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Ua hoʻohana ʻia ka ginseng ʻulaʻula i ka lāʻau lapaʻau kuʻuna ʻĀsia no nā haneli mau makahiki.Ma kēia noiʻi ʻana, ua loiloi mākou i ka hiki o nā ʻano ʻehā ʻulaʻula ʻulaʻula (ʻulaʻula Kina, ginseng ʻulaʻula Korean A, ginseng ʻulaʻula Korean B, a me ginseng ʻulaʻula Korea C) i ulu ʻia ma nā ʻāpana like ʻole e pale i ka hoʻokumu ʻana a me ka ulu ʻana o ka māmā kino kino. nā maʻi koko.Ua mālama ʻia kahi hoʻāʻo benzo(a)pyrene (B(a)P) ma luna o nā ʻiole A/J, a ua ʻike ʻia ʻo Korea ʻulaʻula B ginseng ka mea maikaʻi loa i ka hōʻemi ʻana i ke kaumaha ma waena o nā ʻano ginseng ʻulaʻula ʻehā.Eia kekahi, ua nānā mākou i nā mea o nā ginsenosides like ʻole (Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 a me Rg5) i loko o ʻehā mau ʻāpana ginseng ʻulaʻula a ʻike ʻia ua loaʻa ka ginseng ʻulaʻula Korean B. nā pae kiʻekiʻe loa o ka ginsenoside Rg3 (G-Rg3), e hōʻike ana he kuleana koʻikoʻi paha ka G-Rg3 i kāna hana therapeutic.Hōʻike kēia hana he haʻahaʻa haʻahaʻa ko G-Rg3.Eia nō naʻe, i ka wā i hui pū ʻia ai ʻo G-Rg3 me ka verapamil inhibitor P-gp, ua hoʻemi ʻia ka efflux o G-Rg3 i loko o nā pūnae Caco-2, ua hoʻonui ʻia ka nui o ka absorption intestinal o G-Rg3 i kahi ʻano ʻiole, a me G-Rg3 ua hoonuiia.Ma nā pūnaewele Caco-2, emi iho ka puka o Rg3, a emi iho ka pae o Rg3.Hoʻonui ʻia ka G-Rg3 i loko o ka ʻōpū a me ka plasma, a ua hoʻonui ʻia kona hiki ke pale i nā maʻi maʻi ma kahi ʻano ʻiole o B (a) P-induced tumorigenesis.Ua ʻike pū mākou ua hoʻemi ʻo G-Rg3 i ka B (a) P-induced cytotoxicity a me ka hoʻokumu ʻana i ka hoʻohui ʻana o DNA i loko o nā pūnaʻi māmā kanaka, a hoʻihoʻi i ka ʻōlelo a me ka hana o nā enzymes phase II ma o ke ala Nrf2, e pili ana paha i ke ʻano o ka hana. o G kaohi -Rg3..E pili ana i ka hiki ʻana mai o nā maʻi ʻōpū.Hōʻike kā mākou aʻo ʻana i kahi kuleana koʻikoʻi no G-Rg3 i ka huli ʻana i nā ʻōpū o ka pūpū i nā ʻiole.Hoʻonui ʻia ka bioavailability waha o kēia ginsenoside e ka huli ʻana i ka P-glycoprotein, e ʻae ana i ka mole e hana i nā hopena anticancer.
ʻO ke ʻano maʻamau maʻamau o ka maʻi maʻi maʻi ʻaʻole liʻiliʻi (NSCLC), ʻo ia kekahi o nā kumu nui o ka make maʻi maʻi ma Kina a me ʻAmelika ʻĀkau1,2.ʻO ke kumu nui e hoʻonui ai i ka pilikia o ka hoʻomohala ʻana i ka maʻi maʻi maʻi ʻaʻai liʻiliʻi liʻiliʻi ʻo ka puhi paka.Loaʻa i ka uahi paka ma mua o 60 mau carcinogens, me benzo(a)pyrene (B(a)P), nitrosamines, a me radioactive isotopes mai ka palaho o radon. uahi.Ma ka ʻike ʻana iā B(a)P, hoʻololi ka cytochrome P450 iā B(a)P-7,8-dihydrodiol-9,10-epoxide (BPDE), ka mea e hana me DNA e hana i ka BPDE-DNA addduct 4. nā mea hoʻohui i hoʻoulu i ka tumorigenesis o ka māmā i nā ʻiole me ka pae maʻi maʻi a me ka histopathology e like me nā maʻi ʻōpū o ke kanaka5.ʻO kēia hiʻohiʻona ka B(a)P-induced lung cancer model i ʻōnaehana kūpono no ka loiloi ʻana i nā pūhui me nā waiwai anticancer.
ʻO kahi hoʻolālā e hiki ai ke pale i ka ulu ʻana o ka maʻi maʻi maʻi i loko o nā pūʻulu kiʻekiʻe, ʻoi aku ka poʻe puhi paka, ʻo ia ka hoʻohana ʻana i nā lāʻau chemopreventive e hoʻopau i ka ulu ʻana o nā maʻi neoplastic intraepithelial a no laila e pale ai i ko lākou piʻi ʻana i ka malignancy.Hōʻike nā haʻawina holoholona i nā ʻano lāʻau lapaʻau chemopreventive maikaʻi loa6.Ua hōʻike kā mākou hōʻike mua7 i nā hopena pale maikaʻi o ka ginseng ʻulaʻula i ka maʻi kanesa.Ua hoʻohana ʻia kēia lau nahele no nā kenekulia i ka lāʻau lapaʻau kuʻuna ʻĀsia e hoʻolōʻihi i ke ola a me ke olakino, a ua kākau ʻia he hopena antitumor8.
ʻO ka mea ikaika o ka ginseng he ginsenoside, kahi i hoʻohana ʻia ma ke ʻano he hōʻailona composite e loiloi i ka maikaʻi o nā extract ginseng.Hoʻohana maʻamau ka hoʻohana ʻana i kekahi mau ginsenosides, me ka RK1, Rg1, F1, Re, Rb1, Rb2, Rb3, Rd, Rh1, Rh2, Rg3, Rg5, a me Rc9,10.ʻAʻole hoʻohana liʻiliʻi nā Ginsenosides ma muli o kā lākou bioavailability waha maikaʻi loa11.ʻOiai ʻaʻole maopopo ka mīkini no kēia bioavailability maikaʻi ʻole, ʻo ka efflux o ginsenosides i hana ʻia e P-glycoprotein (P-gp)12 ke kumu.ʻO P-gp kekahi o nā mea lawe efflux koʻikoʻi i loko o ka ATP-binding cassette transporter superfamily, e hoʻohana ana i ka ikehu o ATP hydrolysis e hoʻokuʻu i nā mea intracellular i loko o ke kaiapuni o waho.Hoʻolaha nui ʻia nā mea lawe P-gp i loko o ka ʻōpū, ka puʻupaʻa, ke ake a me ka pale koko-lolo13.He kuleana koʻikoʻi ka P-gp i ka absorption intestinal, a hoʻonui ka inhibition o P-gp i ka absorption waha a me ka loaʻa ʻana o kekahi mau lāʻau anticancer12,14.ʻO nā laʻana o nā mea paʻa i hoʻohana mua ʻia ma ka palapala ʻo verapamil a me cyclosporine A15.Aia kēia hana i ka hoʻokumu ʻana i kahi ʻōnaehana ʻiole no ke aʻo ʻana i ka maʻi maʻi maʻi maʻi maʻi B (a) P-induced no ka loiloi i ka hiki o nā ʻāpana ginseng ʻulaʻula like ʻole mai Kina a me Korea e hoʻopilikia i nā maʻi ʻino.Hoʻokaʻawale ʻia nā ʻāpana e ʻike i nā ginsenosides kikoʻī e pili ana i ka carcinogenesis.A laila ua hoʻohana ʻia ʻo Verapamil e hoʻopaʻa i ka P-gp a hoʻomaikaʻi i ka bioavailability o ka waha a me ka pono therapeutic o nā ginsenosides e huli ana i ka maʻi kanesa.
ʻAʻole maopopo ke ʻano o ka hana ʻana o ginseng saponins i nā hopena therapeutic i ka carcinogenesis.Ua hōʻike ʻia ka noiʻi e hiki i nā ginsenosides like ʻole ke hōʻemi i ka pōʻino DNA i hana ʻia e nā carcinogens ma o ka hoʻohaʻahaʻa ʻana i ke kaumaha oxidative a me ka hoʻoulu ʻana i nā enzyme detoxification phase II, no laila e pale ai i ka pōʻino o nā cell.ʻO Glutathione S-transferase (GST) kahi enzyme maʻamau maʻamau II e koi ʻia e hōʻemi i ka pōʻino DNA i hana ʻia e nā carcinogens17.ʻO ka nuclear erythroid 2-related factor 2 (Nrf2) he mea transcription koʻikoʻi e hoʻoponopono i ka homeostasis redox a hoʻoulu i ka hōʻike o nā enzymes phase II a me nā pane antioxidant cytoprotective18.Ua nānā pū kā mākou noiʻi i nā hopena o nā ginsenosides i ʻike ʻia ma ka hōʻemi ʻana i ka B (a) P-induced cytotoxicity a me ka BPDE-DNA adduct formation, a me ka hoʻoulu ʻana i nā enzymes phase II ma ka hoʻololi ʻana i ke ala Nrf2 i loko o nā pūpū māmā maʻamau.
ʻO ka hoʻokumu ʻana i kahi ʻano ʻiole o ka maʻi maʻi B(a)P-induced e kūlike me ka hana mua5.Hōʻike ka Figure 1A i ka hoʻolālā hoʻokolohua o ka mālama ʻana i nā pule he 20 pule o ke ʻano maʻi maʻi ʻiole i hoʻoulu ʻia e B(a)P, wai (mana), ʻāpana ʻulaʻula ʻulaʻula Kina (CRG), ʻāpana ʻulaʻula ʻulaʻula Korean A (KRGA), a me ka ʻulaʻula Korea. ginseng.Unuhi B (KRGB) a me Korean Red Ginseng Extract C (KRGC).Ma hope o 20 pule o ka mālama ʻana i ka ginseng ʻulaʻula, ua mōhai ʻia nā ʻiole e ka asphyxiation CO2.Hōʻike ka Figure 1B i nā ʻōpū māmā macroscopic i loko o nā holoholona i mālama ʻia me nā ʻano ginseng ʻulaʻula like ʻole, a hōʻike ka Figure 1C i kahi micrograph māmā o kahi laʻana tumo.ʻO ke kaumaha nui o nā holoholona i mālama ʻia e KRGB (1.5 ± 0.35) he haʻahaʻa ma mua o nā holoholona hoʻomalu (0.82 ± 0.2, P <0.05), e like me ka hōʻike ʻana ma ke Kiʻi 1D.ʻO ka awelika degere o ka hoʻopaʻa ʻana i ke koko he 45%.ʻAʻole i hōʻike ʻia nā hoʻololi koʻikoʻi o ke kaumaha o ke koko (P > 0.05) i nā ʻāpana ginseng ʻulaʻula ʻē aʻe i hoʻāʻo ʻia.ʻAʻole i ʻike ʻia nā hopena ʻaoʻao i ʻike ʻia ma ke ʻano ʻiole i nā pule he 20 o ka mālama ʻana i ka ginseng ʻulaʻula, me ka ʻole o ka hoʻololi ʻana i ke kaumaha o ke kino (ʻike ʻole i hōʻike ʻia) a ʻaʻohe mea ʻawaʻawa a i ʻole nā kiʻi (Figure 1E,F).
Hoʻomaʻamaʻa ʻia ka extract ginseng ʻulaʻula i ka ulu ʻana o ka maʻi maʻi maʻi i nā ʻiole A/J.(A) Hoʻolālā hoʻokolohua.(B) ʻO nā maʻi ʻaʻai nui o ka ʻiole.Hōʻike ʻia nā ʻōpū e nā pua.a: Hui ʻulaʻula Kina.b: pūʻulu A o ka ginseng ʻulaʻula Korean.c: Pūʻulu ginseng ʻulaʻula Korean B. d: Pūʻulu ginseng ʻulaʻula Korean C. d: Pūʻulu mana.(C) ʻO ka micrograph māmā e hōʻike ana i kahi maʻi ʻōpū.Hoʻonui: 100. b: 400. (D) ʻO ka hoʻouka ʻana o ka Tumor i ka hui ʻulaʻula ginseng extract.(E) Nā pae plasma o ka ate enzyme ALT.(F) Nā pae plasma o ka enzyme renal Cr.Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± kaʻe maʻamau.*P <0.05.
Ua kālailai ʻia nā ʻāpana ginseng ʻulaʻula i ʻike ʻia ma kēia noiʻi e ka ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) e helu i kēia mau ginsenosides: Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3 , Rh2, F1, Rk1 a me Rg5.ʻO nā kūlana UPLC a me MS i hoʻohana ʻia e ana i nā analytes i wehewehe ʻia ma kahi hōʻike mua19.Hōʻike ʻia nā chromatograms UPLC-MS/MS o ʻehā mau ʻāpana ginseng ʻulaʻula ma ke Kiʻi 2A.Aia nā ʻokoʻa nui i ka nui o ka maʻiʻo ginsenoside, me ka nui o ka nui o ka maʻi ginsenoside ma CRG (590.27 ± 41.28 μmol/L) (Figure 2B).I ka loiloi ʻana i nā ginsenosides pākahi (Figure 2C), ua hōʻike ʻo KRGB i ka pae kiʻekiʻe o G-Rg3 i hoʻohālikelike ʻia me nā ginsenosides ʻē aʻe (58.33 ± 3.81 μmol/L no G-Rg3s a me 41.56 ± 2.88 μmol/L no G -Rg3r).L).ʻano ginseng ʻulaʻula (P <0.001).Hana ʻia ʻo G-Rg3 ma ke ʻano he mau stereoisomers G-Rg3r a me G-Rg3s, ʻokoʻa ke kūlana o ka hui hydroxyl ma carbon 20 (Fig. 2D).Hōʻike nā hopena e loaʻa paha iā G-Rg3r a i ʻole G-Rg3 ka mana anticancer koʻikoʻi i loko o kahi kumu hoʻohālike ʻiole maʻi maʻi maʻi B (a) P-induced.
Maʻiʻo o nā ginsenosides i loko o nā ʻāpana ginseng ʻulaʻula.(A) UPLC-MS/MS chromatograms o ʻehā mau ʻāpana ginseng ʻulaʻula.(B) Ka manaʻo o ka huina ginsenoside ma nā unuhi i hōʻike ʻia.(C) Ka ʻike ʻana i nā ginsenosides pākahi ma nā ʻāpana i hōʻailona ʻia.(D) Nā hana o nā stereoisomers ginsenoside G-Rg3r a me G-Rg3s.Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± ka ʻokoʻa maʻamau o nā hoʻoholo triplicate.***P <0.001.
Pono ka noiʻi UPLC-MS/MS i ka helu ʻana o nā ginsenosides i loko o ka ʻōpū a me ke koko ma hope o 20 mau pule o ka mālama ʻana.Ua hōʻike ʻia ka mālama ʻana me KRGB i ke alo o 0.0063 ± 0.0005 μg/ml Rg5 wale nō i loko o ke koko.ʻAʻole i ʻike ʻia nā koena ginsenosides, e hōʻike ana i ka bioavailability waha a no laila ua hoʻemi ʻia ka ʻike ʻana i kēia mau ginsenosides.
ʻO ka laina cell adenocarcinoma colon Caco-2 he morphologically a me ka biochemically like me nā cell epithelial intestinal epithelial, e hōʻike ana i kona pono i ka loiloi ʻana i ka lawe ʻana i nā enterocyte ma waena o ka pale epithelial intestinal.Ua hoʻokumu ʻia kēia loiloi ma kahi haʻawina mua 20.Hōʻike nā kiʻi 3A,B,C,D,E,F i nā kiʻi hōʻike o ka lawe transcellular o G-Rg3r a me G-Rg3 me ka hoʻohana ʻana i kahi kumu hoʻohālike Caco-2 monolayer.ʻO ka halihali transcellular o G-Rg3r a i ʻole G-Rg3 ma waena o nā monolayers Caco-2 mai ka basolateral i ka ʻaoʻao apical (Pb-a) i ʻoi aku ka kiʻekiʻe ma mua o ka apical a hiki i ka ʻaoʻao basolateral (Pa-b).No G-Rg3r, he 0.38 ± 0.06 ka mean Pa-b, i hoʻonui i ka 0.73 ± 0.06 ma hope o ka mālama ʻana me 50 μmol/L verapamil a i ka 1.14 ± 0.09 ma hope o ka mālama ʻana me 100 μmol/L verapamil (p <0.01, a me 0.01, Kiʻi 2).3A).ʻO nā nānāʻana no G-Rg3 i hahai i kahiʻano like (Fig. 3B), a ua hōʻike nā hopena i ka hoʻonuiʻana o ka verapamil i ka laweʻana o G-Rg3r a me G-Rg3.ʻO ka hoʻomaʻamaʻa ʻana iā Verapamil ka mea i hoʻemi nui i ka mean Pb-a a me G-Rg3r a me G-Rg3s efflux ratios (Figure 3C,D,E,F), e hōʻike ana i ka mālama ʻana i ka verapamil e hōʻemi i ka ʻike ginsenoside i loko o nā pūnaewele efflux Caco-2..
ʻO ka halihali transcellular o G-Rg3 i nā monolayers Caco-2 a me ka absorption o ka ʻōpū i kahi hoʻomaʻamaʻa perfusion assay.(A) waiwai Pa-b o ka hui G-Rg3r ma Caco-2 monolayer.(B) Pa-b waiwai o G-Rg3s hui ma Caco-2 monolayer.(C) waiwai Pb o ka hui G-Rg3r ma Caco-2 monolayer.(D) Waiwai Pb o nā pūʻulu G-Rg3s ma Caco-2 monolayer.(E) Lakiō hua o nā pūʻulu G-Rg3r i kahi monolayer Caco-2.(F) Lakiō hua o nā pūʻulu G-Rg3 i kahi monolayer Caco-2.(G) Paneka o ka intestinal absorption o G-Rg3r i ka perfusion assay i na iole.(H) Ka pākēneka o ka absorption intestinal o G-Rg3 i loko o kahi ho'āʻo perfusion i nā ʻiole.Hoʻohālikelike ʻia ka permeability a me ka absorption me ka hoʻohui ʻole o ka verapamil.Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± ka wehe ʻana o nā hoʻokolohua kūʻokoʻa ʻelima.*P <0.05, **P <0.01, ***P <0.001.
E like me ka hana mua20, ua hana ʻia ka ʻiole orthotopic intestinal perfusion o nā ʻiole no ka hoʻoholo ʻana inā piʻi ka absorption G-Rg3 i loko o ka ʻōpū ma hope o ka mālama ʻana i ka verapamil.Hōʻike nā kiʻi 3G,H i nā hōʻike perfusion hōʻike e loiloi i ka pākēneka o ka absorption o ka ʻōpū o G-Rg3r a me G-Rg3 i nā ʻiole hoʻohālike maʻi maʻi i nā manawa o luna.ʻO ka pākēneka mua o ka lawe ʻana o G-Rg3r nāwaliwali ma kahi o 10% i hoʻonui ʻia ma mua o 20% ma hope o ka mālama ʻana me 50 μM verapamil a i ʻoi aku ma mua o 25% ma hope o ka mālama ʻana me 100 μM verapamil.Pēlā nō, ʻo G-Rg3, ka mea i loaʻa mua he 10%, ua hōʻike pū i kahi kiʻekiʻe ma mua o 20% ma hope o ka mālama ʻana me 50 μM verapamil a kokoke i 30% ma hope o ka mālama ʻana me 100 μM verapamil, e hōʻike ana i ka hoʻonui ʻana i ka P-gp e verapamil. ʻO ka ʻōpū G-absorption Rg3 i loko o kahi ʻano ʻiole o ka maʻi maʻi ʻaʻai.
E like me ke ʻano o luna, ua māhele like ʻia nā ʻiole hoʻohālike o ka maʻi maʻi B(a)P i ʻeono pūʻulu, e like me ka hōʻike ʻana ma ke Kiʻi 4A.ʻAʻole i ʻike ʻia ka pohō kaumaha nui a i ʻole nā hōʻailona lapaʻau o ka toxicity i ka hui mālama G-Rg3 i hoʻohālikelike ʻia me ka pūʻulu mana (ʻaʻole i hōʻike ʻia ka ʻikepili).Ma hope o 20 pule o ka mālama ʻana, ʻohi ʻia nā māmā o kēlā me kēia ʻiole.Hōʻike ka Figure 4B i nā ʻōpū o ka ʻōpū macroscopic i nā ʻiole ma nā hui lapaʻau ma luna, a ʻo ka Figure 4C e hōʻike ana i kahi micrograph māmā ʻokoʻa o kahi maʻi ʻokoʻa.E pili ana i ke kaumaha o ka maʻi maʻi i kēlā me kēia hui (Fig. 4D), nā waiwai no nā ʻiole i mālama ʻia me G-Rg3r a me G-Rg3s he 0.75 ± 0.29 mm3 a me 0.81 ± 0.30 mm3, ʻoiai nā waiwai no G Mice i mālama ʻia. me -Rg3s he 1.63 pakahi ± 0.40 mm3.nā ʻiole hoʻomalu (p <0.001), e hōʻike ana i ka mālama ʻana o G-Rg3 i hōʻemi i ke kaumaha maʻi maʻi i nā ʻiole.Hoʻonui hou ka hoʻohana ʻana i ka verapamil i kēia hoʻemi: ua emi nā waiwai o ka verapamil+ G-Rg3r mice mai 0.75 ± 0.29 mm3 a i 0.33 ± 0.25 mm3 (p <0.01), a ua emi nā waiwai no ka verapamil+ mai 0.81 ± 0.30 mm3 i 0.81 ± 0.30 mm3 i 0.81 ± 0.30 mm3. mm3 i nāʻiole G. -Rg3s i mālamaʻia (p <0.05), e hōʻike ana e hiki i ka verapamil ke hoʻonui i ka hopena o ka G-Rg3 ma ka tumorigenesis.ʻAʻole i hōʻike ʻia ke kaumaha o ka tumor i nā ʻokoʻa nui ma waena o ka pūʻulu mana a me ka hui verapamil, ka hui G-Rg3r a me ka hui G-Rg3s, a me ka hui verapamil + G-Rg3r a me ka hui verapamil + G-Rg3s.Eia kekahi, ʻaʻohe mea ʻawaʻawa koʻikoʻi o ke akepaʻa a i ʻole ka puʻupaʻa i pili i nā lāʻau i loiloi ʻia (Figure 4E,F).
ʻO ke kaumaha ma hope o ka mālama ʻana G-Rg3 a me ka plasma a i ʻole ka intestinal G-Rg3r a me nā pae G-Rg3 i nā hui i hōʻike ʻia.(A) Hoʻolālā hoʻokolohua.(B) ʻO nā maʻi maʻi macroscopic i loko o kahi ʻano ʻiole.Hōʻike ʻia nā ʻōpū e nā pua.a: G-Rg3r.b: G-Rg3s.c: G-Rg3r me ka verapamil.d: G-Rg3 i hui pū me ka verapamil.d: Verapamil.e: hoʻomalu.(C) Micrograph Optical o ke koko ma ka hoʻonui.Pane: 100x.b: 400X.(D) Ka hopena o ka mālama ʻana o G-Rg3 + verapamil i ke kaumaha o ka maʻi maʻi i nā ʻiole A / J.(E) Nā pae plasma o ka ate enzyme ALT.(F) Nā pae plasma o ka enzyme renal Cr.(G) Nā pae plasma o G-Rg3r a i ʻole G-Rg3 o nā hui i hōʻike ʻia.(H) Nā pae o G-Rg3r a i ʻole G-Rg3s i loko o ka ʻōpū o nā hui i hōʻike ʻia.Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± ka ʻokoʻa maʻamau o nā hoʻoholo triplicate.*P <0.05, **P <0.01, ***P <0.001.
Ua loiloi ʻia nā pae G-Rg3 ma ka B(a)P-induced cancer model mice e UPLC-MS/MS ma hope o ka manawa mālama 20-wiki e like me ke ʻano i wehewehe ʻia ma ka pauku Methods.Hōʻike nā kiʻi 4G a me H i ka plasma a me nā pae G-Rg3 intestinal, kēlā me kēia.ʻO ka pae plasma G-Rg3r he 0.44 ± 0.32 μmol / L a hoʻonui i ka 1.17 ± 0.47 μmol / L me ka hoʻohana like ʻana o verapamil (p <0.001), aʻo nā pae G-Rg3r intestinal he 0.53 ± 0.08 µg.Ke hui pū me ka verapamil, hoʻonui ʻia ka g i 1.35 ± 0.13 μg/g (p <0.001).No G-Rg3, ua hahai nā hopena i ke ʻano like, e hōʻike ana ua hoʻonui ka lāʻau verapamil i ka bioavailability waha o G-Rg3 i nā ʻiole A / J.
Ua hoʻohana ʻia ka hoʻāʻo ʻana o ka cell viability e loiloi i ka cytotoxicity o B (a) P a me G-Rg3 ma nā cell hEL.Hōʻike ʻia ka cytotoxicity i hoʻokomo ʻia e B (a) P i loko o nā pūnaewele hEL ma ka Figure 5A, aʻo nā waiwai nontoxic o G-Rg3r a me G-Rg3 e hōʻike ʻia ma nā Kiʻi 5A a me 5B.5B, C. No ka loiloi ʻana i ka hopena cytoprotective o G-Rg3, ua hui pū ʻia ʻo B (a) P me nā ʻano ʻokoʻa like ʻole o G-Rg3r a i ʻole G-Rg3 i loko o nā cell hEL.E like me ka mea i hōʻike ʻia ma ka Figure 5D, ʻo G-Rg3r ma nā ʻāpana o 5 μM, 10 μM, a me 20 μM i hoʻihoʻi ʻia i ka viability cell i 58.3%, 79.3%, a me 77.3%.Hiki ke ʻike ʻia nā hopena like ma ka hui G-Rg3s.I ka manawa o 5 µM, 10 µM a me 20 µM ka nui o nā G-Rg3s, ua hoʻihoʻi ʻia ke ola o ka cell i 58.3%, 72.7% a me 76.7%, i kēlā me kēia (Figure 5E).).Ua ana ʻia ka hele ʻana o nā mea hoʻohui BPDE-DNA me ka hoʻohana ʻana i kahi pahu ELISA.Ua hōʻike ʻia kā mākou mau hopena ua hoʻonui ʻia nā pae hoʻohui BPDE-DNA i ka hui B (a) P i hoʻohālikelike ʻia me ka pūʻulu mana, akā i hoʻohālikelike ʻia me G-Rg3 co-treatment, BPDE-DNA adduct pae i ka hui B (a) P. B i ka hui i mālama ʻia, ua hoʻemi nui ʻia nā pae hoʻohui DNA.Hōʻikeʻia nā hopena o ka mālamaʻana me B (a) P wale nō ma ka Figure 5F (1.87 ± 0.33 vs. 3.77 ± 0.42 no G-Rg3r, 1.93 ± 0.48 vs. 3.77 ± 0.42 no G -Rg3s, p <0.001).
ʻO ke ola o ke kelepona a me ka hoʻohui ʻana o BPDE-DNA i nā cell hEL i mālama ʻia me G-Rg3 a me B (a) P.(A) Hiki ke ola o nā keena hEL i mālama ʻia me B(a)P.(B) Hiki ke ola o nā pūnaewele hEL i mālama ʻia me G-Rg3r.(C) Hiki ke ola o nā pūnaewele hEL i mālama ʻia me G-Rg3.(D) Hiki ke ola o nā mea hel i mālama ʻia me B(a)P a me G-Rg3r.(E) Hiki ke ola o nā mea hel i mālama ʻia me B(a)P a me G-Rg3.(F) Nā pae o ka BPDE-DNA hoʻohui ʻia i loko o nā keena hEL i mālama ʻia me B(a)P a me G-Rg3.Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± ka ʻokoʻa maʻamau o nā hoʻoholo triplicate.*P <0.05, **P <0.01, ***P <0.001.
Ua ʻike ʻia ka hōʻike enzyme GST ma hope o ka mālama pū ʻana me 10 μM B (a) P a me 10 μM G-Rg3r a i ʻole G-Rg3s.Ua hōʻike ʻia kā mākou mau hopena ua kāohi ʻo B (a) P i ka ʻōlelo GST (59.7 ± 8.2% i ka hui G-Rg3r a me 39 ± 4.5% i ka hui G-Rg3s), a ua pili pū ʻo B (a) P me G-Rg3r. , a i ʻole me G-Rg3r, a i ʻole me G-Rg3r.Hoʻoponopono hou ʻia me G-Rg3s i ka hōʻike GST.Hōʻike GST (103.7 ± 15.5% i ka hui G-Rg3r a me 110 ± 11.1% i ka hui G-Rg3s, p <0.05 a me p <0.001, kēlā me kēia, Fig. 6A, B, a me C).Ua loiloi ʻia ka hana GST me ka hoʻohana ʻana i kahi pahu hōʻike hana.Ua hōʻike ʻia kā mākou mau hopena i ʻoi aku ka nui o ka hana GST o ka hui hui pū ʻana i ka hui B (a) P wale nō (96.3 ± 6.6% vs. 35.7 ± 7.8% i ka hui G-Rg3r vs. 92.3 ± 6.5 i ka hui G-Rg3r. ).% vs 35.7 ± 7.8% i ka hui G-Rg3s, p <0.001, Kiʻi 6D).
Hōʻike o GST a me Nrf2 i loko o nā pūnaewele hEL i mālama ʻia me B (a) P a me G-Rg3.(A) Ka ʻike ʻana i ka hōʻike GST ma o ke kāhili ʻana o ke Komohana.(B) Hōʻike nui o GST i loko o nā pūnaewele hEL i mālama ʻia me B(a)P a me G-Rg3r.(C) Hōʻike nui o GST i nā pūnaewele hEL i mālama ʻia me B(a)P a me G-Rg3s.(D) ka hana GST i loko o nā keena hEL i mālama ʻia me B(a)P a me G-Rg3.(E) Ka ʻike ʻana i ka ʻōlelo Nrf2 e ka hoʻopau ʻana i ke Komohana.(F) Hōʻike nui o Nrf2 i nā pūnaewele hEL i mālama ʻia me B (a) P a me G-Rg3r.(G) Hōʻike nui o Nrf2 i nā pūnaewele hEL i mālama ʻia me B (a) P a me G-Rg3s.Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± ka ʻokoʻa maʻamau o nā hoʻoholo triplicate.*P <0.05, **P <0.01, ***P <0.001.
No ka wehewehe ʻana i nā ala i komo i ka G-Rg3-mediated suppression o B (a) P-induced tumorigenesis, ua loiloi ʻia ka ʻōlelo Nrf2 e ka Western blotting.E like me ka mea i hōʻike ʻia ma nā Kiʻi 6E, F, G, i hoʻohālikelike ʻia me ka pūʻulu mana, ʻo ke kiʻekiʻe o Nrf2 wale nō i ka hui mālama B (a) P i hoʻemi ʻia;akā naʻe, ke hoʻohālikelike ʻia me ka hui mālama B (a) P, ua hoʻonui ʻia nā pae B (a) Nrf2 i ka hui PG-Rg3 (106 ± 9.5% no G-Rg3r vs. 51.3 ± 6.8%, 117 ± 6. 2% no ka G-Rg3r vs. 41 ± 9.8% no G-Rg3s, p <0.01).
Ua hōʻoia mākou i ka hana pale o Nrf2 ma ke kāohi ʻana i ka ʻōlelo Nrf2 me ka hoʻohana ʻana i ka RNA liʻiliʻi liʻiliʻi liʻiliʻi (siRNA).Ua hōʻoia ʻia ʻo Nrf2 knockdown e ka Western blotting (Fig. 7A, B).E like me ka mea i hōʻike ʻia ma nā Kiʻi 7C, D, ʻo ka hoʻomaʻamaʻa like ʻana o nā cell hEL me B (a) P a me G-Rg3 i hōʻemi i ka helu o nā hoʻohui BPDE-DNA (1.47 ± 0.21) i hoʻohālikelike ʻia me ka mālama ʻana me B (a) P wale nō i ka pūʻulu siRNA mana.) ʻO G-Rg3r he 4.13 ± 0.49, G-Rg3s he 1.8 ± 0.32 a me 4.1 ± 0.57, p <0.01).Eia naʻe, ua hoʻopauʻia ka hopena pale o G-Rg3 ma ka hoʻokumuʻana o BPDE-DNA e Nrf2 knockdown.Ma ka hui siNrf2, ʻaʻohe ʻokoʻa koʻikoʻi i ka hoʻokumu ʻana o BPDE-DNA ma waena o B (a) P a me G-Rg3 co-treatment a me B (a) P mālama wale nō (3.0 ± 0.21 no G-Rg3r vs. 3.56 ± 0.32 ).no ka G-Rg3r me ka 3.6 no ka G-Rg3 me ka ± 0.45 me ka 4.0±0.37, p > 0.05).
Ka hopena o Nrf2 knockdown ma BPDE-DNA hoʻohui i ka hoʻokumu ʻana i nā cell hEL.(A) Ua hōʻoia ʻia ʻo Nrf2 knockdown e ka Western blotting.(B) Ka helu o ka ikaika o ka band Nrf2.(C) Ka hopena o ka Nrf2 knockdown ma BPDE-DNA adduct pae i loko o nā pūnaewele hEL i mālama ʻia me B (a) P a me G-Rg3r.(D) Ka hopena o Nrf2 knockdown ma BPDE-DNA hoʻohui i nā pae i loko o nā cell hEL i mālama ʻia me B (a) P a me G-Rg3.Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± ka ʻokoʻa maʻamau o nā hoʻoholo triplicate.*P <0.05, **P <0.01, ***P <0.001.
Ua loiloi kēia haʻawina i nā hopena pale o nā ʻāpana ʻulaʻula ʻulaʻula ma ke kumu hoʻohālike ʻiole o ka maʻi maʻi maʻi maʻi B(a)P-i hoʻokomo ʻia, a ʻo ka mālama ʻana iā KRGB i hōʻemi nui i ke kaumaha maʻi.Ke noʻonoʻo nei ʻo G-Rg3 ka mea kiʻekiʻe loa i loko o kēia extract ginseng, ua aʻo ʻia ke kuleana koʻikoʻi o kēia ginsenoside i ka pale ʻana i ka tumorigenesis.ʻO G-Rg3r a me G-Rg3 (ʻelua epimers o G-Rg3) i hōʻemi nui i ke kaumaha o ka maʻi maʻi i loko o kahi kumu ʻiole o B (a) P-induced cancer.Hoʻohana ʻo G-Rg3r a me G-Rg3 i nā hopena anticancer ma o ka hoʻoulu ʻana i ka apoptosis o nā cell tumor21, ke kāohi ʻana i ka ulu ʻana o ka tumor22, ka hopu ʻana i ka pōʻai cell23 a me ka hoʻopili ʻana i ka angiogenesis24.Ua hōʻike pū ʻia ʻo G-Rg3 e kāohi i ka metastasis cellular25, a ua kākau ʻia ka hiki o G-Rg3 e hoʻonui i nā hopena o ka chemotherapy a me ka radiotherapy i kākau ʻia26,27.Ua hōʻike ʻo Poon et al e hiki i ka lāʻau G-Rg3 ke hōʻemi i nā hopena genotoxic o B(a)P28.Hōʻike kēia haʻawina i ka hiki ke therapeutic o G-Rg3 i ka huli ʻana i nā molekala carcinogenic kaiapuni a me ka pale ʻana i ka maʻi kanesa.
ʻOiai ko lākou pono prophylactic maikaʻi, ʻo ka maikaʻi o ka bioavailability waha o ginsenosides he mea paʻakikī no ka hoʻohana ʻana i kēia mau mole.ʻO ka loiloi Pharmacokinetic o ka hoʻokele waha o ginsenosides i nā ʻiole i hōʻike ʻia ʻo kona bioavailability ʻoi aku ka liʻiliʻi ma mua o 5%29.Ua hōʻike ʻia kēia mau hoʻāʻo ʻana ma hope o ka mālama ʻana i nā pule he 20 pule, ua emi wale nā pae koko o Rg5.ʻOiai ke hoʻomau nei ka wehewehe ʻana i ke ʻano kumu o ka bioavailability maikaʻi ʻole, ua manaʻo ʻia ʻo P-gp e pili ana i ka efflux o ginsenosides.Ua hōʻike ʻia kēia hana no ka manawa mua i ka hoʻohana ʻana i ka verapamil, kahi P-gp blocker, hoʻonui i ka bioavailability waha o G-Rg3r a me G-Rg3s.No laila, hōʻike kēia ʻike e hana ʻo G-Rg3r a me G-Rg3s ma ke ʻano he substrates o P-gp e hoʻoponopono i kāna efflux.
Hōʻike kēia hana i ka hoʻonui ʻia ʻana o ka lāʻau lapaʻau me ka verapamil i ka bioavailability waha o G-Rg3 i loko o kahi ʻano ʻiole o ka maʻi kanesa o ka maʻi.Kākoʻo ʻia kēia ʻike e ka hoʻonui ʻana i ka transcellular transport intestinal o G-Rg3 ma luna o P-gp blockade, a laila e hoʻonui ai i kona absorption.Ua hōʻike ʻia nā hoʻāʻo ʻana i nā cell Caco2 i ka mālama ʻana i ka verapamil i hōʻemi i ka efflux o G-Rg3r a me G-Rg3s i ka hoʻomaikaʻi ʻana i ka membrane permeability.ʻO kahi haʻawina na Yang et al.Ua hōʻike nā haʻawina i ka mālama ʻana me ka cyclosporine A (kekahi P-gp blocker) e hoʻonui i ka bioavailability o ginsenoside Rh2 mai kahi kumu waiwai o 1%20 a ʻoi aku ma mua o 30%.Ua hōʻike pū nā hui Ginsenosides K a me Rg1 i nā hopena like30,31.I ka hoʻohana pū ʻia ʻana o ka verapamil a me ka cyclosporin A, ua hoʻemi nui ʻia ka efflux o ka pūhui K i loko o nā pūnaewele Caco-2 mai ka 26.6 a i lalo o 3, ʻoiai ua hoʻonui ʻia nā pae intracellular i 40-fold30.Ma ke alo o ka verapamil, ua hoʻonui ʻia nā pae Rg1 i loko o nā pūnaewele epithelial lung rat, e hōʻike ana i kahi kuleana no P-gp i ka ginsenoside efflux, e like me ka hōʻike ʻana e Meng et al.31.Eia naʻe, ʻaʻole i like ka hopena o ka verapamil i ka efflux o kekahi mau ginsenosides (e like me Rg1, F1, Rh1 a me Re), e hōʻike ana ʻaʻole lākou i hoʻopilikia ʻia e nā substrates P-gp, e like me ka hōʻike ʻana e Liang et al.32 .Pili paha kēia nānā ʻana i ke komo ʻana o nā mea halihali ʻē aʻe a me nā hale ginsenoside ʻē aʻe.
ʻAʻole maopopo ke ʻano o ka hopena pale o G-Rg3 ma ke kanesa.Ua hōʻike ʻia nā haʻawina mua e pale aku ʻo G-Rg3 i ka pōʻino DNA a me ka apoptosis ma ka hoʻemi ʻana i ke kaumaha oxidative a me ka mumū16,33, ʻo ia paha ke kumu kumu no ka pale ʻana i ka B (a) P-induced tumorigenesis.Hōʻike kekahi mau hōʻike e hiki ke hoʻemi ʻia ka genotoxicity i hoʻokomo ʻia e B(a)P ma o ka hoʻololi ʻana i nā enzymes phase II e hana i ka BPDE-DNA34.ʻO GST kahi enzyme maʻamau maʻamau II e kāohi ana i ka hoʻohui ʻana o BPDE-DNA ma o ka paipai ʻana i ka hoʻopaʻa ʻana o GSH i BPDE, a laila e hōʻemi ana i ka pōʻino DNA i hoʻokomo ʻia e B(a)P35.Hōʻike kā mākou hopena i ka mālama ʻana o G-Rg3 e hōʻemi i ka B (a) P-induced cytotoxicity a me ka BPDE-DNA adduct hoʻokumu i loko o nā cell hEL a hoʻihoʻi i ka hōʻike GST a me ka hana in vitro.Eia naʻe, ʻaʻole i loaʻa kēia mau hopena i ka loaʻa ʻole o Nrf2, e manaʻo ana e hoʻoulu ʻo G-Rg3 i nā hopena cytoprotective ma o ke ala Nrf2.ʻO Nrf2 kahi kumu transcription nui no ka pae II detoxification enzymes e hāpai ana i ka hoʻomaʻemaʻe o xenobiotics36.ʻO ka hoʻouluʻana o ke ala Nrf2 e hoʻoulu i ka cytoprotection a ho'ēmi i kaʻino kino37.Eia kekahi, ua kākoʻo kekahi mau hōʻike i ke kuleana o Nrf2 ma ke ʻano he suppressor tumor i ka carcinogenesis38.Hōʻike kā mākou haʻawina i ka hoʻokomo ʻana o ke ala Nrf2 e G-Rg3 i kahi kuleana koʻikoʻi i ka B (a) P-induced genotoxicity ma o ka hana ʻana i ka detoxification B (a) P ma ka hoʻoulu ʻana i nā enzymes phase II, a laila e kāohi ai i ke kaʻina tumorigenesis.
Hōʻike kā mākou hana i ka hiki o ka ginseng ʻulaʻula i ka pale ʻana i ka maʻi maʻi maʻi maʻi B (a) P-induced i nā ʻiole ma o ke komo ʻana nui o ginsenoside G-Rg3.ʻO ka maikaʻi ʻole o ka bioavailability waha o kēia mole ke hoʻopilikia i kāna noi lapaʻau.Eia naʻe, hōʻike kēia haʻawina no ka manawa mua ʻo G-Rg3 kahi substrate o P-gp, a ʻo ka hoʻokele ʻana o kahi mea hoʻopale P-gp e hoʻonui i ka bioavailability o G-Rg3 in vitro a in vivo.Hoʻemi ʻo G-Rg3 i ka cytotoxicity i hoʻokomo ʻia i ka B (a) P ma o ka hoʻoponopono ʻana i ke ala Nrf2, ʻo ia paha kahi hana kūpono no kāna hana pale.Ua hōʻoia kā mākou noiʻi i ka hiki o ginsenoside G-Rg3 no ka pale ʻana a me ka mālama ʻana i ka maʻi maʻi maʻi.
Ua loaʻa nā ʻiole A/J wahine ʻeono pule (20 ± 1 g) a me nā ʻiole Wistar kāne 7 wiki (250 ± 20 g) mai The Jackson Laboratory (Bar Harbor, USA) a me ka Wuhan Institute of Zoology.Kulanui (Wuhan, Kina).Ua hāʻawi mai ka Chinese Type Culture Collection Center (Wuhan, Kina) iā mākou me nā pūnaewele Caco-2 a me nā hEL.ʻO Sigma-Aldrich (St. Louis, USA) kahi kumu o B (a) P a me tricaprine.Ua kūʻai ʻia nā ginsenosides G-Rg3r a me G-Rg3s, dimethyl sulfoxide (DMSO), CellTiter-96 proliferation assay kit (MTS), verapamil, minimal essential medium (MEM), a me fetal bovine serum (FBS) mai Chengdu Must Bio-Technology. .Co.,Ltd.(Chengdu, Kina).Ua kūʻai ʻia ka QIAamp DNA mini kit a me BPDE-DNA addduct ELISA kit mai Qiagen (Stanford, CA, USA) a me Cell Biolabs (San Diego, CA, USA).Ua kūʻai ʻia mai Solarbio (Beijing, Kina) ka pahu hōʻike hana GST a me ka pahu hoʻāʻo protein holoʻokoʻa (keʻano BCA maʻamau).Hoʻopaʻa ʻia nā ʻāpana ʻulaʻula ʻulaʻula a pau ma Mingyu Laboratory 7. ʻO ke Kulanui Baptist Hong Kong (Hong Kong, Kina) a me Korea Cancer Center (Seoul, Korea) nā kumu kūʻai kālepa o ka CRG extract a me nā ʻano ʻāpana ʻulaʻula ʻulaʻula o nā kumu Korean like ʻole (me KRGA, KRGB. a me KRGC).Hana ʻia ka ginseng ʻulaʻula mai nā aʻa o ka ginseng hou 6 makahiki.Loaʻa ka extract ginseng ʻulaʻula ma ka holoi ʻana i ka ginseng me ka wai ʻekolu mau manawa, a laila hoʻopaʻa ʻia i ka aqueous extract, a hoʻomaloʻo hope i ka haʻahaʻa haʻahaʻa e loaʻa ai ka pauka ginseng extract.Ua kūʻai ʻia nā antibodies (anti-Nrf2, anti-GST, a me β-actin), horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG), transfection reagent, control siRNA, a me Nrf2 siRNA mai Santa Cruz Biotechnology (Santa Cruz, CA). .), USA).
Hoʻoulu ʻia ʻo Caco2 a me hEL i loko o nā kīʻaha moʻomeheu cell 100 mm2 me MEM e loaʻa ana ka 10% FBS ma 37 °C i kahi ea humidified o 5% CO2.No ka hoʻoholoʻana i ka hopena o nā kūlana lapaʻau, ua hoʻokomoʻia nā pūnaewele hEL me nā manaʻo likeʻole o B (a) P a me G-Rg3 i MEM no 48 h.Hiki ke nānā hou ʻia a hōʻiliʻili ʻia nā kelepona no ka hoʻomākaukau ʻana i nā ʻāpana kelepona ʻole.
Ua ʻae ʻia nā hoʻokolohua a pau e ka Experimental Animal Ethics Committee o Tongji Medical College, Huazhong University of Science and Technology (Approval No. 2019; Registration No. 4587TH).Ua hana ʻia nā hoʻokolohua āpau e like me nā alakaʻi a me nā hoʻoponopono kūpono, a ua mālama ʻia ke aʻo ʻana e like me ka Animal Research: Reporting of In Vivo Experiments (ARRIVE) alakaʻi.Ua hoʻokomo muaʻia nāʻiole A / J iʻewalu mau pule me ka B (a) P i ka solution tricaprine (100 mg / kg, 0.2 ml).Ma hope o hoʻokahi pule, ua hoʻokaʻawale ʻia nā ʻiole i nā pūʻulu hoʻomalu a me nā pūʻulu lapaʻau like ʻole, 15 mau ʻiole i kēlā me kēia hui, a hoʻopaʻa ʻia i hoʻokahi manawa i ka lā.Ma hope o 20 pule o ka mālama ʻana, ua mōhai ʻia nā holoholona e CO2 asphyxia.Ua hōʻiliʻili ʻia nā māmā a hoʻopaʻa ʻia no 24 mau hola.Ua helu ʻia ka nui o nā maʻi maʻi pāpaʻu a me ka nui o kēlā me kēia maʻi no kēlā me kēia māmā ma lalo o kahi microscope dissecting.Ua helu ʻia nā manaʻo manaʻo nui o ka puʻupuʻu (V) me ka hoʻohana ʻana i kēia huaʻōlelo: V (mm3) = 4/3πr3, kahi r ʻo ia ke anawaena tumor.ʻO ka huina ʻupena o nā puʻupuʻu maʻi maʻi a pau i loko o nā māmā o nā ʻiole e hōʻike ana i ka nui o ka nui o ka tumo, a ʻo ka awelika o ka nui o ka nui o ka maʻi maʻi i kēlā me kēia pūʻulu e hōʻike ana i ka haʻawe tumo.Ua hōʻiliʻili ʻia a mālama ʻia nā koko holoʻokoʻa a me nā ʻōpū ma −80 ° C no ka hoʻoholo ʻana o UPLC-MS/MS.Ua hōʻiliʻili ʻia ka serum a ua hoʻohana ʻia kahi mea loiloi kemika automated no ka nānā ʻana i nā pae alanine aminotransferase (ALT) a me ka serum creatinine (Cr) e loiloi i ka hana o ka ate a me ke kīkī.
Wehe ʻia nā laʻana i hōʻiliʻili ʻia mai kahi waiho anuanu, hoʻoheheʻe ʻia, kaupaona ʻia, a waiho ʻia i loko o nā paipu e like me ka mea i hōʻike ʻia ma luna.Ua hoʻohui ʻia kēia 0.5 μM phlorizin (maʻamau i loko) i 0.8 ml methanol solution.Hoʻohui ʻia ka ʻiʻo me ka hoʻohana ʻana i ka Tissue-Tearor a ua hoʻoili ʻia ka homogenate i kahi 1.5 ml microcentrifuge tube.Hoʻopili ʻia ka hui ʻana ma 15500 rpm no 15 mau minuke.Ma hope o ka wehe ʻana i 1.0 ml o ka supernatant, maloʻo me ka nitrogen.ʻElua haneli microliters o ka methanol i hoʻohana ʻia no ka hoʻōla.ʻOhi ʻia ke koko a hana ʻia ma ka laina hoʻokahi a hoʻohana ʻia ma ke ʻano he kuhikuhi no nā ana āpau.
ʻO 24-well Transwell plates i hua ʻia me 1.0 × 105 Caco-2 cell i kēlā me kēia punawai e loiloi i ka hiki ke hoʻonui ʻia o ka halihali G-Rg3 ma o ka hoʻohui ʻana o verapamil.Ma hope o 3 mau pule o ka moʻomeheu, holoi ʻia nā cell me HBSS a preincubated ma 37 ° C.400 μL o 10 μM G-Rg3 (G-Rg3r, G-Rg3s, a i ʻole kahi hui me 50 a i ʻole 100 μM verapamil) i hoʻokomo ʻia ma ka ʻaoʻao basolateral a i ʻole apical o ka monolayer, a ua hoʻohui ʻia ka 600 μL o ka solution HBSS i kekahi. aoao.E hōʻiliʻili i 100 µl o ka moʻomeheu moʻomeheu i nā manawa i koho ʻia (0, 15, 30, 45, 60, 90 a me 120 mau minuke) a hoʻohui i 100 µl o HBSS e hana i kēia leo.Ua mālama ʻia nā laʻana ma −4 °C a hiki i ka ʻike ʻia e UPLC-MS/MS.Hoʻohana ʻia ka huaʻōlelo Papp = dQ/(dT × A × C0) no ka helu ʻana i ka ʻike unidirectional apical a me basolateral permeability a me ka hope (Pa-b a me Pb-a, kēlā me kēia);ʻO dQ/dT ka hoʻololi ʻana i ka neʻe ʻana, ʻo A (0.6 cm2) ka ʻili o ka monolayer, a ʻo C0 ka mea hāʻawi mua.Ua helu ʻia ka ratio efflux e like me Pb-a/Pa-b, e hōʻike ana i ka helu efflux o ka lāʻau noiʻi.
Ua hoʻokē ʻai ʻia nā ʻiole Wistar kāne no 24 mau hola, inu wai wale nō, a hoʻomaʻamaʻa ʻia me kahi hoʻoheheʻe intravenous o 3.5% pentobarbital solution.ʻO ka pahu silicone intubated ka hopena o ka duodenum i ke komo ʻana a ʻo ka hope o ka ileum ka puka.E hoʻohana i ka pauma peristaltic no ka paila ʻana i ka puka komo me 10 µM G-Rg3r a i ʻole G-Rg3s i loko o ka isotonic HBSS ma kahi kahe kahe o 0.1 ml/min.Ua loiloi ʻia ka hopena o ka verapamil ma ka hoʻohui ʻana i 50 μM a i ʻole 100 μM o ka hui i 10 μM G-Rg3r a i ʻole G-Rg3s.Ua hana ʻia ʻo UPLC-MS/MS ma nā ʻāpana perfusion i hōʻiliʻili ʻia i nā manawa manawa 60, 90, 120, a me 150 mau minuke ma hope o ka hoʻomaka ʻana o ka perfusion.Hoʻohālikelike ʻia ka pākēneka o ka absorption e ka formula % absorption = (1 – Cout/Cin) × 100%;ua hōʻike ʻia ka manaʻo o G-Rg3 ma ka puka a me ka inlet e Cout a me Cin.
Ua kanu ʻia nā pūnae hel i loko o nā papa 96-well ma kahi ʻano o 1 × 104 cell i kēlā me kēia pūnāwai a mālama ʻia me B (a) P (0, 1, 5, 10, 20, 30, 40 μM) a i ʻole G-Rg3 i hoʻoheheʻe ʻia i DMSO .Ua hoʻoheheʻe ʻia nā lāʻau lapaʻau me ka moʻomeheu moʻomeheu i nā ʻano ʻokoʻa (0, 1, 2, 5, 10, 20 μM) ma luna o 48 mau hola.Me ka hoʻohana ʻana i kahi kit assay MTS i kūʻai ʻia, ua hoʻokau ʻia nā cell i kahi protocol maʻamau a laila ana ʻia me ka mea heluhelu microplate ma 490 nm.ʻO ka pae viability cell o nā hui i hui pū ʻia me B (a) P (10 μM) a me G-Rg3 (0, 1, 5, 10, 20 μM) i loiloi ʻia e like me ke ʻano o luna a hoʻohālikelike ʻia me ka hui i mālama ʻole ʻia.
Ua kanu ʻia nā pūnaewele hEL i loko o nā papa 6-well ma kahi kiko o 1 × 105 cell / well a mālama ʻia me 10 μMB (a) P i ke alo a i ʻole ka nele o 10 μM G-Rg3.Ma hope o 48 mau hola o ka mālama ʻana, ua unuhi ʻia ka DNA mai nā cell hEL me ka hoʻohana ʻana i ka QIAamp DNA Mini Kit e like me ka protocol a ka mea hana.Ua ʻike ʻia ka hoʻokumu ʻana o nā mea hoʻohui BPDE-DNA me ka hoʻohana ʻana i kahi kit BPDE-DNA addduct ELISA.Ua ana ʻia nā pae pili o ka BPDE-DNA adduct me ka hoʻohana ʻana i ka microplate reader ma ke ana ʻana i ka absorbance ma 450 nm.
Ua kanu ʻia nā pūnae hel i loko o nā pā 96-well ma kahi ʻano o 1 × 104 cell i kēlā me kēia pūnāwai a mālama ʻia me 10 μMB (a) P i ka ʻole a i ʻole ke alo o 10 μM G-Rg3 no 48 mau hola.Ua ana ʻia ka hana GST me ka hoʻohana ʻana i kahi pahu hōʻike hana GST pāʻoihana e like me ka protocol a ka mea hana.Ua ana ʻia ka hoʻoulu ʻana o GST e ka absorbance ma 450 nm me ka hoʻohana ʻana i kahi mea heluhelu microplate.
Ua holoi ʻia nā pūnaewele hEL me ka PBS anu anuanu a laila lysed me ka radioimmunoprecipitation assay buffer i loaʻa nā protease inhibitors a me phosphatase inhibitors.Ma hope o ka helu ʻana i ka protein me ka hoʻohana ʻana i kahi pahu hōʻike protein holoʻokoʻa, ua hoʻokaʻawale ʻia ka 30 μg o ka protein i kēlā me kēia hāpana e 12% SDS-PAGE a hoʻololi ʻia i kahi membrane PVDF e ka electrophoresis.Hoʻopaʻa ʻia nā membrane me ka waiū skim 5% a hoʻokomo ʻia me nā antibodies mua i ka pō ma 4 ° C.Ma hope o ka hoʻoulu ʻana me ka horseradish peroxidase-conjugated antibodies lua, ua hoʻonui ʻia nā reagents chemiluminescence e nānā i ka hōʻailona paʻa.Ua helu ʻia ka ikaika o kēlā me kēia pūʻulu protein me ka hoʻohana ʻana i ka polokalamu ImageJ.
Ua hoʻohana ʻia ka polokalamu GraphPad Prism 7.0 e kālailai i nā ʻikepili a pau, i hōʻike ʻia ma ke ʻano he mean ± maʻamau deviation.Ua loiloi ʻia ka ʻokoʻa ma waena o nā pūʻulu lapaʻau me ka hoʻohana ʻana i ka hōʻike t Student a i ʻole ka nānā ʻana o ka ʻokoʻa, me ka waiwai P <0.05 e hōʻike ana i ke koʻikoʻi helu.
Aia nā ʻikepili a pau i loaʻa a i ʻike ʻia i loko o kēia noiʻi ʻana i loko o kēia ʻatikala i paʻi ʻia a me nā faila ʻike hou.
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