Tag Nrho Glycosides Ntawm Cistanche Deserticola Txhawb Kev Ua Haujlwm Zoo Tshaj Plaws Los Ntawm Inducing Neurovascular Regeneration Via Nrf- 2/Keap-1 Txoj Kev hauv MCAO/R Rats-Ⅰ

Taw qhia

Strokes raug suav hais tias yog qhov ua rau tuag thiab kev tsis taus nyob hauv ntiaj teb. Ze li ntawm 87% ntawm tag nrho cov mob stroke yog tshwm sim los ntawm ischemic stroke. Tam sim no, tus neeg sawv cev zoo tshaj plaws thiab tsuas yog FDA pom zoo tshuaj siv rauKev kho mob stroke ischemicyog recombinant cov ntaub so ntswg plasminogen activator. Txawm li cas los xij, ntau tus neeg mob stroke tsis teb rau cov tshuaj no, vim nws txoj kev kho lub sijhawm nqaim thiab muaj kev pheej hmoo loj ntawm cov teeb meem hemorrhagic. Ib qho kev sib tw loj ntawm kev kho mob thrombolytic yog ischemia / reperfusion (I / R) raug mob, uas yog suav tias yog qhov ua rau lub hlwb raug mob thiab ua haujlwm puas tsuaj. Reperfusion tom qab cerebral ischemia ua rau muaj kev pheej hmoo ntawm lub paj hlwb hemorrhage thaum ua rau muaj kev raug mob neurovascular thiab ua rau ntau hom reactive oxygen (ROS) uas ua rau cov ntshav-hlwb barrier. Ntau qhov kev tshawb fawb tau lees paub tias kev cuam tshuam ntawm BBB yog qhov ua rau muaj kev cuam tshuam ntawm ischemic stroke.

NATURAL CISTANCHE TUBULOSA rau Kev Kho Mob ISCHEMIC STROKE PHGS75% ECH 30% ACT 12%

BBB muaj feem ntau ntawm cov hlwb endothelial, pericytes, astrocytes, neurons, thiab hauv qab daus daim nyias nyias. Cov ntsiab lus tseem ceeb ntawm BBB yog cerebral microvascular endothelial hlwb uas koom nrog nruj junctions, yog li txwv exogenous molecules rau hauv lub hlwb. Cov kev hloov pauv pathological ntawm kev sib tw nruj-tshwj xeeb tshaj yog occludin, claudin-5, thiab zonula occludens-1 (ZO-1)-tshwj xeeb cuam tshuam rau BBB kev ua haujlwm thaum lub sij hawm ischemic stroke, tshwj xeeb tshaj yog kev thaiv permeability. Thaum lub sij hawm I / R, ntau ROS yog ib qho ntawm cov ntsiab lus tseem ceeb uas ua rau muaj kev puas tsuaj ncaj qha ntawm lub hlwb neurons. ROS overproduction ua rau degradation ntawm tej yam junctions thiab BBB cuam tshuam, uas ua rau exogenous molecules nkag mus rau hauv lub hlwb los ntawm BBB, ua rau lub hlwb puas aggravation. Yog li ntawd, kev tiv thaiv ntawm BBB los ntawm cov tshuaj tiv thaiv oxidants tau raug suav tias yog txoj hauv kev los tiv thaiv kev raug mob reperfusion.

Dhau li ntawm qhov tawg ntawm BBB, I / R tuaj yeem ua rau muaj kev raug mob neurovascular thiab kev tuag neuronal. Thaum mob stroke, nce neuronal cell tuag yuav tshwm sim los ntawm oxidative kev nyuaj siab thiab ntau cov kev tshawb fawb pom tau hais tias ROS aggravates mob stroke thiab neurological puas. Txawm hais tias kev sim tshuaj tsis tau txais txiaj ntsig zoo, kev tiv thaiv neuroprotection tseem yog ib lub tswv yim zoo rau kev kho mob ntawm ischemic stroke. Yog li, nrhiav cov tshuaj zoo neuroprotection los kho mob stroke yog ib qho txiaj ntsig rau cov neeg mob stroke.

Cov tshuaj suav tshuaj suav tshuaj (TCM) siv cov kev ntsuas los cuam tshuam rau lub cev tsis sib xws. Raws li cov txheej txheem nyuaj ntawm ischemic strokes, cov nyhuv multifactorial ntawm TCM thiab nws cov koom haum nquag ua lub luag haujlwm tseem ceeb hauv kev kho mob stroke.Cistanchedeserticola YC Ma, thoob plaws hauv thaj chaw qhuav lossis ib nrab arid thoob plaws hauv Mongolia thiab Northwest Tuam Tshoj, tau siv dav siv tshuaj ntsuab TCM rau kev kho mob ntawm ntau yam kab mob xws li tsis nco qab thiab kev nyuaj siab rau ntau tshaj 1,000 xyoo hauv Suav teb. Cov kev tshawb fawb pharmacological niaj hnub tau qhia tias cov khoom siv raw ntawm C. deserticola tau pom ntau yam kev ua haujlwm tshuaj, xws liTxhim khu kev kawm thiab kev nco muaj nuj nqi, neuroprotection, tiv thaiv kab mob, antioxidant, anti-aging, thiab antifatigue los. Tshuaj ntsuam xyuas ntawm C. deserticola pom tias nws cov ntsiab lus tseem ceeb suav nrogphenylethanoid glycosides, iridoid glycosides, polysaccharides, thiab oligosaccharides. Txawm li cas los xij, cov khoom nquag ntawm C. deserticola rau kev tiv thaiv lub hlwb tsis meej heev.

Cov cuab yeej tiv thaiv neuroprotective ntawm C. deserticola txhais tau hais tias nws cov peev xwm kho tau hauv kev paub txog kev mob nkeeg xws li mob stroke thiab kev nyuaj siab, nrog rau Alzheimer's disease. Txawm li cas los xij, kev tshawb fawb txog qhov cuam tshuam ntawm C. deserticola ntawm strokes, nrog rau nws cov khoom nquag thiab cov txheej txheem ua haujlwm, muaj tsawg heev. Hauv kev ua haujlwm tam sim no, peb tau tshawb fawb txog kev tiv thaiv ntawm peb qhov kev rho tawm los ntawm C. deserticola, tag nrho glycosides (TGs, phenylethanoid glycosides, thiab lwm yam glycosides), polysaccharides (PSs), thiab oligosaccharides (OSs) ntawm cerebral I/R raug mob. Peb qhov kev tshawb pom tuaj yeem ua rau muaj qhov tseeb daim ntawv thov kev kho mob ntawm C. deserticola thiab muab tus neeg sawv cev sib tw rauKev kho mob stroke ischemic.

KHOOM PLIG THIAB MUAB

Tshuaj thiab Reagents

Cov stems ntawm Cistanche deserticola tau yuav los ntawm Alashan, Inner Mongolia, thiab txheeb xyuas los ntawm ib tus kws sau ntawv (P.-F. Tu). TGs, PSs, thiab OSs tau npaj raws li peb txoj kev qhia yav dhau los. Kev txheeb xyuas ntau ntawm TGs tau ua los ntawm kev ua haujlwm siab ua kua chromatography (HPLC) raws li tau piav qhia dhau los, thiab nws cov chromatogram tau qhia hauv daim duab 1. Cov khoom tseem ceeb ntawm TGs yog echinacoside, tubuloside A, acteoside, isoacteoside, thiab 2'-acetylacteoside; Lawv cov ntsiab lus yog 163.05 mg / g, 4.125 mg / g, 41.66 mg / g, 22.655 mg / g, thiab 12.045 mg / g, feem. Cov ntsiab lus ntawm PSs thiab OSs yog 69.42% thiab 65.24%, feem, raws li kev txiav txim los ntawm HPLC thiab phenol-sulfuric acid tsom xam, feem.

NATURAL CISTANCHE TUBULOSA rau kev kho mob ISCHEMIC STROKE PHGS75% ECH 30% ACT 12%

Cov qauv siv ntawm echinacoside (A0282), tubuloside A (A0942), acteoside (A0280), isoacteoside (A0281), thiab 2'- acetylacteoside (A0943) tau yuav los ntawm Chengdu Yuav Tsum Biotechnology. Lub purities ntawm tag nrho cov qauv yog ntau tshaj 98%. Nissl stain H&E cov khoom siv tau yuav los ntawm Boster. Edaravone (T0407-1) ​​tau yuav los ntawm Target Mol (Shanghai, Suav). Rabbit anti-rat MAP-2 (ab32454), Nrf-2 (ab31163), PDGFRb (ab32570), Keap-1 (ab66620), thiab nas anti-rat CD31 (ab24590) tau muas los ntawm Abcam Inc. Rabbit anti-rat Claudin5 (BS1069), ZO-1 (BS9802M), thiab Occludin (BS72035) tau yuav los ntawm Bioworld Technology. Cell Signaling Technology Inc. (Boston, MA, USA) yog lub hauv paus ntawm luav anti-rat Synapsin-1 (SYN,5297T), PSD95 (3450T), a-Smooth Muscle Actin (a-SMA,19245T). GAPDH (HRP-60004) tau yuav los ntawm Proteintech Group, Inc.Secondary antibodies tau muab los ntawm Zhongshan Golden Choj Biotechnology (Beijing, Suav). Hoechst 33258 tau txais los ntawm Beyotime.

Tsiaj txhu

Sprague-Dawley nas (txiv neej, hnyav 250-300g) tau txais los ntawm Vital River Laboratory Tsiaj Technology thiab muab tso rau hauv chav cua txias cia rau ntawm 12 teev lub teeb / tsaus ntuj. Txhua qhov kev sim tsiaj tau ua los ntawm kev tshawb fawb tsiaj txhu ARRIVE, thiab tau pom zoo los ntawm Pawg Saib Xyuas Tsiaj Tsiaj thiab Siv Pawg ntawm Peking University Health Science Center.

Animal Experimental Protocols

Cov nas tau raug rau MCAO/R, raws li tau piav qhia yav dhau los (Wang et al., 2018). Luv luv, sab laug ntawm cov hlab ntsha carotid (CCA), sab nraud carotid artery (ECA), thiab sab hauv carotid artery (ICA) raug nthuav tawm, thiab ib qho 3-0 nylon monofilament suture tau ntxig los ntawm ECA rau hauv ICA kom txog rau thaum nruab nrab. cerebral artery (MCA). Tom qab 1.5 teev ntawm MCA occlusion, reperfusion tau simulated los ntawm kev tshem cov filament. Thaum lub sij hawm kev phais, lub cev kub ntawm tag nrho cov nas tau khaws cia ntawm 37.0 degree. Tshuaj

Kev tswj hwm

Cov nas tau muab cais ua rau rau pawg siv SPSS software version 22.0 raws li tau piav qhia: pawg ib txwm (NOR); pawg qauv (MOD); edaravone pawg (cov tshuaj zoo, 6 mL / kg, EDI); TGs (280 mg/kg, TGs); PSs group (280 mg/kg, PSs), and OSs group (280 mg/kg, OSs). TGs, PSs, thiab OSs raug tswj hwm ib hnub ib hnub tom qab MCAO/R rau 14 hnub. Cov pab pawg NOR thiab MOD tau kho nrog saline ib txwm. Cov nab npawb tsiaj muaj nyob hauv Table 1.

Kev ntsuas qhov hnyav thiab hloov cov qhab nias Neurological Deficit (mNSS)

Lub cev hnyav tau saib xyuas hnub 14 uas siv ADVENTURE™ Digital Scale (OHAUS, New Jersey, USA). Lub mNSS tau raug soj ntsuam raws li txoj kev piav qhia los ntawm FJ Wang (Wang li al., 2018), nrog rau kev hloov kho me me.

2, 3, 5-Triphenyltetrazolium Chloride (TTC) Staining

Infarct ntim tau ntsuas raws li tau piav qhia dhau los. Hauv luv luv, lub hlwb tau muab faib ua xya qhov sib npaug sib npaug coronal blocks (2 mm). Cov ntu no tau stained nrog 2% TTC ntawm 37 degree rau 15 min. Infarct ntim (%)=(ipsilateral ischemic hemisphere ntim −contralateral ischemic hemisphere ntim)/contralateral ischemic hemisphere ntim × 100.

Nissl thiab H&E Staining

Cov nas tsuag tau mob siab rau, thiab tag nrho lub hlwb tau sai sai tshem tawm ntawm pob txha taub hau kho siv 4% paraformaldehyde embedded nyob rau hauv paraffin wax thiab muab faib mus rau hauv slices ntawm 7 µm thickness. Cov seem tau stained nrog Nissl thiab H&E. Nyob rau hauv txoj kev tshawb no, rau random 200 × 200 µm teb tau raug ntes nyob rau hauv txhua cov ntaub so ntswg nrog lub teeb lub teeb. Tus naj npawb ntawm Nissl lub cev raug suav nrog IPP software version 6.0.

Evans Blue Assay

Cov nas tsuag tau txhaj nrog 2% EB tom qab MCAO/R. Ob teev tom qab ntawd, cov nas tsuag tau tshuaj loog thiab tag nrho lub hlwb raug tshem tawm sai sai thiab homogenized hauv acetone. Cov supernatants tau soj ntsuam ntawm 620 nm los ntawm 800 TS absorbance nyeem ntawv.

Kev ntsuas kev ua haujlwm ntawm Catalase (CAT), Superoxide Dismutase (SOD), Malondialdehyde (MDA), thiab Glutathione Peroxidase (GSH-Px)

Tag nrho cov qauv kuaj ntshav tau centrifuged ntawm 4, 000 × rpm rau 15 min ntawm 4 degree thiab tom qab ntawd tshuaj xyuas kom pom cov dej num ntawm MDA, CAT, SOD, thiab GSH-Px ua raws li cov chaw tsim khoom cov lus qhia.

Western Blotting Analysis

Cov ntaub so ntswg hauv hlwb (100 mg) sau los ntawm txhua tus nas tau ua homogenized thiab lysed hauv RIPA lysis tsis thiab tom qab ntawd tshuaj xyuas kom pom cov protein ntau siv cov khoom siv BCA. Cov ntaub so ntswg tag nrho cov protein tau ntim rau ntawm 10% SDS-PAGE gels thiab xa mus rau ib daim nyias nyias ntawm nitrocellulose. Lub membrane raug thaiv siv 5% skim mis nyuj, ces incubated hmo ntuj nrog thawj cov tshuaj tiv thaiv ntawm 4 degree. Tom qab ntawd lub membrane tau incubated nrog ib tug thib ob antibody. Western blot tsom xam tau txheeb xyuas siv Kodak Digital Imaging System.

Immunofluorescent Analysis

Immunofluorescence staining rau CD31, a-SMA, ZO-1, claudin5, occludin, PDGFRb, SYN, PSD95, MAP-2, Nrf-2 thiab Keap-1 tau ua. Thawj cov tshuaj tiv thaiv tiv thaiv Nrf-2, CD31, a-SMA, ZO-1, claudin5, occludin, PDGFRb, SYN, PSD95, MAP-2 thiab Keap-1 tau diluted rau 1 :200 thiab 1:100, feem. Cov tshuaj tiv thaiv thib ob ntawm Alexa Flur 488 nas los tiv thaiv luav IgG thiab rhodamine (TRITC) tshis los tiv thaiv luav IgG yog ob qho tib si diluted rau 1: 200. Lub nuclei raug stained los ntawm Hoechst 33258. Cov duab tau raug siv Vectra® Polaris™ Automated Quantitative Pathology Imaging System (PerkinElmer, USA). Cov lus qhia protein tau txheeb xyuas siv IPP software version 6.0. Kev txheeb cais

Tag nrho cov ntaub ntawv tau piav raws li txhais tau tias ± SD. SPSS software version 22.0 tau ua rau kev txheeb xyuas cov lej. Ib txoj kev ANOVA tau siv thaum sib piv cov pab pawg sib txawv. P < 0.05 tau suav tias yog qhov sib txawv ntawm cov lej.

NATURAL CISTANCHE TUBULOSA FOR ANTI-ISCHEMIC DISEASE PHGS75% ECH 30% ACT 12%

TSEEM CEEB

TGs nce lub cev qhov hnyav thiab txo qis kev puas hlwb hauv MCAO/R nas

Tom qab 14 hnub ntawm kev kho mob nrog TGs, PSs, Oss, thiab EDI, lub cev hnyav, qhov tsis txaus ntawm lub paj hlwb, thiab cov kab mob infarct ntawm I / R nas raug soj ntsuam. Cov txiaj ntsig tau pom tias lub cev hnyav hauv pab pawg MOD tau poob qis heev, thaum txo qis hauv TGs, PSs, thiab EDI pawg tau nce (Daim duab 2A). Neurological deficit cov qhab nia tau txo qis los ntawm EDI thiab TGs (Daim duab 2B). Lub hlwb hlais hauv NOR pab pawg nas tau sib sib zog nqus liab thiab tsis muaj infarctions, thaum cov nas ntawm MOD pab pawg pom qhov loj ipsilateral cerebral infarction. Tom qab kev kho TGs, qhov ntim infarct tau txo qis (Daim duab 2C, D). Kev kho PSs thiab OSs tsis pom muaj txiaj ntsig zoo rau cov ntsuas saum toj no. Cov ntaub ntawv saum toj no tau qhia tias TGs tuaj yeem txo qhov I / R ua rau lub hlwb raug mob, tab sis PSs thiab OSs ua tsis tau.

TGs Ameliorate Histopathological puas nyob rau hauv MCAO/R nas

Txhawm rau txiav txim siab qee qhov cuam tshuam ntawm TGs, PSs, thiab OSs kev kho mob ntawm kev puas tsuaj histopathological, H&E staining tau ua los qhia txog kev puas tsuaj. Cov txheej txheem histomorphological ntawm lub hlwb hauv NOR pawg tau teem caij tsis tu ncua. Cov kev hloov pauv ntawm morphology hauv pawg TGs tau me dua li cov hauv pawg MOD. Txawm li cas los xij, cov pab pawg kho mob PSs thiab OSs tsis pom qhov tseem ceeb ntawm kev hloov pauv morphology (Daim duab 3).

TGs Attenuate Neuronal Injury Tom qab I / R-Induced nas

Nissl staining pom cov kev hloov pauv histopathological ntawm neurons hauv penumbra ntawm thaj chaw ischemic. Raws li pom hauv daim duab 4, cov neurons ib txwm muaj cov nucleolus meej thiab cov qauv tsis zoo. Hauv pab pawg MOD, cov neurons tau nthuav dav qhov chaw sib txuas. Lub cev nissl ploj lawm, shrunken thiab stained heev. Txawm li cas los xij, cov kev hloov no tsis tshua pom nyob hauv EDI, TGs, thiab PS pawg. Cov txiaj ntsig no qhia tau tias TGs thiab PSs tuaj yeem ua tau zooattenuate ischemia/reperfusion-induced neuronal raug mob.

TGs Attenuate BBB cuam tshuam tom qab kuv / R-kho nas

Evans blue assay yog ib txoj hauv kev tshawb fawb txog kev hloov pauv ntawm BBB permeability. Cov txiaj ntsig kev sim tau pom tias nce Evans xiav tau pom nyob rau hauv pab pawg MOD, thaum muaj qhov txo qis Evans xiav hauv TGs thiab EDI-kho nas. Ntxiv mus, tsis muaj qhov sib txawv tseem ceeb ntawm PSs thiab OSs kho pawg (Daim duab 5). Cov txiaj ntsig no tau qhia tias TGs tuaj yeem txo qis BBB cuam tshuam.

TGs Txhawb nqa Angiogenesis hauv I/R Injured nas

Cov kev tshawb fawb tsis ntev los no qhia tau hais tias angiogenesis plays lub luag haujlwm tseem ceeb hauv kev ua haujlwm ntawm lub paj hlwb thiab cov txiaj ntsig tau tshwm sim tom qab mob stroke ischemic (Yuen li al., 2015). Txhawm rau ntsuas qhov cuam tshuam ntawm TGs, PSs, thiab OSs ntawm angiogenesis, CD31 thiab a-SMA tau siv los ntsuas cov lej capillary. Immunofluorescence staining pom tias pab pawg MOD tau ua rau muaj kev txo qis hauv CD31 (Figures 6A, B) thiab a-SMA (Figures 6C, D) nyob rau hauv lub penumbra ntawm ischemic cheeb tsam ntawm I / R nas, piv nrog rau cov nas ib txwm. . Cov txiaj ntsig no qhia tau tias I / R tuaj yeem ua rau cov hlab ntsha puas tsuaj hauv lub cortex penumbra ntawm ischemic hemispheres. Txawm li cas los xij, kev kho TGs thiab EDI zoo kawg li nce capillary ntom, angiogenesis, thiab arteriogenesis raws li qhia los ntawm kev nthuav qhia ntawm CD31 thiab a-SMA. Cov txiaj ntsig no qhia tias TGs tuaj yeem txhawb nqa angiogenesis hauv ischemic penumbra ntawm I / R nas, tab sis PSs thiab OSs tsis tuaj yeem.

TGs nce kev nthuav qhia ntawm Tight Junction Proteins hauv I / R Cov nas raug mob

Kev cuam tshuam BBB tuaj yeem ua rau cov dej hauv hlwb thiab cov ntaub so ntswg o, ua rau lub hlwb raug mob. Tight junction proteins yog cov khoom tseem ceeb ntawm BBB. Txhawm rau kuaj seb TGs, PSs, thiab OSs kev kho mob tom qab mob stroke tuaj yeem cuam tshuam rau BBB kev ncaj ncees, cov lus hais ntawm ZO-1, claudin-5, thiab occludin tau ua los ntawm kev tshuaj ntsuam xyuas immunofluorescence. Cov txiaj ntsig tau qhia tias cov kab lus ntawm claudin-5, occludin, thiab ZO-1 tau poob qis hauv pawg MOD. Txawm li cas los xij, lawv tau nce ntxiv tom qab 14 hnub ntawm nws cov thawj coj. PSs thiab OSs pawg tsis pom qhov hloov pauv tseem ceeb hauv cov kab lus protein no (Daim duab 7). Cov ntaub ntawv no tau qhia tias TGs tuaj yeem tswj hwm cov kab lus sib txuas ceev ceev thiab tej zaum yuav tswj tau BBB kev ncaj ncees tom qab I / R raug mob.

TGs Nce Pericyte Kev Pabcuam ntawm Capillaries hauv I / R Cov nas raug mob

Pericyte kev pab them nqi ntawm capillaries ua lub luag haujlwm tseem ceeb hauv kev tswj hwm BBB kev ncaj ncees. Yog li, peb tau sim seb puas muaj kev tiv thaiv pericyte tuaj yeem nce los ntawm TGs, PSs, thiab OSs kho. Immunofluorescence kev siv tshuaj ntsuam xyuas tau pom tias ob qho tib si PDGFRb thiab CD31 kab lus tau txo qis hauv pawg MOD. Kev tswj hwm ntawm TGs rau cov nas I / R tau zoo rov qab los yog nce qhov kev siv zog ntawm PDGFRb thiab CD31, tab sis tsis muaj qhov sib txawv tau pom nyob rau hauv pawg PSs thiab OSs kho (Daim duab 8). Yog li, kev kho mob ntawm TGs tuaj yeem nce pericyte kev pab them nqi. Cov kev tshawb pom no tau lees paub ntxiv tias TGs tuaj yeem tswj hwm kev ncaj ncees ntawm BBB tom qab I / R.

NATURAL CISTANCHE TUBULOSA KEV PAB CUAM ISCHEMIC STROKE PHGS75% ECH 30% ACT 12%