Preconditioning-Activated AKT Controls Neuronal Tolerance To Ischemia Los Ntawm MDM2–p53 PathwayⅡ

3. Kev sib tham

Peb cov txiaj ntsig tau nthuav tawm tias IPC-kev sib kho kom sib haum xeeb ntawm PI3K / AKT teeb liab txoj hauv kev ua rau lub hlwb IT los ntawm kev tswj hwm MDM2-p53 complex hauv thawj cortical neurons. Peb thawj zaug tau lees paub qhov ua tau zoo ntawm preconditioning nyob rau hauv cov nqe lus ntawm neuroprotection [33,39–41] siv ib tug validated IPC kev sim qauv.

Nyem rau cistanche tubulosa phiv rau Neuron

Peb pom tias kev sim IPC raug ntxias los ntawm luv luv (20 min) oxygen thiab qabzib deprivation (OGD) ua raws li 2 h ntawm reoxygenation ua rau neuroprotection, raws li qhia los ntawm kev tiv thaiv ntawm ob qho tib si neuronal apoptosis thiab caspase -3 ua kom induced los ntawm ntev. OGD (90 min) ua raws li 4 h ntawm reoxygenation (OGD / R). Peb qhia tau tias IPC txo cov caspase-3 ua kom muaj zog hauv cortical neurons, uas cuam tshuam nrog apoptosis tsawg dua tom qab kev thuam ntawm ischemic thiab hnyav dua.

Qhov sib npaug ntawm pro- thiab antiapoptotic signals yog qhov tseem ceeb los xyuas kom meej tias muaj sia nyob neuronal tom qab ischemia [3,33,38,42–44]. Txawm hais tias qhov tseem ceeb ntawm cov xwm txheej no tau pom nyob rau hauv ob qho tib si hemorrhagic thiab ischemic hauv vivo stroke qauv [43,45], cov txheej txheem uas tswj cov kev taw qhia no tseem tsis tau nkag siab tag nrho hauv cov ntsiab lus ntawm ischemic kam rau ua. Lub luag haujlwm ntawm antiapoptotic AKT thiab nws txoj kev cuam tshuam tau raug tshawb fawb ntau hauv cov qog nqaij hlav cancer [46,47] thiab cov ntaub so ntswg hlwb [38,48]; Txawm li cas los xij, txog tam sim no, lub luag haujlwm ntawm AKT / MDM2- p53 qhia txoj hauv kev hauv IPC-mediated neuronal kam rau siab tiv thaiv ischemic raug mob tseem tsis pom.

Ntawm no, peb pom tias kev ua kom lub PI3K / AKT teeb liab txoj hauv kev tshwm sim los ntawm IPC txhawb nqa phosphorylation ntawm MDM2 ntawm Ser166, uas ua rau nws txoj kev hloov pauv hauv nuclear thiab protein stabilization, tiv thaiv p53-induced apoptosis ntawm caspase-3 ua kom muaj zog tom qab ischemia. Kev ua haujlwm ntawm AKT ntawm phosphorylation txhawb kev muaj sia nyob ntawm neuronal [24,49] thiab tuaj yeem ua rau muaj kev cuam tshuam ntawm IT [25,50]. Peb cov txiaj ntsig tau pom tias cov txheeb ze ntau ntawm AKT protein tsis hloov pauv raws li ischemic lossis preconditioning stimuli. Interestingly, peb pom tias thaum ntxov PI3K-mediated phosphorylation ntawm AKT ntawm Ser473 tiv thaiv ischemia-induced p53 stabilization nyob rau hauv preconditioned neurons.

Cov nyhuv tsis yog vim kev hloov kho hauv p53 mRNA qib [33,34,44] tab sis kom txo qis p53 protein ntau vim IPC ua ntej OGD / R. Txij li thaum MDM2 yog tus tswj hwm lub ntsiab ntawm p53 stabilization thiab tseem yog lub hom phiaj ncaj qha ntawm AKT, peb cov txiaj ntsig tau taw qhia lub luag haujlwm ntawm AKT / MDM2-p53 teeb liab txoj hauv kev hauv neuronal kam rau ischemia. MDM2 mRNA nce sai sai tom qab OGD [34], tab sis MDM2 kev ua haujlwm feem ntau yog tswj hwm los ntawm kev hloov pauv tom qab, tshwj xeeb tshaj yog phosphorylation [51]. Nyob rau hauv kev pom zoo nrog qhov no, peb pom tias ib zaug qhib los ntawm phosphorylation tom qab IPC, AKT, nyob rau hauv lem, phosphorylates MDM2 ntawm residue Ser166, uas yog nyob ze ntawm lub nuclear localization teeb liab [52], thiab cov nyhuv no yog tswj tom qab OGD / R raug mob.

Peb cov txiaj ntsig tau pom tias phosphorylation ntawm MDM2 ntawm Ser166 yog txaus los tawm tsam IPC-mediated neuroprotective nyhuv ntawm p53 destabilization. Yog li, ntawm no, peb tau txheeb xyuas lub sijhawm ua haujlwm ntawm AKT / MDM2-p53 txoj hauv kev tom qab ischemic raug mob thiab, qhov tseeb, peb pom tau tias IPC-activated AKT ua rau muaj kev hloov pauv ntawm ectopic MDM2, nrog rau cov protein stabilization endogenous.

Ntxiv mus, AKT tseem ua haujlwm nyob rau hauv lub nucleus, qhov twg PI3K kuj tuaj yeem txav mus los teb rau oxidative kev nyuaj siab thiab tom qab ntawd suav rau AKT phosphorylation [53]. Qhov inhibition ntawm PI3K-mediated phosphorylation ntawm AKT lossis AKT knockdown txhawb kev tuav ntawm MDM2 protein nyob rau hauv cytoplasm, thiab nws tiv thaiv Ser166 phosphorylation ntawm MDM2, nrog rau IPC-mediated neuroprotection tiv thaiv ischemia-induced neuronal apoptosis. Ntawm qhov tsis sib xws, peb tau pom tias nquag AKT khi rau nuclear MDM2 protein.

Raws li qhov tshwm sim, AKT nquag txhawb nqa ob qho tib si phosphorylations ntawm MDM2 thiab nws cov stabilization nuclear, uas pab txhawb rau IPC-mediated neuroprotection. Peb cov txiaj ntsig tau qhia tias IPC-txhim kho neuroprotection yog nyob ntawm PI3K-mediated AKT ua kom, uas phosphorylated MDM2 ntawm Ser166, txhawb nqa MDM2 nuclear tsub zuj zuj tom qab kev thuam ntawm ischemic.

Raws li, inhibition ntawm PI3K / AKT los ntawm wortmannin lossis AKT depletion los ntawm siRNA tshem tawm IPC-txhim kho neuroprotection, ua rau p53 stabilization thiab cov neuronal apoptosis tom qab ischemia. Yog li, peb cov txiaj ntsig tau pab qhia meej txog lub luag haujlwm tseem ceeb ntawm IPC-raws li kev ua haujlwm ntawm AKT-MDM2 txoj hauv kev muaj sia nyob neuronal tiv thaiv kev raug mob ischemic.

Cov p53 protein tau koom nrog hauv kev tswj hwm ntawm neuronal kev tuag / kev ciaj sia txiav txim siab rau cov neeg mob stroke [34,42,54], nrog rau cov neeg mob TIA [3]. P53 stabilization cuam tshuam rau preconditioning-mediated neuroprotection rau ischemia/reperfusion raug mob [33]. Kev sib cuam tshuam MDM2-p53 yuav, yog li ntawd, tseem ceeb heev rau kev muaj sia nyob ntawm neuronal nyob rau hauv cov ntsiab lus no [34] thiab rau IPC-mediated tolerance tiv thaiv ischemic raug mob [33].

Thus, the control of such interaction will also have an impact on stroke outcomes. In this context, we recently found that a single-nucleotide polymorphism (SNP) 309T>G hauv MDM2 tus neeg txhawb nqa txiav txim siab qhov kev qhia ntawm MDM2 thiab, dhau los, hloov kho cov neeg mob rov qab los ntawm kev mob stroke [34].

Tsis tas li ntawd, peb tau pom tias Tp53 gene SNP (rs1042522) modulates mitochondrial p53 stabilization thiab neuronal kam rau ischemia thaum kwv yees kev ua haujlwm zoo ntawm cov neeg mob uas raug kev txom nyem TIA ua ntej mob stroke [3]. Yog li ntawd, kev tswj ntawm p53 apoptotic txoj kev yuav yog qhov tseem ceeb los xyuas kom meej cov nyhuv neuroprotective ntawm IPC.

Cov txiaj ntsig no muab txoj hauv kev txhais lus rau txoj kev tshawb fawb uas tuaj yeem siv rau yav tom ntej rau cov txiaj ntsig ntawm cov neeg mob, thiab lawv tsim PI3K / AKT-MDM2-p53 teeb liab txoj hauv kev uas yog lub hom phiaj tseem ceeb rau cov tswv yim ua ntej-txhim kho IT hauv ischemic stroke. Hauv cov ntsiab lus, peb pom tau tias IPC-txhim kho PI3K / AKT teeb liab txoj hauv kev txhawb nqa phosphorylation ntawm MDM2 ntawm Ser166, ua rau MDM2 nuclear translocation thiab nws stabilization, uas ua rau neuronal IT los ntawm kev txhawb p53 destabilization thiab tom qab inactivation inactivation.

Peb cov txiaj ntsig tau qhia txog cov txiaj ntsig zoo ntawm kev ua kom ntxov ntawm AKT hauv IPC-mediated neuronal kam rau ua, uas tswj txoj hauv kev MDM2-p53 apoptotic nyob rau hauv ischemic raug mob. Cov kev tshawb pom no qhia txog lub sijhawm los nkag siab txog cov txheej txheem uas tswj cov neuronal AKT-MDM2-p53 qhia txoj hauv kev los tsim cov tswv yim tshiab neuroprotective rau IT-txog kev mob.

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Thawj kab lis kev cai ntawm Cortical Neurons

Neuronal kab lis kev cai tau npaj los ntawm C57Bl/6J lossis p53-null (Tp53−/−, B6.129S2, The Jackson Laboratory) nas embryo (14.5E) cortices. Neurons tau noob ntawm 1.8 × 105 hlwb / cm2 nyob rau hauv ib tug Neurobasal nruab nrab supplemented nrog 2 feem pua ​​B27 thiab 2 mM glutamine (Invitrogen, Madrid, Spain) thiab incubated ntawm 37 ◦C nyob rau hauv ib tug humidified 5 feem pua ​​CO2-muaj cua [ 55] ib.

4.2. Oxygen Glucose Deprivation thiab Preconditioning Models

Tom qab 9–1{{1{19}}}} hnub hauv vitro (DIV), cov neurons raug oxygen thiab qabzib deprivation (OGD) los ntawm incubating hlwb ntawm 37 ◦C rau 90 min nyob rau hauv lub incubator nruab nrog lub airlock thiab tsis tu ncua gassed nrog 95 feem pua ​​N2/5 feem pua ​​CO2. Qhov nruab nrab incubation (buffered Hanks 'solution tsis muaj qabzib: 5.26 mM KCl, 0.43 mM KH2PO4, 132.4 mM NaCl, 4.09 mM NaHCO3, 0.33 mM Na2HPO4, 2 mM CaCl2, thiab 20 mM HEP4 9%), pH yav dhau los / 5% CO2 rau 30 min. Nyob rau hauv cov xwm txheej no, cov pa oxygen concentration hauv qhov nruab nrab incubation yog 6.7 ± 0.5 µM raws li ntsuas nrog Clark-type oxygen electrode [56,57].

Thaum qhia, cov neurons raug cuam tshuam rau ischemic preconditioning (IPC; luv OGD rau 20 min ua raws li 2 h ntawm reoxygenation) ua ntej txuas ntxiv ischemia (OGD, 90 min) thiab 4 h ntawm reoxygenation (IPC ntxiv rau OGD / R) (Daim duab S1B. ). Nyob rau hauv parallel, neurons tau incubated nyob rau hauv normoxia (Nx) ntawm 37 ◦C nyob rau hauv ib tug humidified huab cua ntawm 95 feem pua ​​​​cua / 5 feem pua ​​CO2 los yog ischemic preconditioning (IPC). Thaum qhia, neurons tau incubated 30 min ua ntej IPC nyob rau hauv buffered Hanks 'xov (pH 7.4), thaum tsis muaj los yog muaj wortmannin (100 nmol / L), raws li tau piav yav tas los [19].

4.3. Kev hloov ntawm tes

Neurons (8 DIV) los yog HEK-293T hlwb tau kis nrog plasmid vector qhia YFP-tagged Mdm2 los ntawm MDM2 tib neeg txhawb nqa. MDM2p/Mdm2-YFP yog khoom plig los ntawm Uri Alon & Galit Lahav (Addgene plasmid # 53962, Watertown, MA, USA) [58]. Thaum xav tau, ib qho vector khoob (pYFP) tau siv los ua kev tswj hwm hauv tib lub sijhawm. Plasmid transfection tau ua tiav siv Lipofectamine® LTX (Invitrogen, Carlsbad, MA, USA), raws li cov chaw tsim khoom cov lus qhia. Cells tau kis nrog 1.5 µg / µL ntawm plasmid vectors thiab siv tom qab 24 teev.

AKT knockdown nyob rau hauv 6 DIV neurons tau ua tiav los ntawm kev hloov pauv nrog me me cuam tshuam ob-stranded ribonucleotides (siRNA). Targeted sequences yog raws li nram no: 50–CUCAAGUAACUCAUUCCAGAtt–30, antisense: 5 0–UCUGGAAUGAGUACUUGAGgg–30 (nas, s62216, sib xws rau nucleotides 1006–1025, GenBank accession number.} NM{ Raws li kev tswj tsis zoo, peb siv Silencer ™ Xaiv Tsis Zoo Tswj No. 1 siRNA (siControl). Txhua siRNAs tau yuav los ntawm Ambion®, Invitrogen®, thiab Thermo Fischer Scientific (Offenbach, Lub Tebchaws Yelemees). Raws li qib ntawm cov protein knockdown, qhov ua tau zoo ntawm kev hloov pauv ntawm siRNA tau kwv yees li 70-80 feem pua ​​​​ntawm 3 hnub tom qab hloov pauv. Rau kev sim ua kom ntsiag to, cov neurons tau kis nrog siRNA (10 nM) siv Lipofectamine® RNAiMAX (Invitrogen), ua raws li cov chaw tsim khoom cov lus qhia. Neurons tau ntxiv rau hauv qhov nruab nrab Neurobasal rau 72 teev ua ntej lawv siv.

4.4. Flow Cytometric Detection ntawm Apoptotic Cell Tuag

Neurons tau ua tib zoo cais tawm ntawm daim hlau siv 1 mM EDTA tetrasodium ntsev hauv PBS (pH 7.4) thiab tau stained nrog annexin V/APC thiab 7-AAD, ua raws nraim li tau piav qhia yav dhau los [55].

4.5. Caspase-3 Kev Ntsuam Xyuas Kev Ua Haujlwm

Caspase-3 kev ua haujlwm tau raug soj ntsuam hauv cell lysates [33] thiab raws li cov chaw tsim khoom cov lus qhia siv cov khoom siv Fluorimetric Assay CASP3F los ntawm SIGMA thiab nyeem ntawm emission ntawm wavelength ntawm 405 nm. Cov txheej txheem yog ua raws li kev tso tawm ntawm fluorescent 7-amino4-methyl coumarin (AMC) moiety. AMC concentration yog xam nrog tus qauv AMC.

4.6. Immunoblots thiab Co-Immunoprecipitation Assay

Neurons tau lysed nyob rau hauv tsis muaj 1 feem pua ​​SDS, 2 mM EDTA, 150 mM NaCl, 12.5 mM Na2HPO4, thiab 1 feem pua ​​Triton X-100 (NP40: 1 feem pua ​​​​NP40, EDTA diK ntxiv rau 5 mM, Tris pH{{13} }} mM, NaCl 135 mM, thiab 10 feem pua ​​glycerol) ntxiv nrog phosphatase inhibitors (1 mM Na3VO4 thiab 50 mM NaF) thiab protease inhibitors (100 mM phenylmethylsulfonyl fluoride, 50 µg / mL anti-papain, 50 μpstat, µg/mL amastatin, 50 µg/mL leupeptin, 50 µg/mL bestatin, thiab 50 µg/mL soybean trypsin inhibitor), muab cia rau hauv dej khov rau 30 feeb thiab boil rau 5 feeb. Aliquots ntawm lysed extracts tau raug SDS polyacrylamide gel (MiniProtean®, Bio-Rad) thiab blotted nrog cov tshuaj tiv thaiv thaum hmo ntuj ntawm 4 ◦C. Cov tshuaj tiv thaiv uas siv tau yog anti-AKT (9272), anti-p (Ser473) AKT (9271), anti-cleaved caspase-3 (Asp175, 9661) (Cell Signaling, Danvers, MA, USA), anti-p53 ( 554157, BD Biosciences), anti-MDM2 (2A10, ab-16895), anti (Ser166)MDM2 (ab131355), anti-GFP (ab290; kuj kuaj tau YFP) (Abcam, Cambridge, UK), anti-LAMIN B (sc-374015, Santa Cruz Biotechnology, Heidelberg, Lub teb chaws Yelemees), thiab antiGAPDH (Ambion, Cambridge, UK) hmo ntuj ntawm 4 ◦C. Tom qab incubation nrog horseradish peroxidase-conjugated tshis los tiv thaiv luav IgG (Pierce, Thermo Scientific) los yog tshis anti-nas IgG (Bio-Rad), cov membranes tau tam sim incubated nrog enhanced chemiluminescence SuperSignal West Dura (Pierce) rau 5 min ua ntej raug rau Kod XAR-5 zaj duab xis rau 1 mus rau 5 feeb thiab autoradiograms tau luam theej duab. Band siv tau suav nrog siv ImageJ 1.48v software, raws li tau piav qhia yav dhau los [60].

Rau qhov kev ntsuam xyuas co-immunoprecipitation, neurons tau lysed nyob rau hauv cov dej khov-txias tsis muaj 50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 feem pua ​​​​NP -40) ​​ntxiv nrog phosphatase inhibitors tau piav qhia saum toj no. Tom qab tshem tawm cov khib nyiab los ntawm centrifugation, neuronal lysates (100 mg) tau incubated nrog 1 mg ntawm cov tshuaj tiv thaiv rau 24 teev ntawm 4 ◦C tom qab ntawd ntxiv 10 mL ntawm cov protein A-agarose (GE Healthcare Life Sciences) rau 2 h ntawm 4. ◦ C. Immunoprecipitates tau ntxuav nrog lysis tsis thiab daws los ntawm SDS-PAGE thiab immunoblotted nrog qhia cov tshuaj tiv thaiv [61]. Cov txheeb ze protein ntau muaj nyob rau hauv daim duab S1. Tag nrho blots thiab gel scans muaj nyob rau hauv daim duab S3.

4.7. Immunocytochemistry thiab Kev Ntsuas Duab

Neurons tau loj hlob ntawm cov iav npog thiab kho nrog 4 feem pua ​​(w/v, hauv PBS) paraformaldehyde rau 30 min thiab tiv thaiv kab mob luav anti-phosphoAKT (Ser473; 9271; Cell Signaling, MA, USA), nas anti-MDM2 (2A10, ab-16895), nas anti-MAP2 (1:500; M#1406, Sigma-Aldrich, St. Louis, MO, USA) [55], mouse anti-p53 (1:200; 554157, BD Pharmingen , San Diego, CA, USA), thiab anti-GFP (1: 1000; ab290; kuj validated los ntes YFP). Immunolabeling tau kuaj pom siv cov tshuaj tiv thaiv kab mob thib ob los tiv thaiv luav IgG-Cy3 lossis tiv thaiv nas IgG-Cy2 (1: 500; Jackson ImmunoResearch. Cambridge, UK).

Nuclei tau stained nrog 40, 6-diamidino-2 phenylindole (DAPI; D9542, Sigma-Aldrich). Cov ntawv npog tau ntxuav, ntsia hauv SlowFade lub teeb antifade reagent (Invitrogen) ntawm cov iav slides, thiab tshuaj xyuas siv lub tshuab microscope (Nikon Inverted microscope Eclipse Ti-E, (NY, TEB CHAWS USA)) nruab nrog 40 × lub hom phiaj, lub pre-centered fiber illuminator Nikon Intensilight C-HGFI, thiab dub-thiab-dawb them-coupled ntaus ntawv digital lub koob yees duab Hamamatsu ORCAER lossis scanning laser confocal microscope ("Spinning Disk" Roper Scientific Olympus IX81, Tokyo, Nyiv) nrog peb lasers 405, 491, thiab 561 nm, nruab nrog 40 ×, 63 ×, thiab 100 × PL Apo roj-immersion lub hom phiaj rau cov duab daws teeb meem siab thiab cov cuab yeej digital koob yees duab Evolve Photometrics.

Txhua qhov chaw tsom iav tau teeb tsa kom sau cov duab fluorescent hauv qab qhov saturation thiab tau khaws cia tas mus li rau tag nrho cov duab thaij hauv qhov kev sim. Cov duab tau txheeb xyuas nrog ImageJ 1.48v software (National Institutes of Health). Qhov feem pua ​​​​ntawm p (Ser473) AKT ntxiv thiab p53 ntxiv rau cov neurons thiab qhov ntau ntawm cov protein ntau fluorescence siv ntawm p (Ser473) AKT thiab p53 yog qhia hauv daim duab S2A. Hauv MDM2-GFP-transfected neurons, nucleocytoplasmic tis ntawm MDM2-GFP raug xam raws li qhov piv ntawm cov ntsiab lus nuclear fluorescence rau cytoplasmic txhais tau tias fluorescence ntawm endogenous MDM2, ntsuas hauv 24 neurons (rau neurons ib. mob nyob rau hauv plaub yam kab mob sib txawv) (Daim duab S2B) [62].

Txhawm rau txhawm rau ntsuas qhov siab tshaj plaws nuclear fluorescence siv ntawm endogenous MDM2 staining thiab pSer473AKT, 40 neurons (10 neurons ib yam mob hauv plaub kab lis kev cai) raug ntsuas (Daim duab S2C), raws li tau piav qhia yav dhau los [44]. Nyob rau hauv tus neeg sawv cev qhov kev siv qhov sib txawv ntawm daim duab 5B, qhov feem pua ​​​​ntawm p (Ser473) AKT thiab MDM2 qhia hauv qab no txhua qhov xwm txheej tau suav tias yog nuclear txhais tau tias fluorescence. Nyob rau hauv txhua rooj plaub, nuclei raug txheeb xyuas los ntawm DAPI staining. Qhov siab tshaj plaws nuclear MDM2 fluorescence siv nyob rau hauv neurons kho nrog wortmannin lossis siAkt yog qhia hauv daim duab S2D.

4.8. Kev txheeb cais

Cov txiaj ntsig tau raug soj ntsuam los ntawm ib txoj kev tsom xam ntawm qhov sib txawv, ua raws li Bonferroni post hoc test, siv los sib piv cov txiaj ntsig ntawm ntau pawg. Cov txiaj ntsig tau qhia raws li txhais tau tias ± SEM. Cov tub ntxhais kawm qhov t-test tau siv rau kev sib piv ntawm ob pawg ntawm cov txiaj ntsig. Hauv txhua qhov xwm txheej, p < 0.05 tau suav tias yog qhov tseem ceeb (* p < 0.05 versus Nx; # p<0.05 versus OGD). Statistical analyses were performed using SPSS Statistics 24.0 for Macintosh (IBM).

Cistanche tiv thaiv neurons li cas?

Muaj qee cov pov thawj los qhia tias Cistanche tuaj yeem tiv thaiv cov neurons los ntawm kev txo qis apoptosis (programmed cell tuag) thiab txhawb kev muaj sia nyob. Apoptosis yog txheej txheem ntuj tsim uas tshwm sim hauv lub cev kom tshem tawm cov hlwb puas lossis tsis xav tau, tab sis nws tuaj yeem tsim kev puas tsuaj thaum nws tshwm sim ntau dhau lossis tsis tsim nyog. Cistanche tau pom tias inhibit apoptosis hauv kev tshawb fawb hauv chav kuaj, thiab cov nyhuv no yuav pab tiv thaiv cov neurons los ntawm kev puas tsuaj.

Tsis tas li ntawd, Cistanche muaj ntau yam bioactive compounds uas tau pom tias muaj cov teebmeem neuroprotective. Piv txwv li, nws muaj echinacoside, uas tau pom tias tiv thaiv neurons los ntawm oxidative kev nyuaj siab thiab o. Nws kuj muaj cov acteoside, uas tau pom tias muaj cov tshuaj tiv thaiv kab mob thiab antioxidant.

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