Herba Cistanche Extract Enhances Mitochondrial Glutathione Status Thiab Ua pa hauv nas Lub Plawv, Nrog Muaj peev xwm Induction Ntawm Uncoupling Proteins

Taw qhia

Reperfusion ntawm yav dhau los ischemic myocardium ua rau muaj kev hloov pauv ntawm mitochondrial, suav nrog kev tsim cov pa oxygen ntau ntxiv (ROS), Ca.2+overload, thiab poob ntawm adenosine triphosphate (ATP) ntau lawm, tag nrho cov no tuaj yeem ua rau necrosis thiab / lossis apoptosis-mediated cell tuag (Redegeld li al., 1992; Lemasters et al., 1998). Raws li mitochondria ua lub luag haujlwm tseem ceeb hauv kev txiav txim siabnyob rau hauv txoj hmoo ntawm cardiomyocytes tom qab ib qho kev sib tw ischemia / reperfusion (I / R), kev tiv thaiv ntawm myocardial I / R raug mob yuav tsum tau tsom mus rau kev khaws cia ntawm mitochondrial cov qauv thiab kev ua haujlwm zoo. Kev saib xyuas ntawm mitochondrial zog metabolism tom qab I / R kev sib tw yog qhov tseem ceeb tshwj xeeb vim tias lub plawv contractile muaj nuj nqi yuav luag tag nrho nyob ntawm mitochondria-tsim ATP. Cardiomyocytes uas ciaj sia tab sis tsis cog lus yog siv tsawg heev.

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Herba Cistanche, tag nrho cov nroj tsuag qhuav ntawmCistanche deserticolaYC Ma (Orobanchaceae), yog cov kab mob cab loj hlob hauv thaj chaw suab puam ntawm sab qaum teb thiab sab hnub tuaj ntawm Tuam Tshoj. Herba Cistanche, muab faib ua "Yang-invigorating" tshuaj ntsuab hauv Suav tshuaj, yog tshuaj rau lub raum tsis muaj peev xwm, poj niam tsis muaj menyuam, thiab cem quav tshwm sim los ntawm plab hnyuv qhuav hauv cov neeg laus (Chen, 1998). Cov kev tshawb fawb yav dhau los hauv peb chav kuaj tau pom tias muaj methanolExtract ntawm Herba Cistanchence myocardial ATP tiam muaj peev xwm (Leung & Ko, 2008) thiab tiv thaiv myocardial I/R raug mob hauv nas (Leung, 2006). Whereas stimulation ntawm ATP tiam muaj peev xwm yog paralleled los ntawm kev txhim kho nyob rau hauv mitochondrial electron thauj (Leung & Ko, 2008), cardioprotection tiv thaiv I / R raug mob los ntawm Herba Cistanche kev kho mob yog txuam nrog ib tug txo nyob rau hauv myocardial ATP depletion (Leung, 2006). Hauv kev tshawb fawb tam sim no, txhawm rau txheeb xyuas lub luag haujlwm ntawm mitochondria hauv kev ua haujlwm cardioprotective ntawm Herba Cistanche, peb tau tshawb xyuas cov txiaj ntsig ntawm Herba Cistanche kev kho mob ntawm mitochondrial glutathione raws li txoj cai, Ca2+ cov ntsiab lus, daim nyias nyias, thiab kev ua pa ntawm lub siab, hauv nas plawv. .

Cov ntaub ntawv thiab cov txheej txheem

Cov khoom siv tshuaj ntsuab

Herba Cistanche tau yuav los ntawm ib lub zos (Hong Kong-raws li) tshuaj ntsuab (Lee Hoong Kee). Cov tshuaj ntsuab tau lees paub los ntawm tus neeg muag khoom thiab daim ntawv pov thawj (HKUST00301) tau muab tso rau hauv Department of Biochemistry, Hong Kong University of Science & Technology (HKUST). Kev rho tawm methanol ntawm cov tshuaj ntsuab tau ua, raws li cov kev tshawb fawb yav dhau los qhia tau hais tias xws li cov extract txhim kho mitochondrial ATP tiam thiab tiv thaiv I / R raug mob hauv lub siab nas (Leung, 2006; Leung & Ko, 2008). Nyob rau hauv luv luv, Herba Cistanche (400 g) raug txiav mus rau hauv me me pieces thiab muab rho tawm los ntawm cua sov nyob rau hauv reflux nyob rau hauv 300mL ntawm methanol ntawm 60 degree rau 2h, raws li yav tas los tau piav (Leung & Ko, 2008). Cov txheej txheem tau rov ua dua ob zaug. Lub pas dej ua ke tau qhuav los ntawm evaporating cov kuab tshuaj nyob rau hauv lub siab txo; yield yog 42% (w/w) txog tus nqi ntawm cov tshuaj ntsuab crude. Cov extract tau khaws cia ntawm 4 degree kom txog thaum siv. Txawm hais tias suav tshuaj ntsuab yog ib txwm siv cov dej rho tawm rau qhov ncauj, methanol tau siv rau hauv peb txoj kev tshawb fawb ua ntej kom yooj yim rau kev ua thiab khaws cia cov qauv (Yim & Ko, 2002), thiab peb pom methanol extraction kom txaus siab rau txhua yam.

Kev tu tsiaj thiab kev kho tshuaj

Cov neeg laus cov poj niam Sprague-Dawley nas (8-10 lub lis piam ntawm hnub nyoog; 200-230 g) tau nyob hauv chav tswj cov av noo, nrog lub voj voog 12h tsaus ntuj / lub teeb, ntawm kwv yees li 22 degree, thiab tso cai rau zaub mov thiab dej ad libitum. Tsiaj txhu tau random muab rau ntau pab pawg, nrog tsib tsiaj nyob rau hauv txhua pab pawg. Cov nas tau txaisHerba Cistanche Extractintragastrically ({{0}}.2 g/mL hauv dej) ntawm 0.5g/kg lossis 1.0 g/kg rau 3 hnub sib law liag. Tswj (tsis kho) tsiaj tau txais dej xwb. Nees nkaum plaub teev tom qab txhaj tshuaj kawg, lub plawv ventricular cov ntaub so ntswg tau txais los ntawm anesthetized tsiaj rau biochemical tsom xam. Tag nrho cov txheej txheem kev sim tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Tshawb Fawb, HKUST.

Kev npaj ntawm mitochondrial feem

Minced lub plawv ventricular cov ntaub so ntswg (~{{0}}.6g) tau homogenized nyob rau hauv ib tug 10- quav (w/v) tshaj ntawm cov dej khov-txias sucrose tsis [0.32M sucrose, 1mM ethylene diamine tetraacetic acid (EDTA), 50mM Tris / HCl; pH 7.4] siv Teflon-iav homogenizer, ntawm 4000 rpm, nrog 25-30 strokes. Mitochondrial pellets tau npaj los ntawm cov ntaub so ntswg homogenates los ntawm centrifugation ntawm 800 × g ntawm 4 degree rau 30min, thiab purity yog txiav txim los ntawm kev ntsuas ntawm cov txheeb ze cov dej num ntawm succinate dehydrogenase thiab lactate dehydrogenase nyob rau hauv lub supernatant thiab pellet, raws li tau piav ua ntej (Evans, 1992). Mitochondrial pellets raug tshem tawm hauv 1mL ntawm homogenizing tsis.

Kev ntsuas ntawm mitochondrial txo glutathione thiab oxidized glutathione qib

Mitochondrial txo glutathione (GSH) qib tau txiav txim siab enzymatically siv 5,59-dithiobis(2- nitrobenzoic acid) (DTNB) thiab glutathione reductase (GR), nyob rau hauv ib tug raws tu qauv hloov los ntawm Griffith (198{{17}) }). Ib qho aliquot (210 µL) ntawm mitochondrial feem yog tov nrog 90 µL ntawm 10% (v/v) 5-sulfosalicylic acid (SSA) thiab cov sib tov yog centrifuged ntawm 600 × g rau 10min. Txhawm rau ntsuas oxidized glutathione (GSSG) qib, 100µL cov nyiaj SSA supernatants tau tov nrog 10 µL ntawm 20% (w/v) 2-vinylpyridine thiab 10 µL 60% (v/v) triethanolamine hauv microfuge raj. Txhua lub raj raug tso cai kom sawv ntawm chav tsev kub tsawg kawg 1h. Cov tshuaj tiv thaiv sib xyaw uas muaj 0.63mM DTNB thiab 0.053mM NADPH (txo nicotinamide adenine dinucleotide phosphate) hauv phosphate tsis (0.1M, nrog 5mM Na2 EDTA; pH7.5) yog pre-incubated ntawm 2.30 degree. Ib qho aliquot (30 µL) ntawm ib qho SSA qauv supernatant (tag nrho glutathione) los yog GSSG qauv tau ntxiv rau ib lub qhov dej ntawm 96-zoo microtiter phaj, thiab 180 µL ntawm cov tshuaj tiv thaiv ua ntej sov, muaj 0.525U / mL GR, ntxiv tom ntej. Kev hloov pauv ntawm 412nm tau saib xyuas spectrophotometrically rau 5min. Cov concentrations ntawm GSH thiab GSSG tau kwv yees los ntawm calibration curves siv GSH thiab GSSG [kua nyob rau hauv 3% (w/v) SSA] raws li qauv thiab qhia raws li nmol / mg protein. GSH qib tau kwv yees los ntawm rho tawm ob npaug ntawm GSSG los ntawm tag nrho GSH.

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Kev ntsuas ntawm mitochondrial Ca2+ cov ntsiab lus

Mitochondrial Ca{{0}} cov ntsiab lus raug ntsuas los ntawm Ca2+-sensitive fluorescence sojntsuam, Fluo-5N AM ester (Molecular Probes, Eugene, OR) ntiav Victor2 Multi-Label Counter (Model 1420; PerkinElmer, Turku, Finland), raws li tau piav qhia hauv Menze li al. (2005). Ca2+ dissociation constants (Kd qhov tseem ceeb) tau txiav txim siab siv Ca2+ calibration kit siv tau nyob rau hauv qhov concentration range 1–1000 µM; kwv yees Kd qhov tseem ceeb yog 90-100 µM, nrog kev ntseeg siab, raws li cov khoom siv cov ntaub ntawv. Ib qho aliquot (25µL) ntawm mitochondrial feem (0.5mg protein / mL zaum kawg concentration) yog tov nrog 25 µL incubation buffer {100mM KCl thiab 30mM MOPS [3-(N-morpholino)propanesulfonic acid]; pH7.2} hauv 96-zoo microtiter phaj. Qhov sib tov yog incubated ntawm 25 degree rau 15 min; 25 µL digitonin (50 µg/mL) thiab 25 µL Fluo-5N AM ester (1 µM hauv 0.005% (w/v) Pluronic F-127) tau ntxiv. Los ntawm kev yooj yim rau daim nyias nyias permeation, digitonin mediated mitochondrial nkag ntawm fluorescent dye. Peb pom tias Fluo-5N theem nyob rau hauv nas plawv mitochondrial npaj tau nce 20-fold nyob rau hauv lub xub ntiag ntawm digitonin. Cov tshuaj tiv thaiv sib xyaw tau tsim nyob rau ntawm 25 degree rau 30 feeb, thiab kev nyeem ntawv fluorescence tau ntsuas ntawm qhov excitation wavelength ntawm 488nm thiab emission wavelength ntawm 532nm. Mitochondrial Ca2+ cov ntsiab lus tau kwv yees siv qhov ntsuas qhov nkhaus thiab qhia tias yog µmol/mg protein.

Kev ntsuas ntawm mitochondrial membrane muaj peev xwm

Membrane peev xwm (ΔΨm) tau soj ntsuam los ntawm ib txoj kev hloov kho los ntawm Bonavita li al. (2003), siv cov xim fluorescent, lipophilic cationic sojntsuam 5,596,69- tetrachloro-1,193,39-tetraethylbenzimidazolylcarbocyanine iodide, feem ntau hu ua JC-1. Aliquots (50 µL) ntawm mitochondrial feem (hloov mus rau 1mg protein / mL) tau incubated ntawm 37 degree nrog 50 µL ntawm substrate tov (muaj 6mM pyruvate thiab 6mM malate), 25 µL ntawm pretreated adenosine diphosphate (30 μL) daws (AD) thiab 25 µL ntawm 3 µM JC-1, nyob rau hauv qhov tsaus ntuj. Cov txiaj ntsig ΔΨm tau txais los ntawm kev ntsuas fluorescence ntawm 535nm (FL1) piv rau 580nm (FL2), siv Victor2 MultiLabel Counter. JC-1 cov ntaub ntawv sib sau ua ke hauv mitochondria, ua rau muaj FL2 fluorescence siab thiab qhia tias muaj peev xwm mitochondrial. Kev poob ntawm ΔΨm ua rau txo qis hauv FL2 fluorescence (JC-1 aggregates mus rau qhov tsawg dua) thiab qhov sib txuas ntawm FL1 fluorescence (los ntawm JC-1 monomers). Cov ntaub ntawv tau nthuav tawm raws li qhov sib piv ntawm FL1 / FL2 fluorescence qhov tseem ceeb. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), tus neeg sawv cev ΔΨm-collapsing, tau siv los ntsuas qhov kev xeem.

Kev ntsuas ntawm mitochondrial ua pa

Mitochondrial ua pa tau ntsuas ntawm 37 degree siv Hansatech Oxygraph-Plus ntsuas nruab nrog Clark-hom oxygen electrode (Sarasota, FL, USA). Mitochondrial fractions (0.6mg protein/mL) raug tshem tawm hauv kev ua pa tsis muaj 125mM KCl, 20mM MOPS, 10mM Tris, 0.5mM ethylene glycol tetraacetic acid (EGTA), thiab PO4M; pH 7.2. Tom qab incubation ntawm 37 degree rau 2min, txhua tus qauv tau txais 50 µL substrate tov (5mM pyruvate thiab 5mM malate). Kev ua pa tau ntsuas nyob rau hauv lub xub ntiag (State 3) ntawm 1mM ADP thiab tom qab phosphorylation ntawm tag nrho ADP rau ATP (State 4). Qhov piv ntawm Lub Xeev 3: Lub Xeev 4respiration tus nqi yog qhov ntsuas kev ua pa, uas qhia txog qhov nruj ntawm kev sib txuas ntawm kev ua pa thiab phosphorylation (Javadov li al., 2005; Kuwabara et al., 1997). Ua ntej qhov sib ntxiv ntawm cov tshuaj substrate, Lub Xeev 1 kev ua pa tau ntsuas kom txog thaum pib ntawm Lub Xeev 2 ua pa, thiab Lub Xeev 2 ua pa tau kwv yees ua ntej qhov sib ntxiv ntawm ADP.

Kev txheeb cais

Cov ntaub ntawv tau txheeb xyuas los ntawm ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) rau ntau pab pawg sib piv. Yam tsawg kawg nkaus qhov sib txawv (LSD) qhov tseem ceeb tau siv los txheeb xyuas qhov sib txawv tseem ceeb ntawm ob pawg thaum p qhov tseem ceeb < 0.05.

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Cov txiaj ntsig

Raws li pom hauv daim duab 1 thiab 2, kev kho mob nrog methanolExtract ntawm Herba Cistanche({{0}}.5 lossis 1.0g/kg/hnub × 3) txhim kho mitochondrial glutathione nyob rau hauv nas plawv, raws li pov thawj los ntawm koob tshuaj-nyob ntawm qhov nce hauv GSH qib (11-30%) thiab txo qis hauv GSSG theem (31%).

Herba Cistanche kev kho koob tshuaj-raws li txo qis mitochondrial Ca2+ qib hauv nas plawv piv nrog kev tswj hwm; kev tawm dag zog yog 41% ntawm koob tshuaj 1.0 g / kg (Daim duab 3).

Kev kho mob Herba Cistanche nce mitochondrial ΔΨm, raws li pom los ntawm kev txo qis (14-16%) hauv FL1 / FL2 piv. CCCP (100 µM) ua rau ΔΨm vau, raws li qhia los ntawm kev nce hauv FL1 / FL2 piv (Daim duab 4).

Kev kho mob Herba Cistanchetxhim kho mitochondrial ua pa, raws li pov thawj los ntawm tus nqi siab dua ntawm Lub Xeev 3 ua pa (84%) hauv cov tsiaj sim piv rau kev tswj hwm (Table 1). Lub Xeev 2 thiab Lub Xeev 4 ua pa tau nce los ntawm 47 thiab 80%, feem, tab sis lub zog ntawm lub xeev 4 kev ua pa tau raug tshem tawm tag nrho los ntawm 1mM guanosine-5'-diphosphate, ib qho inhibitor ntawm uncoupling protein 2/3. Kev kho mob Herba Cistanche tsis cuam tshuam rau qhov ntsuas kev ua pa, raws li kwv yees los ntawm qhov piv ntawm Lub Xeev 3 / Lub Xeev 4 qhov ua pa.

Kev sib tham

Mitochondria cais los ntawm Herba Cistanche-kho nas lub siab pom tau tias muaj kev txhim kho glutathione, raws li qhia los ntawm qib GSH nce thiab txo GSSG qib. Kev saib xyuas ntawm mitochondrial glutathione redox xwm txheej yog qhov tseem ceeb rau cardioprotection tiv thaiv I/R raug mob (Chiu & Ko, 2003). Cytosolic Ca2+ cov ntsiab lus nce thaum lub sij hawm myocardial ischemia, los ntawm uptake los ntawm lub puab membrane Ca2+ uniporter (Ataka li al., 1992; Grover li al., 1990), ua rau mitochondrial Ca{{6} } sib sau. Peb cov txiaj ntsig tau qhia tias kev kho mob nrog Herba Cistanche extract txhim kho mitochondrial glutathione redox raws li txoj cai thiab txo qis mitochondrial Ca2+ qib, uas tuaj yeem tiv thaiv kev raug mob myocardial I/R (Leung, 2006). Qhov tsis muaj peev xwm ntawm Herba Cistanche kev kho mob, ntawm qhov koob tshuaj ntau dua ntawm 1.0 g / kg, txhawm rau txo qis GSSG thiab Ca{11}} qib, tej zaum yuav cuam tshuam nrog kev muaj tus kheej txwv kev tswj hwm tus kheej cuam tshuam rau glutathione redox peev xwm thiab calcium. hauv mitochondria.

Mitochondria tsim ib qho electrochemical proton gradient thoob plaws hauv daim nyias nyias, los ntawm electron thauj. Qhov gradient no yog siv los ntawm ATP synthase rau phosphorylate ADP rau ATP. Txo cov mitochondrial membrane muaj peev xwm, xws li hauv hypoxia / reoxygenation tom qab raug mob ntawm kab lis kev cai cardiomyocytes (Chiu li al., 2008), ua rau txo qis hauv ATP tiam. Herba Cistanche kev kho mob, uas yuav ua rau kom myocardial mitochondrial membrane muaj peev xwm, tejzaum nws txhim kho ATP tiam, tshwj xeeb tshaj yog thaum post-ischemic reperfusion. Ib zaug ntxiv, qhov muaj nyob ntawm tus kheej-txheej txheem kev tswj hwm tus kheej yuav piav qhia vim li cas Herba Cistanche kev kho mob cuam tshuam rau mitochondrial membrane qhov peev xwm tsis yog koob tshuaj.

Kev kho mob Herba Cistanche ua rau lub xeev 3 ua pa ntau dua hauv nas plawv mitochondria, raws li kev soj ntsuam los ntawm kev noj cov pa. Qhov kev tshawb pom no zoo ib yam nrog cov txiaj ntsig tau los ntawm kev ntsuas hauv vitro thiab qhov ntsuas ntawm mitochondrial ATP tiam muaj peev xwm (Leung & Ko, 2008). Intriguingly, txawm hais tias Herba Cistanche kev kho mob txo qis hauv lub xeev ntawm myocardial ATP (cov ntaub ntawv tsis qhia), kev kho mob txhim kho mitochondrial ATP tiam. Kev koom tes ntawm kev ua pa tsis sib xws tuaj yeem piav qhia cov kev soj ntsuam tsis sib xws. Uncoupled oxidative phosphorylation tshwm sim thaum protons tau tsiv mus thoob plaws mitochondrial inner membrane rov qab mus rau hauv mitochondrial matrix, yog li dissipating lub membrane muaj peev xwm tsim los ntawm electron thauj saw. Qhov no ua rau txo qis ntawm proton-motive quab yuam uas tsav ATP tsim (Garvey, 2003). Raws li qhov tshwm sim, Ca2+ concentration thiab ROS ntau lawm hauv mitochondria raug txo qis (Teshima li al., 2003). Cov lus qhia no yog ua raws li cov txiaj ntsig ntawm txoj kev tshawb fawb tam sim no, uas pom tau tias txo qis hauv mitochondrial Ca2+ qib hauv Herba Cistanche-kho siab. Txog kev ua pa tsis sib xws, kev sib txuas ntawm cov protein tsis sib txuas (UCPs) tuaj yeem raug ntxias los ntawm kev kho mob Herba Cistanche. UCPs paub txog kev sib koom ua ke oxidative phosphorylation, thiab kev qhia ntawm UCP1 hauv cov nas transgenic lub siab tiv thaiv I / R-induced myocardial puas (Hoerter li al., 2004). Cov nyhuv uncoupling ntawm UCPs yog inhibited los ntawm purine nucleotide diphosphates thiab triphosphates (xws li ADP, ATP, guanosine diphosphate (GDP), thiab GTP) (Echtay li al., 2002). Nyob rau hauv txoj kev tshawb no, mitochondrial tsis xws li daim nyias nyias muaj peev xwm thiab ua pa tus nqi tau ntsuas nyob rau hauv lub xub ntiag ntawm ADP (raws li substrate); Yog li ntawd, cov nyhuv uncoupling ntawm UCPs tau inhibited. Txawm li cas los xij, thaum mitochondrial State 4 kev ua pa ntsuas tau ntsuas thaum tsis muaj ADP, tus nqi no siab dua hauv mitochondria npaj los ntawm Herba Cistanche-kho lub siab dua hauv lub siab ntawm kev tswj tsis tau kho. Lub Xeev 4 kev ua pa ntawm lub cev qhia txog kev siv oxygen los ntawm mitochondria thaum protons xau rov qab rau hauv mitochondrial matrix ntawm lub tshuab uas tsis koom nrog F1 F0 -ATPase (Garvey, 2003). Qhov muaj peev xwm koom tes ntawm UCPs hauv Herba Cistanche-induced nce hauv Xeev 4 kev ua pa yog txhawb los ntawm qhov kev tshawb pom tias qhov kev txhim kho no tau raug txwv los ntawm GDP (Bento li al., 2007).

Hauv kev xaus, kev kho mob nrog Herba Cistanche tuaj yeem txhim kho mitochondrial glutathione raws li txoj cai, txo mitochondrial Ca2+ qib, thiab ua kom cov mitochondrial membrane muaj peev xwm thiab ua pa ntawm nas plawv. Kev txhim kho ntawm mitochondrial glutathione raws li txoj cai thiab kev muaj peev xwm ua haujlwm, thiab kev tso tawm ntawm UCPs, tuaj yeem cuam tshuam nrog kev tiv thaiv cardioprotection los ntawmKev kho mob Herba Cistanchetom qab I/R raug mob.

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