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Ginseng yofiira yakhala ikugwiritsidwa ntchito mu mankhwala azikhalidwe aku Asia kwazaka mazana ambiri.Mu kafukufukuyu, tidawunika kuthekera kwa mitundu inayi ya ginseng yofiyira (ginseng yofiira yaku China, ginseng yofiira yaku Korea A, ginseng yofiira yaku Korea B, ndi ginseng yofiira yaku Korea C) yokulira m'magawo osiyanasiyana kuti aletse mapangidwe ndi kukula kwa mapapo opangidwa ndi carcinogen. zotupa.Kuyezetsa kwa benzo (a) pyrene (B (a) P) kunachitika pa mbewa za A/J, ndipo ginseng yofiira ya ku Korea B inapezeka kuti ndi yothandiza kwambiri pochepetsa chotupa pakati pa mitundu inayi yofiira ya ginseng.Kuphatikiza apo, tidasanthula zomwe zili m'ma ginsenosides osiyanasiyana (Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 ndi Rg5) m'zigawo zinayi zofiira za ginseng ndikupeza kuti ginseng yofiira yaku Korea inali ndi Magulu apamwamba kwambiri a ginsenoside Rg3 (G-Rg3), kutanthauza kuti G-Rg3 ikhoza kukhala ndi gawo lofunikira pakuchiritsa kwake.Ntchitoyi ikuwonetsa kuti G-Rg3 ili ndi bioavailability yochepa.Komabe, G-Rg3 itagwiritsidwa ntchito limodzi ndi P-gp inhibitor verapamil, kutuluka kwa G-Rg3 m'maselo a Caco-2 kunachepa, kuchuluka kwa mayamwidwe a G-Rg3 m'matumbo kunawonjezeka ngati makoswe, ndipo G-Rg3. chinawonjezeka.M'maselo a Caco-2, kutuluka kwa Rg3 kumachepa, ndipo mlingo wa Rg3 umachepa.G-Rg3 imachulukitsidwa m'matumbo ndi m'madzi a m'magazi, ndipo mphamvu yake yoletsa zotupa imalimbikitsidwanso mu chitsanzo cha makoswe a B (a) P-induced tumorigenesis.Tinapezanso kuti G-Rg3 inachepetsa B (a) P-induced cytotoxicity ndi DNA adduct mapangidwe m'maselo a m'mapapo aumunthu, ndikubwezeretsanso mawu ndi ntchito za ma enzymes a gawo lachiwiri kudzera mu njira ya Nrf2, yomwe ingakhale yokhudzana ndi njira yogwiritsira ntchito. Kuletsa kwa G -Rg3..Za kupezeka kwa zotupa m'mapapo.Kafukufuku wathu akuwonetsa gawo lofunikira la G-Rg3 pakulondolera zotupa zam'mapapo mumitundu ya mbewa.Oral bioavailability ya ginsenoside iyi imakulitsidwa poyang'ana P-glycoprotein, kulola molekyu kukhala ndi zotsatira za anticancer.
Mtundu wofala kwambiri wa khansa ya m'mapapo ndi khansa ya m'mapapo yosakhala yaying'ono (NSCLC), yomwe ndi imodzi mwazomwe zimayambitsa kufa kwa khansa ku China ndi North America1,2.Chinthu chachikulu chomwe chimawonjezera chiopsezo chokhala ndi khansa ya m'mapapo yosakhala yaying'ono ndiyo kusuta.Utsi wa ndudu uli ndi ma carcinogens opitilira 60, kuphatikiza benzo(a)pyrene (B(a)P), nitrosamines, ndi ma radioactive isotopi ochokera pakuwola kwa radon.3 Polycyclic aromatic hydrocarbons B(a)P ndizomwe zimayambitsa poizoni mu ndudu. kusuta.Ikafika ku B(a)P, cytochrome P450 imaisintha kukhala B(a)P-7,8-dihydrodiol-9,10-epoxide (BPDE), yomwe imachita ndi DNA kupanga BPDE-DNA adduct 4. Kuphatikiza apo, izi Adducts imapangitsa mapapu tumorigenesis mu mbewa zokhala ndi chotupa ndi histopathology yofanana ndi zotupa za m'mapapo amunthu5.Izi zimapangitsa mtundu wa khansa ya m'mapapo ya B(a)P kukhala njira yoyenera yowunikira mankhwala omwe ali ndi mphamvu zothana ndi khansa.
Njira imodzi yomwe ingalepheretse kukula kwa khansa ya m'mapapo m'magulu omwe ali pachiwopsezo chachikulu, makamaka osuta, ndikugwiritsa ntchito mankhwala a chemopreventive kuletsa kukula kwa zotupa za intraepithelial neoplastic ndipo potero zimalepheretsa kupita patsogolo kwawo kukhala zilonda.Kafukufuku wa zinyama akuwonetsa kuti ma chemopreventive agents osiyanasiyana amagwira ntchito6.Lipoti lathu lapitalo7 lidawunikira zabwino zopewera za red ginseng pa khansa ya m'mapapo.Chitsambachi chakhala chikugwiritsidwa ntchito kwazaka zambiri muzamankhwala aku Asia kuti atalikitse moyo ndi thanzi, ndipo adalembedwa kuti ali ndi antitumor effects8.
Zomwe zimagwiritsidwa ntchito pa ginseng ndi ginsenoside, yomwe imagwiritsidwa ntchito ngati cholembera kuti iwunikire mtundu wa zotulutsa za ginseng.Kusanthula kachulukidwe kazinthu zamagulu a ginseng nthawi zambiri kumaphatikizapo kugwiritsa ntchito ma ginsenosides angapo, kuphatikiza RK1, Rg1, F1, Re, Rb1, Rb2, Rb3, Rd, Rh1, Rh2, Rg3, Rg5, ndi Rc9,10.Ma Ginsenosides sagwiritsidwa ntchito pang'ono pachipatala chifukwa cha kusapezeka kwawo pakamwa pakamwa11.Ngakhale kuti njira ya bioavailability yosaukayi sizodziwika bwino, kutuluka kwa ginsenosides chifukwa cha P-glycoprotein (P-gp)12 kungakhale chifukwa.P-gp ndi imodzi mwazinthu zofunika kwambiri zonyamula efflux mu ATP-binding cassette transporter superfamily, yomwe imagwiritsa ntchito mphamvu ya ATP hydrolysis kutulutsa zinthu zam'thupi kupita ku chilengedwe chakunja.P-gp transporters amagawidwa kwambiri m'matumbo, impso, chiwindi ndi chotchinga chamagazi13.P-gp imakhala ndi gawo lofunika kwambiri pakuyamwa m'mimba, ndipo kuletsa kwa P-gp kumawonjezera kuyamwa mkamwa ndi kupezeka kwa mankhwala ena oletsa khansa12,14.Zitsanzo za zoletsa zomwe zidagwiritsidwa ntchito kale m'mabuku ndi verapamil ndi cyclosporine A15.Ntchitoyi ikuphatikiza kukhazikitsa njira ya mbewa yophunzirira khansa ya m'mapapo ya B(a)P kuti awone kuthekera kwamitundu yosiyanasiyana ya ginseng yofiira kuchokera ku China ndi Korea kukhudza zilonda.Zotulutsazo zidawunikidwa payekhapayekha kuti zizindikire ma ginsenosides omwe angakhudze carcinogenesis.Verapamil ndiye adagwiritsidwa ntchito kutsata P-gp ndikuwongolera pakamwa bioavailability ndi chithandizo chamankhwala cha ginsenosides omwe amalimbana ndi khansa.
Njira yomwe ma ginseng saponins amapangira chithandizo chamankhwala pa carcinogenesis sichidziwika bwino.Kafukufuku wasonyeza kuti ma ginsenosides osiyanasiyana amatha kuchepetsa kuwonongeka kwa DNA chifukwa cha ma carcinogens pochepetsa kupsinjika kwa okosijeni ndi kuyambitsa ma enzymes a gawo lachiwiri la detoxification, potero kupewa kuwonongeka kwa maselo.Glutathione S-transferase (GST) ndi gawo lachiwiri la enzyme lomwe limafunikira kuchepetsa kuwonongeka kwa DNA komwe kumachitika chifukwa cha ma carcinogens17.Nuclear erythroid 2-related factor 2 (Nrf2) ndi chinthu chofunikira cholembera chomwe chimayang'anira redox homeostasis ndikuyambitsa kufotokozera kwa ma enzymes a phase II ndi mayankho a cytoprotective antioxidant18.Phunziro lathu linayang'ananso zotsatira za ginsenosides zomwe zimadziwika pochepetsa B (a) P-induced cytotoxicity ndi BPDE-DNA adduct mapangidwe, komanso kuyambitsa ma enzymes a gawo lachiwiri mwa kusintha njira ya Nrf2 m'maselo achibadwa a m'mapapo.
Kukhazikitsidwa kwa mtundu wa mbewa wa khansa ya B (a) P-induced ikugwirizana ndi ntchito yam'mbuyomu5.Chithunzi 1A chikuwonetsa mawonekedwe oyesera a chithandizo cha milungu 20 cha mtundu wa khansa ya mbewa wopangidwa ndi B (a) P, madzi (control), Chinese red ginseng extract (CRG), Korea red ginseng extract A (KRGA), and Korea red ginseng.Kutulutsa B (KRGB) ndi Korea Red Ginseng Extract C (KRGC).Pambuyo pa milungu 20 ya chithandizo cha ginseng yofiira, mbewa zinaperekedwa nsembe ndi CO2 asphyxiation.Chithunzi 1B chikuwonetsa zotupa zam'mapapo zam'mapapo m'zinyama zomwe zimathandizidwa ndi mitundu yosiyanasiyana ya ginseng yofiira, ndipo Chithunzi 1C chikuwonetsa choyimira chowala chamtundu wa chotupacho.Kulemera kwa chotupa cha nyama zothandizidwa ndi KRGB (1.5 ± 0.35) kunali kochepa kusiyana ndi zinyama zolamulira (0.82 ± 0.2, P <0.05), monga momwe tawonetsera pa Chithunzi 1D.Avereji ya kuchuluka kwa chotupa choletsa kuledzera kunali 45%.Zotulutsa zina zofiira za ginseng zomwe zidayesedwa sizinawonetse kusintha kwakukulu kwa chotupa (P> 0.05).Palibe zotsatira zodziwikiratu zomwe zidawonedwa mu chitsanzo cha mbewa mkati mwa masabata a 20 a chithandizo cha ginseng chofiira, kuphatikizapo kusasintha kwa kulemera kwa thupi (deta yosasonyezedwa) komanso palibe chiwindi kapena poizoni wa impso (Chithunzi 1E, F).
Kutulutsa kwa ginseng kofiira kumathandizira kukula kwa chotupa cha m'mapapo mu mbewa za A/J.(A) Mapangidwe oyesera.(B) Zotupa zazikulu za m'mapapo pamtundu wa mbewa.Zotupa zimawonetsedwa ndi mivi.a: Chinese red ginseng gulu.b: gulu A la Korean red ginseng.c: Gulu la ginseng lofiira la Korea B. d: Gulu la ginseng lofiira la Korea C. d: Gulu lolamulira.(C) Micrograph yopepuka yowonetsa chotupa cha m'mapapo.Kukula: 100. b: 400. (D) Chotupa chotupa mu gulu lofiira la ginseng.(E) Milingo ya plasma ya enzyme ya chiwindi ALT.(F) Miyezo ya plasma ya enzyme ya aimpso Cr.Deta imawonetsedwa ngati ± kupatuka kokhazikika.*P <0.05.
Zotulutsa za ginseng zofiira zomwe zadziwika mu phunziroli zidawunikidwa ndi ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) kuti adziwe ginsenosides zotsatirazi: Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 ndi Rg5.Mikhalidwe ya UPLC ndi MS yomwe idagwiritsidwa ntchito kuyeza owunika idafotokozedwa mu lipoti lapitalo19.Ma chromatogram a UPLC-MS/MS a zotulutsa zinayi zofiira za ginseng akuwonetsedwa Chithunzi 2A.Panali kusiyana kwakukulu muzinthu zonse za ginsenoside, zomwe zili ndi ginsenoside yapamwamba kwambiri mu CRG (590.27 ± 41.28 μmol/L) (Chithunzi 2B).Poyesa ma ginsenosides (Chithunzi 2C), KRGB idawonetsa kuchuluka kwa G-Rg3 poyerekeza ndi ma ginsenosides (58.33 ± 3.81 μmol/L kwa G-Rg3s ndi 41.56 ± 2.88 μmol/L kwa G -Rg3r).mtundu wa ginseng wofiira (P <0.001).G-Rg3 imapezeka ngati ma stereoisomers G-Rg3r ndi G-Rg3s, omwe amasiyana ndi malo a gulu la hydroxyl pa carbon 20 (mkuyu 2D).Zotsatira zikuwonetsa kuti G-Rg3r kapena G-Rg3 atha kukhala ndi kuthekera kofunikira kothana ndi khansa mu mtundu wa mbewa ya khansa ya B(a)P.
Zomwe zili mu ginsenosides mumitundu yosiyanasiyana yofiira ya ginseng.(A) Ma chromatogram a UPLC-MS/MS a zigawo zinayi zofiira za ginseng.(B) Kuyerekeza kuchuluka kwa ginsenoside zomwe zili muzowonjezera zomwe zawonetsedwa.(C) Kuzindikira kwa ginsenosides pawokha muzolemba zolembedwa.(D) Mapangidwe a ginsenoside stereoisomers G-Rg3r ndi G-Rg3s.Deta imawonetsedwa ngati ± kupotozedwa koyenera kwa kutsimikiza kwapatatu.***P <0.001.
Kafukufuku wa UPLC-MS/MS anafunikira kuchulukitsidwa kwa ginsenosides m'matumbo ndi magazi pambuyo pa milungu 20 yamankhwala.Kuchiza ndi KRGB kunawonetsa kupezeka kwa 0.0063 ± 0.0005 μg/ml Rg5 yokha m'magazi.Palibe ma ginsenosides otsala omwe adapezeka, zomwe zikuwonetsa kusapezeka kwapakamwa kwapakamwa ndipo chifukwa chake kuchepetsedwa kwa ma ginsenosides awa.
The colon adenocarcinoma cell line Caco-2 ndi morphologically ndi biochemically ofanana ndi matumbo a m'mimba epithelial maselo, kusonyeza ntchito yake powunika kayendedwe ka enterocyte kudutsa matumbo epithelial chotchinga.Kusanthula uku kunachokera ku kafukufuku wakale 20.Zithunzi 3A,B,C,D,E,F zikuwonetsa zithunzi zoyimira zoyendera za G-Rg3r ndi G-Rg3 pogwiritsa ntchito Caco-2 monolayer model.Transcellular transport ya G-Rg3r kapena G-Rg3 kudutsa Caco-2 monolayers kuchokera ku basolateral kupita ku apical side (Pb-a) inali yokwera kwambiri kuposa kuchokera ku apical kupita ku basolateral side (Pa-b).Kwa G-Rg3r, tanthauzo la Pa-b linali 0.38 ± 0.06, lomwe linakwera mpaka 0.73 ± 0.06 mutalandira chithandizo ndi 50 μmol/L verapamil ndi 1.14 ± 0.09 mutalandira mankhwala ndi 100 μmol/L verapamil (p <0.001 ndi motsatana; Chithunzi 2).3 A).Kuwona kwa G-Rg3 kunatsatira chitsanzo chofanana (mkuyu 3B), ndipo zotsatira zinasonyeza kuti chithandizo cha verapamil chinalimbikitsa kunyamula G-Rg3r ndi G-Rg3.Chithandizo cha Verapamil chinapangitsanso kuchepa kwakukulu kwa Pb-a ndi G-Rg3r ndi G-Rg3s efflux ratios (Chithunzi 3C,D,E,F), kusonyeza kuti mankhwala a verapamil amachepetsa zomwe zili mu ginsenoside m'maselo a Caco-2 efflux..
Mayendedwe a Transcellular a G-Rg3 mu Caco-2 monolayers ndi kuyamwa m'matumbo mu kuyesa kwa makoswe.(A) Mtengo wa Pa-b wa gulu la G-Rg3r mu Caco-2 monolayer.(B) Mtengo wa Pa-b wamagulu a G-Rg3s mu Caco-2 monolayer.(C) Mtengo wa Pb wa gulu la G-Rg3r mu Caco-2 monolayer.(D) Mtengo wa Pb wamagulu a G-Rg3s mu Caco-2 monolayer.(E) Chiŵerengero cha zokolola za magulu a G-Rg3r mu Caco-2 monolayer.(F) Chiŵerengero cha zokolola za magulu a G-Rg3 mu Caco-2 monolayer.(G) Peresenti ya mayamwidwe a m'mimba a G-Rg3r mu kuyesa kotulutsa makoswe.(H) Peresenti ya mayamwidwe a m'mimba a G-Rg3 mu kuyesa kwa perfusion mu makoswe.Permeability ndi mayamwidwe anayerekezedwa popanda kuwonjezera verapamil.Deta imafotokozedwa ngati njira ± yopatuka muyeso zisanu zodziyimira pawokha.*P <0.05, **P <0.01, ***P <0.001.
Mogwirizana ndi ntchito yoyambirira20, matumbo a orthotopic a makoswe adapangidwa kuti adziwe ngati kuyamwa kwa G-Rg3 m'matumbo kumawonjezeka pambuyo pa chithandizo cha verapamil.Zithunzi za 3G, H zikuwonetsa kuyesa koyimilira kuti awone kuchuluka kwa mayamwidwe am'mimba a G-Rg3r ndi G-Rg3 mu makoswe amtundu wa khansa pazaka zomwe zatchulidwazi.Chiwopsezo choyambirira cha kufooka kwa G-Rg3r pafupifupi 10% chinawonjezeka kufika kupitirira 20% mutalandira mankhwala ndi verapamil 50 μM ndi kupitirira 25% mutalandira mankhwala ndi 100 μM verapamil.Momwemonso, G-Rg3, yomwe idamwa koyamba ndi 10%, idawonetsanso chiwopsezo chopitilira 20% mutalandira chithandizo ndi verapamil 50 μM ndipo pafupifupi 30% mutalandira chithandizo ndi 100 μM verapamil, kutanthauza kuti kuletsa kwa P-gp ndi verapamil kumawonjezera. m'mimba G-mayamwidwe Rg3 mu mtundu wa mbewa wa khansa ya m'mapapo.
Malingana ndi njira yomwe ili pamwambayi, mbewa za mtundu wa khansa ya B (a) P-zoyambitsa khansa zinagawidwa mwachisawawa m'magulu asanu ndi limodzi, monga momwe tawonetsera pa Chithunzi 4A.Palibe kulemera kwakukulu kapena zizindikiro zachipatala za poizoni zomwe zinawonedwa mu gulu lachipatala la G-Rg3 poyerekeza ndi gulu lolamulira (deta yosasonyezedwa).Pambuyo pa milungu 20 ya chithandizo, mapapu a mbewa iliyonse adatengedwa.Chithunzi 4B chikuwonetsa zotupa zam'mapapo zazikulu mu mbewa m'magulu ochizira omwe ali pamwambapa, ndipo Chithunzi 4C chikuwonetsa choyimira chowala cha chotupa choyimira.Ponena za kuchuluka kwa chotupa pagulu lililonse (mkuyu 4D), mikhalidwe ya mbewa zomwe zimathandizidwa ndi G-Rg3r ndi G-Rg3s zinali 0.75 ± 0.29 mm3 ndi 0.81 ± 0.30 mm3, motsatana, pomwe mbewa za G mbewa zimathandizira. ndi -Rg3s anali 1.63 motsatira ± 0.40 mm3.kuwongolera mbewa (p <0.001), kusonyeza kuti chithandizo cha G-Rg3 chimachepetsa kuchuluka kwa chotupa mu mbewa.Kuwongolera kwa verapamil kunathandiziranso kuchepetsa uku: mbewa za verapamil+ G-Rg3r zidatsika kuchoka pa 0.75 ± 0.29 mm3 mpaka 0.33 ± 0.25 mm3 (p <0.01), ndipo mitengo ya verapamil+ kuchoka pa 0.81 ± 0.30 mm29 yatsika mpaka ± 0.30 mm291 mm3 mu mbewa zothandizidwa ndi G. -Rg3s (p <0.05), kusonyeza kuti verapamil akhoza kupititsa patsogolo zotsatira zoletsa za G-Rg3 pa tumorigenesis.Kulemera kwa chotupa sikunawonetse kusiyana kwakukulu pakati pa gulu lolamulira ndi gulu la verapamil, gulu la G-Rg3r ndi gulu la G-Rg3s, ndi gulu la verapamil + G-Rg3r ndi gulu la verapamil + G-Rg3s.Komanso, panalibe chiwopsezo chachikulu cha chiwindi kapena impso chokhudzana ndi mankhwala omwe adayesedwa (Chithunzi 4E, F).
Kulemera kwa chotupa pambuyo pa chithandizo cha G-Rg3 ndi plasma kapena matumbo a G-Rg3r ndi G-Rg3 m'magulu osonyezedwa.(A) Mapangidwe oyesera.(B) Zotupa zazikulu mumtundu wa mbewa.Zotupa zimawonetsedwa ndi mivi.ndi :g3r.b: G-Rg3s.c: G-Rg3r pamodzi ndi verapamil.d: G-Rg3 kuphatikiza verapamil.d: Verapamil.e: kulamulira.(C) Optical micrograph ya chotupa pakukula.Yankho: 100x.b:400x.(D) Zotsatira za G-Rg3 + mankhwala a verapamil pa chotupa mu A/J mbewa.(E) Miyezo ya plasma ya enzyme ya chiwindi ALT.(F) Miyezo ya plasma ya enzyme ya aimpso Cr.(G) Miyezo ya plasma ya G-Rg3r kapena G-Rg3 yamagulu osonyezedwa.(H) Miyezo ya G-Rg3r kapena G-Rg3s m'matumbo amagulu osonyezedwa.Deta imawonetsedwa ngati ± kupotozedwa kokhazikika kwa kutsimikiza kwapatatu.*P <0.05, **P <0.01, ***P <0.001.
Miyezo ya G-Rg3 mu B (a) P-induced mbewa za khansa zinayesedwa ndi UPLC-MS / MS pambuyo pa nthawi ya chithandizo cha masabata a 20 malinga ndi njira yomwe ikufotokozedwa mu gawo la Njira.Zithunzi za 4G ndi H zimasonyeza plasma ndi matumbo a G-Rg3, motsatira.Miyezo ya Plasma G-Rg3r inali 0.44 ± 0.32 μmol/L ndipo idakwera mpaka 1.17 ± 0.47 μmol/L ndi makonzedwe amodzimodzi a verapamil (p <0.001), pamene matumbo a G-Rg3r anali 0.53 ± 0.08 µµ.Ikaphatikizidwa ndi verapamil, g idakwera mpaka 1.35 ± 0.13 μg/g (p <0.001).Kwa G-Rg3, zotsatirazo zinatsatira chitsanzo chofanana, chosonyeza kuti chithandizo cha verapamil chinawonjezera bioavailability wapakamwa wa G-Rg3 mu A/J mbewa.
Kuyesa kwamphamvu kwa ma cell kunagwiritsidwa ntchito kuyesa cytotoxicity ya B (a) P ndi G-Rg3 pama cell a hEL.Cytotoxicity yoyambitsidwa ndi B(a)P m'maselo a hEL ikuwonetsedwa mu Chithunzi 5A, pomwe zinthu zopanda poizoni za G-Rg3r ndi G-Rg3 zikuwonetsedwa mu Zithunzi 5A ndi 5B.5B, C. Kuti muwone zotsatira za cytoprotective za G-Rg3, B (a) P zinagwiritsidwa ntchito limodzi ndi magulu osiyanasiyana a G-Rg3r kapena G-Rg3 mu maselo a hEL.Monga momwe tawonetsera pa Chithunzi 5D, G-Rg3r pamagulu a 5 μM, 10 μM, ndi 20 μM anabwezeretsa mphamvu ya selo ku 58.3%, 79.3%, ndi 77.3%, motero.Zotsatira zofananira zitha kuwonekanso mu gulu la G-Rg3s.Pamene kuchuluka kwa G-Rg3s kunali 5 µM, 10 µM ndi 20 µM, mphamvu ya selo inabwezeretsedwa ku 58.3%, 72.7% ndi 76.7%, motsatira (Chithunzi 5E) .).Kukhalapo kwa BPDE-DNA adducts kunayesedwa pogwiritsa ntchito zida za ELISA.Zotsatira zathu zasonyeza kuti BPDE-DNA adduct milingo inawonjezeka mu B (a) P-treated gulu poyerekeza ndi gulu lolamulira, koma poyerekeza ndi G-Rg3 co-treatment, BPDE-DNA adduct levels mu B (a) P gulu. B m'gulu lothandizidwa, ma DNA adduct milingo adachepetsedwa kwambiri.Zotsatira za mankhwala ndi B (a) P okha zikuwonetsedwa mu Chithunzi 5F (1.87 ± 0.33 vs. 3.77 ± 0.42 kwa G-Rg3r, 1.93 ± 0.48 vs. 3.77 ± 0.42 kwa G -Rg3s, p <0.001).
Kutheka kwa ma cell ndi BPDE-DNA adduct mapangidwe m'maselo a hEL omwe amathandizidwa ndi G-Rg3 ndi B(a)P.(A) Kutheka kwa maselo a hEL omwe amathandizidwa ndi B(a)P.(B) Kutheka kwa maselo a hEL omwe amathandizidwa ndi G-Rg3r.(C) Kutheka kwa maselo a hEL omwe amathandizidwa ndi G-Rg3.(D) Kutheka kwa maselo a hEL omwe amathandizidwa ndi B (a) P ndi G-Rg3r.(E) Kutheka kwa maselo a hEL omwe amathandizidwa ndi B(a)P ndi G-Rg3.(F) Miyezo ya BPDE-DNA adduct m'maselo a hEL omwe amathandizidwa ndi B (a) P ndi G-Rg3.Deta imawonetsedwa ngati ± kupotozedwa koyenera kwa kutsimikiza kwapatatu.*P <0.05, **P <0.01, ***P <0.001.
Kufotokozera kwa enzyme ya GST kunadziwika pambuyo pothandizidwa ndi 10 μM B (a) P ndi 10 μM G-Rg3r kapena G-Rg3s.Zotsatira zathu zidawonetsa kuti B(a)P idapondereza mawu a GST (59.7 ± 8.2% mu gulu la G-Rg3r ndi 39 ± 4.5% mu gulu la G-Rg3s), ndipo B(a)P idalumikizidwa ndi G-Rg3r , kapena ndi G-Rg3r, kapena ndi G-Rg3r.Kuchitira limodzi ndi G-Rg3s kubwezeretsedwa kwa mawu a GST.Mawu a GST (103.7 ± 15.5% mu gulu la G-Rg3r ndi 110 ± 11.1% mu gulu la G-Rg3s, p <0.05 ndi p <0.001, motsatira, Mkuyu 6A, B, ndi C).Ntchito ya GST idayesedwa pogwiritsa ntchito zida zoyeserera.Zotsatira zathu zinasonyeza kuti gulu la mankhwala ophatikizana linali ndi ntchito yapamwamba ya GST poyerekeza ndi B (a) P gulu lokha (96.3 ± 6.6% vs. 35.7 ± 7.8% mu gulu la G-Rg3r vs. 92.3 ± 6.5 mu gulu la G-Rg3r ).% vs 35.7 ± 7.8% mu gulu la G-Rg3s, p <0.001, Chithunzi 6D).
Kufotokozera kwa GST ndi Nrf2 m'maselo a hEL omwe amathandizidwa ndi B (a) P ndi G-Rg3.(A) Kuzindikira mawu a GST ndi Western blotting.(B) Kuchulukitsidwa kwa GST m'maselo a hEL omwe amachitidwa ndi B (a) P ndi G-Rg3r.(C) Kuchulukitsidwa kwa GST m'maselo a hEL omwe amathandizidwa ndi B (a) P ndi G-Rg3s.(D) Ntchito ya GST m'maselo a hEL omwe amathandizidwa ndi B(a)P ndi G-Rg3.(E) Kuzindikira mawu a Nrf2 ndi Western blotting.(F) Kuchulukitsa kwa Nrf2 m'maselo a hEL omwe amachitidwa ndi B (a) P ndi G-Rg3r.(G) Kuchulukitsa kwa Nrf2 m'maselo a hEL omwe amachitidwa ndi B (a) P ndi G-Rg3s.Deta imawonetsedwa ngati ± kupotozedwa koyenera kwa kutsimikiza kwapatatu.*P <0.05, **P <0.01, ***P <0.001.
Pofuna kufotokozera njira zomwe zimakhudzidwa ndi G-Rg3-mediated kuponderezedwa kwa B (a) P-induced tumorigenesis, mawu a Nrf2 adayesedwa ndi Western blotting.Monga momwe tawonetsera mu Zithunzi 6E, F, G, poyerekeza ndi gulu lolamulira, mlingo wokha wa Nrf2 mu gulu la mankhwala la B (a) P unachepetsedwa;komabe, poyerekeza ndi gulu lachipatala la B (a) P, B (a) Nrf2 mu gulu la PG-Rg3 linawonjezeka (106 ± 9.5% kwa G-Rg3r vs. 51.3 ± 6.8%, 117 ± 6. 2% kwa G-Rg3r vs. 41 ± 9.8% kwa G-Rg3s, p <0.01).
Tidatsimikizira gawo loletsa la Nrf2 popondereza mawu a Nrf2 pogwiritsa ntchito RNA (siRNA) yaying'ono yosokoneza.Nrf2 knockdown inatsimikiziridwa ndi Western blotting (Mkuyu 7A, B).Monga momwe tawonetsera mu Zithunzi 7C, D, kuchitira limodzi ma cell a hEL ndi B (a) P ndi G-Rg3 kunapangitsa kuchepa kwa chiwerengero cha BPDE-DNA adducts (1.47 ± 0.21) poyerekeza ndi chithandizo ndi B (a) P yekha mu gulu lolamulira la siRNA.) G-Rg3r inali 4.13 ± 0.49, G-Rg3s inali 1.8 ± 0.32 ndi 4.1 ± 0.57, p <0.01).Komabe, kulepheretsa kwa G-Rg3 pa BPDE-DNA mapangidwe kunathetsedwa ndi Nrf2 knockdown.Mu gulu la siNrf2, panalibe kusiyana kwakukulu mu BPDE-DNA mapangidwe adduct pakati pa B (a) P ndi G-Rg3 co-treatment ndi B (a) P mankhwala okha (3.0 ± 0.21 kwa G-Rg3r vs. 3.56 ± 0.32 ).kwa G-Rg3r motsutsana ndi 3.6 ya G-Rg3s motsutsana ndi ± 0.45 motsutsana ndi 4.0±0.37, p > 0.05).
Zotsatira za Nrf2 knockdown pa BPDE-DNA adduct mapangidwe m'maselo a hEL.(A) Kugwetsa kwa Nrf2 kunatsimikiziridwa ndi Western blotting.(B) Kuchulukitsa kwa Nrf2 band intensity.(C) Zotsatira za Nrf2 knockdown pa BPDE-DNA adduct levels mu maselo a hEL omwe amachitidwa ndi B (a) P ndi G-Rg3r.(D) Zotsatira za Nrf2 kugogoda pamagulu a BPDE-DNA adduct m'maselo a hEL omwe amachitidwa ndi B (a) P ndi G-Rg3.Deta imawonetsedwa ngati ± kupotozedwa koyenera kwa kutsimikiza kwapatatu.*P <0.05, **P <0.01, ***P <0.001.
Kafukufukuyu adawunika zodzitetezera zamitundu yosiyanasiyana yofiira ya ginseng pamtundu wa mbewa ya B(a) P-khansa ya m'mapapo, ndipo chithandizo cha KRGB chachepetsa kwambiri chotupa.Poganizira kuti G-Rg3 ili ndi zomwe zili pamwamba pa ginseng iyi, gawo lofunikira la ginsenoside iyi poletsa tumorigenesis laphunziridwa.Onse G-Rg3r ndi G-Rg3 (ma epimers awiri a G-Rg3) amachepetsa kwambiri chotupa mu mtundu wa mbewa wa khansa ya B(a) P-induced.G-Rg3r ndi G-Rg3 zimakhala ndi zotsatira za anticancer poyambitsa apoptosis ya chotupa maselo21, kuletsa chotupa kukula22, kumanga cell cycle23 ndi kukhudza angiogenesis24.G-Rg3 yasonyezedwanso kuti imalepheretsa ma metastasis25 a ma cell, ndipo kuthekera kwa G-Rg3 kupititsa patsogolo zotsatira za chemotherapy ndi radiotherapy kwalembedwa26,27.Poon et al adawonetsa kuti chithandizo cha G-Rg3 chikhoza kuchepetsa zotsatira za genotoxic za B(a)P28.Kafukufukuyu akuwonetsa kuthekera kochizira kwa G-Rg3 poyang'ana mamolekyu achilengedwe a carcinogenic ndikupewa khansa.
Ngakhale ali ndi kuthekera kodziletsa, kusapezeka bwino kwapakamwa kwa ma ginsenosides kumabweretsa zovuta pakugwiritsa ntchito kwachipatala kwa mamolekyuwa.Kusanthula kwa pharmacokinetic pakuwongolera pakamwa kwa ginsenosides mu makoswe kunawonetsa kuti bioavailability yake ikadali yochepera 5% 29.Mayeserowa adawonetsa kuti pambuyo pa nthawi ya chithandizo cha masabata 20, magazi okha a Rg5 adachepa.Ngakhale kuti njira yochepetsera kuwonongeka kwa bioavailability ikadali yodziwika bwino, P-gp imaganiziridwa kuti ikuphatikizidwa mu efflux ya ginsenosides.Ntchitoyi idawonetsa koyamba kuti kugwiritsa ntchito verapamil, blocker ya P-gp, kumawonjezera kupezeka kwapakamwa kwa G-Rg3r ndi G-Rg3s.Chifukwa chake, zomwe zapezazi zikuwonetsa kuti G-Rg3r ndi G-Rg3s amagwira ntchito ngati magawo a P-gp kuti aziwongolera kutulutsa kwake.
Ntchitoyi ikuwonetsa kuti chithandizo chophatikizana ndi verapamil chimawonjezera kupezeka kwapakamwa kwa G-Rg3 mu mtundu wa mbewa wa khansa ya m'mapapo.Kupeza uku kumathandizidwa ndi kuchulukitsidwa kwamatumbo am'mimba a G-Rg3 pa blockade ya P-gp, motero kumakulitsa kuyamwa kwake.Kuyesa m'maselo a Caco2 kunawonetsa kuti chithandizo cha verapamil chinachepetsa kutulutsa kwa G-Rg3r ndi G-Rg3s kwinaku akuwongolera kutulutsa kwa membrane.Kafukufuku wopangidwa ndi Yang et al.Kafukufuku wasonyeza kuti chithandizo cha cyclosporine A (chotchinga china cha P-gp) chimawonjezera bioavailability wa ginsenoside Rh2 kuchokera pamtengo woyambira wa 1% 20 mpaka 30%.Mankhwala a Ginsenosides K ndi Rg1 adawonetsanso zotsatira zofanana30,31.Pamene verapamil ndi cyclosporin A adagwiritsidwa ntchito limodzi, kutuluka kwa K m'maselo a Caco-2 kunachepetsedwa kwambiri kuchokera ku 26.6 mpaka kuchepera 3, pamene miyeso yake ya intracellular inakula 40-fold30.Pamaso pa verapamil, mlingo wa Rg1 unawonjezeka m'maselo a epithelial a makoswe, kutanthauza kuti P-gp imagwira ntchito mu ginsenoside efflux, monga momwe Meng et al.31 akuwonetsera.Komabe, verapamil sanakhudzidwenso chimodzimodzi ndi ma ginsenosides ena (monga Rg1, F1, Rh1 ndi Re), kusonyeza kuti sakhudzidwa ndi magawo a P-gp, monga momwe Liang et al.32 .Kuwona uku kungakhale kokhudzana ndi kukhudzidwa kwa onyamula ena ndi zida zina za ginsenoside.
Njira yodzitetezera ya G-Rg3 pa khansa sikudziwika bwino.Kafukufuku wam'mbuyo wasonyeza kuti G-Rg3 imalepheretsa kuwonongeka kwa DNA ndi apoptosis mwa kuchepetsa kupsinjika kwa okosijeni ndi kutupa16,33, yomwe ingakhale njira yotetezera B (a) P-induced tumorigenesis.Malipoti ena akuwonetsa kuti genitotoxicity yoyambitsidwa ndi B(a)P imatha kuchepetsedwa posintha ma enzymes a gawo II kupanga BPDE-DNA34.GST ndi gawo lachiwiri la enzyme yomwe imalepheretsa BPDE-DNA adduct mapangidwe mwa kulimbikitsa kumanga kwa GSH ku BPDE, motero kuchepetsa kuwonongeka kwa DNA kochititsidwa ndi B(a)P35.Zotsatira zathu zikuwonetsa kuti chithandizo cha G-Rg3 chimachepetsa B (a) P-induced cytotoxicity ndi BPDE-DNA adduct mapangidwe m'maselo a hEL ndikubwezeretsa GST kufotokoza ndi ntchito mu vitro.Komabe, zotsatirazi zinalibe popanda Nrf2, kutanthauza kuti G-Rg3 imayambitsa zotsatira za cytoprotective kudzera mu njira ya Nrf2.Nrf2 ndi chinthu chachikulu cholembera ma enzymes a gawo lachiwiri la detoxification omwe amalimbikitsa kuchotsedwa kwa xenobiotics36.Kutsegula kwa njira ya Nrf2 kumapangitsa cytoprotection ndikuchepetsa kuwonongeka kwa minofu37.Komanso, malipoti angapo athandizira udindo wa Nrf2 ngati chotupa chopondereza mu carcinogenesis38.Kafukufuku wathu akuwonetsa kuti kulowetsedwa kwa njira ya Nrf2 ndi G-Rg3 kumagwira ntchito yofunika kwambiri yolamulira mu B (a) P-induced genotoxicity poyambitsa B (a) P detoxification poyambitsa ma enzymes a gawo lachiwiri, motero amaletsa njira ya tumorigenesis.
Ntchito yathu imawulula kuthekera kwa ginseng yofiyira popewa khansa ya m'mapapo ya B (a) P mu mbewa kudzera mukutengapo gawo kofunikira kwa ginsenoside G-Rg3.Kusakwanira kwapakamwa kwa molekyuluyi kumalepheretsa kugwiritsa ntchito kwake kuchipatala.Komabe, kafukufukuyu akuwonetsa kwa nthawi yoyamba kuti G-Rg3 ndi gawo laling'ono la P-gp, ndipo kuyendetsa P-gp inhibitor kumawonjezera bioavailability wa G-Rg3 mu vitro ndi mu vivo.G-Rg3 imachepetsa B(a) P-induced cytotoxicity poyendetsa njira ya Nrf2, yomwe ingakhale njira yodzitetezera.Kafukufuku wathu amatsimikizira kuthekera kwa ginsenoside G-Rg3 popewa komanso kuchiza khansa ya m'mapapo.
Makoswe a A/J azaka zisanu ndi chimodzi (20 ± 1 g) ndi makoswe aamuna a Wistar a masabata 7 (250 ± 20 g) adapezedwa kuchokera ku The Jackson Laboratory (Bar Harbor, USA) ndi Wuhan Institute of Zoology.University (Wuhan, China).China Type Culture Collection Center (Wuhan, China) idatipatsa ma cell a Caco-2 ndi hEL.Sigma-Aldrich (St. Louis, USA) ndi gwero la B (a) P ndi tricaprine.Oyeretsedwa ginsenosides G-Rg3r ndi G-Rg3s, dimethyl sulfoxide (DMSO), CellTiter-96 proliferation assay kit (MTS), verapamil, minimal essential medium (MEM), ndi fetal bovine serum (FBS) anagulidwa ku Chengdu Must Bio-Technology. .Co., Ltd.(Chengdu, China).The QIAamp DNA mini kit ndi BPDE-DNA adduct ELISA kit zidagulidwa ku Qiagen (Stanford, CA, USA) and Cell Biolabs (San Diego, CA, USA).Zida zoyezera zochita za GST ndi zida zonse zoyezera mapuloteni (njira yokhazikika ya BCA) zidagulidwa kuchokera ku Solarbio (Beijing, China).Zotulutsa zonse zofiira za ginseng zimasungidwa ku Mingyu Laboratory 7. Hong Kong Baptist University (Hong Kong, China) ndi Korea Cancer Center (Seoul, Korea) ndi magwero a malonda a CRG extract ndi mitundu yosiyanasiyana ya ginseng yofiira yamitundu yosiyanasiyana yaku Korea (kuphatikiza KRGA, KRGB). ndi KRGC).Ginseng yofiira imapangidwa kuchokera ku mizu ya ginseng wazaka 6 watsopano.Tizilombo tofiira ta ginseng timapezedwa potsuka ginseng ndi madzi katatu, kenako ndikuyika pamadzi amadzimadzi, kenako ndikuwumitsa kutentha pang'ono kuti mupeze ufa wa ginseng.Ma antibodies (anti-Nrf2, anti-GST, ndi β-actin), horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG), transfection reagent, control siRNA, ndi Nrf2 siRNA anagulidwa ku Santa Cruz Biotechnology (Santa Cruz, CA) .), USA).
Ma cell a Caco2 ndi hEL adakulitsidwa mu mbale za 100 mm2 cell culture ndi MEM yomwe ili ndi 10% FBS pa 37 °C m'malo onyowa a 5% CO2.Kuti mudziwe zotsatira za mankhwala, maselo a hEL adayikidwa ndi magulu osiyanasiyana a B (a) P ndi G-Rg3 mu MEM kwa 48 h.Maselo amatha kuwunikidwanso kapena kusonkhanitsidwa kuti akonze zotulutsa zopanda ma cell.
Zoyeserera zonse zidavomerezedwa ndi Komiti Yoyeserera ya Zanyama Zanyama ku Tongji Medical College, Huazhong University of Science and Technology (Chivomerezo No. 2019; Registration No. 4587TH).Zoyesera zonse zidachitidwa motsatira malangizo ndi malamulo oyenerera, ndipo kafukufukuyu adachitika motsatira malangizo a Animal Research: Reporting of In Vivo Experiments (ARRIVE).Makoswe a A/J a masabata asanu ndi atatu anayamba kubayidwa intraperitoneally ndi B(a)P mu njira ya tricaprine (100 mg/kg, 0.2 ml).Pambuyo pa sabata, mbewazo zinagawidwa mwachisawawa m'magulu olamulira ndi magulu osiyanasiyana ochizira, mbewa za 15 pagulu lililonse, ndipo zimawombera kamodzi patsiku.Pambuyo pa milungu 20 ya chithandizo, nyama zidaperekedwa nsembe ndi CO2 asphyxia.Mapapo adasonkhanitsidwa ndikukonzedwa kwa maola 24.Chiwerengero cha zotupa zapamtunda ndi kukula kwake kwa chotupacho chinawerengedwera m'mapapo aliwonse pansi pa maikulosikopu yophatikizira.Kuyerekeza kuchuluka kwa chotupa (V) kunawerengedwa pogwiritsa ntchito mawu awa: V (mm3) = 4/3πr3, pomwe r ndi m'mimba mwake.Kuchulukirachulukira kwa chotupa chonse m'mapapu a mbewa kumayimira kuchuluka kwa chotupacho, ndipo kuchuluka kwathunthu kwa chotupa pagulu lililonse kumayimira kuchuluka kwa chotupacho.Magazi athunthu ndi matumbo athunthu adasonkhanitsidwa ndikusungidwa pa -80 ° C kuti adziwe UPLC-MS/MS.Seramu inasonkhanitsidwa ndipo makina opangira chemistry analyzer adagwiritsidwa ntchito kusanthula ma alanine aminotransferase (ALT) ndi serum creatinine (Cr) kuti aone ntchito ya chiwindi ndi impso.
Zitsanzo zosonkhanitsidwa zinachotsedwa kusungirako kuzizira, kusungunuka, kuyeza, ndikuyikidwa mu machubu monga tafotokozera pamwambapa.Izi zinawonjezeredwa 0,5 μM phlorizin (muyezo wamkati) mu 0,8 ml ya methanol solution.Minofuyo idasinthidwa pogwiritsa ntchito Tissue-Tearor ndipo homogenate idasamutsidwa ku chubu cha 1.5 ml microcentrifuge.Kusakaniza kunali centrifuged pa 15500 rpm kwa mphindi 15.Mukachotsa 1.0 ml ya supernatant, youma ndi nayitrogeni.Ma microliters mazana awiri a methanol adagwiritsidwa ntchito pochira.Mwazi umasonkhanitsidwa ndikukonzedwa pamzere umodzi ndipo umagwiritsidwa ntchito ngati chizindikiritso cha miyeso yonse.
Ma mbale a 24-chitsime a Transwell adabzalidwa ndi ma cell a 1.0 × 105 Caco-2 pachitsime chilichonse kuti athe kuwunika momwe angayendetsere G-Rg3 powonjezera verapamil.Pambuyo pa masabata atatu a chikhalidwe, ma cell adatsukidwa ndi HBSS ndikuyikidwa pa 37 ° C.400 μL ya 10 μM G-Rg3 (G-Rg3r, G-Rg3s, kapena osakaniza ndi 50 kapena 100 μM verapamil) anabayidwa pa basolateral kapena apical mbali ya monolayer, ndipo 600 μL wa HBSS yankho anawonjezedwa kwa ena. mbali.Sonkhanitsani 100 µl ya sing'anga ya chikhalidwe munthawi yake (0, 15, 30, 45, 60, 90 ndi 120 mphindi) ndikuwonjezera 100 µl ya HBSS kuti mupange voliyumuyi.Zitsanzo zinasungidwa pa −4 °C mpaka zitadziwika ndi UPLC-MS/MS.Mawu akuti Papp = dQ/(dT × A × C0) amagwiritsidwa ntchito kuwerengera momwe zimawonekera za unidirectional apical and basolateral permeability ndi mosemphanitsa (Pa-b ndi Pb-a, motsatana);dQ/dT ndikusintha kwa ndende, A (0.6 cm2) ndi gawo lapamwamba la monolayer, ndipo C0 ndiye ndende yoyambira yopereka.Chiyerekezo cha efflux chimawerengedwa ngati Pb-a/Pa-b, chomwe chikuyimira kuchuluka kwa efflux kwamankhwala ophunzirira.
Makoswe aamuna a Wistar adasala kudya kwa maola a 24, amamwa madzi okha, ndikuwotchedwa anesthetized ndi jekeseni wa 3.5% pentobarbital solution.The intubated silikoni chubu ali mapeto a duodenum monga khomo ndi mapeto a ileamu ngati potuluka.Gwiritsani ntchito mpope wa peristaltic kupopera cholowera ndi 10 µM G-Rg3r kapena G-Rg3s mu isotonic HBSS pamlingo wothamanga wa 0.1 ml/min.Zotsatira za verapamil zidayesedwa powonjezera 50 μM kapena 100 μM ya pawiri ku 10 μM G-Rg3r kapena G-Rg3s.UPLC-MS/MS inkachitika pazigawo za perfusion zomwe zimasonkhanitsidwa panthawi ya 60, 90, 120, ndi mphindi 150 pambuyo poyambira kutulutsa.Kuchuluka kwa mayamwidwe kumayesedwa ndi chilinganizo% mayamwidwe = (1 - Cout / Cin) × 100%;Kuchulukira kwa G-Rg3 potuluka ndi polowera kumawonetsedwa ndi Cout ndi Cin, motsatana.
Maselo a hEL adabzalidwa m'mbale za 96-chitsime pa kachulukidwe ka maselo a 1 × 104 pachitsime ndipo amathandizidwa ndi B (a) P (0, 1, 5, 10, 20, 30, 40 μM) kapena G-Rg3 kusungunuka mu DMSO .Mankhwalawa adachepetsedwa ndi chikhalidwe chapakati pazigawo zosiyanasiyana (0, 1, 2, 5, 10, 20 μM) pa maola 48.Pogwiritsa ntchito zida zoyeserera za MTS zomwe zimapezeka pamalonda, ma cell adayikidwa pa protocol yokhazikika ndikuyezedwa pogwiritsa ntchito chowerengera cha microplate pa 490 nm.Maselo a maselo a magulu omwe amathandizidwa ndi B (a) P (10 μM) ndi G-Rg3 (0, 1, 5, 10, 20 μM) adayesedwa molingana ndi njira yomwe ili pamwambayi ndikuyerekeza ndi gulu losagwiritsidwa ntchito.
Maselo a hEL adabzalidwa m'mbale za 6-chitsime pamtundu wa 1 × 105 maselo / bwino ndikuchiritsidwa ndi 10 μMB (a) P pamaso kapena kusowa kwa 10 μM G-Rg3.Pambuyo pa chithandizo cha maola 48, DNA inachotsedwa m'maselo a hEL pogwiritsa ntchito QIAamp DNA Mini Kit molingana ndi ndondomeko ya wopanga.Mapangidwe a BPDE-DNA adducts adapezeka pogwiritsa ntchito BPDE-DNA adduct ELISA kit.Miyezo yofananira ya BPDE-DNA adduct idayezedwa pogwiritsa ntchito chowerengera cha microplate poyesa kuyamwa pa 450 nm.
Maselo a hEL adayikidwa mu mbale za 96-chitsime pa kachulukidwe ka 1 × 104 maselo pachitsime ndipo amachiritsidwa ndi 10 μMB (a) P popanda kapena kukhalapo kwa 10 μM G-Rg3 kwa 48 h.Ntchito ya GST idayezedwa pogwiritsa ntchito zida zoyeserera za GST molingana ndi ndondomeko ya wopanga.Kutsegula kwa GST kwachibale kunayesedwa ndi kuyamwa pa 450 nm pogwiritsa ntchito microplate reader.
Maselo a hEL adatsukidwa ndi PBS yozizira kwambiri ndipo kenako amapukutidwa pogwiritsa ntchito radioimmunoprecipitation assay buffer yokhala ndi protease inhibitors ndi phosphatase inhibitors.Pambuyo pa kuchuluka kwa mapuloteni pogwiritsa ntchito zida zonse zoyesa mapuloteni, 30 μg ya mapuloteni mu chitsanzo chilichonse adasiyanitsidwa ndi 12% SDS-PAGE ndikusamutsira ku nembanemba ya PVDF ndi electrophoresis.Mamembala anali otsekedwa ndi 5% mkaka wa skim ndi kuikidwa ndi ma antibodies oyambirira usiku pa 4 ° C.Pambuyo pa kukulitsidwa ndi ma antibodies achiwiri a horseradish peroxidase-conjugated, zowonjezera zowonjezera za chemiluminescence zinawonjezeredwa kuti ziwone chizindikiro chomangirira.Kukula kwa gulu lililonse la mapuloteni adayesedwa pogwiritsa ntchito pulogalamu ya ImageJ.
Pulogalamu ya GraphPad Prism 7.0 idagwiritsidwa ntchito kusanthula deta yonse, yowonetsedwa ngati ± kupatuka kokhazikika.Kusiyanasiyana pakati pa magulu ochizira kunayesedwa pogwiritsa ntchito mayeso a Wophunzira t kapena kusanthula kwa njira imodzi yosiyana, ndi mtengo wa P <0.05 wosonyeza kufunikira kwa chiwerengero.
Deta zonse zomwe zapezedwa kapena kufufuzidwa panthawi ya phunziroli zikuphatikizidwa m'nkhani yofalitsidwayi ndi mafayilo owonjezera owonjezera.
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Nthawi yotumiza: Sep-17-2023