Cistanche Tubulosa Phenylethanoid Glycosides Induce Apoptosis hauv Eca-109 Cells Ntawm Txoj Kev Mitochondria-dependent
Mar 06, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
CHANGSHUANG FU, JINYU LI, ADILA AIPIRE, LIJIE XIA, YI YANG, QIUYAN CHEN, JIE LV, XINHUI WANG thiab JINYAO LI
Abstract
Cistanche tubulosamuaj ntau yam kev ua haujlwm lom neeg. Hauv kev tshawb fawb tam sim no, cov nyhuv antitumor ntawm dej-soluble phenylethanoid glycosides ntawm C. tubulosa (CTPG-W) ntawm kev mob qog noj ntshav tau tshawb xyuas. Eca-109 hlwb raug kho nrog CTPG-W thiab lub cell viability raug ntsuas los ntawm MTT kev soj ntsuam. Lub apoptosis, cell voj voog, mitochondrial membrane muaj peev xwm (Δψm), thiab cov pa oxygen reactive tau txheeb xyuas los ntawm kev ntws cytometry. Cov qib ntawm cov proteins hauv txoj hauv kev apoptotic tau kuaj pom los ntawm kev txheeb xyuas western blot. Nws tau txiav txim siab tias CTPG-W txo qis kev muaj peev xwm ntawm Eca-109 hlwb los ntawm kev cuam tshuam ntawm apoptosis thiab cell voj voog raug ntes. Tom qab CTPG-W kev kho mob, qhov Δψm ntawm Eca-109 tau txo qis, uas cuam tshuam nrog kev tswj hwm qib ntawm B-cell lymphoma-2 (Bcl-2)-xws li X thiab qis qis. ntawm Bcl-2. Yog li ntawd, cov theem ntawm cytochrome c thiab c-Jun NH2-terminal kinase tau nce, uas txhawb cov qib ntawm cleaved-poly (ADP-ribose) polymerase thiab cleaved-caspase-3, {{18} } thiab -9, tab sis tsis yog caspase-8. Raws li qhov tshwm sim, qib ntawm cov pa oxygen reactive hauv Eca-109 cov hlwb tau pom cov kev hloov pauv tseem ceeb. Cov txiaj ntsig no tau qhia tias CTPG-W induced apoptosis ntawm Eca{23}} hlwb los ntawm txoj kev mitochondrial-dependent.
Taw qhia
Kev mob qog noj ntshav yog ib hom mob qog noj ntshav ntau tshaj plaws nrog 11 tus neeg mob siab tshaj plaws thiab 6 tus neeg tuag siab tshaj plaws hauv ntiaj teb thiab ua rau ~ 439,000 kev tuag hauv 2015 (1). Qhov tshwm sim ntawm kev mob qog noj ntshav tshwj xeeb yog txawv ntawm ntau thaj chaw, nrog rau sab hnub tuaj Asia thiab sab hnub tuaj thiab sab qab teb ntawm Africa nthuav tawm cov neeg mob siab tshaj plaws thiab sab hnub poob teb chaws Africa uas pom tus nqi qis tshaj hauv xyoo 2012 (1,2). Hauv Suav teb, kwv yees cov neeg mob qog noj ntshav thiab cov neeg tuag yog 477,{10}} thiab 375,{12}}, raws li, xyoo 2015 (3). Txawm hais tias tus kab mob qog nqaij hlav hauv plab tau txo qis hauv nruab nrab thiab nruab nrab ntawm cov teb chaws sociodemographic Performance index ntawm xyoo 2005 thiab 2015, qhov kev tuag tseem nyob siab vim qhov tsis zoo (1,4). Kev sib xyaw ua ke ntawm kev phais kev phais nrog tshuaj khomob lossis kev siv hluav taws xob tau siv los kho mob qog noj ntshav, txawm li cas los xij, nws tau raug tshaj tawm tias nyob nruab nrab ntawm 2003 thiab 2014 qhov 5-xyoo ciaj sia nyob.<20% in="" china,="" the="" usa,="" and="" europe="" (4,5).="" for="" these="" reasons,="" it="" is="" urgent="" to="" develop="" novel="" therapeutic="" agents="" to="" treat="" esophageal="">
Cov tshuaj suav tshuaj ntsuab tshuaj ntsuab (CHM) tau siv los kho ntau yam mob qog noj ntshav, suav nrog cov qog nqaij hlav tsis me hauv lub ntsws (6), mob qog noj ntshav (7), mob qog nqaij hlav hepatocellular (8). Tsis ntev los no, cov kev sim tshuaj ntsuam xyuas tau tshaj tawm tias kev sib xyaw ntawm CHM nrog kev siv tshuaj khomob lossis kev siv hluav taws xob tsis yog tsuas yog ua kom pom ntau cov txiaj ntsig ntawm lub neej zoo thiab txo cov kev mob tshwm sim los ntawm kev siv tshuaj khomob lossis xov tooj cua (9,10), tab sis kuj tseem txhim kho cov ciaj sia ntawm cov neeg mob. nrog rau lub ntsws uas tsis yog me me hauv lub ntsws, lub siab, lub plab, plab hnyuv, nasopharyngeal lossis mob qog noj ntshav (9). Txawm li cas los xij, muaj pov thawj tsis sib haum xeeb txog kev ua tau zoo ntawm CHM kev kho mob ntawm txoj hlab pas (10,11). Ntau cov kev tshawb fawb tau txiav txim siab tias ntau cov tshuaj ntsuab los yog cov khoom xyaw tuaj yeem cuam tshuam kev loj hlob ntawm cov qog nqaij hlav hauv plab hauv vitro thiab hauv vivo, suav nrog Andrographis paniculata (12,13), Daikenchuto (14), icariin (15), Rosa Roxburghii Tratt, thiab Fagopyrum. Cymosum (16), Jaridonin (17), Marsdenia tenacissima (18), OP16 (ib qho tshiab ent-kaurene diterpenoid) (19), Qigesan (20) thiab Tonglian decoction (21). Cistanche yog hom CHM thiab siv ntau yam kev ua haujlwm lom neeg, suav nrog kev tiv thaiv oxidation, tiv thaiv kab mob, thiab kev tiv thaiv neuroprotection (22,23). Peb qhov kev tshawb fawb yav dhau los tau pom tiasCistanche tubulosa phenylethanoid glycosides(CTPG) tuaj yeem cuam tshuam kev loj hlob ntawm melanoma B16-F10 hlwb hauv vitro thiab hauv vivo (24). Txawm li cas los xij, qhov tsis zoo ntawm cov dej solubility ntawm CTPG yav dhau los siv txwv tsis pub siv tshuaj (24). Yog li ntawd, cov dej-soluble CTPG (CTPG-W) tau siv thiab cov tshuaj tiv thaiv kab mob ntawm cov qog nqaij hlav hauv plab (Eca-109) tau tshawb xyuas. Nws tau txiav txim siab tias CTPG-W tuaj yeem txhaj tshuaj-raws li inhibit qhov kev muaj peev xwm ntawm Eca-109 hlwb los ntawm induction ntawm apoptosis ntawm txoj kev mitochondrial-dependent.
Cov ntaub ntawv thiab txoj kev Tsiaj.
Poj niam C57BL/6 nas ({2}} lub lis piam, ~ 25 g) tau yuav los ntawm Beijing Laboratory Tsiaj Research Center (Beijing, Tuam Tshoj) thiab nyob hauv qhov ntsuas kub (25˚C), lub teeb-cycled (12/ 12) Tsiaj Facility ntawm Xinjiang University (Urumqi, Suav). Txhua tus tsiaj tau txais dej tsis muaj kab mob thiab zaub mov.
Cell kab thiab kab lis kev cai. Tib neeg esophageal carcinoma cell kab (Eca{0}}) tau khaws cia los ntawm Xinjiang Key Laboratory of Biological Resources and Genetic Engineering (College of Life Science and Technology, Xinjiang University, Urumqi, China) thiab kab lis kev cai hauv RPMI{{1} } nruab nrab (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) ntxiv nrog 10 feem pua ntawm cov cua sov-inactivated fetal bovine serum (FBS; MRC, EN MOASAI Biological Technology Co., Ltd, Jiangsu, Tuam Tshoj), 1 feem pua L -glutamine (100 mM), 100 U / ml penicillin thiab 100 µg / ml streptomycin ntawm 37˚C nyob rau hauv ib qho chaw humidified uas muaj 5 feem pua CO2.
High-performance kua chromatography (HPLC). CTPG-W (cat. no. SGJG20170410) tau yuav los ntawm Shanghai Upbio Tech Co., Ltd. (Shanghai, Suav). Cov ntsiab lus tseem ceeb ntawm CTPG tau tsim nyog thiab ntsuas los ntawm HPLC raws li peb txoj kev tshawb fawb dhau los (24). Luv luv, HPLC tau ua tiav ntawm ZORBAX SB-C18 Kem (250x4.6 mm; 5 µm) ntawm 30˚C thiab eluted nrog 0.2 feem pua formic acid tov thiab gradient ntawm methanol pib ntawm 23 feem pua, raws li 1 ml tau ntxiv txhua min rau 45 min mus txog rau 31 feem pua. Tag nrho ntawm 10 µl qauv raug txhaj thiab kuaj pom ntawm 330 nm. Tus qauv echinacoside tau yuav los ntawm Shanghai Baoban Biotech Co., Ltd. (Shanghai, Tuam Tshoj), thiab tus qauv acteoside tau yuav los ntawm Sigma-Aldrich (Merck KGaA, Darmstadt, Lub teb chaws Yelemees). Cov qauv siv los txheeb xyuas cov khoom ntawm CTPG-W.
MTT kev xeem. Kev loj hlob ntawm tes tau ntsuas nrog MTT kev soj ntsuam. Eca{{0}} cov hlwb raug inoculated rau hauv 96-cov phaj zoo ntawm qhov ceev ntawm 5x103 hlwb hauv 100 µl RPMI{{5 }} nruab nrab / zoo thiab kab lis kev cai ntawm 37˚C rau 24 teev, tom qab ntawd kho los ntawm ntau qhov sib txawv (0, 200, 400, 600, thiab 800 µg / ml) ntawm CTPG-W lossis 0.4 feem pua dimethyl sulfoxide (DMSO) rau 24, 48i72 ib. DMSO tau siv los ua hnyav tswj (800 µg / ml CTPG-W muaj 0.4 feem pua DMSO). Cisplatin (20 µg / ml) tau siv los ua kev tswj hwm zoo. Tom qab ntawd, cov supernatant raug muab pov tseg tom qab centrifugation ntawm 225 xg rau 5 min ntawm chav tsev kub, thiab 100 µl MTT tov (0.5 mg / ml hauv RPMI-1640 nruab nrab tsis muaj FBS) tau ntxiv rau txhua qhov dej thiab incubated ntawm 37˚C. rau 3h. Cov formazan crystals tau yaj hauv 200 µl DMSO. Qhov ntsuas qhov muag pom qhov ntom ntom (OD) tau ntsuas ntawm lub wavelength ntawm 490 nm los ntawm 96-zoo microplate nyeem ntawv (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Tus txheeb ze ntawm kev muaj sia nyob tau raug xam raws li cov qauv: Cell viability (%)=(ODtreated/ODuntreated) x100 feem pua . Lub morphology ntawm Eca-109 hlwb raug soj ntsuam nrog lub inverted fluorescence microscope (magnification, x200) (Nikon Eclipse Ti‑E; Nikon Corporation, Tokyo, Nyiv).
Rau kev loj hlob ntawm splenocytes, C57BL / 6 nas tau euthanized los ntawm ncauj tsev menyuam dislocation, thiab spleens raug cais. Kev tshem tawm ib leeg ntawm tes tau tsim thiab splenocytes tau cog rau hauv 96- cov phiaj zoo ntawm qhov ceev ntawm 1x105 hlwb / zoo hauv 100 µl RPMI-1640 nruab nrab, thiab tom qab ntawd kho nrog ntau qhov sib txawv (0, 200, 400, 600, thiab 800 µg / ml) ntawm CTPG-W rau 24, 48 thiab 72 h ntawm 37˚C nrog 5% CO2. Kev loj hlob ntawm splenocytes tau ntsuas los ntawm MTT kev soj ntsuam, raws li cov txheej txheem hais saum toj no. Stimulatory index=ODtreated/ODuntreated.
Kev ntsuas ntawm apoptosis thiab lub voj voog ntawm tes. Eca{{0}} cov hlwb raug coj los rau hauv 60 hli lauj kaub tais diav ntawm qhov ceev ntawm 2.5x105 hlwb / tais rau 24 teev thiab kho nrog ntau qhov sib txawv ({{31} }, 200, 400, 600, thiab 800 µg/ml) ntawm CTPG-W lossis 0.4 feem pua DMSO rau 24 teev ntawm 37˚C nrog 5 feem pua CO2. Cells tau sau thiab stained nrog Annexin V-fluorescein isothiocyanate (FITC) / Propidium iodide (PI) Apoptosis Detection kit (Shanghai Shengsheng Biotechnology Co., Ltd., Shanghai, Tuam Tshoj), raws li cov chaw tsim khoom raws tu qauv. Cov qauv raug sau los ntawm kev ntws cytometry (BD FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) thiab tshuaj xyuas los ntawm FlowJo 7.6 (Tree Star, Inc., Ashland, OR, USA). Txhawm rau txheeb xyuas cov txiaj ntsig ntawm CTPG-W ntawm lub voj voog ntawm tes, 2.5x105 Eca-109 cov hlwb tau muab noob rau hauv 60 hli kab lis kev cai tais diav thiab kho nrog CTPG-W (0, 100, 200, thiab 400 µg/ml) lossis 0.4 feem pua DMSO rau 24 teev ntawm 37˚C nrog 5 feem pua CO2. Tag nrho cov hlwb raug sau thiab ntxuav ob zaug nrog dej khov-txias PBS (Gibco; Thermo Fisher Scientific, Inc.), tom qab ntawd kho hauv 70 feem pua ntawm cov dej khov-txias ethanol ntawm 4˚C rau 30 min. Tom qab ntxuav ob zaug nrog dej khov-txias PBS, cov hlwb tau rov ua dua hauv 300 µl PI / RNase staining buffer (BD Biosciences, San Jose, CA, USA) rau 10 min ntawm chav tsev kub. Lub voj voog ntawm tes faib tau txheeb xyuas nrog ModFit LT 3.0 software los ntawm flow cytometry (BD FACSCalibur).
Kev soj ntsuam ntawm mitochondrial membrane muaj peev xwm (Δψm) thiab hom reactive oxygen (ROS). Txhawm rau txheeb xyuas Δψm, Eca{{0}} cov hlwb raug kho nrog CTPG-W (0, 400, 600 thiab 800 µg/ml) lossis 0.4 feem pua DMSO rau 24 h ntawm 37˚C nrog 5 feem pua CO2, thiab stained nrog mitochondrial membrane muaj peev xwm kuaj cov khoom siv nrog JC-1 (Beyotime Institute of Biotechnology, Shanghai, Suav), raws li cov chaw tsim khoom raws tu qauv, rau 20 min ntawm 37˚C . Tom qab ntxuav ob zaug nrog JC-1 ntxhua khaub ncaws tsis (Beyotime Institute of Biotechnology), cov qauv tau rov ua dua nrog 300 µl JC-1 ntxhua khaub ncaws thiab tshuaj xyuas los ntawm kev ntws cytometry (BD FACSCalibur). Lub fluorescence ntawm JC-1 dye nyob rau hauv Eca-109 hlwb kuj tau pom nrog ib tug inverted fluorescence microscope (magnification, x200; Nikon Eclipse Ti‑E). Rau kev soj ntsuam ntawm ROS, Eca{21}} hlwb raug kho nrog CTPG-W (0, 400, 600, thiab 800 µg/ml) rau 2, 4, 6, 12, thiab 24 h, thiab stained nrog Reactive Oxygen Hom Assay Kit (Beyotime Institute of Biotechnology), raws li cov chaw tsim khoom raws tu qauv, rau 20 min ntawm 37˚C. Tom qab ntxuav peb zaug nrog dej khov-txias PBS, cov qauv tau sau los ntawm flow cytometry (BD FACSCalibur) thiab soj ntsuam los ntawm FlowJo 7.6 software.
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging kev ua si. Cov dawb radical scavenging kev ua si ntawm CTPG-W tau txiav txim siab nrog DPPH dawb radical assay raws li cov txheej txheem luam tawm nrog kev hloov me me, raws li methanol tau hloov nrog ethanol kom yaj DPPH (25,26). Rau kev ntsuas tsis tu ncua, 150 µl DPPH (100 mmol / l) hauv ethanol tau sib xyaw nrog ntau qhov sib txawv ntawm CTPG-W (25, 50, 75, 100, 250, 300, 400, 500, thiab 600 µg / ml) hauv 50 µl PBS, thiab incubated nyob rau hauv qhov tsaus ntuj rau 30 min ntawm chav tsev kub. Lub absorbance ntawm 517 nm tau kuaj pom nyob rau hauv lub xub ntiag thiab tsis muaj CTPG-W. Tag nrho ntawm 50 µl ntawm vitamin C tau siv los ua kev tswj xyuas zoo. DPPH radical scavenging kev ua si tau suav nrog siv cov qauv: Scavening (%)=[1-(A sample-Ablank)/A0] x100, qhov twg Ablank yog qhov absorbance ntawm kev tswj (tsis muaj DPPH), Ib qho piv txwv yog qhov nqus ntawm cov qauv thiab A0 yog qhov nqus ntawm PBS nrog DPPH.
Western blot tsom xam. Eca{{0}} hlwb raug kho nrog CTPG-W (0, 200, 600 µg/ml) lossis 0.4 feem pua DMSO rau 24 teev ntawm 37˚C nrog 5 feem pua CO2. Cov nram qab no ntxuav ob zaug nrog PBS, hlwb tau lysed nyob rau hauv Radioimmunoprecipitation Assay Lysis buffer (Beijing ComWin Biotech Co., Ltd., Beijing, Suav teb) rau 20 min rau dej khov. Tom qab centrifugation ntawm 10, 000 xg rau 10 min ntawm 4˚C, cov supernatants tau sau, thiab cov protein ntau tau kuaj pom nrog cov khoom siv bicinchoninic acid (Thermo Fisher Scientific, Inc.) raws li cov chaw tsim khoom cov txheej txheem. Western blot tsom xam tau ua raws li peb cov lus piav qhia dhau los (24). Antibodies tiv thaiv caspase-7 (cat. no. D120077), caspase-8 (cat. no. D155240), caspase-9 (cat. no. D220078), B-cell lymphoma{ {24}} (Bcl-2)-associated X (Bax) (cat. no. D220073) thiab Bcl-2 (cat. no. D260117), thiab anti-nas IgG-horseradish peroxidase (HRP ) (cat. no. D111050) thiab anti-luav IgG-HRP (cat. no. D110058) tau yuav los ntawm BBI Life Sciences (Shanghai, Suav). Cov tshuaj tiv thaiv kab mob tiv thaiv caspase-3 (cat. no. E-AB-10050) thiab active caspase-3 (cat. no. E-AB-22115) tau yuav los ntawm Elabscience ( Wuhan, Suav teb). Lwm cov tshuaj tiv thaiv kab mob caspase-7 (cat. no. 9492), poly (ADP-ribose) polymerase (PARP) (cat. no. 9542), cytochrome c (cat. no. AC909), c-Jun NH{ {48}}terminal kinase (JNK) (cat. no. 9252S), thiab -actin (cat. no. 58169) tau txais los ntawm Cell Signaling Technology, Inc. (Danvers, MA, USA). Tag nrho cov tshuaj tiv thaiv thawj thiab theem nrab tau diluted ntawm 1: 1, 000. Thawj cov tshuaj tiv thaiv tau muab tso rau ntawm 4˚C thaum hmo ntuj thiab cov tshuaj tiv thaiv thib ob tau muab tso rau ntawm 37˚C rau 1 teev.
Lub hom phiaj cov proteins tau kuaj pom siv cov khoom siv kho mob zoo dua qub (Beyotime Institute of Biotechnology), raws li cov chaw tsim khoom raws tu qauv.
Kev txheeb cais. Kev txheeb xyuas qhov tseem ceeb yog xam los ntawm ib txoj kev tsom xam ntawm qhov sib txawv nrog Tukey's post hoc test thiab cov txiaj ntsig tau txheeb xyuas siv GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA) ntawm cov pab pawg kho mob thiab tswj. Tag nrho cov ntaub ntawv tau nthuav tawm raws li qhov nruab nrab ± tus qauv sib txawv. P<0.05 was="" considered="" to="" indicate="" a="" statistically="" significant="">
Cistanche tubulosa phenylethanoid glycosides(CTPG)
Cov txiaj ntsig
CTPG-W inhibits kev loj hlob ntawm Eca-109 hlwb. Cov khoom ntawm CTPG-W tau tsim nyog thiab ntsuas los ntawm HPLC (Daim duab 1), uas tau muab piv nrog cov qauv ntawm echinacoside thiab acteoside. Raws li lub sijhawm khaws cia siab tshaj plaws thiab thaj chaw ncov, CTPG-W muaj 39.16 feem pua ntawm echinacoside thiab 2.44 feem pua ntawm acteoside. Ua ntej, cov txiaj ntsig ntawm CTPG-W ntawm kev muaj peev xwm ntawm Eca-109 hlwb tau txiav txim siab nrog kev ntsuam xyuas MTT. CTPG-W tau yaj hauv DMSO ntawm 200 mg / ml thiab diluted nrog RPMI -1640 nruab nrab uas muaj 10 feem pua ntawm cov cua sov-inactivated FBS los qhia cov concentrations. Eca{15}} cov hlwb tau kho nrog CTPG-W thiab kev muaj peev xwm ntawm tes tau txheeb xyuas nrog MTT kev soj ntsuam ntawm lub sijhawm qhia. CTPG-W kev kho mob txo qis kev muaj peev xwm ntawm Eca{17}} cov hlwb hauv qhov koob tshuaj thiab lub sijhawm nyob ntawm lub sijhawm (P<0.001; fig.="" 2a).="" the="" morphology="" of="" eca-109="" cells="" was="" observed="" with="" an="" inverted="" fluorescence="" microscope="" (magnification,="" x200)="" following="" ctpg‑w="" treatment="" for="" 24="" h,="" which="" changed="" notably="" in="" a="" dose-dependent="" manner,="" with="" the="" cells="" shrinking="" and="" becoming="" round="" following="" ctpg-w="" treatment="" (fig.="" 2b).="" these="" results="" indicate="" that="" ctpg-w="" suppresses="" the="" growth="" of="" eca-109="" cells.="" the="" effect="" of="" ctpg-w="" on="" the="" proliferation="" of="" splenocytes="" was="" also="" detected="" with="" an="" mtt="" assay.="" ctpg-w="" significantly="" promoted="" the="" proliferation="" of="" splenocytes="" in="" a="" dose-dependent="" manner="" (fig.="" 2c),="" indicating="" that="" it="" has="" no="" cytotoxic="" effect="" on="">
CTPG-W induces apoptosis hauv Eca-109 hlwb. Txhawm rau tshawb xyuas seb CTPG-W cuam tshuam qhov kev loj hlob ntawm Eca-109 cov hlwb los ntawm kev cuam tshuam ntawm apoptosis lossis necrosis, cov hlwb tau kho nrog ntau qhov sib txawv ntawm CTPG-W. Tom qab 24 teev, apoptosis thiab necrosis ntawm Eca{5}} hlwb raug kuaj pom nrog Annexin V/PI staining. Raws li tau piav qhia hauv daim duab 3A, Annexin V-/PI ntxiv cov hlwb raug gated li necrotic hlwb, thiab Annexin V ntxiv / PI ntxiv thiab Annexin V ntxiv / PI- hlwb raug gated li apoptotic hlwb. CTPG-W feem ntau induced apoptosis ntawm Eca{10}} hlwb nyob rau hauv ib koob tshuaj-nyob ntawm seb, txawm hais tias cov necrotic Eca{12}} hlwb kuj nce ntau nyob rau hauv kev kho mob ntawm 600 thiab 800 µg/ml CTPG-W (P<0.001 at="" 600="" µg/ml="" and=""><0.05 at="" 800="" µg/ml).="" consistently,="" the="" levels="" of="" pro-apoptotic="" bax="" and="" anti-apoptotic="" bcl-2="" in="" eca-109="" cells="" were="" upregulated="" and="" downregulated,="" respectively,="" upon="" ctpg-w="" treatment="" (fig.="" 3b).="" the="" results="" indicated="" that="" ctpg-w="" primarily="" inhibited="" the="" growth="" of="" eca-109="" cells="" through="" the="" induction="" of="">
CTPG-W induces cell voj voog ntes ntawm S theem hauv Eca-109 hlwb. Kev cuam tshuam ntawm cov kab mob qog noj ntshav yuav txo qis kev loj hlob ntawm tes thiab txhawb nqa apoptosis (27). Kev faib tawm ntawm lub voj voog ntawm tes hauv Eca-109 hlwb tau kuaj pom nrog PI staining tom qab CTPG-W kho rau 24 teev. Nws tau pom tias cov hlwb hauv S theem tau nce thiab cov hlwb hauv G0 / G1 theem qhia tau hais tias qhov kev txo qis tag nrho thaum kho CTPG-W (P<0.05; fig.="" 4),="" indicating="" that="" ctpg-w="" arrests="" the="" eca-109="" cell="" cycle="" at="" the="" s="">
CTPG-W txo Δψm thiab induces tso tawm cytochrome c. Apoptosis tuaj yeem raug ntxias los ntawm txoj kev mitochondrial-dependent (28,29). Cov tswv cuab tiv thaiv thiab tiv thaiv apoptotic ntawm BCL-2 tsev neeg cov protein ua haujlwm tseem ceeb hauv kev tswj hwm ntawm mitochondrial membrane kev ncaj ncees (30,31). Tom qab CTPG-W kev kho mob rau 24 teev, Δψm tau soj ntsuam siv JC-1 staining. JC-1 aggregate (liab fluorescence kuaj pom hauv FL-2) yuav tawg mus rau hauv monomer (ntsuab fluorescence kuaj pom hauv FL-1) thaum Δψm txo (32). Raws li tau piav qhia hauv daim duab 5A, qhov zaus ntawm FL-1 ntxiv rau FL{17}}/ ntxiv rau cov hlwb tau nce ntau hauv cov koob tshuaj, qhia tias Δψm ntawm Eca-109 hlwb txo qis. Kev hloov fluorescence hauv Eca-109 hlwb kuj tau pom nrog lub tshuab hluav taws xob inverted fluorescence (Fig. 5B). Nrog rau qhov nce ntxiv ntawm CTPG-W, cov fluorescence liab tau txo qis thiab ntsuab fluorescence nce, uas zoo ib yam nrog cov ntaub ntawv los ntawm cov cytometry ntws. Nws kuj tau pom tias qib ntawm cytochrome c hauv cytosol tau nce ntxiv (Daim duab 5C), uas yog qhov tshwm sim ntawm kev txo qis ntawm Δψm. Qhov no txhawb qhov kev txiav txim siab los ntawm kev suav ntau ntxiv ntawm FL-1 ntxiv rau FL-2-/ ntxiv rau cov hlwb uas Δψm txo qis vim yog kev kho CTPG-W. Nws tau tshaj tawm tias JNK tuaj yeem tswj hwm kev ua kom BCL-2 tsev neeg cov protein ua rau tso tawm cytochrome c ({26}}). Nws kuj tau txiav txim siab tias qib ntawm JNK yog qhov tshwj xeeb tshaj tawm kev kho mob tom qab CTPG-W kev kho mob (Daim duab 5C). Cov txiaj ntsig tau qhia tias CTPG-W tuaj yeem ua rau apoptosis ntawm Eca{30}} hlwb los ntawm txoj kev mitochondrial-dependent.
Cov nyhuv ntawm CTPG-W ntawm intracellular ROS tiam. ROS tuaj yeem txo Δψm kom induce apoptosis (36). Txhawm rau tshawb xyuas seb CTPG-W tuaj yeem nce ROS ntau lawm, Eca-109 hlwb raug kho nrog ntau qhov sib txawv ntawm CTPG-W. Cells tau sau los ntawm lub sijhawm teev cov ntsiab lus thiab stained nrog DCFH-DA. Kev tsim tawm ntawm intracellular ROS hauv Eca-109 hlwb tau txiav txim los ntawm kev ntws cytometry. Raws li tau piav qhia hauv daim duab 6A, 800 µg/ml CTPG-W tau nce ROS ntau lawm los ntawm 2-6h thiab txo los ntawm 12-24 h. Tsis tas li ntawd, 400 µg/ml CTPG-W nce ROS ntau lawm los ntawm 12-24 h. Tsis tas li ntawd, 200 µg / ml CTPG-W tsis tseem ceeb hloov ROS ntau lawm. Cov kev hloov pauv hloov pauv ntawm ROS ntau lawm tuaj yeem cuam tshuam nrog Eca{15}} cell apoptosis. Nws kuj tau txiav txim siab tias CTPG-W muaj cov dawb radical scavenging kev ua si (Fig. 6B), uas tej zaum yuav cuam tshuam nrog qhov txo qis ROS ntau lawm hauv Eca{18}} hlwb kho nrog 800 µg/ml CTPG-W tom qab 12 teev.
CTPG-W txhawb kev ua haujlwm ntawm caspase-3, caspase-7, caspase-9 thiab PARP. Kev tso tawm ntawm cytochrome c vim Δψm txo tuaj yeem qhib cov caspase proteases kom induce apoptosis (29,30,37). Tom qab CTPG-W kev kho mob rau 24 teev, kev ua kom lub cev ntawm caspase-3, 7, 8, 9, thiab PARP tau kuaj pom los ntawm kev txheeb xyuas sab hnub poob. Piv nrog rau kev tswj, qib ntawm cleaved-caspase-9, cleaved-caspase-7, cleaved-caspase-3, thiab cleaved-PARP, tab sis tsis yog qib ntawm cleaved-caspase{{ 20}}, raug kho los ntawm CTPG kev kho mob raws li koob tshuaj (Daim duab 7). Cov txiaj ntsig no tau qhia tias CTPG-W txo Δψm thiab txhawb nqa cytochrome c tso tawm kom qhib cov caspases uas ua rau apoptosis ntawm Eca{24}} hlwb.
Cistanche tubulosa extract
Kev sib tham
Ib txwm CHM tuaj yeem ua rau apoptosis ntawm esophageal cancer hlwb los ntawm ntau txoj hauv kev, suav nrog cov extrinsic death receptor, intrinsic mitochondrial thiab endoplasmic reticulum stress pathways (29). Peb txoj kev tshawb fawb yav dhau los tau pom tias CTPG, uas yog qhov tseem ceeb ntawm C. tubulosa, inhibited kev loj hlob ntawm melanoma B16-F10 hlwb los ntawm induction ntawm apoptosis ntawm txoj kev mitochondrial-dependent (24). Hauv kev tshawb fawb tam sim no, cov nyhuv antitumor ntawm CTPG-W ntawm Eca-109 hlwb tau tshawb xyuas thiab nws tau txiav txim siab tias CTPG-W inhibited kev loj hlob ntawm Eca-109 hlwb, induced apoptosis, thiab cell voj voog ntes, txo Δψm, nce qhov tso tawm ntawm cytochrome c thiab activated caspases. CTPG thiab CTPG-W tuaj yeem ua rau apoptosis thiab cell voj voog ntes hauv cov qog nqaij hlav cancer. Txawm li cas los xij, cov txheej txheem raug sib txawv vim muaj cov khoom sib txawv ntawm CTPG (26.64% echinacoside, 10.19% acteoside, thiab 1.71% isoacteoside) thiab CTPG-W (39.16% echinacoside thiab 2.44% acteoside). CTPG ntes B16-F10 hlwb ntawm G0/G1 theem, tab sis CTPG-W ntes Eca{26}} hlwb ntawm S theem (24). ROS ntau lawm yog koob tshuaj-raws li nce los ntawm CTPG, tab sis nws qhia tau hais tias ib tug hloov nyob rau hauv ib lub sij hawm-nyob ntawm seb los ntawm ib tug siab koob tshuaj CTPG-W, uas tau nce ntau heev thaum pib ntawm CTPG-W kev kho mob (2-6 h) thiab txo qis tom qab 12 teev, piv nrog kev tswj hwm. Ib qho laj thawj yog vim li cas qhov tseem ceeb ntawm CTPG-W yog echinacoside. Ntau cov kev tshawb fawb tau tshaj tawm tias echinacoside tuaj yeem cuam tshuam ROS ntau lawm thiab ROS-induced apoptosis los ua nws cov teebmeem neuroprotective thiab anti-aging (38-40). Ib yam li ntawd, cov dawb radical scavenging kev ua tau pom nyob rau hauv txoj kev tshawb no. Yog li ntawd, nws tau kwv yees tias qee cov khoom xyaw, suav nrog verbascoside, iso-verbascoside, thiab salidroside nyob rau hauv cov koob tshuaj ntau ntawm CTPG-W tuaj yeem ua rau ROS tiam tam sim ua rau apoptosis ntawm Eca-109 hlwb (41,42), thiab tom qab ntawd ROS tau scavenged los ntawm echinacoside. Lwm qhov laj thawj rau qhov sib txawv ntawm ROS ntau lawm los ntawm CTPG thiab CTPG-W yog cov kab sib txawv ntawm tes tau siv hauv txoj kev tshawb no thiab kev kawm yav dhau los (24). Dong et al (43) tau tshaj tawm tias echinacoside tuaj yeem ua rau apoptosis ntawm tib neeg SW480 kab mob qog nqaij hlav hauv plab los ntawm kev tsim cov oxidative DNA puas yam tsis muaj ROS ntau ntxiv.
CTPG-W kev kho mob txo Δψm thiab ua rau muaj kev tso tawm ntawm cytochrome c, uas txhawb nqa qhov cleavage ntawm caspase-9 (28). Raws li qhov tshwm sim, cov qib ntawm cleaved-caspase-9 tau raug kho los ntawm kev kho CTPG-W. Tom qab ntawd, lub active caspase -9 tuaj yeem qhib tau caspase -3 kom induce apoptosis (44). Cov theem ntawm cleaved-caspase-3 kuj tau tswj hwm los ntawm kev kho CTPG-W. Txawm li cas los xij, caspase-8 tsis tau qhib los ntawm CTPG-W, qhia tias txoj kev tuag ntawm tus neeg sab nrauv tsis koom nrog apoptosis tshwm sim los ntawm CTPG-W. Cov kev soj ntsuam no qhia tau hais tias CTPG-W induces apoptosis ntawm Eca{16}} hlwb los ntawm kev ua kom muaj txoj hauv kev mitochondrial-dependent.
PARP ua haujlwm tseem ceeb hauv kev ruaj ntseg genomic thiab tuaj yeem raug tshem tawm los ntawm cov caspases nquag, tshwj xeeb yog caspase-3 thiab -7 (45). Nws tau txiav txim siab tias CTPG-W kev kho mob tau qhib lub caspase-3 thiab -7, uas tuaj yeem cuam tshuam PARP los tiv thaiv DNA kho thiab ua rau apoptosis.
CTPG-W kuj tau koob tshuaj- thiab lub sij hawm-dependently suppresses kev loj hlob ntawm tib neeg hepatocellular carcinoma BEL-7404 hlwb (cov ntaub ntawv tsis tau tshaj tawm). Txawm hais tias CTPG-W inhibits kev loj hlob ntawm Eca-109 thiab BEL-7404 hlwb, nws txhawb kev loj hlob ntawm splenocytes, uas tej zaum yuav yog vim cov ntsiab lus ntawm polysaccharides (~50 feem pua) hauv CTPG-W (46. ). Ib yam li ntawd, ntau cov kev tshawb fawb tau qhia tias polysaccharides tuaj yeem txhawb kev loj hlob ntawm splenocytes (46-49). Nyob rau hauv tus qauv nas, nws tau txiav txim siab tias CTPG-W nce qhov spleen Performance index, piv nrog rau pawg tswj hwm, tab sis tsis cuam tshuam rau lub cev qhov hnyav thiab lwm yam khoom nruab nrog cev suav nrog lub plawv, daim siab, lub raum, thiab lub ntsws (cov ntaub ntawv tsis tau tshaj tawm), qhia tias CTPG-W tsis muaj cov nyhuv cytotoxic rau cov hlwb li qub.
Ua ke, cov ntaub ntawv qhia tias CTPG-W inhibits kev loj hlob ntawm Eca-109 hlwb los ntawm induction ntawm apoptosis ntawm txoj kev mitochondrial-dependent.
Cov txiaj ntsig cistanche: anti-apoptosis
Kev lees paub
Cov kws sau ntawv xav ua tsaug rau Dr. Jianhua Yang (Baylor College of Medicine) rau polishing cov ntawv sau.
Nyiaj txiag
Txoj kev tshawb no tau txais kev txhawb nqa los ntawm 13th Tsib Xyoo Kev Npaj rau Txoj Haujlwm Tseem Ceeb Biology Bidding Project (grant no. 17SDKD0202), Xinjiang Normal University thiab Key Laboratory of Special Environment Biodiversity Application and Regulation in Xinjiang (grant no. XJTSWZ-2017-04) rau JL thiab Suav National Natural Science Foundation Grant (grant no. 31760260) rau XW.
Muaj cov ntaub ntawv thiab cov ntaub ntawv
Cov ntaub ntawv thiab cov ntaub ntawv siv thiab tshuaj xyuas thaum lub sijhawm kawm tam sim no muaj los ntawm tus kws sau ntawv raws li kev thov tsim nyog.
Cov neeg sau ntawv pab txhawb
CF, AA, YY, thiab QC ua qhov kev sim. LX, JLv, thiab XW txheeb xyuas cov ntaub ntawv thiab cov duab npaj. JinyuL thiab JinyaoL tsim qhov project thiab sau cov ntawv sau.
Kev pom zoo ntawm kev ncaj ncees thiab kev tso cai los koom
Tag nrho cov kev sim tsiaj tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Tsiaj Kev sim ntawm Xinjiang Key Laboratory ntawm Biological Resources thiab Genetic Engineering (kev pom zoo, no. BRGE-AE001; Xinjiang University).
Tus neeg mob tso cai rau kev tshaj tawm
Tsis siv tau.
Kev nyiam sib tw
Cov kws sau ntawv tshaj tawm tias lawv tsis muaj kev sib tw nyiam.
Cov ntaub ntawv
Wu CR, Lin HC, thiab Su MH: Reversal by aqueousExtract ntawm Cistanche 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 14: 202, 2014.
Li J, Aspire A, Gao L, Huo S, Luo J, Zhang F.Phenylethanoid glycosides los ntawm Cistanche tubulosainhibits kev loj hlob ntawm B16-F10 hlwb ob leeg hauv vitro thiab hauv vivo los ntawm induction ntawm apoptosis ntawm mitochondria-dependent Pathway. J Cancer 7: 1877-1887, 2016.
Brand-Williams W, Culver ME, thiab Berset C: Siv cov txheej txheem dawb radical los ntsuas kev ua haujlwm antioxidant. LWT-Food Sci Technol 28: 25-30, 1995.
