Cov teebmeem Cistanche Ntawm Apoptosis Dopaminergic Neurons Induced Los Ntawm Neurotoxin 1-Methyl-4 -Phenylpyridinium (MPP)

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

Tian Jiyu, Chen Jianzong

(Traditional suav tshuaj thiab Suav Materia Medica Research Center ntawm Xijing Tsev Kho Mob, FMMU, Xi'an 710032)

Abstract

Lub hom phiaj: Tshawb xyuas cov teebmeem ntawmCistanche ntawm thawj kab lis kev cai dopaminergic neurons apoptosis cell induced los ntawm 1-methyl-4-phenylpyridinium (MPP ntxiv). Txoj kev: Txhawm rau npaj tus luav cov ntshav suav nrogCistanchentiav cov txheej txheem ntawm cov tshuaj serum. Tom qab ntawd soj ntsuam cov teebmeem ntawm Cistanche ntawm apoptosis cell ntawm DA neurons los ntawmMPP ntxivinduced qauv siv morphological, ntws cytometry, TUNEL staining.Kev tshwm sim: Kev kho mob ntawm 200μmol / LMPP ntxivrau 24h lossis ntev dua induced apoptosis hauv DA neurons. Tom qabMPP ntxivkho, qhov sib piv apoptosis ntawm cov pab pawg uas muaj qhov sib npaug ntawm cov ntshav ntawm Cistanche yog 6.3 feem pua,MPP ntxivkho ib leeg yog 30.2 feem pua. Tus naj npawb ntawm apoptosis hlwb ntawm pawg B thiab C tsawg dua li pawg E (P<0.05).>Xaus: Cov tshuaj muaj tshuajCistanchetiv thaiv apoptosis DA neurons induced los ntawmMPP ntxiv.

Ntsiab lus: Cistanche;1-methyl-4-phenylpyridinium(MPP ntxiv ); dopaminergic neurons; apoptosis

Tam sim no, lub ntsiab kev kho mob rauTus kab mob Parkinsonyog kev hloov kho ntawm compound levodopa (L-dopa) kev npaj, uas tswj tsis tau txoj kev loj hlob ntawm tus kab mob. Raws li tus kab mob loj hlob zuj zus, cov koob tshuaj tau nce ntxiv, thiab feem ntau hais lus, hauv 3 mus rau 5 xyoos, cov nyhuv kho tau txo qis, cov tsos mob ntawm lub cev hloov pauv, thiab cov tshuaj dyskinesia tshwm sim. Cov kev tshawb fawb tsis ntev los no tau pom tias L-dopa muaj cov txiaj ntsig ntawm inducing apoptosis ntawm neurons kab lis kev cai hauv vitro [1, 2], thiab tom qab ntawd nws tau pom tias cov nyhuv neurotoxic ntawm endogenous dopamine ua rau poob ntawm dopaminergic neurons hauvTus kab mob Parkinson. Ib qho laj thawj [3]. Hauv kev soj ntsuam mus sij hawm ntev, peb tau siv tshuaj suav raunourishing lub siab thiab lub raumua ke nrog levodopa npaj los kho PD thiab ua tiav cov txiaj ntsig zoo [4-6]. Qhov kev sim no tshawb nrhiav kev tiv thaiv ntawm cov tshuaj suav tshuaj ntawm dopaminergic neuron apoptosis thiab nws cov txheej txheem los ntawm qib molecular thiab cellular.

1. Cov ntaub ntawv thiab cov txheej txheem

1.1 Cov khoom siv

DMEM nruab nrab,1-methyl-4-phenylpyridini um, MPP plus, fetal bovine serum muas los ntawm lub tuam txhab Gibco.Cytarabine, poly-l-lysine, PLLtau yuav los ntawm Sigma Company. 24-zoo kab lis kev cai phaj, 25cm2

Yas kab lis kev cai khobtau yuav los ntawm NUNC Company.Suav tshuajpeb tau siv tau los ntawm Xi'an Decoction Piece Factory. Cov tsiaj uas peb tau siv yog muab los ntawm lub tsev kawm ntawv lub chaw tsiaj.https://www.xjcistanche.com/products

1.2 Kev npaj thiab kev sim ua pab pawg ntawm cov tshuaj kho mob

Cistanche yog npaj ua cov hmoov qhuav qhuav rau kev khaws cia thiab diluted nrog dej distilled thaum siv. Cov tsiaj ntshav tau npaj los ntawm New Zealand tus loj-eared dawb luavntshav qab zib, tag nrho cov no yog txiv neej noj qab haus huv, hnyav 2.5-30 kg. Cov koob tshuaj sib npaug ntawm cov tsiaj yog hloov pauv raws li thaj chaw ntawm lub cev thiab muab faib ua pawg tswj hwm dawb paug; 1/2 sib npaug koob tshuaj pab pawg ntshav (gavage koob ntawm Cistanche raw cov khoom siv tshuaj 0.8g/kg); pawg koob tshuaj sib npaugntshav qab zib(Cov koob tshuaj yog Cistanche cistanche thawj cov khoom siv tshuaj 1.6g / kg); 2 npaug ntawm qhov sib npaug ntawm koobntshav qab zib(cov koob tshuaj yog Cistanche thawj cov khoom siv tshuaj 3.2g / kg). Gavage ob zaug ib hnub rau tag nrho 3 hnub, 1 teev tom qab qhov kawg gavage, carotid bloodletting, hmo ntuj ntawm 4 degree, centrifugation ntawm 2500 r / min rau 25 feeb, ceev faj sib cais ntawm cov ntshav, inactivation ntawm 56 degree rau 30 feeb, thiab nqus los ntawm 0.22μm lim membrane Lim tawm cov kab mob thiab khaws cia ntawm -20 degree . Nyob rau hauv tib yam kev mob, ib tug dawb paug luav tswj serum tau npaj los ntawm gavage nrog ib tug sib npaug ntim ntawm ib txwm saline. Qhov kev sim tau muab faib ua 5 pawg. Pawg A yog ib tug luav dawbntshav qab zibpawg tswj kab lis kev cai; Group B yog aserum culture plus MPPpab pawg kho mob uas muaj 2 zaug ntawm qhov sib npaug; Pawg C yog ibserum culture plus MPPpab pawg kho mob uas muaj cov koob tshuaj sib npaug; Pab pawg D yog pawg tswj hwm kab lis kev cai uas muaj 1/2 sib npaug ntawm cov kab lis kev cai ntawm cov kab lis kev cai ntxiv rau MPP pab pawg kho mob; E pab pawg yog cov kab lis kev cai ntawm luav dawb paug ntxiv rau MPP pab pawg kho mob.

Cov txiaj ntsig Cistanche

1.3 Kev cog qoob loo ntawm midbrain dopaminergic neurons

Peb SD nas ntawm 14 hnub ntawm cev xeeb tub raug xaiv thiab tua los ntawm kev txhaj tshuaj ncaj qha ntawm huab cua embolism rau hauv plawv; Cov nas embryonic tau muab tshem tawm nyob rau hauv aseptic mob thiab muab tso rau hauv ib lub tais petri uas muaj dej khov D-Hanks tov. Lub fetal nas hlwb cov ntaub so ntswg tau dissected thiab sib cais nyob rau hauv ib tug stereomicroscope. Cov txheej txheem ntawm kev siv [7] tau txhim kho me ntsis. Cov qog nqaij hlav thiab cov hlab ntsha tau raug tshem tawm, thiab cov ntaub so ntswg hauv A8, A9, thiab A10 thaj chaw ntawm lub ventral midbrain raug cais. Ntxiv 1mL ntawm 0.125 feem pua ​​​​ntawm pancreatin thiab muab tso rau hauv 37 degree incubator rau li 20 feeb, ntxiv qhov tsim nyog tus nqi ntawm cov kab lis kev cai nruab nrab uas muaj 10 feem pua ​​​​fetal bovine serum kom tshem tawm cov plab zom mov, thiab siv cov aspiration.

Pipette lub raj rau hauv lub cev tsis sib xws, centrifuge ntawm 1500r / min rau 5 feeb ntawm chav tsev kub, ua tib zoo tshem cov supernatant, pipette 15 zaug nrog lub caj dab nyias, thiab cia nws sawv ntsug li 5 feeb. Hauv 24-zoo kab lis kev cai phaj ntawm lysine, ntxiv kab lis kev cai nruab nrab rau txhua lub qhov dej los kho cov cell ceev rau 2 × 105 hlwb / mL, ntxiv kab lis kev cai nruab nrab rau 0.5mL ib qhov dej, thiab ntxiv cytarabine tom qab 3 hnub kab lis kev cai ( Qhov kawg concentration yog 10μmol / L), inhibit qhov kev loj hlob ntawm non-neurons.

1.4 Tyrosine hydroxylase (TH) immunocytochemical staining

Siv txoj kev SABC, cov tshuaj tiv thaiv kab mob diluent muaj bovine serum albumin 1g/100mL (Boster Company, Wuhan), 0.3 feem pua ​​Triton-100 (Sigma, US), NaN3 80mg/ 100mL, diluted nrog PBS rau 100mL, kho pH rau 7.6. Cov kab lis kev cai tau raug txiav rau hnub 7th, ntxuav nrog D-PBS rau 5min 3 zaug, tshiab tau npaj 4 feem pua ​​​​ntawm paraformaldehyde tau kho ntawm chav sov li 30min thiab ntxuav nrog D-PBS rau 5min 3 zaug; nrog 1: 1: 8 hydrogen peroxide, methanol thiab PBS tov Incubate rau 30 feeb ntawm chav tsev kub, yaug nrog D-PBS rau 5 feeb, ntxiv 5 feem pua ​​​​ntawm cov tshis cov ntshav rau 30 feeb ntawm chav tsev kub, yaug nrog D-PBS rau 5 feeb. feeb, thiab ntxiv tshis anti-nas TH antibody diluted nrog antibody diluent (1: 1000 dilution, Calbiochem)), thaum hmo ntuj ntawm 4 degree, yaug nrog D-PBS rau 3 min 3 zaug, ntxiv biotinylated nas anti-rat IgG diluted nrog antibody. diluent (diluted 1:200, Sigma USA), ua nyob rau hauv chav tsev kub rau 4 teev, yaug nrog D-PBS rau 3 min 3 zaug, Ntxiv cov HRP complex diluted nrog antibody diluent rau 2h ntawm chav tsev kub, thiab yaug nrog D-PBS. rau 3m3x. Yaug lub DAB diluent rau 3 feeb, ntxiv cov npaj txhij-rau-siv DAB chromogenic reagent (Wuhan Boster Company) nyob rau hauv lub hlwb, thiab saib cov xim nyob rau hauv ib tug inverted microscope.https://www.xjcistanche.com/

1.5 Kev tuav tshuaj

Kev soj ntsuam morphological ntawm cov kab lis kev cai neurons tau ua nyob rau hauv ib theem-qhov sib piv ntawm lub tshuab ntsuas txhua hnub thiab cov kev hloov pauv tau kaw nrog cov duab. Tom qab culturing rau 12 teev, lub hlwb tau pom nyob rau hauv ib tug inverted microscope thiab cov tshuaj tau ntxiv ua ntej confluence. Cov ntsiab lus ntshav ntawm txhua pab pawg yog 10 feem pua. Tom qab culturing rau 3 hnub, MPP ntxiv tau ntxiv rau qhov kawg concentration ntawm 200 μmol / L. Tom qab incubating nyob rau hauv ib tug 37 degree, 5 feem pua ​​CO2 incubator rau 24 teev, ua ntau yam kev soj ntsuam.

Cistanche

1.6 Flow cytometry nrhiav pom

Cov tshuaj kho tau zoo ib yam li yav dhau los. Tom qab txhua pab pawg tau kho, cov hlwb uas txuas nrog PC12 tau zom nrog 0.125 feem pua ​​​​ trypsin, thiab cov hlwb tau noj txog 1 × 106 PBS. Cov hlwb raug ntxuav ob zaug, thiab 490 μL ntawm kev sib khi tsis tau ntxiv, thiab cov hlwb tau sib xyaw kom zoo, thiab 5 μL annexin V tau ntxiv. FITC thiab 10μL ntawm yaj PI nyob rau hauv lub cell ncua kev kawm ntawv, maj mam sib tov, muab lub xeem raj ntawm 4 degree, incubate rau 10 min nyob rau hauv qhov tsaus ntuj nti, thiab ntsuam xyuas ntawm lub COULTER EPICS flow cytometer (Beckman Coulter, USA).

1.7 Nyob rau hauv situ kawg labeling (TUNEL) txhawm rau kuaj cell apoptosis

Lub DNA nyob rau hauv situ kawg daim ntawv lo ntawm kab lis kev cai neurons siv Boster's apoptosis detection kit (MK10}22), thiab cov kauj ruam yog raws li nram no: Siv cov dopaminergic neuron hlwb ntawm txhua pab pawg tom qab MPP ntxiv kev kho mob (cov swb ua ntej thawj kab lis kev cai yog pre-cultured nrog polymer Lysine kev kho mob), kho nrog 4{{30}}g / L paraformaldehyde rau 1h, tshiab 3 feem pua ​​​​acetic acid (pH2.5) ntawm chav tsev kub rau 10min, neutralize alkaline phosphatase, ntxiv 0.1mol/L TBS (pH7.5) 1:200 freshly diluted proteinase K digestion ntawm 37 degree rau 20s, ntxiv 1μL ntawm TdT thiab DIG-d-UTP rau txhua tus swb thiab 18μL ntawm cov tshuaj tsis haum rau 2h ntawm 37 degree; ntxiv 50μL ntawm kev thaiv cov tshuaj rau txhua qhov swb, ntawm chav tsev kub rau 30min; Ntxiv 50μL ntawm biotinylated anti-digoxigenin antibody diluted nrog thaiv kev daws; react rau 30min nyob rau hauv ib lub thawv ntub dej ntawm 37 degree; ntxiv 50μL ntawm SABC-AP diluted nrog 0.1mol / L TBS rau txhua tus swb; BCIP/NBT (0.1mol ntawm pH9.5 /L TBS diluted 1:20) tau ntxiv rau cov qauv tsim xim rau 30 feeb; Nyob rau tib lub sijhawm, lwm txoj kev tswj hwm (TdT tsis yog siv los hloov TdT / biotin-dUTP sib tov) rau photomicrography thiab inverted theem contrast microscope (200 ×) Saib cov hlwb apoptotic. Suav cov hlwb zoo nyob rau hauv lub tshuab ntsuas theem-qhov sib piv, suav 5 qhov chaw rau ib qhov dej hauv txhua pab pawg, rau tag nrho ntawm 6 qhov dej.

2. Cov txiaj ntsig kev sim

2.1 Kev soj ntsuam Morphological ntawm kab lis kev cai neurons

Pom tau los ntawm OlympusIX70 theem-qhov sib piv ntawm lub tshuab ntsuas: 5h tom qab inoculation, feem ntau ntawm cov hlwb ua rau ntawm phab ntsa, cov hlwb tawg, lub cev ntawm lub cev yog puag ncig, lub nucleus tsis pom, thiab qhov ntsuas pom muaj zog. Tom qab culturing rau 24 teev, cell protuberances loj hlob thiab maj mam elongated. Thaum 48h, feem ntau ntawm cov hlwb protruded nrog branched filamentous protrusions. Lub cell morphology yog ntau haiv neeg, uas yog bipolar, tripolar, thiab multipolar, thiab maj mam tsim ib tug sparse network. Lub cev ntawm tes tau nthuav dav, puag ncig, thiab elliptical. Tom qab MPP ntxiv rau kev raug mob, cov hlwb ua ntej swelled, lub refractive index nce, lub hlwb nyob rau hauv ib tug degenerative lub xeev, thiab lub hlwb thaum kawg shrink thiab poob tawm. Cov koob tshuaj sib txawv ntawm cov tshuaj suav tshuaj suav nrog cov ntshav qab zib muaj kev tiv thaiv MPP ntxiv rau kev raug mob. Tom qab kab lis kev cai tau stained nrog TH lub cev tiv thaiv kab mob, nws tau pom tias TH-zoo hlwb yog xim av nrog neurite outgrowth. Tom qab ntxiv qhov sib npaug ntawm cov tshuaj suav tshuaj suav tshuaj, qhov feem pua ​​​​ntawm TH-zooneuronstuaj yeem ncav cuag 55 feem pua ​​-60 feem pua ​​.

2.2 Kev soj ntsuam cov txiaj ntsig cytometry flow (saib daim duab 1)

Phosphatidylserine (PS) nyob rau hauv lub cell membrane nyob rau hauv thaum ntxov theem ntawm apoptosis migrate mus rau sab nraud ntawm lub cell membrane. Phosphatidyl binding protein V (Annexin V) yog calcium-dependent phospholipid-binding protein, uas muaj affinity siab rau PS. Yog li, Annexin V tuaj yeem siv los ua kev sojntsuam txhawm rau txheeb xyuas PS nthuav tawm ntawm lub cell membrane. Nyob rau tib lub sijhawm, ua ke nrog PI exclusion txoj kev rau ob npaugstainingntawm apoptotic hlwb, apoptotic hlwb tuaj yeem kuaj tau los ntawm kev ntws cytometry. Nyob rau sab xis sab xis ntawm Fig.1 (FITC ntxiv / PI-), cov ntsiab lus apoptosis ntawm txhua pab pawg tau tshwm sim. Qhov feem pua ​​​​ntawm cov hlwb, tus nqi apoptosis ntawm txhua pawg yog 4.1 feem pua, 5.8 feem pua, 6.3 feem pua, 28.6 feem pua, 30.2 feem pua.

2.3 Kev tshuaj xyuas ntawm TUNEL staining cov txiaj ntsig

Cov uas muaj xiav-ntsuab liab hais nyob rau hauv lub nucleus yog cov hlwb zoo, uas yog, apoptotic hlwb. Qhov feem pua ​​​​ntawm cov hlwb apoptotic hauv txhua pab pawg tau suav nrog hauv lub teeb pom kev zoo. Tus naj npawb ntawm apoptotic cells ib qhov chaw saib hauv txhua pab pawg yog: Pawg A 2.13 ±0.34; Pab pawg B 4.92±0.67; Pab pawg C 6.20 ± 1.47; Pab pawg D 10.33 ± 1.51; Pab pawg E 12.74 ± 2.51. Tom qabt-kev soj ntsuam,pawg B thiab C tau muab piv nrog pawg E (P<0.05). the="">tshwm simyog qhov yooj yim raws li covCov txiaj ntsig ntawm flow cytometry.

3. Kev sib tham

Tus kab mob Parkinsonbelongs rau qeb ntawm "tremor syndrome" nyob rau hauv Suav tshuaj. Cov tshuaj suav tshuaj suav hais tias qhov pib ntawm PD feem ntau yog qhov tshwm sim ntawm kev maj mam depletion ntawm daim siab thiab lub raum hauv cov neeg laus thiab tsis muaj kev noj haus ntawm lub hlwb pob txha meridian. Lub ntsiab kev kho mob yog mustxhawb lub siab thiab lub raum, calm lub siab thiab tshem tawm cua. Cov tshuaj suav tshuaj tuaj yeem ua lub hom phiaj ntawm "txo toxicity thiab nce kev ua tau zoo"nyob rau hauv kev kho mob ntawmParkinson tus kab mob, tab sis nws cov mechanism ntawm kev txiav txim tsis meej heev tam sim no. Ntau qhov kev sim ntawm cov pov thawj hauv cov tshuaj niaj hnub no qhia tias oxidative kev nyuaj siab yog ib yam uas ua rau kev tuag ntawm PD paj hlwb [3]. Cov kev tshawb fawb tau pom tias tag nrho glycosides ntawm cistanche tuaj yeem tshem tawm ntau cov pa oxygen dawb radicals thiab tiv thaiv DNA los ntawm oxidation los ntawm OH[8]. Cistanche extract tubuloside B tuaj yeem txo qhov cytotoxicity ntxias los ntawmMPP ntxiv,Tsis muaj zog lub tsub zuj zuj ntawm reactive oxygen hom nyob rau hauv lub cell, thiab muaj ib tug antagonistic nyhuv ntawm apoptosis thiab oxidative kev nyuaj siab induced los ntawmMPP ntxiv[9]. Cov tshuaj suav tshuaj tuaj yeem qhib thiab ntxiv dag zog rau nws tus kheej txoj kev tiv thaiv kev tiv thaiv, tiv thaiv kev puas tsuaj sab nraud, thiab rov ua kom nws qhov sib npaug. Qhov no yog ib lub hom phiaj ntawm modernization ntawm tsoos suav tshuaj. Tom qab dopamine hloov kho, tib neeg tam sim no feem ntau muaj nuj nqis rau kev kho mob neuroprotective, uas yog hu ua lub kiv puag ncig thib ob hauv kev kho mob.Tus kab mob Parkinson. Hauv qhov kev sim no, cov kab mob midbrain dopaminergic paj hlwb raug cais nrog cov tshuaj suav nrog cov tshuaj suav, thiab nws tau pom tias Cistanche muaj qee yam kev tiv thaiv neuroprotective. Hauv qhov kev sim no, ob txoj kev tshawb pom ntawm cov dej cytometry thiab TUNEL staining tau siv los ua ib txhij pom tias qhov sib npaug ntawm cov tshuaj pab pawg ntshav qab zib thiab 2 npaug ntawm cov tshuaj sib npaug ntawm cov tshuaj hauv Cistanche cov kab mob ntshav qab zib tuaj yeem txo cov kab mob hauv nruab nrab hauv nruab nrab dopamine paj hlwb los ntawmMPP ntxivTus naj npawb ntawm apoptotic hlwb muaj qee yam kev tiv thaiv ntawm apoptotic hlwb. Cov tshuaj suav tshuaj tuaj yeem tiv thaiv dopaminergic neurons los ntawm kev puas tsuaj thaum ntxov, uas yog ib qho kev ntsuas tseem ceeb tshaj li lwm cov kev kho mob. Nyob rau hauv qhov kev nkag siab no, kev kho mob ntawm Suav tshuaj yog qhov kho rau lub hauv paus, thiab nws tuaj yeem suav tias yog ib qho kev kho mob neuroprotective uas tib neeg muaj txiaj ntsig tam sim no.

Cov ntaub ntawv

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9. Sheng G, Pu X, Li L, et al. Tubuloside B los ntawm Cistanche salsa Rescues PC12 neuronal hlwb los ntawm 1-Methyl-4-phenylpyridinium ion-induced apoptosis thiab oxidative.Stress.Planta Med,2002,68(11):966