NGF Modulates Cholesterol Metabolism Thiab Stimulates ApoE Secretion hauv Glial Cells Conferring Neuroprotection Tawm tsam Oxidative Stress Part 2
Aug 31, 2023
2.6. NGF-Mediated Secretion ntawm ApoE los ntawm U373 Tiv Thaiv Neuronal Cells los ntawm Oxidative Insult
Cov ntaub ntawv pov thawj qhia tau hais tias ApoE nce neuronal resilience rau oxidative insults, yog li tiv thaiv apoptosis thiab neurodegeneration [17,18,37]. Hauv cov ntsiab lus no, peb tau soj ntsuam seb NGF-induced ApoE secretion los ntawm U373 hlwb tuaj yeem tiv thaiv N1E-115 neurons los ntawm oxidative stress. Kev sib txawv tag nrho N1E-115 tau kho ua ntej nrog rotenone, uas ua rau oxidative kev nyuaj siab los ntawm inhibiting mitochondrial complex I.
Apoptosis yog tus txheej txheem ntawm kev puas tsuaj rau tus kheej, thiab nws yog ib txoj hauv kev tseem ceeb rau cov hlwb kom tswj lub cev homeostasis ntawm lub cev los ntawm kev ua kom nws tus kheej puas tsuaj nyob rau qee qhov xwm txheej. Apoptosis plays lub luag haujlwm sib txawv hauv ntau theem ntawm lub neej.
Cov kev tshawb fawb tsis ntev los no tau pom tias muaj qee qhov kev sib raug zoo ntawm apoptosis thiab nco. Peb twb paub lawm hais tias lub cim xeeb yog ib tug complex neural txheej txheem nyob rau hauv lub hlwb, nrog rau kev sib txuas thiab kev sib koom tes ntawm ntau lub paj hlwb. Apoptosis tuaj yeem ua lub luag haujlwm hauv cov txheej txheem neural no.
Ntawm qhov tod tes, apoptosis tuaj yeem txhim kho lub network qauv ntawm cov neurons los ntawm kev tshem cov neurons tsis tsim nyog thiab synapses, ua rau kev sib txuas ntawm cov neurons zoo dua qub, yog li txhim kho qhov zoo thiab ua haujlwm ntawm kev nco. Ntawm qhov tod tes, ntau apoptosis kuj tseem tuaj yeem ua rau txo qis ntawm cov hlwb hlwb, uas tuaj yeem cuam tshuam tsis zoo rau kev khaws cia thiab khaws cia.
Yog li ntawd, thaum tswj hwm qib tsim nyog ntawm apoptosis, peb tuaj yeem txhim kho kev nco los ntawm kev coj cwj pwm thiab kev txawj ntse, xws li kev nyeem ntawv, kev kawm, kev tawm dag zog, thiab kho peb lub siab. Cov cwj pwm thiab kev txawj ntse no tuaj yeem txhawb txoj kev loj hlob thiab kev sib txuas ntawm cov hlwb hlwb thiab txhim kho qhov zoo thiab ua haujlwm ntawm kev nco los ntawm kev txhawb nqa kev sib txuas thiab kev sib koom tes ntawm lub hlwb neurons.
Hauv kev xaus, kev sib raug zoo ntawm apoptosis thiab kev nco yog nyuaj. Tsuas yog thaum tswj hwm apoptosis kom zoo tuaj yeem txhawb peb lub hlwb thiab txhim kho peb lub cim xeeb los ntawm ntau yam kev coj cwj pwm thiab kev txawj ntse. Los ntawm qhov kev xav no, peb yuav tsum txhim kho peb lub cim xeeb. Cistanche deserticola tuaj yeem pab peb txhim kho peb lub cim xeeb, vim Cistanche deserticola tseem tuaj yeem tswj hwm qhov sib npaug ntawm cov tshuaj neurotransmitters, xws li nce qib ntawm acetylcholine thiab kev loj hlob. Cov tshuaj no tseem ceeb rau kev nco. Nws tseem ceeb heev rau kev kawm thiab kev kawm. Tsis tas li ntawd, cov nqaij ntawm cov nqaij kuj tuaj yeem txhim kho cov ntshav fluidity thiab txhawb kev thauj mus los ntawm oxygen, uas tuaj yeem ua kom lub hlwb tau txais cov as-ham txaus thiab lub zog, yog li txhim kho lub hlwb tseem ceeb thiab kev ua siab ntev.
Nyem paub ntxiv los txhim kho kev nco
Tom qab 16 h ntawm rotenone raug, N1E-115 neurons tau coj mus rau hauv nruab nrab tshiab (Ctrl), txias nruab nrab ntawm U373 hlwb (U373-Ctrl), nruab nrab nruab nrab ntawm U373 ua ntej kho nrog NGF rau 48 h (U373-NGF), lossis nruab nrab nruab nrab ntawm ApoE-silenced U373 ua ntej kho nrog NGF rau 48 teev (U373-NGF ApoE siRNA). Kev kho mob Rotenone ua ntej txo qis tus naj npawb ntawm N1E-115 kab lis kev cai hauv kev tswj hwm nruab nrab, qhia tias kev tswj hwm ntawm cov tshuaj tua kab no cuam tshuam rau N1E-115 ciaj sia.
Rotenone cytotoxicity kuj txiav txim siab khaus neurite retraction, raws li pom los ntawm qhov ntev ntawm neurites. Neurite puas elicited los ntawm kev kho rotenone yog tshwj xeeb tshaj yog tshwm sim thaum kev ntsuam xyuas ntawm neurite-cov kabmob hlwb, raws li tus naj npawb ntawm N1E-115 nrog neurites txo mus rau 50%. Cov txiaj ntsig zoo sib xws tau txais thaum N1E-115 cov hlwb raug coj los ua hauv U373-Ctrl txias nruab nrab, qhia tias muaj cov supernatant muab los ntawm cov astrocytes tsis txaus los tiv thaiv cov hlwb neuronal los ntawm rotenone-mediated toxicity. Nyob rau hauv contrite ary, daim ntawv thov n ntawm conditioned nruab nrab muab los ntawm NGF-kho U373 zoo tiv thaiv neuronal tuag, nrog rau kev txo qis ntawm neurite-cov kabmob hlwb thiab neurite ntev los ntawm rotenone tswj (Daim duab 5).
Qhov tseem ceeb tshaj y, qhov nruab nrab txias tau muab los ntawm ApoE-silenced U373 hlwb tag nrho poob cov txiaj ntsig zoo ua rau d los ntawm U373-NGF conditioned nruab nrab, qhia tias neuroprotection yog tsav los ntawm NGF-mediated ApoE tso tawm hauv kab lis kev cai nruab nrab. Qhov tseem ceeb, qhov nruab nrab nruab nrab ntawm NGF-kho U373 hloov pauv nrog scrambled siRNA tag nrho khaws cov kev tiv thaiv los ntawm U373-NGF conditioned nruab nrab, ua kom pom tias qhov poob ntawm neuroprotection pom nyob rau hauv nruab nrab conditioned los ntawm ApoE-silenced hlwb yog zoo nyob ntawm ApoE. downregulation (Daim duab S3C).
Daim duab 5. Cov teebmeem ntawm U373 conditioned nruab nrab ntawm oxidative kev nyuaj siab nyob rau hauv neurons. Cov duab sawv cev hauv qhov kaj ci thiab kev ntsuas ntau ntawm neuronal morphology ntawm N1E-115. Cov hlwb yav dhau los tau kho (+) lossis tsis yog (−) nrog rotenone (0.1 µM) rau 16 h, thiab tom qab ntawd coj mus rau hauv DMEM tshiab (Ctrl), hauv qhov nruab nrab uas tau txais los ntawm kev tswj U373 (U{{ 9}}Ctrl), NGF-pre-treated U373 (U373-NGF), thiab NGF-pre-treated U373 silenced rau apoE (U373-NGF ApoE siRNA) rau 48 h. n=5 kev sim sib txawv. Cov ntaub ntawv sawv cev txhais tau tias ± SD. Kev txheeb xyuas kev txheeb xyuas tau raug ntsuas los ntawm kev siv ib-txoj kev ANOVA, ua raws li Tukey's post hoc test. ** p < 0 01, *** p < 0.001.
Txhawm rau ntxiv dag zog rau cov txiaj ntsig tau los ntawm qhov nruab nrab nruab nrab, peb tau ua U373 / N1E115 co-cultures. Txhawm rau pib, U373 hlwb tau kho ua ntej lossis tsis nrog NGF rau 48 teev. Tsis tas li ntawd, sib txawv N1E-115 hlwb tau kho ua ntej lossis tsis nrog rotenone rau 16 teev. Tom qab ntawd, U373 nruab nrab tau hloov nrog cov nruab nrab tshiab uas muaj 0.5% FBS, thiab npog cov noob nrog N1E-115 raug xa mus rau U373 txheej los npaj kev sib koom ua ke (Daim duab 6A).
Oxidative puas elicited los ntawm rotenone kev kho mob considerably cuam tshuam cell viabilineurite bearingring hlwb thiab neurite ntev ntawm kev tswj N1E-115 thiab N1E-115 co-cultured nrog U373-Ctrl astrocytes (Daim duab 6B). Raws li cov kev sim ntsuas nruab nrab, N1E-115 hlwb co-cultured nrog U373-NGF hlwb raug tiv thaiv los ntawm oxidative puas tsuaj, whereas ApoE silencing nyob rau hauv U373 tshem tawm cov neuroprotection txhawb nqa los ntawm NGF.
Daim duab 6. ApoE secretion los ntawm NGF-kho U373 hlwb muab neuroprotection los ntawm oxidative kev nyuaj siab. (A) Cov txheej txheem sawv cev ntawm astrocyte-neuron co-cultures teeb tsa raws li tau piav qhia hauv Cov Khoom Siv thiab Cov Txheej Txheem. Neurite-cov kabmob hlwb, ntev neurite, thiab xov tooj ntawm tes tau soj ntsuam cov xuab moos tom qab tsim kev sib koom ua ke. (B) Cov duab sawv cev hauv thaj chaw ci ntsa iab thiab ntau qhov kev ntsuam xyuas ntawm neuronal morphology ntawm N1E-115 hlwb, yav tas los kho (+) lossis tsis (−) nrog rotenone (0.1 µM) rau 16 h.
N1E-115 tom qab ntawd tau khaws cia hauv DMEM tshiab (Ctrl), lossis koom nrog kev tswj hwm U373 (U373-Ctrl), NGF-pre-treated U373 (U373-NGF), thiab NGF-pre-treated U373 silenced rau ApoE (U373-NGF ApoE siRNA) rau 48 teev. n=5 kev sim sib txawv. Cov ntaub ntawv sawv cev txhais tau tias ± SD. Kev txheeb xyuas txheeb cais tau ua los ntawm kev siv ib-txoj kev ANOVA, ua raws li Tukey's post hoc test. * p < 0 05, ** p < 0.01, *** p < 0.001.
3. Kev sib tham
Kev tsis txaus ntseeg hauv neurotrophin signaling txoj hauv kev, nrog rau kev hloov pauv hauv lub hlwb cov roj cholesterol metabolism, muaj kev cuam tshuam nrog cov kab mob neurologic thiab neurodegenerative, xws li Rett syndrome, HD, Alzheimer's disease (AD), thiab Parkinson's disease (PD) [2,38,39] . Hauv cov ntsiab lus no, kev tshawb fawb tseem ceeb ntawm kev ua haujlwm lom neeg ntawm cov neurotrophins yog qhov tseem ceeb rau kev txiav cov txheej txheem molecular txuas cov roj cholesterol metabolism thiab lub hlwb physiopathology. Ob peb xyoos dhau los, kev tshawb fawb nthuav qhia txog lub luag haujlwm rau BDNF hauv kev tswj hwm cov roj cholesterol metabolism hauv astrocytes [38].
Txawm hais tias qhov kev xav no, tsis muaj ntaub ntawv qhia txog kev koom tes ntawm NGF hauv kev tswj hwm ntawm astrocytic cholesterol. Yog li ntawd, hauv txoj haujlwm no, peb tau tsom mus rau lub luag haujlwm yav tom ntej uas tau ua los ntawm NGF hauv kev tswj hwm cov roj cholesterol metabolism, nrog rau kev siv tshwj xeeb rau kev cuam tshuam ntawm ApoE secretion ntawm neuronal sib txawv thiab muaj sia nyob. U373, astrocytoma cell kab siv los ua tus qauv sim ntawm tib neeg astrocytes, khaws lub peev xwm los teb rau NGF, raws li nws qhia txog qib txaus siab ntawm ob qho tib si TrkA thiab p75NTR.
Cov txiaj ntsig tseem ceeb tau qhia tias NGF txhawb nqa cov qib ntawm cov proteins tseem ceeb koom nrog hauv cov roj cholesterol biosynthesis (HMGCR), kev lag luam hauv lub cev (NPC1), thiab zais cia (ABCA1). Cov xwm txheej no tau nrog los ntawm kev nce ntxiv ntawm ApoE thiab cov roj cholesterol lein rau hauv kab lis kev cai nruab nrab, qhia tias NGF nce cov roj cholesterol biosynthesis thiab extrusion los ntawm glial hlwb. Nws tau raug tshaj tawm tias NGF induces ApoE protein qhia los ntawm kev nce nws cov ntawv sau [40]. Txawm li cas los xij, peb tsis tau pom ib qho kev hloov pauv tseem ceeb hauv intracellular ApoE qhia. Qhov kev tsis sib haum xeeb no tuaj yeem piav qhia tau txij li ApoE buildup tsis tuaj yeem txaus siab vim tias nws cov dej ntau ntxiv los ntawm NGF-kho UIn
Nyob rau hauv ib qho kev sim kom zoo dua dissect lub molecular mechanisms, peb kho U373 nrog LM11A-31 thiab pom tias p75NTR modulator tsuas yog tuaj yeem ua raws li qhov nce hauv HMGCR kev qhia kho los ntawm NGF. Kev koom tes ntawm p75NTR hauv kev hloov pauv ntawm HMGCR qib yog ua raws li cov ntaub ntawv dhau los tau txais hauv lwm hom ntawm tes, qhia tias cov receptor no tswj cov kev hloov pauv ntawm cov cholesterol enzymes [41]. Txawm li cas los xij, LM11A-31 tsis tau ua rau muaj kev hloov pauv ntawm lwm cov proteins uas tau soj ntsuam hauv txoj kev tshawb no, suav nrog ABCA1, lossis hauv cov roj cholesterol thiab ApoE qib rau hauv kab lis kev cai nruab nrab, qhia tias kev xaiv ntawm p75NTR tsis txaus los txhawb cov roj cholesterol. extrusion los ntawm ApoE-nplua nuj hais pom nyob rau hauv NGF-kho hlwb. Cov txiaj ntsig no ua rau peb xav tias NGF tswj hwm cov roj cholesterol metabolism hauv glial hlwb los ntawm kev koom tes ntawm ob qho tib si p75NTR thiab TrkA receptors.
Nws paub zoo tias neurotrophin khi rau Trk receptors qhib ERK1/2, yog li tswj ntau txoj hauv kev koom nrog hauv kev sib txawv ntawm tes, kev loj hlob, thiab kev ciaj sia [2,38]. Interestingly, nws tau pom tias ERK1/2 ua kom muaj zog kuj tswj ABCA1 thiab ApoE qhia hauv ntau hom cell [38,42,43]. Cov kev tshawb pom no, ua ke nrog cov pov thawj uas BDNF / TrkB / ERK axis txhawb nqa ApoE extrusion thiab ABCA1 kev qhia hauv glial hlwb [38], qhia tias txoj kev teeb liab hloov pauv cuam tshuam nrog TrkA / ERK axis tuaj yeem piav qhia txog ib feem ntawm qhov cuam tshuam los ntawm NGF. hauv peb txoj kev kawm.
Txawm hais tias tsis muaj kev hloov pauv hauv qhov kev loj hlob ntawm neurite, kev ua haujlwm ntawm U373- qhov nruab nrab nruab nrab thiab tsim kev sib koom ua ke kev sim qhia tau hais tias qhov nce ApoE secretion los ntawm glial hlwb mediated los ntawm NGF yog txiav txim siab los tiv thaiv qhov tshwm sim tsis zoo los ntawm oxidative. kev ntxhov siab hauv cov hlwb neuronal. Txhawm rau ua kom muaj cytotoxicity, peb ua haujlwm rotenone, tshuaj tua kab feem ntau siv hauv kev tshawb fawb ua ntej los ua rau mitochondria-derived reactive oxygen hom (ROS) thiab tom qab apoptosis [44].
Peb cov txiaj ntsig tau hais qhia tias NGF txhawb nqa ApoE zais cia los ntawm cov hlwb glial, uas yog qhov tsim nyog los lav cov teebmeem neuroprotective tiv thaiv rotenone kev tswj hwm hauv kev sib txawv N1E-115 hlwb. Lub luag haujlwm ntawm ApoE hauv kev tiv thaiv oxidative kev nyuaj siab yog cov ntaub ntawv zoo. Piv txwv li, ApoE depletion nyob rau hauv nas qauv exacerbates oxidative raug mob nyob rau hauv lub paj hlwb [45,46]. Raws li, kev tswj hwm ntawm exogenous ApoE tiv thaiv irreversible oxidative kev puas tsuaj los ntawm hydrogen peroxide kho los ntawm counteracting theem nrab glutamate toxicity [18]. Tsis tas li ntawd, ApoE-muaj lipoproteins muaj txiaj ntsig zoo hauv kev txo qis apoptosis tshwm sim los ntawm kev tshem tawm ntawm kev txhawb nqa trophic hauv retinal ganglion neurons [47].
Hauv peb txoj kev tshawb fawb, kev ua haujlwm ntawm rotenone qhia tias ApoE-mediated neuroprotection, txhawb nqa los ntawm NGF-stimulated glial hlwb, tej zaum yuav cuam tshuam rau hauv cov ntsiab lus ntawm neurodegenerative mob xws li PD. Qhov tseem ceeb, rotenone reproduces dopaminergic neuronal degeneration zoo ib yam li uas tau pom hauv PD, ob qho tib si hauv tsiaj qauv thiab kab lis kev cai ntawm tes, suav nrog N1E-115- muab los ntawm neurons [48–50]. Coherently, peb cov txiaj ntsig tau txhawb nqa cov pov thawj yav dhau los uas qhia tias ApoE elicits neuroprotection raws li kev tswj hwm ntawm 6-hydroxydopamine (6-OHDA), lwm cov neurotoxin muaj peev xwm ua kom muaj PD-zoo li phenotype [51]. Ua ke, peb txoj kev tshawb fawb pom tau tias NGF yog qhov tseem ceeb ntawm kev hloov pauv ntawm cov roj cholesterol metabolism thiab ApoE extrusion los ntawm glial hlwb. Qhov tseem ceeb tshaj plaws, kev txhim kho hauv ApoE secretion txhawb nqa los ntawm cov neurotrophin no yuav tsum tau ua kom muaj kev tiv thaiv neuro tiv thaiv rotenone cytotoxicity.
BDNF yog qhov muaj ntau tshaj neurotrophin hauv cov neeg laus lub hlwb. Txawm hais tias muaj pov thawj no, nws tau pom tias NGF tuaj yeem ua lub luag haujlwm tseem ceeb tsis yog thaum lub sijhawm kev loj hlob nkaus xwb tab sis kuj tseem nyob hauv cov neeg laus hauv nruab nrab lub paj hlwb. Piv txwv li, NGF cuam tshuam rau hippocampal kev ua ub no thiab, raws li qhov tshwm sim, spatial nco thiab kev kawm [52]. Lwm qhov kev tshawb pom qhia tias NGF nthuav tawm cov txiaj ntsig neuroprotective ntawm nigrostriatal dopaminergic neurons thiab tias qhov haujlwm no tuaj yeem tsim lub hauv paus rau NGF-raws li kev kho tshiab rau PD [53].
Raws li qhov kev xav no, nws tau pom tias kev tsim cov NGF tau tswj hwm nruj hauv astrocytes thaum muaj hnub nyoog laus thiab ua haujlwm modulatory rau neuroinflammation, mob hlwb, thiab neurodegeneration [54-56]. Nyob rau hauv cov ntsiab lus no, nws yuav nthuav rau kev soj ntsuam, hauv kev tshawb fawb yav tom ntej, seb cov teebmeem no puas tuaj yeem kho, yam tsawg kawg hauv ib feem, los ntawm kev hloov pauv ntawm glial cholesterol txhawb los ntawm NGF. Txoj haujlwm tam sim no nthuav tawm qee qhov kev txwv uas yuav tsum tau kov yeej hauv kev tshawb nrhiav tom ntej; Piv txwv li, nws yuav yog ib qho tseem ceeb kom zoo dua rau kev txiav tawm cov txheej txheem molecular txuas NGF kev kho mob thiab cov roj cholesterol metabolism hauv glial hlwb.
Kev nkag siab dav dav yog qhov muaj txiaj ntsig los txheeb xyuas cov hom phiaj molecular amenable rau pharmacological manipulation uas tuaj yeem ua haujlwm hauv cov ntsiab lus neurodegenerative. Tsis tas li ntawd, txawm hais tias U373 thiab N1E-115 hlwb tau dav siv los ua cov kab ntawm tes tswj tau raws li kev tsim tawm astrocytic thiab neuronal nta [27–29,57], cov kev tshawb pom no yuav tsum tau lees paub ntxiv hauv cov kab lis kev cai hauv lub cev. Txawm hais tias kev siv zog ntxiv yuav tsum tau ua kom ntxiv dag zog rau kev koom tes ntawm neurotrophin signaling nyob rau hauv lub hlwb cholesterol homeostasis, qhov kev ua haujlwm no qhia tau hais tias thawj zaug uas NGF tuaj yeem cuam tshuam kev tiv thaiv neuroprotect los ntawm kev cuam tshuam glial cholesterol metabolism.
4. Cov khoom siv thiab cov txheej txheem
4.1. Cell Culture
N1E-115 cov hlwb neuroblastoma thiab U373 hlwb tau coj los ntawm 5% CO2 hauv DMEM ntawm cov piam thaj siab (Merck Life Science, Milan, Ltalis), nrog 10% (v/v) fetal bovine serum (FBS) (Merck Life Science , Milan, Ltalis), thiab ntxiv nrog penicillin / streptomycin tov.
Txhawm rau ua kom muaj kev sib txawv ntawm cov paj hlwb, N1E-115 cov hlwb tau cog rau ntawm 30% kev sib txuam thiab tau hloov mus rau DMEM nrog 0.5% FBS rau 96 teev.
Txhawm rau ua kom muaj kev sib txawv ntawm cov paj hlwb, N1E-115 cov hlwb tau cog rau ntawm 30% kev sib txuam thiab tau hloov mus rau DMEM nrog 0.5% FBS rau 96 teev.
Astrocyte-neuron co-cultures tau teeb tsa raws li cov txheej txheem piav qhia los ntawm Ioannou thiab cov npoj yaig, nrog kev hloov kho [59]. Paraffin spacers tau nias rau ntawm daim npog kom paub tseeb tias kev ua raws; peb siv spacers ntawm neuronal npog npog kom zoo tiv thaiv kev puas tsuaj thaum lub hlwb raug muab tso rau ib leeg. Cov ntawv npog tom qab ntawd tau ua kom tsis muaj menyuam hauv ethanol thiab coated nrog poly-D-lysine (Merck Life Science, Milan, Ltalis). N1E-115 tau cog rau ntawm daim npog npog ntawm qhov xav tau ceev thiab, ib zaug npaj rau kev coj noj coj ua, tsis muaj zog forceps tau siv los nqa cov npog npog nrog N1E-115 neurons thiab muab lawv ntsej muag rau hauv { {10}}qhov dej noob nrog U373 astrocytes.
Kev kho ua ntej ntawm N1E-115 hlwb tau ua los ntawm kev tswj hwm 0.1 µM ntawm rotenone (Sigma-Aldrich, Milan, Ltalis, R8875) rau 16 teev. Cov hlwb tswj tau txais DMSO (dilution 1: 1000 hauv cell culture nruab nrab) ua lub tsheb. U373 hlwb raug kho nrog NGF (Alomone Labs, Yeluxalees, Israel, N{11}}) ntawm koob tshuaj 100 ng / mL rau 48 teev hauv txhua qhov kev sim.
4.2. Kev Npaj Lysate thiab Western Blot Analysis
U373 hlwb tau sonicated (lub voj voog ua haujlwm 20%, tso zis 3) hauv cov qauv tsis (0.1Tris-HClisHCl uas muaj 10% SDS, protease inhibitor cocktail, pH 6.8) rau toer kom tau txais tag nrho lysate, raws li tau piav qhia yav dhau los [ 60, 61] ib. Laemmli tsis tau ntxiv, thiab cov qauv tau muab tso rau ntawm 95 ◦C rau 5 feeb. Protein extracts (nees nkaum micrograms ntawm cov proteins) tau daws ntawm SDS-PAGE, thiab hloov mus rau nitrocellulose daim nyias nyias tau ua los ntawm kev siv lub trans-blot turbo hloov system (Biorad Laboratories, Milan, Ltalis), raws li yav dhau los qhia [62].
Tom qab ntawd, daim nyias nyias tau muab tso rau hauv chav tsev kub rau 1 teev nrog 5% tsis muaj roj qhuav mis hauv Tris-buffered saline (25 mM Tris-HCl, 138 mM NaCl, 27 mM KCl, 0.05% Tween{ {10}}, pH 6.8) thiab soj ntsuam ib hmos ntawm 4 ◦C nrog cov tshuaj tiv thaiv hauv qab no: anti-p75NTR (Santa Cruz Biotechnology, Dallas, TX, USA, sc-271708, dilution 1:500), anti- TrkA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-118, dilution 1:500), anti-SREBP-1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-8984 , dilution 1:1000), anti-SREBP-2 (Abcam, Cambridge, UK, ab30682, dilution 1:1000), anti-HMGCR (Abcam, Cambridge, UK, ab242315, dilution 1:1000), anti-LDLr (Abcam, Cambridge, UK, ab30532, dilution 1:500), anti-NPC1 (Novus Biologicals, Centennial, CO, USA, NB400-148, dilution 1:3000 ), antiABCA1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-58219, dilution 1:400), anti-ApoE (Santa Cruz Biotechnology, Dallas, TX, USA, sc-53570, dilution 1 :400), anti-vinculin (SigmaAldrich, Milan, Ltalis, V9264, dilution 1:10,000), thiab anti- -actin (Santa Cruz Biotechnology, Dallas, TX, USA, sc{60}}, dilution 1 : 10,000).
Membranes tau ua tiav incubated rau 1 h nyob rau hauv chav tsev kub nrog HRP-conjugated theem nrab anti-nas los yog tshuaj tiv thaiv luav (Bio-Rad Laboratories, Milan, Ltalis). Protein-bound antibodies tau pom los ntawm qhov tseeb ECL Western blotting (Bio-Rad Laboratories, Milan, Ltalis, # 1705061), thiab chemiluminescence tau sau npe los ntawm ChemiDoc MP system (Bio-Rad Laboratories, Milan, Ltalis). Densitometric tsom xam tau los ntawm Western blots tau ua tiav los ntawm kev siv ImageJ version 1.52t (National Institutes of Health, Bethesda, MD, USA) software rau Windows. Vinculin lossis -actin tau siv los ua cov khoom noj hauv tsev, uas tau ua haujlwm hauv kev tswj hwm rau kev thauj khoom protein. Densitometric xam tau txais raws li arbitrary units, muab los ntawm qhov sib piv ntawm qhov siv ntawm cov protein band thiab cov protein nyob hauv tsev.
4.3. Roj Liab O Staining
U373 hlwb tau cog rau ntawm poly-L-lysine (Sigma-Aldrich, P6282-5MG)-coated coverlips nyob rau hauv 6-zoo daim hlau, thiab Roj liab O staining tau ua raws li yav dhau los qhia [60]. Luv luv, cov hlwb raug kho rau hauv paraformaldehyde (4% tov) rau 10 min thiab maj mam yaug peb zaug nrog PBS. Cov hlwb ruaj khov tau tsim nrog 60% isopropanol rau 5 feeb thiab tom qab ntawd ntxuav nrog dej distilled. Tom qab ntawd, cov hlwb raug soj ntsuam nrog 1 mL ntawm Cov Roj Liab O ua haujlwm daws teeb meem (Sigma-Aldrich, O1391-250ML) rau 15 min ntawm chav tsev kub los ntawm kev muab lub phaj 6- zoo rau lub orbital rotator shaker. Tom qab incubation nrog staining tov, qhov dej tau ntxuav peb zaug nrog distilled dej kom tshem tawm cov stain ntau dhau.
Cov ntawv npog thaum kawg tau nruab nrog Fluoroshield mounting nruab nrab (Sigma-Aldrich, F6182), thiab Roj liab O autofluorescence tau pom los ntawm confocal microscopy (TCS SP8; Leica, Wetzlar, Lub teb chaws Yelemees). Cov duab raug ntes siv Leica TCS SP8 nruab nrog 63 × magnification thiab Leica LAS X Software (Milan, Ltalis). Quantification ntawm Roj liab O staining tau ua los ntawm ImageJ software rau Windows thiab xam raws li txhais tau tias fluorescence siv ib cheeb tsam ntawm tes.
4.4. Filipin Staining
Filipin staining yog ua los ntawm Filipin complex (Sigma-Aldrich, F9765). Filipin stock solution (10 mg/mL in PBS) yeej ib txwm npaj tam sim ua ntej siv. Cells raug tsau rau hauv paraformaldehyde (4% tov) rau 10 min thiab ntxuav nrog PBS. U373 ces stained nrog 1 mL Filipin ua hauj lwm tov (0.05 mg / mL hauv PBS) rau 1 h nyob rau hauv qhov tsaus ntuj nti, nyob rau hauv chav tsev kub. Tom qab ntawd, cov hlwb raug ntxuav peb zaug nrog PBS, thiab cov ntawv npog tau muab mounted nrog Fluoroshield mounting nruab nrab thiab tam sim ntawd soj ntsuam los ntawm confocal microscopy siv lub UV lim teeb (340-380 nm excitation). Cov duab tau txais ntawm 40X magnification. Filipin quantification raug xam raws li txhais tau tias fluorescence siv ib lub xov tooj ntawm tes los ntawm kev siv ImageJ software rau Windows.
4.5. Cholesterol Quantification
Kev ntsuas cov roj (cholesterol) tau ua los ntawm kev siv tshuaj ntsuam xyuas xim (Cholesterol Quantitation Kit, MAK043, Sigma-Aldrich, Milan, Ltalis) ua raws li cov chaw tsim khoom cov lus qhia.
4.6. ELISA
ApoE theem hauv kab lis kev cai nruab nrab raug soj ntsuam los ntawm kev siv Human Apolipoprotein ELISA Kit (Abcam, Cambridge, UK, ab108813), raws li cov chaw tsim khoom cov lus qhia.
4.7. Immunofluorescence
Immunofluorescence ntawm U373 hlwb raug tua raws li tau piav yav dhau los [60]. U373 tau kho nyob rau hauv paraformaldehyde (4% hauv PBS) thiab soj ntsuam ib hmos nrog thawj cov tshuaj tiv thaiv: anti-p75NTR (Santa Cruz Biotechnology, Dallas, TX, USA, sc-271708, dilution 1:100), thiab anti-TrkA ( Santa Cruz Biotechnology, Dallas, TX, USA, sc{10}}, dilution 1:100). Tom qab ntawd, cov hlwb raug incubated rau 1 h nrog tshis anti-nas thib ob antibody Alexa Fluor 555 (ThermoFisher Scientific, Waltham, MA, USA, A28180) thiab nrog tshis anti-luav theem nrab antibody Alexa Fluor 488 (ThermoFisher Waltham, USA, MA, USA, MA. A27034, ib.). DAPI staining tau ua kom pom cov nuclei, thiab cov ntawv npog thaum kawg tau nruab nrog Fluoroshield mounting nruab nrab. Cov qauv tau txheeb xyuas los ntawm confocal microscopy, raws li tau piav qhia saum toj no.
4.8. ApoE Silence
ApoE mRNA silencing tau ua rau U373 hlwb siv ApoE siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-29708) raws li cov chaw tsim khoom cov lus qhia. Kev hloov pauv tau raug teeb tsa hauv 6-cov ntaub so ntswg kab lis kev cai phaj qhov twg 100,000 cov cell ntawm ib qhov dej tau muab noob rau hauv DMEM tshuaj tua kab mob tsis pub loj hlob nruab nrab ntxiv nrog 10% ntawm FBS rau 24 teev. Rau txhua qhov dej, siRNA Transfection Medium (Santa Cruz Biotechnology, Dallas, TX, USA, sc-36868) tau ntxiv rau siRNA Transfection Reaction tov, npaj siv siRNA duplex thiab siRNA Transfection Reagent (Santa Cruz Biotechnology, Dallas, TX, USA, sc29528). Cells raug ntxuav ib zaug nrog siRNA Transfection Medium thiab npog nrog cov khoom sib xyaw ua ke ua ntej. Tom qab ntawd, cov kab lis kev cai phaj tau incubated rau 7 h ntawm 37 ◦C hauv 5% CO2 incubator.
DMEM kev loj hlob nruab nrab nrog 20% ntawm FBS thiab tshuaj tua kab mob (2-fold qhov qub concentration) tau ntxiv rau hauv cov dej sib tov sib xyaw nrog 1: 1 piv. Cells tau incubated rau 18 h, thiab ces qhov nruab nrab yog tawm thiab hloov nrog tshiab ib txwm loj hlob nruab nrab; 24 teev tom qab cov kauj ruam kawg no, cov hlwb tau npaj los siv rau kev sim ntxiv. Kev ua tau zoo ntawm ApoE silencing tau soj ntsuam los ntawm RT-qPCR hauv U373 hlwb thiab los ntawm ELISA hauv cell supernatant; ApoE siRNA ua tau zoo tiv thaiv ApoE induction los ntawm NGF yog piv rau scramble siRNA (ApoE mRNA: 83% txo; ApoE ELISA: 78% txo). Qhov tseem ceeb, ApoE qhia raws li siRNA silencing yog qis dua cov theem qhia hauv paus tau pom hauv kev tswj U373 (ApoE mRNA: 72% txo; ApoE ELISA: 58% txo) (Daim duab S 3A, B).
4.9. RNA Extraction thiab Real-Time PCR
mRNA tsom xam tau ua raws li tau tshaj tawm yav dhau los [22,63]. Luv luv, tag nrho RNA los ntawm U373 hlwb raug rho tawm nrog TRI Reagent (Merck Life Science, Milan, Ltalis) raws li cov chaw tsim khoom cov lus qhia. Kev kho DNAse (Ambion, Thermo Fisher Scientific, Milan, Ltalis) tau ua tiav, thiab tom qab ntawd RNA tau ua kom huv si siv RNA ntxuav cov khoom siv (Zymo, Ltalis). RNA tau ua tiav rov qab xa mus rau cDNA los ntawm Cov Khoom Siv Muaj Peev Xwm CDNA Rov Qab Rov Qab Cell Cov Khoom Siv (Applied Bio-System, Foster City, CA, USA) thiab siv rau kev tshuaj xyuas qPCR.
Primers siv nyob rau hauv qRT-PCR rau apoE yog: rau pem hauv ntej 5 0 -GGGTCGCTTTTTGGGATTACCTG-30 thiab thim rov qab 50 -CAACTCCTTTCATGGTCTCGTCC3 0 . Primers siv nyob rau hauv qRT-PCR rau gapdh (xaiv raws li cov noob siv) yog: rau pem hauv ntej 50 - GTCTCCTCTGACTTTCAACAGCG-30 thiab thim rov qab 50 -ACCACCCTGTTGCTGTAGCCA-30. Kev tsim tawm ntawm qhov tseeb amplicon raug soj ntsuam los ntawm kev ntsuam xyuas ntawm qhov nkhaus melting. Txhua tus qauv lom neeg tau khiav hauv triplicate nrog SYBR ntsuab IQ reagent (Bio-Rad Laboratories, Milan, Ltalis) thiab siv CFX Connect system kuaj xyuas (Bio-Rad Laboratories, Milan, Ltalis).
4.10. Quantitative Assessment ntawm Neuronal Morphology
Neuronal morphology nyob rau hauv N1E-115 hlwb tau kwv yees los ntawm kev ntes cov duab nrog lub tshuab ntsuas inverted. Kev soj ntsuam Morphometric tau ua tiav los ntawm ImageJ software rau Windows, thiab cov xov tooj ntawm tag nrho cov hlwb (qhia raws li feem pua ntawm kev tswj hwm), qhov nruab nrab neurite ntev (qhia raws li feem pua ntawm kev tswj), thiab cov hlwb neurite-cov kabmob tau kwv yees. Rau cov kab mob neurite-cov kabmob, qhov feem pua yog xam los ntawm kev faib cov cell sib txawv los ntawm tag nrho cov xov tooj ntawm ib daim teb. N1E-115 tau devas sib txawv thaum lawv muaj tsawg kawg yog ib qho neurite nrog qhov ntev sib npaug los yog ntau dua li txoj kab uas hla ntawm soma. Kev ntsuam xyuas morphological tau ua rau tsawg kawg tsib qhov kev sim ywj pheej, uas tsawg kawg peb cov duab raug tshuaj xyuas.
4.11. Kev txheeb cais
Tag nrho cov txiaj ntsig tau nthuav tawm hauv txoj kev tshawb fawb no tau qhia tias txhais tau tias ± SD (tus qauv sib txawv ntawm cov ntaub ntawv ib txwm raug kuaj xyuas los ntawm kev siv Shapiro-Wilk test. Thaum ob pawg kev sim tau muab piv, cov kev xeem tsis sib xws tau siv. Txawm li cas los xij, thaum peb lossis ntau pawg. Tau muab piv, ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) ua raws li Tukey's post hoc test tau ua haujlwm.Hais txog cov kev sim nrog rotenone, qhov twg muaj ob qhov sib txawv, ob txoj kev ANOVA ua raws li Bonferroni post-test tau siv. p < 0.05 raug suav hais tias yog qhov sib txawv ntawm qhov sib txawv. Kev txheeb xyuas txheeb cais tau ua tiav siv GraphPad Prism 5 (GraphPad, La Jolla, CA, USA) rau Windows.
Tus sau kev koom tes:
Conceptualization, MS; methodology, MS thiab MC; validation, MS thiab MC; kev soj ntsuam, MS, MC, DP, thiab MP; kev tshawb nrhiav, MC, NM, LL, MP, thiab DP; cov peev txheej, MS thiab VP; cov ntaub ntawv curation, MS thiab MC; kev sau ntawv-kev npaj ua ntej, MS thiab MC; kev sau ntawv- tshuaj xyuas thiab kho, MP, LL, DP, NM thiab VP; kev pom, MS, MC, thiab MP; kev saib xyuas, MS; kev tswj xyuas qhov project, MS; Kev nrhiav nyiaj txiag, MS 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 tau txais nyiaj los ntawm Cov Nyiaj Pab rau Kev Tshawb Fawb Kev Tshawb Fawb 2021 (University of Molise) rau MS, thiab los ntawm Jerome Lejeune Foundation hu rau Grant session 2021a, # 2043 rau MS
Institutional Review Board Statement:
Tsis siv tau.
Cov Lus Qhia Txog Kev Pom Zoo:
Tsis siv tau.
Cov ntaub ntawv muaj nyob:
Tsis siv tau.
Kev lees paub:
Peb ua tsaug Claudia Tonini rau kev pab nrog kev ntsuas cov roj cholesterol
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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