Cistanche Tubulosa Phenylethanoid Glycosides Induce Apoptosis nyob rau hauv H22 Hepatocellular Carcinoma Cells Los ntawm Ob Txoj Kev Extrinsic Thiab Intrinsic Signaling Pathways

Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com

Pengfei Yuan, Jinyu Li, Adila Aipire, Yi Yang, Lijie Xia, Xinhui Wang, Yijie Li thiab Jinyao Li

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 ntawmC. 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 peev xwm (Δψm) ntawm H22 hlwb raug soj ntsuam los ntawm inverted microscopy, MTT assay, thiab ntws cytometry, feem. Kev qhia thiab ua kom cov proteins nyob rau hauv txoj kev apoptosis 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 tau txo qis H22 cell loj hlob nyob rau hauv ib koob- thiab lub sij hawm-nyob ntawm seb, uas yog correlated nrog nce apoptosis thiab cell voj voog ntes 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 txoj kev tau nce loj uas ua raws li caspase ua kom nquag plias -7 thiab -3 kom cleave PARP. Thaum kawg, CTPG (Cistanchetubulosaphenylethanoid glycosides) inhibited kev loj hlob ntawm H22 hlwb nyob rau hauv nas thiab txhim kho cov ciaj sia taus ntawm cov nas nas. Cov ntsiab lus: Cov txiaj ntsig no tau qhia tias CTPG cuam tshuam H22 cell loj hlob los ntawm ob qho tib si extrinsic thiab intrinsic apoptosis txoj hauv kev.

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 HCC ua tau raws li cov txheej txheem ntawm kev kho cov kab mob siab hepatic resection, thiab tag nrho 5- xyoo muaj sia nyob tus nqi tseem tsawg li 35-50 feem pua ​​​​ntawm qhov kev rov ua dua siab 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 hom TCM, muaj ntau yam kev ua haujlwm lom neeg, xws liAnti-oxidation, tiv thaiv kab mob, tiv thaiv kev laus,thiab neuroprotection [10, 11]. Phenylethanoid glycosides tau suav hais tias yog cov khoom tseem ceeb ntawmCistanche, 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 ua rau apoptosis hauv melanoma B16-F10 hlwb thiab inhibited 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 induced apoptosis hauv 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 tau yuav los ntawm Hetian Dichen Biotech Co., Ltd. (Hetian, Xinjiang, Tuam Tshoj) thiab cov ntsiab lus tseem ceeb ntawm CTPG tau tsim nyog thiab ntsuas los ntawm kev ua haujlwm siab ua 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 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- tus nyeem ntawv zoo microplate (Bio-Rad Laboratories, CA, USA). Lub cell viability raug xam raws li cov qauv: Cell viability (%)=(ODtreated/ODuntreated) × 100 feem pua ​​.

Kev kuaj mob apoptosis

H22 hlwb raug kho nrog ntau qhov sib txawv ntawm CTPG (0, 100, 200, 300, thiab 400 ug/ml) lossis 0.3 feem pua ​​DMSO rau 24 teev, thiab tom qab ntawd stained nrog Annexin V FITC/ Propidium iodide (PI) Apoptosis Detection Kit (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 raug kho nrog ntau qhov sib txawv ntawm CTPG (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 h ua. 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.

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 ib qho 6-zoo phaj ntawm qhov concentration ntawm 1 × 105 hlwb/zoo hauv 2 ml nruab nrab. Tom qab 60 feem pua ​​​​~ 70 feem pua ​​​​ntawm qhov sib txuam, cov hlwb tau kho nrog CTPG (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, anti-nas 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 (0, 100, 200, 300 thiab 400 ug/ml) lossis 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,{11}} g rau 15 min ntawm 4 degree) txhawm rau 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 hauv 0.1 ml DMSO nyob ib ncig ntawm cov 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 xam raws li cov qauv: qog ntim (mm3 )=(ntev × dav2 )/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 txo qhov muaj peev xwm ntawm H22 hlwb hauv vitro

Txhawm rau tshawb nrhiav cov nyhuv antitumor ntawm CTPG 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 txo qis H22 cell viability nyob rau hauv ib koob tshuaj thiab nyob ntawm lub sij hawm (Fig. 1b). CTPG ntawm 300 ug / ml tuaj txog ntawm qhov zoo tshaj plaws inhibitory tus nqi (Fig. 1c). Cov txiaj ntsig ntawm IC50 ntawm CTPG rau H22 hlwb yog 236 ug / ml ntawm 24 teev thiab 169.8 ug / ml ntawm 48 teev.

CTPG induced apoptosis hauv H22 hlwb

Txhawm rau tshawb xyuas seb qhov txo qis ntawm H22 hlwb puas yog kho los ntawm induction ntawm apoptosis, 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 ua rau muaj kev cuam tshuam apoptosis ntawm H22 hlwb (xws li apoptosis ntxov thiab lig) nyob rau hauv ib koob tshuaj (Daim duab 2a). Txawm hais tias cov koob tshuaj ntau ntawm CTPG kuj tseem ua rau muaj kev nce hauv necrosis ntawm H22 hlwb, necrosis plays lub luag haujlwm tseem ceeb hauv inhibition ntawm H22 cell loj hlob vim nws qhov qis dua (8.3 feem pua) piv rau apoptosis (52.6 feem pua). Tsis tas li ntawd, tag nrho cov proteins ntawm H22 hlwb raug cais tawm tom qab CTPG kev kho mob thiab cov lus qhia ntawm anti-apoptotic B cell lymphoma 2 (Bcl-2) thiab pro-apoptotic BCL-2-associated 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. Qhov piv ntawm Bax/Bcl{24}} tau nce ntau heev (Fig. 2b). Cov txiaj ntsig no qhia tias CTPG induces apoptosis hauv H22 hlwb.

CTPG 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 rau apoptosis hauv cov qog hlwb [17, 18]. Txhawm rau txheeb xyuas qhov morphology ntawm nuclei hauv H22 hlwb tom qab kev kho CTPG rau 24 teev, H22 hlwb raug stained los ntawm Hoechst 33,342 thiab pom siv inverted fluorescent microscopy. CTPG kho cov hlwb tau pom qhov nce ntawm koob tshuaj ntawm qhov ci ntsa iab chromatin ntawm nuclei, thaum cov hlwb tsis kho tau pom cov nuclei uas muaj homogeneously stained (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 kev kho mob tau nce qhov feem ntawm G0/G{13}} thiab G2/M-phase hlwb thiab txo qis feem ntawm S-phase hlwb, qhia tias CTPG induced G 0/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 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 ntawm apoptosis [19, 20]. Cov kev hloov ntawm mitochondrial membrane muaj peev xwm (Δψm) tuaj yeem 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 kev kho mob rau 24 teev, 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 thaum kho CTPG. Qhov kev faib ua feem ntawm PE- FITC ntxiv rau cov hlwb tau nce ntxiv (Daim duab 4a), qhia tias CTPG txo qhov Δψm hauv H22 hlwb. Qhov no yog raws li qhov nce Bax/Bcl-2 piv. Yog li ntawd, peb tau pom tias qhov tso tawm ntawm cytochrome c tau nce ntxiv thaum kho CTPG (Fig. 4b). Cov txiaj ntsig no tau qhia tias CTPG tuaj yeem ua rau qee qhov ua rau apoptosis hauv H22 hlwb ntawm txoj kev mitochondrial-dependent (intrinsic).

CTPG qhib txoj kev caspase thiab tiv thaiv DNA kho

Tom ntej no, kev ua kom cov caspase induced los ntawm CTPG ntawm ob qho tib si extrinsic thiab intrinsic signaling txoj kev raug soj ntsuam. Tom qab CTPG 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 kev kho tsis tau lossis DMSO tswj, CTPG kev kho mob tseem ceeb nce-tswj tsis yog qib ntawm cleaved caspase -8 (extrinsic pathway) tab sis kuj yog qib ntawm cleaved caspase -9 (txoj kev sab hauv) (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 induced apoptosis hauv H22 hlwb los ntawm ob qho tib si extrinsic thiab intrinsic signaling pathways.

CTPG inhibits 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 ntawm HCC tau soj ntsuam hauv cov 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 8 zaug. 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 uas tau xaiv tsis muaj qhov tshwm sim pom tseeb. 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 ntawm CTPG kev kho mob zoo heev 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 koob tshuaj (Daim duab 6c), qhia tias CTPG 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 ua rau apoptosis nyob rau hauv ntau hom qog hlwb los ntawm ob qho tib si extrinsic (tuag receptor-mediated) thiab intrinsic (mitochondria-dependent) 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 cuam tshuam H22 cell loj hlob los ntawm induction ntawm apoptosis thiab cell voj voog raug ntes. Cov theem ntawm cleaved caspase-8 thiab -9 tau nce siab- tswj los ntawm CTPG kev kho mob, qhia tias ob qho tib si sab hauv thiab sab hauv lub cev qhia txoj hauv kev tau koom nrog hauv induction ntawm apoptosis. Peb txoj kev tshawb fawb yav dhau los tau pom tias CTPG induced apoptosis hauv melanoma B16-F10 hlwb los ntawm mitochondria-dependent txoj kev uas nce qib ntawm cleaved caspase{15}} tab sis tsis caspase-8 [16]. CTPG 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-dependent apoptosis txoj kev [29]. Hauv H22 hlwb kho los ntawm CTPG, Bax/Bcl-2 piv tau nce-tswj, uas yuav ua rau txo qis ntawm Δψm thiab tso tawm cytochrome c pom 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 tau qhia tias CTPG induced apoptosis hauv H22 hlwb los ntawm ob qho tib si extrinsic thiab intrinsic signaling txoj kev.

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 koob tshuaj-raws li txhawb kev loj hlob ntawm splenocytes los ntawm Kunming nas, uas yog raws li 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.

Cov lus xaus

CTPG inhibited kev loj hlob ntawm H22 hlwb ob leeg hauv vitro thiab hauv vivo thiab induced apoptosis hauv H22 hlwb los ntawm ob qho tib si extrinsic thiab intrinsic signaling pathways. Cov ntaub ntawv no tau qhia tias CTPG tej zaum yuav yog tus neeg sib tw muaj peev xwm los kho HCC.