Gossypitrin, Ib txwm tshwm sim Flavonoid, Attenuates Iron-Induced Neuronal Thiab Mitochondrial Damage Part 1

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

Abstract:Kev cuam tshuam ntawm cov hlau homeostasis yog ib qho tseem ceeb hauv kev poob ntawm mitochondrial kev ua haujlwm hauv cov hlwb neural, ua rau neurodegeneration. Ntawm no, peb tau soj ntsuam qhov kev tiv thaiv ntawm gossypitrin (Gos), uas tshwm sim los ntawm flavonoid, ntawm hlau-induced neuronal cell puas siv nas hippocampal HT-22 hlwb thiab mitochondria cais tawm ntawm nas hlwb. Gos muaj peev xwm cawm HT22 hlwb los ntawm kev puas tsuaj los ntawm 100 uM Fe()-citrate (EC50 8.6 uM). Qhov kev tiv thaiv no tau txuas nrog kev tiv thaiv ntawm ob qho tib si hlau-induced mitochondrial membrane muaj peev xwm dissipation thiab ATP depletion. Hauv kev sib cais mitochondria, Gos (50uM) elicited yuav luag tag kev tiv thaiv tiv thaiv hlau-induced mitochondrial o, poob ntawm mitochondrial transmembrane peev xwm, thiab ATP depletion. Gos kuj tiv thaiv Fe(Ⅱ)-citrate-induced mitochondrial lipid peroxidation nrog ICs0 tus nqi (12.45 μM) uas yog kwv yees li cuaj zaus qis dua li ntawm tert-butyl hydroperoxide-induced.oxidation. Tsis tas li ntawd, cov flavonoid tau zoo nyob rau hauv inhibiting degradation ntawm 15 thiab 1.5 mM 2-deoxyribose. Nws kuj txo qis Fe (II) concentration nrog lub sijhawm, thaum nce O2 noj tus nqi, thiab txo qis ntawm Fe (II) los ntawm ascorbate. Gos-Fe (I) complexes tau kuaj pom los ntawm UV-VIS thiab IR spectroscopies, nrog pom meej Gos-hlau stoichiometry ntawm 2: 1. Cov txiaj ntsig tau qhia tias Gos feem ntau tsis ua raws li cov tshuaj tiv thaiv kab mob classical, tab sis nws ncaj qha cuam tshuam rau cov hlau, los ntawm kev tswj nws hauv nws daim ntawv ferric tom qab stimulating Fe (II) oxidation. Cov hlau ions yog li tsis tuaj yeem koom nrog Fenton-hom tshuaj tiv thaiv thiab lipid peroxidation propagation theem. Li no, Gos tuaj yeem siv los kho cov kab mob neuronal cuam tshuam nrog cov hlau-vim oxidative kev nyuaj siab thiab kev puas tsuaj mitochondrial.

Thov nias ntawm no kom paub ntxiv

Ntsiab lus: lus gossypitrin; hlau; HT-22 hlwb; mitochondria;neurodegeneration; neurotiv thaiv

1. Taw qhia

Hlau yog ib qho tseem ceeb cofactor nyob rau hauv ntau yam tseem ceeb lom cov txheej txheem xws li DNA synthesis thiab kho, thauj oxygen, cellular respiration, xenobiotic metabolism, thiab cov tshuaj hormone synthesis [1. Txawm li cas los xij, ntawm qib siab intracellular, nws tuaj yeem nce cellular reactive oxygen hom nrog kev hloov kho hauv cellular redox peev xwm [2,3]. Iron dyshomeostasis tau pom nyob rau hauv yuav luag txhua yam mob paj hlwb xws li Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), ntau yam sclerosis, Friedreich's ataxia, qaug dab peg, tsis xis nyob ceg, thiab mob stroke [4,5]. Tshwj xeeb tshaj yog, nyob rau hauv intracerebral hemorrhage, qhov twg muaj ib tug rupture ntawm lub microvasculatures thiab cov ntshav tso tawm ua rau cov ntshav-derived hlau raug ntawm neural hlwb, lub ntsiab mechanisms ntawm neurodegeneration yog hlau- thiab heme-induced ROS ntau lawm, o, thiab excitotoxicity [6] ,7] ib. Hauv vitro thiab hauv vivo kev sim ua qauv ntawm lub hlwb hemorrhage tau pom tias muaj ferroptosis, ib hom hlau-nyob ntawm kev tswj hwm necrosis [8]. Tsis tas li ntawd, cov pov thawj nce ntxiv qhia tias kev cuam tshuam ntawm cov hlau homeostasis yog qhov tseem ceeb hauv kev poob ntawm mitochondrial kev ua haujlwm hauv cov hlwb neural [9,10], uas tuaj yeem ua rau cov neurodegeneration [11-14. Ib qho kev sib raug zoo tau tshaj tawm tsis ntev los no ntawm qhov pib ntxov ntawm Parkinson tus kab mob nrog hlau metabolism dysregulation, thiab mitochondrial impairment [15]. Kev sib koom ua ke ntawm cov nyiaj ntau ntawm ob qho tib si hlau thiab ROS hauv mitochondrial compartments ua rau cov organelle no raug rau hydroxyl radical-mediated puas. Pharmacological cov neeg ua haujlwm tsom rau kev tswj lub hlwb hlau thiab cov hlau-mediated mitochondrial impairment yog li ntawd yuav pab tau zoo tiv thaiv neurodegeneration. Ntau tus neeg ua haujlwm hlau-chelating tau pom tias muaj cov tshuaj tiv thaiv neuroprotective hauv cov qauv sib txawv [16-19]. Ntawm qhov tod tes, tau muaj ntau cov lus ceeb toom txog qhov muaj peev xwm tiv thaiv neuroprotective ntawm ib txwm tshwm sim polyphenols vim lawv cov tshuaj tiv thaiv antioxidant raws li kev sib xyaw ntawm hlau chelation thiab dawb radical scavenging kev ua ub no [20,21]. Peb pab pawg tau pom tias catechol-muaj polyphenols zoo li mangiferin, guttiferone-A, thiab rapanone cuam tshuam nrog hlau, ob qho tib si hauv vitro thiab hauv vivo, yog li tshem tawm nws lub luag haujlwm catalytic hauv kev txhawb ROS [22-28]. Peb kuj tsis ntev los no tau tshaj tawm cov teebmeem antioxidant muaj zog ntawm gossypitrin (Gos), uas tshwm sim los ntawm flavonoid (Daim duab 1), qhia tias nws muaj peev xwm siv tau los ua tus neeg saib xyuas neuroprotective [29]. Txawm li cas los xij, me ntsis paub txog cov txheej txheem cuam tshuam nrog cov teebmeem antioxidant. Lub xub ntiag ntawm ib tug planar rau-tus tswv cuab cyclic system nrog electron delocalization, ib tug catechol moiety, thiab peb ntau aromatic hydroxyl pawg (ob tug ntawm lawv nyob ib sab ntawm ib pab pawg neeg carbonyl), xav hais tias cov molecule no yuav ua raws li ib tug hlau-chelating neeg sawv cev txij li thaum no. yog cov qauv nta uas nyiam hlau chelation los ntawm flavonoids 30]. Li no, lub hom phiaj ntawm txoj kev tshawb no yog los ntsuas lub peev xwm ntawm Gos los tiv thaiv HT-22neuronal hlwbthiab cais nas lub hlwb mitochondria los ntawm hlau-mediatedoxidative kev puas tsuaj. Lwm cov kev tshawb fawb hauv cell / mitochondria-dawb systems tau ua tiav los ua kom pom tus yam ntxwv ntawm Gos-hlau sib cuam tshuam.

2. Cov txiaj ntsig

2.1. Gos Rescues HT-22 Cells los ntawm Fe(I)-Induced Cell Death

Fe (II) (100 μM) txo HT-22 cell ciaj sia mus rau 17.4 feem pua ​​​​tom qab 24 teev ntawm qhov raug, raws li kev kwv yees los ntawm MTT kev soj ntsuam. Gos tiv thaiv hippocampal cell tuag nyob rau hauv ib koob tshuaj raws li. EC50 yog 8.64 ± 1.58 μM (Daim duab 2A, inset). Kev tiv thaiv tag nrho ntawm tes tau pom nrog 100 μM Gos tau zoo dua Trolox (500 uM), uas paub zoo hydrosoluble antioxidant, uas tiv thaiv cell tuag los ntawm 74 feem pua.

HT-22 raug rau hlau rau 2 h (ua ntej kev tuag ntawm tes) induced lub dissipation ntawm mitochondrial membrane muaj peev xwm, raws li kwv yees los ntawm kev tuav ntawm rhodamine 123 nyob rau hauv lub organelles (Daim duab 2B), thiab ATP depletion (Daim duab 2C ). Qhov no qhia tau hais tias muaj kev sib koom ua ke ntawm cov tshuaj lom hlau-mediated los ntawm mitochondria thiab cell viability. Gos tiv thaiv cov hlau-induced dose-dependent impairment ntawm mitochondria, ua raws li cov qauv ntawm neuron viability uas pom tau tias nws yuav tiv thaiv tau hlau-induced HT-22 cell tuag los ntawm kev tiv thaiv mitochondrial.

Daim duab 2.Gos tiv thaiv hlau-induced neuronal cell puas.Effects ntawm Gos ntawm HT-22 nas hippocampal neuron ciaj sia taus (A), cellular mitochondrial membrane peev xwm (B), thiab cellular ATP theem (C). Gos (0-100 μM) lossis Trolox (500 μM) tau sib koom ua ke rau 24 h (kev vam meej) lossis 2h (mitochondrial membrane peev xwm thiab ATP qib) nrog 100 μM FeCl2 / 1 mM citrate. Cov kev kuaj mob tau piav qhia nyob rau hauv Tshooj 4. Cov kev tswj xyuas puas hlwb (tsis muaj Gos) muaj DMSO (0.001 feem pua) ntxiv rau cov hlau-citrate sib tov. Cov txiaj ntsig tau nthuav tawm raws li qhov feem pua ​​​​ntawm cov xov tooj ntawm tes tuag nyob rau hauv kev sib raug zoo nrog kev tswj tsis raug (muaj DMSO0.001 feem pua ​​​​ntawm lub tsheb). Tus nqi yog qhov txhais tau tias ± SD ntawm peb qhov kev sim sib txawv. Cov cim sib txawv qhia txog qhov sib txawv ntawm p<0.05 when="" comparing="" all="" pairs="" of="" experimental="" groups="" according="" to="" the="" anova="" and="" post="" hoc="" newman-keuls="" tests.="" (if="" the="" symbols="" are="" different="" among="" groups="" indicate="" statistical="" differences,="" when="" they="" are="" equal,="" there="" is="">

Cistanche tuaj yeem txhim kho kev tiv thaiv

2.2. Gos Rescues Rat-Brain Mitochondria from Iron-Induced Mitochondrial Lipoperoxidation Brain mitochondria incubated with Fe（）-citrate （50 μM） underwent a marked lipoperoxidation expressed by an increase in the formation of the thiobarbituric acid reactive substance(Figure 3A). Gos inhibited the iron-mediated mitochondrial lipid peroxidation in a concentration-dependent manner （IC50 value of 12.45±1.24 μM） （Figure 3A, inset）. The effects of Gos on tert-butyl hydroperoxide（300 μM）-induced mitochondrial lipid peroxidation（IC50 value>100 μM) qhia tau hais tias nws muaj txiaj ntsig zoo hauv kev tiv thaiv cov hlau-induced lipid peroxidation dua li peroxyl induced lipid peroxidation, qhia nws lub peev xwm los ua ncaj qha rau hlau (Daim duab 3B).

Daim duab 3. Gos inhibits malondialdehyde tsim nyob rau hauv mitochondria, induced los ntawm 50 uM Fe()-citrate (A) los yog 300μM tert-butyl hydroperoxide (B). Ratbrain mitochondria (1 mg / mL) tau tsim nyob rau hauv cov tshuaj tiv thaiv nruab nrab uas muaj 125 mM sucrose, 65 mM KCl, 10mM HEPES tsis muaj (pH 7.4), 2 mM succinate, thiab 2.5 uM rotenone, nrog lossis tsis muaj Gos(1-50 uM). Cov kev sim pib los ntawm qhov sib ntxiv ntawm 50μM Fe (II) lossis 300 μM tert-butyl hydroperoxide, tshwj tsis yog rau kev tswj tsis raug (cov kab grey). Lub sijhawm incubation yog 20 min ntawm 28 degree C. Qhov tseem ceeb yog txhais tau tias ± SD (n=6). Cov cim sib txawv qhia txog qhov sib txawv ntawm p<0.05 when="" comparing="" all="" experimental="" groups="" according="" to="" the="" anova="" and="" posthoc="" newman-keuls="" tests.="" (if="" the="" symbols="" are="" different="" among="" groups="" indicate="" statistical="" differences,="" when="" they="" are="" equal,="" there="" is="">

2.3.Gos Tiv thaiv Iron-Mediated Mitochondrial Swelling Dissipation, thiab ATP Depletion

Daim duab 4A qhia tau hais tias 50μM Fe (I)-citrate complex induced mitochondrial o raws li qhia los ntawm kev txo qis ntawm qhov nqus ntawm 540nm (kab f piv rau kab a). Nws tau cuam tshuam nrog qhov tshwm sim thiab ua tiav △Y depolarization (Daim duab 4B, linef piv rau kab a), thiab ATP depletion (Daim duab 4C, Fe() / citrate kem). Gos inhibited cov txheej txheem o ntawm hlau thiab AY dissipation nyob rau hauv ib tug concentration-dependent yam (Daim duab 4A, B kab). Tus nqi absorbance (txhais tau tias ± SD ntawm 7 min) los ntawm Vaj Huam Sib Luag A yog: 0.482±{17}}.013 (kab a), 0.459± { {23}}. kab d), 0.360 ± 0.011 (kab e), thiab 0.338 ± 0.012 (kab f). fluorescence qhov tseem ceeb ntawm vaj huam sib luag B (txhais tau tias ± SD ntawm 7 min) yog 15.03 ± 1.71 (kab a), 19.41 ± 2.07 (kab b), 22.29 ± 2.58 (kab c), 45.505 ± 4.47 (kab d), 118.668 ± 5.7 (kab e), thiab 146.201 ± 5.83 (kab f). Kev txheeb xyuas qhov sib txawv tseem ceeb tau pom ntawm kab f thiab lwm kab, ntawm p<0.05. the="" naturally="" occurring="" flavonoids="" also="" prevented="" iron-mediated="" atp="" depletion="" in="" a="" concentration-dependent="" manner(figure="" 4c).="" a="" strong="" interaction="" of="" gos="" with="" iron="" is="" suggested="" as="" the="" key="" mechanism="" for="" neuroprotection="" against="" iron-induced="" damage,="" both="" in="" ht-22="" neuronal="" cells="" and="" in="" brain="" mitochondria.="" to="" further="" characterize="" this="" gos-iron="" interaction,="" different="" experiments="" on="" a="" cell/mitochondria-free="" system="" were="">

Daim duab 4. (A) Mitochondrial permeability transition induction, (B) mitochondrial membrane peev xwm (AH) poob, thiab (C) txo cov qib ATP induced los ntawm 50 μM Fe (II)-citrate. A tau kwv yees siv fluorescence sojntsuam safranine (5 uM). Cov teebmeem inhibitory ntawm Gos 50,25, 10, thiab 1μM tau qhia hauv kab (b), (c), (d), thiab (e), feem. Kab (a) thiab (f) sawv cev tsis muaj kev puas tsuaj (tsis muaj hlau), thiab kev puas tsuaj (tsis muaj Gos), feem. Rat hlwb mitochondria (RBM-0.5 mg/mL), Fe (II) thiab 1 μM CCCP tau ntxiv qhov uas qhia los ntawm cov xub. Cov cim sib txawv hauv daim duab (C) qhia txog qhov sib txawv ntawm qhov atp<0.05 when="" comparing="" all="" pairs="" of="" experimental="" groups="" according="" to="" the="" anova="" and="" post="" hoc="" newman-keuls="" tests.(if="" the="" symbols="" are="" different="" among="" groups="" indicate="" statistical="" differences,="" when="" they="" are="" equal,="" there="" is="">

2.4. Gos Induces Fe(II)-Citrate Autoxidation thiab O, Kev siv

Gos txo Fe() concentration (1-min preincubation) nyob rau hauv cov tshuaj tiv thaiv nruab nrab uas muaj 2 mM citrate (tsis muaj mitochondria) nyob rau hauv ib koob tshuaj (Daim duab 5A, dub voj voog).A 5-min preincubation lub sij hawm (Daim duab 5A, lub thawv liab) txo Fe (II) kev xav ntau dua. Gos kuj nce tus nqi ntawm O2 noj, tej zaum vim oxidation ntawm Fe (ll) rau Fe (ⅡII) (Daim duab 5B, C). Cov txiaj ntsig no qhia tias Gos tuaj yeem tshem tawm Fe (II) los ntawm citrate complex thiab oxidizing nws mus rau ferric daim ntawv nyob rau hauv ib tug txheej txheem uas yuav tsum tau O2 raws li lub electron txais. Raws li, Gos tuaj yeem cuam tshuam qhov tsim ntawm "OH radicals los ntawm inactivating Fe (Il) tsim nyog rau Fenton-Haber-Weiss-hom tshuaj tiv thaiv, uas tuaj yeem pib lipid peroxidation ntawm mitochondrial membrane.

Daim duab 5. (A)Gos stimulates Fe(Ⅲl) autoxidation thaum tsis muaj nas hlwb mitochondria nyob rau hauv tus qauv nruab nrab ntxiv nrog 1 mM citrate, thiab 5 mM1, 10-phenanthroline.1,10-phenanthroline yog ntxiv 1 lossis 5 min tom qab Gos-Fe (II) incubation lub sij hawm, thiab cov absorbances tau nyeem ntawm 510nm. Tus nqi yog qhov nruab nrab ntawm peb qhov kev txiav txim. * Kev txheeb cais sib txawv ntawm p<0.05 when="" comparing="" 1="" vs.="" 5min="" incubation.="" (b)effects="" of="" gos="" on="" o,="" consumption="" mediated="" fe(il)="" autoxidation="" in="" a="" standard="" medium="" supplemented="" with="" 1="" mm="" citrate="" under="" the="" following="" conditions:(a)="" no="" gos,(b)10="" um="" gos,="" （c）="" 30μm="" gos,="" （d）50="" μm="" gos,and="" （e）100μm="" gos.="" fe（ii）="" （50μm）="" was="" added="" where="" indicated="" by="" the="" arrow.="" results="" are="" representative="" of="" three="" experiments.="" (c)="" oxygen="" consumption="" rate="" (nmol="" o2/min/ml)1="" min="" after="" fe()="" addition.legends="" are="" the="" same="" as="" in(b).="" different="" symbols="" indicate="" statistical="" differences="" at=""><0.05 when="" comparing="" all="" pairs="" of="" experimental="" groups="" (in="" the="" same="" cell="" type)="" according="" to="" the="" anova="" and="" post="" hoc="" newman-keuls="" tests.="" (if="" the="" symbols="" are="" different="" among="" groups="" indicate="" statistical="" differences,="" when="" they="" are="" equal,="" there="" is="">

Kab lus no yog muab rho tawm los ntawm Molecules 2021, 26, 3364. https://doi.org/10.3390/molecules26113364 https://www.mdpi.com/journal/molecules