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I-ginseng ebomvu isetyenziswe kumayeza emveli aseAsia amakhulu eminyaka.Kolu phononongo, sivavanye isakhono seentlobo ezine zeginseng ebomvu (iginseng ebomvu yaseTshayina, iginseng ebomvu yaseKorea, iginseng ebomvu yaseKorea, kunye neginseng ebomvu yaseKorea C) ekhule kwimimandla eyahlukeneyo ukunqanda ukwakheka kunye nokukhula kwemiphunga ebangelwa yi-carcinogen. amathumba.Uvavanyo lwe-benzo (a) pyrene (B (a) P) lwenziwa kwiigundane ze-A / J, kunye ne-Korean ebomvu ye-ginseng B yafunyanwa iyona nto isebenzayo ekunciphiseni umthwalo we-tumor phakathi kweentlobo ezine ze-ginseng ezibomvu.Ukongeza, sihlalutye imixholo yee-ginsenosides ezahlukeneyo (Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 kunye neRg5) kwizicatshulwa ezine ezibomvu zeginseng kwaye safumanisa ukuba iginseng ebomvu yaseKorea awona manqanaba aphezulu e-ginsenoside Rg3 (G-Rg3), ebonisa ukuba i-G-Rg3 inokudlala indima ebalulekileyo ekusebenzeni kwayo kwonyango.Lo msebenzi ubonisa ukuba i-G-Rg3 ine-bioavailability ephantsi ngokwentelekiso.Nangona kunjalo, xa i-G-Rg3 ilawulwa kunye ne-P-gp inhibitor verapamil, ukuphuma kwe-G-Rg3 kwiiseli ze-Caco-2 zehla, izinga lokufunxa kwamathumbu e-G-Rg3 lonyuswa kwimodeli yegundane, kunye ne-G-Rg3. yandiswa.Kwiiseli ze-Caco-2, ukuphuma kwe-Rg3 kunciphisa, kwaye izinga le-concentration ye-Rg3 liyancipha.I-G-Rg3 inyuswe emathunjini kunye ne-plasma, kwaye amandla ayo okuthintela amathumba nawo aphuculwe kwimodeli yegundane ye-B (a) P-induced tumorigenesis.Siphinde safumanisa ukuba i-G-Rg3 yanciphisa i-B (a) i-P-induced cytotoxicity kunye ne-DNA adduct formation kwiiseli zemiphunga yomntu, kwaye ibuyisele intetho kunye nomsebenzi we-enzyme yesigaba se-II ngokusebenzisa indlela ye-Nrf2, enokuthi ihambelane nendlela yokusebenza. ye-G inhibition -Rg3..Malunga nokuvela kwamathumba emiphungeni.Uphononongo lwethu lubonisa indima enokubakho ebalulekileyo ye-G-Rg3 ekujoliseni amathumba emiphunga kwiimodeli zempuku.I-bioavailability yomlomo yale ginsenoside iphuculwe ngokujolisa kwi-P-glycoprotein, ivumela i-molecule ukuba yenze iziphumo ze-anticancer.
Olona hlobo luxhaphakileyo lomhlaza wemiphunga ngumhlaza wemiphunga ongengowomncinane (NSCLC), ongoyena nobangela wokusweleka komhlaza eTshayina nakuMntla Melika1,2.Eyona nto yonyusa umngcipheko wokuba nomhlaza wemiphunga ongeyonxalenye encinci kukutshaya.Umsi wecuba uqulethe ngaphezu kwe-60 carcinogens, kuquka i-benzo(a)pyrene (B(a)P), i-nitrosamines, kunye ne-isotopi ye-radioactive evela ekuboleni kwe-radon.3 I-Polycyclic aromatics hydrocarbons B(a)P ngoyena nobangela wetyhefu kwicuba. ukutshaya.Emva kokuvezwa kwi-B (a) P, i-cytochrome P450 iyiguqulela kwi-B (a) P-7,8-dihydrodiol-9,10-epoxide (BPDE), ehambelana ne-DNA ukwenza i-BPDE-DNA adduct 4. Ukongezelela, ezi I-adducts yenza i-tumorigenesis ye-lung kwiimpuku ezinenqanaba lethumba kunye ne-histopathology efana namathumba emiphunga yomntu5.Olu phawu lwenza imodeli yomhlaza wemiphunga ye-B(a)-P ibe yinkqubo efanelekileyo yokuvavanya iikhompawundi ezineempawu zokulwa nomhlaza ezinokwenzeka.
Esinye isicwangciso esinokwenzeka sokuthintela ukuphuhliswa komhlaza wemiphunga kumaqela asemngciphekweni omkhulu, ngakumbi abatshayayo, kukusetyenziswa kwee-chemopreventive agents ukucinezela uphuhliso lwezilonda ze-intraepithelial neoplastic kwaye ngaloo ndlela zithintele ukuqhubela phambili kwabo okulandelayo kwi-malignancy.Izifundo zezilwanyana zibonisa ukuba ii-agent ezahlukeneyo ze-chemopreventive ziyasebenza6.Ingxelo yethu yangaphambili7 ibonise iziphumo ezilungileyo zokukhusela i-ginseng ebomvu kumhlaza wemiphunga.Le mifuno isetyenziswe iinkulungwane kumayeza emveli aseAsia ukwandisa ubomi kunye nempilo, kwaye ibhalwe ukuba ine-antitumor effects8.
Into esebenzayo ye-ginseng yi-ginsenoside, esetyenziswa njengemakishi edibeneyo yokuvavanya umgangatho we-ginseng extracts.Uhlalutyo lobungakanani bencindi yeginseng ekrwada lubandakanya ukusetyenziswa kweginsenosides ezininzi, kubandakanya i-RK1, Rg1, F1, Re, Rb1, Rb2, Rb3, Rd, Rh1, Rh2, Rg3, Rg5, kunye neRc9,10.I-Ginsenosides inosetyenziso oluncinci lwezonyango ngenxa yokungafumaneki kwabo ngomlomo11.Nangona umatshini wale bioavailability embi ayicacanga, i-efflux ye-ginsenosides ebangelwa yi-P-glycoprotein (P-gp) 12 ingaba yimbangela.I-P-gp yenye yezona zinto zibalulekileyo zokuthutha i-efflux kwi-ATP-binding cassette transporter superfamily, esebenzisa amandla e-ATP hydrolysis ukukhulula izinto ze-intracellular kwindawo yangaphandle.Abathuthi be-P-gp basasazwa ngokubanzi emathunjini, kwizintso, isibindi kunye nesithintelo segazi-ingqondo13.I-P-gp idlala indima ebalulekileyo ekufakweni kwamathumbu emathunjini, kwaye ukuvinjelwa kwe-P-gp kwandisa ukuxutywa komlomo kunye nokufumaneka kwezinye izidakamizwa ze-anticancer12,14.Imizekelo ye-inhibitors esetyenziswe ngaphambili kwiincwadi yi-verapamil kunye ne-cyclosporine A15.Lo msebenzi ubandakanya ukusekwa kwenkqubo yempuku yokufunda umhlaza wemiphunga we-B(a)P-P-ukuvavanya amandla eencindi zeginseng ebomvu ezahlukeneyo ezivela eTshayina naseKorea ukuchaphazela ubugwenxa.Izicatshulwa zahlalutywa ngabanye ukuchonga i-ginsenosides ethile enokuthi ichaphazele i-carcinogenesis.I-Verapamil yayisetyenziselwa ukujolisa kwi-P-gp kunye nokuphucula i-bioavailability yomlomo kunye nokusebenza konyango lwe-ginsenosides ekujoliswe kuyo ngumhlaza.
Indlela apho i-ginseng saponins yenza ngayo iziphumo zonyango kwi-carcinogenesis ayicacanga.Uphando luye lwabonisa ukuba i-ginsenosides eyahlukeneyo inokunciphisa umonakalo we-DNA obangelwa yi-carcinogens ngokunciphisa uxinzelelo lwe-oxidative kunye nokuvula i-enzyme ye-detoxification yesigaba se-II, ngaloo ndlela ikhusela umonakalo weseli.I-Glutathione S-transferase (GST) yi-enzyme yesigaba se-II esifunekayo ukunciphisa umonakalo we-DNA obangelwa yi-carcinogens17.I-Nuclear erythroid 2-related factor 2 (Nrf2) yinto ebalulekileyo yokubhala elawula i-redox homeostasis kwaye isebenze ukubonakaliswa kwesigaba se-II enzymes kunye ne-cytoprotective antioxidant responses18.Uphononongo lwethu luphinde luhlolisise imiphumo ye-ginsenosides echongiweyo ekunciphiseni i-B (a) i-P-induced cytotoxicity kunye ne-BPDE-DNA i-adduct formation, kunye nokunyanzeliswa kwe-enzyme yesigaba se-II ngokumodareyitha indlela ye-Nrf2 kwiiseli zemiphunga eziqhelekileyo.
Ukusekwa kwemodeli yemouse ye-B (a) ye-P-induced cancer ihambelana nomsebenzi wangaphambili5.Umzobo we-1A ubonisa uyilo lovavanyo lweveki ye-20 yonyango lwemodeli yomhlaza wempuku eyenziwa yi-B (a) P, amanzi (ukulawula), i-Chinese ebomvu ye-ginseng extract (CRG), i-ginseng ebomvu yaseKorea isicatshulwa A (KRGA), kunye nobomvu baseKorea. iginseng.Isicatshulwa B (KRGB) kunye neKorea Red Ginseng Extract C (KRGC).Emva kweeveki ze-20 zonyango lwe-ginseng ebomvu, iigundane zabingelelwa yi-CO2 asphyxiation.Umzobo 1B ubonisa amathumba emiphunga macroscopic kwizilwanyana ziphathwa ngeentlobo ezahlukeneyo zeginseng ebomvu, kwaye Umfanekiso 1C ubonisa ummeli ukukhanya micrograph isampuli ithumba.Umthwalo we-tumor wezilwanyana eziphathwe nge-KRGB (1.5 ± 0.35) yayingaphantsi kwezilwanyana zokulawula (0.82 ± 0.2, P <0.05), njengoko kuboniswe kuMfanekiso 1D.Umyinge weqondo lokuvinjelwa komthwalo wethumba lalingama-45%.Ezinye izicatshulwa ze-ginseng ezibomvu ezivavanyiweyo azizange zibonise utshintsho oluphawulekayo kumthwalo we-tumor (P> 0.05).Akukho miphumo ecacileyo yabonwa kwimodeli yegundane kwiiveki ze-20 zonyango lwe-ginseng ebomvu, kubandakanywa akukho tshintsho kwisisindo somzimba (idatha engaboniswa) kwaye akukho sibindi okanye ubuthi bezintso (Umfanekiso 1E, F).
Isicatshulwa se-ginseng esibomvu siphatha ukuphuhliswa kwethumba lemiphunga kwi-A / J iimpuku.(A) Uyilo lovavanyo.(B) Amathumba amakhulu emiphunga kwimodeli yempuku.Amathumba aboniswa ngeentolo.a: Iqela leginseng ebomvu yaseTshayina.b: iqela A le-ginseng ebomvu yaseKorea.c: Iqela le-ginseng ebomvu yaseKorea B. d: Iqela le-ginseng ebomvu yaseKorea C. d: Iqela lokulawula.(C) I-micrograph yokukhanya ebonisa ithumba lemiphunga.Ukwandiswa: 100. b: 400. (D) Umthwalo wethumba kwiqela elikhupha i-ginseng ebomvu.(E) Amanqanaba ePlasma ye-enzyme yesibindi i-ALT.(F) Amanqanaba e-Plasma ye-enzyme ye-renal Cr.Idatha ichazwa njengentsingiselo ± ukutenxa okusemgangathweni.*P <0.05.
Izicatshulwa ezibomvu ze-ginseng ezichongiweyo kolu phononongo zihlalutywe yi-ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) ukulinganisa ezi ginsenosides zilandelayo: Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 kunye neRg5.Iimeko ze-UPLC kunye ne-MS ezisetyenziselwa ukulinganisa abahlalutyi zichazwe kwingxelo yangaphambili19.Iikhromatogram ze-UPLC-MS/MS zezicatshulwa ezine ezibomvu ze-ginseng ziboniswa kuMfanekiso 2A.Kwakukho umahluko obalulekileyo kumxholo we-ginsenoside, kunye nomxholo ophezulu we-ginsenoside kwi-CRG (590.27 ± 41.28 μmol / L) (Umfanekiso 2B).Xa kuhlolwa i-ginsenosides nganye (Umfanekiso 2C), i-KRGB ibonise izinga eliphezulu le-G-Rg3 xa kuthelekiswa namanye ama-ginsenosides (58.33 ± 3.81 μmol / L ye-G-Rg3 kunye ne-41.56 ± 2.88 μmol / L ye-G -Rg3r).uhlobo lwe-ginseng ebomvu (P <0.001).I-G-Rg3 yenzeka njengesibini se-stereoisomers i-G-Rg3r kunye ne-G-Rg3s, eyahlukileyo kwindawo yeqela le-hydroxyl kwi-carbon 20 (Umfanekiso we-2D).Iziphumo zibonisa ukuba i-G-Rg3r okanye i-G-Rg3 inokuba namandla abalulekileyo e-anticancer kwimodeli yegundane ye-B(a)P eyenziwe ngumhlaza.
Umxholo we-ginsenosides kwiincindi ezahlukeneyo ezibomvu ze-ginseng.(A) Iikhromatogram ze-UPLC-MS/MS zezicatshulwa ezine ezibomvu ze-ginseng.(B) Uqikelelo lomxholo we-ginsenoside kwiincindi ezibonisiweyo.(C) Ukufunyanwa kwe-ginsenosides nganye kwiicatshulwa ezibhaliweyo.(D) Izakhiwo ze-ginsenoside stereoisomers G-Rg3r kunye ne-G-Rg3s.Idatha ichazwa njengentsingiselo ± ukutenxa komgangatho wokumisela ngokuphindwe kathathu.***P <0.001.
Uphononongo lwe-UPLC-MS / MS ludinga ubungakanani be-ginsenosides emathunjini kunye neesampuli zegazi emva kweeveki ze-20 zonyango.Unyango nge-KRGB lubonise ubukho be-0.0063 ± 0.0005 μg/ml Rg5 kuphela egazini.Akukho ginsenosides eseleyo efunyenweyo, ebonisa ukuba i-bioavailability yomlomo imbi kwaye ngoko ke iyancipha ukubonakaliswa kwezi ginsenosides.
I-colon adenocarcinoma cell line Caco-2 i-morphologically kunye ne-biochemically efana neeseli ze-epithelial zamathumbu omntu, ebonisa ukusetyenziswa kwayo ekuhloleni ukuthuthwa kwe-enterocyte kuwo wonke umqobo we-epithelial wamathumbu.Olu hlalutyo lwalusekelwe kwisifundo sangaphambili se-20.Amanani 3A, B, C, D, E, F abonisa imifanekiso emele yothutho lwe-transcellular ye-G-Rg3r kunye ne-G-Rg3 usebenzisa i-Caco-2 imodeli ye-monolayer.Ukuthuthwa kwe-Transcellular ye-G-Rg3r okanye i-G-Rg3 ngaphesheya kwe-Caco-2 i-monolayers ukusuka kwi-basolateral ukuya kwicala le-apical (Pb-a) yayiphezulu kakhulu kune-apical side ukuya kwi-basolateral side (Pa-b).Kwi-G-Rg3r, ixabiso eliphakathi kwe-Pa-b laliyi-0.38 ± 0.06, elonyuka laya ku-0.73 ± 0.06 emva konyango nge-50 μmol/L ye-verapamil kunye ne-1.14 ± 0.09 emva konyango nge-100 μmol/L ye-verapamil (p <0.001 kunye ne-verapamil) ngokulandelelanayo;3A).Ukuqwalaselwa kwe-G-Rg3 kulandele umzekelo ofanayo (Umfanekiso 3B), kwaye iziphumo zibonise ukuba unyango lwe-verapamil luphucule ukuthuthwa kwe-G-Rg3r kunye ne-G-Rg3.Unyango lwe-Verapamil luphinde lwaphumela ekunciphiseni okukhulu kwi-Pb-a kunye ne-G-Rg3r kunye ne-G-Rg3s efflux ratios (Umfanekiso 3C, D, E, F), ebonisa ukuba unyango lwe-verapamil lunciphisa umxholo we-ginsenoside kwiiseli ze-Caco-2 efflux..
Ukuthuthwa kwe-Transcellular ye-G-Rg3 kwi-Caco-2 i-monolayers kunye ne-intestinal absorption kwi-rat perfusion assay.(A) Ixabiso le-Pa-b leqela le-G-Rg3r kwiCaco-2 monolayer.(B) Ixabiso le-Pa-b lamaqela e-G-Rg3s kwiCaco-2 monolayer.(C) Ixabiso lePb leqela le-G-Rg3r kwiCaco-2 monolayer.(D) Ixabiso lePb lamaqela e-G-Rg3s kwiCaco-2 monolayer.(E) Umlinganiselo wesivuno samaqela e-G-Rg3r kwiCaco-2 monolayer.(F) Umlinganiselo wesivuno samaqela e-G-Rg3 kwiCaco-2 monolayer.(G) Ipesenti ye-intestinal absorption ye-G-Rg3r kwi-perfusion assay kwiigundane.(H) Ipesenti ye-intestinal absorption ye-G-Rg3 kwi-perfusion assay kwiigundane.I-Permeability kunye ne-absorption yathelekiswa ngaphandle kokongezwa kwe-verapamil.Idatha ichazwa njengentsingiselo ± ukutenxa okusemgangathweni kwemifuniselo emihlanu ezimeleyo.*P <0.05, **P <0.01, ***P <0.001.
Ngokuhambelana nomsebenzi wangaphambili we-20, i-orthotopic intestinal perfusion yeegundane yenziwa ukugqiba ukuba i-G-Rg3 i-absorption emathunjini iyanda emva kokunyangwa kwe-verapamil.Amanani 3G, H abonisa abameli bovavanyo lokuvavanya ipesenti yokufunxa amathumbu e-G-Rg3r kunye ne-G-Rg3 kwimodeli yomhlaza wegundane kwixesha elingasentla.Ipesenti yokuqala ye-G-Rg3r ebuthathaka yokuthatha malunga ne-10% inyuke ukuya ngaphezu kwe-20% emva konyango nge-50 μM ye-verapamil kunye nangaphezulu kwe-25% emva konyango nge-100 μM yeverapamil.Ngokunjalo, iG-Rg3, ebinokuthathwa okokuqala nge-10%, ikwabonise incopho engaphezulu kwe-20% emva konyango nge-50 μM yeverapamil kwaye phantse i-30% emva konyango nge-100 μM yeverapamil, ecebisa ukuba ukuvinjwa kwe-P-gp ngeverapamil kuyaphucula. amathumbu e-G-absorption Rg3 kwimodeli yempuku yomhlaza wemiphunga.
Ngokwale ndlela ingentla, iigundane zemodeli yomhlaza we-B (a) ye-P zahlulwa ngokungenamkhethe zibe ngamaqela amathandathu, njengoko kubonisiwe kuMfanekiso 4A.Akukho kuncipha kwesisindo esibalulekileyo okanye iimpawu zeklinikhi zetyhefu zabonwa kwiqela lonyango le-G-Rg3 xa kuthelekiswa neqela lokulawula (idatha engaboniswa).Emva kweeveki ezingama-20 zonyango, imiphunga yempuku nganye yaqokelelwa.Umzobo 4B ubonisa amathumba emiphunga macroscopic kwiimpuku kumaqela unyango angentla, kwaye Figure 4C ibonisa ummeli ukukhanya micrograph of ithumba ummeli.Ngokumalunga nomthwalo we-tumor kwiqela ngalinye (Fig. 4D), amaxabiso eempuku eziphathwe nge-G-Rg3r kunye ne-G-Rg3s yayiyi-0.75 ± 0.29 mm3 kunye ne-0.81 ± 0.30 mm3, ngokulandelelana, ngelixa amaxabiso e-G Mice aphathwa. nge -Rg3s yayiyi-1.63 ngokulandelelanayo ± 0.40 mm3.iigundane zokulawula (p <0.001), ebonisa ukuba unyango lwe-G-Rg3 lunciphisa umthwalo we-tumor kwiigundane.Ulawulo lweverapamil luphucule ngakumbi olu kuncipha: amaxabiso everapamil+ G-Rg3r ehle ukusuka ku-0.75 ± 0.29 mm3 ukuya ku-0.33 ± 0.25 mm3 (p <0.01), kwaye amaxabiso everapamil+ ukusuka ku-0.81 ± 0.30 mm3 ehle ukuya ku-± 0.30 mm29 mm3 kwi-G. -Rg3s-ephathwe iigundane (p <0.05), ebonisa ukuba i-verapamil inokunyusa umphumo we-inhibitory we-G-Rg3 kwi-tumorigenesis.Umthwalo we-tumor awubonisi nantlukwano ebalulekileyo phakathi kweqela lolawulo kunye neqela le-verapamil, iqela le-G-Rg3r kunye neqela le-G-Rg3s, kunye neqela le-verapamil + G-Rg3r kunye neqela le-verapamil + G-Rg3s.Ngaphezu koko, bekungekho nto ibalulekileyo yesibindi okanye ityhefu yezintso ezinxulumene nonyango oluvavanyiweyo (Umfanekiso 4E, F).
Umthwalo we-tumor emva kokunyanga kwe-G-Rg3 kunye ne-plasma okanye intestinal G-Rg3r kunye ne-G-Rg3 amanqanaba kumaqela abonisiwe.(A) Uyilo lovavanyo.(B) Izicubu zeMacroscopic kwimodeli yemouse.Amathumba aboniswa ngeentolo.a: G-Rg3r.b: G-Rg3s.c: G-Rg3r idibene neverapamil.d: G-Rg3 idibene neverapamil.d: iVerapamil.e: ulawulo.(C) I-Micrograph ye-Optical ye-tumor ekukhuliseni.Impendulo: 100x.b: 400X.(D) Umphumo we-G-Rg3 + unyango lwe-verapamil kumthwalo we-tumor kwiigundane ze-A/J.(E) Amanqanaba ePlasma ye-enzyme yesibindi i-ALT.(F) Amanqanaba e-Plasma ye-enzyme ye-renal Cr.(G) Amanqanaba e-Plasma ye-G-Rg3r okanye i-G-Rg3 yamaqela abonisiwe.(H) Amanqanaba e-G-Rg3r okanye i-G-Rg3 emathunjini amaqela abonisiwe.Idatha ichazwa njengentsingiselo ± ukutenxa komgangatho wokumisela ngokuphindwe kathathu.*P <0.05, **P <0.01, ***P <0.001.
Amanqanaba e-G-Rg3 kwi-B (a) ye-P-induced imodeli yeegundane zavavanywa yi-UPLC-MS / MS emva kwexesha lokunyanga kweeveki ze-20 ngokwendlela echazwe kwicandelo leNdlela.Amanani e-4G kunye ne-H abonisa i-plasma kunye ne-intestinal G-Rg3 amanqanaba, ngokulandelanayo.Amanqanaba e-Plasma G-Rg3r ayeyi-0.44 ± 0.32 μmol/L kwaye anyuke aye kwi-1.17 ± 0.47 μmol/L kunye nolawulo oluhambelanayo lweverapamil (p <0.001), ngelixa amanqanaba e-G-Rg3r emathunjini ayeyi-0.53 ± 0.08 µµ.Xa idibene neverapamil, i-g inyuke ukuya kwi-1.35 ± 0.13 μg/g (p <0.001).Kwi-G-Rg3, iziphumo zilandele iphethini efanayo, ebonisa ukuba unyango lwe-verapamil lwandise i-bioavailability yomlomo we-G-Rg3 kwiigundane ze-A/J.
Uvavanyo lokusebenza kweeseli lusetyenziselwa ukuvavanya i-cytotoxicity ye-B (a) P kunye ne-G-Rg3 kwiiseli ze-hEL.I-cytotoxicity eyenziwa yi-B (a) P kwiiseli ze-hEL iboniswe kwi-Figure 5A, ngelixa iipropati ezingenabungozi ze-G-Rg3r kunye ne-G-Rg3 ziboniswa kwiMifanekiso 5A kunye ne-5B.I-5B, C. Ukuvavanya umphumo we-cytoprotective we-G-Rg3, i-B (a) P ilawulwa ngokubambisana kunye neentlobo ezahlukeneyo ze-G-Rg3r okanye i-G-Rg3 kwiiseli ze-hEL.Njengoko kuboniswe kwi-Figure 5D, i-G-Rg3r ekugxininiseni kwe-5 μM, i-10 μM, kunye ne-20 μM ibuyiselwe ukusebenza kweeseli kwi-58.3%, 79.3%, kunye ne-77.3%, ngokulandelanayo.Iziphumo ezifanayo zinokubonwa kwiqela le-G-Rg3s.Xa izigxina ze-G-Rg3 ziyi-5 µM, i-10 µM kunye ne-20 µM, ukusebenza kweeseli kwabuyiselwa kwi-58.3%, 72.7% kunye ne-76.7%, ngokulandelanayo (Umfanekiso 5E).).Ubukho be-BPDE-DNA adducts bulinganiswe kusetyenziswa ikiti ye-ELISA.Iziphumo zethu zibonise ukuba amanqanaba e-adduct ye-BPDE-DNA anyuswe kwiqela le-B (a) P-ephathwayo xa kuthelekiswa neqela lokulawula, kodwa xa kuthelekiswa ne-G-Rg3 yonyango, amanqanaba e-BPDE-DNA adduct kwiqela le-B (a) P. B kwiqela eliphathwayo, amanqanaba e-adduct e-DNA ancitshiswa kakhulu.Iziphumo zonyango kunye ne-B (a) P kuphela ziboniswe kuMfanekiso 5F (1.87 ± 0.33 vs. 3.77 ± 0.42 ye-G-Rg3r, 1.93 ± 0.48 vs. 3.77 ± 0.42 ye-G -Rg3s, p <0.001).
Ukusebenza kweseli kunye ne-BPDE-DNA yokwenziwa kwe-adduct kwiiseli ze-hEL eziphathwa nge-G-Rg3 kunye ne-B (a) P.(A) Ukusebenza kweeseli ze-hEL eziphathwa nge-B (a) P.(B) Ukusebenza kweeseli ze-hEL eziphathwa nge-G-Rg3r.(C) Ukusebenza kweeseli ze-hEL eziphathwa nge-G-Rg3.(D) Ukusebenza kweeseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3r.(E) Ukusebenza kweeseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3.(F) Amanqanaba e-BPDE-DNA adduct kwiiseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3.Idatha ichazwa njengentsingiselo ± ukutenxa komgangatho wokumisela ngokuphindwe kathathu.*P <0.05, **P <0.01, ***P <0.001.
Ukubonakaliswa kwe-enzyme ye-GST kwafunyanwa emva kokunyangwa ngokubambisana kunye ne-10 μM B (a) P kunye ne-10 μM G-Rg3r okanye i-G-Rg3s.Iziphumo zethu zibonise ukuba i-B(a)P icinezele intetho ye-GST (i-59.7 ± 8.2% kwiqela le-G-Rg3r kunye ne-39 ± 4.5% kwiqela le-G-Rg3s), kwaye i-B(a)P yayinxulumene nokuba yi-G-Rg3r , okanye nge-G-Rg3r, okanye nge-G-Rg3r.Unyango kunye ne-G-Rg3s ibuyiselwe intetho ye-GST.Ukubonakaliswa kwe-GST (103.7 ± 15.5% kwiqela le-G-Rg3r kunye ne-110 ± 11.1% kwiqela le-G-Rg3s, p <0.05 kunye ne-p <0.001, ngokulandelanayo, i-Fig. 6A, B, kunye ne-C).Umsebenzi we-GST wavavanywa kusetyenziswa ikiti yovavanyo lomsebenzi.Iziphumo zethu zibonise ukuba iqela lonyango elidibeneyo linomsebenzi ophezulu we-GST xa kuthelekiswa ne-B (a) P kuphela iqela (96.3 ± 6.6% vs. 35.7 ± 7.8% kwiqela le-G-Rg3r vs. 92.3 ± 6.5 kwiqela le-G-Rg3r ).% vs 35.7 ± 7.8% kwiqela le-G-Rg3s, p <0.001, Umfanekiso 6D).
Ukubonakaliswa kwe-GST kunye ne-Nrf2 kwiiseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3.(A) Ukufunyanwa kwentetho ye-GST ngokucinywa kweNtshona.(B) Ukubonakaliswa kobungakanani be-GST kwiiseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3r.(C) Ukubonakaliswa kobungakanani be-GST kwiiseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3s.(D) Umsebenzi we-GST kwiiseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3.(E) Ukufunyanwa kwentetho ye-Nrf2 ngokucinywa kweNtshona.(F) Ukubonakaliswa kobungakanani be-Nrf2 kwiiseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3r.(G) Ukubonakaliswa kobungakanani be-Nrf2 kwiiseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3s.Idatha ichazwa njengentsingiselo ± ukutenxa komgangatho wokumisela ngokuphindwe kathathu.*P <0.05, **P <0.01, ***P <0.001.
Ukucacisa iindlela ezibandakanyekayo kwi-G-Rg3-mediated suppression ye-B (a) P-induced tumorigenesis, i-Nrf2 ibonakaliso ihlolwe yi-Western blotting.Njengoko kuboniswe kwiMifanekiso 6E, F, G, xa kuthelekiswa neqela lolawulo, kuphela inqanaba le-Nrf2 kwiqela le-B (a) P lonyango liye lancipha;nangona kunjalo, xa kuthelekiswa neqela le-B (a) P lonyango, i-B (a) amanqanaba e-Nrf2 kwiqela le-PG-Rg3 anyuswa (106 ± 9.5% ye-G-Rg3r vs. 51.3 ± 6.8%, 117 ± 6. 2% ngenxa I-G-Rg3r vs. 41 ± 9.8% ye-G-Rg3s, p <0.01).
Siqinisekise indima yokuthintela ye-Nrf2 ngokucinezela intetho ye-Nrf2 usebenzisa i-RNA encinci ephazamisayo (siRNA).Ukubetha kwe-Nrf2 kwaqinisekiswa ngokucinywa kweNtshona (Umfanekiso 7A, B).Njengoko kuboniswe kwiMifanekiso ye-7C, D, ukunyangwa ngokubambisana kweeseli ze-hEL kunye ne-B (a) P kunye ne-G-Rg3 kubangele ukuhla kwenani le-BPDE-DNA adducts (1.47 ± 0.21) xa kuthelekiswa nonyango nge-B (a) P yedwa kwiqela le-siRNA lolawulo.) I-G-Rg3r yayingu-4.13 ± 0.49, i-G-Rg3s yayingu-1.8 ± 0.32 kunye ne-4.1 ± 0.57, p <0.01).Nangona kunjalo, impembelelo yokuthintela ye-G-Rg3 kwi-BPDE-DNA yokwenziwa yapheliswa yi-Nrf2 knockdown.Kwiqela le-siNrf2, kwakungekho mmahluko omkhulu kwi-BPDE-DNA yokwakhiwa kwe-adduct phakathi kwe-B (a) P kunye ne-G-Rg3 yonyango kunye ne-B (a) P yonyango yodwa (3.0 ± 0.21 ye-G-Rg3r vs. 3.56 ± 0.32 ).kwi-G-Rg3r xa kuthelekiswa ne-3.6 ye-G-Rg3s ngokuchasene ne-± 0.45 ngokuchasene ne-4.0±0.37, p > 0.05).
Impembelelo ye-Nrf2 yokunkqonkqoza kwi-BPDE-DNA yokwenziwa kwe-adduct kwiiseli ze-hEL.(A) Ukubetha kwe-Nrf2 kwaqinisekiswa ngokucinywa kweNtshona.(B) Ubalo lobungakanani bebhendi ye-Nrf2.(C) Impembelelo ye-Nrf2 yokugoqa kwi-BPDE-DNA adduct amanqanaba kwiiseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3r.(D) Impembelelo ye-Nrf2 yokugoqa kwi-BPDE-DNA adduct amanqanaba kwiiseli ze-hEL eziphathwa nge-B (a) P kunye ne-G-Rg3.Idatha ichazwa njengentsingiselo ± ukutenxa komgangatho wokumisela ngokuphindwe kathathu.*P <0.05, **P <0.01, ***P <0.001.
Olu phononongo luvavanye iziphumo zothintelo lwezicatshulwa ezahlukeneyo zeginseng ebomvu kwimodeli yempuku ye-B (a) P-eyenziwe ngumhlaza wemiphunga, kunye nonyango lwe-KRGB lwehlise kakhulu umthwalo wethumba.Ukuqwalasela ukuba i-G-Rg3 inomxholo ophezulu kule ngqungquthela ye-ginseng, indima ebalulekileyo yale ginsenoside ekuvimbeleni i-tumorigenesis ifundwe.Bobabini i-G-Rg3r kunye ne-G-Rg3 (ii-epimers ezimbini ze-G-Rg3) zanciphisa kakhulu umthwalo we-tumor kwimodeli ye-mouse ye-B (a) ye-P-induced cancer.I-G-Rg3r kunye ne-G-Rg3 zenza iziphumo ze-anticancer ngokubangela i-apoptosis yeeseli ze-tumor21, inqanda ukukhula kwe-tumor22, ukubamba i-cell cycle23 kunye nokuchaphazela i-angiogenesis24.I-G-Rg3 nayo ibonakaliswe ukuba inqanda i-metastasis25 yeselula, kunye nokukwazi kwe-G-Rg3 ukunyusa imiphumo ye-chemotherapy kunye ne-radiotherapy ibhalwe26,27.U-Poon et al ubonise ukuba unyango lwe-G-Rg3 lunokunciphisa iziphumo ze-genotoxic ze-B(a)P28.Olu phononongo lubonisa amandla onyango e-G-Rg3 ekujoliseni iimolekyuli ze-carcinogenic zokusingqongileyo kunye nokuthintela umhlaza.
Nangona amandla abo afanelekileyo eprophylactic, i-bioavailability yomlomo engafanelekanga ye-ginsenosides ibeka umngeni wokusetyenziswa kweklinikhi kwezi molekyuli.Uhlalutyo lwe-Pharmacokinetic yokulawulwa komlomo we-ginsenosides kwiigundane lubonise ukuba i-bioavailability yayo isengaphantsi kwe-5% ye-29.Olu vavanyo lubonise ukuba emva kwexesha lokunyanga iiveki ezingama-20, kuphela amanqanaba egazi e-Rg5 ehlileyo.Nangona indlela esisiseko ye-bioavailability embi isahlala icaciswa, i-P-gp icingelwa ukuba ibandakanyeka kwi-efflux ye-ginsenosides.Lo msebenzi ubonise okokuqala ukuba ukulawulwa kwe-verapamil, i-blocker ye-P-gp, kwandisa i-bioavailability yomlomo we-G-Rg3r kunye ne-G-Rg3s.Ngaloo ndlela, oku kufunyanisiweyo kubonisa ukuba i-G-Rg3r kunye ne-G-Rg3s zisebenza njenge-substrates ye-P-gp ukulawula ukuphuma kwayo.
Lo msebenzi ubonisa ukuba unyango oludityanisiweyo kunye neverapamil lunyusa i-bioavailability yomlomo ye-G-Rg3 kwimodeli yempuku yomhlaza wemiphunga.Oku kufunyaniswayo kuxhaswa ngokunyuka kwe-intestinal transcellular transport ye-G-Rg3 kwi-blockade ye-P-gp, ngaloo ndlela inyusa ukufunxa kwayo.Ii-assays kwiiseli ze-Caco2 zibonise ukuba unyango lwe-verapamil lunciphisa ukukhutshwa kwe-G-Rg3r kunye ne-G-Rg3 ngelixa iphucula i-membrane permeability.Uphononongo olwenziwe nguYang et al.Uphononongo lubonise ukuba unyango nge-cyclosporine A (enye i-blocker ye-P-gp) yandisa i-bioavailability ye-ginsenoside Rh2 ukusuka kwixabiso lesiseko se-1% ukuya kwi-30%.I-Ginsenosides i-K kunye ne-Rg1 ibonise iziphumo ezifanayo30,31.Xa i-verapamil kunye ne-cyclosporin A idityaniswe ngokubambisana, i-efflux ye-compound K kwiiseli ze-Caco-2 yancitshiswa kakhulu ukusuka kwi-26.6 ukuya ngaphantsi kwe-3, ngelixa amanqanaba ayo e-intracellular anda i-40-fold30.Xa kukho i-verapamil, amanqanaba e-Rg1 anda kwiiseli ze-rat lung epithelial, ebonisa indima ye-P-gp kwi-ginsenoside efflux, njengoko kuboniswe nguMeng et al.31.Nangona kunjalo, i-verapamil ayizange ibe nefuthe elifanayo kwi-efflux yezinye i-ginsenosides (ezifana ne-Rg1, F1, Rh1 kunye ne-Re), ebonisa ukuba azichatshazelwa yi-P-gp substrates, njengoko kuboniswe nguLiang et al.32 .Oku kuqwalaselwa kunokunxulumana nokubandakanyeka kwabanye abathuthi kunye nezakhiwo ezizezinye ze-ginsenoside.
Indlela yokuthintela i-G-Rg3 kumhlaza ayicacanga.Izifundo zangaphambili zibonise ukuba i-G-Rg3 ikhusela umonakalo we-DNA kunye ne-apoptosis ngokunciphisa uxinzelelo lwe-oxidative kunye nokuvuvukala16,33, enokuthi ibe yindlela ephantsi yokukhusela i-B (a) P-induced tumorigenesis.Ezinye iingxelo zibonisa ukuba i-genotoxicity eyenziwa yi-B (a)P inokuncitshiswa ngokumodareyitha i-enzyme yesigaba se-II ukwenza i-BPDE-DNA34.I-GST yi-enzyme yesigaba se-II esiqhelekileyo esithintela i-BPDE-DNA i-adduct formation ngokukhuthaza ukubophelela kwe-GSH kwi-BPDE, ngaloo ndlela inciphisa umonakalo we-DNA obangelwa yi-B (a) P35.Iziphumo zethu zibonisa ukuba unyango lwe-G-Rg3 lunciphisa i-cytotoxicity ye-B (a) ye-P kunye ne-BPDE-DNA yokubunjwa kwe-adduct kwiiseli ze-hEL kwaye ibuyisela ukubonakaliswa kwe-GST kunye nomsebenzi we-vitro.Nangona kunjalo, ezi ziphumo zazingekho ngokungabikho kwe-Nrf2, ebonisa ukuba i-G-Rg3 yenza imiphumo ye-cytoprotective ngokusebenzisa indlela ye-Nrf2.I-Nrf2 iyona nto ibalulekileyo yokukhutshelwa kwisigaba se-II se-enzymes ye-detoxification ekhuthaza ukucocwa kwe-xenobiotics36.Ukusebenza kwendlela ye-Nrf2 kubangela i-cytoprotection kwaye inciphisa umonakalo wezicubu37.Ngaphezu koko, iingxelo ezininzi ziye zaxhasa indima ye-Nrf2 njenge-tumor suppressor kwi-carcinogenesis38.Uphononongo lwethu lubonisa ukuba ukufakwa kwendlela ye-Nrf2 nge-G-Rg3 idlala indima ebalulekileyo yokulawula kwi-B (a) i-P-induced genotoxicity ngokubangela i-B (a) P detoxification ngokuvula i-enzyme yesigaba se-II, ngaloo ndlela inqanda inkqubo ye-tumorigenesis.
Umsebenzi wethu utyhila amandla e-ginseng ebomvu ekuthinteleni umhlaza wemiphunga we-B (a) P-owenziwe kwiimpuku ngokubandakanyeka okubalulekileyo kwe-ginsenoside G-Rg3.Ukungafumaneki kakuhle kwe-bioavailability yomlomo kule molekyuli kuthintela ukusetyenziswa kwayo kwezonyango.Nangona kunjalo, olu phononongo lubonisa okokuqala ukuba i-G-Rg3 i-substrate ye-P-gp, kwaye ukulawulwa kwe-P-gp inhibitor kwandisa i-bioavailability ye-G-Rg3 kwi-vitro kunye ne-vivo.I-G-Rg3 yehlisa i-B(a)P-induced cytotoxicity ngokulawula indlela ye-Nrf2, enokuba yinto enokubakho kumsebenzi wayo wokuthintela.Uphononongo lwethu luqinisekisa ukubanakho kweginsenoside G-Rg3 kuthintelo kunye nonyango lomhlaza wemiphunga.
Iimpuku eziziimazi ezineeveki ezintandathu ze-A/J (20 ± 1 g) kunye neempuku ze-Wistar ezineveki ezisi-7 ubudala (250 ± 20 g) zafunyanwa kwi-Jackson Laboratory (i-Bar Harbour, e-USA) kunye ne-Wuhan Institute of Zoology.IYunivesithi (eWuhan, China).IZiko leNgqokelela yeNkcubeko yaseTshayina (i-Wuhan, e-China) yasinika i-Caco-2 kunye neeseli ze-hEL.I-Sigma-Aldrich (eSt. Louis, eU.SA) ngumthombo we-B (a) P kunye ne-tricaprine.I-ginsenosides ecocekileyo i-G-Rg3r kunye ne-G-Rg3s, i-dimethyl sulfoxide (DMSO), i-CellTiter-96 proliferation assay kit (MTS), i-verapamil, i-minimential medium (MEM), kunye ne-fetal bovine serum (FBS) zathengwa kwi-Chengdu Must Bio-Technology .Co.,Ltd.(eChengdu, eTshayina).I-QIAamp DNA mini kit kunye ne-BPDE-DNA adduct ELISA kit yathengwa kwi-Qiagen (Stanford, CA, USA) kunye ne-Cell Biolabs (San Diego, CA, USA).Ikiti yokuvavanya umsebenzi we-GST kunye nekiti yovavanyo lweprotheyini iyonke (indlela eqhelekileyo ye-BCA) yathengwa kwiSolarbio (eBeijing, eChina).Zonke izicatshulwa ezibomvu ze-ginseng zigcinwe kwi-Mingyu Laboratory 7. IYunivesithi yase-Hong Kong yaseBhaptizi (i-Hong Kong, i-China) kunye ne-Korea Cancer Centre (i-Seoul, eKorea) yimithombo yorhwebo ye-CRG isicatshulwa kunye nezicatshulwa ezahlukeneyo ezibomvu ze-ginseng zemvelaphi eyahlukeneyo yaseKorea (kuquka i-KRGA, i-KRGB kunye ne-KRGC).I-ginseng ebomvu yenziwe kwiingcambu ze-ginseng entsha eneminyaka emi-6 ubudala.Isicatshulwa esibomvu se-ginseng sifunyanwa ngokuhlamba i-ginseng ngamanzi amaxesha amathathu, emva koko igxininise isicatshulwa esinamanzi, kwaye ekugqibeleni yome kwiqondo eliphantsi lobushushu ukufumana i-ginseng isicatshulwa somgubo.Ama-Antibodies (i-anti-Nrf2, i-anti-GST, kunye ne-β-actin), i-horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG), i-reagent transfection, i-siRNA yokulawula, kunye ne-Nrf2 siRNA zathengwa kwi-Santa Cruz Biotechnology (Santa Cruz, CA) .), I-USA).
I-Caco2 kunye neeseli ze-hEL zaye zakhuliswa kwi-100 mm2 izitya zenkcubeko yeeseli kunye ne-MEM equkethe i-10% ye-FBS kwi-37 ° C kwindawo enomswakama we-5% CO2.Ukumisela umphumo weemeko zonyango, iiseli ze-hEL zaye zaxutywa ngeentlobo ezahlukeneyo ze-B (a) P kunye ne-G-Rg3 kwi-MEM ye-48 h.Iiseli zinokuphinda zihlalutywe okanye ziqokelelwe ukuze kulungiswe izicatshulwa ezingenaselseli.
Zonke iimvavanyo zivunyiwe yiKomidi yokuSebenza yeZilwanyana zoVavanyo kwiKholeji yezoNyango yaseTongji, iYunivesithi yaseHuazhong yeSayensi kunye neTeknoloji (iNombolo yokuVumela i-2019; iNombolo yoBhaliso ye-4587TH).Zonke iimvavanyo zenziwa ngokuhambelana nezikhokelo kunye nemimiselo efanelekileyo, kwaye isifundo senziwe ngokuhambelana noPhando lwezilwanyana: Ukunika ingxelo ye-In Vivo Experiments (ARRIVE) izikhokelo.Iigundane ze-A / J ezineveki ezisibhozo zaqala ukujova nge-intraperitoneally nge-B (a) P kwisisombululo se-tricaprine (100 mg / kg, 0.2 ml).Emva kweveki, iigundane zahlulwe ngokungenamkhethe ngokwamaqela olawulo kunye namaqela ahlukeneyo onyango, iigundane ze-15 kwiqela ngalinye, kwaye zahluthwa kanye ngosuku.Emva kweeveki ezingama-20 zonyango, izilwanyana zabingelelwa yi-CO2 asphyxia.Imiphunga yaqokelelwa yaza yalungiswa iiyure ezingama-24.Inani lamathumba angaphezulu kunye nobukhulu bethumba ngalinye babalwa kumphunga ngamnye phantsi kwe-microscope yokuhlambulula.Uqikelelo lomthamo wethumba (V) lubalwe kusetyenziswa le ntetho ilandelayo: V (mm3) = 4/3πr3, apho r yi-diameter yethumba.I-net sum of all ithumba imithamo kwimiphunga yeempuku imele umthamo wethumba lilonke, kunye ne-avareji yomthamo wethumba lilonke kwiqela ngalinye limele umthwalo wethumba.Igazi elipheleleyo kunye neesampulu zamathumbu zaqokelelwa kwaye zagcinwa kwi-−80 ° C ukwenzela ukuzimisela kwe-UPLC-MS/MS.I-Serum yaqokelelwa kwaye i-automated chemistry analyzer isetyenziselwa ukuhlalutya i-alanine aminotransferase (ALT) kunye ne-serum creatinine (Cr) amanqanaba okuvavanya ukusebenza kwesibindi kunye nezintso.
Iisampulu eziqokelelweyo zisuswe kwindawo yokugcina ebandayo, zinyibilikisiwe, zilinganiswe, kwaye zifakwe kwiibhubhu njengoko kuchaziwe ngasentla.Kule nto yongezwa i-0.5 μM i-phhlorizin (umgangatho wangaphakathi) kwi-0.8 ml isisombululo se-methanol.I-tissue yabe i-homogenized isebenzisa i-Tissue-Tearor kwaye i-homogenate yathunyelwa kwi-tube ye-microcentrifuge ye-1.5 ml.Umxube wawuyi-centrifuged kwi-15500 rpm imizuzu eyi-15.Emva kokususa i-1.0 ml ye-supernatant, yome nge-nitrogen.Amakhulu amabini e-microliters e-methanol asetyenziselwa ukubuyisela.Igazi liqokelelwa kwaye licutshungulwe kumgca omnye kwaye lisetyenziswe njengesalathisi kuyo yonke imilinganiselo.
Iipleyiti ze-24-qula ze-Transwell zifakwe kwi-1.0 × 105 iiseli ze-Caco-2 kwiqula ngalinye ukuvavanya ukuphuculwa okunokwenzeka kwe-G-Rg3 yothutho ngokongezwa kwe-verapamil.Emva kweeveki ezi-3 zenkcubeko, iiseli zahlanjwa nge-HBSS kwaye zafakwa ngaphambili kwi-37 ° C.I-400 μL ye-10 μM G-Rg3 (G-Rg3r, G-Rg3s, okanye umxube onama-50 okanye i-100 μM i-verapamil) yatofwa kwicala elisezantsi okanye le-apical le-monolayer, kunye ne-600 μL yesisombululo se-HBSS yongezwa kwelinye. icala.Qokelela i-100 µl yesikhulumi senkcubeko ngamaxesha amisiweyo (0, 15, 30, 45, 60, 90 kunye ne-120 imizuzu) uze udibanise i-100 µl ye-HBSS ukwenza lo mqulu.Iisampulu zagcinwa ku-−4 °C de zabhaqwa yiUPLC-MS/MS.Ibinzana elithi Papp = dQ/(dT × A × C0) lisetyenziselwa ukulinganisa i-apical unidirectional ebonakalayo kunye ne-basolateral permeability kunye vice versa (Pa-b kunye ne-Pb-a, ngokulandelanayo);I-dQ/dT lutshintsho ekugxininiseni, i-A (0.6 cm2) yindawo engaphezulu ye-monolayer, kwaye i-C0 yingxininiso yokuqala yabanikeli.Umyinge we-efflux ubalwa njenge-Pb-a/Pa-b, emele iqondo lokukhukula iyeza lophononongo.
Iigundane ze-Male Wistar zazizila ukutya kwiiyure ze-24, zasela amanzi kuphela, kwaye zifakwe i-anesthetized nge-intravenous injection ye-3.5% yesisombululo se-pentobarbital.Ithubhu ye-silicone ene-intubated inesiphelo se-duodenum njengomnyango kunye nesiphelo se-ileum njengokuphuma.Sebenzisa impompo yeperistaltic ukumpompa i-inlet nge-10 µM G-Rg3r okanye i-G-Rg3s kwi-isotonic HBSS ngesantya sokuhamba kwe-0.1 ml/min.Umphumo we-verapamil uhlolwe ngokongeza i-50 μM okanye i-100 μM yekhompawundi kwi-10 μM G-Rg3r okanye i-G-Rg3s.I-UPLC-MS / MS yenziwa kwii-perfusion extracts eziqokelelwe ngexesha lamanqaku e-60, i-90, i-120, kunye ne-150 imizuzu emva kokuqala kwe-perfusion.Ipesenti yokufunxa ibalwa ngefomula % ukufunxa = (1 - Cout / Cin) × 100%;Ugxininiso lwe-G-Rg3 kwindawo yokuphuma kunye ne-inlet iboniswa yi-Cout kunye ne-Cin, ngokulandelanayo.
Iiseli ze-hEL zahlwayelwa kwiiplate ze-96-well kubuninzi be-1 × 104 iiseli ngequla kwaye ziphathwe nge-B (a) P (0, 1, 5, 10, 20, 30, 40 μM) okanye i-G-Rg3 echithwe kwi-DMSO .Amachiza ke aye axutywa nge-culture medium ukuya kwii-concentrations ezahlukeneyo (0, 1, 2, 5, 10, 20 μM) ngaphezu kweeyure ezingama-48.Ukusebenzisa ikhithi yokuvavanya ye-MTS ekhoyo ngokurhweba, iiseli ziphantsi kweprotocol esemgangathweni kwaye emva koko zilinganiswe ngokusebenzisa i-microplate reader kwi-490 nm.Inqanaba lokusebenza kweeseli zamaqela aphathwe ngokubambisana ne-B (a) P (10 μM) kunye ne-G-Rg3 (0, 1, 5, 10, 20 μM) yavavanywa ngokwendlela engentla kwaye ithelekiswa neqela elingaphathwanga.
Iiseli ze-hEL zahlwayelwa kwiiplati ze-6-well kubuninzi be-1 × 105 iiseli / kakuhle kwaye ziphathwa nge-10 μMB (a) P phambi okanye ukungabikho kwe-10 μM G-Rg3.Emva kweeyure ze-48 zonyango, i-DNA ikhutshwe kwiiseli ze-hEL isebenzisa i-QIAamp DNA Mini Kit ngokweprotocol yomenzi.Ukuqulunqwa kwe-BPDE-DNA adducts kwafunyanwa kusetyenziswa i-BPDE-DNA adduct ELISA kit.Amanqanaba anxulumene ne-BPDE-DNA adduct alinganiswa kusetyenziswa i-microplate reader ngokulinganisa i-absorbence kwi-450 nm.
Iiseli ze-hEL zahlwayelwa kwiiplati ze-96-well kubuninzi be-1 × 104 iiseli ngequla kwaye ziphathwe nge-10 μMB (a) P ngokungabikho okanye ubukho be-10 μM G-Rg3 kwi-48 h.Umsebenzi we-GST ulinganiswe kusetyenziswa ikiti yokuvavanya umsebenzi we-GST ngokwenkqubo yomenzi.Ukusebenza kwe-GST ehambelanayo kulinganiswa ngokuxhamla kwi-450 nm usebenzisa i-microplate reader.
Iiseli ze-hEL zahlanjwa nge-PBS ebandayo ye-ice kwaye emva koko i-lysed usebenzisa i-radioimmunoprecipitation assay buffer equkethe i-protease inhibitors kunye ne-phosphatase inhibitors.Emva kokulinganisa iprotheni usebenzisa i-protein assay kit, i-30 μg yeprotheni kwisampuli nganye yahlulwe yi-12% ye-SDS-PAGE kwaye idluliselwe kwi-membrane ye-PVDF nge-electrophoresis.Iimembranes zavalwa nge-5% yobisi lweskim kwaye zifakelwe izilwa-buhlungu eziphambili ngobusuku obukwi-4°C.Emva kokufakwa kunye ne-horseradish peroxidase-conjugated antibodies yesibini, i-chemiluminescence reagents ephuculweyo yongezwa ukuze ibone umqondiso wokubopha.Ubunzulu bebhendi yeprotheni nganye yalinganiswa kusetyenziswa isoftware ye-ImageJ.
Isoftware yeGraphPad Prism 7.0 yasetyenziselwa ukuhlalutya yonke idatha, ichazwe njengentsingiselo ± ukutenxa okuqhelekileyo.Ukwahluka phakathi kwamaqela onyango kwavavanywa kusetyenziswa uvavanyo loMfundi okanye uhlalutyo lwendlela enye yokwahluka, ngexabiso le-P <0.05 ebonisa ukubaluleka kwamanani.
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Ixesha lokuposa: Sep-17-2023