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Kev paub

Ntu Ⅰ Tus Hlau Chelator, PBT434, Modulates Transcellular Iron Trafficking hauv Brain Microvascular Endothelial Cells

Apr 28, 2023

Abstract

Hlau thiab lwm yam kev hloov pauv hlau, xws li tooj liab thiab manganese, yog qhov tseem ceeb rau kev txhawb nqa lub hlwb, tab sis kev sib sau ntau dhau yog cytotoxic. Qhov no ntau dhau ntawm cov hlau, tshwj xeeb tshaj yog hlau, yog ib qho tshwm sim rau ntau yam kab mob neurological; Cov no suav nrog Alzheimer's disease, Parkinson's disease, Friedrich's ataxia, thiab lwm yam kab mob uas tshwm sim nrog neurodegeneration thiab txuam nrog lub hlwb hlau tsub zuj zuj. Kev tswj cov hlau flux los ntawm cov ntshav-hlwb barrier muab thawj kab ntawm kev tiv thaiv tiv thaiv tshaj-sau ntawm hlau nyob rau hauv ib txwm physiology thiab cov pathological tej yam kev mob. Hauv txoj kev tshawb no, peb tau txiav txim siab tias cov hlau chelator PBT434, uas tam sim no tau tsim kho rau kev kho mob Parkinson's kab mob thiab ntau lub cev atrophy, hloov kho cov hlau los ntawm tib neeg lub hlwb microvascular endothelial hlwb (hBMVEC) los ntawm chelation ntawm extracellular Fe.2 plus. Kev kho mob ntawm hBMVEC nrog PBT434 ua rau muaj kev nce ntxiv ntawm cov ntawv sau tseg rau kev hloov pauv receptor (TfR) thiab ceruloplasmin (Cp). Western blot thiab ELISA kev txheeb xyuas qhia txog qhov sib txuas ntawm cov proteins thiab. Nyob rau hauv lub xovtooj ntawm tes, PBT434 tsub kom qhov kuaj pom ntawm charitable, labile Fe2 plus; cov ntaub ntawv qhia tias qhov no Fe2 plusyog tso tawm los ntawm ferritin. Tsis tas li ntawd, PBT434 potentiates hlau efflux yuav yog vim qhov nce hauv cytosolic ferrous hlau, lub substrate rau hlau exporter, ferroportin. PBT434 sib npaug sai thiab ob-txoj kev hla ntawm hBMVEC ntshav-hlwb teeb meem. Cov txiaj ntsig no qhia tau tias PBT434- hlau complex tsis yog substrate rau hBMVEC uptake thiab yog li txhawb nqa tus qauv uas PBT434 yuav chelate interstitial hlau thiab inhibit re-uptake ntawm hlau los ntawm endothelial hlwb ntawm cov ntshav-hlwb barrier, raws li zoo li inhibit nws uptake los ntawm lwm lub hlwb ntawm lub neurovascular unit. Zuag qhia tag nrho, qhov no nthuav tawm cov txheej txheem tshiab thiab cog lus rau kev kho cov hlau chelation.

Cistanche benefits

Nyem qhov no kom tau txaisCov txiaj ntsig ntawm Cistanche yog dab tsi

Taw qhia

Hlau chelation therapy (MCT) tau ntev tau siv los ua kev kho mob rau kev hloov pauv hlau lom thiab rau cov kab mob caj ces hauv cov metabolism ntawm ib qho tseem ceeb hlau ion uas ua rau cov hlau ntau dhau [1–3]. Ob qho piv txwv ntawm cov tom kawg yog qhov hyper-sau ntawm tooj liab hauv Wilson tus kab mob [4] thiab ntawm cov hlau nyob rau hauv hereditary hemochromatosis [5]. Ob qho tib si tooj liab thiab hlau yog catalysts ntawm oxidative kev nyuaj siab thiab yog li yog cytotoxic ntawm concentrations uas tshaj lub peev xwm ntawm lub cell thiab kab mob mus rau 'chaperone' cov redox-active kev hloov pauv hlau [6, 7]. Hlau tsub zuj zuj, tshwj xeeb, yog dav idiopathic; Qhov tseeb, qhov nce hauv cov hlau yog qhov cim ntawm lub hlwb laus [8–10]. Pathologically, no lub hlwb hlau tsub zuj zuj yog ib tug feature ntawm kev hloov nyob rau hauv cov noob tsis cuam tshuam rau hlau metabolism [11-15] nrog rau ntau yam ntawm lwm yam neurodegenerative kab mob, ib co ntawm cov uas tsis muaj ib tug tshwj xeeb genetic txuas xws li kev laus [16], Alzheimer's Disease [ 17], Friedreich's Ataxia [18] thiab Parkinson's Disease [19]. Raws li ib pab pawg, cov kab mob no tuaj yeem xav tias yog neurodegeneration nrog lub hlwb hlau tsub zuj zuj (NBIA) txawm hais tias cov lus no feem ntau tau txwv rau cov neeg uas tau txheeb xyuas cov kab mob sib txuas [11, 13, 14].

Nyob rau hauv cov ntaub ntawv ntawm hlau overload, lub hom phiaj yog 'tshuaj' lub cev ntawm cov hlau ntau dhau vim muaj qhov tsis xws luag hauv cov xov tooj ntawm tes los yog efflux. Ntawm no lub hom phiaj yog tawm-kev sib tw physiologic hlau chelators nrog cov tshuaj; ib qho chaw uas muaj cov tshuaj pharmacokinetics zoo thiab muaj kev sib raug zoo rau ferrous hlau yog lub hom phiaj tshuaj. Txij li thaum lub cev yog over-replete nrog cov hlau tseem ceeb, muaj kev txhawj xeeb me ntsis txog inducing ib tug deficiency nyob rau hauv lub chav kawm ntawm kev kho mob. Kev kho mob hlwb nrog kev kho mob hlau chelation yuav tsum muaj lub tswv yim sib txawv. Qhov no tsis yog ib qho teeb meem ntawm lub cev hlau overload, tab sis ntawm hlau tsub zuj zuj nyob rau hauv cov cheeb tsam ntawm pathology nrog kev puas tsuaj downstream sequelae. Piv txwv li, muaj hnub nyoog txuam nrog hlau tsub zuj zuj hauv Parkinson's disease (PD), piv txwv li, muaj feem cuam tshuam rau oxidative stress-related cellular puas [20]. Ntau labile hlau txhawb cov misfolding ntawm -synuclein hauv substantia nigral neurons. Kev siv cov chelator siab tuaj yeem ua rau qee qhov txo qis hauv lub hlwb hlau tab sis yuav ua rau muaj cov hlau tsis muaj zog uas nyob rau hauv cov neeg laus, tsawg kawg, yog contraindicated muab lub cev tsis muaj hlau ntau rau cov hnub nyoog ntawd [21] . Lub chelator nrog kev pom kev sib raug zoo muaj peev xwm txo tau cov hlau tsub zuj zuj ntxiv nrog rau cov neeg koom nrog oxidative kev nyuaj siab vim muaj ntau labile hlau thiab cov txheej txheem kab mob.

Cistanche benefits

Cistanche tubulosathiabCistanche cov teebmeem

Ib qho chelator tau pom zoo siv los kho cov kab mob uas ua rau cov hlau ntau dhau hauv cov neeg mob thalassemia yog deferiprone (DFP, hom npe Ferriprox) [5, 22]. DFP kuj tau siv los kho Friedreich's ataxia [23] thiab Parkinson's disease [24, 25]. Hauv kev tshuaj ntsuam xyuas meta, DFP tau pom tias muaj kev txo qis hauv cov ntsiab lus ntawm cov hlau myocardial nrog rau kev tiv thaiv plawv ntau dua hauv cov neeg mob thalassemia dua li deferoxamine, classical hlau chelating tus neeg sawv cev [5]. Ntawm qhov tod tes, DFP tau nrawm nrawm los ntawm daim siab [26] thiab kev ua haujlwm tsis ntev los no tau pom tias nws chelates Fe2 ntxiv rau ntawm qhov chaw ua haujlwm ntawm cov hlau-dependent histone lysine demethylase, ib qho haujlwm uas cuam tshuam nrog yav dhau los tsis paub txog cytotoxicity [27]. Qhov kev tshawb pom no qhia txog qhov kev txwv tseem ceeb hauv kev siv cov hlau chelation kho, uas yog kev sib tw los ntawm cov tshuaj rau lub cev-tseem ceeb hlau, txawm tias nyob rau hauv ib lub khw muag hlau lossis cov protein uas muaj cov kab mob prosthetic hlau. Txawm li cas los xij, DFP, piv txwv li, tau pom tias muaj txiaj ntsig hauv Phase 2 sim kho mob Parkinson tus kab mob raws li qhia los ntawm ob qho tib si analytic (txo lub hlwb hlau thauj khoom los ntawm T2- hnyav MRI) thiab cov ntsuas kev coj cwj pwm (kev txawj ntse thiab lub cev muaj zog neuron muaj nuj nqi) [ 24, 25] ib.

Qhov kev sib raug zoo ntawm DFP rau Fe3 ntxiv tseem muaj kev txhawj xeeb, txawm li cas los xij. Qhov ruaj khov DFP-hlau hom yog tris-complex, [Fe(DFP)3] 0 [28]. Txawm hais tias qhov nruab nrab ntawm qhov kev ua haujlwm no yog qhov zoo tshaj plaws rau kev txav hlau tawm ntawm lub xov tooj ntawm tes, qhov kev ruaj ntseg tsis tu ncua rau nws, ~ 1037, ua rau DFP qhov tseeb hlau scavenger; Nyob rau hauv cov ntsiab lus no, nws inhibition ntawm ib tug hlau enzyme zoo li lysine demethylase yog kwv yees [27]. Qhov kev txhawj xeeb no qhia txog qhov xav tau los tsim cov hlau chelators uas muaj cov membrane permeability ntawm DFP tab sis ib qho tsis muaj zog affinity rau ob qho tib si Fe2 ntxiv thiab Fe3 ntxiv. Qhov tom kawg no txwv tsis pub siv tshuaj tshem tawm ntawm cov hlau prosthetic thiab lub peev xwm thermodynamic ntawm tus neeg sawv cev chelating kom ua rau cov hlau ferrous oxidation uas ua rau tsim cov pa oxygen reactive. Hauv cov ntsiab lus, muaj zog ferric hlau chelators catalyze cov khoom pro-oxidant ntawm Fe2 ntxiv [29]. Nyob rau hauv txoj kev tshawb no, peb qhia li cas xws li ib tug hlau chelator nrog nruab nrab ferric thiab ferrous hlau affinities modulates lub flux ntawm hlau nyob rau hauv lub hlwb microvascular endothelial hlwb uas tsim cov ntshav-hlwb barrier (BBB).

Cistanche benefits

Cistanche ntsiav tshuaj

Cov tshuaj no, PBT434 [5,7-dichloro-2-((methylamino)methyl)-8-hydroxy-3-methylquinazolin-4 (3H)-ib, Fig 1A] , tsim cov bis-hlau complex nrog cov log ruaj khov ntawm ~ 11 thiab ~ 15 rau Fe2 plusthiab Fe3 plus ibib., [30]. PBT434 tiv thaiv kev poob ntawm substantia nigra pars compacta (SNpc) neurons, txo qis nigral -synuclein tsub zuj zuj, txo tus kab mob PD tus qauv hais txog cov ntsiab lus hlau hauv nruab nrab, thiab cawm lub cev muaj zog hauv ob tus qauv nas ntawm Parkinson tus kab mob yam tsis muaj qhov pom tseeb ntawm cov khw muag khoom hlau. [30]. PBT434 kuj tseem muaj txiaj ntsig zoo hauv cov qauv murine ntawm Ntau Qhov System Atrophy (MSA) [30, 31], lub cev muaj zog zoo ib yam li kev nthuav qhia rau Parkinson's tab sis uas yog tus cwj pwm los ntawm -synuclein misfolding thiab tom qab tsub zuj zuj ua rau tsim cov glial cytoplasmic inclusions uas yog cov cim qhia. pathology ntawm tus kab mob [32] Qhov tseem ceeb, PBT434 txo cov cim ntawm oxidative kev nyuaj siab hauv nas PD qauv [30] qhia tias 1) PBT434 tsom cov khw muag khoom hlau uas txwv tsis pub ua haujlwm ua cov tshuaj tiv thaiv oxidants thiab 2) PBT434 tsis muaj zog no oxidation-based cytotoxicity. PBT434 tau ua tiav Phase 1 txoj kev kawm txaus siab [33].

Figure 1

Cov hauj lwm nthuav tawm ntawm no yog tsim los soj ntsuam qhov cuam tshuam PBT434 muaj rau kev lag luam hlau nyob rau hauv lub hlwb lub hlwb barrier cells, microvascular endothelial hlwb uas ua ke nrog hauv qab glia tsim cov ntshav-hlwb barrier. Cov kev tshawb fawb no tau siv cov kab mob endothelial cell kab mob uas muaj txiaj ntsig zoo hauv ob qho tib si monolayer thiab transwell kab lis kev cai hom [34–37]. Lub hom phiaj tseem ceeb ntawm cov kev tshawb fawb no yog los txiav txim siab cov kinetics ntawm hlau uptake thiab efflux los ntawm cov hlwb thiab lawv cov kev hloov kho los ntawm PBT434. Tus qauv transwell BBB kuj tau siv los ua qauv qhia ob-txoj kev PBT434 transcellular flux hla endothelial cell barrier. Cov qauv ua qauv qhia nyob rau hauv cov ntsiab lus molecular uas PBT434 inhibits hlau uptake los ntawm chelation thaum stimulating hlau efflux. Cov kev tshawb fawb ntawm tes qhia tau hais tias PBT434 nkag mus rau tib lub labile hlau pas dej ua ke soj ntsuam los ntawm classic Fe2 plusChelating tus neeg sawv cev, 2,2'-bipyridine los yog bipyridyl, thiab fluorescent sojntsuam rau ferrous hlau. Cov txiaj ntsig tau qhia txog qhov ua tau ntawm kev ua haujlwm rau PBT434 uas suav nrog inhibition ntawm kev nqus ntawm cov hlau hauv BBB, thiab tom qab sequestration ntawm lub hlwb hlau nyob rau hauv qhov chaw interstitial.

Cov txiaj ntsig

1. PBT434 tsis muaj cytotoxic cuam tshuam rau lub hlwb microvascular endothelial hlwb

Txhawm rau txiav txim siab qhov tsim nyog ntawm kev ua haujlwm ntau rau PBT434 hauv peb cov kab lis kev cai hauv vitro cell, peb siv MTT kev soj ntsuam los saib xyuas hBMVEC mitochondrial muaj nuj nqi hauv kev teb rau PBT434. Raws li cov ntaub ntawv dhau los [30], tau raug kho nrog ntau yam ntawm PBT434 concentrations txog 100 μM rau 24 teev. Peb pom tsis muaj kev hloov pauv tseem ceeb hauv hBMVEC kev muaj peev xwm nrog txhua qhov kev ntsuam xyuas concentration (Fig 2).

Figure 2

2. PBT434 tau nrawm nrawm thiab ua lag luam thoob plaws hBMVEC teeb meem

PBT434 yog ib qho tshuaj bioavailable hauv qhov ncauj uas tuaj yeem nkag mus rau BBB, raws li pom hauv cov kev tshawb fawb tau ua hauv nas thiab tib neeg [30, 38, 39]. Peb tau saib xyuas qhov sib txuam ntawm PBT434 hauv hBMVEC loj hlob hauv monolayers siv 14C-labeled PBT434 ua cov xov tooj cua. Cov ntaub ntawv qhia tau hais tias nyob rau hauv thawj theem, 14C-PBT434 sai equilibated ntawm lub uptake nruab nrab thiab lub cell. Qhov kev txhawb nqa thawj zaug no tau ua raws li kev sib txuas ntxiv qeeb dua 3 h uas nthuav tawm tus nqi ntawm 30.1 ± 9.8 pmol / mg / h (Fig 3A). Nyob rau hauv txoj cai uptake, uptake yog quenched thiab hlwb raug ntxuav ntawm 4˚C ua ntej ua rau 14C-PBT434 tsub zuj zuj (Txoj Kev). Hauv kev sim cais, peb tau tshuaj xyuas qhov efflux ntawm 14C-PBT434 los ntawm hBMVEC tom qab lub sijhawm thauj khoom 30 min. Hauv cov txheej txheem efflux, cov hlwb raug ntxuav ntawm 25˚C. Cov ntaub ntawv hauv Fig 3B qhia tias hauv 25˚C ntxuav, kwv yees li 92 feem pua ​​​​ntawm cov cell-accumulated 14C-PBT434 tau ploj (cf 550 pmol 14C-PBT434 / mg protein hauv 3A ntawm 30 min rau 43 pmol 14C-PBT434 / mg. protein ntawm t=0 hauv 3B). Muaj qhov poob qeeb ntxiv ntawm qhov seem 14C-PBT434 (Fig 3B). Cov ntaub ntawv qhia ob yam ntawm kev txuam nrog thiab efflux ntawm PBT434 los ntawm hBMVEC. Flux thoob plaws plasma membrane yog ceev mus txog qhov zoo li qhov sib npaug txawm tias thaum lub sij hawm uptake los yog efflux. Txawm li cas los xij, nyob rau hauv ob qho tib si txheej txheem, muaj lwm txoj kev qeeb qeeb. Qhov no qhia tau hais tias nyob rau hauv lub cell, qee feem ntawm cell PBT434 yog nyob rau hauv ib cheeb tsam / xeev uas yog nyob rau hauv ib tug kinetic khov kho-xeev kev sib raug zoo nrog cov feem nyob rau hauv equilibrium nrog lub extra-cellular milieu. Kev soj ntsuam kinetic tau sau tseg hauv daim duab 3B kwv yees lub pas dej ntawm PBT434 yog sawv cev los ntawm 27 ± 4 pmol / mg protein nyob rau hauv lub cell lysate thaum cov hlwb raug kho nrog 20 μM reagent.

Figure 3

Txhawm rau tshuaj xyuas cov kab mob sib kis ntawm PBT434, peb tau ua haujlwm tau zoo hauv vitro BBB qauv siv tau loj hlob nyob rau sab apical ntawm daim nyias nyias [35, 36, 40, 41]. Cov khoom pov thawj ntawm cov kab lis kev cai ntawm cov kab lis kev cai no tau txheeb xyuas los ntawm kev ua kom muaj nuj nqis ntawm lawv cov hluav taws xob transendothelial hluav taws xob (TEER) thiab impermeability rau FITC-labeled dextran (S1 Fig). Peb piv 14C-PBT434 uptake ntawm lub luminal (los yog apical, ntshav sab) (Daim duab 4A) rau uptake ntawm lub abluminal (los yog basolateral, hlwb sab) (Fig 4C) membrane. Hauv tib qhov kev sim, qhov sib thooj efflux (transcellular flux) tau ntsuas los ntawm qhov pom ntawm 14C-PBT434 hauv efflux chamber (Daim duab 4 panels B thiab D). Tus nqi ntawm cov txheej txheem no tau muab rau hauv Table 1. Cov ntaub ntawv loj uas tau piav qhia hauv daim duab 4 (cov vaj huam sib luag B thiab D) qhia tau tias cov net flux ntawm PBT434 hla tus qauv ntshav-hlwb barrier yog tib yam hauv ob qho kev qhia. Muaj 976 ± 185 pmol 14C-PBT434 sau nyob rau hauv basal chamber (Fig 4B) thiab 1033 ± 210 pmol quantified nyob rau hauv lub basal chamber (Fig 4D). Qhov ze ntawm qhov sib npaug no kuj tau tshwm sim nyob rau hauv cov nqi zoo sib xws ntawm PBT434 efflux ntawm ob daim kab xev thaiv (Table 1). Txawm li cas los xij, muaj qhov cuam tshuam loj dua ntawm PBT434 ntawm basolateral daim nyias nyias hauv cov qauv teeb meem no raws li tau piav qhia los ntawm ~ 50 feem pua ​​​​tsawg dua ntawm cov khoom sib txuas los ntawm cov basal chamber (Daim duab 4C) uas sib xws rau ~ 40 feem pua ​​​​siab dua ntawm qhov pom tseeb ntawm tes. (Table 1). Kev ua kom muaj zog ntau dua yuav raug kwv yees kom ua rau muaj ntau dua. Kev soj ntsuam ntawm cov hlwb ntawm 3h tau pom tias lawv khaws cia ~ 6 μM PBT434 tsis hais txog kev taw qhia flux. Qhov tseem ceeb yog 8.1 ± 1.3 μM (apical rau basal) thiab 4.7 ± 1.2 μM (basal rau apical). Raws li tau sau tseg saum toj no, qhov kev tshuaj xyuas no ua raws li kev ntxuav cov hlwb ua ntej lysis thiab kom muaj nuj nqis ntawm tag nrho cov cell protein thiab 14C-PBT434. Tsis tas li ntawd, qhov nruab nrab hauv lub apical chamber muaj RPMI ntxiv rau 10 feem pua ​​​​FBS thiab 10 feem pua ​​​​NuSerum whereas lub basal, 'hlwb' chamber tsuas muaj RPMI (Txoj Kev). Ib qho kev xav tsim nyog yog tias qhov ntau dua 'uptake' ntawm lub basal membrane tau cuam tshuam lub cell nto adsorption ntawm PBT434 uas txwv tsis pub nyob rau hauv lub apical chamber los ntawm muaj protein ntau nyob rau hauv cov ntshav. Thaum ntxuav lub hlwb kom tsub zuj zuj ntawm PBT434, cov khoom siv adsorbed (uas tau sau npe ua 'uptake') raug tshem tawm. Rov ua dua qhov kev sim flux no tab sis nrog cov ntshav hauv cov basal chamber tau pom tias, qhov tseeb, cov ntshav tau txwv qhov zoo li ntawm tes ntawm PBT434 adsorption (S2 Fig).

Figure 4

Table 1

3. PBT434, tsis zoo li bipyridyl, tsis txwv qhov muaj nyob hauv lub cev ntawm labile hlau

Txij li thaum PBT434 muaj qhov sib txawv nruab nrab ntawm cov hlau piv rau cov hlau chelators xws li deferiprone lossis bipyridyl, peb tau tshuaj xyuas seb qhov sib txawv ntawd tau cuam tshuam li cas hauv PBT434 cov nyhuv ntawm lub cellular labile hlau pas dej (LIP) ntawm hBMVEC. Ua li no, peb coj kom zoo dua ntawm permeable, Fe2 plus-Specific fluorescent zas xim FerroOrange, uas reacts nrog charitable cytoplasmic hlau. Peb pom ib qho tseem ceeb ablation ntawm fluorescence nyob rau hauv cov hlwb thaum kho nrog bipyridyl, raws li chelation ntawm LIP los ntawm no high-affinity ferrous hlau chelator thiab yog li thaiv qhov kev txiav txim ntawm fluorescent hlau qhia (Fig 5A). Hauv qhov sib piv, PBT434 tsis tau sib tw nrog FerroOrange rau Fe2 plus, tus cwj pwm zoo ib yam nrog nws cov affinity ntau nruab nrab [30]. Cov txiaj ntsig tau pom tias PBT434, tab sis tsis yog PBT434- tau ntsib cov khoom siv tsis zoo, ua rau 34 ± 9 feem pua ​​​​nce hauv FerroOrange-accessible Fe2 plusqhia tias tus neeg sawv cev chelating no tau tsa cov hlau nyob hauv lub xov tooj ntawm tes yam tsis muaj kev sib xyaw ua ke. Cov ntaub ntawv qhia hauv qab no qhia tias cov hlau no los ntawm ferritin.

Figure 5

PBT434 tau qhia yav dhau los los kho cov kab mob ferroportin cov protein uas tsis muaj nyob hauv MPTP-kho nas mus rau qib zoo ib yam li cov nas uas tsis muaj kab mob [30]. Qhov txiaj ntsig no, nrog rau kev nce hauv cov kab mob ferrous hlau staining nyob rau hauv cov lus teb rau PBT434, qhia tias muaj peev xwm cuam tshuam rau lub cellular hlau teb system thiab kev ua haujlwm ntawm cov hlau txuas nrog cov protein. Txhawm rau ntsuam xyuas qhov no, peb thawj zaug ua qhov ntsuas PCR (qPCR) ntawm PBT434 cuam tshuam rau kev nplua nuj ntawm cov ntawv sau tseg rau ntau cov hlau tuav cov protein (Fig 6). Thaum cov ntawv sau tseg rau cov hlau efflux protein, ferroportin (Fpn), thiab ob lub cytoplasmic hlau chaperones, PCBP1 thiab 2 tsis muaj kev cuam tshuam, kev nplua nuj ntawm mRNAs rau cov hloov pauv receptor (TfR), thiab ferroxidase, ceruloplasmin (Cp), tau ua. hloov. TfR thiab Cp cov ntawv sau tseg tau nce los ntawm 2.8 thiab 3.6-fold, feem. Transferrin receptor (TfR) kev qhia yog txuas rau cov hlau teb (IRE) / hlau tswj cov protein (IRP) system [42–44]. Qhov nce hauv TfR mRNA qhia tias PBT434 sib tw nrog PCBP1- nyob ntawm kev xa cov hlau rau kev sib dhos ntawm Fe, S pawg uas hloov cov kev tswj hwm IREBP los ntawm RNA-binding protein rau cytosolic aconitase [45]. Yog li, PBT434 hloov qhov kev tswj hwm kev tswj hwm ntawm RNA-binding thiab sib cuam tshuam inhibition ntawm TfR mRNA degradation. Hauv cell hlau tsis muaj peev xwm, Cp qhia yog, ib feem, tswj hwm los ntawm HIF-1 [46]. Kev nce hauv HIF-1 muaj nuj nqi ua raws los ntawm kev poob qis ntawm nws cov hydroxylation los ntawm kev ua haujlwm ntawm prolyl hydroxylase hauv cov tshuaj tiv thaiv hlau-dependent [47]. Raws li nyob rau hauv cov ntaub ntawv ntawm IREBP, PBT434 zoo li txo qis lub pas dej ua ke ntawm hlau uas ua hauj lwm raws li ib tug co-factor nyob rau hauv HIF-1 hydroxylation thiab degradation. Nyob rau hauv cov qauv no, qhov nce nyob rau hauv khov kho-lub xeev theem ntawm no transcriptional activator nce Cp transcription.

Figure 6

Siv kev sib xyaw ua ke ntawm ELISA kev tshuaj xyuas thiab kev pleev xim sab hnub poob, peb tau tshawb xyuas qhov kev qhia ntawm cov hlau tuav cov proteins hauv PBT434 lossis PBT434- tau ntsib hBMVEC; piv txwv ntawm WB kev tshuaj ntsuam tau muab rau hauv daim duab 7A. Cov ntaub ntawv qhia tau hais tias qhov ntau ntawm TfR monomer thiab dimer tau nce ntxiv los ntawm 24h raws li Cp (Daim duab 7B thiab 7C). Ob qho kev nce ntxiv ntawm PBT434- nyob ntawm qhov nce hauv cov ntawv sau tseg (Daim duab 6), Qhov sib txawv, qhov kev qhia ntawm cov hlau efflux protein, Fpn, tsis hnov ​​​​qab rau PBT434 kev kho mob (Fig 7D).

Figure 7

Peb siv ELISA ua ib txoj hauv kev ntxiv los ntsuas qhov kev hloov pauv uas qhia los ntawm cov ntaub ntawv western blot. Yog li, hBMVEC tau kho nrog PBT434 rau 24h thiab cell lysates tau soj ntsuam los ntawm ELISA rau TfR (Fig 8A). Qhov kev sib tw nce hauv TfR hauv kev teb rau PBT434 kev kho mob kom muaj nuj nqis los ntawm ELISA yog sib npaug rau qhov uas tau muab los ntawm kev tshuaj xyuas ntawm sab hnub poob blots (Daim duab 7B). ELISA kuj tseem siv los ntsuas qhov zais cia thiab GPI-txuas Cp protein ntau, siv HepG2 hlwb los tswj qhov zoo. Hais txog Cp zais cia rau hauv xov xwm kev loj hlob, txoj hauv kev no tau txwv nyob rau hauv qhov sCp abundance nyob rau hauv ob qho tib si HepG2 thiab hBMVEC conditioned media nyob rau hauv los yog qis dua qhov rhiab heev txwv ntawm qhov kev ntsuam xyuas no (S3 Fig). Txawm li cas los xij, nws tau ua kom qhov kev ntsuam xyuas ntawm GPI-Cp abundance. Hauv cov qauv no, cov hlwb tau kho nrog phosphatidylinositol-specific phospholipase C (PI-PLC), uas cleaves GPI thauj tog rau nkoj; cov xov xwm yog li conditioned yog concentrated thiab soj ntsuam los ntawm Cp-ELISA. Thaum txoj hauv kev no pom tau tias PBT434 nce tus nqi ntawm GPI-Cp hauv HepG2 hlwb, nws rov ua tsis tau tej yam Cp tso tawm los ntawm PI-PLC (Fig 8B). ELISA kuj tau them ib txoj hauv kev ncaj qha rau kom muaj nuj nqis ntawm ferritin. Ua li no, hBMVEC tau ntim nrog 1 uM Fe-citrate rau 24h, ua raws li kev kho mob thaum tsis muaj lossis muaj PBT434 ntxiv rau 1 h. Cov txiaj ntsig ntawm cov lysates tau raug kuaj xyuas ELISA rau ferritin (Fig 8C). Nyob rau hauv sib piv rau qhov nce ntawm TfR, kev kho mob nrog PBT434 knocked down ferritin (Ft) protein los ntawm ~ 18 feem pua. Tseeb, qhov poob ntawm Ft protein tau pom meej tom qab tsuas yog 1h kev kho mob nrog cov tshuaj reagent. Qhov xwm txheej ntawm lub cev ntawm qhov tshwm sim no tuaj yeem cuam tshuam nrog kev nce hauv kev siab hlub Fe2 ntxiv rau sau tseg saum toj no tom qab 30 min kho nrog PBT434. Raws li tau tham tom qab, qhov kev poob qis ntawm ferritin tau pom tias tom qab kev kho mob nrog lwm cov cell-permeant Fe2 ntxiv rau cov tshuaj chelating [48].

Figure 8

4. 55Fe2 plusuptake yog inhibited los ntawm complexation nrog PBT434

Muab qhov sib npaug nrawm ntawm PBT434 hauv hBMVEC hauv 30 min, piv rau qhov qeeb, biphasic uptake thiab sib npaug ntawm Fe2 ntxiv rau 24h [49], peb xav tias PBT434 thiab Fe2 ntxiv tsis sib koom tib lub tswv yim txhawb nqa. Txhawm rau kuaj qhov no, monolayers tau tsim nrog radiolabeled 55Fe2 ntxiv rau hauv qhov tsis muaj lossis muaj PBT434 lossis PBT434- tau ntsib, thiab 55Fe2 ntxiv rau qhov nce siab tshaj 3 teev tau saib xyuas (Daim duab 9A). PBT434 txo qis tus nqi ntawm 55Fe2 ntxiv rau kev nce ntxiv, nrog rau kev txo qis tag nrho ntawm 55Fe2 ntxiv rau hauv cell lysates (Fig 9C). Cov nyhuv no tsis pom nrog PBT434- ntsib. Kev sib piv ntawm PBT434 txog 55Fe-uptake tus nqi qhia tau hais tias PBT434 thiab Fe2 ntxiv yog coj los ntawm txoj kev thauj mus los. Tsis tas li ntawd, qhov inhibition ntawm 55Fe-uptake nyob rau hauv lub xub ntiag ntawm PBT434 tab sis tsis PBT434- tau ntsib qhia tias ib tug extracellular PBT434- hlau complex tsis yog ib tug ligand rau ferrous hlau thauj nyob rau hauv hBMVEC, uas yog ZIP8, thiab ZIP 14.

Cistanche benefits

Cistanche ntxiv

Txhawm rau tshuaj xyuas ntxiv txog lub luag haujlwm ntawm PBT434 hauv kev sib xyaw hlau, peb tau sim cov txiaj ntsig nws qhov ua ntej muaj nyob rau ntawm 55Fe2 ntxiv rau kev nce ntxiv. Cells pre-kho nrog PBT434 uas yog, tom qab ntxuav, raug rau 55Fe2 ntxiv rau pom qhov nce ntawm tus nqi ntawm uptake thiab tsub zuj zuj ntawm 55Fe2 ntxiv tom qab 3h (Fig 9, panels B thiab D). Qhov kev nce ntxiv no tau khaws cia tsawg kawg 24 teev. Cov ntaub ntawv no qhia tau hais tias ua ntej-exposure ntawm hlwb rau PBT434 ib ntus potentiates hlau uptake. Kev npaj txhij txog, PBT434- tau ntsib kev kho mob ua ntej kuj tau pom tias muaj kev nce ntxiv ntawm kev nce thiab nce ntxiv (Daim duab 9B), tab sis qhov txiaj ntsig no tsis yog qhov tseem ceeb lossis tsis tu ncua raws li qhia los ntawm PBT434.

Peb tau pom tias qhov nce ntawm cov hlau los ntawm 59Fe-transferrin tau txais kev txhawb nqa los ntawm ferri-txo thiab ferro-permeation ntawm plasma membrane ntawm muaj [50, 51]. Ib qho kev sim tau tshwm sim hauv kev txhawb nqa ntawm tus qauv TBI hlau uptake no yog lub khob ntawm qhov kev txhawb nqa los ntawm inhibition ntawm kev ua haujlwm ntxiv-cytoplasmic ferrireductase; Lwm qhov tshwm sim yog 60 feem pua ​​​​ inhibition ntawm TBI hlau uptake los ntawm ferrozine, muaj zog ferrous hlau chelating tus neeg sawv cev [50]. Cov tswv yim tom kawg no tau siv los ua kom pom tias PBT434, tab sis tsis yog PBT434- tau ntsib, kuj tseem cuam tshuam TBI hlau uptake (Fig 10).

Figure 10

5. PBT434 stimulates Fpn-dependent 55Fe2 ntxiv rau efflux

PBT434 muaj kwv yees li 20 feem pua ​​​​ntawm lub peev xwm ntawm deferiprone los tsim qhov pom tseeb stimulation ntawm Fe2 ntxiv rau efflux los ntawm neuronal hlwb [30]. Peb tau soj ntsuam cov efflux ntawm 55Fe2 ntxiv los ntawm hBMVEC thaum tsis muaj lossis muaj PBT434 hauv cov hlwb tswj lossis cov hlwb kho nrog mini-hepcidin, PR73. Hepcidin yog ib qho tshuaj peptide pom ob qho tib si hauv lub cev thiab hauv lub hlwb interstitium uas khi rau Fpn thiab lub hom phiaj ntawm kev thauj mus los rau kev degradation. Cov teebmeem ntawm hepcidin ntawm cov hlau xa tawm ua haujlwm ntawm Fpn tau kawm ntau heev [52–54]. Peb tau pom yav dhau los efflux ntawm Fe2 ntxiv los ntawm hBMVEC yog Fpn-dependent [35, 49]. PR73 muaj EC50 ntawm ~ 4 nM rau Fpn degradation hauv GFP reporter assay [55]. hBMVEC hauv monolayers tau ntim nrog 55Fe2 ntxiv rau 24 teev thaum tsis muaj lossis muaj PR73. 55Fe-efflux tom qab ntawd suav nrog lub sijhawm 5h nyob rau hauv qhov txuas ntxiv lossis muaj PR73 nrog rau qhov tsis muaj thiab muaj PBT434 (Daim duab 11). Thaum PR73 tsoo 55Fe efflux los ntawm ob qho tib si tswj thiab PBT434- kho kab lis kev cai, PBT434 ib feem cuam tshuam qhov inhibition vim yog mini-hepcidin. Thaum tsis muaj PBT434, hlau efflux los ntawm PR73- cov kab lis kev cai kho tau raug tsoo los ntawm ~ 75 feem pua ​​​​thaum knockdown hauv PBT434- kev kho kab lis kev cai tsuas yog ~ 50 feem pua ​​(Fig 11 thiab Table 2). Ob qhov kev xav tuaj yeem kos los ntawm cov txiaj ntsig no. Ua ntej, knockdown ntawm Fpn los ntawm PR73 down-regulates 55Fe-efflux nyob rau hauv lub xub ntiag thiab tsis muaj PBT434. Thib ob, nyob rau hauv ob qho xwm txheej, PBT434 txhawb nqa qhov tseem ceeb txawm tias muaj kev txhawb nqa me me ntawm cov hlau efflux.

Figure 11

table 2

Cov ntaub ntawv

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Danielle K. BaileyID, Whitney Clark, Daniel J. Kosman

Department of Biochemistry, Jacobs School of Medicine thiab Biomedical Sciences, State University of New York ntawm Buffalo, Buffalo, NY, United States of America

Koj Tseem Yuav Zoo Li