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A na-eji ginseng na-acha uhie uhie mee ihe na nkà mmụta ọgwụ ndị Eshia kemgbe ọtụtụ narị afọ.N'ime ọmụmụ ihe a, anyị nyochara ikike nke ụdị ginseng anọ na-acha uhie uhie (ginseng na-acha uhie uhie nke China, ginseng red Korean, ginseng red Korean, na Korean red ginseng C) nke a na-eto na mpaghara dị iche iche iji gbochie nhazi na uto nke ngụgụ na-akpata carcinogen. etuto ahụ.A na-eme ule benzo (a) pyrene (B(a)P) na ụmụ oke A/J, na ginseng na-acha uhie uhie Korea bụ nke kacha dị irè n'ibelata ibu arọ n'etiti ụdị ginseng anọ na-acha uhie uhie.Na mgbakwunye, anyị nyochara ọdịnaya nke ginsenosides dị iche iche (Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 na Rg5) na mkpụrụ ginseng anọ na-acha uhie uhie wee chọpụta na ginseng B nke Korea nwere. ọkwa kachasị elu nke ginsenoside Rg3 (G-Rg3), na-atụ aro na G-Rg3 nwere ike ịrụ ọrụ dị mkpa na ọgwụgwọ ọgwụgwọ ya.Ọrụ a na-egosi na G-Rg3 nwere obere bioavailability.Otú ọ dị, mgbe G-Rg3 na-ejikọta ya na P-gp inhibitor verapamil, mpụta nke G-Rg3 n'ime sel Caco-2 belatara, ọnụ ọgụgụ nke ntinye eriri afọ nke G-Rg3 na-abawanye na ụdị oke, na G-Rg3. e mụbara.Na mkpụrụ ndụ Caco-2, ọpụpụ nke Rg3 na-ebelata, ọkwa nke itinye uche Rg3 na-ebelata.G-Rg3 na-abawanye na eriri afọ na plasma, na ike ya igbochi etuto ahụ na-akwalitekwa n'ụdị oke nke B(a) P-induced tumorigenesis.Anyị chọpụtakwara na G-Rg3 belatara B (a) cytotoxicity nke P na-akpata na DNA na-emepụta n'ime mkpụrụ ndụ akpa ume mmadụ, ma weghachite okwu na ọrụ nke enzymes nke abụọ nke abụọ site na ụzọ Nrf2, nke nwere ike jikọta ya na usoro nwere ike ime ihe. nke G mgbochi -Rg3..Banyere ihe omume nke etuto akpa ume.Ọmụmụ ihe anyị na-egosipụta ọrụ dị mkpa maka G-Rg3 n'ịchụso etuto akpa ume n'ụdị òké.A na-akwalite bioavailability nke ginsenoside a site na ịchụ P-glycoprotein, na-ekwe ka mkpụrụ ndụ ahụ nwee mmetụta anticancer.
Ụdị ọrịa cancer akpa ume na-emekarị bụ ọrịa cancer akpa ume na-abụghị obere cell (NSCLC), nke bụ otu n'ime ihe ndị na-ebute ọnwụ cancer na China na North America1,2.Isi ihe na-eme ka ohere nke ịmepụta ọrịa cancer akpa ume na-abụghị obere cell bụ ise siga.Anwụrụ sịga nwere ihe karịrị carcinogen 60, gụnyere benzo (a) pyrene (B(a)P), nitrosamines, na isotopes redioactive sitere na ire ere nke radon.3 Polycyclic aromatic hydrocarbons B(a) P bụ isi ihe na-akpata nsị na sịga. anwụrụ.Mgbe ikpughe na B (a) P, cytochrome P450 na-atụgharị ya na B (a) P-7,8-dihydrodiol-9,10-epoxide (BPDE), nke na-emeghachi omume na DNA iji mepụta BPDE-DNA ntinye 4. Ọzọkwa, ndị a adducts na-ebute tumorigenesis nku ume n'ime ụmụ oke nwere ọkwa tumo na histopathology yiri etuto akpa ume mmadụ5.Njirimara a na-eme ka ụdị ọrịa cancer akpa ume na-ebute B (a) bụrụ usoro kwesịrị ekwesị maka ịlele ogige nwere ihe mgbochi ọrịa cancer.
Otu usoro enwere ike iji gbochie mmepe nke ọrịa cancer akpa ume na ìgwè ndị nwere nnukwu ihe ize ndụ, karịsịa ndị na-ese siga, bụ iji chemopreventive eme ihe iji gbochie mmepe nke ọnya neoplastic intraepithelial ma si otú ahụ gbochie ọganihu ha na-esote na njọ.Ọmụmụ anụmanụ na-egosi na ụdị chemopreventive dị iche iche dị irè6.Akụkọ anyị gara aga7 gosipụtara ezigbo mgbochi mgbochi nke ginseng uhie na ọrịa kansa akpa ume.A na-eji ahịhịa a eme ihe kemgbe ọtụtụ narị afọ na ọgwụ ọdịnala ndị Eshia iji mee ka ndụ na ahụike dị ogologo, ma edepụtala ya na ọ nwere mmetụta antitumor8.
Ihe na-arụ ọrụ nke ginseng bụ ginsenoside, nke a na-eji dị ka ihe nrịbama mejupụtara iji nyochaa àgwà nke ginseng wepụ.Nyocha ọnụọgụ nke ginseng crude na-agụnyekarị iji ọtụtụ ginsenosides, gụnyere RK1, Rg1, F1, Re, Rb1, Rb2, Rb3, Rd, Rh1, Rh2, Rg3, Rg5, na Rc9,10.Ginsenosides nwere obere ojiji ụlọ ọgwụ n'ihi na ha adịghị mma bioavailability ọnụ11.Ọ bụ ezie na usoro maka bioavailability a na-adịghị mma abụghị nke doro anya, mgbapụta nke ginsenosides nke P-glycoprotein (P-gp) 12 kpatara nwere ike ịbụ ihe kpatara ya.P-gp bụ otu n'ime ndị na-ebufe efflux kachasị mkpa na ATP-binding cassette transporter superfamily, nke na-eji ike nke ATP hydrolysis hapụ ihe intracellular n'ime mpụga.A na-ekesa ndị na-ebu P-gp na eriri afọ, akụrụ, imeju na mgbochi ụbụrụ ọbara13.P-gp na-arụ ọrụ dị oke egwu na mmịnye eriri afọ, na mgbochi nke P-gp na-abawanye nnabata ọnụ na nnweta ụfọdụ ọgwụ mgbochi ọrịa cancer12,14.Ihe atụ nke ndị na-emechi ihe na-ejibu eme ihe na akwụkwọ ndị ahụ bụ verapamil na cyclosporine A15.Ọrụ a gụnyere ịmepụta usoro òké maka ịmụ B (a) ọrịa cancer akpa ume na-akpata P iji nyochaa ike nke dị iche iche ginseng na-acha uhie uhie sitere na China na Korea na-emetụta ọrịa ọjọọ.A na-enyocha ihe ndị ahụ n'otu n'otu iji chọpụta kpọmkwem ginsenosides nke nwere ike imetụta carcinogenesis.A na-eji Verapamil lekwasịrị anya P-gp ma melite bioavailability nke ọnụ na ọgwụgwọ ọgwụgwọ nke ginsenosides na-eche cancer.
Usoro nke ginseng saponins na-egosipụta mmetụta ọgwụgwọ na carcinogenesis ka edobeghị anya.Nnyocha egosila na ginsenosides dị iche iche nwere ike ibelata mmebi DNA nke carcinogens kpatara site na ibelata nrụgide oxidative na ime ka enzymes detoxification nke II na-arụ ọrụ, si otú a na-egbochi mmebi cell.Glutathione S-transferase (GST) bụ ụdị enzyme a na-ahụkarị nke II nke achọrọ iji belata mmebi DNA nke carcinogens17 kpatara.Ngwurugwu erythroid 2 metụtara 2 (Nrf2) bụ ihe dị mkpa ederede nke na-achịkwa redox homeostasis ma na-eme ka nkwupụta nke enzymes nke II na cytoprotective antioxidant nzaghachi18.Ọmụmụ ihe anyị nyochakwara mmetụta nke ginsenosides achọpụtara na ibelata B (a) P-induced cytotoxicity na BPDE-DNA adduct formation, yana ime ka usoro II enzymes site n'ịgbanwe ụzọ Nrf2 na sel akpa ume nkịtị.
Ịmepụta ụdị òké nke B(a) ọrịa cancer na-akpata P dabara na ọrụ gara aga5.Ọnụ ọgụgụ 1A na-egosi nhazi nnwale nke ọgwụgwọ izu 20 nke ụdị ọrịa cancer òké nke B (a) P, mmiri (nchịkwa), ginseng red ginseng (CRG), Korean red ginseng wepụ A (KRGA), na Korean red. ginseng.Wepụ B (KRGB) na Korean Red Ginseng Extract C (KRGC).Mgbe izu 20 nke ọgwụgwọ ginseng na-acha uhie uhie, a na-achụ ụmụ oke site na CO2 asphyxiation.Ọgụgụ 1B na-egosi etuto akpa ume macroscopic na anụmanụ na-eji ụdị ginseng na-acha uhie uhie dị iche iche na-emeso ya, na eserese 1C na-egosi micrograph ìhè nnọchite anya nke ihe atụ etuto.Ibu akpụ nke anụmanụ ndị a na-emeso KRGB (1.5 ± 0.35) dị ala karịa nke anụmanụ na-achịkwa (0.82 ± 0.2, P <0.05), dị ka egosiri na Figure 1D.Nkezi ogo nke mgbochi ibu tumor bụ 45%.Ihe ndị ọzọ ginseng na-acha uhie uhie a nwalere egosighi mgbanwe dị ukwuu na ibu akpụ (P> 0.05).Enweghị mmetụta doro anya na-ahụ anya na ụdị òké n'ime izu 20 nke ọgwụgwọ ginseng na-acha uhie uhie, gụnyere enweghị mgbanwe na ịdị arọ nke ahụ (data egosighi) na ọ dịghị imeju ma ọ bụ akụrụ toxicity (Figure 1E, F).
Mwepu ginseng na-acha uhie uhie na-agwọ mmepe akpụ nku ume na ụmụ oke A / J.(A) Nhazi nnwale.(B) Nnukwu etuto ngụgụ n'ụdị òké.A na-egosi etuto ahụ site na akụ.a: Chinese red ginseng otu.b: otu A nke ginseng uhie Korea.c: Korean red ginseng otu B. d: Korean red ginseng otu C. d: otu njikwa.(C) Igwe okwu ọkụ na-egosi akpụ ngụgụ.Nkwalite: 100. b: 400. (D) Ibu ibu n'ime otu ginseng na-acha uhie uhie.(E) Ọkwa Plasma nke imeju enzyme ALT.(F) Ọkwa Plasma nke renal enzyme Cr.A na-akọwapụta data dị ka ihe pụtara ± ọkọlọtọ.P <0.05.
A na-enyocha ihe ndị na-acha uhie uhie ginseng ndị a chọpụtara na nchọpụta a site na ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) iji chọpụta ginsenosides ndị a: Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 na Rg5.Akọwara ọnọdụ UPLC na MS ejiri tụọ nyocha ndị ahụ na akụkọ gara aga19.UPLC-MS/MS chromatograms nke mpụta ginseng uhie anọ ka egosiri na eserese 2A.Enwere ọdịiche dị ịrịba ama na mkpokọta ginsenoside, yana ọdịnaya ginsenoside kachasị elu na CRG (590.27 ± 41.28 μmol / L) (Figure 2B).Mgbe ị na-enyocha onye ginsenosides (Ọnọdụ 2C), KRGB gosipụtara ọkwa kachasị elu nke G-Rg3 ma e jiri ya tụnyere ginsenosides ndị ọzọ (58.33 ± 3.81 μmol / L maka G-Rg3s na 41.56 ± 2.88 μmol / L maka G -Rg3r).L.ụdị ginseng uhie (P <0.001).G-Rg3 na-eme dị ka ụzọ stereoisomers G-Rg3r na G-Rg3s, nke dị iche na ọnọdụ nke hydroxyl otu na carbon 20 (Fig. 2D).Nsonaazụ na-egosi na G-Rg3r ma ọ bụ G-Rg3 nwere ike ịnwe ikike mgbochi ọrịa cancer dị mkpa na ụdị òké ọrịa cancer B (a) P.
Ọdịnaya nke ginsenosides dị n'ụdị ginseng uhie dị iche iche.(A) UPLC-MS/MS chromatograms nke ginseng uhie anọ.(B) Ntụle nke mkpokọta ginsenoside dị na ntinye egosiri.(C) Nchọpụta nke ginsenosides n'otu n'otu na mpempe akwụkwọ akara.(D) Ọdịdị nke ginsenoside stereoisomers G-Rg3r na G-Rg3s.A na-akọwapụta data dị ka ihe pụtara ± ọkọlọtọ ngbanwe nke mkpebi atọ.*** P <0.001.
Ọmụmụ UPLC-MS / MS chọrọ nyocha nke ginsenosides na eriri afọ na ọbara mgbe izu 20 gasịrị.Ọgwụgwọ na KRGB gosipụtara ọnụnọ naanị 0.0063 ± 0.0005 μg/ml Rg5 n'ime ọbara.Ọ dịghị ginsenosides fọdụrụnụ achọpụtara, na-egosi adịghị mma bioavailability ọnụ ya mere ibelata ikpughe na ginsenosides ndị a.
Akara cell adenocarcinoma colon Caco-2 bụ morphologically na biochemically yiri mkpụrụ ndụ epithelial intestinal nke mmadụ, na-egosipụta uru ya n'ịtụle njem enterocyte gafere ihe mgbochi eriri afọ nke eriri afọ.Nyocha a dabere na nyocha mbụ nke 20.Ọnụọgụ 3A,B,C,D,E,F na-egosi onyonyo nnọchite anya njem transcellular nke G-Rg3r na G-Rg3 site na iji ụdị monolayer Caco-2.Ụgbọ njem transcellular nke G-Rg3r ma ọ bụ G-Rg3 gafee Caco-2 monolayers site na basolateral gaa n'akụkụ apical (Pb-a) dị elu nke ukwuu karịa site na apical na basolateral (Pa-b).Maka G-Rg3r, uru Pa-b pụtara bụ 0.38 ± 0.06, nke mụbara na 0.73 ± 0.06 mgbe ọgwụgwọ na 50 μmol / L verapamil na 1.14 ± 0.09 mgbe ọgwụgwọ na 100 μmol / L verapamil (p <0.01) gasịrị. n'otu n'otu;3A).Nleba anya maka G-Rg3 gbasoro usoro yiri nke ahụ (Fig 3B), nsonaazụ ya gosiri na ọgwụgwọ verapamil kwalitere njem nke G-Rg3r na G-Rg3.Ọgwụgwọ Verapamil mekwara ka mbelata dị ukwuu na Pb-a na G-Rg3r na G-Rg3s efflux ratios (Njirimara 3C, D, E, F), na-egosi na ọgwụgwọ verapamil na-ebelata ọdịnaya ginsenoside na sel Caco-2 efflux..
Ụgbọ njem transcellular nke G-Rg3 na Caco-2 monolayers na ntinye eriri afọ na nyocha nke oke oke.(A) Pa-b uru nke G-Rg3r otu na Caco-2 monolayer.(B) uru Pa-b nke otu G-Rg3s na Caco-2 monolayer.(C) Pb uru nke G-Rg3r otu na Caco-2 monolayer.(D) uru Pb nke otu G-Rg3s na Caco-2 monolayer.(E) Oke mkpụrụ nke G-Rg3r dị na monolayer Caco-2.(F) Oke mpụta nke otu G-Rg3 na monolayer Caco-2.(G) Pasent nke mmịnye eriri afọ nke G-Rg3r na nyocha nrịbama n'ime oke.(H) Pasent nke mmịnye eriri afọ nke G-Rg3 na nyocha nrịbama n'ime oke.Permeability na absorption na-atụnyere na-enweghị mgbakwunye nke verapamil.Ekwuputara data dị ka ihe pụtara ± ọkọlọtọ nke nnwale ise nọọrọ onwe ya.P <0.05, ** P <0.01, *** P <0.001.
N'ikwekọ na ọrụ mbụ 20, a na-eme orthotopic intestinal perfusion nke oke iji chọpụta ma nnabata G-Rg3 na eriri afọ na-abawanye mgbe ọgwụgwọ verapamil gasịrị.Ọnụọgụ 3G,H na-egosi nyocha nrịanrịa onye nnọchi anya iji nyochaa pasentị mmịnye eriri afọ nke G-Rg3r na G-Rg3 na oke ụdị ọrịa kansa n'oge oge dị n'elu.Pasent mbụ nke nnabata G-Rg3r na-adịghị ike nke ihe dịka 10% mụbara karịa 20% mgbe ọgwụgwọ ya na 50 μM verapamil na karịa 25% mgbe ọgwụgwọ na 100 μM verapamil gasịrị.N'otu aka ahụ, G-Rg3, nke nwere mbido mbụ nke 10%, gosikwara ọnụ ọgụgụ kasị elu nke ihe karịrị 20% mgbe ọgwụgwọ na 50 μM verapamil na ihe fọrọ nke nta ka ọ bụrụ 30% mgbe ọgwụgwọ na 100 μM verapamil, na-atụ aro na mgbochi nke P-gp site na verapamil na-abawanye. eriri afọ G-absorption Rg3 n'ụdị òké nke ọrịa kansa akpa ume.
Dịka usoro a dị n'elu si dị, a na-ekewa ụmụ oke ụdị ọrịa cancer B (a) P na-enweghị usoro n'ime otu isii, dịka egosiri na foto 4A.Achọpụtaghị oke ibu dị arọ ma ọ bụ akara ahụike nke toxicity na G-Rg3 ọgwụgwọ otu ma e jiri ya tụnyere otu njikwa (data egosighi).Mgbe izu 20 ọgwụgwọ gasịrị, a na-anakọta akpa ume nke òké ọ bụla.Ọgụgụ 4B na-egosi etuto akpa ume macroscopic na ụmụ oke na otu ọgwụgwọ dị n'elu, yana eserese 4C na-egosi micrograph ọkụ nnọchite anya nke etuto nnọchite anya.Banyere ibu akpụ na otu ọ bụla (Fig 4D), ụkpụrụ maka ụmụ oke na G-Rg3r na G-Rg3s bụ 0.75 ± 0.29 mm3 na 0.81 ± 0.30 mm3, n'otu n'otu, ebe ụkpụrụ maka G Mice na-emeso ya. na -Rg3s bụ 1.63 n'otu n'otu ± 0.40 mm3.ụmụ oke na-achịkwa (p <0.001), na-egosi na ọgwụgwọ G-Rg3 belatara ibu akpụ na ụmụ oke.Nlekọta nke verapamil mere ka mbelata a dịkwuo elu: ụkpụrụ dị na verapamil + G-Rg3r ụmụ oke na-ebelata site na 0.75 ± 0.29 mm3 ruo 0.33 ± 0.25 mm3 (p <0.01), na ụkpụrụ maka verapamil + site na 0.81 ± 0.30 ± .2 . mm3 na G. -Rg3s na-agwọ ụmụ oke (p <0.05), na-egosi na verapamil nwere ike ịkwalite mmetụta mgbochi nke G-Rg3 na tumorigenesis.Ibu ibu egosighi ọdịiche dị n'etiti ndị na-ahụ maka njikwa na otu verapamil, G-Rg3r na G-Rg3s, na verapamil + G-Rg3r na otu verapamil + G-Rg3s.Ọzọkwa, ọnweghị nnukwu imeju ma ọ bụ akụrụ nsi nke metụtara ọgwụgwọ enyochara (Foto 4E,F).
Ibu ibu mgbe ọgwụgwọ G-Rg3 gasịrị na plasma ma ọ bụ eriri afọ G-Rg3r na G-Rg3 n'ime otu egosipụtara.(A) Nhazi nnwale.(B) Ụbụrụ macroscopic na ụdị òké.A na-egosi etuto ahụ site na akụ.a: g-rg3r.b: g-rg3s.c: G-Rg3r jikọtara ya na verapamil.d: G-Rg3 jikọtara ya na verapamil.d: Verapamil.e: njikwa.(C) Micrograph anya nke etuto ahụ na mmụba.Azịza: 100x.b: 400X.(D) Mmetụta G-Rg3 + ọgwụgwọ verapamil na ibu akpụ na ụmụ oke A/J.(E) Ọkwa Plasma nke imeju enzyme ALT.(F) Ọkwa Plasma nke renal enzyme Cr.(G) Ọkwa Plasma nke G-Rg3r ma ọ bụ G-Rg3 nke otu egosipụtara.(H) Ọkwa G-Rg3r ma ọ bụ G-Rg3 n'ime eriri afọ nke otu ndị egosipụtara.A na-akọwapụta data dị ka ihe pụtara ± ọkọlọtọ nke mkpebi ugboro atọ.P <0.05, ** P <0.01, *** P <0.001.
A na-enyocha ọkwa G-Rg3 na B (a) ụdị ọrịa cancer nke P-ebute site na UPLC-MS / MS mgbe oge ọgwụgwọ 20-izu dịka usoro akọwara na ngalaba Ụzọ.Ọnụọgụ 4G na H na-egosi ọkwa plasma na eriri afọ G-Rg3, n'otu n'otu.Plasma G-Rg3r ọkwa bụ 0.44 ± 0.32 μmol/L wee mụbaa na 1.17 ± 0.47 μmol/L na concomitant nchịkwa nke verapamil (p <0.001), mgbe eriri afọ G-Rg3r ọkwa bụ 0.53 ± 0.08 .µl.Mgbe ejikọtara ya na verapamil, g na-abawanye na 1.35 ± 0.13 μg / g (p <0.001).Maka G-Rg3, nsonaazụ ahụ gbasoro usoro yiri nke ahụ, na-egosi na ọgwụgwọ verapamil mụbara bioavailability ọnụ nke G-Rg3 na ụmụ oke A / J.
A na-eji nyocha ndụ ndụ cell mee ihe iji nyochaa cytotoxicity nke B(a) P na G-Rg3 na sel hel.A na-egosi cytotoxicity nke B (a) P na sel hEL na Figure 5A, ebe ihe ndị na-adịghị egbu egbu nke G-Rg3r na G-Rg3 na-egosi na ọnụ ọgụgụ 5A na 5B.5B, C. Iji nyochaa mmetụta cytoprotective nke G-Rg3, B (a) P na-ejikọta ọnụ ọgụgụ dị iche iche nke G-Rg3r ma ọ bụ G-Rg3 n'ime sel hEL.Dị ka egosiri na eserese 5D, G-Rg3r na ntinye nke 5 μM, 10 μM, na 20 μM weghachiri ikike cell na 58.3%, 79.3%, na 77.3%, n'otu n'otu.Enwere ike ịhụ nsonaazụ ndị yiri ya na otu G-Rg3s.Mgbe mkpokọta nke G-Rg3s bụ 5 µM, 10 µM na 20 μM, eweghachiri ike ndụ cell na 58.3%, 72.7% na 76.7%, n'otu n'otu (Nyocha 5E).).A tụlere ọnụnọ BPDE-DNA site na iji ngwa ELISA.Nsonaazụ anyị gosiri na BPDE-DNA na-agbatị ọkwa na-abawanye na B (a) P-mere otu na-ahụ maka njikwa, ma e jiri ya tụnyere G-Rg3 co-treatment, BPDE-DNA adct level in B (a) P group. B n'ime otu a na-emeso ya, a na-ebelata ọkwa ntinye DNA nke ukwuu.A na-egosipụta nsonaazụ ọgwụgwọ na B (a) P naanị na Figure 5F (1.87 ± 0.33 vs. 3.77 ± 0.42 maka G-Rg3r, 1.93 ± 0.48 vs. 3.77 ± 0.42 maka G -Rg3s, p <0.001).
Nrụpụta mkpụrụ ndụ na BPDE-DNA nguzobe n'ime sel hEL ejiri G-Rg3 na B(a) P.(A) Ikike nke mkpụrụ ndụ hel ejiri B(a) P.(B) Ikike nke mkpụrụ ndụ hel ejiri G-Rg3r gwọọ ya.(C) Ikike nke mkpụrụ ndụ hel ejiri G-Rg3 gwọọ ya.(D) Ịdị ndụ nke mkpụrụ ndụ hel nke ejiri B(a) P na G-Rg3r gwọọ ya.(E) Ikike nke mkpụrụ ndụ hel nke ejiri B(a) P na G-Rg3 gwọọ ya.(F) Ọkwa BPDE-DNA na-etinye n'ime sel hEL ejiri B (a) P na G-Rg3.A na-akọwapụta data dị ka ihe pụtara ± ọkọlọtọ ngbanwe nke mkpebi atọ.P <0.05, ** P <0.01, *** P <0.001.
Achọpụtara okwu enzyme GST ka emechara ya na 10 μM B(a) P na 10 μM G-Rg3r ma ọ bụ G-Rg3s.Nsonaazụ anyị gosipụtara na B (a) P kwụsịrị GST okwu (59.7 ± 8.2% na otu G-Rg3r na 39 ± 4.5% na otu G-Rg3s), yana B (a) P jikọtara ya na G-Rg3r. , ma ọ bụ ya na G-Rg3r, ma ọ bụ ya na G-Rg3r.Usoro ọgwụgwọ na G-Rg3s weghachiri eweghachi okwu GST.Okwu GST (103.7 ± 15.5% na G-Rg3r na 110 ± 11.1% na G-Rg3s, p <0.05 na p <0.001, n'otu n'otu, Fig. 6A, B, na C).A tụlere ọrụ GST site na iji ngwa nyocha ọrụ.Nsonaazụ anyị gosiri na otu ọgwụgwọ nchikota nwere ọrụ GST dị elu ma e jiri ya tụnyere B(a) P naanị otu (96.3 ± 6.6% vs. 35.7 ± 7.8% na G-Rg3r otu vs. 92.3 ± 6.5 na G-Rg3r otu. ).% vs 35.7 ± 7.8% na G-Rg3s otu, p <0.001, Figure 6D).
Ngosipụta nke GST na Nrf2 na sel hEL ejiri B (a) P na G-Rg3.(A) Nchọpụta okwu GST site na ihichapụ Western.(B) Ọnụ ọgụgụ nke GST n'ime sel hEL ejiri B(a) P na G-Rg3r gwọọ ya.(C) Ọnụ ọgụgụ nke GST n'ime sel hel nke ejiri B(a) P na G-Rg3s gwọọ ya.(D) Ọrụ GST na sel hEL ejiri B(a) P na G-Rg3 gwọọ ya.(E) Nchọpụta nke Nrf2 okwu site Western blotting.(F) Ngosipụta ọnụọgụ nke Nrf2 na sel hEL na-emeso ya na B (a) P na G-Rg3r.(G) Ngosipụta ọnụọgụ nke Nrf2 na sel hEL na-emeso ya na B (a) P na G-Rg3s.A na-akọwapụta data dị ka ihe pụtara ± ọkọlọtọ ngbanwe nke mkpebi atọ.P <0.05, ** P <0.01, *** P <0.001.
Iji kọwaa ụzọ ndị dị na G-Rg3-mediated suppression of B (a) P-induced tumorigenesis, a na-enyocha okwu Nrf2 site na Western blotting.Dị ka e gosiri na ọnụ ọgụgụ 6E, F, G, ma e jiri ya tụnyere ndị na-achịkwa, ọ bụ naanị ọkwa Nrf2 na B (a) P na-agwọ ọrịa na-ebelata;Otú ọ dị, ma e jiri ya tụnyere B (a) P na-agwọ ọrịa, B (a) Nrf2 ọkwa na PG-Rg3 otu na-abawanye (106 ± 9.5% maka G-Rg3r vs. 51.3 ± 6.8%, 117 ± 6. 2% maka G-Rg3r vs. 41 ± 9.8% maka G-Rg3s, p <0.01).
Anyị kwadoro ọrụ mgbochi nke Nrf2 site na ịkwụsị okwu Nrf2 site na iji obere RNA (siRNA) na-etinye aka.Nrf2 knockdown kwadoro site na Western blotting (Fig. 7A, B).Dị ka e gosiri na ọnụ ọgụgụ 7C, D, nchịkọta ọgwụgwọ nke mkpụrụ ndụ heEL na B (a) P na G-Rg3 mere ka ọnụ ọgụgụ nke BPDE-DNA adducts (1.47 ± 0.21) ma e jiri ya tụnyere ọgwụgwọ na B (a) P. naanị na otu siRNA njikwa.) G-Rg3r bụ 4.13 ± 0.49, G-Rg3s bụ 1.8 ± 0.32 na 4.1 ± 0.57, p <0.01).Otú ọ dị, mmetụta mgbochi nke G-Rg3 na nhazi BPDE-DNA kwụsịrị site na Nrf2 knockdown.N'ime otu siNrf2, ọ dịghị ihe dị iche iche dị na nhazi BPDE-DNA n'etiti B (a) P na G-Rg3 ngalaba ọgwụgwọ na B (a) ọgwụgwọ naanị (3.0 ± 0.21 maka G-Rg3r vs. 3.56 ± 0.32). ).maka G-Rg3r megide 3.6 maka G-Rg3s megide ± 0.45 megide 4.0± 0.37, p> 0.05).
Mmetụta nke Nrf2 knockdown na BPDE-DNA ntinye ntinye n'ime sel hEL.(A) Nrf2 knockdown kwadoro site na Western blotting.(B) Ntụle ike nke band Nrf2.(C) Mmetụta nke Nrf2 knockdown na BPDE-DNA adct larịị na sel hEL na-emeso B (a) P na G-Rg3r.(D) Mmetụta nke Nrf2 knockdown na ọkwa BPDE-DNA adcts na sel hEL na-emeso B (a) P na G-Rg3.A na-akọwapụta data dị ka ihe pụtara ± ọkọlọtọ ngbanwe nke mkpebi atọ.P <0.05, ** P <0.01, *** P <0.001.
Ọmụmụ ihe a tụlere nsonaazụ mgbochi nke mpụta ginseng dị iche iche na-acha uhie uhie na ụdị òké nke B(a) ọrịa cancer akpa ume na-akpata, yana ọgwụgwọ KRGB belatara ibu arọ nke etuto ahụ.N'iburu n'uche na G-Rg3 nwere ọdịnaya kachasị elu na nchịkọta ginseng a, a mụọla ọrụ dị mkpa nke ginsenoside a na-egbochi tumorigenesis.Ma G-Rg3r na G-Rg3 (epimers abụọ nke G-Rg3) belatara ibu arọ akpụ n'ụdị òké nke B(a) ọrịa cancer kpatara.G-Rg3r na G-Rg3 na-arụ ọrụ anticancer site na ime ka apoptosis nke mkpụrụ ndụ tumo21, na-egbochi uto tumo22, na-ejide cell cycle23 ma na-emetụta angiogenesis24.E gosikwara G-Rg3 ka ọ na-egbochi metastasis cellular25, na ikike G-Rg3 iji kwalite mmetụta nke chemotherapy na redio ka edeela26,27.Poon et al gosipụtara na ọgwụgwọ G-Rg3 nwere ike belata mmetụta genotoxic nke B (a) P28.Ọmụmụ ihe a na-egosipụta ike ọgwụgwọ nke G-Rg3 n'ịchụso mkpụrụ ndụ carcinogenic gburugburu ebe obibi na igbochi ọrịa cancer.
N'agbanyeghị ikike prophylactic ha dị mma, ginsenosides na-adịghị mma nke ọnụ na-adịghị mma na-ebute ihe ịma aka maka ojiji ụlọ ọgwụ nke ụmụ irighiri ihe ndị a.Nyocha ọgwụ ọgwụ nke nchịkwa ọnụ nke ginsenosides na oke gosiri na bioavailability ya ka na-erughị 5%29.Nlele ndị a gosipụtara na mgbe oge ọgwụgwọ izu 20 gasịrị, ọ bụ naanị ọkwa Rg5 na-ebelata.Ọ bụ ezie na usoro dị n'okpuru nke bioavailability na-adịghị mma na-anọgide na-akọwapụta, a na-eche na P-gp na-etinye aka na nsị nke ginsenosides.Ọrụ a gosipụtara na nke mbụ nchịkwa nke verapamil, onye na-egbochi P-gp, na-abawanye bioavailability ọnụ nke G-Rg3r na G-Rg3s.Ya mere, nchoputa a na-egosi na G-Rg3r na G-Rg3s na-arụ ọrụ dị ka ihe ntinye nke P-gp iji chịkwaa nsị ya.
Ọrụ a gosipụtara na ọgwụgwọ njikọta na verapamil na-abawanye bioavailability ọnụ nke G-Rg3 n'ụdị òké nke ọrịa cancer akpa ume.A na-akwado nchoputa a site na mbufe transcellular intestinal nke G-Rg3 na mgbochi P-gp, si otú a na-abawanye nnabata ya.Nyocha na mkpụrụ ndụ Caco2 gosiri na ọgwụgwọ verapamil belatara nsị nke G-Rg3r na G-Rg3 ma na-eme ka akpụkpọ ahụ dịkwuo mma.Ọmụmụ Yang et al.Nnyocha egosiwo na ọgwụgwọ na cyclosporine A (ọzọ P-gp blocker) na-abawanye bioavailability nke ginsenoside Rh2 site na uru ntọala nke 1%20 ruo karịa 30%.Ogige Ginsenosides K na Rg1 gosikwara nsonaazụ yiri ya30,31.Mgbe a na-ejikọta verapamil na cyclosporin A, mpụta nke compound K na mkpụrụ ndụ Caco-2 belatara nke ukwuu site na 26.6 ruo ihe na-erughị 3, ebe ọkwa intracellular ya mụbara 40-fold30.N'ebe verapamil nọ, ọkwa Rg1 mụbara na sel epithelial na-egbu egbu, na-atụ aro ọrụ maka P-gp na ginsenoside efflux, dị ka Meng et al.31 gosipụtara.Otú ọ dị, verapamil enweghị otu mmetụta ahụ na nsị nke ụfọdụ ginsenosides (dị ka Rg1, F1, Rh1 na Re), na-egosi na ọ dịghị emetụta ha site na P-gp substrates, dị ka e gosiri Liang et al.32 .Nlebanya a nwere ike jikọta na ntinye aka nke ndị na-ebugharị ndị ọzọ na usoro ginsenoside ọzọ.
Usoro nke mmetụta mgbochi nke G-Rg3 na ọrịa kansa amabeghị.Nnyocha ndị gara aga egosila na G-Rg3 na-egbochi mmebi DNA na apoptosis site n'ibelata nrụgide oxidative na mbufụt16,33, nke nwere ike ịbụ usoro dị n'okpuru iji gbochie B (a) P-induced tumorigenesis.Ụfọdụ akụkọ na-egosi na genotoxicity nke B(a) P kpatara nwere ike ibelata site n'ịgbanwe usoro nke abụọ enzymes iji mepụta BPDE-DNA34.GST bụ ụdị enzyme nke abụọ nke na-egbochi nhazi BPDE-DNA site n'ịkwalite njikọ nke GSH na BPDE, si otú ahụ na-ebelata mmebi DNA nke B(a) P35 kpatara.Nsonaazụ anyị na-egosi na ọgwụgwọ G-Rg3 na-ebelata cytotoxicity nke B (a) P na BPDE-DNA na-emepụta n'ime sel heEL ma weghachite okwu GST na ọrụ na vitro.Otú ọ dị, mmetụta ndị a adịghị adị na enweghị Nrf2, na-atụ aro na G-Rg3 na-ebute mmetụta cytoprotective site na ụzọ Nrf2.Nrf2 bụ isi ihe ederede maka nkeji II detoxification enzymes nke na-akwalite mkpochapụ nke xenobiotics36.Ịkwalite ụzọ Nrf2 na-ebute cytoprotection ma belata mmebi anụ ahụ37.Ọzọkwa, ọtụtụ akụkọ akwadowo ọrụ Nrf2 dị ka onye na-egbochi tumor na carcinogenesis38.Ọmụmụ ihe anyị na-egosi na ntinye nke ụzọ Nrf2 site na G-Rg3 na-arụ ọrụ nchịkwa dị mkpa na B (a) P-induced genotoxicity site n'ime ka B (a) P detoxification site n'ịgbalite usoro II enzymes, si otú ahụ na-egbochi usoro tumorigenesis.
Ọrụ anyị na-ekpughe ikike nke ginseng na-acha uhie uhie na-egbochi B (a) P na-ebute ọrịa cancer akpa ume na ụmụ oke site na ntinye aka dị mkpa nke ginsenoside G-Rg3.Enweghi ike bioavailability ọnụ nke molekul a na-egbochi ngwa ụlọ ọgwụ ya.Otú ọ dị, ọmụmụ ihe a na-egosi na nke mbụ G-Rg3 bụ mkpụrụ nke P-gp, na nchịkwa nke P-gp inhibitor na-abawanye bioavailability nke G-Rg3 na vitro na na vivo.G-Rg3 na-ebelata cytotoxicity nke B (a) P site na ịhazi ụzọ Nrf2, nke nwere ike ịbụ usoro nwere ike ime maka ọrụ mgbochi ya.Ọmụmụ ihe anyị kwadoro ikike nke ginsenoside G-Rg3 maka mgbochi na ọgwụgwọ ọrịa cancer akpa ume.
Nwa nwanyị A/J dị izu isii (20 ± 1 g) na oke Wistar dị izu asaa (250 ± 20 g) nwetara site na ụlọ nyocha Jackson (Bar Harbor, USA) na Wuhan Institute of Zoology.Mahadum (Wuhan, China).Ụlọ ọrụ mkpokọta ụdị omenala ndị China (Wuhan, China) nyere anyị Caco-2 na sel hEL.Sigma-Aldrich (St. Louis, USA) bụ isi iyi nke B(a) P na tricaprine.Ginsenosides dị ọcha G-Rg3r na G-Rg3s, dimethyl sulfoxide (DMSO), CellTiter-96 proliferation assay kit (MTS), verapamil, minimal important medium (MEM), na fetal bovine serum (FBS) ka zụtara n'aka Chengdu Must Bio-Technology .Co., Ltd.(Chengdu, China).A zụrụ obere ngwa QIAamp DNA na BPDE-DNA ntinye ELISA ngwa site na Qiagen (Stanford, CA, USA) na Cell Biolabs (San Diego, CA, USA).A zụtara ngwa nyocha ọrụ GST na mkpokọta protein nyocha (usoro BCA ọkọlọtọ) site na Solarbio (Beijing, China).A na-echekwa ihe niile ginseng na-acha uhie uhie na Mingyu Laboratory 7. Mahadum Baptist Hong Kong (Hong Kong, China) na Korea Cancer Center (Seoul, Korea) bụ isi mmalite azụmahịa nke CRG wepụ na ụdị ginseng dị iche iche na-acha uhie uhie nke sitere na Korea dị iche iche (gụnyere KRGA, KRGB). na KRGC).A na-eme ginseng na-acha uhie uhie site na mgbọrọgwụ nke ginseng ọhụrụ dị afọ 6.A na-enweta ihe mgbapụta ginseng na-acha uhie uhie site na ịsacha ginseng na mmiri ugboro atọ, wee tinye uche na mmiri mmiri, na n'ikpeazụ ihicha na obere okpomọkụ iji nweta ginseng wepụ ntụ ntụ.A zụrụ ọgwụ mgbochi (anti-Nrf2, anti-GST, na β-actin), horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG), transfection reagent, control siRNA, na Nrf2 siRNA ka zụtara na Santa Cruz Biotechnology (Santa Cruz, CA). .), USA).
Azụlitere mkpụrụ ndụ Caco2 na hEL n'ime efere omenala sel 100 mm2 nwere MEM nwere 10% FBS na 37 Celsius C na ikuku iru mmiri nke 5% CO2.Iji chọpụta mmetụta nke ọnọdụ ọgwụgwọ, a na-etinye mkpụrụ ndụ hEL na ntinye dị iche iche nke B (a) P na G-Rg3 na MEM maka 48 h.Enwere ike nyochaa ma ọ bụ kpokọta mkpụrụ ndụ iji kwado ihe ndị na-enweghị cell.
All nnwale e mma site na Experimental Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Nkwenye Mba. 2019; Ndebanye aha No. 4587TH).Emere nnwale niile dị ka ụkpụrụ na ụkpụrụ dị mkpa si dị, a na-eduzi ọmụmụ ihe ahụ dabere na nyocha anụmanụ: Nkwupụta nke In Vivo Experiments (ARRIVE).A na-ebu ụzọ gbaa ụmụ oke A/J dị izu asatọ n'ime intraperitoneally na B(a) P na ngwọta tricaprine (100 mg/kg, 0.2 ml).Mgbe otu izu gachara, a na-ekewa ụmụ oke ndị ahụ na-enweghị usoro n'ime otu njikwa na otu ọgwụgwọ dị iche iche, ụmụ oke iri na ise n'ime otu ọ bụla, ma na-eri nri otu ugboro n'ụbọchị.Mgbe izu 20 nke ọgwụgwọ gasịrị, CO2 asphyxia na-achụ anụmanụ.A na-anakọta akpa ume ma dozie maka awa 24.Ọnụ ọgụgụ nke etuto ahụ dị elu na nha etuto ahụ dị n'otu n'otu ka akwadoro maka ngụgụ ọ bụla n'okpuru microscope na-ekesa.A na-agbakọ atụmatụ olu tumor (V) site na iji okwu ndị a: V (mm3) = 4/3πr3, ebe r bụ dayameta tumor.Nchikota ụgbụ akpụ niile dị na ngụgụ ụmụ oke na-anọchi anya mkpokọta etuto ahụ, na nkezi mkpokọta etuto ahụ n'otu ọ bụla nọchiri anya ibu etuto ahụ.A nakọtara ma chekwaa ọbara na eriri afọ na -80 Celsius maka mkpebi UPLC-MS/MS.A chịkọtara ọbara ma jiri ihe nyocha kemịkalụ akpaghị aka wee nyochaa ọkwa alanine aminotransferase (ALT) na serum creatinine (Cr) iji chọpụta ọrụ imeju na akụrụ.
E wepụrụ ihe nlele ndị anakọtara na nchekwa oyi, gbazee, tụọ, ma tinye ya n'ime tube dị ka akọwara n'elu.Na nke a, etinyere 0.5 μM phlorizin (ụkpụrụ dị n'ime) na ngwọta methanol 0.8 ml.A na-ejikọta anụ ahụ ahụ site na iji Tissue-Tearor ma mesịa bufee homogenate na tube microcentrifuge 1.5 ml.A na-etinye ngwakọta ahụ na 15500 rpm maka nkeji 15.Mgbe ị wepụsịrị 1.0 ml nke supernatant, kpoo ya na nitrogen.A na-eji methanol narị microliters mee ihe maka mgbake.A na-anakọta ma hazie ọbara ahụ n'otu ahịrị ma jiri ya mee ihe maka nha niile.
A na-eji mkpụrụ ndụ 1.0 × 105 Caco-2 dị 24-nke ọma were mkpụrụ sel kwa nke ọma iji nyochaa nkwalite nkwalite nke njem G-Rg3 site na mgbakwunye nke verapamil.Mgbe izu 3 nke omenala gasịrị, a na-eji HBSS sachaa mkpụrụ ndụ ma tinye ya na 37 Celsius.400 μL nke 10 μM G-Rg3 (G-Rg3r, G-Rg3s, ma ọ bụ ngwakọta na 50 ma ọ bụ 100 μM verapamil) ka agbanyere n'akụkụ basolateral ma ọ bụ apical nke monolayer, na 600 μL nke HBSS ngwọta na-agbakwunyere na nke ọzọ. akụkụ.Chịkọta 100 µl nke usoro omenala n'oge a kara aka (0, 15, 30, 45, 60, 90 na 120 nkeji) ma tinye 100 µl nke HBSS iji mejupụta olu a.A na-echekwa ihe nlele na -4 Celsius ruo mgbe UPLC-MS/MS chọpụtara.A na-eji okwu Papp = dQ / (dT × A × C0) mee ka ọnụ ọgụgụ nke apical na basolateral permeability pụtara ìhè na nke ọzọ (Pa-b na Pb-a, n'otu n'otu);dQ / dT bụ mgbanwe na ntinye uche, A (0.6 cm2) bụ mpaghara elu nke monolayer, na C0 bụ ntinye onyinye mbụ.A na-agbakọ nha nke efflux dị ka Pb-a/Pa-b, nke na-anọchi anya ọnụọgụ mmiri nke ọgwụ ọmụmụ.
A na-ebu ọnụ oke oke Wistar maka awa 24, ṅụọ naanị mmiri, wee were ọgwụ ntụtụ nke 3.5% pentobarbital kụchie ya.The intubated silicone tube nwere njedebe nke duodenum dị ka ọnụ ụzọ na njedebe nke ileum dị ka ụzọ ọpụpụ.Jiri mgbapụta peristaltic were 10 µM G-Rg3r ma ọ bụ G-Rg3s na-ebubata ntinye n'ime HBSS isotonic na ọsọ ọsọ nke 0.1 ml/min.A na-enyocha mmetụta nke verapamil site na ịgbakwunye 50 μM ma ọ bụ 100 μM nke ogige ahụ na 10 μM G-Rg3r ma ọ bụ G-Rg3s.A na-eme UPLC-MS/MS na ihe ndị na-esi ísì ụtọ anakọtara na oge 60, 90, 120, na 150 nkeji mgbe mmalite nke perfusion.A na-atụle pasent nke nnabata site na usoro % absorption = (1 - Cout/Cin) × 100%;A na-egosipụta ntinye nke G-Rg3 na ntinye na ntinye site na Cout na Cin, n'otu n'otu.
A na-akụ mkpụrụ ndụ heEL na efere 96-well na njupụta nke sel 1 × 104 kwa nke ọma ma jiri B (a) P (0, 1, 5, 10, 20, 30, 40 μM) ma ọ bụ G-Rg3 gbazere na DMSO. .Ejiri usoro omenala gbazere ọgwụ ndị ahụ na ntinye dị iche iche (0, 1, 2, 5, 10, 20 μM) karịa awa 48.N'iji ngwa nyocha MTS dị na azụmaahịa, e debere mkpụrụ ndụ n'usoro ọkọlọtọ wee tụọ ya site na iji microplate agụ na 490 nm.A na-enyocha ọkwa ịdị ndụ cell nke ndị otu a jikọtara ya na B (a) P (10 μM) na G-Rg3 (0, 1, 5, 10, 20 μM) dịka usoro a dị n'elu ma jiri ya tụnyere ndị a na-adịghị edozi ya.
A na-akụ mkpụrụ ndụ heEL na efere 6-nke ọma na njupụta nke 1 × 105 sel / nke ọma ma mesoo ya na 10 μMB (a) P na ọnụnọ ma ọ bụ enweghị 10 μM G-Rg3.Mgbe awa 48 gachara ọgwụgwọ, ewepụtara DNA na mkpụrụ ndụ heEL site na iji QIAamp DNA Mini Kit dị ka ụkpụrụ ndị nrụpụta siri dị.Achọpụtara nguzobe BPDE-DNA site na iji ngwa ELISA ntinye BPDE-DNA.A tụlere ọkwa agbakwunyere BPDE-DNA site na iji ihe na-agụ microplate site n'ịtụ nnabata na 450 nm.
A na-akụ mkpụrụ ndụ heEL na efere 96-well na njupụta nke 1 × 104 sel kwa nke ọma ma mesoo ya na 10 μMB (a) P na enweghị ma ọ bụ ọnụnọ nke 10 μM G-Rg3 maka 48 h.A tụrụ ọrụ GST site na iji ngwa nyocha ihe omume GST azụmahịa dịka ụkpụrụ ndị nrụpụta siri dị.A tụrụ mgbakwa GST dịtụ iche site na nnabata na 450 nm site na iji ihe na-agụ microplate.
A na-eji PBS oyi na-asacha sel hEL wee hịchaa ya site na iji redioimmunoprecipitation assay buffer nwere protease inhibitors na phosphatase inhibitors.Mgbe nyochachara protein site na iji ngwa nyocha protein zuru oke, 30 μg nke protein dị na nlele ọ bụla kewapụrụ site na 12% SDS-PAGE wee bufee ya na akpụkpọ anụ PVDF site na electrophoresis.Eji 5% mmiri ara ehi na-amị amị kpọchie akụkụ ahụ ma tinye ya na ọgwụ mgbochi mbụ n'otu abalị na 4 Celsius.Mgbe etinyere ya na ọgwụ mgbochi nke abụọ nke horseradish peroxidase-conjugated, agbakwunyere chemiluminescence reagents emelitere iji hụ na mgbama njikọ ahụ.Achọpụtara ike nke eriri protein ọ bụla site na iji ngwanrọ ImageJ.
Ejiri ngwanro GraphPad Prism 7.0 wee nyochaa data niile, ekwuputara dị ka ọ pụtara ± ọkọlọtọ ọkọlọtọ.A tụlere ọdịiche dị n'etiti otu ọgwụgwọ site na iji nyocha nke Student's t ma ọ bụ nyocha otu ụzọ nke ọdịiche, yana uru P <0.05 na-egosi mkpa ndekọ.
A na-etinye data niile enwetara ma ọ bụ nyochara n'oge ọmụmụ ihe a n'akwụkwọ akụkọ a na faịlụ ozi mgbakwunye.
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Oge nzipu: Sep-17-2023