Tubuloside B Los ntawm Cistanche Salsa cawm PC12 Neuronal Cells Los ntawm 1-Methyl-4-phenylpyridinium Ion-Induced Apoptosis Thiab Oxidative Stress

Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com

Guoqing Zheng, Xiaoping Pu, Li Lei, Pengfei Tu, Changling Li

Abstract:

Cov neuroprotectiveCov teebmeem ntawm tubuloside B los ntawm cistanche, ib qho ntawm phenylethanoids cais los ntawm SuavTshuaj ntsuab Cistanche salsa, ntawm 1-methyl-4-phenylpyridinium ion (MPP plus )-induced apoptosis thiab oxidative stress nyob rau hauv PC12 neuronal hlwb raug soj ntsuam. PC12 hlwb kho nrog MPP ＋ underwent apoptotic tuag raws li tau txiav txim los ntawm MTF assay, ntws cytometry, thiab DNA agarose gel electrophoresis; intracellular tsub zuj zuj ntawm reactive oxygen hom (ROS) yog ntsuas los ntawm DCFH-DA staining nrog laser scanning confocal microscopy (LSCM). Kev kho mob ib txhij nrog tuhuloside B pom tau tias txo qis MPP＋ -induced cytotoxicity, DNA fragmentation, thiab intracellular tsub zuj zuj ntawm ROS. Cov txiaj ntsig no qhia tau hais tias tubuloside B tiv thaiv MPP-induced apoptosis thiab oxidative stress. Tubuloside B tuaj yeem siv ua ib qhoanti-Parkinson tus kab mobtus neeg sawv cev.

Taw qhia

Pathologically, sporadic Parkinson's disease (PD) feem ntau tshwm sim los ntawm kev poob ntawm catecholaminergic neurons, tshwj xeeb tshaj yog dopaminergic neurons ntawm substantia nigra pars compacta (SNc), thiab muaj cov yam ntxwv intraneurona| suav nrog hu ua Lewy lub cev hauv thaj tsam ntawm lub hlwb. Txawm hais tias muaj ntau yam uas tau cuam tshuam rau hauv cov kab mob ntawm PD, cov txheej txheem koom nrog hauv kev pib thiab kev loj hlob ntawm PD tseem tsis paub meej, Txawm tias qhov ua rau PD tseem teb, ntau cov ntaub ntawv pov thawj qhia tau hais tias muaj kev koom tes ntawm oxidative kev nyuaj siab, thaum kawg ua rau. mus rau neuronal tuag los yog apoptosis los ntawm ntau tiam ntawm dawb radicals [1], [2], [3]. Qhov tseeb tias nigral dopaminergic system tuaj yeem tsim cov qib siab ntawm cov dawb radicals thiab muaj cov qib qis ntawm cov tshuaj tiv thaiv antioxidant ntxiv txhawb qhov kev xav no. Ob leeg hauv vivo thiab hauv vitro kev tshawb fawb pom oxidative stress-mediated neurotoxicity ntawm MPP＋, ib qho active metabolite ntawm l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) ntawm dopaminergic neurons [4 ], [5]. Kev koom tes ntawm cov dawb radicals ntau dhau los ntawm oxidative kev nyuaj siab raws li qhov tshwm sim ntawm cov kab mob neurodegenerative xws li PD qhia txog lub luag haujlwm tseem ceeb rau antioxidants. Tubuloside B (Fig. l) yog ib qho ntawm cov phenylethanoid compounds cais los ntawm stems ntawmCistanche salsa. Cov kev tshawb fawb yav dhau los tau pom ntau yam phenylethanoids nrog rau tubuloside B pom muaj zog dawb radical scavenging kev ua si[6], [7].

Xav txog cov kev tshawb pom no, peb tau tshawb xyuas hauv qhov kev tshawb fawb tam sim no cov txiaj ntsig ntawm tubu]nside B ntawm MPP ntxiv -induced neurotoxicity hauv vitro, siv cov paj hlwb pheochromocytoma cell kab PC12 uas tau siv ntau xyoo los kawm txog kev ua haujlwm neuronal [8], [9].

Anti-Parkinson tus kab mob:cistanche

Cov ntaub ntawv thiab cov txheej txheem

Khoom siv

Tubuloside B los ntawmCistanche salsatau ua siab zoo muab los ntawm Dr. Li Lei (Department of Natural Products, School of Pharmaceutical Sciences, Peking University, Beijing, Suav). Lub purity ntawm cov compound yog ntau tshaj 98 feem pua ​​​​ntawm HPLC tsom xam. Dulbecco's modified Eagle's medium (DMEM), horse serum, and fetal calf serum was purchased from GIBCO BRL, 1-methyl~-phenylpyridinium ion (MPP plus), poly-L-lysine, 3-(4, 5 -dimethylthiazol-2 -yl)-2,5- diphenyltetrazolium bromide (MTT), propidium iodide (PI), RNase A, proteinase K, thiab 2;7'-dichlorofluorescein diacetate (DCFH-DA) tau yuav los ntawm Sigma.

Cell kab lis kev cai

PCI2 hlwb tau khaws cia hauv DMEM ntxiv nrog 10 feem pua ​​nees cov ntshav, 5 feem pua ​​​​fetal calf serum, 100U/ml penicillin thiab 100ug/ml streptomycin. Cov hlwb tau coj mus rau hauv lub chaw tsim khoom humidified aerated nrog 95 feem pua ​​​​ntawm huab cua thiab 5 feem pua ​​​​CO2 ntawm 37 degree. Txhua qhov kev sim tau ua tiav 24-48 h tom qab cov noob tau cog. Cov hlwb tau niaj hnub sau los ntawm trypsinization 0.25 feem pua ​​​​thaum cov hlwb mus txog theem subconfluent thiab plated hauv 25 cm kab lis kev cai flasks faib ntawm 1: 8.

Kev tsom xam ntawm cell viability

Lub cell viability tau txiav txim siab los ntawm kev hloov kho MTT kev soj ntsuam raws li tau piav qhia yav dhau los [10], Hauv luv luv, PC12 hlwb tau cog rau hauv 96- cov phiaj zoo ntawm qhov ntom ntawm I × 104 ceils ib qhov dej. Cov kab lis kev cai tau loj hlob rau 48 h, thiab tom qab ntawd qhov nruab nrab tau hloov mus rau qhov uas muaj ntau yam ntawm tubulnside B lossis MPP ＋. Tom qab incubation mus txog 48h thiab 72 h, MTr tov (5 mg / ml hauv DMEM) tau ntxiv rau hauv 96- daim hlau zoo thiab cov hlwb raug tso cai rau incubate rau 4h ntawm 37 degree . Tom qab qhov nruab nrab tau raug tshem tawm, cov xov tooj ntawm tes thiab dye crystals tau solubilized los ntawm kev ntxiv 200μL ntawm DMSO, thiab qhov nqus tau ntsuas ntawm 570nm (540 nm raws li kev siv) nrog tus qauv 550 microplate nyeem ntawv (Bio-Rad). Cell viability kuj tau txheeb xyuas siv trypan blue exclusion method. Cov hlwb tau sau, maj mam pelleted, thiab stained nrog 0.6 feem pua ​​​​tshuaj ntawm trypan xiav rau 3 los nag. Qhov feem pua ​​​​ntawm cov hlwb uas tsis muaj xim tau raug soj ntsuam tom qab hauv kaum qhov kev xaiv xaiv lub tshuab kuaj kab mob. Raws li cov tshuaj zoo, 100 ng / ml epidermal growth factor (EGF) tau siv.

Kev kuaj pom ntawm apoptosis los ntawm kev ntws cytometry

Cov hlwb apoptotic tau kuaj pom los ntawm kev ntws cytometry siv propidium iodide (PI) [11]. Luv luv, txog 1 × 106 hlwb incubated nrog cov tshuaj ntsuam xyuas tau sau los ntawm centrifugation thiab tau ntxuav ib zaug nrog PBS. Cells raug kho nrog 70 feem pua ​​​​ethanol rau 24 teev ntawm - 20 degree thiab ntxuav ib zaug nrog PBS. Cells tau raug tshem tawm hauv 300μL ntawm PBS uas muaj 0.5 mg / ml RNase thiab 0.5 mg / ml PI. Tom qab ntxiv incubation rau 30 min nyob rau hauv qhov tsaus ntuj, ntws cytometry tau ua nrog ib tug FACScan (Becton Dickinson, Heidelberg, lub teb chaws Yelemees).

Peb tau siv Moore tus txheej txheem [12] uas cais tsuas yog tawg ntawm DNA thiab cov kev sim tau normalized los ntawm kev siv cov kab lis kev cai sib npaug ntawm txhua pawg xeem. Luv luv, 3 × 106 neurons los ntawm txhua tus qauv kho tau raug ntxuav hauv Hank cov kua ntsev sib npaug (1000g rau 10 min), thiab cov pellets tsim rau hauv qab ntawm lub raj yog homogenized. nrog 1 ml lysis buffer (10 mM Tris ntawm pH 7.4. 5 mM EDTA, 1 feem pua ​​Triton X-100) rau 20 feeb ntawm dej khov. Tom qab centrifugation ntawm 11, 000 g rau 20 los nag ntawm 4 degree, supernatants uas muaj fragmented DNA raug tshem tawm thiab zom nrog 20 mg / ml RNase A ntawm 37 degree rau 1 h, thiab 0.1 mg / ml proteinase K ntawm 56 degree rau 1h. Cov DNA tawg tau muab rho tawm los ntawm kev siv phenol thiab chloroform thiab centrifuged ntawm 1O, 000g rau 1 los nag ntawm 4 C. Cov theem dej tau hloov mus rau hauv Eppendorf raj tshiab, tov nrog 2 ntim dej khov-txias ethanol, thiab tom qab ntawd muab tso rau ntawm -20 degree tsawg kawg yog 1 h kom precipitate DNA. Tom qab centrifugation ntawm 15, 000g rau 20 min ntawm 4 degree, supernatants raug tshem tawm, thiab DNA pellets raug ntxuav nrog 80 feem pua ​​​​ethanol ib zaug (15,000 g rau 15 min ntawm 4 degree), cua qhuav, thiab yaj hauv 20μL TE Tsis muaj pH 7.6. Tag nrho cov qauv yog tom qab ntawd electrophoresed ntawm 1.5 feem pua ​​​​agarose gel rau 1h ntawm 85 V. Cov gel tau tshuaj xyuas thiab thaij duab los ntawm Ultra Violet Products Gel Documentation System (GELDOC-2000, Bio-Rad, USA).

Kev ntsuas rau intracellular ROS tsim

Kev tsim ntawm intracellular peroxides tau kuaj pom nrog confocal scanning laser microscope siv cov khoom tsis muaj fluorescent, 2 "7"-dichlorofluorescein diacetate (DCFH-DA) [13], uas yog esterified hauv hlwb los ntawm endogenous esterases rau ionized dawb acid, 2 ;7"-dichlorofluorescein. 2",7-Dichlorofiuorescin muaj peev xwm raug oxidized rau fluorescent 2"7"-dichlorofluorescein (DCF) los ntawm hydroantioxidants. Cells tau incubated nrog 10 μM DCFH-DA rau 30 min ntawm 37 degree. Cov kab lis kev cai raug ntxuav ob zaug nrog Hank qhov sib npaug ntawm cov ntsev tov thiab muab tso rau hauv tib qhov nruab nrab. Kev yees duab tau ua tiav los ntawm kev siv confocal scanning laser microscope, DCF tau zoo siab ntawm 488 nm thiab cov emission filter yog 510nm barrier filter, Txhawm rau muab qhov sib piv siv tau, tib qhov kev tau txais tsis tau siv rau txhua qhov kev soj ntsuam. Qhov zoo tshaj plaws txoj hauj lwm ntsug ntawm nruab nrab ntawm cov hlwb tau teeb tsa, thiab tom qab ntawd lub teb tau nrawm nrawm. Vim tias lub teeb pom kev ntawm lub zog siab ntawm 488 nm ua rau muaj zog fluorescence vim oxidation ntawm cov xim no, txhua daim teb tau raug rau Lub Teeb rau tib lub sijhawm. Cov txiaj ntsig tseem ceeb los ntawm cov hlwb uas tsis muaj zog tau siv los ua qhov tseem ceeb rau kev sib piv nrog MPP-kho cov hlwb nyob rau hauv lub xub ntiag lossis tsis muaj tubuloside B. Tom qab kuaj xyuas, qhov nruab nrab txheeb ze fluorescence siv nyob rau hauv 15-20 neuronal cell lub cev ntawm ib lub tshuab tsom iav tau suav rau hauv plaub. cais kab lis kev cai raws li kev kho mob.

cistanche

Kev txheeb cais

Tag nrho cov txiaj ntsig tau txais tau qhia raws li txhais tau tias ± SD. Kev txheeb xyuas qhov tseem ceeb ntawm qhov sib txawv ntawm cov pab pawg tau txiav txim los ntawm Tub Ntxhais Kawm T-test; Muab xam p qhov tseem ceeb tsawg dua 0.05 raug suav hais tias yog qhov tseem ceeb.

Cov txiaj ntsig

Raws li pom nyob rau hauv daim duab 2, MPP ntxiv tau tsim ib koob tshuaj-dependant txo nyob rau hauv lub viability ntawm PCI2 hlwb. Nyob rau hauv sib piv, EGF, ib tug zoo-paub neurotrophic yam, tsim kev tiv thaiv tseem ceeb ntawm cell viability ntawm 100ng / mL MPP ＋-induced cytotoxic teebmeem kuj tau attenuated nyob rau hauv lub xub ntiag ntawm tubuloside B (5, 10, 50 los yog 100ug-ml-1 ), txawm hais tias muaj zog tsawg dua li cov tshuaj siv (Fig .3). Tubuloside B ntawm cov concentrations no tau nthuav tawm cov teebmeem cytoprotective nyob rau hauv kev siv tshuaj ntau npaum li cas thiab qhov sib xyaw ib leeg tsis ua rau pom tseeb cytotoxicity (cov ntaub ntawv tsis qhia). Gel electrophoresis ntawm DNA extract los ntawm PCI 2 hlwb kho nrog 200 µM MPP ntxiv rau 48 h pom DNA laddering. lub cim tseem ceeb ntawm apoptosis, tubuloside B, ntawm koob tshuaj 10 thiab 100μ g·ml-1educedformationoftheDNAladder(Daim duab. nyob rau hauv lub xub ntiag los yog tsis muaj tubuloside B .Quantitation ntawm lub cell voj voog profile nrog PI qhia tus naj npawb ntawm ceils nrog subdiploid yam ntxwv (M1 cheeb tsam) qhia txog apoptotic DNA fragmentation. Hauv cov hlwb tsis kho, qhov apoptotic subdiploid feem tsawg tsawg txog 5.94% ntawm tag nrho 10000 hlwb (Fig .5 A). Tom qab 48 teev raug rau MPP, qhov feem pua ​​​​ntawm apoptosis tau nce mus rau 43.02;j; (Fig .5 B). Thaum ntxiv rau kab lis kev cai ntawm tes, tubuloside B inhibited MPP + induced apoptosis nyob rau hauv ib koob tshuaj. Ntawm qhov siab ntawm 10 ug·ml-1 kev tiv thaiv los ntawm tubuloside B tsis yog qhov tseem ceeb, tab sis ntawm 50 thiab 1 00ug·ml-1, ib qho 45％ thiab 63％ txo qis. tau pom tias ua rau muaj kev nce hauv feem pua ​​​​ntawm cov ciaj sia taus (Daim duab .5D, E).

Kev sib sau ntawm intracellular ROS tuaj yeem kuaj pom los ntawm kev siv DCFH-DA．PC12 hlwb kho nrog 200 uM MPP＋ rau 48 h tso tawm cov fluorescence hnyav hauv cell tom qab staining nrog DCFH-DA dye(Fig .6). Kev kho mob ib txhij nrog tubuloside B (ntawm koob tshuaj 10 thiab 100ug·ml.) txwv tsis pub MPP＋一 induced ROS ntau lawm (qis feem pua ​​​​yog 24.52％ thiab 55.63％, feem).

Kev sib tham

Lub pathogenesis ntawm PD muaj zog oxidative kev nyuaj siab, txo cov antioxidant theem, thiab mitochondrial tsis xws luag. tag nrho cov paub tias induce apoptosis nyob rau hauv ntau lub cellular systems. Tshwj xeeb, cov dawb radicals tau pom tias ua rau lub cev muaj zog tuag hauv cov neurons[14]. MPTP tsim ib qho kev hloov tsis tau thiab mob hnyav zoo li parkinsonian uas ua rau kev xaiv degeneration ntawm nigrostriatal dopaminergic neurons hauv tib neeg. Cov tshuaj neurotoxin no tau siv los tsim cov qauv tsiaj ntawm PD[15] .MPTP hloov los ntawm monoamine oxidase B rau MPP, uas yog khaws cia hauv mitochondria ＋. qhov twg nws inhibits mitochondrial complex kuv thiab cov hnub mitochondrial. muaj nuj nqi ua rau apoptosis[16] .Yog li ntawd, MPP ntxiv rau ib qho kev cuam tshuam neurotoxicity hauv dopaminergic neurons tau ua haujlwm ntau dhau los ua tus qauv etiologic ntawm PD rau kev tshuaj xyuas cov tshuaj tiv thaiv neuroprotective.

Hauv txoj kev tshawb fawb tam sim no, peb tau pom tias MPP ntxiv rau -induced cell tuag ntawm oxidative kev nyuaj siab hauv PC12 hlwb raug txo los ntawm tubuloside B. Ntxiv mus, peb tau pom tias tubuloside B kuj tau tiv thaiv nyob rau hauv ib koob tshuaj tiv thaiv MPP ntxiv -induced DNA laddering thiab apoptosis, raws li ntsuas siv DNA gel electrophoresis thiab ntws cytometric tsom xam. Ntxiv mus, kev kho mob ib txhij nrog ntau dua ntawm tubuloside B inhibited MPP-induced ROS ntau lawm. Yog li, tubuloside B tau qhia ntau yam kev ua haujlwm neuroprotective teebmeem. Ob peb lub tswv yim, sib cais lossis hauv kev koom tes, tuaj yeem koom nrog hauv kev ua ntawm tubuloside B. Cov nyhuv antioxidant yog qhov ua tau rau tubuloside B-mediated neuroprotection. Xiong Q pom tias tubuloside B pom muaj zog dawb radical scavenging kev ua si ntawm 1,1-diphenyl-2-picrylhydrazyl radical thiab xanthine]xanthine oxidase generated superoxide anion radical [6].

Ob peb phenylethanoids tsis ntev los no tau tshaj tawm tias muaj cov dawb radical scavenging zog thiab tiv thaiv oxidative stress-induced toxic raug mob. ROS tsim los ntawm MPP ntxiv rau xws li hydrogen peroxide, superoxide anion, thiab hydroxyl radical tuaj yeem ua rau cov kab mob lom, uas tuaj yeem ua rau apoptotic lossis necrotic cell tuag. Yog li, tubuloside B tuaj yeem muaj qhov cuam tshuam ncaj qha rau ROS. Thaum peb cov ntaub ntawv tsis tsim kom muaj lub vev xaib uas tubuloside B ua rau txhawm rau txhawm rau ROS, lawv tau hais tias cov txheej txheem tiv thaiv neuroprotective cuam tshuam nrog kev txhawb nqa ntawm txoj hauv kev antioxidants. Kev tshawb fawb ntawm cov qauv molecular ntawm tubuloside B kuj qhia tau hais tias nws muaj lub peev xwm los tshem tawm cov dawb radicals (kev sib txuas lus ntawm tus kheej).

Lwm cov txheej txheem kuj tseem tuaj yeem cuam tshuam. Piv txwv li, tubuloside B tuaj yeem muaj peev xwm los tawm tsam qhov toxicity ntawm MPP ＋ los ntawm inhibiting qhov qhib ntawm mitochondrial permeability hloov pore thiab ua tsis taus. Tsis tas li ntawd, seb tubuloside B muaj qhov cuam tshuam ncaj qha rau kev loj hlob ntawm cov hlwb neuronal yuav tsum tau txiav txim siab. Qhov tseeb mechanism ntawm kev ua haujlwm neuroprotective ntawm tubuloside B tseem tsis meej. Cov kev tshawb fawb ntxiv yuav tsum tau piav qhia tag nrho cov duab ntawm nws cov nyhuv neuroprotective.

Qhov tseeb, tubuloside B los ntawm Cistanche salsa extract muaj pov thawj kev tiv thaiv ntawm MPP ntxiv rau -induced apoptosis thiab oxidative kev nyuaj siab. Nws cov teebmeem neuroprotective tiv thaiv MPP ＋ toxicity yuav yog ib qho tseem ceeb thiab qhia tias nws yuav muaj peev xwm kho tau hauv kev tiv thaiv thiab kho PD.

Kev kho mob ntawm PD: cistanche

Cov ntaub ntawv

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