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Pls nyem qhov no mus rau Ntu 2

Cistanche muaj cov nyhuv neuroprotective zoo heev

Gabriel Gonzalez 1,2, Jiˇrí Grz 1, Cosimo Walter D'Acunto 1, Petr Ka ˇnovsk2 thiab Miroslav Strnad1,2,*

1. Laboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences,

thiab Kws qhia ntawv ntawm Science, Palacký University, Šlechtitel ˚u 27, CZ-78371 Olomouc, Czech koom pheej;

Gonzalez.gabriel@seznam.cz (GG); jiri.gruz@upol.cz (JG); waldacun@gmail.com (CWD)

2. Department of Neurology, University Hospital Olomouc thiab Kws Qhia Ntawv ntawm Tshuaj thiab Kho Hniav,

Palacký University Olomouc, CZ-775 20 Olomouc, Czech koom pheej; Petr.Kanovsky@fnol.cz

* Cov ntawv sau: miroslav.strnad@upol.cz; Tel.: plus 420-585-634-850

Abstract: Cytokininsyog adenine-based phytohormones uas tswj cov txheej txheem tseem ceeb hauv cov nroj tsuag, xws li kev faib tawm ntawm tes thiab kev sib txawv, hauv paus thiab tua kev loj hlob, apical dominance, branching, thiab noob germination. Hauv kev tshawb fawb ua ntej, lawv kuj tau qhia txog kev tiv thaiv kev tiv thaiv tib neeg cov kab mob neurodegenerative. Txhawm rau txuas ntxiv kev paub txog kev tiv thaiv (kev tiv thaiv kev tiv thaiv) lawv muab, peb tau tshawb xyuas cov haujlwm ntawm ntujcytokininstiv thaiv salsolinol (SAL)-induced toxicity (aTus kab mob Parkinsonqauv) thiab glutamate (Glu)-vim tuag ntawmneuron-zoo li dopaminergic SH-SY5Y hlwb. Peb pom tias kinetin-3-glucoside, cis-zeatin riboside, thiab N6-isopentenyl adenosine tau ua haujlwm hauv SAL-induced PD qauv. Tsis tas li ntawd, trans-, cis-zeatin, thiab kinetin nrog rau cov hlau chelator deferoxamine (DFO) thiab cov necroptosis inhibitor necrostatin 1 (NEC-1) tseem ceeb txo qis cell tuag nyob rau hauv Glu-induced qauv. Lactate dehydrogenase kev soj ntsuam tau qhia tiascytokininsmuab qis duaneuroprotectivekev ua haujlwm dua li DFO thiab NEC-1. Ntxiv mus, lawv txo cov apoptotic caspase-3/7 cov dej num tsawg dua li DFO. Txawm li cas los xij, lubcytokininsmuaj cov teebmeem zoo sib xws rau DFO thiab NEC-1 ntawm superoxide radical ntau lawm. Zuag qhia tag nrho, lawv tau qhia txog kev tiv thaiv hauv SAL-induced qauv ntawmparkinsonian kevneuronalcell tuag thiab Glu-induced qauv ntawm oxidative puas tsuas yog los ntawm kev txo cov oxidative kev nyuaj siab.

Ntsiab lus: cytokinin; phytohormone; neuroprotection; neuron zoo li SH-SY5Y hlwb; cytotoxicity; salsolinol; glutamate; oxidative kev nyuaj siab; Tus kab mob Parkinson

cistanche cogntawm cov teebmeem ntawmNeuroprotection

1. Taw qhia

Tus kab mob Parkinson(PD) yog qhov thib ob feem ntau ntawm cov kab mob neurodegenerative motor, thiab cov naj npawb ntawm cov neeg mob thoob ntiaj teb raug kuaj pom tias yuav nce los ntawm 6 lab hauv 2015 mus rau ntau dua 12 lab los ntawm 2040 [1]. Nws yog tus cwj pwm los ntawm cov tsos mob ntawm lub cev muaj zog txuas nrog qhov tshwj xeeb degeneration thiab poob ntawm kwv yees li 30-70 feem pua ​​​​ntawm dopaminergic (DA)neuronsnyob rau hauv substantia nigra pars compacta thiab lawv qhov projections mus rau striatum [2,3]. Qee qhov, ntawm ntau, paub txog cov cim molecular ntawm PD suav nrog kev txhim kho oxidative thiab nitrosative kev nyuaj siab, mitochondrial tsis ua haujlwm [4-7], excitotoxicity [8], ubiquitin / proteasomal system tsis ua haujlwm [9], thiab neuroinflammation [10]. Cov kev kho mob tam sim no muaj ntau yam tsis zoo tshwm sim thiab tsuas yog muaj cov tsos mob nyem [11], yog li muaj kev mob siab rau tsim cov tshuaj uas muaj txiaj ntsig zoo rau kev kho mob ntawm degenerating DA.neurons. Cov peev txheej uas yuav pab tau cov kev siv zog no suav nrog cov khoom siv ntuj tsim uas yuav muaj kev phiv tsawg dua. Inter alia, tshuaj los ntawm Ginkgo biloba (ginkgetin, ginkgolide, bilobalide), ginseng (ginsenosides), thiab flavonoids (baicalein, kaempferol, rutin, thiab luteolin) tau pom dav kev tiv thaiv nyob rau hauv ntau yam hauv vitro qauv (xws li kab mob ntawm tib neeg SH. -SY5Y) thiab hauv vivo qauv ntawm PD induced los ntawm 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), 1-methyl- 4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP plus), thiab 6-hydroxydopamine (6-OHDA) [12].

Txoj kev tshawb no nthuav tawm ntawm no tsom mus rau qhov cuam tshuam ntawm ib chav kawm ntawm phytohormones ntuj hu uacytokinins(CKs), thiab lawv cov metabolites, uas yog cov neeg paub zoo ntawm kev faib cell, kev loj hlob, kev sib txawv, thiab nplooj senescence hauv cov nroj tsuag [13]. Raws li txoj cai, CKs yog adenine derivatives hloov ntawm N6- txoj hauj lwm nrog ib tug prenyl (isopentenyl) los yog aromatic sidechain. Cov ntaub ntawv ntuj muaj xws li 6-(E)-4-hydroxy-3-methyl but-2-enylaminopurine (trans- zeatin, tZ), nws 6-(Z)-isomer ( cis-zeatin, cZ), N6-isopentenyl adenine (iP), 6-benzyl amino purine (BAP), 6-furfurylaminopurine (kinetin, K), thiab ortho-, meta-, thiab para-hydroxylated lossis methoxylated derivatives ntawm BAP, hu ua topolins (oT, mT, pT, MeoT, MemT, MepT). Ntau yam 9-ribosides, 9-nucleotides, nrog rau 7-, 9, thiab O-glucosides ntawm cov ntaub ntawv no kuj tshwm sim, raws li qhia hauv Table 1. Ntxiv rau lawv lub luag haujlwm hauv zej zog hauv cov nroj tsuag, CKs tau pom muaj zog ant-oxidant kev ua haujlwm ntawm cov pa oxygen reactive (ROS) uas muab kev tiv thaiv ntau yam hauv vitro kev nyuaj siab qauv ntawm kev laus-txoj kev mob [14].

Rooj 1. Cov qauv ntawmcytokininsthiab cov neeg saib xyuas zoo.

Tshwj xeeb, CKs tau tshaj tawm tias muaj cytoprotective kev ua haujlwm hauv cov qauv xws li H2O2- induced cell tuag ntawm tib neeg fibroblasts [15] thiab D-galactose-induced glycoxidative kev nyuaj siab hauv nas astrocytes [16]. Qhov tseem ceeb tshaj, nyob rau hauv cov ntsiab lus no, lawv tau qhianeuroprotectiveCov teebmeem hauv cov qauv ntsig txog cov kab mob neurodegenerative xws li tsev neeg PD, proteasome inhibitor MG 132-induced lossis H2O2-induced toxicity hauv SH-SY5Y hlwb [17], Glu-induced oxidative puas ntawm HT22 nas hippocampalneuronalhlwb [18], thiab PC12 cell qauv ntawm Huntington tus kab mob [19]. Lwm cov kev tshawb fawb qhia tau hais tias CKs 'kev tiv thaiv kev ua ub no koom nrog ob qho tib si ncaj qha [20,21] thiab tsis ncaj [15,16,22] kev hloov pauv ntawm cellular redox systems. Ntxiv nrog rau CKs 'tus yam ntxwv ua haujlwm antioxidant, lawv tau tshaj tawm tias muaj kev tswj hwm cov teebmeem hauv mitochondria uas txhim kho.neuronalkev ua si [17]. Tsis tas li ntawd, K tuaj yeem txhim kho mitochondrial membrane muaj peev xwm thiab nce ATP ntau lawm, yog li txo qis Glu-induced tuag ntawm HT22 hlwb [18]. Txawm li cas los xij, txawm tias muaj kev tshawb nrhiav txog lawv cov teebmeem hauv ntau tus qauv, muaj kev paub tsawg ntawm CKs 'kev tiv thaiv kev ua si hauv feem ntau (sporadic) daim ntawv ntawm PD.

Txhawm rau hais txog qhov kev paub dhau los uas tau piav qhia saum toj no, peb tau soj ntsuam cov txiaj ntsig ntawm ntuj CKs thiab lawv cov metabolites hauv ob qho qauv hauv vitro: Ib tus qauv salsolinol (SAL)-induced qauv ntawm PD thiab glutamate (Glu)-induced qauv ntawm oxidative puas hauvneuron-zoo li SH-SY5Y hlwb. Cov kab no tau siv vim nws cov dopaminergic phenotype, rhiab heev rau dopaminergic co toxins xws li SAL, thiab yooj yim tsim ntawm cov neeg muaj kev ruaj ntseg ntawm kev sib txawv.neuronalcov hlwb nrog txo qis proliferation tom qab 48 h raug rau 10 uM all-trans retinoic acid (ATRA) [23–25].

Neuron-zoo li cov hlwb raug cuam tshuam rau endo / exotoxin SAL kom ua raws li PD pathology los ntawm kev ua haujlwm ntawm cellular redox system: Depletion ntawm glutathione (GSH), thiab inhibition ntawm ob qho tib si anti-oxidant enzyme (Cu / Zn superoxide dismutase thiab catalase) kev ua ub no thiab mitochondrial complexes (I thiab II), ua rau apoptosis thiab necrosis [26]. Hauv lwm cov qauv, Glu induces muaj peev xwm ua rau tuag taus oxidative puas tsuaj los ntawm kev cuam tshuam ntawm lub redox system. Ob qho qauv hauv SH-SY5Y xov tooj ntawm tes tau siv yav dhau los hauv kev tshawb fawb neuroprotection [26,27].

Cytoprotective thiab/los yog antioxidant kev ua ub no muaj feem xyuam rau degenerative ntshawv siab ntawm K, iP, BAP, iPR, tZR, thiab lawv cov hauv paus dawb tau raug sim, thiab (raws li tau teev tseg saum toj no) qee cov CKs tau pom tias muaj kev tiv thaiv hauvneuronalhlwb. Txawm li cas los xij, tsis muaj kev tshawb fawb luam tawm yav dhau los tau tshuaj xyuas cov qauv-neuroprotectiveKev sib raug zoo (SAR) ntawm natural CKs (Table 1). Yog li ntawd, qhov kev tshawb fawb no tau ua los tshuaj xyuasneuroprotective(anti-parkinsonian kev) kev ua ub no ntawm yuav luag tag nrho cov paub ib txwm tshwm sim CKs hauv cov xaiv SAL- thiab Glu-induced qauv ntawm neurodegeneration. Ua ntej, peb tau soj ntsuam txhua qhov ntawm CKs ' oxygen radical absorbance peev (ORAC) thiab (hauv kev ntsuam xyuas kev nyab xeeb) cytotoxicity ntawmneuron-zoo li SH-SY5Y hlwb. Tom qab ntawd, peb tau soj ntsuam cov tshuaj tiv thaiv cov teebmeem neuroprotective thiab cuam tshuam rau oxidative kev ntxhov siab los ntawm kev ntsuas superoxide (O2 .) ntau lawm (dihy- droethidium, DHE assay) thiab apoptotic caspase -3,7 kev ua ub no. Cov txiaj ntsig tau muab thawj zaug qhia txog kev qhia txog kev sib raug zoo ntawm ntuj CKs cov qauv thiabneuroprotectivekev ua si.

Cistanche tshuaj ntsuabmuaj qhov zoo heevneuroprotectivenyhuv

2. Cov txiaj ntsig thiab kev sib tham

2.1. Cytokinins 'Oxygen Radical Absorbance Peev Xwm (ORAC)

Raws li cov kab mob neurodegenerative cuam tshuam nrog kev ntxhov siab oxidative siab, kev ua haujlwm antioxidant ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv.neuronalhlwb. Txhawm rau ntsuas CKs 'kev muaj peev xwm lom neeg hauv qhov kev hwm no, lub peev xwm antioxidant tau txiav txim siab los ntawm ORAC, uas feem ntau siv los txiav txim siab txog cov khoom muaj peev xwm antioxidant [28]. Antioxidant muaj peev xwm tau qhia raws li Trolox sib npaug (TE), uas txiav txim siab qhov ua tau zoo (qis mus rau siab dua) ntawm cov tebchaw tshaj Trolox ntawm lub hauv paus equimolar. Cov txiaj ntsig, nthuav tawm hauv Table 1, qhia tias topolins (oT, mT, thiab pT) thiab lawv cov ribosides (oTR, mTR, pTR) muaj cov tshuaj tiv thaiv antioxidant siab, uas tej zaum muaj feem cuam tshuam nrog cov khoom siv hluav taws xob ntau ntawm lawv cov C{{ 3}}hydroxy benzyl amino substituent. Txawm hais tias lawv cov txiaj ntsig ORAC siab, cov topolins tsis muaj siabneuroprotectivekev ua si. Txawm li cas los xij, ntau yam heteroaromatic CKs suav nrog K (N6-furfurylaminopurine) thiab tsis muaj ntxhiab cis-zeatin-O-glucoside (cZOG), uas muaj ib qho 4-hydroxy-3-methylbut{{7} }en-1-yl)amino hloov pauv, kuj pom muaj peev xwm antioxidant siab (Table 2). Lwm cov CK metabolites-xws li kinetin{10}}glucoside (K3G), kinetin riboside 5<-monophosphate (kmp),="" kinetin-9-glucoside="" (k9g),="" and="" trans-zeatin=""><- monophosphate="" (tzmp)—had="" moderate="" antioxidant="" activity.="" all="" the="" others="" had="" detectable="" capacity="" except="" bap.="" these="" results="" confirm="" previous="" findings="" that="" ip,="" pt,="" k="" can="" act="" as="" direct="" radical="" scavengers,="" but="" conflict="" with="" the="" previously="" reported="" activity="" of="" bap="" in="" the="" orac="" test="" [20,21].="" to="" conclude,="" these="" compounds="" have="" potential="" in="" the="" treatment="" of="" neurodegenerative="" diseases="" associated="" with="" increased="" oxidative="" stress="">

cistanche qia cov txiaj ntsigntawmanti-Alzheimer tus kab mob

2.2. Kev sib txawv ntawm SH-SY5Y Cells

Mus kawm CKs'neuroprotectivecov teebmeem, SH-SY5Y neuroblastoma hlwb (xaiv rau cov laj thawj twb tau piav qhia [23]) tau sib txawv los ntawm kev raug rau 10 uM ATRA rau 48 h raws li tau piav qhia yav dhau los [23,24]. Tom qab ntawd lawv tau stained siv cov khoom siv membrane staining los tshuaj xyuas qhov sib txawv ntawm morphological ntawm cov hlwb tsis txawv thiab txawv. Raws li pom hauv daim duab 1A, lubneuron-zoo li cov hlwb sib txawv tau loj hlob tsawg dua, tau ntev dua, thiab tsim cov neurite ntau dua (qhia los ntawm cov xub daj hauv daim duab) dua li cov hlwb tsis txawv. Cov kev hloov morphological cuam tshuam nrog kev sib txawv tau pom yav dhau los, txawm tias tom qab raug luv luv (24 h) rau ATRA [24,30]. Qhov tseem ceeb tshaj, tus naj npawb ntawm cov neurite nce siab mus rau theem thaum lawv tuaj yeem tsim cov neurite network. Vim li no, cell viability raug ntsuas los sib piv tus nqi ntawm kev loj hlob ntawm undifferentiated thiab txawv SH-SY5Y hlwb. Qhov kev muaj peev xwm ntawm qhov tsis txawv ntawm SH-SY5Y tau raug coj los ua qhov siab tshaj plaws ntawm kev loj hlob. Cov txiaj ntsig tam sim no hauv

Daim duab 1B qhia tias qhov kev loj hlob ntawm tus nqi (kuaj los ntawm Calcein AM viability assay) ntawm SH-SY5Y raug txo los ntawm 23 feem pua ​​​​tom qab 48 h ATRA kho.

Daim duab 1. (A) Fluorescent micrographs ntawm SH-SY5Y cov hlwb nrog cov membranes stained siv cov khoom siv Neurite outgrowth (Invitro-gen™): Tswj, cov hlwb tsis sib xws (tshwj xeeb rau kev kho cov tshuaj lom neeg:<0.1% dmso);="" cells="" differentiated="" by="" exposure="" to="" 10="" um="" all-trans="" retinoic="" acid="" (atra)="" for="" 48="" h.="" bars="50" um.="" (b)="" proliferation="" rates="" of="" undifferentiated="" and="" differentiated="" sh-sy5y="" cells:="" numbers="" of="" viable="" cells="" after="" 48="" h="" exposure="" to=""><0.1% dmso="" and="" 10="" um="" atra,="" respectively.="" data="" were="" obtained="" from="" five="" independent="" experiments="" with="" triplicate="" cultures:="" asterisks="" show="" the="" significance="" of="" differences="" in="" numbers="" of="" viable="" cells="" (as="" percentages="" of="" numbers="" of="" undifferentiated="" cells)="" between="" the="" cultures:="" *="" p="" <="">

Table 2. Oxygen radical absorbance peev (ORAC) ntawm qhov kev simcytokinins(CKs) qhia raws li Trolox sib npaug (TE) ntawm qhov sib npaug. Cov npe, cov ntawv luv, thiab cov qauv ntawm CKs tau nthuav tawm hauv daim duab 1.

2.3. Cytotoxicity ntawm Cytokinins rau Neuron zoo li SH-SY5Y Cells

Hauv kev ntsuam xyuas ntawm CKs 'muaj peev xwm cytotoxicity nrog Calcein AM viability assay [31] feem ntau pom tias muaj toxicity tsawg rau covneuron-zoo li SH-SY5Y hlwb. Qhov txo qis ntawm kev muaj peev xwm qis dua 90 feem pua ​​​​yog suav tias yog qhov pib rau cov nyhuv neurotoxic. Tsuas yog ob qhov kev zam yog KR (11.9 feem pua) thiab pTR (10.5 feem pua), raws li kev tshawb pom yav dhau los uas qee qhovtshuaj cytokininmetabolites, tshwj xeeb tshaj yog ribosides, tej zaum yuav muaj cytotoxic teebmeem [32]. Lwm cov ribosides, xws li cZR, iPR, oTR, mTR, ua rau tsis pom qhov txo qis hauvneuron-zoo li SH-SY5Y cell 'vability (Table 3). DFO [33,34] thiab NEC-1 [35,36] siv los ua kev tswj xyuas zoo hauv peb cov qauv hauv vitro kuj tau ua pov thawj los ntawm lwm cov kev tshawb fawb ntawm SH-SY5Y hlwb kom tsis muaj tshuaj lom. Hauv kev xaus, feem ntau cov derivatives KR thiab pTR tau pom qhov muaj peev xwm qis dua 90 feem pua ​​​​thiab yog li ntawd tau txiav txim siab tsis tshua txaus siab rau kev ntsuam xyuas ntxiv hauv ob qho tib si hauv vitro qauv ntawm neurodegeneration.

Table 3. Cell viability ntawmneuron-zoo li SH-SY5Y hlwb tom qab raug raucytokininsrau 24h. Viability tau qhia raws li feem pua ​​​​ntawm DMSO tswj.

2.4. Kev txheeb xyuas ntawm Neuroprotective Cytokinins hauv SAL-induced Model of PD

Rau cov kev xeem no,neuronalSH-SY5Y hlwb tau sib txawv rau 48 h ces co-kho nrog 500 uM SAL thiab txhua CK ntawm peb qhov concentrations (0.1, 1, 10 uM). Raws li pom los ntawm cov kab dotted hauv daim duab 2A, daim ntawv thov ntawm neurotoxin SAL ntawm 500 uM txo qhov kev muaj peev xwm ntawm cov hlwb sib txawv SH-SY5Y, raws li Calcein AM assay, los ntawm 30 feem pua. N-acetylcysteine ​​(NAC) tau siv los ua kev tswj hwm zoo hauv cov kev sim no vim nws tau tshaj tawm cov txiaj ntsig neuroprotective hauv tib SH-SY5Y cell-based hauv vitro qauv [37]. Concentrations ntawm 10, 100, thiab 1000 uM NAC tau siv los ntxias ib nrab lossis yuav luag tag rov qab los ntawm SAL qauv. NAC muaj peev xwm ua kom cov cell viability ntawm 100 uM thiab 1 mM concentration, sib xws rau 83.39 ± 1.74 feem pua ​​thiab 89.21 ± 2.89 feem pua, feem. NAC txoj haujlwm tiv thaiv ntawm 100 uM (qhia los ntawm kab dashed hauv daim duab 2A) tau siv los ua qhov pib muaj zog rau xaiv CKs rau kev sim ntxiv. Raws li kev teeb tsa no, cov haujlwm tseem ceeb hauv kev tiv thaiv kab mob neuro-tiv thaiv tau pom nrog K3G ntawm 10 uM (81.84 ± 2.36 feem pua), cZR ntawm 0.1 uM (81.14 ± 2.30 feem pua) thiab 1 uM (81.53 ± 2.36 feem pua) thiab PR ntawm 2.24%. uM (82.43 ± 2.51 feem pua). Yog li, iPR thiab cZR tau txais txiaj ntsig zoo neuroprotectants ntawm qis micro lossis sub-micromolar concentrations dua NAC. Covtshuaj cytokininKev tshuaj ntsuam kuj tau qhia tias ntau lwm cov metabolites tuaj yeem ua rau muaj kev vam meej ntawm cov hlwb sib txawv ntawm SH-SY5Y raug rau SAL. Txawm li cas los xij, qee qhov kev sim CKs (xws li tZR, tZMP, mT, mTR, pT, thiab pTR) muaj kev tiv thaiv me ntsis.

Daim duab 2. (A)Neuroprotectivekev ua ntawmcytokininsthiab N-acetylcysteine ​​(NAC) hauv SAL-induced qauv ntawm PD rauneuron-zoo li SH-SY5Y hlwb. Cov kab dashed qhia tau hais tias NAC cuam tshuam qhov pib ntawm qhov twgcytokininsraug xaiv rau kev sim ntxiv; cov kab dotted ces suav cov hlwb nyob hauv Calcein AM assay tom qab kho cov hlwb nrog 500 uM SAL; Cov hlwb tswj kev noj qab haus huv (CTR, DMSO <0.1 feem="" pua).="" triplicates="" tsawg="" kawg="" yog="" peb="" hnub="" sib="" cais.="" (b)="" normalized="" sh-sy5y="" cell="" tuag="" tom="" qab="" propidium="" iodide="" staining.="" triplicates="" tsawg="" kawg="" tsib="" hnub="" ywj="" pheej.="" *="" p="" piv="" nrog="" lub="" tsheb="" nrog="" 500="" um="" sal,="" #="" p="" piv="" nrog="" lub="" tsheb="" tsis="" muaj="" 500="" um="">

Txhawm rau kom paub meej, qhov ua tau zoo tshaj plaws ntawm CKs 'tiv thaiv PD kev ua ub no, tag nrho cov xov tooj ntawm tes tuag tau raug txheeb xyuas los ntawm propidium iodide (PI) staining, uas (raws li kev ntsuam xyuas ntawm cell metabolism-based viability tests) tsuas yog sau cov hlwb uas tsis muaj kev ntseeg siab, cov hlwb tuag. , thiab twb tuag lawm [38]. Cov txiaj ntsig tau zoo li qub nrog kev hwm ntawm cell tuag tus nqi tom qab kev kho mob nrog SAL ib leeg (tsim li 100 feem pua). Raws li pom nyob rau hauv daim duab 2B, NAC zoo tswj cov khoom tseem ceeb txo cov cell tuag ntawm ob qho tib si 100 thiab 1000 uM (rau 77.3 ± 2.21 feem pua ​​​​thiab 77.5 ± 4.44 feem pua, feem). Zuag qhia tag nrho, NAC ua pov thawj tias yog ib qhoneuroprotectivetus neeg sawv cev nrog cov dej num sib piv rau cov uas tau sau tseg hauv lwm cov kev tshawb fawb hauv kev siv tshuaj ntau npaum li cas (hauv 50-50}0 uM ntau) rau SH-SY5Y hlwb [37]. Qhov kev ntsuam xyuas PI kuj tau pom tias CKs cZR, K3G, thiab iPR muaj kev tiv thaiv kev ua haujlwm, tshwj xeeb tshaj yog cZR, uas txo cov cell tuag rau 71.6 ± 5.08 feem pua ​​​​ntawm 0.1 uM. Hauv qhov sib piv rau cZR, K3G tau thim rov qab cov tshuaj tiv thaiv kab mob, nrog rau kev ua haujlwm siab tshaj plaws ntawm 10 uM (txo cov cell tuag rau 75.0 ± 3.69 feem pua) thiab NPR cov haujlwm tau nce siab ntawm 1 uM (txo tus nqi mus rau 73.9 ± 4.99 feem pua). Ua ke, raws li pom hauv daim duab 2, CKs muab qhov sib pivneuroprotectivekev ua haujlwm rau 100 uM NAC raws li ob qho tib si kev muaj peev xwm thiab kev tshuaj xyuas cytotoxicity. Ntxiv mus, qhov muaj txiaj ntsig zoo ntawm CKs xws li cZR thiab iPR tau qis dua li ntawm NAC, nyob rau hauv sub-micromolar thiab micromolar ranges. Cov kev soj ntsuam yav dhau los tau txais tom qab ob chav staining nrog PI thiab annexin V / PI qhia tias K tuaj yeem txo cov apoptosis [39], yog li peb kuj tau tshawb xyuas qhov cuam tshuam ntawm CKs thiab NAC ntawm oxidative kev nyuaj siab thiab caspase-3,7 ua kom muaj zog (qhov zoo- paub apoptosis marker).

2.5. Cytokinins Txo SAL-induced Superoxide Radical Tsim

Oxidative stress (OS) yog ib qho tseem ceeb pathological contributor rau ntau yam kab mob neurodegenerative, thiab ob qho tib si SAL (ntawm > 100 uM) thiab tetrahydroisoquinolines yog cov muaj zog OS inducers [26,40]. Yog li, peb kuj ntsuas qhov tsim ntawm superoxide (ROS thiab OS cim cim tseem ceeb) nyob rau hauv lub xub ntiag ntawm SAL nrog thiab tsis xaiv CKs lossis NAC. Txhawm rau kom ntseeg tau tias SAL ua rau muaj kev puas tsuaj OS txaus hauv SH-SY5Y hlwb txhawm rau txheeb xyuas cov lus teb, cov hlwb raug nthuav tawm rau 500 uM SAL rau 24 teev, ib yam li hauv kev ua haujlwm dhau los [37] thiab ua raws li cov lus pom tau hais los saum toj no. Cov hlwb tom qab ntawd tau stained los ntawm dihydroethidium (DHE) txhawm rau kuaj pom superoxide radical tsim [41,42]. Raws li tuaj yeem pom hauv daim duab 3A, cov hlwb tau pom qhov muag pom tom qab sau npe nrog DHE (uas muab cov cim liab fluorescence tom qab cov tshuaj tiv thaiv nrog superoxide). SAL induced ib tug ntshiab nce nyob rau hauv DHE fluorescence, txheeb ze rau theem ntawm kev tswj thiab NAC-kho hlwb. Ntxiv mus, peb CKs (cZR, K3G, thiab iPR) muaj qhov pom kev zoo sib xws rau NAC (100 uM) ntawm DHE fluorescence. Tsis tas li ntawd, qhov spectrophotometric quantification nrog rau qib kuaj pom hauv cov hlwb uas kho los ntawm SAL ib leeg (tsim li 100 feem pua), tau ua raws li kev soj ntsuam microscopy. Raws li pom nyob rau hauv daim duab 3B, lub normalized superoxide theem nyob rau hauv lub noj qab haus huv tswj hlwb (CTR) yog tsawg tshaj li 39 feem pua, thiab cov zoo tswj NAC muab nruab nrab-rau-ua tiav txo ntawm SAL-induced ROS ntau lawm ntawm 100 thiab 1000 uM (rau 76.3). ± 4.33 thiab 44.3 ± 5.12 feem pua), qhia tias glutathione (GSH) depletion plays lub luag haujlwm tseem ceeb hauv tus qauv [26]. Interestingly, SAL induced dramatically txo nyob rau hauv GSH ntsiab lus ntawm SH-SY5Y hlwb nrog elevation ntawm OS, mus rau theem zoo ib yam li cov yav tas los soj ntsuam nyob rau hauv ib txoj kev tshawb no uas tau sau tseg NAC-mediated teebmeem ntawm cell viability, cell tuag, thiab glutathione cov ntsiab lus [43] . Cov txiaj ntsig tau nthuav tawm ntawm no qhia tau hais tias NAC kuj txo qis superoxide radical tsim rau qib basal (piv txwv li, qib hauv DMSO-kho tswj). CK ribosides raug kuaj ntawm cov nquag nquag (0.1-1 uM) nrog rau K3G, thiab txo qis cov cell 'superoxide radical ntsiab lus mus rau qib hauv qab no (xws li cov hlwb kho nrog SAL ib leeg): cZR 80.34 ± 5.99 feem pua ​​​​ntawm 0.1 uM ; K3G 77.1 ± 4.89 feem pua ​​​​ntawm 10 uM; iPR 79.2 ± 5.91 feem pua ​​​​ntawm 1 uM, piv rau cov teebmeem ntawm 100 uM NAC. Sib sau ua ke, cov kev ua yeeb yam orthogonal qhia tau hais tias muaj zog tiv thaiv OS ua si lub luag haujlwm tseem ceeb hauv kev tiv thaiv los ntawm NAC thiab CKs hauv SAL-induced PD qauv. Ib qho kev sib raug zoo ntawm OS amelioration thiab neuroprotection kuj tau sau tseg los ntawm lwm tus kws sau ntawv [29], thiab ntau qhov kev tshawb fawb tau pom tias K thiab BAP tuaj yeem ncaj qha rau OS cov dej num [44] los ntawm kev tsim cov complexes nrog Cu2 ntxiv rau ions, ua rau superoxide dismutase- zoo li kev ua si [45,46]. Txawm li cas los xij, CKs kuj tau piav qhia tias yog cov tshuaj tiv thaiv kab mob tsis ncaj nrog cov teebmeem cuam tshuam los ntawm kev cuam tshuam ntawm cov khoom siv nuclear erythroid 2-txog yam 2 (NRF2) antioxidant teb txoj hauv kev (iPR) [22] los yog ib feem ntawm glutathione peroxidase thiab SOD cov dej num ( K) [16]. Tsis tas li ntawd, K tshaj tawm muajneuroprotectiveCov dej num tiv thaiv OS raug mob los ntawm H2O2 hauv SH-SY5Y hlwb [17]. Ob hom kev tshaj tawm los tiv thaiv kev ua haujlwm ntawm CKs tuaj yeem piav qhia txog cov teebmeem ntawm cZR, K3G, thiab iPR hauv kev txo cov superoxide radicals hauv SAL-induced SH-SY5Y cell PD qauv [47–50].

Daim duab 3. (A) Microphotographs qhia SAL-induced oxidative stress thiab oxidative stress-txo cov dej num ntawmcytokininsnyob rau hauv tib neeg txawvneuron-zoo li SH-SY5Y hlwb pom los ntawm fluorescence microscopy tom qab dihydroethidium (DHE) labeling. Bars=50 um. Cov duab qhia cov hlwb kho nrog DMSO tov (tswj), 500 uM salsolinol (SAL) ib leeg, thiab kev sib txuas ntawm 500 uM SAL thiab 1000 uM NAC (ntxiv rau NAC), 0.1 uM cZR (ntxiv rau cZR); 10 uM K3G (ntxiv rau K3G), 1uM iPR (ntxiv rau iPR) rau 24 teev ua ntej staining nrog DHE. (B) SAL-induced superoxide radical tsim thiabtshuaj cytokininlos yog N-acetylcysteine ​​(NAC) kev tiv thaiv. Daim duab qhia txog qhov muaj pes tsawg leeg ntawm DHE stained hlwb siv Infinite M200 Pro microplate nyeem ntawv (Tecan, Austria). Triplicates tsawg kawg tsib hnub ywj pheej. * P piv nrog lub tsheb nrog 500 uM SAL, # P piv nrog lub tsheb tsis muaj 500 uM SAL.