Ua tsaug rau koj tuaj xyuas Nature.com.Cov version ntawm browser koj siv tau txwv CSS kev txhawb nqa.Txhawm rau kom tau txais txiaj ntsig zoo tshaj plaws, peb pom zoo kom siv lub vev xaib tshiab ntawm koj lub browser (lossis kaw hom kev sib raug zoo hauv Internet Explorer).Nyob rau lub sijhawm no, txhawm rau ua kom muaj kev txhawb nqa tsis tu ncua, peb tab tom nthuav tawm lub xaib tsis muaj styling lossis JavaScript.
Liab ginseng tau siv nyob rau hauv cov tshuaj Asian tsoos rau ntau pua xyoo.Hauv txoj kev tshawb no, peb tau soj ntsuam lub peev xwm ntawm plaub hom ginseng liab (Suav liab ginseng, Kauslim liab ginseng A, Korean liab ginseng B, thiab Korean liab ginseng C) zus nyob rau hauv txawv cheeb tsam los inhibit qhov tsim thiab loj hlob ntawm carcinogen-induced ntsws. qog nqaij hlav.Kev kuaj benzo(a)pyrene (B(a)P) tau ua rau A / J nas, thiab Korean liab ginseng B tau pom tias muaj txiaj ntsig zoo tshaj plaws hauv kev txo cov qog nqaij hlav ntawm plaub hom ginseng liab.Tsis tas li ntawd, peb tau soj ntsuam cov ntsiab lus ntawm ntau yam ginsenosides (Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 thiab Rg5) hauv plaub liab ginseng extracts thiab pom tias Korean liab ginseng B muaj. cov qib siab tshaj plaws ntawm ginsenoside Rg3 (G-Rg3), qhia tias G-Rg3 tuaj yeem ua lub luag haujlwm tseem ceeb hauv nws txoj kev kho mob.Txoj haujlwm no qhia tau hais tias G-Rg3 muaj bioavailability tsawg.Txawm li cas los xij, thaum G-Rg3 tau koom ua ke nrog P-gp inhibitor verapamil, efflux ntawm G-Rg3 rau hauv Caco-2 hlwb raug txo, tus nqi ntawm txoj hnyuv nqus ntawm G-Rg3 tau nce hauv tus qauv nas, thiab G-Rg3. tau nce.Hauv Caco-2 hlwb, qhov tawm ntawm Rg3 txo qis, thiab qib ntawm Rg3 concentration txo.G-Rg3 tau nce hauv cov hnyuv thiab ntshav, thiab nws lub peev xwm los tiv thaiv qog nqaij hlav kuj tau txhim kho hauv cov qauv nas ntawm B(a) P-induced tumorigenesis.Peb kuj pom tias G-Rg3 txo B(a) P-induced cytotoxicity thiab DNA adduct tsim nyob rau hauv tib neeg lub ntsws hlwb, thiab rov qab qhia thiab kev ua ub no ntawm theem II enzymes los ntawm txoj kev Nrf2, uas tej zaum yuav muaj feem xyuam rau lub peev xwm mechanism ntawm kev ua. G inhibition -Rg3..Hais txog qhov tshwm sim ntawm lub ntsws qog.Peb txoj kev tshawb fawb pom tau tias muaj lub luag haujlwm tseem ceeb rau G-Rg3 hauv kev tsom mus rau lub ntsws qog hauv cov qauv nas.Lub qhov ncauj bioavailability ntawm no ginsenoside yog txhim kho los ntawm kev tsom P-glycoprotein, tso cai rau cov molecule rau exert anticancer.
Hom mob ntsws cancer ntau tshaj plaws yog non-me me cell lung cancer (NSCLC), uas yog ib qho ua rau mob qog noj ntshav hauv Suav teb thiab North America1,2.Lub ntsiab tseem ceeb uas ua rau muaj kev pheej hmoo ntawm kev tsim cov qog nqaij hlav qog nqaij hlav me me yog kev haus luam yeeb.Cov pa luam yeeb muaj ntau dua 60 cov carcinogens, suav nrog benzo(a) pyrene (B(a)P), nitrosamines, thiab radioactive isotopes los ntawm kev lwj ntawm radon.3 Polycyclic aromatic hydrocarbons B(a)P yog lub ntsiab ua rau muaj toxicity hauv luam yeeb. haus luam yeeb.Thaum raug B(a)P, cytochrome P450 hloov nws mus rau B(a)P-7,8-dihydrodiol-9,10-epoxide (BPDE), uas reacts nrog DNA los tsim BPDE-DNA adduct 4. Tsis tas li ntawd, cov no adducts induce ntsws qog hlav hauv nas nrog qog theem thiab histopathology zoo ib yam li tib neeg lub ntsws qog 5.Cov yam ntxwv no ua rau B(a) P-induced ntsws cancer qauv ib qho tsim nyog rau kev soj ntsuam cov tebchaw uas muaj peev xwm tiv thaiv kab mob.
Ib lub tswv yim ua tau los tiv thaiv kev txhim kho mob qog noj ntshav hauv cov pab pawg muaj kev pheej hmoo siab, tshwj xeeb tshaj yog cov neeg haus luam yeeb, yog kev siv cov tshuaj tua kab mob txhawm rau txhawm rau txhawm rau txhim kho cov kab mob intraepithelial neoplastic thiab yog li tiv thaiv lawv txoj kev loj hlob mus rau malignancy.Tsiaj cov kev tshawb fawb qhia tau hais tias ntau yam chemopreventive agents ua tau zoo6.Peb daim ntawv tshaj tawm yav dhau los 7 tau qhia txog kev tiv thaiv zoo ntawm cov ginseng liab ntawm mob ntsws cancer.Cov tshuaj ntsuab no tau siv rau ntau pua xyoo hauv cov tshuaj Esxias cov tshuaj niaj hnub los ua kom lub neej ntev thiab kev noj qab haus huv, thiab tau raug sau tseg kom muaj cov nyhuv antitumor8.
Lub luag haujlwm tseem ceeb ntawm ginseng yog ginsenoside, uas yog siv los ua cov cim sib xyaw los ntsuas qhov zoo ntawm ginseng extracts.Kev txheeb xyuas ntau ntawm cov ginseng extracts feem ntau suav nrog kev siv ntau yam ginsenosides, suav nrog RK1, Rg1, F1, Re, Rb1, Rb2, Rb3, Rd, Rh1, Rh2, Rg3, Rg5, thiab Rc9,10.Ginsenosides muaj kev siv tshuaj me me vim lawv qhov ncauj tsis zoo heev11.Txawm hais tias cov txheej txheem rau qhov tsis zoo bioavailability tsis meej, qhov efflux ntawm ginsenosides tshwm sim los ntawm P-glycoprotein (P-gp)12 yuav yog qhov ua rau.P-gp yog ib qho tseem ceeb tshaj plaws efflux transporters nyob rau hauv ATP-binding cassette transporter superfamily, uas siv lub zog ntawm ATP hydrolysis tso tawm intracellular tshuaj mus rau hauv ib puag ncig sab nraud.P-gp transporters feem ntau yog dav dav hauv txoj hnyuv, lub raum, daim siab thiab ntshav-hlwb barrier13.P-gp ua lub luag haujlwm tseem ceeb hauv plab hnyuv, thiab inhibition ntawm P-gp ua rau kom qhov ncauj nqus thiab muaj qee cov tshuaj tiv thaiv kab mob 12,14.Piv txwv ntawm inhibitors yav dhau los siv hauv cov ntaub ntawv yog verapamil thiab cyclosporine A15.Qhov kev ua haujlwm no suav nrog kev tsim cov kab ke nas rau kev kawm B(a) P-induced ntsws cancer los ntsuas lub peev xwm ntawm cov ginseng liab sib txawv los ntawm Tuam Tshoj thiab Kauslim los cuam tshuam rau malignancies.Cov extracts tau raug txheeb xyuas tus kheej los txheeb xyuas cov ginsenosides tshwj xeeb uas yuav cuam tshuam rau carcinogenesis.Verapamil tau siv rau lub hom phiaj P-gp thiab txhim kho qhov ncauj bioavailability thiab kho cov txiaj ntsig ntawm kev mob qog noj ntshav-targeting ginsenosides.
Cov txheej txheem uas ginseng saponins siv tshuaj kho cov teebmeem ntawm carcinogenesis tseem tsis meej.Kev tshawb fawb tau pom tias ntau yam ginsenosides tuaj yeem txo DNA kev puas tsuaj los ntawm cov kab mob carcinogens los ntawm kev txo qis oxidative kev nyuaj siab thiab ua kom cov theem II detoxification enzymes, yog li tiv thaiv cell puas.Glutathione S-transferase (GST) yog ib theem II enzyme uas yuav tsum tau txo cov DNA puas los ntawm carcinogens17.Nuclear erythroid 2-related factor 2 (Nrf2) yog qhov tseem ceeb ntawm kev hloov pauv uas tswj hwm redox homeostasis thiab ua kom muaj kev qhia ntawm theem II enzymes thiab cytoprotective antioxidant teb.Peb txoj kev tshawb fawb kuj tau tshuaj xyuas qhov cuam tshuam ntawm kev txheeb xyuas ginsenosides ntawm kev txo B(a) P-induced cytotoxicity thiab BPDE-DNA adduct tsim, nrog rau inducing theem II enzymes los ntawm modulating Nrf2 txoj hauv kev hauv lub ntsws li qub.
Kev tsim tsa tus qauv nas ntawm B(a) P-induced cancer yog zoo ib yam nrog rau yav dhau los ua haujlwm5.Daim duab 1A qhia txog kev sim tsim ntawm 20-lub lim tiam kev kho mob ntawm tus nas mob qog noj ntshav uas tshwm sim los ntawm B(a)P, dej (tswj), Suav liab ginseng extract (CRG), Korean liab ginseng extract A (KRGA), thiab Korean liab ginseng.Extract B (KRGB) thiab Korean Red Ginseng Extract C (KRGC).Tom qab 20 lub lis piam ntawm kev kho mob ginseng liab, nas tau txi los ntawm CO2 asphyxiation.Daim duab 1B qhia txog cov qog nqaij hlav hauv lub ntsws hauv cov tsiaj kho nrog ntau hom ginseng liab, thiab daim duab 1C qhia txog lub teeb micrograph ntawm tus qauv qog.Cov qog nqaij hlav ntawm KRGB-kho tsiaj (1.5 ± 0.35) qis dua li cov tsiaj tswj (0.82 ± 0.2, P < 0.05), raws li qhia hauv daim duab 1D.Qhov nruab nrab qib ntawm qog load inhibition yog 45%.Lwm cov tshuaj ginseng liab tau kuaj tsis pom qhov hloov pauv tseem ceeb hauv cov qog nqaij hlav (P> 0.05).Tsis muaj kev phiv tshwm sim pom tseeb hauv tus qauv nas thaum 20 lub lis piam ntawm kev kho mob ginseng liab, suav nrog tsis muaj kev hloov pauv hauv lub cev hnyav (cov ntaub ntawv tsis qhia) thiab tsis muaj lub siab lossis lub raum toxicity (Daim duab 1E, F).
Red ginseng extract kho mob ntsws hlav hauv A / J nas.(A) Kev sim tsim.(B) Cov qog nqaij hlav loj hauv tus qauv nas.Cov qog yog qhia los ntawm cov xub.a: Suav liab ginseng pawg.b: pab pawg A ntawm Korean liab ginseng.c: Kauslim liab ginseng pawg B. d: Kauslim liab ginseng pawg C. d: Pab pawg tswj.(C) Lub teeb micrograph qhia lub ntsws qog.Magnification: 100. b: 400. (D) Tumor load nyob rau hauv lub liab ginseng extract pawg.(E) Plasma qib ntawm daim siab enzyme ALT.(F) Plasma qib ntawm lub raum enzyme Cr.Cov ntaub ntawv yog qhia raws li txhais tau tias ± tus qauv sib txawv.* P <0.05.
Cov tshuaj ginseng liab pom nyob rau hauv txoj kev tshawb no tau soj ntsuam los ntawm ultra-performance kua chromatography tandem huab hwm coj spectrometry (UPLC-MS/MS) kom muaj nuj nqis hauv qab no ginsenosides: Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 thiab Rg5.UPLC thiab MS cov xwm txheej siv los ntsuas cov ntsuas tau piav qhia hauv tsab ntawv tshaj tawm dhau los19.UPLC-MS/MS chromatograms ntawm plaub liab ginseng rho tawm tau pom hauv daim duab 2A.Muaj qhov sib txawv tseem ceeb ntawm tag nrho cov ntsiab lus ginsenoside, nrog cov ntsiab lus ginsenoside siab tshaj plaws hauv CRG (590.27 ± 41.28 μmol / L) (Daim duab 2B).Thaum ntsuas ib tus neeg ginsenosides (Daim duab 2C), KRGB pom qhov siab tshaj plaws ntawm G-Rg3 piv rau lwm cov ginsenosides (58.33 ± 3.81 μmol / L rau G-Rg3s thiab 41.56 ± 2.88 μmol / L rau G -Rg3r).ginseng liab hom (P <0.001).G-Rg3 tshwm sim raws li ib khub ntawm stereoisomers G-Rg3r thiab G-Rg3s, uas txawv nyob rau hauv txoj hauj lwm ntawm hydroxyl pawg ntawm carbon 20 (Fig. 2D).Cov txiaj ntsig tau qhia tias G-Rg3r lossis G-Rg3 yuav muaj peev xwm tiv thaiv kab mob tseem ceeb hauv B(a)P-induced cancer nas qauv.
Cov ntsiab lus ntawm ginsenosides nyob rau hauv ntau yam liab ginseng extracts.(A) UPLC-MS/MS chromatograms ntawm plaub liab ginseng rho tawm.(B) Kev kwv yees ntawm tag nrho cov ntsiab lus ginsenoside nyob rau hauv cov ntaub ntawv qhia.(C) Kev kuaj xyuas tus kheej ginsenosides hauv cov ntawv sau cia.(D) Cov qauv ntawm ginsenoside stereoisomers G-Rg3r thiab G-Rg3s.Cov ntaub ntawv raug qhia raws li qhov nruab nrab ± tus qauv sib txawv ntawm kev txiav txim triplicate.***P <0.001.
Txoj kev tshawb fawb UPLC-MS/MS xav kom muaj cov ginsenosides hauv cov hnyuv thiab cov ntshav kuaj tom qab 20 lub lis piam ntawm kev kho mob.Kev kho mob nrog KRGB pom tias tsuas muaj 0.0063 ± 0.0005 μg / ml Rg5 hauv cov ntshav.Tsis muaj cov tshuaj ginsenosides ntxiv lawm, qhia tias qhov ncauj tsis zoo thiab yog li txo qhov raug rau cov ginsenosides.
Cov kab mob adenocarcinoma cell kab Caco-2 yog morphologically thiab biochemically zoo ib yam li tib neeg txoj hnyuv epithelial hlwb, ua kom pom nws cov txiaj ntsig hauv kev ntsuas kev thauj mus los ntawm enterocyte hla txoj hnyuv epithelial barrier.Qhov kev ntsuam xyuas no yog los ntawm kev tshawb fawb ua ntej 20.Cov duab 3A,B,C,D,E,F qhia cov duab sawv cev ntawm kev thauj mus los ntawm G-Rg3r thiab G-Rg3 siv Caco-2 monolayer qauv.Kev thauj mus los ntawm G-Rg3r lossis G-Rg3 hla Caco-2 monolayers los ntawm basolateral mus rau sab apical (Pb-a) tau nce siab dua li ntawm apical mus rau basolateral sab (Pa-b).Rau G-Rg3r, qhov nruab nrab Pa-b tus nqi yog 0.38 ± 0.06, uas nce mus rau 0.73 ± 0.06 tom qab kho nrog 50 μmol / L verapamil thiab mus rau 1.14 ± 0.09 tom qab kho nrog 100 μmol / L verapamil (p <0.01, thiab raws li daim duab 2).3A).Kev soj ntsuam rau G-Rg3 ua raws li cov qauv zoo sib xws (Daim duab 3B), thiab cov txiaj ntsig tau pom tias kev kho verapamil txhim kho kev thauj mus los ntawm G-Rg3r thiab G-Rg3.Kev kho mob Verapamil kuj ua rau muaj qhov txo qis ntawm Pb-a thiab G-Rg3r thiab G-Rg3s efflux ratios (Daim duab 3C, D, E, F), qhia tias kev kho verapamil txo cov ntsiab lus ginsenoside hauv Caco-2 efflux hlwb..
Transcellular thauj ntawm G-Rg3 nyob rau hauv Caco-2 monolayers thiab plab hnyuv nqus nyob rau hauv ib tug nas perfusion assay.(A) Pa-b tus nqi ntawm G-Rg3r pawg hauv Caco-2 monolayer.(B) Pa-b tus nqi ntawm G-Rg3s pawg hauv Caco-2 monolayer.(C) Pb tus nqi ntawm G-Rg3r pawg hauv Caco-2 monolayer.(D) Pb tus nqi ntawm G-Rg3s pawg hauv Caco-2 monolayer.(E) Yield ratio ntawm G-Rg3r pawg hauv Caco-2 monolayer.(F) Yield ratio ntawm G-Rg3 pawg hauv Caco-2 monolayer.(G) Feem pua ntawm cov hnyuv nqus ntawm G-Rg3r hauv cov tshuaj perfusion hauv nas.(H) Feem pua ntawm cov hnyuv nqus ntawm G-Rg3 hauv cov tshuaj perfusion hauv nas.Permeability thiab nqus tau muab piv nrog tsis muaj qhov sib ntxiv ntawm verapamil.Cov ntaub ntawv raug qhia raws li qhov nruab nrab ± tus qauv sib txawv ntawm tsib qhov kev sim ywj pheej.*P <0.05, **P <0.01, ***P <0.001.
Raws li kev ua haujlwm dhau los 20, orthotopic plab hnyuv perfusion ntawm nas tau ua los txiav txim siab seb qhov nqus ntawm G-Rg3 hauv cov hnyuv nce ntxiv tom qab kev kho mob verapamil.Cov duab 3G, H qhia cov neeg sawv cev perfusion tshuaj ntsuam xyuas qhov feem pua ntawm plab hnyuv nqus ntawm G-Rg3r thiab G-Rg3 hauv cov qauv nas mob qog noj ntshav thaum lub sijhawm saud.Thawj feem pua ntawm cov tsis muaj zog G-Rg3r uptake ntawm kwv yees li 10% nce mus rau ntau tshaj 20% tom qab kev kho mob nrog 50 μM verapamil thiab ntau tshaj 25% tom qab kev kho mob nrog 100 μM verapamil.Ib yam li ntawd, G-Rg3, uas tau txais thawj zaug ntawm 10%, kuj tau pom qhov siab tshaj ntawm 20% tom qab kho nrog 50 μM verapamil thiab ze li 30% tom qab kev kho mob nrog 100 μM verapamil, qhia tias inhibition ntawm P-gp los ntawm verapamil txhim kho. plab hnyuv G-absorption Rg3 hauv tus qauv nas ntawm lub ntsws cancer.
Raws li cov txheej txheem saum toj no, B(a) P-induced cancer qauv nas tau muab faib ua rau pawg, raws li qhia hauv daim duab 4A.Tsis muaj qhov hnyav hnyav lossis cov tsos mob tshwm sim ntawm toxicity tau pom nyob rau hauv pawg kho mob G-Rg3 piv rau pawg tswj hwm (cov ntaub ntawv tsis qhia).Tom qab 20 lub lis piam ntawm kev kho mob, lub ntsws ntawm txhua tus nas tau sau.Daim duab 4B qhia txog cov qog nqaij hlav hauv lub ntsws hauv cov nas hauv cov pab pawg kho mob saum toj no, thiab daim duab 4C qhia pom lub teeb micrograph ntawm tus neeg sawv cev qog.Hais txog cov qog nqaij hlav hauv txhua pab pawg (Fig. 4D), qhov tseem ceeb rau cov nas kho nrog G-Rg3r thiab G-Rg3s yog 0.75 ± 0.29 mm3 thiab 0.81 ± 0.30 mm3, raws li, thaum qhov tseem ceeb rau G Mice kho. nrog -Rg3s yog 1.63 feem ± 0.40 mm3.tswj cov nas (p <0.001), qhia tias kev kho G-Rg3 txo cov qog nqaij hlav hauv cov nas.Kev tswj hwm ntawm verapamil ntxiv txhim kho qhov kev txo qis no: qhov tseem ceeb hauv verapamil + G-Rg3r nas txo los ntawm 0.75 ± 0.29 mm3 txog 0.33 ± 0.25 mm3 (p < 0.01), thiab qhov tseem ceeb rau verapamil + ntawm 0.81 ± 0.29 mm3 txo. mm3 hauv G. -Rg3s-kho nas (p <0.05), qhia tias verapamil tuaj yeem txhim kho cov nyhuv inhibitory ntawm G-Rg3 ntawm cov qog nqaij hlav.Cov qog nqaij hlav tsis muaj qhov sib txawv tseem ceeb ntawm pawg tswj hwm thiab pawg verapamil, pawg G-Rg3r thiab pawg G-Rg3s, thiab pawg verapamil + G-Rg3r thiab pawg verapamil + G-Rg3s.Ntxiv mus, tsis muaj lub siab tseem ceeb lossis lub raum toxicity cuam tshuam nrog kev soj ntsuam kev kho mob (Daim duab 4E, F).
Cov qog nqaij hlav tom qab kho G-Rg3 thiab ntshav ntshav lossis plab hnyuv G-Rg3r thiab G-Rg3 qib hauv pawg qhia.(A) Kev sim tsim.(B) Macroscopic qog nyob rau hauv tus qauv nas.Cov qog yog qhia los ntawm cov xub.ib: g3r.b:g 3s.c: G-Rg3r ua ke nrog verapamil.d: G-Rg3 ua ke nrog verapamil.d: Verapamil ua.e: tswj.(C) Optical micrograph ntawm cov qog ntawm magnification.Teb: 100x.ib: 400x.(D) Cov nyhuv ntawm G-Rg3 + verapamil kev kho mob ntawm cov qog nqaij hlav hauv A / J nas.(E) Plasma qib ntawm daim siab enzyme ALT.(F) Plasma qib ntawm lub raum enzyme Cr.(G) Plasma qib ntawm G-Rg3r lossis G-Rg3 ntawm pawg qhia.(H) Qib G-Rg3r lossis G-Rg3s hauv cov hnyuv ntawm pawg qhia.Cov ntaub ntawv raug qhia raws li qhov nruab nrab ± tus qauv sib txawv ntawm kev txiav txim triplicate.*P <0.05, **P <0.01, ***P <0.001.
G-Rg3 qib hauv B(a)P-induced cancer qauv nas raug soj ntsuam los ntawm UPLC-MS / MS tom qab 20-lub lim tiam kev kho mob raws li txoj kev tau piav qhia hauv Tshooj Lus.Cov duab 4G thiab H qhia plasma thiab plab hnyuv G-Rg3 qib, feem.Plasma G-Rg3r qib yog 0.44 ± 0.32 μmol / L thiab nce mus rau 1.17 ± 0.47 μmol / L nrog kev tswj hwm ntawm verapamil (p <0.001), thaum lub plab hnyuv G-Rg3r qib yog 0.53 ± 0.08 µg / l.Thaum ua ke nrog verapamil, g nce mus rau 1.35 ± 0.13 μg / g (p <0.001).Rau G-Rg3, cov txiaj ntsig tau ua raws li tus qauv zoo sib xws, qhia tias kev kho mob verapamil nce qhov ncauj bioavailability ntawm G-Rg3 hauv A / J nas.
Cell viability assay tau siv los ntsuas cytotoxicity ntawm B(a)P thiab G-Rg3 ntawm hEL hlwb.Lub cytotoxicity induced los ntawm B(a)P nyob rau hauv hEL hlwb yog qhia nyob rau hauv daim duab 5A, thaum lub nontoxic zog ntawm G-Rg3r thiab G-Rg3 yog qhia nyob rau hauv daim duab 5A thiab 5B.5B, C. Txhawm rau ntsuas qhov cytoprotective nyhuv ntawm G-Rg3, B(a)P tau koom ua ke nrog ntau qhov ntau ntawm G-Rg3r lossis G-Rg3 rau hauv hEL hlwb.Raws li pom nyob rau hauv daim duab 5D, G-Rg3r ntawm concentrations ntawm 5 μM, 10 μM, thiab 20 μM rov qab cell viability mus rau 58.3%, 79.3%, thiab 77.3%, feem.Cov txiaj ntsig zoo sib xws kuj tuaj yeem pom hauv pawg G-Rg3s.Thaum cov concentrations ntawm G-Rg3s yog 5 µM, 10 µM thiab 20 µM, cell viability tau rov qab los rau 58.3%, 72.7% thiab 76.7%, feem (Daim duab 5E).).Lub xub ntiag ntawm BPDE-DNA adducts tau ntsuas siv cov khoom siv ELISA.Peb cov txiaj ntsig tau pom tias qib BPDE-DNA adduct tau nce hauv B(a) P-kho pab pawg piv nrog pawg tswj hwm, tab sis piv nrog G-Rg3 co-kho, BPDE-DNA adduct qib hauv pawg B(a)P B hauv pab pawg kho mob, DNA adduct theem tau txo qis.Cov txiaj ntsig ntawm kev kho mob nrog B(a)P ib leeg yog pom hauv daim duab 5F (1.87 ± 0.33 vs. 3.77 ± 0.42 rau G-Rg3r, 1.93 ± 0.48 vs. 3.77 ± 0.42 rau G -Rg3s, p <0.001).
Cell viability thiab BPDE-DNA adduct tsim nyob rau hauv hEL hlwb kho nrog G-Rg3 thiab B(a)P.(A) Viability ntawm hEL hlwb kho nrog B(a)P.(B) Viability ntawm hEL hlwb kho nrog G-Rg3r.(C) Viability ntawm hEL hlwb kho nrog G-Rg3.(D) Viability ntawm hEL hlwb kho nrog B(a)P thiab G-Rg3r.(E) Viability ntawm hEL hlwb kho nrog B(a)P thiab G-Rg3.(F) Qib ntawm BPDE-DNA adduct hauv hEL hlwb kho nrog B(a)P thiab G-Rg3.Cov ntaub ntawv raug qhia raws li qhov nruab nrab ± tus qauv sib txawv ntawm kev txiav txim triplicate.*P <0.05, **P <0.01, ***P <0.001.
GST enzyme qhia tau kuaj pom tom qab kev kho mob nrog 10 μM B(a)P thiab 10 μM G-Rg3r lossis G-Rg3s.Peb cov txiaj ntsig tau pom tias B(a)P txwv tsis pub GST qhia (59.7 ± 8.2% hauv pawg G-Rg3r thiab 39 ± 4.5% hauv pawg G-Rg3s), thiab B(a)P tau cuam tshuam nrog G-Rg3r. , lossis nrog G-Rg3r, lossis nrog G-Rg3r.Kev kho mob nrog G-Rg3s rov qab GST qhia.GST qhia (103.7 ± 15.5% hauv pawg G-Rg3r thiab 110 ± 11.1% hauv pawg G-Rg3s, p < 0.05 thiab p < 0.001, feem, Fig. 6A, B, thiab C).GST kev ua ub no raug soj ntsuam los ntawm kev siv cov khoom ntsuas kev ua ub no.Peb cov txiaj ntsig tau pom tias pab pawg kho mob sib xyaw ua ke muaj cov haujlwm GST ntau dua piv rau B(a) P tsuas yog pawg (96.3 ± 6.6% vs. 35.7 ± 7.8% hauv pawg G-Rg3r vs. 92.3 ± 6.5 hauv pawg G-Rg3r ).% vs 35.7 ± 7.8% hauv pawg G-Rg3s, p < 0.001, Daim duab 6D).
Kev nthuav qhia ntawm GST thiab Nrf2 hauv hEL hlwb kho nrog B(a)P thiab G-Rg3.(A) Tshawb nrhiav GST qhia los ntawm Western blotting.(B) Quantitative nthuav qhia ntawm GST hauv hEL hlwb kho nrog B(a)P thiab G-Rg3r.(C) Quantitative nthuav qhia ntawm GST hauv hEL hlwb kho nrog B(a)P thiab G-Rg3s.(D) GST kev ua hauv hEL hlwb kho nrog B(a)P thiab G-Rg3.(E) Kev Tshawb Nrhiav Nrf2 qhia los ntawm Western blotting.(F) Quantitative nthuav qhia ntawm Nrf2 hauv hEL hlwb kho nrog B(a)P thiab G-Rg3r.(G) Kev qhia ntau ntawm Nrf2 hauv hEL hlwb kho nrog B(a)P thiab G-Rg3s.Cov ntaub ntawv raug qhia raws li qhov nruab nrab ± tus qauv sib txawv ntawm kev txiav txim triplicate.*P <0.05, **P <0.01, ***P <0.001.
Txhawm rau nthuav tawm txoj hauv kev koom nrog G-Rg3-kho kev sib kho ntawm B(a) P-induced tumorigenesis, Nrf2 qhia tau raug soj ntsuam los ntawm Western blotting.Raws li pom hauv daim duab 6E,F,G, piv nrog rau pawg tswj hwm, tsuas yog theem ntawm Nrf2 hauv pawg B(a)P kho tau txo qis;Txawm li cas los xij, piv nrog pawg B(a)P kho mob, B(a) Nrf2 qib hauv pawg PG-Rg3 tau nce (106 ± 9.5% rau G-Rg3r vs. 51.3 ± 6.8%, 117 ± 6. 2% rau G-Rg3r vs. 41 ± 9.8% rau G-Rg3s, p < 0.01).
Peb tau lees paub lub luag haujlwm tiv thaiv ntawm Nrf2 los ntawm kev txwv Nrf2 kev qhia siv tshwj xeeb cuam tshuam me me RNA (siRNA).Nrf2 knockdown tau lees paub los ntawm Western blotting (Fig. 7A,B).Raws li pom nyob rau hauv daim duab 7C,D, kev sib koom ua ke ntawm hEL hlwb nrog B(a)P thiab G-Rg3 ua rau txo qis ntawm BPDE-DNA adducts (1.47 ± 0.21) piv rau kev kho nrog B(a)P ib leeg hauv pawg tswj siRNA.) G-Rg3r yog 4.13 ± 0.49, G-Rg3s yog 1.8 ± 0.32 thiab 4.1 ± 0.57, p < 0.01).Txawm li cas los xij, cov nyhuv inhibitory ntawm G-Rg3 ntawm BPDE-DNA tsim tau raug tshem tawm los ntawm Nrf2 knockdown.Hauv pab pawg siNrf2, tsis muaj qhov sib txawv tseem ceeb hauv BPDE-DNA adduct tsim ntawm B(a)P thiab G-Rg3 co-treatment thiab B(a)P kev kho mob ib leeg (3.0 ± 0.21 rau G-Rg3r vs. 3.56 ± 0.32 ).rau G-Rg3r piv rau 3.6 rau G-Rg3s piv rau ± 0.45 piv rau 4.0 ± 0.37, p > 0.05).
Qhov cuam tshuam ntawm Nrf2 knockdown ntawm BPDE-DNA adduct tsim hauv hEL hlwb.(A) Nrf2 knockdown tau lees paub los ntawm Western blotting.(B) Quantification ntawm Nrf2 band siv.(C) Qhov cuam tshuam ntawm Nrf2 knockdown ntawm BPDE-DNA adduct theem hauv hEL hlwb kho nrog B(a)P thiab G-Rg3r.(D) Qhov cuam tshuam ntawm Nrf2 knockdown ntawm BPDE-DNA adduct theem hauv hEL hlwb kho nrog B(a)P thiab G-Rg3.Cov ntaub ntawv raug qhia raws li qhov nruab nrab ± tus qauv sib txawv ntawm kev txiav txim triplicate.*P <0.05, **P <0.01, ***P <0.001.
Txoj kev tshawb no tau soj ntsuam cov kev tiv thaiv ntawm ntau yam liab ginseng rho tawm ntawm tus nas qauv ntawm B(a) P-induced ntsws cancer, thiab KRGB kev kho mob txo cov qog nqaij hlav.Xav tias G-Rg3 muaj cov ntsiab lus siab tshaj plaws hauv cov ginseng extract, lub luag haujlwm tseem ceeb ntawm no ginsenoside hauv inhibiting qog nqaij hlav tau kawm.Ob leeg G-Rg3r thiab G-Rg3 (ob lub epimers ntawm G-Rg3) txo cov qog nqaij hlav hauv tus nas qauv ntawm B(a) P-induced cancer.G-Rg3r thiab G-Rg3 siv cov tshuaj tiv thaiv kab mob los ntawm inducing apoptosis ntawm cov qog hlwb21, inhibiting qog loj hlob22, ntes lub cell cycle23 thiab cuam tshuam rau angiogenesis24.G-Rg3 kuj tau pom tias inhibit cellular metastasis25, thiab lub peev xwm ntawm G-Rg3 los txhim kho cov teebmeem ntawm kev kho mob thiab xov tooj cua tau sau tseg 26,27.Poon et al tau qhia tias kev kho G-Rg3 tuaj yeem txo cov teebmeem genotoxic ntawm B(a)P28.Txoj kev tshawb no qhia txog kev kho lub peev xwm ntawm G-Rg3 hauv kev tsom mus rau ib puag ncig carcinogenic molecules thiab tiv thaiv qog noj ntshav.
Txawm hais tias lawv muaj peev xwm prophylactic zoo, qhov tsis zoo ntawm qhov ncauj bioavailability ntawm ginsenosides ua rau muaj kev sib tw rau kev siv cov tshuaj molecules.Pharmacokinetic tsom xam ntawm qhov ncauj kev tswj hwm ntawm ginsenosides hauv nas pom tias nws cov bioavailability tseem tsawg dua 5% 29.Cov kev ntsuam xyuas no tau pom tias tom qab lub sijhawm kho mob 20 lub lis piam, tsuas yog cov ntshav ntawm Rg5 txo qis.Txawm hais tias lub hauv paus txheej txheem ntawm bioavailability tsis zoo tseem yuav tsum tau piav qhia, P-gp tau xav tias yuav koom nrog hauv kev tawm ntawm ginsenosides.Qhov kev ua haujlwm no tau pom thawj zaug tias kev tswj hwm ntawm verapamil, P-gp blocker, nce qhov ncauj bioavailability ntawm G-Rg3r thiab G-Rg3s.Yog li, qhov kev tshawb pom no qhia tias G-Rg3r thiab G-Rg3s ua raws li cov substrates ntawm P-gp los tswj nws cov efflux.
Qhov kev ua haujlwm no qhia tau tias kev kho mob ua ke nrog verapamil nce qhov ncauj bioavailability ntawm G-Rg3 hauv tus qauv nas ntawm lub ntsws cancer.Qhov kev tshawb pom no tau txais kev txhawb nqa los ntawm kev nce hauv plab hnyuv transcellular thauj ntawm G-Rg3 raws li P-gp blockade, yog li nce nws qhov nqus.Kev soj ntsuam hauv Caco2 hlwb tau pom tias kev kho mob verapamil txo cov efflux ntawm G-Rg3r thiab G-Rg3s thaum txhim kho daim nyias nyias permeability.Kev kawm los ntawm Yang et al.Cov kev tshawb fawb tau pom tias kev kho mob nrog cyclosporine A (lwm tus P-gp blocker) ua rau kom muaj bioavailability ntawm ginsenoside Rh2 los ntawm tus nqi pib ntawm 1% 20 mus rau ntau dua 30%.Ginsenosides compounds K thiab Rg1 kuj tau pom zoo sib xws 30,31.Thaum verapamil thiab cyclosporin A tau koom ua ke, cov efflux ntawm compound K hauv Caco-2 hlwb tau txo qis los ntawm 26.6 mus rau tsawg dua 3, thaum nws cov qib intracellular nce 40-fold30.Nyob rau hauv lub xub ntiag ntawm verapamil, Rg1 qib nce nyob rau hauv nas ntsws epithelial hlwb, qhia lub luag hauj lwm rau P-gp nyob rau hauv ginsenoside efflux, raws li qhia los ntawm Meng li al.31.Txawm li cas los xij, verapamil tsis muaj qhov cuam tshuam zoo ib yam ntawm efflux ntawm qee cov ginsenosides (xws li Rg1, F1, Rh1 thiab Re), qhia tias lawv tsis cuam tshuam los ntawm P-gp substrates, raws li qhia los ntawm Liang li al.32.Qhov kev soj ntsuam no yuav muaj feem xyuam nrog kev koom tes ntawm lwm tus neeg thauj khoom thiab lwm cov qauv ginsenoside.
Cov txheej txheem ntawm kev tiv thaiv cov nyhuv ntawm G-Rg3 ntawm kev mob qog noj ntshav tsis meej.Cov kev tshawb fawb yav dhau los tau pom tias G-Rg3 tiv thaiv DNA kev puas tsuaj thiab apoptosis los ntawm kev txo qis oxidative kev nyuaj siab thiab o 16,33, uas tej zaum yuav yog lub hauv paus txheej txheem los tiv thaiv B(a) P-induced tumorigenesis.Qee cov ntawv ceeb toom qhia tias genotoxicity tshwm sim los ntawm B(a)P tuaj yeem txo qis los ntawm kev hloov pauv theem II enzymes los ua BPDE-DNA34.GST yog theem II enzyme uas inhibits BPDE-DNA adduct tsim los ntawm kev txhawb nqa kev khi ntawm GSH rau BPDE, yog li txo cov DNA puas tsuaj los ntawm B(a) P35.Peb cov txiaj ntsig tau pom tias kev kho G-Rg3 txo B(a) P-induced cytotoxicity thiab BPDE-DNA adduct tsim nyob rau hauv hEL hlwb thiab rov kho GST qhia thiab kev ua hauv vitro.Txawm li cas los xij, cov teebmeem no tsis tuaj yeem tshwm sim thaum tsis muaj Nrf2, qhia tias G-Rg3 induces cytoprotective teebmeem los ntawm txoj kev Nrf2.Nrf2 yog qhov tseem ceeb tshaj plaws rau theem II detoxification enzymes uas txhawb nqa kev tshem tawm ntawm xenobiotics36.Ua kom txoj hauv kev Nrf2 induces cytoprotection thiab txo cov ntaub so ntswg puas tsuaj37.Ntxiv mus, ntau cov ntawv ceeb toom tau txhawb nqa lub luag haujlwm ntawm Nrf2 ua cov qog nqaij hlav hauv carcinogenesis38.Peb txoj kev tshawb fawb qhia tau hais tias induction ntawm Nrf2 txoj hauv kev los ntawm G-Rg3 plays lub luag haujlwm tseem ceeb hauv B(a) P-induced genotoxicity los ntawm kev ua rau B(a)P detoxification los ntawm activating theem II enzymes, yog li inhibiting cov txheej txheem tumorigenesis.
Peb txoj haujlwm qhia txog lub peev xwm ntawm ginseng liab hauv kev tiv thaiv B(a) P-induced ntsws cancer hauv nas los ntawm kev koom tes tseem ceeb ntawm ginsenoside G-Rg3.Qhov tsis zoo ntawm qhov ncauj bioavailability ntawm cov molecule hampers nws daim ntawv thov kev kho mob.Txawm li cas los xij, qhov kev tshawb fawb no qhia thawj zaug tias G-Rg3 yog ib qho substrate ntawm P-gp, thiab kev tswj hwm ntawm P-gp inhibitor ua rau kom muaj bioavailability ntawm G-Rg3 hauv vitro thiab hauv vivo.G-Rg3 txo B(a)P-induced cytotoxicity los ntawm kev tswj txoj hauv kev Nrf2, uas tej zaum yuav yog lub peev xwm rau nws txoj haujlwm tiv thaiv.Peb txoj kev tshawb fawb tau lees paub lub peev xwm ntawm ginsenoside G-Rg3 rau kev tiv thaiv thiab kho mob qog noj ntshav.
Rau 6-lub lim tiam-laus poj niam A/J nas (20 ± 1 g) thiab 7-lub lim tiam-txiv neej Wistar nas (250 ± 20 g) tau txais los ntawm Jackson Laboratory (Bar Harbor, USA) thiab Wuhan Institute of Zoology.University (Wuhan, Suav).Tuam Tshoj Hom Kab lis kev cai Collection Center (Wuhan, Tuam Tshoj) muab peb cov Caco-2 thiab hEL hlwb.Sigma-Aldrich (St. Louis, USA) yog ib qho ntawm B(a)P thiab tricaprine.Purified ginsenosides G-Rg3r thiab G-Rg3s, dimethyl sulfoxide (DMSO), CellTiter-96 proliferation assay kit (MTS), verapamil, tsawg yam tseem ceeb nruab nrab (MEM), thiab fetal bovine serum (FBS) tau yuav los ntawm Chengdu Yuav Tsum Bio-Technology .Co., Ltd.(Chengdu, Suav teb).Cov khoom siv QIAamp DNA mini thiab BPDE-DNA adduct ELISA cov khoom siv tau yuav los ntawm Qiagen (Stanford, CA, USA) thiab Cell Biolabs (San Diego, CA, USA).GST cov khoom siv tshuaj ntsuam xyuas thiab tag nrho cov khoom siv tshuaj ntsuam xyuas protein (tus qauv BCA) tau yuav los ntawm Solarbio (Beijing, Suav).Tag nrho cov ginseng liab extracts yog khaws cia nyob rau hauv Mingyu Laboratory 7. Hong Kong Baptist University (Hong Kong, Tuam Tshoj) thiab Kauslim Cancer Center (Seoul, Kauslim) yog coj mus muag qhov chaw ntawm CRG extract thiab ntau yam liab ginseng extracts ntawm ntau yam Korean keeb kwm (xws li KRGA, KRGB. thiab KRGC).Liab ginseng yog tsim los ntawm cov hauv paus hniav ntawm 6-xyoo-laus tshiab ginseng.Liab ginseng extract yog tau los ntawm kev ntxuav ginseng nrog dej peb zaug, ces tsom rau cov aqueous extract, thiab thaum kawg ziab ntawm qhov kub thiab txias kom tau ginseng extract hmoov.Antibodies (anti-Nrf2, anti-GST, thiab β-actin), horseradish peroxidase-conjugated anti-luv immunoglobulin G (IgG), transfection reagent, tswj siRNA, thiab Nrf2 siRNA tau muas los ntawm Santa Cruz Biotechnology (Santa Cruz, CA) .), USA).
Caco2 thiab hEL hlwb raug coj mus rau hauv 100 mm2 cell kab lis kev cai tais diav nrog MEM muaj 10% FBS ntawm 37 ° C nyob rau hauv ib qho chaw humidified ntawm 5% CO2.Txhawm rau txiav txim siab cov txiaj ntsig ntawm kev kho mob, cov hlwb hEL tau tsim nrog qhov sib txawv ntawm B(a)P thiab G-Rg3 hauv MEM rau 48 teev.Cells tuaj yeem txheeb xyuas ntxiv lossis sau los npaj cov cell-free extracts.
Txhua qhov kev sim tau pom zoo los ntawm Pawg Neeg Saib Xyuas Tsiaj Cuam Tshuam ntawm Tongji Medical College, Huazhong University of Science thiab Technology (Kev Pom Zoo No. 2019; Kev Sau Npe No. 4587TH).Txhua qhov kev sim tau ua tiav raws li cov lus qhia thiab cov kev cai, thiab txoj kev tshawb fawb tau ua raws li Kev Tshawb Fawb Tsiaj: Kev Tshaj Tawm ntawm In Vivo Experiments (ARRIVE) cov lus qhia.Yim-lub lim tiam A / J nas tau txhaj tshuaj B(a)P hauv cov tshuaj tricaprine (100 mg / kg, 0.2 ml).Tom qab ib lub lim tiam, cov nas tau muab faib ua pawg tswj hwm thiab pab pawg kho mob sib txawv, 15 nas tsuag hauv txhua pab pawg, thiab muab faib ua ib hnub.Tom qab 20 lub lis piam ntawm kev kho mob, tsiaj txhu tau txi los ntawm CO2 asphyxia.Lub ntsws tau sau thiab kho rau 24 teev.Tus naj npawb ntawm cov qog nqaij hlav thiab cov qog nqaij hlav ib leeg tau ntsuas rau txhua lub ntsws nyob rau hauv lub tshuab kuaj kab mob.Cov qog ntim kwv yees (V) tau suav nrog cov lus qhia hauv qab no: V (mm3) = 4/3πr3, qhov twg r yog lub qog txoj kab uas hla.Cov txiaj ntsig ntawm tag nrho cov qog nqaij hlav hauv lub ntsws ntawm cov nas sawv cev rau tag nrho cov qog nqaij hlav, thiab qhov nruab nrab tag nrho cov qog nqaij hlav hauv txhua pab pawg sawv cev rau cov qog nqaij hlav.Tag nrho cov ntshav thiab cov hnyuv tau sau thiab khaws cia ntawm -80 ° C rau kev txiav txim siab UPLC-MS/MS.Cov ntshav tau sau tseg thiab cov khoom siv hluav taws xob tsis siv neeg tshuaj ntsuam xyuas tau siv los tshuaj xyuas alanine aminotransferase (ALT) thiab ntshav creatinine (Cr) qib los ntsuas lub siab thiab lub raum ua haujlwm.
Cov qauv sau tau raug tshem tawm ntawm qhov chaw txias, thawed, hnyav, thiab muab tso rau hauv cov raj raws li tau piav qhia saum toj no.Rau qhov no tau ntxiv 0.5 μM phlorizin (tus qauv sab hauv) hauv 0.8 ml methanol tov.Tom qab ntawd cov ntaub so ntswg yog homogenized siv Tissue-Tearor thiab homogenate tom qab tau xa mus rau 1.5 ml microcentrifuge raj.Qhov sib tov yog centrifuged ntawm 15500 rpm rau 15 feeb.Tom qab tshem tawm 1.0 ml ntawm supernatant, qhuav nrog nitrogen.Ob puas microliters ntawm methanol tau siv rau kev rov qab los.Cov ntshav tau sau thiab ua tiav ntawm ib kab thiab siv los ua ib qho kev siv rau txhua qhov kev ntsuas.
24-zoo Transwell daim hlau tau cog nrog 1.0 × 105 Caco-2 hlwb ntawm ib qhov dej los ntsuas qhov muaj peev xwm txhim kho ntawm G-Rg3 thauj los ntawm kev ntxiv ntawm verapamil.Tom qab 3 lub lis piam ntawm kab lis kev cai, cov hlwb raug ntxuav nrog HBSS thiab preincubated ntawm 37 ° C.400 μL ntawm 10 μM G-Rg3 (G-Rg3r, G-Rg3s, lossis sib xyaw nrog 50 lossis 100 μM verapamil) tau txhaj rau ntawm basolateral lossis apical sab ntawm monolayer, thiab 600 μL ntawm HBSS tov tau ntxiv rau lwm qhov. sab.Sau 100 µl ntawm kab lis kev cai nruab nrab ntawm lub sijhawm (0, 15, 30, 45, 60, 90 thiab 120 feeb) thiab ntxiv 100 µl ntawm HBSS los tsim cov ntim no.Cov qauv raug khaws cia ntawm -4 ° C kom txog thaum kuaj pom los ntawm UPLC-MS / MS.Cov kev qhia Papp = dQ / (dT × A × C0) yog siv los ntsuas qhov pom tseeb unidirectional apical thiab basolateral permeability thiab vice versa (Pa-b thiab Pb-a, feem);dQ / dT yog qhov kev hloov pauv hauv concentration, A (0.6 cm2) yog qhov chaw ntawm lub monolayer, thiab C0 yog qhov pib pub dawb concentration.Tus efflux piv yog xam raws li Pb-a/Pa-b, uas sawv cev rau tus nqi eflux ntawm cov tshuaj kawm.
Txiv neej Wistar nas tau yoo mov rau 24 teev, haus dej xwb, thiab tshuaj loog nrog kev txhaj tshuaj ntawm 3.5% pentobarbital daws.Intubated silicone tube muaj qhov kawg ntawm duodenum raws li qhov nkag thiab qhov kawg ntawm ileum raws li qhov tawm.Siv lub twj tso kua mis peristaltic los tso cov dej nkag nrog 10 µM G-Rg3r lossis G-Rg3s hauv isotonic HBSS ntawm tus nqi ntws ntawm 0.1 ml / min.Cov nyhuv ntawm verapamil tau soj ntsuam los ntawm kev ntxiv 50 μM lossis 100 μM ntawm cov tshuaj sib xyaw rau 10 μM G-Rg3r lossis G-Rg3s.UPLC-MS / MS tau ua tiav ntawm perfusion rho tawm ntawm lub sijhawm cov ntsiab lus 60, 90, 120, thiab 150 feeb tom qab pib perfusion.Qhov feem pua ntawm kev nqus yog ntsuas los ntawm cov mis % absorption = (1 – Cout/Cin) × 100%;Qhov concentration ntawm G-Rg3 ntawm qhov hluav taws xob thiab qhov hluav taws xob yog qhia los ntawm Cout thiab Cin, feem.
hEL hlwb tau muab noob rau hauv 96-zoo daim hlau ntawm qhov ntom ntawm 1 × 104 hlwb ntawm ib qhov dej thiab kho nrog B(a)P (0, 1, 5, 10, 20, 30, 40 μM) lossis G-Rg3 yaj hauv DMSO .Tom qab ntawd cov tshuaj tau diluted nrog kab lis kev cai nruab nrab mus rau ntau qhov ntau (0, 1, 2, 5, 10, 20 μM) dhau 48 teev.Siv cov khoom siv tshuaj ntsuam xyuas MTS uas muaj nyob hauv kev lag luam, cov hlwb tau raug tus qauv raws tu qauv thiab tom qab ntawd ntsuas siv tus nyeem ntawv microplate ntawm 490 nm.Lub cell viability theem ntawm cov pab pawg sib koom ua ke nrog B(a) P (10 μM) thiab G-Rg3 (0, 1, 5, 10, 20 μM) raug soj ntsuam raws li cov txheej txheem saum toj no thiab muab piv nrog cov pab pawg tsis kho.
hEL hlwb tau muab noob rau hauv 6-zoo daim hlau ntawm qhov ceev ntawm 1 × 105 hlwb / zoo thiab kho nrog 10 μMB(a)P nyob rau hauv lub xub ntiag los yog tsis muaj 10 μM G-Rg3.Tom qab 48 teev ntawm kev kho mob, DNA tau muab rho tawm los ntawm hEL hlwb siv QIAamp DNA Mini Kit raws li cov chaw tsim khoom raws tu qauv.Kev tsim ntawm BPDE-DNA adducts tau kuaj pom siv BPDE-DNA adduct ELISA cov khoom siv.Cov txheeb ze qib ntawm BPDE-DNA adduct raug ntsuas los ntawm kev nyeem ntawv microplate los ntawm kev ntsuas qhov nqus ntawm 450 nm.
hEL hlwb tau muab noob rau hauv 96-zoo daim hlau ntawm qhov ntom ntawm 1 × 104 hlwb ntawm ib qhov dej thiab kho nrog 10 μMB(a)P thaum tsis muaj lossis muaj 10 μM G-Rg3 rau 48 h.GST kev ua haujlwm tau ntsuas los ntawm kev lag luam GST cov khoom siv tshuaj ntsuam xyuas raws li tus neeg tsim khoom raws tu qauv.Tus txheeb ze GST ua kom raug ntsuas los ntawm kev nqus ntawm 450 nm siv tus nyeem ntawv microplate.
HEL hlwb raug ntxuav nrog dej khov-txias PBS thiab tom qab ntawd lysed siv radioimmunoprecipitation assay tsis muaj protease inhibitors thiab phosphatase inhibitors.Tom qab cov protein kom muaj nuj nqis siv tag nrho cov khoom siv tshuaj ntsuam xyuas protein, 30 μg ntawm cov protein hauv txhua tus qauv raug cais los ntawm 12% SDS-PAGE thiab xa mus rau PVDF membrane los ntawm electrophoresis.Membranes raug thaiv nrog 5% skim mis nyuj thiab incubated nrog thawj cov tshuaj tiv thaiv thaum hmo ntuj ntawm 4 ° C.Tom qab incubation nrog horseradish peroxidase-conjugated thib ob cov tshuaj tiv thaiv, txhim kho chemiluminescence reagents tau ntxiv kom pom cov teeb liab khi.Qhov kev siv ntawm txhua pawg protein tau suav nrog siv ImageJ software.
GraphPad Prism 7.0 software tau siv los txheeb xyuas tag nrho cov ntaub ntawv, qhia tias txhais tau tias ± tus qauv sib txawv.Kev sib txawv ntawm pab pawg kho mob tau raug soj ntsuam los ntawm Kev Ntsuas Tub Ntxhais Kawm t lossis kev tshuaj xyuas ib leeg ntawm qhov sib txawv, nrog tus nqi P <0.05 qhia qhov tseem ceeb.
Tag nrho cov ntaub ntawv tau los yog tshuaj xyuas thaum lub sijhawm kawm no suav nrog hauv tsab xov xwm no thiab cov ntaub ntawv qhia ntxiv.
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Post lub sij hawm: Sep-17-2023