A Novel Netrin-1-Drived Peptide Enhances Protection Against Neuronal Death Thiab Mitigates Of Intracerebral Hemorrhage in Mos Part 2

Tsis tas li ntawd, peb pom tias peptide E1 tshwj xeeb qhib rau FAK thiab SFK txoj kev taw qhia. Yog li, peptide E1 tuaj yeem muaj cov teebmeem tshwj xeeb thiab ua haujlwm zoo hauv cov txheej txheem rov qab los ntawm ICH.

Muaj kev sib raug zoo ntawm qhov tshwj xeeb thiab kev nco. Qhov tshwj xeeb siab yog hais txog kev ua haujlwm muaj zog hauv qee qhov chaw ntawm lub hlwb thaum tib neeg ua haujlwm. Kev nco yog hais txog tib neeg lub peev xwm los kawm thiab nco qab cov ntaub ntawv. Thaum peb kawm siv qhov tshwj xeeb los ua haujlwm, peb lub cim xeeb kuj yuav nce ntxiv.

Kev tshawb fawb qhia tau hais tias thaum peb muaj kev txawj ntse tshwj xeeb thiab kev paub dhau los hauv kev ua haujlwm, lub hlwb yuav maj mam tsim tshwj xeeb neural circuits thiab cim xeeb qauv. Cov kab hluav taws xob thiab cov qauv no yuav muaj kev cuam tshuam tshwj xeeb rau cov lus teb rau kev ua haujlwm, yog li txhim kho kev ua haujlwm ntawm kev ua haujlwm. Nyob rau tib lub sijhawm, thaum ua cov haujlwm zoo sib xws yav tom ntej, cov circuits thiab cov qauv no tseem yuav qhib kom pab peb nco qab zoo dua thiab ua haujlwm.

Cov haujlwm uas muaj qhov tshwj xeeb muaj xws li suab paj nruag, lus, kis las, thiab lwm yam, thiab kev kawm cov txuj ci no tuaj yeem txhim kho peb lub cim xeeb. Piv txwv li, thaum tus pianist xyaum ua si, lub hlwb yuav kawm txog cov kev txawj ntse ntawm piano thiab ntxiv dag zog rau cov circuits no thaum lub sij hawm xyaum ua. Kev tsim thiab ntxiv dag zog rau cov circuits no tseem yuav txhim kho cov neeg pianist kev nkag siab thiab nco txog cov suab paj nruag.

Yog li ntawd, muaj kev sib raug zoo ntawm qhov tshwj xeeb thiab kev nco. Kev kawm tshwj xeeb thiab cov kev paub dhau los tuaj yeem tsis tsuas yog txhim kho peb cov kev ua tau zoo tab sis kuj ntxiv dag zog rau peb lub cim xeeb, pab peb kom kawm tau zoo dua thiab nco txog ntau yam ntaub ntawv. Nws tuaj yeem pom tias peb yuav tsum txhim kho kev nco, thiab Cistanche tuaj yeem txhim kho kev nco zoo vim tias nws muaj antioxidant, tiv thaiv kev mob, thiab tiv thaiv kev laus, uas tuaj yeem pab txo qis oxidation thiab inflammatory tshwm sim hauv lub hlwb, yog li tiv thaiv kev noj qab haus huv. paj hlwb. Tsis tas li ntawd, Cistanche tseem tuaj yeem txhawb kev loj hlob thiab kho cov paj hlwb, yog li txhim kho kev sib txuas thiab kev ua haujlwm ntawm neural networks. Cov teebmeem no tuaj yeem pab txhim kho kev nco, kev kawm muaj peev xwm, thiab kev xav nrawm, thiab tseem tuaj yeem tiv thaiv qhov tshwm sim ntawm kev paub tsis meej thiab cov kab mob neurodegenerative.

Nyem paub cov tshuaj ntxiv los txhawb kev nco

Tsis zoo li peptide E1, peptide E2 tuaj yeem qhib ERK signaling, uas txhawb kev tuag neuronal. Qhov no tuaj yeem tshwm sim los ntawm Cx(1–2)Cx(3–4)Tx(0 -1)G motifin E2 uas tuaj yeem qhib ERK signaling pathway [28].

Qhov no qhia tau hais tias txawvNetrin-1-peptides muab tau los ua haujlwm sib txawv.Lub ntsiab receptors ntawm Netrin-1 yog DCC thiab cov tswv cuab ntawm UNC5 tsev neeg [50]. Lub luag haujlwm ntawm DCC thiab UNC5H2 hauv ICH tseem muaj teeb meem [51,52].

Cov kab ke ntawm Netrin-1 peb tau txheeb xyuas yog qhov tseem ceeb rau kev sib cuam tshuam ntawm Netrin-1 nrog DCC [19]. Cov txiaj ntsig no qhia tias DCC tuaj yeem koom nrog hauv Netrin-1-vim ua haujlwm rov qab los tom qab ICH.

Cov kev tshawb fawb ntxiv yog xav tau los tshawb xyuas cov txheej txheem hauv qab ntawm cov txheej txheem no thiab cov kev cuam tshuam txog kev taw qhia. Hauv kev sim yav tom ntej, peb yuav ntsuas cov khoom siv roj ntsha ntawm peptide E1 los tshawb txog nws cov tshuaj lom thiab ib nrab-lub neej rau kev siv tshuaj kho mob.

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Tsiaj txhu

Lub chaw kuaj mob yog ib lub chaw kuaj kab mob tsis muaj kab mob (SPF). Txhua tus nas tau nyob hauv 12-h lub voj voog tsaus ntuj nrog lub teeb ntawm 6 teev sawv ntxov txog 6 teev tsaus ntuj thiab muab kev nkag mus rau zaub mov ad libitum. DCC +/- nas tau tsim tawm raws li tau piav qhia yav dhau los [53].

Embryosof DCC +/- nas tau siv rau cortical neuronal kab lis kev cai. Genotyping tau ua los ntawm polymerase saw cov tshuaj tiv thaiv (PCR) raws li tau piav qhia yav dhau los [53]. Cov nas C57BL/6J tau yuav los ntawm Beijing Huafukang Bioscience Co., Ltd. (Beijing, Suav).

4.2. Kev tsim kho

Kev nthuav qhia tsim encoding tib neeg DCC (HGNC: 2701), tib neeg Netrin-1 (HGNC: 8029), thiab tib neeg UNC5A (HGNC: 12567) tau tsim tawm hauv peb chav kuaj [14,32,45].

pcDNA 3. cDNA ntu ntawm hNetrin-1 (∆407–443) tau pom nyob rau hauv Cov Lus Ntxiv S2.

cDNA cov kab ke sib raug rau FN5 lub npe ntawm DCC thiab cov amino acids 407–443 ntawm Netrin-1 tau tsim los ntawm ligation rau hauv pGEX-5x-1vector los tsim GST fusion proteins.

4.3. Sau los ntawm Netrin-1 Kho Mob Nruab Nrab

Lub netrin-1-qhov nruab nrab qhov nruab nrab tau sau thiab siv tau raws li tau piav qhia hauv peb tsab ntawv tshaj tawm dhau los [13,14]. Luv luv, Myc-Nws-tagged tib neeg Netrin-1 tau qhia hauv HEK293Tcells.

Tom qab 24 teev, cov hlwb raug ntxuav 3 zaug nrog Opti-MEM (Invitrogen, 31985070, Carlsbad, CA, USA) thiab tom qab ntawd incubated rau peb hnub nyob rau hauv serum-dawb Opti-MEM.

Cov xov xwm tau txhawb nqa los ntawm centrifugal filtration (Millipore, UFC903096, Billerica, MA, USA) thiab khaws cia ntawm −80 ◦C. Enriched Netrin-1 tau immunoblotted nrog anti-Nws thiab kom muaj nuj nqis los ntawm kev sib piv nrog BSA raws li kev tswj kev thauj khoom.

Cov nyhuv ntawm Netrin{{0}} tom qab ntawd tau soj ntsuam los ntawm phosphorylation ntawm FAK. Nquag, 0.1 mg / mL ntawm Netrin-1 ua rau muaj txiaj ntsig zoo ntawm FAK phosphorylation, thiab qhov concentration no tau siv hauv peb cov kev tshawb fawb. Lub sijhawm ntawm Netrin-1kev txhawb nqa yog 20 feeb hauv txhua qhov kev sim tshwj tsis yog qhia lwm yam.

4.4. Western Blot Analysis

Western blotting tau ua raws li tau piav qhia yav dhau los [54]. Cov qauv tau muab pov tseg hauv lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 100 mM NaF, 1 mM vanadate, thiab protease inhibitors) rau lyse hlwb thiab cov ntaub so ntswg tshiab.

Thawj cov tshuaj tiv thaiv yog: nas anti-DCC (1: 500, #554223, BD, San Jose, CA, USA), nas anti-His (1: 1000, TA-02, ZSGB-BIO, Beijing, Suav teb ), nas anti-myc (1:1000, 22E8, Sungene Biotech, Tianjin, Tuam Tshoj), nas anti-Netrin-1 (1:1000, ALX804838, Enzo Life Sciences, Farmingdale, New York, USA), nas anti-flag (1:1000, F3165, Sigma Aldrich, St Louis, MO, USA), luav anti-phospho-FAK(pTyr861) (1:1000, F9176, Sigma Aldrich, St Louis, MO, USA), luav anti-FAK (1:200, sc-557, Santa Cruz, Santa Cruz, CA, USA), luav anti-phosphoSRC (Tyr418)(1:1000, Invitrogen, Carlsbad, CA, USA) , luav anti-SRC Tsev Neeg (1: 1000, # 2123, CST, Danvers, MA, USA), nas anti-GAPDH (1: 5000, TransGene Biotech, Beijing, Suav), nas anti-beta actin (1: 3000, A5441, Sigma Aldrich, St Louis, MO, USA), luav anti-phospho-ERK (Thr202/Tyr204) (1:1000, #9101, CST, Danvers, MA, USA), rabbitanti-ERK (1:1000, # 4695, CST, Danvers, MA, USA), HRP-conjugated Secondary antibody-gainst nas, tshis lossis luav tau yuav los ntawm Jackson ImmunoResearch (West Grove, PA, USA).

4.5. Cell Surface Binding

HEK293 hlwb plated ntawm daim npog npog tau hloov pauv nrog Flag-DCC plasmids siv cov txheej txheem calcium phosphate raws li tau piav qhia yav dhau los [55]. Kwv yees li 40 h tom qab hloov pauv, cov hlwb tau tsim nrog 100 µg/mL myc-Netrin-1 lossis myc-Netrin-1 (∆407–443) rau 30 min ua ntej ntxuav rau 5 min nrog HBHA (20 mM HEPES, pH 7.0, thiab 0.5 mg / mLBSA hauv HBSS), yaug nrog 1 × PBS, thiab tsau nrog 4% paraformaldehyde inPBS rau 10 min. Thawj cov tshuaj tiv thaiv uas tau siv yog luav anti-myc (1: 200, Abcam, ab9106, Cambridge, MA, USA) thiab nas anti-flag (1: 100, Sigma Aldrich, F3165) antibodies.

Cov tshuaj tiv thaiv kab mob thib ob uas tau siv yog Alexa Fluor 488-conjugated donkeyanti-Lavbit (1:1000; Invitrogen, A21206) thiab Alexa Fluor 546-conjugated tshis anti-nas (1:1000; Invitrogen, A10036) antibodies . Lub nuclei tau stained nrog 40,6-diamino-2-phenylindole (DAPI) (Thermo Fisher, Waltham, MA, USA). Tag nrho cov duab tau txais nrog Zeiss LSM 880 confocal microscope.

4.6. GST rub-down

GST-DCC fusion protein tau ntxuav nrog glutathione-SepharoseTM 4B hlaws (GE Healthcare, Little Chalfont, Buckinghamshire, UK) los ntawm cov chaw tsim khoom raws tu qauv.

Kwv yees li 500 µg ntawm cell lysate los ntawm HEK293T hlwb hloov nrog cov qhia plasmids tau incubated nrog 2-5 µg ntawm GST-DCC-FN5 fusion protein inRIPA tsis nyob ntawm 4 ◦C thaum hmo ntuj. Glutathi-one-SepharoseTM 4B hlaws tau siv los ntes GST-DCC-FN5 fusion protein thiab nws cov protein sib cuam tshuam. Cov proteins uas khi tau raug tso tawm rau hauv cov protein thauj khoom tsis pub dhau los ntawm kev kub denaturation.

4.7. Primary Cortical Neuron Culture

Lub hauv paus cortical neurons tau coj los ua raws li tau piav qhia yav dhau los [56]. Hauv luv luv, embryos (E17) tau raug tshem tawm los ntawm cov nas uas cev xeeb tub. Cov cerebral cortices tau muab cais thiab txiav mus rau hauv me me pieces.

Tom qab incubation nyob rau hauv {{0}}.125% Trypsin Plus nrog 0.05% DNase nyob rau hauv HBSS ntawm 37 ◦C rau 20 min, cov hlwb tau triturated nrog hluav taws-polished iav Pasteur pipettes thiab lim nrog ib tug 40 µm lim.

Dissociated cells raug tshem tawm hauv DMEM nrog 10% FBS thiab plated ntawm poly-D-lysine-coated lauj kaub tais diav lossis iav npog ntawm 37 ◦C hauv 5% CO2 atmosphere. Tom qab 4 teev, qhov nruab nrab tau hloov nrog ib qho nruab nrab neurobasal supplemented nrog B27 thiab GlutaMAX.

4.8. NLT Cell Culture thiab Treatment Protocols

Mouse GnRH-expressing neuronal cells (NLT) tau khaws cia los ntawm peb lub chaw kuaj mob. NLTcells tau loj hlob hauv DMEM (Sigma Aldrich, St Louis, MO, USA) muaj 10% FBS (BiologicalIndustries, Kibbutz Beit Haemek, Israel) thiab 1% penicillin / streptomycin (Thermo FisherScientific, Waltham, MA, USA) ntawm 37 hauv qab no ◦ ib qho av humidified ntawm 95% cua thiab 5% CO2.

Txhawm rau txiav txim siab txog kev ua haujlwm tsis zoo ntawm hemin, ntau qhov ntau (0, 10, 30, 60, 90, thiab 120 µM) ntawm hemin (Sigma-Aldrich) tau ntxiv rau NLT hlwb thiab kho rau 6 h. Ntawm cov concentrations no, 60 µM (LD50) raug xaiv thiab siv hauv cov kev sim tom ntej.

Cov pab pawg tswj tau raug kho nrog lub tsheb (DMSO) nkaus xwb. Cell viability thiab Live/Deadstaining tau ua 6 teev tom qab kho. Peb lub qhov dej tau siv rau txhua pab pawg.Cov kev sim tau rov ua dua tsawg kawg 3 zaug.

4.9. Hauv Vitro qauv ntawm Hemin-Induced Cell Death

Cell tuag raug ntxias hauv thawj cortical neurons thiab NLT hlwb los ntawm kev kho mob nrog 50µM thiab 60 µM hemin, feem, raws li tau piav qhia yav dhau los [57,58]. Hemin tau yaj 0.1 M NaOH los tsim cov tshuaj hauv Tshuag. Rau cov kev tshawb fawb neuroprotection, hlwb raug kho nrog hemin 6h nyob rau hauv lub xub ntiag ntawm cov neeg sawv cev los yog sodium selenite yaj indouble distilled dej.

4.10. CCK-8 Assay

Cell viability raug ntsuas los ntawm CCK{0}} assay (CK04, Dojindo, Kumamoto, Nyiv) raws li tau piav qhia yav dhau los [59]. Nyob rau hauv luv luv, Neuronal tau noob nyob rau hauv 96- zoo daim hlau ntawm ib tug ceev ntawm 6000 hlwb/zoo nyob rau hauv 100 µL kab lis kev cai nruab nrab thiab pub nyob rau hauv lub incubator thaum hmo ntuj.

Cells raug kho nrog Hemin rau 6 teev. Cov hlwb neuronal raug ntxuav hauv prewarmed PBS (37 ◦C) thiab CCK-8 reagent (10 µL ib 100 µL nruab nrab) hauv txhua qhov dej.

Tom qab ntawd, peb incubated theplates rau 2 h nyob rau hauv ib tug 5% CO2 huab cua, thiab lub absorbance ntawm 450 nm yog ntsuas los ntawm kev siv cov amicroplate nyeem ntawv. Cell viability tau sawv cev raws li ib feem pua ​​​​ntawm kev tswj hwm (cov hlwb tsis kho).

4.11. Nyob / Tuag Staining

Lub peev xwm ntawm CCK-8 kev soj ntsuam los ntsuas qhov muaj peev xwm tau txheeb xyuas los ntawm staining nrog calceinAM/PI hauv PBS (Live/Dead Assay, KeyGEN BioTECH, Nanjing, China) raws li tus neeg tsim khoom cov lus qhia.

Cov tshuaj staining muaj 2 µM calcein AM reagent thiab 8 µM PI reagent sib xyaw hauv PBS. Cov qauv raug tsim rau 30 feeb thiab ua duab siv lub 20xobjective lens ntawm lub fluorescence microscope (Olympus FSX100, Tokyo, Nyiv). Yam tsawg kawg ntawm 3 cov kab lis kev cai ywj pheej tau siv rau hauv vitro qauv ntawm hemin-induced cell tuag.

4.12. Peptide Synthesis thiab Administration

Pep Ctrl (NH2-TPCCGKTDVGI-CONH2), Pep E1 (NH2- HPVGAAGKTCNQTTGQ-CONH2), Pep E2 (NH2- CPCKDGVTGIT-CONH2), Tat (NH{{11}) }YGRKKRRQRRR-CONH2), TE1 (NH2- YGRK-KRRQRRR-HPVGAAGKTCNQTTGQ-CONH2), FITC sau npe TE1 (NH2-YGRK-KRRQRRR-HPVGAAGKTCNQTTGQ-CONH2), FITC{21} 27}}HPVGAAGKTCNQTTGQCPCKDGVTGITCNRCAKGYQQ-CONH2), Chij sau npe Pep Ctrl(NH2-DYKDHDGDYKDHDIDYKDDDDK-GGGGS-TPCRCGKTYDVGI-CONH2), Chij labeled Pep E1KD (NIDG{DDYKDGHD) GGS-HPVGAAGKTCNQTTGQ-CONH2) thiab Chij sau npe Pep E2 (NH2- DYKDHDGDYKDHDIDYKDDDDK-GGGGSCPCKDGVTGIT-CONH2) peptides nrog 99% purity tau yuav los ntawm Shanghai GL Biochem (Shanghai, Tuam Tshoj) tuam txhab thiab Wuhan Bioyeargene Biosciences (Wuhan, Tuam Tshoj).

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