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I-ginseng ebomvu isetshenziswe emithini yendabuko yase-Asia amakhulu eminyaka.Kulolu cwaningo, sihlole ikhono lezinhlobo ezine ze-ginseng ebomvu (i-ginseng ebomvu yaseShayina, i-ginseng ebomvu yaseKorea, i-ginseng ebomvu yaseKorea B, ne-ginseng ebomvu yaseKorea C) etshalwe ezifundeni ezahlukene ukuvimbela ukwakheka nokukhula kwamaphaphu abangelwa umdlavuza. izimila.Ukuhlolwa kwe-benzo(a)pyrene (B(a)P) kwenziwa kumagundane e-A/J, futhi i-ginseng ebomvu yaseKorea itholwe iphumelela kakhulu ekwehliseni isimila phakathi kwezinhlobo ezine ze-ginseng ebomvu.Ngaphezu kwalokho, sihlaziye okuqukethwe kwama-ginsenosides ahlukahlukene (i-Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 ne-Rg5) ekukhishweni okune kwe-ginseng ebomvu sathola ukuthi i-ginseng ebomvu yaseKorea amazinga aphezulu kakhulu e-ginsenoside Rg3 (G-Rg3), okuphakamisa ukuthi i-G-Rg3 ingase idlale indima ebalulekile ekusebenzeni kwayo kokwelapha.Lo msebenzi ubonisa ukuthi i-G-Rg3 inokutholakala kwe-bioavailability okuphansi uma kuqhathaniswa.Kodwa-ke, lapho i-G-Rg3 isetshenziswa kanyekanye ne-P-gp inhibitor verapamil, ukuphuma kwe-G-Rg3 kumaseli e-Caco-2 kwehliswa, izinga lokumuncwa kwamathumbu e-G-Rg3 lenyuka ngemodeli yamagundane, kwathi i-G-Rg3. kwandiswa.Emaseli e-Caco-2, ukuphuma kwe-Rg3 kuncipha, futhi izinga lokuhlushwa kwe-Rg3 liyancipha.I-G-Rg3 iyanda emathunjini nakuplasma, futhi ikhono layo lokuvimbela izimila liphinde lithuthukiswe kumodeli yegundane ye-B(a)P-induced tumorigenesis.Siphinde sathola ukuthi i-G-Rg3 inciphise i-B(a)P-induced cytotoxicity kanye nokwakheka kwe-adduct ye-DNA kumaseli wamaphaphu omuntu, futhi yabuyisela inkulumo nomsebenzi wama-enzyme esigaba II ngokusebenzisa indlela ye-Nrf2, engase ihlobane nendlela yokusebenza engaba khona. ye-G inhibition -Rg3..Mayelana nokuvela kwezimila zamaphaphu.Ucwaningo lwethu lubonisa indima engaba namandla ye-G-Rg3 ekuqondiseni izimila zamaphaphu kumamodeli wamagundane.I-oral bioavailability yale ginsenoside ithuthukiswa ngokukhomba i-P-glycoprotein, okuvumela i-molecule ukuthi yenze imiphumela yokulwa nomdlavuza.
Uhlobo oluvame kakhulu lomdlavuza wamaphaphu umdlavuza wamaphaphu ongewona omncane (NSCLC), ongesinye sezimbangela eziphambili zokufa komdlavuza eChina naseNyakatho Melika1,2.Isici esiyinhloko esandisa ingozi yokuba nomdlavuza wamaphaphu ongewona omncane ukubhema.Intuthu kagwayi iqukethe ngaphezu kuka-60 carcinogens, kuhlanganise benzo(a)pyrene (B(a)P), nitrosamines, futhi isotopes radioactive kusukela ukubola radon.3 Polycyclic aromatics hydrocarbons B(a)P iyimbangela eyinhloko yobuthi kugwayi. bhema.Ngemva kokuchayeka ku-B(a)P, i-cytochrome P450 iyiguqulela ku-B(a)P-7,8-dihydrodiol-9,10-epoxide (BPDE), ehlangana ne-DNA yakhe i-BPDE-DNA adduct 4. Ukwengeza, lezi Ama-adducts afaka i-tumorigenesis yamaphaphu kumagundane anesiteji sesimila kanye ne-histopathology efana nezimila zamaphaphu omuntu5.Lesi sici senza imodeli yomdlavuza wamaphaphu e-B(a)P ibe yisistimu efanelekile yokuhlola izinhlanganisela ezinezici zokulwa nomdlavuza ezingaba khona.
Elinye isu elingenzeka lokuvimbela ukuthuthukiswa komdlavuza wamaphaphu emaqenjini asengozini enkulu, ikakhulukazi ababhemayo, ukusetshenziswa kwezidakamizwa ze-chemopreventive ukucindezela ukuthuthukiswa kwezilonda ze-intraepithelial neoplastic futhi ngaleyo ndlela kuvimbele ukuqhubekela phambili kwabo ekuguleni.Ucwaningo lwezilwane lukhombisa ukuthi ama-chemopreventive agents ahlukahlukene ayasebenza6.Umbiko wethu wangaphambilini7 wagqamisa imiphumela emihle yokuvimbela i-ginseng ebomvu kumdlavuza wamaphaphu.Lesi sitshalo sisetshenziswe amakhulu eminyaka emithini yendabuko yase-Asia ukwandisa impilo nempilo, futhi kubhalwe ukuthi kunemiphumela ye-antitumor8.
Isici esisebenzayo se-ginseng yi-ginsenoside, esetshenziswa njengomaka oyinhlanganisela ukuhlola ikhwalithi ye-ginseng extracts.Ukuhlaziywa okulinganiselwe kokukhishwe kwe-ginseng engahluziwe ngokuvamile kuhilela ukusetshenziswa kwama-ginsenosides amaningana, okuhlanganisa i-RK1, Rg1, F1, Re, Rb1, Rb2, Rb3, Rd, Rh1, Rh2, Rg3, Rg5, kanye ne-Rc9,10.Ama-ginsenosides anokusetshenziswa okuncane emtholampilo ngenxa yokungabi bikho kwawo kahle ngomlomo11.Nakuba indlela yalokhu kutholakala kwe-bioavailability empofu ingacacile, ukuphuma kwe-ginsenosides okubangelwa i-P-glycoprotein (P-gp)12 kungase kube imbangela.I-P-gp ingesinye sezithuthi ezibaluleke kakhulu ze-efflux emndenini omkhulu we-ATP-binding cassette transporter, esebenzisa amandla e-ATP hydrolysis ukukhulula izinto ezingaphakathi kweseli endaweni yangaphandle.Izithuthi ze-P-gp zivame ukusatshalaliswa kabanzi emathunjini, ezinso, esibindini nasengqondweni yegazi13.I-P-gp idlala indima ebalulekile ekumunceni emathunjini, futhi ukuvinjelwa kwe-P-gp kwandisa ukumuncwa komlomo nokutholakala kwezinye izidakamizwa ezilwa nomdlavuza12,14.Izibonelo zama-inhibitor asetshenziswe ngaphambilini ezincwadini yi-verapamil ne-cyclosporine A15.Lo msebenzi uhilela ukusungula uhlelo lwegundane lokutadisha umdlavuza wamaphaphu obangelwa u-B(a)P ukuze kuhlolwe ikhono lezindatshana ezihlukene ze-ginseng ebomvu ezivela e-China nase-Korea ukuze zithinte izifo.Okukhishwayo kwahlaziywa ngakunye ukuze kuhlonzwe ama-ginsenoside athile angase athinte i-carcinogenesis.I-Verapamil yabe isetshenziselwe ukukhomba i-P-gp futhi ithuthukise i-bioavailability yomlomo kanye nempumelelo yokwelapha ye-ginsenosides eqondiswe umdlavuza.
Indlela i-ginseng saponins eyenza ngayo imiphumela yokwelapha ku-carcinogenesis ihlala ingacacile.Ucwaningo luye lwabonisa ukuthi ama-ginsenosides ahlukahlukene anganciphisa ukulimala kwe-DNA okubangelwa ama-carcinogens ngokunciphisa ukucindezeleka okwenziwe nge-oxidative futhi kusebenze ama-enzyme e-detoxification esigaba II, ngaleyo ndlela avimbele ukulimala kwamangqamuzana.I-Glutathione S-transferase (GST) iyi-enzyme yesigaba II esidingekayo ukuze kuncishiswe umonakalo we-DNA obangelwa ama-carcinogens17.I-Nuclear erythroid 2-related factor 2 (Nrf2) iyisici esibalulekile sokuloba esilawula i-redox homeostasis futhi isebenze ukuvezwa kwe-enzyme yesigaba II kanye nezimpendulo ze-cytoprotective antioxidant18.Ucwaningo lwethu luphinde lwahlola imiphumela ye-ginsenosides ehlonziwe ekunciphiseni i-B (a) i-P-induced cytotoxicity kanye nokwakheka kwe-adduct ye-BPDE-DNA, kanye nokufaka ama-enzyme esigaba II ngokumodela indlela ye-Nrf2 kumaseli wamaphaphu avamile.
Ukusungulwa kwemodeli yegundane lomdlavuza we-B(a)P owenziwe kuyahambisana nomsebenzi wangaphambilini5.Umfanekiso 1A ukhombisa idizayini yokuhlola yokwelashwa kwamasonto angama-20 kwemodeli yomdlavuza wegundane elenziwa i-B(a)P, amanzi (control), i-Chinese red ginseng extract (CRG), i-ginseng ebomvu yaseKorea ekhipha A (KRGA), kanye nokubomvu kwaseKorea. i-ginseng.I-Extract B (KRGB) kanye ne-Korean Red Ginseng Extract C (KRGC).Ngemuva kwamaviki angu-20 okwelashwa kwe-ginseng ebomvu, amagundane ahlatshwa yi-CO2 asphyxiation.Umfanekiso 1B ubonisa izimila zamaphaphu ezinkulu ezilwaneni ezilashwa ngezinhlobo ezahlukene ze-ginseng ebomvu, futhi Umfanekiso 1C ubonisa i-micrograph ekhanyayo emele isampula yesimila.Umthwalo wesimila wezilwane ezilashwe yi-KRGB (1.5 ± 0.35) wawuphansi kunalowo wezilwane ezilawulayo (0.82 ± 0.2, P <0.05), njengoba kuboniswe kuMfanekiso 1D.I-avareji yezinga lokuvinjwa kwe-tumor load kwaba ngu-45%.Okunye okukhishwe kwe-ginseng ebomvu okuhloliwe akuzange kubonise izinguquko ezinkulu kangako kumthwalo wesimila (P > 0.05).Ayikho imiphumela emibi ecacile eyabonwa kumodeli yegundane phakathi namaviki angu-20 wokwelashwa kwe-ginseng ebomvu, okuhlanganisa ukungabi nashintsho esisindweni somzimba (idatha engabonisiwe) futhi akukho ubuthi besibindi noma bezinso (Umfanekiso 1E, F).
Ukukhishwa kwe-ginseng ebomvu kuphatha ukukhula kwesimila samaphaphu kumagundane e-A/J.(A) Idizayini yokuhlola.(B) Izimila zamaphaphu ezinkulu kumodeli yegundane.Amathumba aboniswa ngemicibisholo.a: Iqembu le-ginseng elibomvu laseShayina.b: iqembu A le-ginseng ebomvu yaseKorea.c: Iqembu le-ginseng ebomvu yaseKorea B. d: Iqembu le-ginseng ebomvu yaseKorea C. d: Iqembu lokulawula.(C) I-micrograph elula ebonisa isimila samaphaphu.Ukukhulisa: 100. b: 400. (D) Umthwalo wethumba eqenjini elikhipha i-ginseng elibomvu.(E) Amazinga e-Plasma we-enzyme yesibindi i-ALT.(F) Amazinga e-Plasma we-enzyme ye-renal Cr.Idatha ichazwa njengokushiwo ± ukuchezuka okujwayelekile.*P <0.05.
Izingcaphuno ze-ginseng ezibomvu ezikhonjwe kulolu cwaningo zahlaziywa nge-ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) ukuze kulinganiswe ama-ginsenoside alandelayo: Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3 , I-Rh2, F1, Rk1 kanye ne-Rg5.Izimo ze-UPLC ne-MS ezisetshenziselwa ukukala abahlaziyi zichazwe embikweni wangaphambilini19.Ama-chromatogram e-UPLC-MS/MS ezingxenye ezine ezibomvu ze-ginseng aboniswa kuMfanekiso 2A.Kube nomehluko obalulekile kokuqukethwe kwe-ginsenoside isiyonke, enenani eliphakeme kakhulu lokuqukethwe kwe-ginsenoside ku-CRG (590.27 ± 41.28 μmol/L) (Umfanekiso 2B).Lapho ihlola ama-ginsenosides angawodwana (Umfanekiso 2C), i-KRGB ibonise izinga eliphakeme kakhulu le-G-Rg3 uma liqhathaniswa namanye ama-ginsenosides (58.33 ± 3.81 μmol/L ku-G-Rg3s kanye no-41.56 ± 2.88 μmol/L ku-G -Rg3r).uhlobo lwe-ginseng ebomvu (P <0.001).I-G-Rg3 yenzeka njengepheya lama-stereoisomers i-G-Rg3r kanye ne-G-Rg3s, ahluke ngokuma kweqembu le-hydroxyl ku-carbon 20 (Fig. 2D).Imiphumela ibonisa ukuthi i-G-Rg3r noma i-G-Rg3 ingase ibe namandla abalulekile okulwa nomdlavuza kumodeli yegundane lomdlavuza elibangelwa u-B(a)P.
Okuqukethwe kwama-ginsenosides ekukhishweni okuhlukahlukene kwe-ginseng ebomvu.(A) I-UPLC-MS/MS chromatograms yezingcaphuno ezine ezibomvu ze-ginseng.(B) Isilinganiso sengqikithi yokuqukethwe kwe-ginsenoside kuma-extracts abonisiwe.(C) Ukutholwa kwama-ginsenosides ngamanye kuma-extracts anelebula.(D) Izakhiwo ze-ginsenoside stereoisomers G-Rg3r kanye ne-G-Rg3s.Idatha ichazwa njengencazelo ± ukuchezuka okujwayelekile kokunquma okuphindwe kathathu.***P <0.001.
Ucwaningo lwe-UPLC-MS/MS lwaludinga ukulinganiswa kwe-ginsenosides emathunjini nakumasampula egazi ngemva kwamasonto angu-20 okwelashwa.Ukwelashwa nge-KRGB kubonise ukuba khona kuka-0.0063 ± 0.0005 μg/ml Rg5 kuphela egazini.Awekho ama-ginsenoside asele atholiwe, abonisa ukungatholakali kahle kwe-oral bioavailability ngakho-ke ukunciphisa ukuchayeka kulawa ma-ginsenosides.
I-colon adenocarcinoma cell line i-Caco-2 i-morphologically kanye ne-biochemically efana namangqamuzana e-epithelial amathumbu omuntu, okubonisa ukusebenza kwawo ekuhloleni ukuthuthwa kwe-enterocyte kuwo wonke umgoqo we-epithelial wamathumbu.Lokhu kuhlaziya kwakusekelwe ocwaningweni lwangaphambili lwe-20.Izibalo 3A,B,C,D,E,F zibonisa izithombe ezimele ezokuthutha ezidlula amaselula ze-G-Rg3r ne-G-Rg3 kusetshenziswa imodeli ye-Caco-2 monolayer.Ukuthuthwa kwe-transcellular ye-G-Rg3r noma i-G-Rg3 kunqamula ama-monolayers e-Caco-2 ukusuka ohlangothini oluphansi kuya ohlangothini lwe-apical (Pb-a) bekuphakeme kakhulu kunokusuka ohlangothini lwe-apical kuya ohlangothini lwe-basolateral (Pa-b).Ku-G-Rg3r, inani elimaphakathi le-Pa-b lalingu-0.38 ± 0.06, elikhuphuke lafinyelela ku-0.73 ± 0.06 ngemva kokwelashwa nge-verapamil engu-50 μmol/L futhi laya ku-1.14 ± 0.09 ngemva kokwelashwa nge-verapamil engu-100 μmol/L (p <0.001 kanye ne-verapamil) ngokulandelana; Umfanekiso 2).3A).Ukubuka kwe-G-Rg3 kulandele iphethini efanayo (Fig. 3B), futhi imiphumela yabonisa ukuthi ukwelashwa kwe-verapamil kwathuthukisa ukuthuthwa kwe-G-Rg3r ne-G-Rg3.Ukwelashwa kwe-Verapamil kuphinde kwaholela ekwehleni okukhulu kwesilinganiso se-Pb-a ne-G-Rg3r kanye ne-G-Rg3s efflux (Umfanekiso 3C,D,E,F), okubonisa ukuthi ukwelashwa kwe-verapamil kunciphisa okuqukethwe kwe-ginsenoside kumaseli e-Caco-2 efflux..
Ukuthuthwa kwe-Transcellular kwe-G-Rg3 kuma-monolayers e-Caco-2 kanye nokumuncwa kwamathumbu esivivinyweni sokuxuba amagundane.(A) Inani le-Pa-b leqembu le-G-Rg3r ku-Caco-2 monolayer.(B) Inani le-Pa-b lamaqembu e-G-Rg3s ku-Caco-2 monolayer.(C) Inani le-Pb leqembu le-G-Rg3r ku-Caco-2 monolayer.(D) Inani le-Pb lamaqembu e-G-Rg3s ku-Caco-2 monolayer.(E) Isilinganiso sesivuno samaqembu e-G-Rg3r ku-Caco-2 monolayer.(F) Isilinganiso sesivuno samaqembu e-G-Rg3 ku-Caco-2 monolayer.(G) Iphesenti lokumuncwa kwamathumbu e-G-Rg3r ekuhlolweni kwe-perfusion kumagundane.(H) Iphesenti lokumuncwa kwamathumbu e-G-Rg3 ekuhlolweni kwe-perfusion kumagundane.I-Permeability kanye nokumuncwa kwaqhathaniswa ngaphandle kokwengezwa kwe-verapamil.Idatha ichazwa njengencazelo ± ukuchezuka okujwayelekile kwezivivinyo ezinhlanu ezizimele.*P <0.05, **P <0.01, ***P <0.001.
Ngokuvumelana nomsebenzi wangaphambilini20, ukufakwa kwamathumbu kwamathumbu amagundane kwenziwa ukuze kunqunywe ukuthi ukumuncwa kwe-G-Rg3 emathunjini kuyenyuka ngemva kokwelashwa kwe-verapamil.Amanani angu-3G, H abonisa ama-assay amelela ama-perfusion ukuhlola iphesenti lokumuncwa kwamathumbu e-G-Rg3r kanye ne-G-Rg3 kumagundane ayimodeli yomdlavuza phakathi nenkathi yesikhathi engenhla.Iphesenti lokuqala lokuthatha okubuthakathaka kwe-G-Rg3r elicishe libe ngu-10% likhuphuke laba ngaphezu kwama-20% ngemva kokwelashwa nge-verapamil engu-50 μM futhi laya ngaphezu kwama-25% ngemva kokwelashwa nge-verapamil eyi-100 μM.Ngokunjalo, i-G-Rg3, eqale yathathwa ngo-10%, iphinde yakhombisa inani eliphakeme elingaphezu kwama-20% ngemuva kokwelashwa nge-verapamil engama-50 μM futhi cishe ama-30% ngemuva kokwelashwa nge-verapamil eyi-100 μM, okuphakamisa ukuthi ukuvinjelwa kwe-P-gp nge-verapamil kuyathuthukisa. i-G-absorption Rg3 yamathumbu kumodeli yegundane yomdlavuza wamaphaphu.
Ngokwale ndlela engenhla, amagundane ayimodeli yomdlavuza we-B(a) P-aye ahlukaniswa ngokungahleliwe abe amaqembu ayisithupha, njengoba kukhonjisiwe kuMfanekiso 4A.Akukho ukuncipha okuphawulekayo kwesisindo noma izimpawu zomtholampilo zobuthi ezibonwe eqenjini lokwelapha le-G-Rg3 uma kuqhathaniswa neqembu lokulawula (idatha engaboniswa).Ngemva kwamasonto angu-20 okwelashwa, amaphaphu egundane ngalinye aqoqwa.Umfanekiso 4B ubonisa izimila zamaphaphu ezinkulu kumagundane kumaqembu okwelapha angenhla, futhi Umfanekiso 4C ubonisa i-micrograph ekhanyayo emele isimila esimele.Mayelana nomthwalo wesimila eqenjini ngalinye (Fig. 4D), amanani amagundane aphathwe nge-G-Rg3r kanye ne-G-Rg3s abengu-0.75 ± 0.29 mm3 kanye no-0.81 ± 0.30 mm3, kuyilapho amanani e-G Amagundane ephathwa. nama-Rg3 ayengu-1.63 ngokulandelana kwawo ±0.40 mm3.lawula amagundane (p <0.001), okubonisa ukuthi ukwelashwa kwe-G-Rg3 kwehlisa umthwalo wesimila kumagundane.Ukuphathwa kwe-verapamil kuphinde kwathuthukisa lokhu kwehliswa: amanani e-verapamil+ G-Rg3r ehle esuka ku-0.75 ± 0.29 mm3 aya ku-0.33 ± 0.25 mm3 (p <0.01), futhi amanani e-verapamil+ asuka ku-0.81 ± 0.30 mm3 ehle ukuya ku-± 0.30 mm29 mm3 kumagundane alashwe nge-G. -Rg3s (p <0.05), okubonisa ukuthi i-verapamil ingase ithuthukise umphumela ovimbelayo we-G-Rg3 ku-tumorigenesis.Umthwalo wamathumba awukhombisanga mehluko omkhulu phakathi kweqembu elilawulayo neqembu le-verapamil, iqembu le-G-Rg3r neqembu le-G-Rg3s, kanye neqembu le-verapamil+G-Rg3r kanye neqembu le-verapamil+G-Rg3s.Ngaphezu kwalokho, abukho ubuthi obubalulekile besibindi noma bezinso obuhlobene nokwelashwa okuhloliwe (Umfanekiso 4E, F).
Umthwalo we-tumor ngemva kokwelashwa kwe-G-Rg3 kanye ne-plasma noma amazinga we-G-Rg3r wamathumbu kanye ne-G-Rg3 emaqenjini abonisiwe.(A) Idizayini yokuhlola.(B) Izimila ze-Macroscopic kumodeli yegundane.Amathumba aboniswa ngemicibisholo.a: G-Rg3r.b: G-Rg3s.c: I-G-Rg3r ihlanganiswe ne-verapamil.d: I-G-Rg3 ihlanganiswe ne-verapamil.d: Verapamil.e: ukulawula.(C) I-Optical micrograph yesimila ekukhulisweni.Impendulo: 100x.b: 400x.(D) Umthelela we-G-Rg3 + ukwelashwa kwe-verapamil kumthwalo wesimila kumagundane e-A/J.(E) Amazinga e-Plasma we-enzyme yesibindi i-ALT.(F) Amazinga e-Plasma we-enzyme ye-renal Cr.(G) Amazinga e-Plasma e-G-Rg3r noma e-G-Rg3 yamaqembu abonisiwe.(H) Amazinga we-G-Rg3r noma G-Rg3s emathunjini amaqembu akhonjisiwe.Idatha ichazwa njengencazelo ± ukuchezuka okujwayelekile kokunquma okuphindwe kathathu.*P <0.05, **P <0.01, ***P <0.001.
Amazinga e-G-Rg3 kumagundane oyimodeli yomdlavuza we-B(a)P ahlolwe yi-UPLC-MS/MS ngemva kwesikhathi sokwelashwa samasonto angu-20 ngokuvumelana nendlela echazwe esigabeni Sezindlela.Izibalo ze-4G ne-H zibonisa amazinga e-plasma kanye namathumbu e-G-Rg3, ngokulandelanayo.Amazinga e-Plasma G-Rg3r abengu-0.44 ± 0.32 μmol/L futhi akhuphukela ku-1.17 ± 0.47 μmol/L ngokuphathwa kanyekanye kwe-verapamil (p <0.001), kuyilapho amazinga e-G-Rg3r emathunjini ayengu-0.53 ± 0.08 µµ.Lapho ihlanganiswa ne-verapamil, i-g yenyuka yafika ku-1.35 ± 0.13 μg/g (p <0.001).Ku-G-Rg3, imiphumela ilandele iphethini efanayo, ebonisa ukuthi ukwelashwa kwe-verapamil kwenyusa i-oral bioavailability ye-G-Rg3 kumagundane e-A/J.
Ukuhlolwa kokusebenza kweseli kwasetshenziswa ukuhlola i-cytotoxicity ye-B(a)P ne-G-Rg3 kumaseli e-hEL.I-cytotoxicity evezwa i-B(a)P kumaseli e-hEL iboniswa kuMfanekiso 5A, kuyilapho izici ezingenabuthi ze-G-Rg3r ne-G-Rg3 ziboniswa kuMfanekiso 5A no-5B.5B, C. Ukuze kuhlolwe umthelela we-cytoprotective we-G-Rg3, i-B(a)P isetshenziswe ngokuhlanganyela nokugxilisa okuhlukahlukene kwe-G-Rg3r noma i-G-Rg3 kumaseli e-hEL.Njengoba kuboniswe ku-Figure 5D, G-Rg3r ekugxilweni kuka-5 μM, 10 μM, kanye no-20 μM kubuyiselwe ukusebenza kweseli ku-58.3%, 79.3%, kanye no-77.3%, ngokulandelana.Imiphumela efanayo ingabonakala naseqenjini le-G-Rg3s.Lapho ukugxiliswa kwama-G-Rg3 kungu-5 µM, 10 µM no-20 µM, ukusebenza kweseli kwabuyiselwa ku-58.3%, 72.7% no-76.7%, ngokulandelanayo (Figure 5E) .).Ukuba khona kwe-BPDE-DNA adducts kukalwa kusetshenziswa ikhithi ye-ELISA.Imiphumela yethu ibonise ukuthi amazinga e-BPDE-DNA adduct anyusiwe eqenjini elilashwe i-B(a)P uma kuqhathaniswa neqembu elilawulayo, kodwa uma kuqhathaniswa nokwelashwa ngokubambisana kwe-G-Rg3, amazinga okungezwa kwe-BPDE-DNA eqenjini le-B(a)P. B eqenjini elilashiwe, amazinga e-DNA adduct ancishiswa kakhulu.Imiphumela yokwelashwa nge-B(a)P iyodwa iboniswa ku-Figure 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 kanye nokwakheka kwe-adduct ye-BPDE-DNA kumaseli e-hEL aphathwa nge-G-Rg3 kanye ne-B(a)P.(A) Ukusebenza kwamaseli e-hEL aphathwe nge-B(a)P.(B) Ukusebenza kwamaseli e-hEL aphathwe nge-G-Rg3r.(C) Ukusebenza kwamaseli e-hEL aphathwe nge-G-Rg3.(D) Ukusebenza kwamaseli e-hEL aphathwe nge-B(a)P ne-G-Rg3r.(E) Ukusebenza kwamaseli e-hEL aphathwe nge-B(a)P ne-G-Rg3.(F) Amazinga we-BPDE-DNA adduct kumaseli e-hEL aphathwa nge-B(a)P ne-G-Rg3.Idatha ichazwa njengencazelo ± ukuchezuka okujwayelekile kokunquma okuphindwe kathathu.*P <0.05, **P <0.01, ***P <0.001.
Inkulumo ye-enzyme ye-GST itholwe ngemva kokwelashwa ngokubambisana nge-10 μM B(a)P no-10 μM G-Rg3r noma i-G-Rg3s.Imiphumela yethu ibonise ukuthi i-B(a)P icindezele isisho se-GST (59.7 ± 8.2% eqenjini le-G-Rg3r kanye no-39 ± 4.5% eqenjini le-G-Rg3s), futhi u-B(a)P wahlotshaniswa ne-G-Rg3r , noma nge-G-Rg3r, noma nge-G-Rg3r.Ukwelashwa ngokubambisana nge-G-Rg3s kubuyiselwe isisho se-GST.Isisho se-GST (103.7 ± 15.5% eqenjini le-G-Rg3r kanye no-110 ± 11.1% eqenjini le-G-Rg3s, p <0.05 kanye no-p <0.001, ngokulandelanayo, i-Fig. 6A, B, ne-C).Umsebenzi we-GST uhlolwe kusetshenziswa ikhithi yokuhlola umsebenzi.Imiphumela yethu ibonise ukuthi iqembu lokwelapha elihlanganisiwe linomsebenzi we-GST ophezulu uma kuqhathaniswa neqembu le-B(a)P kuphela (96.3 ± 6.6% vs. 35.7 ± 7.8% eqenjini le-G-Rg3r vs. 92.3 ± 6.5 eqenjini le-G-Rg3r ).% vs 35.7 ± 7.8% eqenjini le-G-Rg3s, p <0.001, Umfanekiso 6D).
Ukuvezwa kwe-GST ne-Nrf2 kumaseli e-hEL aphathwa nge-B(a)P ne-G-Rg3.(A) Ukutholwa kwesisho se-GST ngokucishwa kwaseNtshonalanga.(B) Ukuvezwa komthamo we-GST kumaseli e-hEL aphathwa nge-B(a)P ne-G-Rg3r.(C) Ukuvezwa komthamo we-GST kumaseli e-hEL aphathwa nge-B(a)P ne-G-Rg3s.(D) Umsebenzi we-GST kumaseli e-hEL aphathwa nge-B(a)P ne-G-Rg3.(E) Ukutholwa kwesisho se-Nrf2 nge-Western blotting.(F) Ukuvezwa komthamo we-Nrf2 kumaseli e-hEL aphathwa nge-B(a)P ne-G-Rg3r.(G) Ukuvezwa komthamo we-Nrf2 kumaseli e-hEL aphathwa nge-B(a)P kanye ne-G-Rg3s.Idatha ichazwa njengencazelo ± ukuchezuka okujwayelekile kokunquma okuphindwe kathathu.*P <0.05, **P <0.01, ***P <0.001.
Ukuze kucaciswe izindlela ezihilelekile ekucindezelweni kwe-G-Rg3-mediated ye-B(a)P-induced tumorigenesis, isisho se-Nrf2 sihlolwe ukuchithwa kwe-Western.Njengoba kuboniswe ku-Figure 6E, F, G, uma kuqhathaniswa neqembu lokulawula, kuphela izinga le-Nrf2 eqenjini lokwelapha le-B (a) P lehlisiwe;nokho, uma kuqhathaniswa neqembu lokwelapha le-B(a)P, amazinga e-B(a) e-Nrf2 eqenjini le-PG-Rg3 anyusiwe (i-106 ± 9.5% ye-G-Rg3r vs. 51.3 ± 6.8%, 117 ± 6. 2% ye I-G-Rg3r vs. 41 ± 9.8% ye-G-Rg3s, p <0.01).
Siqinisekise indima yokuvimbela ye-Nrf2 ngokucindezela isisho se-Nrf2 sisebenzisa i-RNA encane ephazamisayo (siRNA).I-Nrf2 knockdown iqinisekiswe yi-Western blotting (Fig. 7A,B).Njengoba kuboniswe ku-Figure 7C, D, ukwelashwa ngokubambisana kwamaseli e-hEL ane-B(a)P ne-G-Rg3 kubangele ukwehla kwenani le-BPDE-DNA adducts (1.47 ± 0.21) uma kuqhathaniswa nokwelashwa nge-B(a)P iyodwa eqenjini le-siRNA lokulawula.) I-G-Rg3r yayingu-4.13 ± 0.49, i-G-Rg3s yayingu-1.8 ± 0.32 kanye no-4.1 ± 0.57, p <0.01).Kodwa-ke, umthelela ovimbelayo we-G-Rg3 ekwakhekeni kwe-BPDE-DNA uye waqedwa yi-Nrf2 knockdown.Eqenjini le-siNrf2, awukho umehluko obalulekile ekwakhekeni kwe-adduct ye-BPDE-DNA phakathi kokwelashwa okuhlangene kwe-B(a)P ne-G-Rg3 kanye nokwelashwa kwe-B(a)P kuphela (3.0 ± 0.21 ye-G-Rg3r vs. 3.56 ± 0.32 ).ku-G-Rg3r uma kuqhathaniswa no-3.6 we-G-Rg3s uma kuqhathaniswa no-±0.45 uma kuqhathaniswa no-4.0±0.37, p > 0.05).
Umthelela we-Nrf2 knockdown ekwakhekeni kwe-adduct ye-BPDE-DNA kumaseli e-hEL.(A) I-Nrf2 knockdown iqinisekiswe ukuchithwa kwe-Western.(B) Ukulinganisa ubukhulu bebhendi ye-Nrf2.(C) Umthelela we-Nrf2 knockdown kumazinga we-BPDE-DNA adduct kumaseli e-hEL aphathwa nge-B(a)P ne-G-Rg3r.(D) Umthelela we-Nrf2 knockdown kumazinga we-BPDE-DNA adduct kumaseli e-hEL aphathwa nge-B(a)P ne-G-Rg3.Idatha ichazwa njengencazelo ± ukuchezuka okujwayelekile kokunquma okuphindwe kathathu.*P <0.05, **P <0.01, ***P <0.001.
Lolu cwaningo luhlole imiphumela yokuvimbela ye-ginseng ehlukahlukene ekhishwe kumodeli yegundane lomdlavuza wamaphaphu owenziwe ngu-B(a)P, kanye nokwelashwa kwe-KRGB kwehlise kakhulu umthwalo wesimila.Uma kucatshangelwa ukuthi i-G-Rg3 inokuqukethwe okuphezulu kakhulu kulokhu kukhishwe kwe-ginseng, indima ebalulekile yale ginsenoside ekuvimbeleni i-tumorigenesis iye yahlolisiswa.Kokubili i-G-Rg3r ne-G-Rg3 (ama-epimers amabili e-G-Rg3) kwehlise kakhulu umthwalo wesimila kumodeli yegundane lomdlavuza obangelwa u-B(a)P.I-G-Rg3r ne-G-Rg3 zisebenzisa imiphumela yokulwa nomdlavuza ngokudala i-apoptosis yamaseli wesimila21, ivimbele ukukhula kwesimila22, ibophe umjikelezo weseli23 futhi ithinte i-angiogenesis24.I-G-Rg3 iphinde yaboniswa ukuthi ivimbela i-metastasis25 yeselula, kanye nekhono le-G-Rg3 lokuthuthukisa imiphumela yokwelapha ngamakhemikhali kanye ne-radiotherapy kubhalwe phansi26,27.U-Poon et al ubonise ukuthi ukwelashwa kwe-G-Rg3 kungehlisa imiphumela ye-genotoxic ye-B(a)P28.Lolu cwaningo lukhombisa amandla okwelapha e-G-Rg3 ekuqondiseni ama-molecule emvelo e-carcinogenic kanye nokuvimbela umdlavuza.
Naphezu kwamandla awo amahle okuvimbela, ukungatholakali kahle komlomo kwe-ginsenosides kubangela inselele ekusetshenzisweni komtholampilo kwala ma-molecule.Ukuhlaziywa kwe-Pharmacokinetic yokuphathwa komlomo kwe-ginsenosides kumagundane kubonise ukuthi i-bioavailability yayo isengaphansi kwe-5% 29.Lokhu kuhlola kwabonisa ukuthi ngemva kwesikhathi sokwelashwa samasonto angu-20, amazinga egazi kuphela e-Rg5 ehla.Nakuba indlela eyisisekelo ye-bioavailability embi isazocaciswa, i-P-gp kucatshangwa ukuthi ihileleke ekuphumeni kwe-ginsenosides.Lo msebenzi ubonise okokuqala ukuthi ukuphathwa kwe-verapamil, isivimbeli se-P-gp, kwandisa i-bioavailability yomlomo ye-G-Rg3r ne-G-Rg3s.Ngakho, lokhu okutholakele kusikisela ukuthi i-G-Rg3r kanye ne-G-Rg3s zisebenza njengama-substrates e-P-gp ukuze zilawule ukuphuma kwayo.
Lo msebenzi ukhombisa ukuthi ukwelashwa okuhlangene nge-verapamil kwandisa i-bioavailability yomlomo ye-G-Rg3 kumodeli yegundane lomdlavuza wamaphaphu.Lokhu okutholakele kusekelwa ukwanda kokuthutha kwe-transcellular emathunjini kwe-G-Rg3 phezu kokuvinjelwa kwe-P-gp, ngaleyo ndlela kukhulise ukumuncwa kwayo.Ukuhlola kumaseli e-Caco2 kubonise ukuthi ukwelashwa kwe-verapamil kwehlise ukuphuma kwe-G-Rg3r kanye ne-G-Rg3 ngenkathi kuthuthukisa ukukwazi ukungena kulwelwesi.Ucwaningo lukaYang et al.Ucwaningo luye lwabonisa ukuthi ukwelashwa nge-cyclosporine A (esinye isivimbeli se-P-gp) kwandisa i-bioavailability ye-ginsenoside Rh2 isuka enanini lokuqala elingu-1%20 iye ngaphezu kuka-30%.I-Ginsenosides compounds K kanye ne-Rg1 nayo ibonise imiphumela efanayo30,31.Lapho i-verapamil ne-cyclosporin A isetshenziswa ngokubambisana, ukuphuma kwe-compound K kumaseli e-Caco-2 kwancipha kakhulu ukusuka ku-26.6 kuya ngaphansi kuka-3, kuyilapho amazinga ayo e-intracellular anda ngama-40-fold30.Lapho kukhona i-verapamil, amazinga e-Rg1 anda kumaseli e-epithelial yamagundane, ephakamisa indima ye-P-gp ku-ginsenoside efflux, njengoba kuboniswe ngu-Meng et al.31.Nokho, i-verapamil ayizange ibe nomthelela ofanayo ekuphumeni kwayo kwamanye ama-ginsenosides (afana ne-Rg1, F1, Rh1 ne-Re), okubonisa ukuthi awathintwa ama-substrates e-P-gp, njengoba kuboniswe ngu-Liang et al.32 .Lokhu kuqaphela kungase kuhlobane nokubandakanyeka kwabanye abathuthi nezinye izakhiwo ze-ginsenoside.
Indlela yokuvimbela umthelela we-G-Rg3 kumdlavuza ayicacile.Ucwaningo lwangaphambili lubonise ukuthi i-G-Rg3 ivimbela ukulimala kwe-DNA kanye ne-apoptosis ngokunciphisa ukucindezeleka kwe-oxidative nokuvuvukala16,33, okungase kube indlela eyisisekelo yokuvimbela i-tumorigenesis ye-B (a) P.Eminye imibiko ibonisa ukuthi i-genotoxicity ebangelwa i-B(a)P ingancishiswa ngokushintsha ama-enzyme esigaba II ukuze akhe i-BPDE-DNA34.I-GST iyi-enzyme yesigaba II esivamile evimbela ukwakheka kwe-adduct ye-BPDE-DNA ngokukhuthaza ukubophezela kwe-GSH ku-BPDE, ngaleyo ndlela yehlise umonakalo we-DNA obangelwa yi-B(a)P35.Imiphumela yethu ibonisa ukuthi ukwelashwa kwe-G-Rg3 kunciphisa i-B(a)P-induced cytotoxicity kanye nokwakheka kwe-adduct ye-BPDE-DNA kumaseli e-hEL futhi ibuyisela ukusho kwe-GST nomsebenzi ku-vitro.Kodwa-ke, le miphumela yayingekho lapho ingekho i-Nrf2, iphakamisa ukuthi i-G-Rg3 idala imiphumela ye-cytoprotective ngokusebenzisa indlela ye-Nrf2.I-Nrf2 iyisici esikhulu sokuloba sesigaba II se-enzyme detoxification esikhuthaza ukucaciswa kwe-xenobiotics36.Ukusebenza kwendlela ye-Nrf2 kubangela i-cytoprotection futhi kunciphisa ukulimala kwezicubu37.Ngaphezu kwalokho, imibiko eminingana isekele indima ye-Nrf2 njenge-tumor suppressor ku-carcinogenesis38.Ucwaningo lwethu lubonisa ukuthi ukufakwa kwendlela ye-Nrf2 nge-G-Rg3 idlala indima ebalulekile yokulawula ku-B(a)P-induced genotoxicity ngokubangela ukukhishwa kwe-B(a)P ngokwenza kusebenze ama-enzyme esigaba II, ngaleyo ndlela kuvimbela inqubo ye-tumorigenesis.
Umsebenzi wethu wembula amandla e-ginseng ebomvu ekuvimbeleni umdlavuza wamaphaphu owenziwe ngu-B(a)P kumagundane ngokubamba iqhaza okubalulekile kwe-ginsenoside G-Rg3.Ukungatholakali kahle kwe-oral bioavailability yale molecule kuphazamisa ukusebenza kwayo emtholampilo.Nokho, lolu cwaningo lubonisa okokuqala ukuthi i-G-Rg3 iyingxenye engaphansi ye-P-gp, futhi ukuphathwa kwe-P-gp inhibitor kwandisa i-bioavailability ye-G-Rg3 in vitro kanye ne-vivo.I-G-Rg3 yehlisa i-B(a)P-induced cytotoxicity ngokulawula indlela ye-Nrf2, okungenzeka kube indlela yokusebenza kwayo yokuvimbela.Ucwaningo lwethu luqinisekisa amandla e-ginsenoside G-Rg3 ekuvimbeleni nasekwelapheni umdlavuza wamaphaphu.
Amagundane e-A/J esifazane anamasonto ayisithupha ubudala (20 ± 1 g) kanye namagundane angama-Wistar angamaviki angu-7 ubudala (250 ± 20 g) atholwe e-The Jackson Laboratory (Bar Harbor, USA) kanye ne-Wuhan Institute of Zoology.Inyuvesi (Wuhan, China).I-Chinese Type Culture Collection Center (Wuhan, China) isinikeze amaseli e-Caco-2 namaseli e-hEL.I-Sigma-Aldrich (e-St. Louis, e-USA) ingumthombo we-B(a)P kanye ne-tricaprine.Ama-ginsenosides ahlanzekile i-G-Rg3r ne-G-Rg3s, i-dimethyl sulfoxide (DMSO), i-CellTiter-96 proliferation assay kit (MTS), i-verapamil, i-minimal essential medium (MEM), kanye ne-fetal bovine serum (FBS) ithengwe kwa-Chengdu Must Bio-Technology .Co., Ltd.(Chengdu, China).Ikhithi encane ye-QIAamp DNA kanye nekhithi ye-BPDE-DNA adduct ELISA ithengwe kwa-Qiagen (Stanford, CA, USA) kanye ne-Cell Biolabs (San Diego, CA, USA).Ikhithi yokuhlola umsebenzi we-GST kanye nekhithi ephelele yokuhlola amaprotheni (indlela ye-BCA evamile) ithengwe kwa-Solarbio (Beijing, China).Zonke izingcaphuno ze-ginseng ezibomvu zigcinwe ku-Mingyu Laboratory 7. I-Hong Kong Baptist University (Hong Kong, China) kanye ne-Korea Cancer Center (Seoul, Korea) ziyimithombo yokuhweba yokukhishwa kwe-CRG kanye nezikhishwe ezihlukahlukene ezibomvu ze-ginseng zemvelaphi ehlukahlukene yaseKorea (kuhlanganise ne-KRGA, i-KRGB kanye ne-KRGC).I-ginseng ebomvu yenziwe ngezimpande ze-ginseng entsha eneminyaka engu-6 ubudala.Ukukhishwa kwe-ginseng ebomvu kutholwa ngokugeza i-ginseng ngamanzi izikhathi ezintathu, bese kugxilwa ekukhishweni okunamanzi, futhi ekugcineni kome ekushiseni okuphansi ukuze kutholwe impushana ekhishwe yi-ginseng.Ama-antibodies (anti-Nrf2, anti-GST, kanye ne-β-actin), i-horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG), i-transfection reagent, i-siRNA yokulawula, ne-Nrf2 siRNA ithengwe ku-Santa Cruz Biotechnology (Santa Cruz, CA) .), USA).
I-Caco2 namaseli e-hEL akhuliswe ezitsheni ze-cell culture ezingu-100 mm2 nge-MEM equkethe u-10% FBS ku-37 °C endaweni enomswakama engu-5% CO2.Ukuze kutholakale umphumela wezimo zokwelashwa, amaseli e-hEL afakwe ngokugxila okuhlukile kwe-B(a)P ne-G-Rg3 ku-MEM amahora angu-48.Amaseli angabuye ahlaziywe noma aqoqwe ukuze kulungiswe okukhishwe ngaphandle kwamaseli.
Konke ukuhlola kuvunywe Ikomidi Lokuziphatha Lezilwane Lokuhlola lase-Tongji Medical College, i-Huazhong University of Science and Technology (Inombolo yokugunyaza yango-2019; Inombolo Yokubhalisa 4587TH).Konke ukuhlola kwenziwa ngokuhambisana neziqondiso ezifanele kanye nemithethonqubo, futhi ucwaningo lwenziwa ngokuhambisana nemihlahlandlela ye-Animal Research: Reporting of In Vivo Experiments (ARRIVE).Amagundane e-A/J anamasonto ayisishiyagalombili ubudala ajovwe okokuqala nge-intraperitoneally nge-B(a)P kusisombululo se-tricaprine (100 mg/kg, 0.2 ml).Ngemva kwesonto, amagundane ahlukaniswa ngokungahleliwe abe amaqembu okulawula kanye namaqembu ahlukene okwelapha, amagundane angu-15 eqenjini ngalinye, futhi agayiwe kanye ngosuku.Ngemva kwamasonto angu-20 ukwelashwa, izilwane zanikelwa nge-CO2 asphyxia.Amaphaphu aqoqwa futhi alungiswa amahora angama-24.Inani lamathumba angaphezulu kanye nosayizi wesimila ngasinye kwalinganiswa iphaphu ngalinye ngaphansi kwesibonakhulu esihlinzayo.Izilinganiso zevolumu yesimila (V) zibalwe kusetshenziswa inkulumo elandelayo: V (mm3) = 4/3πr3, lapho u-r eyi-diameter yesimila.Isamba sesamba sawo wonke amavolumu esimila emaphashini amagundane imele isamba sevolumu yesimila, futhi isamba sengqikithi yesimila eqenjini ngalinye imele umthwalo wesimila.Amasampula egazi eliphelele namathumbu aqoqwa futhi agcinwa ku-−80°C ukuze kunqunywe i-UPLC-MS/MS.I-Serum yaqoqwa futhi kwasetshenziswa i-automated chemistry analyzer ukuze kuhlaziywe amazinga e-alanine aminotransferase (ALT) kanye ne-serum creatinine (Cr) ukuze kuhlolwe ukusebenza kwesibindi nezinso.
Amasampula aqoqiwe akhishwa endaweni ebandayo, ancibilika, akalwa, afakwa kumashubhu njengoba kuchazwe ngenhla.Kulokhu kwengezwe i-phhlorizin engu-0.5 μM (izinga langaphakathi) kusixazululo se-methanol esingu-0.8 ml.Isicubu sabe sesenziwa i-homogenized kusetshenziswa i-Tissue-Tearor futhi i-homogenate yabe isidluliselwa ku-1.5 ml microcentrifuge tube.Ingxube ifakwe i-centrifuged ku-15500 rpm imizuzu engu-15.Ngemuva kokukhipha i-1.0 ml ye-supernatant, yomisa nge-nitrogen.Amakhulu amabili ama-microliters e-methanol asetshenziselwa ukululama.Igazi liyaqoqwa futhi licutshungulwe emugqeni owodwa futhi lisetshenziswa njengereferensi yazo zonke izilinganiso.
Amapuleti e-Transwell anemithombo engu-24 afakwe imbewu ngamaseli e-Caco-2 angu-1.0 × 105 emthonjeni ngamunye ukuze kuhlolwe ukuthuthukiswa okungenzeka kokuthutha kwe-G-Rg3 ngokufaka i-verapamil.Ngemuva kwamaviki ama-3 wesiko, amaseli ahlanzwa nge-HBSS futhi afakwa ku-37°C.U-400 μL ka-10 μM G-Rg3 (G-Rg3r, G-Rg3s, noma ingxube enama-50 noma 100 μM i-verapamil) yajovwa ohlangothini oluyisisekelo noma lwe-apical lwe-monolayer, kwathi u-600 μL wesixazululo se-HBSS wengezwa kwenye. ohlangothini.Qoqa u-100 µl we-culture medium ngezikhathi ezimisiwe (0, 15, 30, 45, 60, 90 kanye nemizuzu engu-120) bese wengeza u-100 µl we-HBSS ukuze wenze le volumu.Amasampula agcinwe ku-−4 °C kuze kube yilapho etholwa yi-UPLC-MS/MS.Inkulumo ethi Papp = dQ/(dT × A × C0) isetshenziselwa ukulinganisa ukufinyeleleka okubonakalayo kwe-apical kanye ne-basolateral permeability futhi ngokuphambene nalokho (Pa-b kanye ne-Pb-a, ngokulandelana);I-dQ/dT iwushintsho ekugxiliseni ingqondo, u-A (0.6 cm2) indawo engaphezulu ye-monolayer, futhi i-C0 iyindawo yokuqala yokugxilisa abanikeli.Isilinganiso se-efflux sibalwa njenge-Pb-a/Pa-b, emele izinga le-efflux lomuthi wocwaningo.
Amagundane angama-Wistar abesilisa azila ukudla amahora angu-24, aphuza amanzi kuphela, futhi afakwa i-anesthetized ngomjovo we-intravenous we-3.5% ye-pentobarbital solution.Ishubhu ye-silicone efakwe ngaphakathi inomkhawulo we-duodenum njengomnyango kanye nesiphetho se-ileum njengendlela yokuphuma.Sebenzisa iphampu ye-peristaltic ukumpompa i-inlet ngo-10 µM G-Rg3r noma i-G-Rg3s ku-isotonic HBSS ngesilinganiso sokugeleza esingu-0.1 ml/min.Umthelela we-verapamil wahlolwa ngokwengeza u-50 μM noma u-100 μM wenhlanganisela ku-10 μM G-Rg3r noma G-Rg3s.I-UPLC-MS/MS yenziwe ekukhishweni kwe-perfusion okuqoqwe ngesikhathi samaphoyinti angu-60, 90, 120, kanye nemizuzu engu-150 ngemva kokuqala kokufaka amanzi.Iphesenti lokumuncwa lilinganiswa ngefomula % ukumuncwa = (1 – Cout/Cin) × 100%;ukugxila kwe-G-Rg3 endaweni yokuphuma nokungena kuvezwa u-Cout no-Cin, ngokulandelana.
Amaseli e-hEL atshalwe kumapuleti anemithombo engu-96 ekumineni kwamaseli angu-1 × 104 emthonjeni ngamunye futhi aphathwe nge-B(a)P (0, 1, 5, 10, 20, 30, 40 μM) noma i-G-Rg3 ehlakazwe ku-DMSO .Izidakamizwa zabe sezihlanjululwa nge-culture medium kuya ekugxilweni okuhlukahlukene (0, 1, 2, 5, 10, 20 μM) ngaphezu kwamahora angu-48.Kusetshenziswa ikhithi yokuhlola ye-MTS etholakalayo kwezohwebo, amaseli angaphansi kwephrothokholi ejwayelekile futhi alinganiswa kusetshenziswa isifundi se-microplate esingu-490 nm.Izinga lokusebenza kwamaseli lamaqembu aphathwe ngokubambisana ne-B(a)P (10 μM) kanye ne-G-Rg3 (0, 1, 5, 10, 20 μM) lihlolwe ngokuvumelana nendlela engenhla futhi liqhathaniswa neqembu elingalashwa.
Amaseli we-hEL afakwe ku-6-well plates ku-1 × 105 amaseli / kahle futhi aphathwe nge-10 μMB (a) P lapho kukhona noma ukungabikho kwe-10 μM G-Rg3.Ngemva kwamahora angu-48 okwelashwa, i-DNA yakhishwa kumaseli e-hEL kusetshenziswa i-QIAamp DNA Mini Kit ngokuya ngephrothokholi yomkhiqizi.Ukwakhiwa kwe-BPDE-DNA adducts kutholwe kusetshenziswa ikhithi ye-ELISA ye-BPDE-DNA.Amazinga ahlobene we-BPDE-DNA adduct akalwe kusetshenziswa isifundi se-microplate ngokulinganisa ukumunca ku-450 nm.
Amaseli we-hEL afakwe ku-96-well plates ekumineni kwe-1 × 104 amaseli emthonjeni ngamunye futhi aphathwe nge-10 μMB (a) P ngokungabikho noma ukuba khona kwe-10 μM G-Rg3 ku-48 h.Umsebenzi we-GST ukalwe kusetshenziswa ikhithi yokuhlola umsebenzi we-GST yezentengiso ngokuya ngephrothokholi yomkhiqizi.Ukusebenza kwe-GST okuhlobene kukalwe ngokumunca ku-450 nm kusetshenziswa isifundi se-microplate.
Amaseli e-hEL ahlanzwa nge-PBS ebanda eqhweni abese elayishwa kusetshenziswa i-radioimmunoprecipitation assay buffer equkethe ama-protease inhibitors nama-phosphatase inhibitors.Ngemva kokulinganisa amaprotheni kusetshenziswa ikhithi yokuhlola amaprotheni ephelele, u-30 μg weprotheyini kusampula ngayinye yahlukaniswa ngo-12% SDS-PAGE futhi yadluliselwa kulwelwesi lwe-PVDF nge-electrophoresis.Amamembrane abevinjwe ngo-5% we-skim milk futhi afakwa amasosha omzimba ayisisekelo ngobusuku obungu-4°C.Ngemva kokufukamela nge-horseradish peroxidase-conjugated antibodies yesibili, ama-reagents e-chemiluminescence athuthukisiwe angeziwe ukuze abone ngeso lengqondo isignali yokubopha.Amandla ebhande ngalinye lephrotheni alinganiswa kusetshenziswa isofthiwe ye-ImageJ.
Isofthiwe ye-GraphPad Prism 7.0 isetshenziselwe ukuhlaziya yonke idatha, evezwe njengokuchezuka okujwayelekile ± okujwayelekile.Ukwehluka phakathi kwamaqembu okwelapha kwahlolwa kusetshenziswa ukuhlolwa kwe-t koMfundi noma ukuhlaziywa kwendlela eyodwa yokuhluka, ngevelu engu-P engu-<0.05 ebonisa ukubaluleka kwezibalo.
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Isikhathi sokuthumela: Sep-17-2023