Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791

Cistanche tubulosa phenylethanoid glycosides induce apoptosis hauv H22 hepatocellular carcinoma cells los ntawm ob qho tib si extrinsic thiab intrinsic signaling pathways

Pengfei Yuan¹ ua al

Abstract

Keeb kwm: Cistanche tubulosa(Schenk) R. Wight yog ib hom tshuaj suav tshuaj uas ua rau cov hauv paus hniav ntawm cov nroj tsuag Tamarix thiab tau siv los kho cov txiv neej impotence, sterility, lub cev tsis muaj zog, thiab ua ib qho tonic. Txawm li cas los xij, nws cov nyhuv antitumor ntawm hepatocellular carcinoma tseem tsis pom. Ntawm no, peb tshawb xyuas cov nyhuv antitumor ntawmCistanche tubulosa phenylethanoid glycosides(CTPG) ntawm H22 hepatocellular carcinoma cells ob leeg hauv vitro thiab hauv vivo thiab nws cov txheej txheem.

Txoj kev:Lub morphology, viability,apoptosis, cell voj voog, thiab mitochondrial membrane muaj peev xwm (Δψm) ntawm H22 hlwb raug tshuaj xyuas los ntawm inverted microscopy, MTT assay, thiab ntws cytometry, raws li. Kev nthuav qhia thiab ua kom cov proteins nyob rau hauv lubapoptosisTxoj kev tau kuaj pom los ntawm Western blot. Cov nyhuv hauv vivo antitumor tau soj ntsuam hauv cov qog nas qauv tsim siv txiv neej Kunming nas.

Cov txiaj ntsig:CTPG kev kho mob tseem ceeb txo qis H22 cell loj hlob nyob rau hauv ib koob- thiab lub sij hawm-raws li txoj kev, uas yog correlated nrog nce.apoptosisthiab kaw lub voj voog ntawm tes ntawm G0/G1 thiab G2/M theem. Ntxiv mus, chromosomal condensation tau pom nyob rau hauv CTPG-kho H22 hlwb. CTPG kev kho mob tau nce Bax/Bcl-2 piv, txo Δψm, thiab txhim kho qhov tso tawm ntawm cytochrome c. Cov theem ntawm cleaved caspase-8 thiab caspase-9 nyob rau hauv ob qho tib si extrinsic thiab intrinsic signaling pathways tau loj hlob ntxiv uas sequentially activated caspase -7 thiab -3 cleave PARP. Thaum kawg, CTPG inhibited kev loj hlob ntawm H22 hlwb hauv cov nas thiab txhim kho qhov ciaj sia ntawm cov nas nas.

Cov lus xaus: Cov txiaj ntsig no tau qhia tias CTPG suppressed H22 cell loj hlob los ntawm ob qho tib si extrinsic thiab intrinsicapoptosistxoj kev.

Ntsiab lus: Cistanche tubulosa, Phenylethanoid glycosides, Apoptosis, Teeb liab txoj kev, Tumor nas qauv

Cistanche tubulosaphenylethanoid glycosides

Keeb kwm

Mob qog noj ntshav nyob rau qib thib rau rau kev mob qog noj ntshav thiab thib plaub rau cov neeg mob qog noj ntshav thoob ntiaj teb. Ntxiv mus, nws nyob rau qib plaub rau kev mob qog noj ntshav thiab thawj zaug rau cov neeg mob qog noj ntshav hauv cov tebchaws uas tsis tshua muaj kev cuam tshuam txog kev noj qab haus huv [1]. Hauv Suav teb, mob qog noj ntshav yog qhov thib peb ua rau mob qog noj ntshav hauv xyoo 2015 [2]. Ntau tshaj li 90 feem pua ​​​​ntawm cov qog nqaij hlav hauv siab yog cov kab mob hepatocellular carcinoma (HCC) hauv ntiaj teb [3]. Tam sim no, hepatic resection yog qhov kev xaiv tseem ceeb rau kev kho HCC. Txawm li cas los xij, tsawg dua 30 feem pua ​​​​ntawm cov neeg mob uas muaj HCC tau ua raws li cov txheej txheem ntawm kev kho mob hepatic resection thiab tag nrho 5- xyoo muaj sia nyob tus nqi tseem tsawg li 35-50 feem pua ​​​​vim qhov rov muaj dua [4, 5] . Muaj cov kev xaiv kho mob rau cov neeg mob uas muaj qhov nruab nrab mus rau HCC siab heev yog txwv. Sorafenib, tshuaj tiv thaiv molecularly, tau pom zoo los ntawm FDA ua thawj kab kev kho mob rau HCC siab heev. Txawm li cas los xij, sorafenib tsuas yog ncua txog 3 lub hlis ntawm kev ciaj sia thiab cov lus teb tsawg dua 4 feem pua ​​[6, 7]. Nws yog qhov tseem ceeb los tsim cov tshuaj tshiab lossis cov tswv yim tawm tsam HCC.

Cov tshuaj suav tshuaj suav tshuaj (TCM) ib leeg lossis ua ke nrog lwm cov tswv yim tau siv los kho HCC thiab qhia cov txiaj ntsig kho mob nrog rau lub sijhawm muaj sia nyob ntev, txhim kho lub neej zoo, txo qis kev phiv, thiab lwm yam [8, 9]. Cistanche, ib yam ntawm TCM, muaj ntau yam kev ua haujlwm lom neeg, xws li tiv thaiv oxidation, tiv thaiv kab mob, tiv thaiv kev laus, thiab neuroprotection [10, 11].Phenylethanoid glycosidestau raug suav hais tias yog cov khoom tseem ceeb ntawm Cistanche, uas muaj ntau yam kev ua ub no suav nrog kev tiv thaiv oxidation, tiv thaiv kab mob, hepatoprotection, thiab neuroprotection [12–15]. Peb pawg tau tshaj tawm tiasCistanche tubulosa phenylethanoid glycosides(CTPG) tuaj yeem txhawb nqaapoptosisnyob rau hauv melanoma B16-F10 hlwb thiab inhibit qhov kev loj hlob ntawm cov qog nyob rau hauv nas [16]. Hauv txoj kev tshawb no, peb tau ntsuas cov nyhuv antitumor ntawm CTPG ntawm HCC H22 hlwb ob leeg hauv vitro thiab hauv vivo thiab tshawb xyuas nws cov txheej txheem. Peb pom tias CTPG(Cistanche tubulosa phenylethanoid glycosides)inducedapoptosishauv H22 hlwb los ntawm ob qho tib si extrinsic thiab intrinsic signaling pathways thiab suppressed kev loj hlob ntawm H22 qog nyob rau hauv nas.

Cov txheej txheem

Cell kab

Cov nas H22 hepatocellular carcinoma hlwb tau txais los ntawm Xinjiang Key Laboratory ntawm Biological Resources thiab Genetic Engineering, Xinjiang University (Urumqi, Xinjiang, Tuam Tshoj) thiab kab lis kev cai hauv RPMI 1640 nruab nrab (Gibco) ntxiv nrog 100 U / ml penicillin thiab 100 ug / ml. streptomycin, thiab 10 feem pua ​​​​ntawm cov cua sov-inactivated fetal bovine serum (Gibco) ntawm 37 degree nyob rau hauv ib tug humidified cua ntawm 5 feem pua ​​CO2.

MTT kev xeem

CTPG(Cistanche tubulosa phenylethanoid glycosides)tau yuav los ntawm Hetian Dichen Biotech Co., Ltd. (Hetian, Xinjiang, Tuam Tshoj), thiab cov tebchaw loj ntawm CTPG(Cistanche tubulosa phenylethanoid glycosides)tau tsim nyog thiab ntsuas los ntawm high-performance kua chromatography [16]. Cell viability raug soj ntsuam los ntawm 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO , USA) assay. H22 hlwb raug inoculated rau hauv 96-cov phiaj zoo ntawm qhov ntom ntawm 2 × 104 hlwb hauv 100 ul nruab nrab ntawm ib qhov dej thiab kab lis kev cai ntawm 37 degree . Tom qab 24 teev, cov hlwb raug kho nrog ntau qhov sib txawv ntawm CTPG (0,100, 200, 300 thiab 400 ug / ml) lossis 0.3 feem pua ​​​​DMSO (sib npaug li 400 ug / ml CTPG) rau 24, 48 thiab 72 h, feem. Tom qab centrifugation ntawm 1000 rpm rau 7 min, lub supernatant raug muab pov tseg thiab 100 ul ntawm MTT tov (5 mg / ml hauv PBS) tau ntxiv rau txhua qhov dej. Cov phaj tau incubated ntawm 37 degree rau 4 h thiab 100 ul DMSO tau ntxiv kom yaj cov formazan crystals. Tus nqi OD490 tau kuaj pom los ntawm 96-zoo microplate nyeem ntawv (Bio-Rad Laboratories, CA, USA). Lub cell viability raug xam raws li tus qauv: Cell viability (%)=(ODtreated/ODuntreated) x 100 feem pua ​​.

Cistanche tubulosa phenylethanoid glycosides

Kev kuaj mob apoptosis

H22 hlwb tau kho nrog ntau qhov sib txawv ntawm CTPG(Cistanche tubulosa phenylethanoid glycosides)({0}}, 100, 200, 300, thiab 400 ug/ml) los yog 0.3 feem pua ​​DMSO rau 24 teev, thiab tom qab ntawd stained nrog Annexin VFITC/Propidium iodide (PI)ApoptosisCov khoom siv kuaj pom (YEASEN, Tuam Tshoj) raws li cov chaw tsim khoom cov lus qhia. Cov qauv raug txheeb xyuas los ntawm kev ntws cytometry (BD FACSCalibur, USA).

Kev kuaj pom ntawm mitochondrial membrane muaj peev xwm

H22 hlwb tau kho nrog ntau qhov sib txawv ntawm CTPG(Cistanche tubulosa phenylethanoid glycosides)(0, 200, thiab 400 ug/ml) rau 24 teev, thiab tom qab ntawd stained nrog cov membrane-permeable JC-1 dye (Beyotime, Tuam Tshoj) rau 20 min ntawm 37 degree . Tom qab ntxuav ob zaug nrog JC-1 tsis, cov qauv tau rov ua dua nrog 300 ul ntawm JC-1 tsis thiab tshuaj xyuas los ntawm kev ntws cytometry (BD FACSCalibur, USA).

Kev tsom xam ntawm lub voj voog ntawm tes

H22 hlwb raug inoculated nyob rau hauv 60 hli kab lis kev cai tais diav thiab kho nrog txawv concentrations ntawm CTPG (0, 100,200, 300 thiab 400 ug/ml) los yog 0.3 feem pua ​​DMSO rau 24 teev . Tag nrho cov hlwb tau sau thiab ntxuav ob zaug nrog PBS. Cells tau kho nyob rau hauv 70 feem pua ​​​​cov dej khov-txias ethanol ntawm - 20 degree rau 2 h thiab ntxuav ob zaug nrog PBS, tom qab ntawd rov muab tshem tawm hauv 300 ul Propidium iodide / RNase staining buffer (BD Biosciences). Tom qab 10 min nyob rau hauv chav tsev kub, cov qauv tau sau los ntawm flow cytometry (BD FACSCalibur, USA), thiab cell voj voog faib tau soj ntsuam nrog ModFit LT 3.0 software.

phenylethanoid glycosideshauvCistanche tubulosa

Hoechst 33,258 staining

Cov kev hloov pauv ntawm morphological ntawm H22 cell nuclei tau txheeb xyuas los ntawm daim nyias nyias-permeable DNA-binding dye Hoechst 33,258 staining. H22 hlwb tau muab noob rau hauv 6-zoo phaj ntawm qhov concentration ntawm 1 × 105 hlwb/zoo hauv 2 ml nruab nrab. Tom qab 60 feem pua ​​~ 70 feem pua ​​confluence, cov hlwb raug kho nrog CTPG(Cistanche tubulosa phenylethanoid glycosides)(0, 100, 200, 300, thiab 400 ug/ml) rau 24 teev. Cov hlwb tau sau thiab kho nrog 4 feem pua ​​​​cov dej khov-txias Paraformaldehyde ntawm 4 degree rau 10 min. Tom qab ntxuav nrog PBS, cov hlwb tau stained nrog Hoechst 33,258 (Beyotime, Tuam Tshoj) ntawm 4 degree rau 10 min. Cov qauv tau pom los ntawm lub tshuab hluav taws xob fluorescence inverted (Nikon Eclipse Ti-E, Nyiv).

Western blot

Anti-caspase-3, anti-cleaved caspase-3, Anti-Bcl-2, thiab anti-Bax tau yuav los ntawm Beyotime Biotech Co., Ltd. (Shanghai, Suav). Anti-caspase-7, anti-cleaved-caspase-7, anti-caspase-8, anti-cleaved-caspase-8, anti-caspase-9, tiv thaiv cleaved-caspase-9, anti-PARP, anti-cleaved PARP, antimouse IgG-HRP thiab anti-luv IgG-HRP tau muas los ntawm Cell Signaling Technology. Anti- -actin tau yuav los ntawm Beijing ComWin Biotech Co., Ltd. (Beijing, Suav).

H22 hlwb tau kho nrog ntau qhov sib txawv ntawm CTPG(Cistanche tubulosa phenylethanoid glycosides)({0}}, 100, 200, 300 thiab 400 ug/ml) los yog 0.3 feem pua ​​DMSO rau 24 teev. Cells tau sau thiab lysed nrog Cell Lysis Solution RIPA (Beijing ComWin Biotech Co., Ltd) rau 30 feeb ntawm dej khov. Cov qauv raug spun down (12,{10}} g rau 15 min ntawm 4 degree) los sau cov supernatants thiab cov protein ntau tau ntsuas los ntawm cov khoom siv BCA (Thermo Fisher Scientific, USA). Qhov sib npaug ntawm cov protein nyob hauv txhua tus qauv raug cais los ntawm 12 feem pua ​​SDS-PAGE thiab xa mus rau PVDF daim nyias nyias (Biosharp, Tuam Tshoj). Tom qab thaiv nrog TBST tsis muaj 5 feem pua ​​​​ntawm cov mis nyuj uas tsis muaj rog, daim nyias nyias tau tsim nrog cov tshuaj tiv thaiv kab mob thiab cov tshuaj tiv thaiv kab mob thib ob conjugated rau horseradish peroxidase (HRP), feem. Tom qab ntxuav nrog TBST, lub hom phiaj cov protein tau kuaj pom los ntawm ECL cov khoom kuaj (Beyotime, Tuam Tshoj).

Tsiaj txhu thiab ethics nqe lus

6-8 lub lis piam qub txiv neej Kunming nas tau yuav los ntawm Animal Laboratory Center, Xinjiang Medical University (Urumqi, Xinjiang, Suav). Cov nas tau khaws cia rau hauv qhov ntsuas kub tswj, lub teeb-cycled tsiaj chaw ntawm Xinjiang University. Tag nrho cov kev tshawb fawb tsiaj tau ua raws li cov lus qhia ntawm Tsiaj Saib Xyuas thiab Siv Pawg ntawm Xinjiang University. Cov txheej txheem tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Tsiaj Kev sim ntawm Xinjiang Key Laboratory of Biological Resources and Genetic Engineering (BRGE-AE001), Xinjiang University.

Tumor nas kawm

Rau induction ntawm qog nas qauv, txiv neej Kunming nas tau subcutaneously txhaj nrog 1 × 106 H22 hlwb hauv 100 ul PBS rau hauv txoj cai flank. Tom qab 3 hnub, cov nas tau muab faib ua 3 pawg (7 nas / pawg). Pawg Tswjhwm tau txhaj nrog 0.1 ml DMSO subcutaneously nyob ib ncig ntawm cov qog. CTPG-200 thiab CTPG{10}} pawg tau txhaj tshuaj subcutaneously nrog 200 lossis 400 mg / kg CTPG(Cistanche tubulosa phenylethanoid glycosides)hauv 0.1 ml DMSO nyob ib ncig ntawm lub qog. Cov nas raug kho txhua 2 hnub mus txog 21 hnub. Cov qog loj tau ntsuas los ntawm kev siv calipers mus txog 25 hnub thiab cov qog ntim tau suav raws li cov qauv: qog ntim (mm3)=(ntev × dav 2) / 2. Tom qab 25 hnub, txoj sia nyob ntawm cov nas nas tau raug saib xyuas txhua hnub kom txog thaum kawg ntawm txoj kev tshawb no.

Kev txheeb cais

Kev txheeb xyuas qhov tseem ceeb yog xam los ntawm ib txoj kev tsom xam ntawm qhov sib txawv ntawm cov kev kho mob thiab kev tswj xyuas pawg. Tag nrho cov ntaub ntawv tau qhia raws li qhov nruab nrab ± tus qauv sib txawv (SD). p < 0.05="" tau="" suav="" tias="" yog="" qhov="" tseem="">

Cov txiaj ntsig

CTPG(Cistanche tubulosa phenylethanoid glycosides)txo qhov viability ntawm H22 hlwb hauv vitro

Txhawm rau tshawb nrhiav cov nyhuv antitumor ntawm CTPG(Cistanche tubulosa phenylethanoid glycosides)ntawm HCC, H22 hlwb tau kho nrog ntau qhov sib txawv ntawm CTPG (0, 100, 200, 300, thiab 400 ug / ml) hauv vitro. Tom qab 24 teev, morphology ntawm H22 hlwb raug soj ntsuam siv lub tshuab ntsuas inverted. Peb pom tias morphology ntawm H22 hlwb tau hloov pauv los ntawm kev kho CTPG. Nrog rau kev nce CTPG concentration, hlwb tau me me thiab puag ncig thiab cov xov tooj ntawm tes kuj tau txo qis heev (Fig. 1a). MTT kev soj ntsuam tau siv los txheeb xyuas qhov muaj peev xwm ntawm H22 hlwb tom qab kev kho CTPG rau 24, 48, thiab 72 h, feem. CTPG(Cistanche tubulosa phenylethanoid glycosides)txo qis H22 cell viability nyob rau hauv ib koob tshuaj thiab nyob ntawm lub sij hawm (Fig. 1b). CTPG(Cistanche tubulosa phenylethanoid glycosides)ntawm 300 ug / ml tuaj txog ntawm qhov zoo tshaj plaws inhibitory tus nqi (Fig. 1c). Qhov tseem ceeb ntawm IC50 ntawm CTPG rau H22 hlwb yog 236 ug / ml ntawm 24 teev thiab 169.8 ug / ml ntawm 48h.

CTPG(Cistanche tubulosa phenylethanoid glycosides)induced apoptosis hauv H22 hlwb

Txhawm rau tshawb xyuas seb qhov txo qis ntawm H22 hlwb puas kho los ntawm induction ntawmapoptosis, H22 hlwb raug kho nrog ntau qhov sib txawv ntawm CTPG (0, 100, 200, 300, thiab 400 ug / ml) rau 24 teev thiab stained nrog PI thiab Annexin V. Cov txiaj ntsig cytometry tau pom tias CTPG(Cistanche tubulosa phenylethanoid glycosides)tseem ceeb inducedapoptosisntawm H22 hlwb (nrog rau thaum ntxov thiab ligapoptosis) nyob rau hauv ib koob tshuaj (Daim duab 2a). Txawm hais tias qhov koob tshuaj ntau ntawm CTPG(Cistanche tubulosa phenylethanoid glycosides)kuj tseem nce necrosis ntawm H22 hlwb, necrosis plays lub luag haujlwm me me hauv inhibition ntawm H22 cell loj hlob vim nws qhov feem tsawg (8.3 feem pua) piv rau covapoptosis(52.6 feem pua). Ntxiv mus, tag nrho cov proteins ntawm H22 hlwb raug cais tom qab CTPG(Cistanche tubulosa phenylethanoid glycosides)Kev kho thiab kev qhia ntawm anti-apoptotic B cell lymphoma 2 (Bcl-2) thiab proapoptotic BCL-2-sociated X protein (Bax) tau kuaj pom los ntawm Western blot. Grayscale scanning cov ntaub ntawv qhia tau hais tias theem kev qhia ntawm Bax thiab Bcl -2 tau nce thiab txo qis, feem. Tus Bax/Bcl-2 piv tau nce ntxiv (Daim duab 2b). Cov txiaj ntsig no qhia tias CTPG inducesapoptosishauv H22 hlwb.

CTPG(Cistanche tubulosa phenylethanoid glycosides)induces chromosomal condensation thiab cell voj voog ntes hauv H22 hlwb

Nws tau raug tshaj tawm tias DNA kev puas tsuaj thiab lub voj voog ntawm tes raug ntes los ntawm cov tshuaj tuaj yeem cuam tshuam cov qog cell loj hlob thiab ua rauapoptosishauv cov qog hlwb [17, 18]. Txhawm rau txheeb xyuas qhov morphology ntawm nuclei hauv H22 hlwb tom qab CTPG(Cistanche tubulosa phenylethanoid glycosides)Kev kho mob rau 24 h, H22 hlwb tau stained los ntawm Hoechst 33,342 thiab soj ntsuam siv inverted fluorescent microscopy. CTPG(Cistanche tubulosa phenylethanoid glycosides)cov hlwb kho tau pom tias muaj koob tshuaj ntau ntxiv ntawm qhov ci ntsa iab chromatin ntawm nuclei, thaum cov hlwb tsis kho tau pom cov nuclei zoo sib xws (Fig. 3a). Cell cycle tis nyob rau hauv H22 hlwb tau soj ntsuam ntxiv los ntawm PI staining tom qab CTPG kho rau 24 teev. Raws li qhia hauv daim duab 3b, CTPG(Cistanche tubulosa phenylethanoid glycosides)Kev kho mob tau nce qhov feem ntawm G0/G1- thiab G2/M-phase hlwb thiab txo qhov feem ntawm S-phase hlwb, qhia tias CTPG(Cistanche tubulosa phenylethanoid glycosides)induced G0/G1 thiab G2/M theem ntes hauv H22 hlwb. Cov koob tshuaj ntau ntawm CTPG kuj tseem nce qhov feem ntawm cov sub G1 hlwb.

CTPG(Cistanche tubulosa phenylethanoid glycosides)txo qis mitochondrial membrane muaj peev xwm thiab nce qhov tso tawm ntawm cytochrome c

mitochondrial-dependent txoj hauv kev plays lub luag haujlwm tseem ceeb hauv induction ntawmapoptosis[19, 20] ib. Cov kev hloov hauv mitochondrial membrane muaj peev xwm (Δψm) tuaj yeem ua tau

saib xyuas los ntawm JC-1 staining vim JC-1 aggregate (liab fluorescence) tuaj yeem tawg mus rau hauv monomer (ntsuab fluorescence) nrog kev txo qis ntawm Δψm [21]. Tom qab CTPG(Cistanche tubulosa phenylethanoid glycosides)kev kho mob rau 24 h, H22 hlwb tau stained los ntawm JC-1 dye. Cov ntaub ntawv ntws cytometry tau pom tias liab fluorescence hauv FL-2 channel thiab ntsuab fluorescence hauv FL-1 channel tau txo qis thiab nce ntxiv rau CTPG(Cistanche tubulosa phenylethanoid glycosides)kev kho mob. Qhov kev faib ua feem ntawm PE-FITC ntxiv rau cov hlwb tau nce ntxiv (Daim duab 4a), qhia tias CTPG(Cistanche tubulosa phenylethanoid glycosides)txo cov Δψm hauv H22 hlwb. Qhov no yog raws li qhov nce Bax/Bcl-2 piv. Yog li ntawd, peb tau pom qhov kev tso tawm ntawm cytochrome c tau nce ntau ntxiv thaum CTPG(Cistanche tubulosa phenylethanoid glycosides)kev kho mob (Fig. 4b). Cov txiaj ntsig no tau qhia tias CTPG tuaj yeem cuam tshuam ib nrabapoptosishauv H22 hlwb ntawm txoj kev mitochondrial-dependent (intrinsic).

CTPG qhib txoj hauv kev caspase thiab tiv thaiv DNA kho

Tom ntej no, ua kom cov caspase induced los ntawm CTPG(Cistanche tubulosa phenylethanoid glycosides)ntawm ob qho tib si extrinsic thiab intrinsic signaling pathways raug soj ntsuam. Tom qab CTPG(Cistanche tubulosa phenylethanoid glycosides)Kev kho mob rau 24 teev, tag nrho cov proteins raug cais tawm ntawm H22 hlwb thiab qib ntawm pro-thiab cleaved-caspases tau kuaj pom los ntawm Western blot. Piv nrog rau qhov tsis kho lossis DMSO tswj, CTPG(Cistanche tubulosa phenylethanoid glycosides)kev kho mob tau nce- tswj tsis tau tsuas yog qib ntawm cleaved caspase-8 (extrinsic pathway) tab sis kuj yog qib ntawm cleaved caspase-9 (txoj kev hauv nruab nrab) (Fig. 5). Ua ke, qhib tau caspase-8 thiab -9 cleaved downstream pro-caspase-3 thiab -7 uas tau pom nyob rau hauv daim duab. 5. Activated caspase-3 cleaved lub DNA kho enzyme ntawm poly (ADP-ribose) polymerase (PARP) los tiv thaiv DNA kho thiab sib sau DNA puas raws li pom hauv daim duab 3a. Cov txiaj ntsig no tau qhia tias CTPG(Cistanche tubulosa phenylethanoid glycosides)inducedapoptosishauv H22 hlwb los ntawm ob qho tib si extrinsic thiab intrinsic signaling pathways.

CTPG(Cistanche tubulosa phenylethanoid glycosides)suppresses kev loj hlob ntawm H22 HCC hauv vivo thiab txhim kho qhov ciaj sia ntawm cov nas nas

Thaum kawg, cov nyhuv antitumor ntawm CTPG(Cistanche tubulosa phenylethanoid glycosides)ntawm HCC tau raug soj ntsuam hauv tus qauv qog nas, uas tau tsim los ntawm kev txhaj tshuaj subcutaneous ntawm H22 hlwb. Tom qab 3 hnub ntawm kev txhaj tshuaj H22 ntawm tes, cov nas mob qog tau kho nrog CTPG(Cistanche tubulosa phenylethanoid glycosides)8x ua. Lub cev hnyav ntawm cov nas thiab cov qog loj tau saib xyuas ntawm lub sijhawm qhia. Raws li pom hauv daim duab 6a, lub cev hnyav ntawm cov nas hauv txhua pab pawg tsis muaj qhov txawv txav, qhia tias cov koob tshuaj CTPG raug xaiv.(Cistanche tubulosa phenylethanoid glycosides)tsis muaj teebmeem tshwm sim. Interestingly, cov qog loj hlob nyob rau hauv nas kho nrog ob qho tib si 200 mg / kg thiab 400 mg / kg ntawm CTPG yog inhibited heev (Fig. 6b). Ntxiv mus, ob koob tshuaj CTPG(Cistanche tubulosa phenylethanoid glycosides)Kev kho mob tau zoo txhim kho txoj sia nyob ntawm cov nas nas (3/7, 3/7) piv nrog rau pawg tswj hwm (0/7) thaum kawg ntawm qhov kev sim (Fig. 6b). Peb kuj pom tias CTPG tau txhim kho qhov kev loj hlob ntawm splenocytes cais los ntawm cov txiv neej Kunming nas nyob rau hauv ib qho kev noj tshuaj (Daim duab 6c), qhia tias CTPG(Cistanche tubulosa phenylethanoid glycosides)muaj cov nyhuv immunostimulatory.

Kev sib tham

TCM tau siv los kho ntau yam kab mob xws li mob qog noj ntshav rau keeb kwm ntev. Nws tau raug tshaj tawm tias TCM tuaj yeem txhawb nqaapoptosisnyob rau hauv ntau hom qog hlwb los ntawm ob qho tib si extrinsic (tuag receptor-mediated) thiab intrinsic (mitochondria-dependent) taw qhia txoj hauv kev rau exert antitumor teebmeem [22–25]. Ob txoj hauv kev tuaj yeem qhib caspase-8 thiab -9, raws li [24, 26]. Ntawm no, peb pom tias CTPG(Cistanche tubulosa phenylethanoid glycosides)Nws txo qis H22 cell loj hlob los ntawm induction ntawm apoptosis thiab cell voj voog ntes. Cov theem ntawm cleaved caspase-8 thiab -9 tau nce siab- tswj los ntawm CTPG(Cistanche tubulosa phenylethanoid glycosides)kev kho mob, tawm tswv yim tias ob qho tib si extrinsic thiab intrinsic signaling pathways tau koom nrog induction ntawmapoptosis. Peb txoj kev tshawb fawb yav dhau los tau pom tias CTPG(Cistanche tubulosa phenylethanoid glycosides)induced apoptosis hauv melanoma B16-F10 hlwb los ntawm txoj kev mitochondrial-dependent uas nce qib ntawm cleaved caspase-9 tab sis tsis caspase-8 [16]. CTPG(Cistanche tubulosa phenylethanoid glycosides)tuaj yeem qhib cov kev taw qhia sib txawv hauv ntau hom qog hlwb.

Mitochondrial membrane kev ncaj ncees yog nruj tswj hwm los ntawm cov tswv cuab ntawm BCL-2 protein tsev neeg suav nrog Bax thiab Bcl-2 [27, 28]. Qhov piv ntawm Bax rau Bcl-2 plays lub luag haujlwm tseem ceeb hauv mitochondria-dependentapoptosistxoj kev [29]. Hauv H22 hlwb kho los ntawm CTPG(Cistanche tubulosa phenylethanoid glycosides), Bax/Bcl -2 piv tau nce-tswj, uas yuav ua rau txo qis ntawm Δψm thiab tso tawm ntawm cytochrome c pom nyob rau hauv txoj kev tshawb no. Yog li ntawd, pro-caspase-9 tau cleaved thiab qhib. Thaum kawg, cov neeg pib ua haujlwm ntawm caspase-8 thiab -9 tau qhib lub executioner ntawm caspase-3 kom tshem tawm PARP los tiv thaiv DNA kho. Ua ke, cov txiaj ntsig no qhia tias CTPG inducedapoptosishauv H22 hlwb los ntawm ob qho tib si extrinsic thiab intrinsic signaling pathways. Hauv cov qauv qog nas, CTPG cuam tshuam qhov kev loj hlob ntawm H22 HCC thiab txhim kho txoj sia nyob ntawm cov nas nas. Interestingly, CTPG(Cistanche tubulosa phenylethanoid glycosides)koob tshuaj-nyob ntawm kev txhawb nqa kev loj hlob ntawm splenocytes los ntawm Kunming nas, uas zoo ib yam nrog peb txoj kev tshawb fawb yav dhau los [16]. Cov txiaj ntsig no tau qhia tias CTPG tuaj yeem cuam tshuam kev loj hlob ntawm H22 HCC hauv cov nas los ntawm ob qho tib si ncaj qha tshuaj tiv thaiv kab mob thiab kev tiv thaiv kab mob tsis ncaj.

Cistanche tululosacov khoom

Cov lus xaus

CTPG(Cistanche tubulosa phenylethanoid glycosides)inhibited kev loj hlob ntawm H22 hlwb ob leeg hauv vitro thiab hauv vivo thiab inducedapoptosishauv H22 hlwb los ntawm ob qho tib si extrinsic thiab intrinsic signaling pathways. Cov ntaub ntawv no qhia tias CTPG(Cistanche tubulosa phenylethanoid glycosides)tej zaum yuav yog tus neeg sib tw muaj peev xwm rau kev kho mob ntawm HCC.

Cov ntawv luv

Bax: BCL-2-sociated X protein; Bcl-2: B cell lymphoma 2; CTPG:Cistanche tubulosa phenylethanoid glycosides; HCC: hepatocellular carcinoma; HRP: horseradish peroxidase; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PARP: DNA kho enzyme ntawm poly (ADP-ribose) polymerase; TCM: tshuaj suav tshuaj; Δψm: mitochondrial membrane muaj peev xwm

Nyiaj txiag

Txoj haujlwm no tau txais kev txhawb nqa los ntawm High-Level Talent Introduction Project ntawm Xinjiang Uygur Autonomous Region rau JL, Suav National Natural Science Foundation Grant (31460241) rau JL, thiab Doctoral Start-up Fund ntawm Xinjiang University (BS160261 rau XW thiab BS150236 rau YL).

Muaj cov ntaub ntawv thiab cov ntaub ntawv

Cov ntaub ntawv nyoos rau txoj kev tshawb fawb no muaj nyob rau ntawm qhov kev thov tsim nyog rau tus neeg sau ntawv.

Cov neeg sau ntawv pab txhawb

JL thiab JL tsim cov kev sim. PY, JL, AA, thiab YY ua qhov kev sim. LX, XW, thiab YL tau txheeb xyuas cov ntaub ntawv. PY, JL, thiab JL tau sau cov ntawv sau. Txhua tus kws sau ntawv tau pab txhawb thiab pom zoo rau daim ntawv kawg.

Kev pom zoo ntawm Ethics

Kev tshawb fawb tsiaj tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Tsiaj Kev sim ntawm Xinjiang Key Laboratory of Biological Resources thiab Genetic Engineering, Xinjiang University

Kev nyiam sib tw

Cov kws sau ntawv tshaj tawm tias lawv tsis muaj kev sib tw nyiam.

Publisher's Note

Springer Nature tseem nyob nruab nrab hais txog kev txiav txim plaub ntug hauv daim duab qhia kev tshaj tawm thiab cov koom haum koom nrog.

Sau Paub meej

¹Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, 666 Shengli Road, Urumqi, Xinjiang 830046, Suav teb.²College of Life Science, Xinjiang Normal University, 102 Xinyi Road, Urumqi 830054, Xinjiang, Suav.³Affiliated Tumor Tsev Kho Mob ntawm Xinjiang Medical University, Urumqi 830011, Suav.

Cistanche tululosacov khoom

Los ntawm: 'Cistanche tubulosa phenylethanoid glycosidesinduceapoptosisnyob rau hauv H22 hepatocellular carcinoma hlwb los ntawm ob qho tib si extrinsic thiab intrinsic signaling pathways 'los ntawm Pengfei Yuan1 li al.

---Yuan et al. BMC Complementary and Alternative Medicine (2018) 18:275 https://doi.org/10.1186/s12906-018-2201-1

Cov ntaub ntawv

1. Ntiaj teb no Burden of Disease Cancer Collaboration, Fitzmaurice C, Allen C, Barber RM, Barregard L, Bhutta ZA, Brenner H, Dicker DJ, Chimed-Orchid O, Dandona R, et al. Ntiaj teb no, lub regional, thiab National Cancer tshwm sim, kev tuag, xyoo ntawm lub neej ploj, xyoo nyob nrog kev tsis taus, thiab kev tsis taus-hloov lub neej-xyoo rau 32 Cancer pab pawg, 1990 txog 2015: ib tug systematic tsom xam rau lub ntiaj teb no lub nra ntawm txoj kev tshawb no kab mob. JAMA Oncol. 2017; 3:524–48.

2. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J.Cancer statistics in China, 2015. CA Cancer J Clin. 2016; 66:115–32.3. Lub Koom Haum European Rau Kev Tshawb Fawb Txog Lub Siab, European Lub Koom Haum rau Kev Tshawb Fawb Thiab Kev Kho Mob Cancer. EASL-EORTC cov txheej txheem kev kho mob: kev tswj cov kab mob hepatocellular carcinoma. JHepatol. 2012; 56:908–43.

4. Roayaie S, Obeidat K, Sposito C, Mariani L, Bhoori S, Pellegrinelli A, Labow D, Llovet JM, Schwartz M, Mazzaferro V. Resection of hepatocellular cancer Less than or equal to 2 cm: tau los ntawm ob lub chaw sab hnub poob. Hepatology. 2013; 57:1426–35.

5. Ting CT, Cheng YY, Tsai TH. Tshuaj ntsuab-tshuaj sib cuam tshuam ntawm cov tshuaj Hepatoprotective ib txwm ua thiab Sorafenib ntawm hepatotoxicity, Histopathology, thiab Pharmacokinetics hauv nas. Molecules. Xyoo 2017; 22: E1034.

6. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, et al. Sorafenib nyob rau hauv cov kab mob siab hepatocellular carcinoma. N Engl J Med. 2008; 359:378–90.

7. Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, Luo R, Feng J, Ye S, Yang TS, et al. Kev ua tau zoo thiab kev nyab xeeb ntawm sorafenib hauv cov neeg mob hauv cheeb tsam Asia-Pacific nrog cov kab mob siab hepatocellular carcinoma: theem III randomized, ob-dig muag, kev sim tshuaj placebo. Lancet Oncol. 2009; 10:25–34.

8. Shi Z, Song T, Wan Y, Xie J, Yan Y, Shi K, Du Y, Shang L. Kev tshuaj xyuas thiab kev tshuaj ntsuam xyuas cov tshuaj suav tshuaj suav tshuaj ua ke rau kev kho mob tsis phais mob hepatocellular carcinoma. Sci Rep. 2017; 7:4355.

9. Yang Z, Liao X, Lu Y, Xu Q, Tang B, Chen X, Yu Y. Add-on therapy with traditional Chinese medicine improves results and reduces adverse events in hepatocellular carcinoma: a meta-analysis of randomized controlled trials. Evid Based Complement Alternat Med. Xyoo 2017; 2017: 3428253.

10. Lin LW, Hsieh MT, Tsai FH, Wang WH, Wu CR. Anti-nociceptive thiab anti-inflammatory kev ua los ntawm Cistanche deserticola nyob rau hauv nas. J Ethnopharmacol. 2002; 83:177–82.

11. Wu CR, Lin HC, Su MH. Rov qab los ntawm aqueous extracts ntawmCistanche tubulosalos ntawm kev coj tus cwj pwm tsis zoo hauv Alzheimer tus kab mob zoo li nas qauv: qhov cuam tshuam rau amyloid deposition thiab central neurotransmitter muaj nuj nqi. BMC Complement Altern Med. Xyoo 2014; 14:202.

12. Jiang Y, Tu PF. Kev soj ntsuam ntawm cov tshuaj lom neeg hauv Cistanche hom. JChromatogr A. 2009; 1216: 1970–9.

13. Morikawa T, Pan Y, Ninomiya K, Imura K, Matsuda H, Yoshikawa M, Yuan D, Muraoka O. Acylated phenylethanoid oligo glycosides nrog hepatoprotective kev ua los ntawm cov nroj tsuag suab puamCistanche tubulosa.Bioorg Med Chem. 2010; 18: 1882–90.

14. Nan ZD, Zeng KW, Shi SP, Zhao MB, Jiang Y, Tu PF.Phenylethanoid glycosidesnrog kev tiv thaiv kev ua haujlwm los ntawm cov stems ntawm Cistanche deserticola kab lis kev cai hauv Tarim suab puam. Fitoterapia.2013; 89:167–74.

15. Deng M, Zhao J, Tu P, Jiang Y, Li Z, Wang Y. Echinacoside resues SHSY5Y neuronal hlwb los ntawm TNF -inducedapoptosis. Eur J Pharmacol. 2004; 505:11–8.

16. Li J, Li J, Aipire A, Gao L, Huo S, Luo J, Zhang F.Phenylethanoid glycosideslos ntawmCistanche tubulosainhibits kev loj hlob ntawm B16-F10 hlwb ob leeg hauv vitro thiab hauv vivo los ntawm induction ntawmapoptosisntawm txoj kev mitochondrial-dependent. J Cancer. 2016; 7:1877–87.

17. Chang HS, Chang CH, Chou YR, Yeh MY, Au MK, Lu HF, Chu YL, Chou HM, Chou HC, Shih YL, et al. Curcumin ua rau DNA puas thiab cuam tshuam cov protein qhia hauv HeLa tib neeg cov qog nqaij hlav qog noj ntshav. Oncol Rep. 2016; 36:2207–15.

18. Wang R, Zhang Q, Peng X, Zhou C, Zhong Y, Chen X, Qiu Y, Jin M, Gong M, Kong D. Stellettin B induces G1 ntes,apoptosis, thiab autophagy nyob rau hauv tib neeg uas tsis yog-me me cell ntsws cancer A549 hlwb ntawm thaiv PI3K / Akt / mTOR txoj kev. Sci Rep. 2016; 6:27071.

19. Sinha K, Das J, Pal PB, Sil PC. Oxidative stress: mitochondria-dependent thiab mitochondria-kev ywj pheej txoj hauv kevapoptosis. Arch Toxicol. 2013; 87:1157–80.

20. Zhang YS, Shen Q, Li J. Tsob tshuaj suav tshuaj tsom apoptotic mechanisms rau kev kho mob qog noj ntshav esophageal. Acta Pharmacol Sin. 2016; 37:295–302.

21. Chong ZZ, Lin SH, Li F, Maiese K. Cov sirtuin inhibitor nicotinamide txhim kho neuronal cell ciaj sia taus thaum mob anoxic raug mob los ntawm AKT, BAD, PARP, thiab mitochondrial txuam nrog "anti-apoptotic" pathways. Curr Neurovasc Cov. 2005; 2:271–85.

22. Hu B, Wang SS, Du Q. Tsob tshuaj suav tshuaj tiv thaiv thiab kho kab mob hepatocarcinoma: los ntawm lub rooj zaum mus rau sab txaj. World J Hepatol. 2015; 7:1209–32.

23. Hu B, An HM, Wang SS, Chen JJ, Xu L. Tiv thaiv thiab kho cov teebmeem ntawm suav tshuaj ntsuab tshuaj tiv thaiv kab mob hepatocellular carcinoma. Molecules. Xyoo 2016; 21:142.

24. Xu H, Zhao X, Liu X, Xu P, Zhang K, Lin X. Antitumor los ntawm cov tshuaj suav tshuaj uas tsom rau txoj hauv kev ntawm cellular apoptotic. Drug Des Devel Ther. 2015; 9:2735–44.

25. Li-Weber M. Targetingapoptosistxoj hauv kev mob qog noj ntshav los ntawm Suav tshuaj. Cancer Lett. 2013; 332: 304–12.

26. Xu G, Shi Y.Apoptosiskev taw qhia txoj hauv kev thiab lymphocyte homeostasis. Cell Res. 2007; 17:759–71.

27. Tait SW, Green DR. Mitochondria thiab cell tuag: txheej membrane permeabilization thiab tshaj. Nat Rev Mol Cell Biol. 2010; 11:621–32.

28. Galluzzi L, Kepp O, Kroemer G. Mitochondria: tus tswv regulators ntawm kev txaus ntshai signaling. Nat Rev Mol Cell Biol. 2012; 13:780–8.

29. Martinou JC, Youle RJ. Mitochondria hauvapoptosis: Bcl-2 tsev neeg thiab mitochondrial dynamics. Dev Cell. 2011; 21:92–101.