Aryl Hydrocarbon Receptor-Dependent Thiab -Independent Pathways Mediate Curcumin Anti-Aging Effects

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

Abstract:Lub aryl hydrocarbon receptor (AhR) yog ligand-activated transcription yam uas nws cov dej num tuaj yeem hloov kho los ntawm polyphenols, xws li curcumin. AhR thiab curcumin muaj evolutionarily txuag cov teebmeem ntawm kev laus. Ntawm no, peb tau tshawb xyuas seb thiab yuav ua li cas AhR kho kom haum rau kev tiv thaiv kev laus ntawm curcumin thoob plaws hom. Siv kev sib xyaw ua ke ntawm hauv vivo, hauv vitro, thiab hauv kev tshuaj ntsuam silico, peb tau pom tias curcumin muaj AhR-dependent lossis-ywj siab los ntawm cov ntsiab lus tshwj xeeb. Peb pom tias hauv Caenorhabditis elegans, AhR mediates curcumin-induced lifespan extension, feem ntau yuav dhau los ntawm ligand-ywj siab inhibitory mechanism uas cuam tshuam nrog nws cov haujlwm antioxidant. Curcumin kuj tau qhia AhR-kev ywj pheej los tiv thaiv kev laus, xws li kev tiv thaiv kev sib sau ua ke ntawm cov proteins thiab oxidative kev nyuaj siab hauv C.elegans thiab txhawb nqa lub peev xwm tsiv teb tsaws ntawm tib neeg cov kab mob endothelial. Cov teebmeem ntawm AhR-kev ywj pheej no feem ntau yog kho los ntawm Nrf2/SKN{12}} txoj hauv kev.

Thov nias ntawm no kom paub ntxiv

Ntsiab lus:aryl hydrocarbon receptor; curcumin; oxidative kev nyuaj siab; Caenorhabditis elegans; nas; endothelial hlwb; hauv vivo; hauv vitro; hauv silico

1. Taw qhia

Lub aryl hydrocarbon receptor (AhR) yog ib qho ubiquitous ligand-activated transcription fac-tor qhia tias yog tus txiav txim rau cov tshuaj toxicological teb rau 23,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) hauv cov tsiaj nyeg [1]. AhR cov kev taw qhia txoj hauv kev tau piav qhia tau zoo hauv cov kab mob mammalian. Luv luv, unliganded AhR yog nyob rau hauv lub cytoplasm thiab stabilized los ntawm ntau yam co-factors, xws li 90-kDa heat shock protein (Hsp90), AHR-interacting protein (AIP), thiab chaperone p23. Kev khi rau exogenous (piv txwv li, TCDD) los yog endogenous (piv txwv li, kynurenine) ligands txhawb txoj kev hloov ntawm qhov complex mus rau hauv lub nucleus, qhov twg AhR dissociates los ntawm nws cov co-factors thiab sib sau ua ke hauv heterodimer nrog AHR nuclear translocator (ARNT). Qhov tshwm sim ntawm AHR / ARNT complex khi rau xenobiotic teb cov ntsiab lus (XREs) ntawm lub roj teeb ntawm cov theem I thiab II detoxification noob, nws thiaj li ua rau ligands 'degradation [2]. Sib nrug los ntawm nws lub luag haujlwm hauv xenobiotic teb, ua haujlwm rau AhR hauv ntau cov txheej txheem pathophysiological, xws li kev tiv thaiv kab mob [3,4], o [5,6], lipid thiab qabzib metabolism [7,8] mus rau plawv plawv, siab, thiab lwm yam kab mob hauv nruab nrog cev [9-12], tau raug tshawb pom nyob rau hauv kaum xyoo dhau los. Cov pov thawj loj hlob kuj tseem qhia txog qhov sib txawv thiab zoo li qhov tsis sib haum xeeb ntawm AhR hauv cov txheej txheem kev laus, uas tseem tuaj yeem sib koom ua ke, suav nrog cov ntaub so ntswg-, koob tshuaj- thiab hom kab mob tshwj xeeb [13].cistanche genghis khanLub luag haujlwm tsis zoo rau AhR hauv kev laus thiab cov hnub nyoog muaj feem cuam tshuam tau piav qhia thoob plaws hom [14,15]. Piv rau hom tsiaj qus Caenorhabditis elegans, AhR mutant strain, ahr-1(ju145), muaj lub neej ntev-thiab kev noj qab haus huv; Hauv cov nas, AhR tsis muaj peev xwm txhim kho cov hlab ntsha ua haujlwm thiab nce kev ua haujlwm ntawm nitric oxide synthase thiab, yog li ntawd, tsis muaj bioavailability; thiab, thaum kawg, pom muaj kev sib raug zoo ntawm AhR kev qhia thiab kev nruj ntawm cov hlab ntsha hauv cov hnub nyoog nruab nrab thiab hnub nyoog tib neeg [14]. Tsis tas li ntawd, hauv kev tshawb fawb txog kev kis mob ntawm cov neeg Suav, AhR qhia tau hais txog qhov tshwm sim ntawm tus kab mob coronary arterial [16].

Cistanche tuaj yeem tiv thaiv kev laus

Nco ntsoov, ntau lub tebchaw cuam tshuam rau kev laus lossis cov kab mob muaj hnub nyoog [17-19] tuaj yeem hloov kho AhR kev ua haujlwm [20-22]. Thaum qhov ua rau AhR los ntawm xenobiotics ua rau cov qog nqaij hlav sib txawv hauv cov tsiaj nyeg [23,24], kev noj zaub mov thiab ib puag ncig tau pom tias muaj qhov cuam tshuam ntawm AhR-nyob ntawm C.elegans'health span [25]. Ntawm cov khoom noj AhR modulators, polyphenols xws li curcumin tau kawm ntau rau lawv cov teebmeem kev noj qab haus huv. Curcumin yog xim daj los ntawm Curcuma longa, nrog rau ntau yam kev hloov pauv tau txais txiaj ntsig zoo, suav nrog cov tshuaj tua kab mob antioxidant, tiv thaiv kab mob, thiab kev tiv thaiv kev laus [26]. Curcumin tiv thaiv kev sib sau ua ke ntawm cov protein thiab ua kom lub neej ntev hauv C.elegans thiab Drosophila los ntawm kev hloov pauv ntawm cov protein homeostasis [27-29].cistanche lub neej extensionNtxiv mus, thaum tswj hwm tus kab mob Alzheimer tus kab mob transgenic nas qauv, nws txo qis tag nrho amyloid-beta (A) burden [30]. Nyob rau hauv cov nas qub, curcumin rov qab tsis muaj bioavailability, yog li txo oxidative kev nyuaj siab thiab txhim kho endothelial dysfunction thiab artery stiffness ntsuam xyuas los ntawm aortic pulse yoj tshaj tawm (PWV) - ib qho tseem ceeb tshaj plaws kev soj ntsuam ntsuas los yog cov cim ntawm loj elastic artery stiffness [31]. Ntau qhov kev tshawb fawb hauv tib neeg kuj tau pom tias muaj kev tiv thaiv ntawm curcumin rau kev noj qab haus huv ntawm cov hlab plawv [32]. Txawm li cas los xij, hom kev txiav txim ntawm curcumin tseem tsis tau meej meej, thiab, qhov tseem ceeb tshaj, seb nws cov txiaj ntsig kev noj qab haus huv puas tau kho los ntawm AhR tsis tau tshawb xyuas [33.34]

Cov kab mob ua qauv, xws li nematode C.elegans, tau ua haujlwm hauv kev txheeb xyuas cov caj ces thiab ib puag ncig kev txiav txim siab ntawm kev laus. Qhov no yog vim nws cov khoom muaj txiaj ntsig zoo, suav nrog nws txoj kev sim yooj yim, kev ua neej luv luv, thiab kev tsim tawm ntawm ntau tus menyuam yaus los ntawm nws tus kheej fertilization. C.elegans'genome tau ua tiav tiav, thiab feem ntau ntawm nws cov noob thiab txoj hauv kev tau hloov pauv hloov pauv. Cov protein ntau ntawm AhR tau khaws cia thaum lub sijhawm hloov pauv thiab hauv C.elegans, lub orthologs ntawm AhR thiab ARNT yog encoded los ntawm AhR-txog (ahr-1) thiab ahr-1 txuam (aha{{4} }}) cov noob, feem [35]. Cov proteins sib raug zoo, AHR-1 thiab AHA-1, qhia txog 40 feem pua ​​​​ntawm lawv cov kab ke sib lawv liag nrog cov tsiaj txhu thiab tsim cov heterodimer (tseem nrog cov tsiaj txhu sib txawv), uas tuaj yeem khi XREs ntawm lub hom phiaj. Cov noob hauv vitro [36]ceg nzAHR-1 feem ntau yog qhia nyob rau hauv neuronal cell hom, xws li GABAergic neurons [37l, thiab nws plays lub luag hauj lwm tseem ceeb hauv kev tswj cov neuronal kev loj hlob [38]. Tsis zoo li vertebrates'AhR, AHR-1 tsis khi TCDD thiab lwm yam xenobiotics [35,39], tsis tau nws koom nrog mammalian AhR cov yam ntxwv hauv kev tswj hwm cov txheej txheem neuronal, kev loj hlob, thiab fertility [37,38,{ {8}}], thaum kawg qhia tias poj koob yawm txwv AhR tsis ncaj qha rau kev tswj cov noob rau kev degradation ntawm cov ligands lom [44,45]. Yog li peb lees paub C.elegans ua tus qauv tshwj xeeb thiab muaj zog los txheeb xyuas thiab kawm txog kev ua haujlwm ntawm poj koob yawm txwv ntawm AhR tej zaum tsis cuam tshuam rau nws cov lus teb xenobiotics.

Hauv txoj kev tshawb no, peb tau tshawb xyuas lub luag haujlwm ntawm AhR hauv curcumin qhov kev tiv thaiv kev laus thoob plaws hom. Los ntawm kev sib xyaw ua ke ntawm hauv vivo, hauv vitro, thiab hauv kev tshuaj xyuas silico, peb pom tias curcumin qhia cov txiaj ntsig sib txawv los tiv thaiv kev laus los ntawm uncoupled ahr-1-dependent and -independent mechanisms. Peb pom tias C.elegans ahr-1-cov tsiaj txhu uas ploj lawm yog nyob ntev tab sis muaj kev cuam tshuam rau oxidative kev nyuaj siab. Thaum curcumin tsis tau txuas ntxiv lub neej ntawm C.elegans ahr-1 mutants, nws txhawb nqa lawv cov kev tiv thaiv rau oxidative kev nyuaj siab. Curcumin kuj txhawb nqa cov tshuaj tiv thaiv antioxidant thiab kev muaj peev xwm tsiv teb tsaws ntawm tib neeg cov kab mob endothelial (EC) ntawm nws tus kheej ntawm AhR, ib qho kev cuam tshuam uas feem ntau tso siab rau Nrf2/SKN-1 thoob plaws hom. SKN-1 yog C.elegans ortholog ntawm tib neeg Nrt2 (nuclear factor erythroid 2-txog yam 2) redox transcription factor, uas plays ib tug evolutionarily conserved lub luag hauj lwm nyob rau hauv cell thiab organismal homeostasis thiab teb rau oxidative kev nyuaj siab [ 46,47]Coupling tshwm sim los ntawm ib tug cellular reporter assay thiab nyob rau hauv silico qauv ntawm AHR-1 ligand-binding domain (LBD), peb mam li qhia tias curcumin feem ntau yuav suppressed AHR{17}} kev ua hauv ligand- kev khi-kev ywj pheej yam. Qhov tseem ceeb, thiab nyob rau hauv txoj kab nrog cov ntaub ntawv hauv C.elegans thiab EC, curcumin thiab pro-oxidants tso tawm qhov cuam tshuam ntawm AHR-1 kev ua, txhais tau hais tias curcumin tuaj yeem hloov kho AHR-1 kev ua haujlwm los ntawm nws cov antioxidant muaj peev xwm ncaj qha. los yog tsis ncaj los ntawm kev tswj hwm ntawm Nrf2/SKN-1 lossis lwm yam kev tswj hwm cov protein.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. C.elegan 1s

2.1.1. C. elegans Hom thiab Cultivation

Peb siv C.elegans hom hauv qab no: N2 [wild-type], CZ2485 [ahr-1(jul45)], NV38b [alhr-1(ju145);unc-54p:Q40 :YFP], NV38wt [unc-54p::Q40:YFP] (original strain AM141 [48]), NV42a [unc-54p::alphasymuclein::YFP, ahr-1 (jul45)], NV42wt [unc-54p::alphasynuclein::YFPI (original strain NL5901 [49]), NV35a [ahr-1(ju145);(pAF15)gst-4 p::GFP::NLS], NV35wt (pAF15)st-4p::GFP::NLS]( thawj hom CL2166).Rau kev saib xyuas, worms tau khaws cia synchronized los ntawm qe nteg ntawm 20 degree ntawm Nematode Growth Media (NGM) daim hlau thiab pub nrog E.coli OP50, raws li txoj kev tau piav qhia hauv [25]. Rau cov kev sim, cov cab tau synchronized ntawm daim hlau ntxiv nrog E.coli HT115(DE3) ntawm daim hlau ntxiv nrog 1 mM IPTG (Fisher Scientific, Geel, Belgium).

2.1.2. Gene Silencing los ntawm RNA-Mediated Interference (RNAi)

Gene Silencing tau ua tiav los ntawm kev noj E.coli HT115(DE3) qhia plasmids nrog dsRNA tiv thaiv cov noob tshwj xeeb [50]. Kev pub noj RNAi tau siv tas li txij thaum yug mus txog thaum tuag. Rau kev soj ntsuam juglone tsis kam nrog HT115(skn-1) kab mob, RNAi noj tau siv los ntawm L4 worms rau 24 teev ua ntej hloov mus rau cov ntawv tshiab juglone.

2.1.3. E.coli hom kab mob thiab kev loj hlob

Cov kab mob tau loj hlob hauv LB nruab nrab ntawm 37 degree thaum hmo ntuj. Thaum siv E.coli nqa vectors, LB nruab nrab tau ntxiv nrog 0.01 feem pua ​​​​ntawm ampicillin thiab 0.0005 feem pua ​​​​ntawm tetracycline.E.coli HT115(L4440), HT115(ugt{{8}) }), HT115(skn{10}}), thiab OP50 tau txais los ntawm lub tsev qiv ntawv Ahringer C. elegans RNAi [51].

2.1.4.Lifespan

Kev soj ntsuam lifespan yog pib los ntawm ib tug synchronized pejxeem ntawm worms, uas tau pauv mus rau tshiab NGM daim hlau txhua txhua hnub nyob rau hauv lub fertile lub sij hawm. Cov tsiaj tuag, ciaj sia, thiab censored tau qhab nia thaum lub sijhawm hloov pauv. Tsiaj txhu raug suav tias tuag thaum lawv tsis pom kev txav mus los, lossis cov lus teb rau kev txhawb nqa nrog platinum hlau, los yog pharyngeal twj tso kua mis. Tsiaj txhu nrog sab hauv hatching (lub hnab), thiab tawg qhov chaw mos, los yog tuag desiccated rau ntawm phab ntsa raug censored. Tus nab npawb ntawm cov tsiaj tuag thiab censored tau siv rau kev soj ntsuam ciaj sia nyob hauv OASIS[52] lossis OASIS 2[53]. Rau kev suav ntawm qhov nruab nrab lub neej thiab txoj kev ciaj sia nyob hauv OASIS thiab OASIS 2, Kaplan-Meier kwv yees tau siv, thiab cov txiaj ntsig p-nqi tau suav nrog siv qhov ntsuas ntsuas ntawm cov tsiaj sib xyaw.

2.1.5. Movement/Health Span

Qhov kev txav mus los tau raug teeb tsa los ua qhov tsis zoo rau kev laus noj qab haus huv, thiab theem ntawm kev txav mus los yog hu ua kev noj qab haus huv. Nws tau soj ntsuam nyob rau hauv cov pej xeem siv rau kev ntsuam xyuas lifespan. Tsiaj txhu, uas tau nkag los ntawm tus kheej los yog tom qab phau ntawv stimulus, raug suav hais tias yog tsiv, thaum cov tsiaj tuag lossis cov tsiaj tsis muaj tus cwj pwm nkag mus tau suav tias yog tsis txav. Kev txheeb cais tau ua tiav raws li tau piav qhia rau lub neej.

2.1.6. Curcumin Kev Kho Mob ntawm C. elegans

Curcumin (Sigma Aldrich, Taufkirchen, Lub teb chaws Yelemees) tau yaj nyob rau hauv DMSO (Carl Roth, Karlsruhe, Lub teb chaws Yelemees) nyob rau hauv ib tug concentration ntawm 100 mM thiab muab rau NGM tom qab autoclaving.Qhov kawg concentration ntawm curcumin hauv xov xwm yog 100 μM (0.1 feem pua ​​DMSO). Cov phaj tswj muaj 0.1 feem pua ​​DMSO. Kab mob tau kho tsis tu ncua pib ntawm qe.

2.1.7. Quantification ntawm PolyQ Aggregates

PolyQ40 aggregates tau pom los ntawm fluorescence microscopy (100 × magnification) nyob rau hauv 10-hnub-laus worms anesthetized nrog 15 mM sodium azide (Sigma Aldrich, Taufkirchen, Lub teb chaws Yelemees). Txhawm rau ntsuas tus naj npawb ntawm cov sib sau ua ke, cov duab tau stitched siv Fiji khub-ntse stitching plugin [54] los tsim cov kab mob tag nrho, thiab cov naj npawb ntawm cov aggregates tau suav nrog hauv Fiji [55] siv lub plugin "Tshaj Tawm".

2.1.8 ib. Quantification ntawm x-Synuclein Aggregates

-synuclein aggregates nyob rau hauv lub taub hau cov leeg ntawm 7-hnub-laus kab mob tau pom los ntawm fluorescence microscopy (40}}0 × magnification) nyob rau hauv cov kab mob anesthetized nrog 15 mM sodium azide (Sigma Aldrich, Taufkirchen, Lub teb chaws Yelemees, S2002 ). Cov duab tau segmented siv Plastik (version 1.3.0)(laboratory ntawm Anna Kreshuk ntawm European Molecular Biology Laboratory, Heidelberg, lub teb chaws Yelemees) (muaj nyob rau ntawm https://www.ilastik.org/, nkag mus rau 10 Plaub Hlis Ntuj 2018)[56] . Cov duab segmented tau siv los txheeb xyuas cov naj npawb ntawm cov aggregates hauv Fiji [55] siv lub plugin "Tshaj Tawm".

2.1.9.Microarray thiab GO Term Analysis

Cov qauv los ntawm tsib tus neeg ua haujlwm ywj pheej nrog kwv yees li {{0}} cov kab mob hnub nyoog ib hnub tau sau, thiab RNA tau muab rho tawm thiab thauj mus rau Affymetrix Chip. Cov ntaub ntawv nyoos microarray hauv hom CEL tau txheeb xyuas siv software R (version 3.4.2) (R Foundation, Vienna, Austria) thiab Bioconductor (Lub Bioconductor Project, Bioconductor yog qhov qhib thiab qhib kev txhim kho) [57]. Kev kho keeb kwm yav dhau, normalization, thiab kev qhia suav nrog tau ua nrog oligo pob58] thiab RMA txoj kev. Rau kev tswj kom zoo ntawm cov array, pob arrayQualityMetrics 3.34.{16}} [59] tau siv. Vim tias qhov ntsuas tau zoo, cov qauv ahr-1C5 raug tshem tawm los ntawm kev tshuaj xyuas ntxiv. Cov noob sib txawv tau txheeb xyuas tau siv cov pob limma thiab cov qauv kab thiab nruab nrab t-statistic nrog FDR los kuaj rau ntau qhov sib piv [6{{20}}]. P-tus nqi ntawm 0.1 tau siv. Cov noob sib txawv tau txheeb xyuas rau Gene Ontology term enrichment siv Cytoscape(version 3.6.0)(Lub Cytoscape Consortium, ib lub koom haum ywj pheej tsis muaj txiaj ntsig)[61] nrog lub plugin ClueGo (version 2.5.0)(Labboratory of Integrative Cancer Immunology, Paris, Fabkis) [62]. Cov ntaub ntawv microarray tuaj yeem nkag mus tau los ntawm Gene Expression Omnibus accession no. TSI 195769.

2.1.10.ROS Quantification

MtROS tau kuaj pom nyob rau hauv wt thiab ahr-1 mutants worms siv MitoSOX Liab (Ther-moFisher Scientific, Dreieich, Lub teb chaws Yelemees). Nematodes tau synchronized los ntawm lub qe tso rau hauv IPTGplates siv HT115 (L4440) kab mob ua zaub mov. Tom qab ntawd, 48 teev tom qab ntawd, 50 tus tsiaj nyob rau theem L4 tau hloov mus rau qhov tshiab tau npaj 10μM MitoSOX Liab daim hlau uas muaj UV-tua HT115 (L4440). Cov worms tau incubated nyob rau hauv qhov tsaus ntuj ntawm 20 degree. Tom qab 16 h incubation, lawv tau tsiv mus rau NGM daim hlau tshiab kis nrog nyob HT115 (L4440) rau 1 h kom tshem tawm cov xim seem ntawm cov hnyuv. Rau kev yees duab, nematodes tau mounted rau 2 feem pua ​​agarose pad slides, anesthetized los ntawm ntxiv 10 mM levamisole thiab kho los ntawm ProLongTM iav Antifade Mountant (ThermoFisher Scientific, Dreieich, Lub teb chaws Yelemees). Cov duab tau txais tam sim ntawd nrog Zeiss Axio Imager M1 microscope (Carl Zeiss, Inc., Cologne, Germany) siv lub hom phiaj 40 × thiab DsRed Lim. Tom qab ntawd, thaj tsam worm taub hau raug xaiv manually, thiab kev sib xyaw ua ke tau suav nrog siv cov duab software Fij 55.

2.1.11. Tetramethylrhodamine Ethyl Ester (TMRE) Kev Ntsuas

Txhawm rau ntsuas qhov muaj peev xwm ntawm mitochondrial, nematodes tau synchronized los ntawm lub qe tso rau ntawm IPTG daim hlau uas muaj cov kab mob HT115 (L4440). Nyob rau hnub ntawm kev sim, TMRE (Invitrogen, Eugene, LOSSIS, USA) tau yaj hauv DMSO mus rau qhov siab ntawm 5 mM thiab tom qab ntawd diluted rau 30 μM nrog cov cua sov-inactivated HT115 (L4440) (30 min, 65 degree). Tag nrho ntawm 150 μL ntawm cov tshuaj no tau ntxiv rau ib phaj thiab sab laug kom qhuav hauv qhov tsaus rau kwv yees li 30 min. Rau caum tus neeg laus synchronous worms ntawm 1, 3, los yog 5 hnub ntawm cov neeg laus raug xaiv mus rau TMRE daim phiaj npaj thiab sab laug rau cov stain kom nqus tau 2 h hauv qhov tsaus ntuj ntawm 20 degree. Tom qab staining, worms raug xa mus rau IPTG daim hlau cog nrog tshav kub-inactivated HT115 (L4440) thiab incubated rau 1 h nyob rau hauv qhov tsaus ntuj ntawm 20 degree, kom tshem tawm cov residual dye los ntawm txoj hnyuv.cistanche noov lojRau kev yees duab, 10 nematodes tau mounted rau 2 feem pua ​​agarose pad slides, anesthetized los ntawm ntxiv 10 mM levamisole thiab tsau los ntawm ProLongM iav Antifade Mountant (ThermoFisher Scientific, Dreieich, lub teb chaws Yelemees). Rau txhua qhov kev sim khiav, 5 slides tau npaj rau ib pawg. Cov duab tau txais tam sim ntawd nrog Zeiss Axio Imager M1 microscope (Carl Zeiss, Cologne, Lub Tebchaws Yelemees) siv lub hom phiaj 2.5 × thiab DsRed lim. Kev siv fluorescence tau suav nrog Fiji [55].

2.1.12. Pharyngeal Pumping Rate thiab Motility Assay

N2 thiab CZ2485 nematodes tau synchronized los ntawm bleaching [63] thiab cov qe raug tso rau hatching qe tsis (118mM NaCl, 48 mM KCl, 2 mM CaCl, 2mM MgCl2,25 mM Hepes, pH 7.3) hmo ntuj, ntawm orbital shacking. L1 larvae tau pom mus rau NGM ntxiv nrog 1 mM IPTG thiab muaj 0.1 feem pua ​​​​DMSO lossis 100 μM curcumin. Cov kab mob HT115 (L4440) tau siv los ua zaub mov. Cov menyuam yaus cov kab mob loj tau sau nrog M9 tsis pub, centrifuged (300g × 3 min), thiab ntxuav ob zaug kom tshem tawm cov kab mob. Cov kab mob tau tsim nrog 0-1 mM H, O, (Sigma-Aldrich, Taufkirchen, Lub teb chaws Yelemees) (100 worms / 100 μL), rau 2 teev ntawm orbital co. Cov kab mob tswj tau raug incubated nrog M9 tsis tsuas. Tom qab 2 teev, cov cab tau txav mus rau NGM ntxiv nrog 1 mM IPTG thiab muaj 0.1 feem pua ​​DMSO lossis 100 μM curcumin thiab noob nrog HT115 (L4440) kab mob ua zaub mov. Lub pharyngeal twj tso kua mis, tau qhab nia los ntawm kev suav tus naj npawb ntawm lub davhlau ya nyob twg qhov muag teev ntawm lub pharynx tau cog lus nyob rau 1 min (twj / min), thiab qhov kev soj ntsuam motility, tau qhab nia los ntawm suav cov naj npawb ntawm lub cev thrash (lub cev khoov / min) hauv M9 tsis pub dhau 1 min lub sijhawm, tau qhab nia ntawm 2 txog 20 teev tom qab.

2.1.13.Acute Juglone Sensitivity Assay

N2 thiab CZ2485 nematodes tau synchronized los ntawm qe-ntev rau NGM daim hlau nrog DMSO lossis 100 μM ntawm curcumin. Cov phaj tau ntxiv nrog 1 mM IPTG thiab noob nrog HT115(L4440) lossis HT115(ugt-45) ​​cov kab mob ua zaub mov. Txhawm rau ntsuas qhov tshwm sim ntawm daim tawv nqaij -1 RNAi, cov kab mob tau ua ke los ntawm cov qe tso rau hauv DMSO lossis curcumin daim hlau uas muaj cov kab mob HT115(L4440) cov kab mob. Raws li nematodes mus txog theem L4 larval, lawv raug xa mus rau 24 teev mus rau DMSO lossis curcumin daim hlau uas muaj cov kab mob HT115(skin{16}}) kab mob. Hnub 1 cov kab mob laus laus (25 worms) tau txav mus rau hauv daim phiaj NGM tshiab uas muaj 200 μM Juglone (Merck, Darmstadt, Lub teb chaws Yelemees) thiab noob nrog 25 μL ntawm 10 × concentrated kab mob thaum hmo ntuj. Kab mob ciaj sia nyob rau hauv juglone-induced oxidative kev nyuaj siab raug kuaj los ntawm kov-provoked txav ib teev, rau 6 h. Tsiaj txhu tau qhab nia tuag thaum lawv tsis teb rau kov nrog platinum hlau xaiv. Nematodes desiccated rau ntawm phab ntsa tau censored. Tus naj npawb ntawm cov tsiaj tuag thiab censored tau qhab nia thiab Daim Ntawv Thov Online rau Kev Tshawb Fawb Txog Kev Ciaj sia nyob OASIS2 tau ua haujlwm rau kev soj ntsuam ciaj sia [53].

2.1.14 ib. Quantification ntawm GST -4: GFP Siv

NV35wt thiab NV35a tau synchronized los ntawm qe-ntug mus rau NGM daim hlau ntxiv nrog 1 mM IPTG thiab muaj 0.1 feem pua ​​​​DMSO lossis 100 μMcurcumin. HT115(L4440), HT115(ugt{10}}) thiab HT115(skin{12}}) kab mob tau siv los ua zaub mov. Txhawm rau pom GFP fluorescence, hnub 1 cov kab mob laus laus tau tshuaj loog nrog 10 mM levamisole hydrochloride tov thiab mounted ntawm 2 feem pua ​​agarose pads. Cov duab tau txais tam sim ntawd nrog Zeiss Axio Imager M1 microscope (Carl Zeiss, Cologne, Lub Tebchaws Yelemees), 2.5 × magnification) thiab tom qab ntawd txheeb xyuas siv software CellProfiler (Cov pab pawg CellProfiler, Cimini Lab ntawm Broad Institute of MIT thiab Harvard, MA, USA). Luv luv, cov duab tau ua tiav los ntawm kev siv lub raj xa mus rau ntu cov kab cab hauv txhua daim duab los ntawm cov duab ci ntsa iab thiab cais lawv los ntawm keeb kwm yav dhau. Tom qab ntawd, qhov sib xyaw ua ke GFP siv tau ntsuas ib tus kab mob.

2.1.15.Semi-Quantitative Real-Time PCR (qPCR) hauv C. elegans

Cov qauv los ntawm 3 qhov kev ywj pheej rov ua dua nrog kwv yees li 1000 3- cov kab mob hnub nyoog ib hnub tau sau thiab RNA raug rho tawm. Tom qab ntxuav thiab elution, cov ntsiab lus RNA raug ntsuas los ntawm spectrophotometric, thiab 1-2 ug ntawm RNA tau siv rau cDNA synthesis (Omniscript RT Kit (Qiagen, Hilden, Lub teb chaws Yelemees).cistanche hmoovPrimer khub tau teev nyob rau hauv Table S1. Rau lub sijhawm tiag tiag qPCR, cDNA tau diluted ntawm 1:20 hauv 10 mM TRIS (pH8.0). Rau cov tshuaj tiv thaiv, cov khoom siv qPCR Green Core (Jena Biosciences, Jena, Lub teb chaws Yelemees)) lossis GoTaq⑧ qPCR cov khoom siv (Promega, Walldorf, Germany) tau siv. Cov qauv no tau khiav hauv MyiQ2 cycler (BioRad, Feldkirchen, Lub teb chaws Yelemees), thiab cov lus qhia ntawm txhua tus qauv raug ntsuas hauv qhov sib npaug ntawm tib lub phaj ntau qhov zoo. Cov lus qhia tau suav txheeb ze rau qhov kev siv cov noob ua haujlwm -1 thiab CDC -42 siv iO5 software. Tag nrho cov ntaub ntawv khaws tseg tau qhib rau kev tshawb fawb noob raws li BioRad cov lus qhia rau cov neeg siv, thiab cov lus qhia tau suav nrog siv cov lus qhia (ddCr). Qhov kev ua tau zoo ntawm txhua tus khub primer cov tshuaj tiv thaiv tau ntxiv rau qhov tseeb kom muaj nuj nqis ntawm cov lus qhia normalized. Qhov kev ua tau zoo tau raug soj ntsuam nrog 1:20, 1:100, 1:500, thiab 1:2500 dilutions ntawm cDNA. Los ntawm cov txiaj ntsig kev qhia zoo, qhov hloov pauv hloov pauv piv rau hom tsiaj qus tau suav rau txhua qhov rov ua dua.

2.2. Mammalian Cells

2.2.1.Cultivation of Cos7 Cells

Cos7 hlwb (ATTC, #CRL-1651) tau cog rau ntawm 37 degree thiab 5 feem pua ​​CO, hauv Dulbecco's Modified Eagle's Medium (DMEM) (Gibco/ThermoScientific, Dreieich, Germany) nrog 1 feem pua ​​pyruvate, 1 feem pua ​​Glutamax thiab 1 0 feem pua ​​​​Fetal Bovine Serum (FBS)(Gibco/ThermoScientific, Dreier-ich, Germany) thiab ntxiv Penicillin (10.000 units/mL)/Streptomycin (10.000 ug/ mL) Thaum cov hlwb tsim cov nyom ntawm tes, lawv raug tshem tawm ntawm lub hauv paus los ntawm kev siv 0.05 feem pua ​​Trypsin/EDTA (Thermo Scientific, Dreieich, Lub teb chaws Yelemees).

2.2.2. Transfection Plasmids

Peb siv cov plasmids hauv qab no rau kev hloov pauv ntawm Cos7 hlwb: pcDNA3, pcDNA3-Ahr-1-VP16,pcDNA3-AhA-1-VP16,pcDNA3-AhR -1(jul45)-VP16, p1A1-FL, Perl-TK. Cov plasmids tau piav qhia los ntawm Larigot li al. (xa nrog rau qhov kev kawm no). Lub p1A1-FL plasmid nqa ib qho XRE-inducible luciferase, Perl-TK nqa ib renilla luciferase, thiab pcDNA3-Ahr-1-VP16 thiab pcDNA3-AhA{24 }}VP16 nqa cov kab ke rau kev qhia ntawm C.elegans AHR-1 thiab AHA-1, feem. Rau txoj kev tshawb no, peb tsim pcDNA3-AhR-1(LBD)-VP16 los ntawm qhov chaw-directed mutagenesis ntawm pcDNA3-Ahr-1-VP16 plasmid nrog QuikChange II Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA). Cov khub primer hauv qab no tau siv los tsim qhov kev hloov pauv L rau A ntawm L363, thiab H rau Qsubstitution ntawm H365 ntawm AHR-1}:LBD-F5'-GGAAGCATCGGCGCGACCCAACGGCTGCTGAACGAG 3'andLBD-R5'-CTCGTTCAGCAGCCGTTGTTGCGGGTTCG{39} '.Super-muaj peev xwm XL1-Blue hlwb tau hloov nrog cov tau txais plasmid rau amplification. Lub plasmid sequence tau txheeb xyuas los ntawm Sanger sequencing.

2.2.3.Transient Transfection ntawm Cos7 Cells

Thaum 24 teev ua ntej kev hloov pauv, 20, 000 cov hlwb / lub qhov dej tau muab noob rau hauv 48- lub phaj zoo hauv 400 uL DMEM (ntxiv rau 10 feem pua ​​FBS ntxiv rau cov tshuaj tua kab mob) thiab incubated ntawm 37 degree . Cells tau kis nrog cov plasmid concentrations hauv qab no siv lipofectamine 200 (Invitrogen, Eugene, OR, USA): p1A1-FL(244ng/well), Phil-TK(36ng/well), pcDNA3- AhA-1-VP16(5ng/well), thiab pcDNA3-VP16(10 ng/well), pcDNA3-Ahr-1-VP16(5 ng/well) los yog pcDNA 3-AhR-1(ju145)-VP16(5 ng/well) raws li tau piav los ntawm Larigot li al. (xa nrog rau qhov kev kawm no). Lipofectamine 2000 yog siv nyob rau hauv ib tug concentration ntawm 1 uL / ​​zoo thiab pre-incubated nrog cov plasmids nyob rau hauv DMEM rau 20min ua ntej siv. Rau kev hloov pauv mus ib ntus, Cos7 hlwb tau tsim nrog cov lipofectamine / plasmid mix for3hin DMEM (ntxiv rau 10 feem pua ​​FBS) yam tsis muaj tshuaj tua kab mob kom tsis txhob muaj tshuaj tua kab mob. Tom qab ntawd, qhov nruab nrab kis tau raug tshem tawm thiab hloov los ntawm 400 μL / zoo DMEM (ntxiv rau 10 feem pua ​​FBS ntxiv rau cov tshuaj tua kab mob). Cov kab mob kis tau raug tsim los ntawm 37 degree .Ntawm txhua lub phaj, 2 lub qhov dej ntawm cov hlwb tsis hloov pauv thiab yog li siv rau lub hom phiaj normalization. 2.2.4.Kev kho ntawm Cos7 Cells

Cov kev daws teeb meem ntawm cov concentrations 1000-times siab tshaj qhov xav tau kev kho mob concentration tau npaj rau tag nrho cov tebchaw. Curcumin (Sigma Aldrich, Taufkirchen, Lub teb chaws Yelemees), Benzo (a) pyrene (Sigma Aldrich, Taufkirchen, Lub teb chaws Yelemees), leflunomide (Sigma Aldrich, Taufkirchen, Lub teb chaws Yelemees), thiab lutein (Sigma Aldrich, Taufkirchen, Lub teb chaws Yelemees), tau yaj hauv DMSO ( Carl Roth, Karlsruhe, Lub teb chaws Yelemees), thaum resveratrol (Sigma Aldrich, Taufkirchen, Lub teb chaws Yelemees) thiab rotenone (Sigma Aldrich, Taufkirchen, Lub teb chaws Yelemees) tau dis-solved nyob rau hauv ethanol (Carl Roth, Karlsruhe, lub teb chaws Yelemees). Tom qab 24 teev tom qab hloov pauv, cov kab lis kev cai nruab nrab ntawm cov cell tau hloov nrog cov kab lis kev cai ntawm tes uas muaj 1: 100 dilution ntawm cov khoom sib txuas. Cov hlwb raug kho rau 24 teev ua ntej ntsuas kev ua haujlwm ntawm luciferase.

2.2.5.Luciferase Assay (AhR Activity)

Qhov kev ua haujlwm ntawm AhR transcriptional raug ntsuas los ntawm kev ntsuas kev ua haujlwm ntawm XRE-driven luciferase[64]. Rau qhov no, Dual-Luciferase Reporter Assay System (Promega, Wall Dorf, Germany) tau siv. Tom qab kev kho mob 24 teev, Cos7 hlwb raug ntxuav ob zaug nrog PBS thiab tom qab ntawd lysed rau 15 feeb ntawm RT siv cov passive lysis tsis suav nrog hauv Dual-Luciferase Reporter Assay cov khoom. Tag nrho ntawm 20 μL ntawm cov lysed hlwb tau muab tso rau hauv ib qho dawb 96-zoo phaj thiab siv rau kev ntsuas luminescence. Lub luciferin (LARII) thiab vanilla (Stop&cGlo) substrates tau npaj raws li cov chaw tsim khoom piav qhia. Cov qauv raug thauj khoom ntawm lub luminometer (EG&G Berthold microplate Luminometer LB 96V Microluminomat plus (Berthold Technologies, Bad Wildbad, Lub teb chaws Yelemees), thiab cov substrates tau txuas rau lub raj xa dej ntawm lub luminometer. Ua ntej, 65 uL ntawm LARII tau ntxiv rau txhua qhov dej. ntawm cov qauv, thiab cov luminescence uas tsim los ntawm firefly luciferase raug ntsuas, ces 65 μL ntawm Stop&clo reagent tau ntxiv thiab cov luminescence uas tsim los ntawm Renilla luciferase raug ntsuas. Txhawm rau ntsuas AhR kev ua los ntawm kev ntsuas luminescence, peb tau ua cov nram qab no tom qab ua tiav. Cov kauj ruam: Ua ntej, lub luminescence ntawm cov hlwb tsis hloov pauv tau muab rho tawm los ntawm cov luminescence ntawm txhua tus qauv rau kev kho tom qab, nyob rau hauv cov kauj ruam tom ntej, peb normalized lub luminescence ntawm renilla luminescence ntawm tib tus qauv kom tshem tawm qhov sib txawv ntawm tus nqi transfection thiab cell. Lwm cov kauj ruam normalization rau pcDNA-VP16 hloov cov hlwb tau ua rau txhua pab pawg kho mob kom tshem tawm AhR-indep Cov txiaj ntsig endent ntawm XRE-driven luciferase.

Kab lus no yog muab rho tawm los ntawm Antioxidants 2022, 11, 613. https://doi.org/10.3390/antiox11040613 https://www.mdpi.com/journal/antioxidants