, ouabain ua rau caspase-3-dependent apoptosis hauv senescent A549 hlwb [26]. Tseeb tiag, peb tau nthuav tawm qhov kev nce ntxiv ntawm qhov zoo dua ob npaug thiab/lossis PI-feem feem hauv cov qog nqaij hlav qog noj ntshav uas kho nrog 10-6M ouabain (Fig.7c). Tsis tas li ntawd, siv cov tshuaj fluorescent peb pom kev ua kom cov caspase-3 hauv ouabain-kho senescent A549 hlwb (Fig.7d). Ua ke, cov txiaj ntsig no tau ua tiav tag nrho nrog cov ntaub ntawv piav qhia los ntawm lwm tus kws sau ntawv thiab lees paub cov haujlwm hemolytic ntawm ouabain ntawm A549.

Tsis tas li ntawd, peb tau siv doxorubicin es tsis txhob ntawm etoposide los ua kom tsis muaj zog hauv A549 (Fig.8a-e). Raws li kev cia siab, doxorubicin-kho senescent A549, nrog rau etoposide-kho tau pom tias muaj kev nkag siab ntau dua ob qho tib si rau ouabain thiab bufalin piv rau lawv cov neeg tswj hwm (Fig. 8g thiab Supplemental Fig. S6). Cov tom kawg tau lees paub tias cov teebmeem senolytic ntawm lub plawv glycosides yog ywj pheej ntawm senescence inducing stimuli. Yog li, cardiac glycosides yeej muaj senolytic

Fig.3 Ouabain tsis tuaj yeem ua rau muaj kev tsis haum tshuaj hauv etoposide-kho senescent END-MSCs. Kev lees paub ntawm tus qauv rov ua rau lub cev tsis zoo rau END-MSCs: lub peev xwm loj hlob, b cell loj, c autofluorescence qib thiab d SA- -Gal kev ua, e phosphorylation theem ntawm Rb thiab qhia qib ntawm p21 thiab HMGBI proteins. f Kev txheeb ze ntawm tes muaj peev xwm (feem pua ​​​​) ntawm kev tswj hwm thiab senescent hlwb tom qab kho nrog 10-6 ouabain. Tus nqi yog txhais tau tias ± SD. Rau ntau pab pawg sib piv ntawm ib txoj kev ANOVA tau thov, n=3, ns tsis tseem ceeb, ***p<0.001.for pair="" comparisons="" at="" b,c,d,f="" welch's="" t-test="" was="" used,="" n="3" for="" b,="" c,f="" n="50" for="" d,="" ns="" not="" significant,=""><0.05,><0.001. scale="" bars="" for="" images="" are="" 500="" μm.="" gapdh="" was="" used="" as="" loading="" control="" activity="" towards="" a549,="" but="" these="" compounds="" turned="" out="" to="" be="" ineffective="" for="" targeted="" death="" induction="" in="" senescent="">

Thaum kawg, peb tau txiav txim siab rov tsim dua cov kev sim tshuaj ntsuam xyuas ntawm etoposide-kho lub siab mob qog noj ntshav SK-Help, lwm tus qauv ntawm tes nrog cov pov thawj senolytic cuam tshuam ntawm plawv glycosides raws li Guerrero li al. kawm [25] (Fig.9a-e). Etoposide-kho senescent SK-Help tau pom tias muaj kev nkag siab ntau dua rau ob tus neeg sawv cev piv rau lawv cov neeg tswj hwm, ua pov thawj qhov kev ua ntawm hemolytic ntawm lub plawv glycosides rau hom cell no (Fig.9f thiab Supplemental Fig. S7).

Ua ke cov ntaub ntawv pov thawj tias qhov kev xaiv ntawm hemolysis tshwm sim los ntawm lub plawv glycosides vam khom ntau dua ntawm cov xovtooj ntawm tes dua li ntawm qhov ua kom pom tseeb.

Kab lus no yog muab rho tawm los ntawm Cellular thiab Molecular Life Sciences (2021) 78:7757–7776 https://doi.org/10.1007/s00018-021-03980-x