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Pls nyem qhov no mus rau Part 1

Cistanche tshuaj ntsuab muaj cov nyhuv neuroprotective zoo heev

2.6. Anti-Apoptotic Effects ntawm Cytokinins Txiav Txim los ntawm Caspase-3/7 Kev Ntsuas Kev Ua Haujlwm

Raws li qhia los ntawm PI staining kev soj ntsuam tau piav qhia saum toj no, SAL induced nce hauv SH-SY5Y hlwb cov nqi tuag. Raws li SAL cuam tshuam nrog ob qho tib si apoptosis thiab necrosis [51] peb kuj tau tshawb xyuas qhov ua kom ntawm caspase-3 thiab 7 (casp-3/7) raws li cov cim tshwj xeeb ntawm apoptosis (theem tua) [52] Tom qab nthuav tawm cov hlwb rau CKs. Caspase-3/7 cov dej num uas tau sau tseg tom qab kev kho mob nrog rau txhua qhov kev sim tshuaj sib xyaw ua ke tau ua raws li cov lus teev tseg tom qab kev kho mob nrog 50{{30}} uM SAL (teeb li 100 feem pua). Raws li pom nyob rau hauv daim duab 4, tus paub zoo caspase inhibitor Ac-DEVD-CHO (nrog rau ib qho tshwj xeeb, apoptosis-txog kev tswj) muaj zog inhibited caspase-3/7 kev ua ntawm sub-micromolar concentrations (rau 36.3 ± 2.66 thiab 25.2. ± 2.69 feem pua ​​​​ntawm cov qib hauv cov hlwb kho nrog SAL ib leeg ntawm 0.05 thiab 0.5 uM, feem). Cov qib zoo sib xws ntawm inhibition tau dhau los ua haujlwm [53] hauv qhov sib txawv hauv vitro SH-SY5Y cell-based qauv ntawm neurodegeneration. Qhov zoo tswj NAC kuj tseem ceeb txo qis caspase-3/7 kev ua, mus rau 88.7 ± 1.87 feem pua ​​thiab 78.5 ± 2.56 feem pua ​​​​ntawm SAL-induced theem ntawm 100 uM thiab 1000 uM, feem. Cov CKs kuaj tau muaj ntau yam cuam tshuam rau caspase-3/7 cov haujlwm. Interestingly los ntawm CK ribosides, tsuas yog cZR muaj qhov cuam tshuam tseem ceeb ntawm cov dej num ntawm 0.1 uM (txo lawv mus rau 83.3 ± 4.33 feem pua ​​​​ntawm SAL-induced qib, feem), thaum iPR ntawm 1 uM tsis tseem ceeb txo cov casp -3 ,7 ib. Thaum kawg, K3G txo qis caspase-3/7 kev ua haujlwm nrog qhov siab tshaj plaws ntawm 10 uM (rau 81.7 ± 4.31 feem pua ​​​​ntawm SAL-induced theem). Zuag qhia tag nrho, SAL-induced a 1.6-fold nce hauv caspase-3/7 kev ua haujlwm piv rau cov hlwb tswj kev noj qab haus huv (CTR, Daim duab 4), raws li cov txiaj ntsig ntawm kev tshawb fawb yav dhau los, uas zoo sib xws SAL concentration (400 uM) thiab tib lub xov tooj ntawm tes tau siv [54]. NAC paub tias cuam tshuam caspase-3/7 kev ua haujlwm hauv ntau tus qauv ntawmneuronaltuag [55–58]. Txawm li cas los xij, cov txiaj ntsig tau nthuav tawm ntawm no muab thawj zaug ua qauv qhia ntawm lawv cov caspase-3/7 hauv SH-SY5Y cell-based SAL-induced qauv ntawmneuronalkev tuag. Kev tswj xyuas zoo txo ​​qis cov caspase-3/7 kev ua haujlwm los ntawm 500 uM SAL zoo ib yam li CKs, tab sis cZR thiab K3G tau ua haujlwm tau zoo txawm tias nyob rau ntawm cov micromolar lossis micromolar concentrations. Lwm cov kev tshawb fawb nrog cov qauv kev ntxhov siab cuam tshuam nrog PD (proteasome inhibitor MG 132- los yog H2O2-induced toxicity nyob rau hauv SH-SY5Y hlwb [17] thiab fibroblasts [15]) thiab Hunt-ington tus kab mob (cov qauv tshaib plab hauv PC12 hlwb [19]) kuj tau qhia tias CKs K thiab tZR tuaj yeem muaj kev tiv thaiv apoptotic (caspase-3) cov dej num [17], thiab tias K thiab tZR tuaj yeem muaj cov dej num tiv thaiv kev laus [17,19] . Ntxiv mus, kev koom tes ntawm kev txo qis casp-3,7 ua kom thiabneuroprotectiveKev ua haujlwm tau tshaj tawm rau SAL thiab SAL cov qauv ntsig txog [54,59,60].

Daim duab 4. Caspase-3/7 kev ua hauv SAL-induced qauv ntawm PD. Triplicates tsawg kawg yog plaub hnub ywj pheej. * P piv nrog lub tsheb nrog 500 uM SAL, # P piv nrog lub tsheb tsis muaj 500 uM SAL.

2.7. Kev txheeb xyuas ntawm Cytokinin Neuroprotective Kev Ua Haujlwm hauv Glutamate-induced CellDeath Model

Raws li SH-SY5Y hlwb tsis qhia tag nrho NMDA receptor subunits [61], feem ntau yuav mechanism ntawm glutamate (Glu) toxicity yog blockage ntawm cystine / glutamate Xc-antiporter nrog loj induction ntawm oxidative kev nyuaj siab [62]. Cov kws sau ntawv yav dhau los tau pom tias muaj kev sib koom ua ke ntawm glutamate-induced cell tuag thiab necroptosis hauv SH-SY5Y hlwb [63], thiab qhov tseem ceeb ntawm caspase-3 ua kom muaj glutamate-induced toxicity rau-wards lawv [62,64]. Ntau qhov kev tshawb fawb kuj tau qhia txog qhov tshwm sim ntawm cov hlau-tsav cell tuag (ferroptosis) tom qab glutamate intoxication [62,65,66], thiab qhia txog qhov tshwm sim ntawm peb hom cell tuag (necroptosis, apoptosis, thiab ferroptosis) tom qab blockade ntawm Xc. -antiporter system. Yuav kom ua tiav qhov kev ntsuam xyuas dav dav ntawm covneuroprotectivemuaj peev xwm ntawm kev simcytokinins, glutamate-induced cell death assay system tau suav nrog hauv kev tshuaj ntsuam xyuas nrog cov paubneuroprotectivehlau chelator deferoxamine (DFO) thiab inhibitor ntawm necroptosis necrostatin-1 (NEC-1) [67] raws li kev tswj zoo. Ntawm no, SH-SY5Y hlwb tau sib txawv rau 48 teev, tom qab ntawd kho nrog 160 mM Glu lossis hauv kev kho mob nrog ntau yam.tshuaj cytokininconcentrations ({{0}}.1–10 uM) rau 24 teev thiab stained los ntawm PI. Hauv cov qauv no, qhov cuam tshuam ntawm Glu ntawm kev tuag ntawm tes yog 100 feem pua ​​​​PI teeb liab, yog li kev txo qis hauv cell tuag los ntawm cov tshuaj ntsuam xyuas tau soj ntsuam. Glu induced yuav luag ib tug 4-fold nce hauv cell tuag piv rau noj qab haus huv SH-SY5Y tswj (26.1 ± 0.42 feem pua). Kev tshuaj xyuas cov kev tswj xyuas zoo thiabcytokininsqhia tias ob qho tib si tswj tau zoo NEC-1 (50 uM, 76.6 ± 2.51 feem pua) thiab DFO (10 uM, 84.2 ± 4.54 feem pua) txo cov cell tuag vim glutamate. Raws li pom hauv daim duab 5A, los ntawm lub vaj huam sib luag ntawmcytokinins,3 xwbcytokininsmuaj peev xwm tiv thaivneuron-zoo li SH-SY5Y hlwb nyob rau hauv ib tug zoo xws li cov kev tswj zoo. Qhov nquag nquagcytokininstau trans-zeatin (tZ) (0.1 uM, 79.5 ± 2.91 feem pua; 1 uM, 81.3 ± 2.77 feem pua) thiab cis-zeatin (CZ) (0.1 uM, 82.1 ± 3.29 feem pua ​​; 1 uM, {{20}}.2 ± 2.89 feem pua ​​) thiab kinetin (K) (1 uM, 88.0 ± 3.76 feem pua ​​; 10 uM, 79.9 ± 3.44 feem pua). Txhawm rau lees paub lawv cov kev ua tau zoo, ib qho kev ntsuam xyuas orthogonal tau siv los piav qhia ntau ntxiv txog CKs cov teebmeem lom neeg hauv cov qauv no: Ib qho lactate dehydrogenase (LDH)-tso tshuaj ntsuam xyuas [68], uas kuj tau pom qhov ua tau zoo (4-fold) nce toxicity tom qab kho Glu. Qhov zoo siab, qhov kev txiav txim siab ntawm LDH-tso tso cai rau peb sib txawv ntawm cov dej num ntawm CKs thiab cov kev tswj zoo (NEC-1 thiab DFO). Cov CKs muaj qhov nruab nrab tab sis tseem ceeb tiv thaiv kev ua haujlwm (tZ txo cov neeg tuag mus rau 91.9 ± 1.40 feem pua ​​​​ntawm 0.1 uM; cZ txo lawv mus rau 92.3 ± 1.15 feem pua ​​​​ntawm 0.1 uM thiab 91.7 feem pua ​​± 1.56 feem pua ​​​​ntawm 1 uM2 ± K. 1.06% ntawm 1 uM). Cov kev tswj xyuas zoo NEC-1 thiab DFO muaj kev tiv thaiv zoo dua tab sis ua tau zoo ntawm cov concentration ntau dua (txo cov nqi mus rau 76.9 ± 2.94 feem pua ​​thiab 81.6 ± 3.74 feem pua ​​​​ntawm 50 thiab 10 uM, feem) (Daim duab 5B). Qhov kev ntsuam xyuas LDH tau lees paub cov cim tau txais nrog PI staining thiab cov kev pom zoo ntawm cov kev tswj xyuas zoo tau ua raws li cov ntaub ntawv luam tawm [69,70]. Kev tshawb pom txog cov teebmeem ntawm K hauv Glu-induced cell tuag qauv hauv HT22 hlwb [18] thiab peb cov kev soj ntsuam ntawm cov teebmeem ntawm tus neeg sawv cev CKs xws li tZ, cZ, thiab K qhia tias CKs tau cog lus rau cov neeg sib tw kho cov kab mob neurodegenerative cuam tshuam nrog oxidative stress [ 71] ib. Thaum kawg, tZ thiab cZ tau nyiam vim tias lawv tau txais txiaj ntsig ntawm qhov qis dua thiab raug xaiv, ua ke nrog K, rau kev tshawb fawb ntxiv ntawm lawv cov teebmeem ntawm qib oxidative kev nyuaj siab thiab caspase-3,7 ua kom muaj zog.

cistanchetshuaj xyuasntawm cov teebmeem ntawmneurotiv thaiv

2.8. Cov teebmeem ntawm Cytokinins ntawm Glu-induced Oxidative Stress hauv SH-SY5Y hlwb

Tsis zoo li cov qauv dhau los, Glu tuaj yeem ua rau oxidative kev nyuaj siab hauv SH-SY5Y hlwb los ntawm txoj kev sib txawv. Ib qho yog raws li kev thaiv ntawm Cystine / glutamate (Xc-) antiporter, uas ua rau glutathione (GSH) depletion thiab tsis zoo rau kev ua haujlwm ntawm superoxide dismutase (SOD) [62]. Glu-mediated cell tuag kuj putatively koom nrog Rac-NADPH-oxidase-tsav ROS (tshwj xeeb tshaj yog superoxide radical) tsim nyob rau hauv SH-SY5Y hlwb [72]. Cov kev tshawb pom no qhia tias lub hauv paus ua rau kev tuag ntawm tes hauv Glu-induced qauv yog OS. Nyob rau hauv tus qauv,neuron-zoo li cov hlwb raug kho nrog Glu thiab kuaj cov tebchaw, stained los ntawm DHE, thiab soj ntsuam los ntawm fluorescence microscopy. Raws li tuaj yeem pom hauv daim duab 6A, qhov tshwm sim loj heev hauv DHE fluorescence tau pom nyob rau hauv Glu-kho hlwb, uas tau ua kom zoo los ntawm cov tshuaj tswj tau zoo thiab kuaj CKs. Raws li kev soj ntsuam microscopy, qhov ntsuas ntawm cov qib superoxide tau ua nyob rau hauv Glu-induced cells zoo ib yam, los ntawm DHE assay, rau cov nyob hauv SAL-induced cells. Cov txiaj ntsig tau zoo ib yam nrog rau cov qib uas raug ntxias los ntawm Glu ib leeg (tseem raws li 100 feem pua), uas ua rau muaj kev cuam tshuam loj heev, yuav luag 3-fold nce qib superoxide hauv 4 h hauvneuron-zoo li SH-SY5Y cov hlwb txheeb ze rau qib hauv cov hlwb tswj kev noj qab haus huv (CTR: 33.79 ± 1.45 feem pua). Raws li pom hauv daim duab 6B, CKs xws li tZ, cZ, thiab K tau txo qis superoxide qib hauv Glu-intoxicated hlwb: 0.1 uM tZ, 0.1 uM cZ, 1 uM, thiab 10 uM K txo nws mus rau 81.0 ± 4.03, 80.6 ± 3.89, 81.8 ± 3.39 thiab 83.8 ± 2.32 feem pua ​​​​ntawm cov qib hauv cov hlwb kho nrog Glu ib leeg. Cov qib no yog piv rau cov uas tau txais nrog cov necroptosis inhibitor NEC-1 (80.5 ± 3.19 feem pua ​​​​ntawm 5 uM thiab 80.4 ± 2.70 feem pua ​​​​ntawm 50 uM) thiab hlau-chelator DFO (80.2 ± 3.80 feem pua ​​​​ntawm 10 uM). Cov txiaj ntsig tau txais nrog Glu-induced cell tuag qauv, tshwj xeeb tshaj yog cov txiaj ntsig ntawm kev tswj hwm zoo, yog raws li cov ntaub ntawv dhau los [70,73]. Cov pov thawj dhau los uas CKs muaj OS-txo cov teebmeem hauv Glu-induced lossis cov qauv zoo sib xws yog txwv rau kev ua qauv qhia tias K yog tus muaj zog OS-txo tus neeg sawv cev (ntawm kwv yees li 23 uM) hauv HT22 cell-based Glu-induced cell tuag qauv [18 ]. Peb pom tias K muaj cov txiaj ntsig OS-txo qis qis (1-10 uM) hauv peb lub hlwb SH- SY5Y. Tsis tas li ntawd, cov txiaj ntsig ntev ntev ntawm tZ rau tib neeg cov kab mob, suav nrog kev ua haujlwm hydrogen peroxide-decomposing, tau pom [15], qhia tias tZ thiab cZ yuav muaj kev cuam tshuam tsis ncaj OS-txo hauv Glu-induced cell tuag qauv. Tsis tas li ntawd, lwm cov ntawv ceeb toom tau taw qhia txog kev sib koom ua ke ntawm CKs-mediated OS-txo cov nyhuv thiab kev tiv thaiv kev ua haujlwm [22,74–76]. Coj ua ke, tZ, cZ, thiab K tau pom tias muaj zog OS-txo cov teebmeem, piv rau cov kev tswj zoo, txawm tias muaj qhov sib txawv ntawm qhov sib txawv.neuroprotectivenyhuv.

Daim duab 5. (A) Kev tuag ntawmneuron-xws li SH-SY5Y hlwb hauv glutamate (Glu)-vim tus qauv ntawm cell tuag; (B) Glu-induced toxicity (LDH-tso) ntawmneuron-zoo li SH-SY5Y hlwb. Triplicates tsawg kawg peb hnub ywj pheej.* P piv nrog lub tsheb nrog 160 uM Glu, # P piv nrog lub tsheb tsis muaj 160 uM Glu.

Daim duab 6. (A) Glu-induced oxidative stress (OS) thiab OS-txo kev ua ntawm cov ntsiab lus qhia hauv tib neegneuron-zoo li SH-SY5Y hlwb pom los ntawm fluorescence microscopy tom qab dihydroethidium (DHE) labelling. Bars=50 um. Cov duab qhianeuron-zoo li SH-SY5Y hlwb kho los ntawm DMSO (Control), 160 mM glutamate (Glu) ib leeg, thiab ua ke nrog: 10 uM deferoxamine (ntxiv rau DFO), 50 uM necrostatin{{6} } (ntxiv rau NEC-1), 0.1 uM tZ (ntxiv tZ) thiab 0.1uM cZ (ntxiv cZ) rau 4 h, ces stained los ntawm DHE. (B) Glu-induced superoxide radical tsim nyob rau hauvneuron-zoo li SH-SY5Y hlwb tom qab 4 h. Daim duab qhia txog qhov ntau ntawm DHE stained hlwb siv Infinite M200 Pro microplate nyeem ntawv (Tecan, Austria). Triplicates nyob rau hauv tsib hnub ywj pheej.

* P piv nrog lub tsheb nrog 160 uM Glu, # P piv nrog lub tsheb tsis muaj 160 uM Glu.

2.9. Cov teebmeem ntawm Cytokinins ntawm Caspase-3/7 Kev Ua Haujlwm hauv Glu-induced Cell Death Model

Glu muaj cov txiaj ntsig zoo sib xws ntawm SH-SY5Y hlwb rau yav dhau los qhia txog cov teebmeem ntawm HT22 hlwb cuam tshuam nrog Xc-antiporter blockage. Yog li, tsis yog-apoptotic cell tuag mechanisms (ferroptosis, necroptosis, thiab lwm yam) tau koom nrog hauv ob qho xwm txheej [62] txawm hais tias kev qhia ntawm caspase-3 thiab rau qee qhov, NMDA subunit ntawm NR1 tuaj yeem tshwm sim ntau dua hauv SH. -SY5Y cells [61]. Yog li ntawd, theem kawg ntawm kev tuag ntawm tes hauv ob kab ntawm tes yuav txawv. Lub luag haujlwm pabcuam ntawm caspase-3 kev ua haujlwm tau pom nyob rau hauv ntau qhov kev tshawb fawb hauv Glu-induced qauv ntawm cell tuag ntawm SH-SY5Y hlwb [62,77–79]. Hauv qhov kev ntsuas no,neuron-zoo li SH-SY5Y hlwb tau kho ntawm no nrog 160 mM Glu, uas ua rau nce siab ntawm caspase-3/7 kev ua haujlwm. Hauv kev siv zog los qhia txog lub luag haujlwm ntawm apoptosis hauv Glu-induced tuag ntawm SH-SY5Y hlwb, ib qho tshwj xeeb caspase-3/7 inhibitor, Ac-DEVD-CHO, tau siv thiab pom tias muaj cov nyhuv inhibitory muaj zog: Ntawm 50 nM nws txo caspase-3,7 kev ua haujlwm rau 23.70 ± 1.01 feem pua ​​​​ntawm cov qib hauv cov hlwb kho

nrog Glu ib leeg thiab ntawm 0.5 uM nws yuav luag tag nrho thaiv qhov kev ua si (txo nws mus rau ib theem li qub ntawm 7.22 ± 1.05 feem pua; piv rau qib pom hauv cov hlwb noj qab haus huv:

7.8 ± 0.39 feem pua), raws li qhia hauv daim duab 7. Kev siv cov kev tswj xyuas zoo tau ua rau muaj qhov txo qis ntawm cov koob tshuaj me me hauv caspase-3,7 kev ua haujlwm. Interestingly, NEC-1 muaj qhov cuam tshuam me ntsis nrog cov txiaj ntsig siab tshaj plaws ntawm 50 uM thiab DFO muaj zog inhibitory kev ua haujlwm ntau dua ntawm 10 uM (txo cov haujlwm rau 80.5 ± 1.85 thiab 75.8 ± 3 .00 feem pua, ntsig txog). Ntawm qhov tod tes, tsuas yog me ntsis, tab sis qhov cuam tshuam tseem ceeb ntawm casp -3,7 tau ua tiav los ntawm tZ ntawm 1 uM (89.5 ± 2.50%), cZ ntawm 0.1 uM (87.4 ± 1.62 feem pua) thiab K ntawm 1 uM ( 91.0 ± 2.06 feem pua). Muab ua ke, cov txiaj ntsig tau pom tias ob qho kev tswj hwm zoo tau muaj zog dua caspase-3/7 kev ua haujlwm txo qis dua li cov CKs tau sim, tab sis CKs muaj qhov cuam tshuam tsawg dua. Zuag qhia tag nrho, cov txiaj ntsig tau qhia tias kev hloov pauv ntawm caspase-3/7 kev ua si ua lub luag haujlwm tseem ceeb hauv lub zogneuroprotectivekev ua ntawm cov neeg ua haujlwm xws li DFO [58] hauv Glu-induced qauv ntawm cell tuag [79,80]. Txawm li cas los xij, raws li qhia hauv ntu no, NEC-1 kuj muajneuroprotectivekev ua haujlwm, qhia tias lwm hom kev tuag ntawm tes yuav koom nrog Glu-induced tuag ntawm neuron-zoo li SH-SY5Y hlwb [63,67,81]. Cov cim qhia tias cov txheej txheem caspase-yooj yim koom nrog hauv Glu-induced cell tuag kuj tau txais los ntawm kev txheeb xyuas cov teebmeem ntawm NEC-1 [70], DFO [69], thiab K [18] ntawm HT22 hlwb.

Daim duab 7. Caspase-3/7 kev ua haujlwm hauv Glu-qauv ntawm oxidative puas tsuaj ntawmneuron-zoo li SH-SY5Y hlwb. Triplicates tsawg kawg yog plaub hnub ywj pheej. * P piv nrog lub tsheb nrog 160 uM Glu, # P piv nrog lub tsheb tsis muaj 160 uM Glu.

3. Cov lus xaus

Hauv cov ntsiab lus, peb txoj kev tshawb fawb tau qhia tias CKs thiab lawv cov metabolites muajneuroprotectivecov dej num hauv SAL-induced qauv ntawmTus kab mob Parkinsonthiab Glu-induced oxidative puas tsuaj hauv tib neeg sib txawv SH-SY5Yneuronalhlwb. K3G, cZR, thiab iPR tau pom tias muaj cov khoom noj muaj txiaj ntsig zooneuroprotectivekev ua si. Ntxiv mus, cov CKs nquag siv tau zoo ntawm qis dua (sub-micromolar thiab micromolar) ntau dua li cov tshuaj tswj zoo NAC. K3G, cZR, thiab iPR kuj muaj txiaj ntsig zoo ntawm kev muaj peev xwm, cytotoxicity, oxidative stress, thiab caspase-3,7 kev ua haujlwm (tshwj tsis yog iPR). Cov kev ua yeeb yam orthogonal qhia tau hais tias kev tiv thaiv oxidative kev nyuaj siab ua lub luag haujlwm tseem ceeb hauv CKs 'kev tiv thaiv cov teebmeem hauv SAL-induced cell tuag qauv. Tsuas yog peb cov metabolites hauv lub vaj huam sib luag kuaj ntawm CKs (tZ, cZ, thiab K) tiv thaivneuron-zoo li SH-SY5Y hlwb hauv Glu-induced qauv ntawm oxidative kev puas tsuaj nyob rau hauv ib tug zoo xws li cov hlau chelator NEC1 thiab necroptosis inhibitor DFO. Txhawm rau kom paub meej tias lawv qhov kev ua haujlwm tau zoo, peb tau sim lawv cov teebmeem hauv orthogonal lactate dehydrogenase (LDH)-tso tshuaj ntsuam xyuas. Cov CKs muaj qhov nruab nrab tab sis tseem ceeb ua haujlwm tiv thaiv. Qhov no tsis muaj zog dua li cov kev ua ub no ntawm kev tswj cov tshuaj, tab sis txawm tias qhov sib txawv ntawm kev hloov kho ntawmneuronalkev noj qab haus huv, tag nrho peb qhov kev sim CKs txhawb kev txo qis hauv superoxide radical tsim, zoo ib yam li cov tshuaj tswj tau zoo. Cov CKs kuj muaj qhov sib piv ntawm Glu-induced oxidative stress nyob rau hauv SH-SY5Y hlwb rau cov NEC1 thiab DFO, tab sis cov tebchaw no muaj zog inhibitory cuam tshuam rau caspase-3/7 kev ua ntau tshaj CKs. Raws li Glu-induced cell tuag tsis cia siab rau txoj hauv kev los ntawm caspase, raws li qhia hauv kev tshawb fawb txog kev cuam tshuam ntawm DFO thiab NEC-1 ntawm HT-22 hlwb [70], CKs tuaj yeem pom meej modulate ob qho tib si caspase- dependent thiab - ywj siab ntawm tes tuag los ntawm kev txo oxidative kev nyuaj siab [18,82]. Hauv kev tshawb fawb tsis tu ncua, cov ncauj lus kom ntxaws ntawm kev ua ntawm CKs thiab / lossis lawv cov teebmeem mitochondrial rauneuronallos yog astrocyte hlwb yuav raug soj ntsuam.

cistanchekev paubrau covPDthiabnco

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Tshuaj thiab Reagents

CytokininCov qauv tau txais los ntawm OlChemIm (Olomouc, Czech koom pheej). Calcein AM (1 mg / mL daws), thiab LDH tso cov khoom siv, thiab cov khoom siv Neurite outgrowth tau yuav los ntawm ThermoFisher. Propidium iodide, dihydroethidium, Cheebtsam ntawm caspase- 3/7 assay buffer, DMEM/F12 1: 1 nruab nrab, fetal bovine serum, trypsin, ATRA, salsolinol hydrobromide, glutamate monosodium ntsev, deferoxcystamine, N-acetyl , Ac-DEVD-CHO, thiab DMSO rau kab lis kev cai ntawm tes tau yuav los ntawm Sigma Aldrich (Merck). Ac-DEVD-AMC substrate tau muab los ntawm Enzo Life Science.

4.2. ORAC Radical Scavenging Activity Assay

Lub peev xwm los tshem tawm cov dawb radicals hauv vitro tau txiav txim siab los ntawm Oxygen Radical Absorbance Capacity (ORAC) assay. Luv luv, fluorescein (100 uL, 500 mM), thiab 25 uL ntawm cov tshuaj tov tshuaj tau ntxiv rau 96-zoo microplate pre-incubated ntawm 37oC. Tom ntej no, 25 ul ntawm 250 mM 2,2<-azobis(2-amidino-propane)dihydrochloride (aaph)="" was="" quickly="" added,="" the="" microplate="" was="" shaken="" for="" 5="" s="" and="" red="" fluorescence="" (with="" 485="" and="" 510="" nm="" excitation="" and="" emission="" wavelengths,="" respectively)="" was="" measured="" every="" 3="" min="" over="" 90="" min="" using="" an="" infinite="" 200="" microplate="" reader="" (tecan,="" männedorf,="" switzerland).="" nauc="" (net="" area="" under="" curve)="" values="" were="" used="" to="" express="" antioxidant="" activities="" relative="" to="" that="" of="" trolox="" (a="" synthetic="" hydrophilic="" analog="" of="" α-tocopherol,="" vitamin="" e).="" substances="" with="" orac="" values="" greater="" than="" zero="" are="" deemed="" to="" actively="" trap="" free="">

4.3. SH-SY5Y Cell Culture

Tib neeg neuroblastoma cell kab SH-SY5Y yuav los ntawm ATCC (American Type Culture Collection, Manassas, VA, USA) tau loj hlob hauv Dulbecco's modified Ea-gle's Medium thiab Ham's F12 Nutrient Mixture (DMEM: F-12, 1:1 ), ntxiv nrog 10 feem pua ​​​​fetal bovine serum (FBS) thiab 1 feem pua ​​penicillin thiab streptomycin ntawm 37 oC nyob rau hauv ib tug humidified 5 feem pua ​​CO2 cua. Cells tau dhau mus txog 20 zaug thiab cov xov xwm tau hloov pauv ob zaug lossis peb zaug hauv ib lub lis piam. Cell ceev tau teem raws li qhov tsim nyog rau qhov kev ntsuam xyuas tau npaj (5000, 7000, 10,000, lossis 20,000 cell ib qhov dej) hauv 96-multiwell phaj hauv 100 uL tag nrho cov ntim nruab nrab rau txhua kev sim. Hnub tom qab cov noob, ATRA hauv 1 feem pua ​​​​FBS DMEM / F12 nruab nrab tau ntxiv rau cov hlwb mus rau qhov kawg siab ntawm 10 uM, thiab incubation tau txuas ntxiv rau 48 h ntxiv rau kev sib txawv, tso cai tsim cov neurite ntev dua, thiab txo qis. kev loj hlob.

4.4. Microscopy

Micrographs ntawmneuron-zoo li SH-SY5Y cov hlwb tau txais los ntawm DM IL LED fluorescence microscope (Leica Microsystems, Mannheim, Lub teb chaws Yelemees) nrog rau kev lim dej kom tsim nyog rau kev ntsuam xyuas, lossis teeb tsa lub teeb ci nrog DP73 lub koob yees duab digital (Olympus, Tokyo, Nyiv ). Txij li thaum lub teeb liab-rau-suab nrov piv rau staining yog nruab nrab, qhov sib piv tau hloov kho me ntsis hauv ImageJ software (Fiji) yam tsis muaj kev cuam tshuam rau kev soj ntsuam. Cov duab qub muaj nyob rau hauv Cov Khoom Siv Ntxiv.

4.5. Cell Membrane Staining (Neurite Outgrowth Kit, Invitrogen ™)

Neuron-zoo li SH-SY5Y hlwb (5000 hlwb ib qhov dej) tau los ntawm 48 h cov txheej txheem sib txawv tau stained los ntawm Neurite outgrowth kit (Invitrogen ™) raws li cov chaw tsim khoom cov lus pom zoo nrog kev hloov kho me me. Cov hlwb raug ntxuav nrog PBS, sau nrog kev daws ntawm daim nyias nyias staining dye nrog cov khoom siv (ua raws li kev cai rau 96 multiwell daim hlau) hauv PBS rau 20 min ntawm 37 oC, ntxuav dua nrog PBS, thiab saib hauv qab lub tshuab ntsuas fluorescence ( nrog 533 thiab 585 nm excitation thiab emission wavelengths, ntsig txog).

4.6. Kev kho mob ntawm tes

Tom qab cov txheej txheem sib txawv, cov xov xwm sib txawv tau hloov mus rau 1 feem pua ​​​​DMEM / F12 nruab nrab ntxiv nrog cov tshuaj ntsuam xyuas ntawm qhov ntau ntawm 0.1, 1, thiab 10 uM (nrog 7000 cell per well for cytotoxicity tests), los yog ua ke nrog cov tshuaj lom SAL ntawm 500 uM (nrog 7000-10,000 hlwb ib qhov dej) los yog 160 mM Glu (nrog 20,000 hlwb ib qhov dej) rau lub sij hawm tsim nyog rau hom tshuaj ntsuam (kev vam meej, cell tuag, thiab lwm yam). Cov hlwb tswj tau raug kho nrog qhov nruab nrab uas muaj .0.1 feem pua ​​​​ntawm DMSO.

4.7. Cell Viability thiab Cell Tuag

Lub viability ntawmneuron-zoo li SH-SY5Y hlwb loj hlob nyob rau hauv 96-zoo microplates (nrog ca. 7000 hlwb ntawm ib qhov dej) tom qab 24 teev ntawm kev kho mob tau soj ntsuam los ntawm Calcein AM assay nrog me me kev hloov kho [31]. Cov tshuaj Calcein AM hauv PBS siv muaj 0.75 uM concentration thiab lub sijhawm incubation tau teem rau 50 min. Tus naj npawb ntawm cov hlwb nyob hauv txhua qhov dej tau txiav txim siab siv Infinite M200 Pro microplate nyeem ntawv (Tecan, Austria), nrog rau 495 thiab 517 nm excitation thiab emission wavelengths, feem.

Cell tuag ntawmneuron-zoo li cov hlwb (SAL qauv: 10,000 cell per well; Glu-model: 20,000 cells per well) tau txiav txim siab los ntawm PI assay raws li qhia los ntawm Pob zeb li al. 2003 nrog kev hloov kho me me [83]. Luv luv, qhov nruab nrab ntawm SAL-induced qauv yog aspirated thiab hloov mus rau 1 ug / mL tov ntawm PI hauv PBS, tab sis cov hlwb raug rau Glu-induction muaj zog adherence yog li 1 mg / mL tov ntawm PI hauv PBS tau ntxiv rau. muab qhov kawg concentration ntawm 1 ug / mL. Nyob rau hauv ob qho tib si, hlwb raug incubated rau ib tug ntxiv 15-25 min nyob rau hauv chav tsev kub ces PI fluorescence yog ntsuas los ntawm ib tug Infinite M200 Pro nyeem ntawv (Tecan, Grödig, Austria) nrog 535 thiab 617 excitation thiab emission wavelengths, feem. Qhov tshwm sim PI fluorescence tau nrog cov co toxins ib leeg tau teeb tsa ua 100 feem pua ​​​​ntawm tes tuag.

4.8. Kev ntsuas ntawm Oxidative Stress los ntawm Dihydroethidium (DHE) assay

Cells tau sib txawv thiab kho raws li tau piav nyob rau hauv nqe lus dhau los rau SAL- induction (10, 000 cell per well, 24 h lossis Glu-induction (20, 000 cell per well, 4 h). Tom qab ntawd cov kev kho mob, hlwb raug centrifuged ntawm 500 xg rau 330 s, ces cov kab lis kev cai xov xwm raug hloov (raws li aspiration) nrog 10 uM DHE tov nyob rau hauv PBS. 96-multiwell daim hlau nrog cov hlwb raug incubated nyob rau hauv qhov tsaus ntuj rau 30 min ntawm chav tsev. Qhov kub thiab txias, ces DHE fluorescence raug ntsuas los ntawm Infinnite M200 Pro microplate nyeem ntawv (Tecan, Grödig, Austria) nrog 500 thiab 580 nm excitation thiab emission wavelengths, raws li qhov tshwm sim DHE fluorescence 10 ib leeg tau txais nrog SAL los yog superoxide. Cov duab fluorescence microphotographs ntawm DHE-stained cells tau txais tom qab ntsuas DHE nrog tus nyeem ntawv microplate.

Cistanche cog muaj cov txiaj ntsig zoo neuroprotective

4.9. Kev ntsuas ntawm Caspase-3/7 Kev Ua Si

Ib-kauj ruam caspase-3/7 kev soj ntsuam tau ua raws li cov txheej txheem luam tawm yav dhau los [84]. Rau cov kab lis kev cai raug rau SAL-induction thiab Glu-induction, cov tshuaj tiv thaiv sib xyaw (caspase-3,7 tsis thiab cov khoom siv nrog cov hlwb hauv 96-multiwell plates) tau incubated rau 2 h thiab 3 h ntawm 37 oC, raws. Caspase-3/7 kev ua haujlwm tom qab ntawd ntsuas los ntawm Infinite M200 Pro microplate nyeem ntawv (Tecan, Austria) nrog 346 thiab 438 nm excitation thiab emission wavelengths, feem.

4.10. Kev txheeb cais

Kev sim tau ua nyob rau hauv triplicates thiab rov ua dua hauv peb mus rau tsib (n {{0}}-5) hnub ywj pheej. Tag nrho cov ntaub ntawv raug qhia raws li txhais tau tias ± SEM. Tus nqi rau txhua qhov ntsuas ntsuas tau qhia raws li txhais tau tias ± SEM, uas tau suav nrog Prism 8.4.3 (GraphPad Software, La Jolla, CA, USA), uas kuj tau siv los tsim cov duab. Kev txheeb xyuas txheeb cais tau ua los ntawm PAST (ver. 1.97) software pob [85]. Rau qhov kev sim sib txawv, Cov Tub Ntxhais Kawm T-test tau siv. Lwm qhov kev sim tau raug soj ntsuam los ntawm qhov tsis yog-parametric Kruskal-Wallis test nrog Mann-Whitney post hoc test nrog kev kho Bonferroni raws li ntu. Tus nqi ntawm p <0.05 tau="" suav="" tias="" yog="" qhov="" tseem="">

Cov Khoom Siv Ntxiv: Cov duab qub muaj nyob online.

Sau Cov Kev Pabcuam: Kev koom tes ntawm cov kws sau ntawv tau raws li hauv qab no: Kev Tshawb Fawb, txheej txheem, kev siv tau, kev txheeb xyuas cov ntaub ntawv, GG, CWD, thiab JG; kev sau ntawv—kev npaj ua ntej GG, CWD, thiab MS; Kev kho cov ntaub ntawv, kev nrhiav nyiaj txiag, kev saib xyuas, sau ntawv- tshuaj xyuas thiab kho, CWD, MS, thiab PK Txhua tus kws sau ntawv tau nyeem thiab pom zoo rau cov ntawv luam tawm ntawm cov ntawv sau.

Cov Nyiaj Txiag: Txoj haujlwm no tau txais kev txhawb nqa nyiaj txiag ib feem los ntawm cov nyiaj pab los ntawm Internal Grant Agency ntawm Palacky University hauv Olomouc, Czech Republic (IGA{{0}}PrF_2020_021), Czech Grant Agency ({{ 2}}S), thiab European Regional Development Fund—Project ENOCH (No. CZ.02.1.01/0.0/0.0/ 16_019/ 0000868) thiab cov nyiaj pab rau tub ntxhais kawm los ntawm Palacky University's Endowment Fund.

Institutional Review Board Statement: Tsis siv tau.

Cov Lus Qhia Txog Kev Pom Zoo: Tsis siv tau.

Cov ntaub ntawv muaj nyob: Cov ntaub ntawv muaj nyob rau hauv tsab xov xwm lossis cov khoom siv ntxiv.

Kev lees paub: Cov kws sau ntawv ua tsaug rau Dita Jordovd, Jana Hrubešovd, thiab Lucie Koplikovd rau kev pabcuam zoo heev. Tsis tas li ntawd, cov kws sau ntawv ua tsaug rau Sees-editing Ltd., UK rau kev kho lus Askiv ntawm cov ntawv sau.

Kev tsis sib haum xeeb ntawm kev txaus siab: Cov neeg sau ntawv tshaj tawm tsis muaj kev cuam tshuam ntawm kev txaus siab.

Sample Availability: Cov qauv ntawm cov tebchaw tsis muaj los ntawm cov kws sau ntawv.