NGF Modulates Cholesterol Metabolism Thiab Stimulates ApoE Secretion hauv Glial Cells Conferring Neuroprotection Tawm tsam Oxidative Stress Part 1
Aug 31, 2023
Abstract:
Cov roj (cholesterol) ua lub luag haujlwm tseem ceeb hauv lub hlwb, qhov chaw ua haujlwm astrocytic tshwj xeeb tshaj yog tswj cov metabolism. Tseeb, cov neeg laus neurons txwv lawv cov roj cholesterol biosynthesis thiab xa cov sterol no los ntawm ApoE-nplua nuj cov khoom zais cia los ntawm astrocytes. Cov pov thawj tsis ntev los no qhia tau hais tias cov paj hlwb loj hlob (NGF) tuaj yeem ua rau muaj kev ua haujlwm neurotrophic los ntawm kev cuam tshuam cov cell metabolism.
Neurons yog lub hauv paus units uas tsim los ntawm tib neeg lub hlwb. Los ntawm kev sib txuas ntawm cov neurons, lawv tsim peb lub hlwb cortex, uas nqa tag nrho peb cov kev paub thiab kev nco. Yog li ntawd, cov neurons muaj feem cuam tshuam nrog lub peev xwm nco.
Lub luag haujlwm tseem ceeb ntawm neurons yog xa cov ntaub ntawv. Txhua neuron muaj ib lub synapse, uas txuas cov dendrites thiab axons ntawm lwm cov neurons. Thaum ib tug neuron tau txais ib lub teeb liab, nws hla lub teeb liab mus rau cov neurons uas nws txuas nrog. Qhov kev taw qhia no, hu ua neuronal kis tau tus mob, yog ib qho ntawm cov txheej txheem tseem ceeb hauv kev tsim kev nco, khaws cia, thiab rov qab.
Ua kom peb lub hlwb noj qab nyob zoo, peb tuaj yeem txhawb kev ua haujlwm zoo ntawm peb cov neurons los ntawm kev tawm dag zog. Kev tawm dag zog lub cev txhim kho cov ntshav ncig thiab cov pa oxygen rau lub hlwb, yog li ua kom cov lej thiab kev sib txuas ntawm cov neurons. Nyob rau tib lub sijhawm, ntau yam kev sib raug zoo thiab kev kawm ua haujlwm tuaj yeem tsim cov neurons tshiab hauv cov hlwb tshiab thiab ua kom lub hlwb muaj zog.
Thaum peb muaj ntau dua, muaj zog neurons, peb lub cim xeeb yuav muaj zog. Synchrony ntawm neurons nyob rau hauv lub paj hlwb cortex ua ntau dua, ua kom yooj yim rau peb khaws thiab khaws cov ntaub ntawv, txhim kho peb lub peev xwm kev txawj ntse. Tsis tas li ntawd, ntau tus neurons thiab kev sib txuas ntawm cov neurons kuj tseem yuav txhim kho peb cov tshuaj tiv thaiv kom ceev thiab muaj peev xwm txiav txim siab.
Yog li ntawd, tswj kev noj qab haus huv neurons thiab txhim kho kev nco mus koom tes. Peb tuaj yeem txhim kho peb lub cim xeeb los ntawm kev txhawb nqa cov neurons noj qab haus huv los ntawm kev tsom mus rau kev tawm dag zog lub cev, ntau yam kev sib raug zoo thiab kev kawm, thiab kev noj zaub mov zoo. Cia peb ua haujlwm ua ke los txhawb peb lub hlwb kev noj qab haus huv! Nws pom tau tias peb yuav tsum txhim kho peb lub cim xeeb. Cistanche deserticola tuaj yeem txhim kho kev nco zoo vim Cistanche deserticola yog cov khoom siv tshuaj suav tshuaj suav nrog ntau yam tshwj xeeb, ib qho ntawm kev txhim kho kev nco. Kev ua tau zoo ntawm cov nqaij minced yog los ntawm ntau yam khoom xyaw uas nws muaj, suav nrog carboxylic acid, polysaccharides, flavonoids, thiab lwm yam. Cov khoom xyaw no tuaj yeem txhawb kev noj qab haus huv ntawm lub hlwb los ntawm ntau txoj hauv kev.
Nyem paub txoj hauv kev los txhim kho lub hlwb
Txawm li cas los xij, qhov cuam tshuam ntawm NGF ntawm glial cholesterol homeostasis tseem tsis tau piav qhia. Qhov phiaj xwm no yog txhawm rau txheeb xyuas seb NGF puas tuaj yeem cuam tshuam cov roj cholesterol metabolism hauv glial hlwb. U373 astrocyte-derived cell kab tau siv los ua tus qauv sim kom ncav cuag lub hom phiaj no. Immunoblot thiab ELISA tsom xam pom tias cov proteins thiab enzymes uas yog cov cholesterol metabolism hauv network tau nce ntxiv thaum kho NGF hauv cov hlwb glial. Tsis tas li ntawd, NGF nce ntxiv ApoE secretion thiab tus nqi ntawm cov cholesterol extracellular hauv kab lis kev cai nruab nrab.
Kev sib koom ua ke thiab U373-qhov kev sim nruab nrab tau ua pov thawj tias NGF kev kho mob tau zoo tiv thaiv rotenone-mediated cytotoxicity hauv N1E-115 cov hlwb neuronal. Conversely, neuroprotection mediated los ntawm NGF kev kho mob yog suppressed thaum N1E-115 tau co-cultured nrog ApoE-silenced U373 hlwb. Cov ntaub ntawv no qhia tias NGF tswj cov roj cholesterol homeostasis hauv glial hlwb. Qhov tseem ceeb tshaj, NGF tawm dag zog tiv thaiv kab mob tiv thaiv oxidative kev nyuaj siab, yuav cuam tshuam nrog induction ntawm glial ApoE secretion.
Ntsiab lus:
ApoE; astrocyte; cov roj cholesterol; kev sib txawv; paj hlwb loj hlob; metabolism; neuron; reactive oxygen hom; rotenone ua.
1. Taw qhia
Lub hlwb yog tus cwj pwm los ntawm cov roj (cholesterol) ntau tshaj plaws hauv lub cev [1]. Qhov tshwj xeeb tshwj xeeb ntawm cov sterol no hauv lub hauv nruab nrab paj hlwb (CNS) yog qhov tseeb los ntawm qhov tseeb tias cov roj cholesterol koom nrog ntau yam txheej txheem lom neeg. Nco ntsoov, ib qho tseem ceeb ntawm cov roj cholesterol tau koom ua ke rau hauv cov ntshav plasma daim nyias nyias ntawm astrocytes thiab neurons, qhov uas nws txhais tau hais tias lub cellular morphology thiab kev ua haujlwm raug ntawm daim nyias nyias receptors thiab synaptic neurotransmission [2].
Cov roj (cholesterol) tseem yog qhov tseem ceeb rau kev tsim cov ntaub so ntswg tshiab uas yuav tsum tau muaj rau cov neurite outgrowth, synaptogenesis, thiab neurotransmitter tso tawm, yog li tseem ceeb hauv kev sib txawv neuronal thiab maturation [3-7].
Qhov tseem ceeb, tag nrho cov roj cholesterol hauv lub hlwb tau txais los ntawm de Novo biosynthesis hauv situ. Tshwj xeeb, cov ntshav-hlwb barrier (BBB) tiv thaiv cov roj cholesterol los ntawm lipoproteins thauj los ntawm cov kab mob hauv lub cev [8]. Vim li no, cov paj hlwb tsim ib qho piv txwv ntawm kev tswj hwm kev tswj hwm ntawm nws tus kheej los tswj lawv tus kheej qhov kev thov rau cov lipid no. Ib tus qauv zoo tsim tau tias tom qab embryogenesis, thaum lub sijhawm postnatal, cov roj cholesterol biosynthesis poob qis hauv cov neurons, nrog rau kev sim txuag lub zog ua haujlwm rau tiam ntawm qhov kev ua tau zoo.
Raws li, astrocytes zaum cov kev xav tau ntawm neuronal ntawm cov roj cholesterol, uas nws cov synthesis tau tshaj tawm [1,9,10]. Kev xa cov roj cholesterol tsim los ntawm astrocytes mus rau cov neurons txhais tau hais tias muaj lub luag haujlwm zoo nkauj ntawm kev thauj mus los, uas suav nrog cov protein thiab cov khoom tseem ceeb. Tshwj xeeb, cov roj cholesterol export pib nyob rau hauv astrocytes, qhov twg neo-synthetized cholesterol yog loaded rau hauv nascent lipoprotein hais uas muaj apolipoprotein E (ApoE). Cov ntaub ntawv pov thawj qhia tau hais tias cov lipidation ntawm ApoE-nplua nuj hais yog kho los ntawm tus thauj khoom ATP-binding cassette A1 (ABCA1) [11]. Tom qab ntawd, ib zaug tso rau hauv qhov chaw ntxiv, ApoE-muaj lipoproteinsuptakentaken los ntawm neurons los ntawm receptor-mediated endocytosislow-densitynsity lipoprotein receptor (LDLr) thiab LDLr-related protein 1 (LRP1) [12].
Raws li lub luag haujlwm tseem ceeb ntawm astrocyte-derived cholesterol hauv neuronal homeostasis, kev tshawb fawb pom tau hais tias cov roj cholesterol muaj nyob hauv ApoE-nplua nuj lipoproteins ua rau muaj txiaj ntsig zoo rau kev sib txawv ntawm dendrite thiab synaptic muaj nuj nqi [13,14]. Accordinastrocyte conditionedioned nruab nrab los ntawm Huntington's kab mob (HD) astrocytes, uas tsuas yog tso tawm ib qho me me ntawm ApoE-lipoprotein-bound cholesterol, cuam tshuam rau neurite outgrowth thiab synaptic kev ua si hauv HD neurons [15].
Qhov kev txiav txim ntawm neurotrophic ntawm astrocyte-derived ApoE-muaj cov khoom yog txuas ntxiv los ntawm kev cuam tshuam ntawm ApoE deficiency, uas ua rau muaj kev cuam tshuam rau cov neeg laus neurogenesis hauv hippocampus [16]. Astrocyte-neuron cholesterol crosstalk kuj tseem ceeb heev hauv kev tiv thaiv neuro tiv thaiv oxidative kev nyuaj siab. Piv txwv li, qee cov ntawv ceeb toom qhia tias ApoE cuam tshuam oxidative puas tsuaj thiab apoptosis tshwm sim los ntawm hydrogen peroxide raug [17,18].
Los ntawm cov kev soj ntsuam no, kev saib xyuas ntawm cov roj cholesterol homeostasis hauv lub hlwb yog qhov tsim nyog los lav kev ua haujlwm ntawm cov hlab ntsha hlwb, thiab cov txheej txheem metabolic hauv qab kev tswj hwm ntawm cov lipid no yuav tsum tau tswj kom zoo kom tswj tau qhov tshwm sim ntawm ntau cov txheej txheem neurophysiological.
Nyob rau hauv xyoo tas los no, neurotrophins tau tshwm sim los ua qhov tseem ceeb hauv cov metabolism hauv cell [2,19–22]. Neurotrophins belongs rau tsev neeg me me ntawm kev loj hlob yam tseem ceeb uas muaj lub luag haujlwm tsis sib haum xeeb hauv kev loj hlob neuronal, ciaj sia taus, thiab plasticity [23]. Tsis ntev los no, cov cim qhia ntawm cov khoom no kuj tau lees paub tias yog cov kev hloov pauv tseem ceeb ntawm cov cell metabolism, suav nrog cov roj cholesterol metabolism [2,22].
Cov pov thawj kev sim qhia tau hais tias cov paj hlwb loj hlob (NGF), tus qauv qauv ntawm tsev neeg neurotrophin, txhawb nqa cov roj cholesterol biosynthesis hauv cov hlwb neuronal thiab hepatocytes [24,25]. Interestingly, cov xwm txheej no koom nrog, yam tsawg kawg ntawm ib feem, qhov qis-kev sib raug zoo neurotrophin receptor p75NTR, uas ua rau kev ua kom cov sterol regulatory element binding protein 2 (SREBP-2) thiab, nyob rau hauv lem, induces transcription ntawm cov roj cholesterol. noob [25]. Kev ua haujlwm ntawm NGF ntawm cov roj cholesterol metabolism tsis txwv rau biosynthesis, vim nws kuj txhawb nqa cov roj cholesterol los ntawm kev nce LDLr qhia hauv neurons [26].
Txawm hais tias cov ntaub ntawv no, kev paub txog kev ua tau zoo ntawm NGF hauv kev tswj hwm ntawm cov roj cholesterol homeostasis tseem tshwm sim tshwj xeeb. Qhov tseem ceeb, tsis muaj kev tshawb fawb txog kev ntsuas lub luag haujlwm ua los ntawm NGF hauv kev tswj hwm cov roj cholesterol metabolism hauv astrocytic hlwb. Xav txog tias cov roj (cholesterol) tsim los ntawm astrocytes yog qhov tseem ceeb los lav qhov kev xav ntawm lub cev, nws yog ib qho tseem ceeb kom nkag siab txog sebcanle puas cuam tshuam rau cov roj cholesterol metabolism hauv glial hlwb nrog rau kev thauj mus los ntawm cov cholesterol intercellular, nws cov kev cai tshwj xeeb yog qhov tseem ceeb los xyuas kom lub hlwb noj qab haus huv.
Raws li txoj haujlwm no, lub hom phiaj yog txhawm rau tshawb nrhiav qhov kev koom tes yav tom ntej ntawm NGF hauv kev hloov pauv ntawm cov roj cholesterol metabolism hauv glial hlwb, nrog rau nws qhov cuam tshuam rau kev sib txawv ntawm neuronal thiab neuronal ciaj sia nyob rau hauv oxidative kev nyuaj siab.
2. Cov txiaj ntsig
2.1. U373 Qhia Ob NGF Receptors TrkA thiab p75NTR
NGF tau pib pom tias yog ib qho kev loj hlob nrog rau qhov tshwj xeeb tropism rau cov hlwb neuronal. Txawm li cas los xij, accumulating eviddemonstratestrate hais tias qhov no neurotrophin kuj induces lom teebmeem nyob rau hauv ob peb non-neuronal hlwb thiab cov ntaub so ntswg [22]. Qhov kev ua ntawm NGF tau lees paub los ntawm nws txoj kev khi rau high-affinity receptor TrkAd rau qhov qis-affinity receptor p75NTR [2].
Yog li, peb pib paub tseeb tias U373 hlwb qhia NGF receptors. Qhov tseem ceeb, U373 yog ib qho astrocytoma cell kab dav dav ua haujlwm raws li astrocyte cell kab lis kev cai qauv, raws li ntau cov ntaub ntawv qhia tau hais tias lawv reproseveraler ntawm morphological, metabolic, thiab functional zog ntawm astrocytes [27-29]. Raws li cov ntaub ntawv luam tawm [30], immunofluorescence tsom xam pom tias U373 hlwb nthuav tawm ob qho tib si TrkA thiab p75NTR ntawm qib txaus (Daim duab S1A). Cov txiaj ntsig no kuj tau lees paub los ntawm Western blot tsom xam, qhia tias U373 hlwb muaj peev xwm ua tau zoo rau NGF.
Txij li thaum kev tswj hwm ntawm NGF tuaj yeem cuam tshuam rau qib kev qhia ntawm nws cov receptors, muaj peev xwm hloov pauv qhov sib npaug ntawm cov kev taw qhia txoj hauv kev los ntawm TrkA thiab / lossis p75NTR [31-33], cov lus qhia ntawm ob qho tib si receptors kuj tau kuaj xyuas thaum 48 h ntawm NGF kev kho mob. Txawm li cas los xij, tsis muaj kev hloov pauv hauv cov protein ntau ntawm TrkA thiab p75NTR tom qab kev txhawb nqa nrog cov neurotrophin (Daim duab S1B).
2.3. NGF nce cov roj cholesterol secretion los ntawm U373 rau hauv kab lis kev cai nruab nrab
Cov txiaj ntsig tau ua rau peb kawm seb qhov cuam tshuam los ntawm NGF tuaj yeem cuam tshuam qhov kev hloov pauv ntawm cov roj cholesterol. Roj Liab O staining tau ua kom pom cov lipids nruab nrab, suav nrog cov roj cholesterol esters. Qhov loj thiab tus naj npawb ntawm cov lipid droplets pom hauv U373 hlwb zoo ib yam li cov uas tau tshaj tawm rau thawj astrocytes [34,35].
Txawm li cas los xij, tsis muaj kev hloov pauv raws li kev kho NGF (Figures 2A thiab S2A, B). Ib yam li ntawd, tsis muaj kev hloov pauv ntawm cov roj (cholesterol) dawb, ntsuas los ntawm filipin staining (Figures 2B thiab S2C). Qhov tsis muaj qhov cuam tshuam tseem ceeb ntawm cov roj cholesterol hauv lub cev tau txuas ntxiv los ntawm kev soj ntsuam cov roj (cholesterol) enzymatic, uas qhia tias NGF tsis hloov cov ntsiab lus ntawm tag nrho cov roj (cholesterol), cov roj (cholesterol) dawb, thiab cov roj (cholesterol) esters (Daim duab 2C).
Hloov pauv, cov roj cholesterol (dawb, esters, thiab tag nrho) tau nce ntxiv hauv kab lis kev cai nruab nrab ntawm U373- kho NGF (Daim duab 2D). Thaum ntsuas tag nrho, cov ntaub ntawv no qhia tau hais tias NGF txhawb nqa cov roj cholesterol extrusion los ntawm kev txhim kho ABCA1-kev sib haum xeeb efflux ntawm ApoE-muaj cov khoom.
Daim duab 1. NGF modulates cov lus qhia ntawm cov proteins tseem ceeb koom nrog hauv cov roj cholesterol homeostasis hauv U373 hlwb thiab txhim kho ApoE secretion n hauv U373 kab lis kev cai nruab nrab. (A–G) Tus Neeg Sawv Cev Western blot thiab densitometric tsom xam ntawm SREBP-1, SREBP-2, HMGCR, LDLr, NPC1, ABCA1, thiab ApoE hauv U373 hlwb kho nrog tsheb (Ctrl) thiab NGF (100 ng / mL) rau 48 h. n=6 kev sim sib txawv.
Actin thiab vinculin tau siv los tswj kev thauj khoom. (H) Quantification ntawm ApoE qib (µg/mL) los ntawm ELISA kev soj ntsuam hauv tsheb- thiab NGF-kho U373 kab lis kev cai nruab nrab. n=3 kev sim sib txawv. Cov ntaub ntawv sawv cev txhais tau tias ± SD. Kev txheeb xyuas txheeb cais tau raug soj ntsuam los ntawm ib qho kev sib tw Studet-test test. * p < 0 05, ** p < 0.01, *** p < 0.001.
2.4. p75NTR yog ib feem koom nrog NGF-Mediated Effects ntawm Cholesterol Protein Network
Cov kev tshawb fawb pom tau hais tias NGF cuam tshuam rau cov roj cholesterol metabolism hauv daim siab hlwb los ntawm kev ua kom nws cov affinity qis p75NTR [25]. Txhawm rau kuaj seb txoj kev taw qhia no puas tseem tuaj yeem khaws cia hauv astrocytic hlwb, peb tau kho U373 nrog LM11A-31, lub ligand me me ua raws li qhov tshwj xeeb p75NTR modulator. Qhov sib xyaw no tsis qhib TrkA tab sis, zoo ib yam li NGF, elicits induction ntawm p75NTR downstream signaling pathways [36]. LM11A-31 ua raws li NGF-mediated nce ntawm HMGCR (Daim duab 3A).
Txawm li cas los xij, tsis muaj kev hloov pauv hauv lwm cov protein uas koom nrog hauv cov roj cholesterol metabolism (Daim duab 3B-D), los yog cov nyiaj ntawm extracellular ApoE (Daim duab 3E) thiab cov cholesterol (tag nrho, dawb, thiab esters) tso rau hauv kab lis kev cai nruab nrab (Daim duab 3F. ). Ua ke, cov txiaj ntsig no qhia tau hais tias NGF-induced qhia ntawm HMGCR yog kho los ntawm p75NTR kev ua, qhov cuam tshuam ntawm NPC1, ABCA1, ApoE secretion, thiab cov roj cholesterol extrusion yog nyob ntawm lwm txoj hauv kev hloov pauv, tejzaum nws koom nrog TrkA.
Daim duab 2. NGF cuam tshuam rau cov lipids nruab nrab thiab cov roj cholesterol. (A) U373 hlwb raug kho nrog tsheb (Ctrl) lossis NGF (100 ng / mL) rau 48 h thiab tom qab ntawd pleev xim nrog Roj Liab O kom pom cov ntsiab lus intracellular ntawm cov lipids nruab nrab. n=3 kev sim sib txawv. (B) Cov duab sawv cev ntawm filipin staining ua rau U373 hlwb kho nrog lub tsheb (Ctrl) thiab NGF (100 ng / mL) rau 48 teev. n=3 kev sim sib txawv. (C) Kev ntsuas cov roj (cholesterol) tag nrho (tag nrho cov roj cholesterol, cov roj cholesterol dawb, thiab cov roj cholesterol) hauv lub tsheb- thiab NGF-kho U373 hlwb. n=3 kev sim sib txawv. (D) Kev ntsuas cov roj (cholesterol) tag nrho, cov roj cholesterol dawb, thiab cov roj cholesterol esters hauv kab lis kev cai nruab nrab ntawm U373 hlwb kho nrog lub tsheb (Ctrl) thiab NGF (100 ng / mL) rau 48 teev. n=3 kev sim sib txawv. Cov ntaub ntawv sawv cev txhais tau tias ± SD. Kev txheeb xyuas txheeb cais tau ua los ntawm kev hu nkauj tus tub ntxhais kawm tsis muaj kev sib tw t-test. **p<0.01.
Daim duab 3. Cov teebmeem ntawm p75NTR kev hloov kho los ntawm LM11A-31 ntawm cov roj cholesterol metabolism thiab tso tawm. (A–D) Tus Neeg Sawv Cev Western blot thiab densitometric tsom xam ntawm HMGCR, ABCA1, NPC1, thiab ApoE hauv U373 hlwb kho nrog lub tsheb (Ctrl) thiab LM11A-31 (0.1 µM) rau 48 teev. n=4 kev sim sib txawv.
Vinculin tau txais txiaj ntsig los ua cov khoom noj hauv tsev kom normalize protein ntau. (E) Quantification ntawm ApoE tus nqi (µg/mL) los ntawm ELISA kev soj ntsuam hauv kab lis kev cai nruab nrab ntawm lub tsheb- thiab LM11A-31- kho U373 hlwb. n=3 kev sim sib txawv. (F) Kev ntsuas cov roj (cholesterol) tag nrho, cov roj (cholesterol) dawb, thiab cov roj cholesterol esters hauv kab lis kev cai nruab nrab ntawm U373 hlwb kho nrog tsheb (Ctrl) thiab LM11A-31 (0.1 µM) rau 48 h. n=3 kev sim sib txawv. Cov ntaub ntawv sawv cev txhais tau tias ± SD. Kev txheeb xyuas txheeb cais tau ua tiav los ntawm kev siv ib qho kev sib tw Studet-test test. *** p < 0.001.
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