Kev cuam tshuam ntawm Phenylethanoid Glycosides Los Ntawm Cistanche Tubulosa Nrog MTT Assay

1. Taw qhia

Cov kab mob cabCistanche tubulosa(Schrenk) R. Wight (Orobanchaceaetsev neeg) tau nthuav dav hauv North African, Arab, thiab Asian lub teb chaws [1]. Cov stems los ntawmC. tubulosathiabC. deserticolayog ib qho tseem ceeb hauv cov tshuaj suav tshuaj, tau siv los kho impotence, sterility, lumbago, thiab cem quav vim colonic inertia [2]. Tsis tas li ntawd, ethanolic extract ntawmC. tubulosatau pom tias muaj kev tiv thaiv tseem ceeb tiv thaiv cerebral hypoxia hauv nas [3].

Cov hlwb endothelial ua lub luag haujlwm tseem ceeb hauv kev tsim cov kab mob hypoxic pulmonary hypertension. EA.hy926 endothelial cell kab yog qhov zoo ib yam li thawj cov kab mob endothelial. Thaum peb kawm txog cov tshuaj tiv thaiv hypoxic ntawm C. tubulosa ethanolic extract ntawm EA.hy926 endothelial hlwb, peb pom tias cov 3-(4,5-dimethylthiazol-2-yl){{7 }}, 5-diphenyltetrazolium bromide (MTT) assay, uas feem ntau yog siv los ntsuas kev muaj peev xwm ntawm tes hauv kev tshawb fawb ntawm cytotoxicity thiab cytostatic kev ua haujlwm, ua rau muaj kev tsis sib haum xeeb los ntawm kev nthuav tawm EA.hy926 cell viability tom qab kho.

Kev ntsuas tseeb ntawm kev muaj peev xwm ntawm tes yog qhov tseem ceeb los txheeb xyuas qhov muaj peev xwm cytotoxic lossis cytoprotective los ntawm tus neeg sawv cev tshuaj. Peb, yog li ntawd, tshawb xyuas cov khoom uas muaj nyob rau hauv C. tubulosa ethanolic extract tej zaum yuav yog lub luag hauj lwm rau cov kev tsis sib haum xeeb tau soj ntsuam nrog MTT assay. Peb cais ob lub ntsiabphenylethanoid glycosides (PhGs)C. tubulosa ethanolic extract,echinacoside (tom qab no compound 1) thiab acteoside (tom qab ntawd compound 2). Peb txiav txim siab lawv cov txiaj ntsig ntawm EA.hy926 cell viability siv ntau txoj hauv kev. Tsis tas li ntawd, txhawm rau kom paub meej ntxiv qhov hloov pauv ntawm cov tshuaj 1 thiab 2 tuaj yeem ua lub luag haujlwm rau qhov tsis sib haum xeeb, peb tau sim lawv cov aglycones, caffeic acid (tom qab no compound 3) thiab 3, 4-dihydroxylphenylethanol (tom qab no compound 4), ntawm EA .hy926 cell viability siv tib txoj kev.

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2. Cov txiaj ntsig thiab kev sib tham

2.1. Kev txheeb xyuas cov khoom sib xyaw

Cov qauv ntawm ob lub tebchaw cais los ntawmC. tubulosa ethanolic extracttau txheeb xyuas los ntawm nuclear magnetic resonance (NMR) thiab huab hwm coj spectrometry (MS) analyses, raws li qhia nyob rau hauv daim duab 1. Compound 1 tau txheeb xyuas raws li echinacoside los ntawm kev sib piv nrog yav dhau los qhia NMR (Table 1) thiab MS (m/z 785 [M-H. ]-) data [4]. Compound 2 NMR spectra tau zoo ib yam li cov compound 1, tshwj tsis yog qhov tsis muaj -D-glucopyranosyl pawg (Table 1). Compound 2 (m / z 623 [M−H]− ) tau txheeb xyuas tiasacteoside[5]. Qhov tseem ceeb, ob lub tebchaw muaj tib yam aglycone, uas suav nrog caffeic acid (compound 3) thiab 3, 4-dihydroxylphenylethanol (compound 4).

2.2. Cell Viability Assay

MTT thiab CCK{0}} kev tshuaj xyuas tau siv los txheeb xyuas cov txiaj ntsig ntawm cov tshuaj 1–4 ntawm EA.hy926 cell viability. Qhov kev ntsuam xyuas MTT tau pom tias qhov muaj peev xwm ntawm EA.hy926 hlwb tau nce ntxiv tom qab kev kho mob nrog 25 thiab 50 μM compounds 1, 2, lossis 3 (166.3% thiab 174.1% rau compound 1, 205.8% thiab 224.3% rau compound 2, thiab 164.8% thiab 189.6% rau compound 3, feem; Tsis tas li ntawd, kev tshuaj xyuas morphological ntawm cov hlwb tau txiav txim siab tias kev kho mob nrog 50 μM compound 1, 2, lossis 3 nce kev tsim cov ntshav formazan crystals, uas yog qhov qhia ncaj qha ntawm kev faib ua feem ntawm cov hlwb (Daim duab 3). Conversely, CCK-8 assay qhia tias cell viability txo qis heev tom qab kev kho mob nrog 12.5, 25, thiab 50 μM compound 1, 2, los yog 3 (75.5%, 45.1%, thiab 43.7% rau compound 1, 85.5%, 51.8%, thiab 34.3% rau compound 2, thiab 64.5%, 48.2%, thiab 36.4% rau compound 3, ntsig txog; Compound 4 tsis cuam tshuam txog kev muaj peev xwm ntawm tes hauv MTT lossis CCK-8 kev soj ntsuam. Qhov tsis sib xws ntawm cov txiaj ntsig tau txais nrog ob qhov kev ntsuam xyuas qhia tias ib qho ntawm ob qhov kev ntsuam xyuas yuav tsis cuam tshuam qhov cuam tshuam ntawm cov tebchaw 1 thiab 2 ntawm EA.hy926 cell viability.

2.3. Kev Ntsuam Xyuas Apoptosis los ntawm Hoechst

Staining Hoechst 33342 yog cell-permeable DNA stain uas zoo siab los ntawm ultraviolet lub teeb thiab emits xiav fluorescence ntawm 460 rau 490 nm. Cov condensed chromatin ntawm apoptotic hlwb stain ntau ci ntsa iab dua li cov chromatin ntawm cov hlwb ib txwm [6]. Thaum tswj cov hlwb nthuav tawm cov chromatin tsis sib xws, cov organelles ib txwm muaj, thiab cov cell tsis zoo, cov cell incubated nrog 50 μM cov tebchaw 1, 2, lossis 3 rau 48 h tau pom qhov staining ntawm apoptotic hlwb (Daim duab 4A). Conversely, raug cov hlwb rau 50 μM ntawm compound 4 tsis tau nce tus naj npawb ntawm apoptotic nuclei piv rau kev tswj kev kho mob.

2.4. Flow Cytometric Analysis ntawm Apoptotic Cells

Lub annexin V-FITC/PI ob-staining assay txheeb xyuas apoptotic hlwb los ntawm high-affinity binding ntawm annexin V-FITC rau phosphatidylserine, uas yog externalized nyob rau hauv lub hlwb nyob rau hauv apoptosis [7]. Hauv qhov kev ntsuam xyuas no, cov hlwb siv tau tsis khi rau annexin V lossis PI thiab yog li ntawd tsis muaj xim (tso sab laug quadrant), cov hlwb apoptotic thaum ntxov yog xim ntsuab los ntawm kev khi ntawm annexin V-FITC (txo txoj cai quadrant), thiab lig apoptotic hlwb. yog stained ntsuab thiab liab los ntawm kev khi ntawm annexin V-FITC rau phosphatidylserine thiab PI rau necrotic hlwb, ntsig txog (saum txoj cai quadrant). Raws li pom nyob rau hauv daim duab 4B, hlwb incubated nrog 50 μM tebchaw 1, 2, los yog 3 rau 48 h, cov feem pua ​​​​ntawm apoptotic hlwb tau nce los ntawm 3.74% (tswj hlwb) mus rau 10.32%, 12.95%, thiab 11.30%, feem. Piv rau kev tswj cov hlwb, compound 4 tsis tau nce qhov feem pua ​​​​ntawm apoptotic EA.hy926 hlwb.

Raws li cov txiaj ntsig tau txais nrog CCK-8 kev soj ntsuam thiab los ntawm Hoechst 33342 staining, flow cytometry assay pom tias cov tebchaw 1, 2, thiab 3 induced apoptosis hauv EA.hy926 hlwb. Ua ke, cov kev soj ntsuam no qhia tias qhov nce hauv EA.hy926 cell viability tau soj ntsuam nrog MTT kev soj ntsuam tom qab kev kho mob nrog cov tebchaw 1-3 sawv cev rau qhov tshwm sim tsis tseeb.

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2.5. Kev sib tham

Xyoo 1983, Mosmann tau tsim ib qho kev ntsuam xyuas colorimetric MTT microplate rau kev ntsuas cell proliferation thiab cytotoxicity [8]. Qhov kev ntsuam xyuas yooj yim no, thiab nws cov kev hloov kho, tam sim no tau siv dav hauv cell biology kuaj thoob ntiaj teb. Cov kev tshawb fawb Fractionation nyob rau hauv mammalian hlwb qhia tias txo pyridine nucleotide cofactor, NADH, yog lub luag hauj lwm rau feem ntau MTT txo. Qhov no tau txhawb nqa los ntawm kev tshawb fawb siv tag nrho cov hlwb [9]. MTT txo yog li ntawd cuam tshuam nrog cov hlwb ua haujlwm metabolically thiab tsis tsuas yog ua ke nrog mitochondrial ua pa, tab sis kuj nrog cov cytoplasm thiab non-mitochondrial daim nyias nyias nrog rau endosome / lysosome compartment thiab plasma membrane [10]. Qhov txiaj ntsig zoo ntawm MTT molecule yog qhov tseem ceeb rau nws cov cellular uptake ntawm lub peev xwm plasma membrane.

Qhov tseem ceeb, MTT qhov kev soj ntsuam qee zaum yuav tsis raug cuam tshuam txog qhov tshwm sim tiag tiag uas cov tshuaj ntsuam xyuas muaj rau cov hlwb tshwj xeeb. Txawm li cas los xij, cov qauv tshuaj lom neeg lossis pab pawg uas yuav ua rau muaj kev tsis sib haum xeeb hauv MTT qhov kev ntsuam xyuas tsis tau txheeb xyuas. Cov kev tshawb fawb tsis ntev los no tau pom tias MTT qhov kev ntsuam xyuas tuaj yeem kwv yees qhov kev tiv thaiv kab mob ntawm (−)-epigallocatechin-3-gallate, ntau tshaj polyphenol hauv tshuaj yej ntsuab [11]. Hauv cov kev tshawb fawb siv MTT kev soj ntsuam los kuaj cov qog nqaij hlav, cov tshuaj tiv thaiv kab mob qog noj ntshav, xws li epirubicin, paclitaxel, docetaxel, thiab imatinib mesylate (Gleevec), pom tias muaj zog ntawm tes [12,13]. Ntxiv mus, ntau qhov kev tshawb fawb tau tshaj tawm tias qee cov nroj tsuag rho tawm thiab redox-active polyphenols tuaj yeem cuam tshuam nrog MTT kev soj ntsuam raws li lawv ncaj qha txo MTT tetrazolium ntsev thaum tsis muaj hlwb [14].

Qhov yuav tsum tau solubilize MTT formazan crystals ua ntej spectrophotometric tsom xam nyob rau hauv ib tug microplate nyeem ntawv thiab lub inherent endpoint xwm ntawm cov tshuaj tiv thaiv txwv tsis pub siv MTT assay rau tej daim ntawv thov. Qhov no ua rau kev txhim kho ntawm tetrazolium analogs uas cov phenyl moieties tau dai kom zoo nkauj nrog cov pab pawg tsis zoo sulfonate, xws li 2,3-bis-(2-methoxy-4-nitro- 5- sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), cov ntsev sab hauv tsis zoo [15] thiab 3-(4,5-dimethylthiazol-2-yl) -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), uas tsis muaj zog acidic sab hauv ntsev ze ze rau MTT [16] . Cov kev hloov kho no tau ua rau kev tsim cov kab lis kev cai nruab nrab-soluble formazan cov khoom uas tshem tawm qhov yuav tsum tau ua ntawm cov kauj ruam solubilization ua ntej kom muaj nuj nqis. Raws li qhov tshwm sim, raws li qhov nce nqi tsis zoo ntawm cov molecules txo lawv lub peev xwm txav mus los ntawm cov cell membranes [17], intermediate electron acceptors (IEA), xws li 1-methoxy-5-methyl phenazine methyl sulfate ({{25). }}methoxy PMS) yuav tsum tau pab txhawb tetrazole dye txo lossis txhim kho tus nqi txo.

Tsis ntev los no, ib tiam tshiab ntawm cov dej-soluble tetrazolium ntsev tau tsim los ntawm WST-1 yog tus qauv [18]. WST-1, qhov tsis zoo them nyiaj disulfonated sab hauv ntsev uas muaj cov iodine residue, ruaj khov dua XTT thiab MTS nyob rau hauv muaj 1-methoxy PMS, nws yog qhov yuav tsum tau IEA. Qhov no coj mus rau kev lag luam ntawm WST-1/1-methoxy PMS raws li ib qho yooj yim reagent cell proliferation khoom. Ob peb lwm tetrazolium ntsev hauv WST series tau tsim, qhov tseem ceeb tshaj plaws uas tej zaum yog WST-8 [19]. WST-8 zoo li muaj qhov zoo sib xws ntawm cellular txo cov khoom rau WST-1 thiab tab tom ua lag luam ntawm nws tus kheej raws li CCK-8 kev soj ntsuam. WST-8 yog cell impermeable thiab yog li ntawd txo qis extracellularly, ntawm electron thauj los ntawm intracellular NADH mus rau WST-8 hla cov plasma membrane mediated los ntawm 1-methoxy PMS. Txawm hais tias ob qho tib si MTT thiab WST-8/1-methoxy PMS txo yog tsav los ntawm intracellular NADH, qhov chaw ntawm NADH zoo li txawv nyob rau hauv ob txoj kev no. WST-8/1-methoxy PMS kev txo qis yog nyob ntawm malate/aspartate shuttle uas txuas mitochondrial tricarboxylic acid cycle-NADH nrog rau qhov chaw extramitochondrial [20].

Hauv kev sim ua ntej, peb tau lees paub tias cov tebchaw 1, 2, 3, thiab 4 tuaj yeem txo qis MTT tetrazolium ntsev ncaj qha (tsis qhia). Hauv txoj kev tshawb fawb tam sim no, kev cuam tshuam ncaj qha ntawm cov tebchaw ntawm lawv tus kheej nrog cov reagent raug tshem tawm los ntawm kev ua tib zoo ntxuav cov microplates nrog phosphate-buffered saline (PBS) ua ntej MTT lossis CCK-8 kev daws tau ntxiv, raws li qhia hauv cov chaw tsim khoom cov txheej txheem. Txawm li cas los xij, cov txiaj ntsig tau siv ob txoj hauv kev tseem tsis sib xws (Daim duab 2). Raws li xav tau, incubation ntawm cov hlwb nrog cov tebchaw 1, 2, thiab 3 nce qhov txo ntawm tetrazolium ntsev mus rau hauv ntshav formazan, piv nrog rau pawg tswj hwm (Daim duab 3), qhia tias tsuas yog txo cov tetrazolium ntsev nyob rau hauv lub xub ntiag ntawm ib tug tshwj xeeb compound. tsis txaus los txiav txim qhov sib xyaw ua ke muaj peev xwm cuam tshuam nrog MTT qhov kev xeem.

Txhawm rau txiav txim siab seb cov tebchaw 1, 2, 3, thiab 4 tuaj yeem ua rau apoptosis hauv EA.hy926 hlwb, kev hloov pauv ntawm cellular morphological thiab phosphatidylserine externalization raug soj ntsuam. Raws li qhov txo qis hauv cellular viability tau soj ntsuam nrog CCK-8 kev soj ntsuam, cov txiaj ntsig ntawm Hoechst staining thiab flow cytometry tsom siv annexin V-FITC / PI qhia tias qhov kev loj hlob inhibition tau pom tom qab kev kho mob 1, 2, thiab 3. vim, yam tsawg kawg ntawm ib feem, rau EA.hy926 cell apoptosis. Peb cov txiaj ntsig kuj tau txhawb lub tswv yim tias pawg caffeyl tam sim no nyob rau hauv ob qho tib si sib txuas 1 thiab 2 yog lub luag haujlwm rau cov txiaj ntsig tsis sib haum xeeb tau txais nrog MTT kev soj ntsuam.

Zoo ib yam li peb qhov kev tshawb pom, Rottlerin, lub zog loj conductance potassium channel qhib, tsis pom muaj kev cuam tshuam rau MTT hauv vitro. Txawm li cas los xij, nws tau txhim kho qhov tsim ntawm formazan crystals hauv cov hlwb, qhov tshwm sim uas tau tshwm sim hauv qhov tsis sib haum xeeb nrog Rottlerin inhibition ntawm NF-κB thiab cell proliferation pom los ntawm kev tsom xam ntawm [3 H]-thymidine koom ua ke rau hauv DNA [21]. Lub tshuab uas Rottlerin txhim kho MTT txo tau raug ntaus nqi rau nws cov nyhuv mitochondrial uncoupling [22]. Rottlerin muaj ib pab pawg neeg cinnamoyl acid hloov. Piv nrog cinnamoyl acid, compound 3 muaj ob qho ntxiv hydroxyl residues, uas nyob ntawm C-3 thiab C-4. Yog li, peb xav tias cov txheej txheem los ntawm cov khoom sib txuas 1 thiab 2 ua rau muaj kev tsis sib haum xeeb hauv MTT qhov kev ntsuam xyuas kuj tseem cuam tshuam nrog lawv cov teebmeem mitochondrial uncoupling. Txhawm rau kuaj qhov kev xav no, kev tshawb fawb sib piv ntawm cov khoom sib txuas 1, thiab 2, thiab cov tshuaj uncoupler, xws li trifluorocarbonylcyanide phenylhydrazone (FCCP), xav tau.

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3. Ntu kev sim

3.1. Reagents thiab tshuaj

Compound 3 (caffeic acid-powder, purity=98.6%) yog yuav los ntawm Chengdu Push Bio-Technology Co., Ltd. (Chengdu, Suav). Compound 4 (3, 4-dihydroxylphenylethanol-roj, purity=98.5%) yog tau los ntawm Chengdu Must Bio-Technology Co., Ltd. (Chengdu, Suav). MTT thiab dimethyl sulfoxide (DMSO) tau txais los ntawm Sigma-Aldrich (St. Louis, MO, USA). CCK-8 kev xeem tau yuav los ntawm Dojin Laboratories (Kumamoto, Nyiv). Hoechst 33342 thiab annexin V-FITC / PI apoptosis kuaj cov khoom siv tau yuav los ntawm Beyotime Institute of Biotechnology (Haimen, Suav).

3.2. Khoom siv cog

C. tubulosastems tau sau los ntawm Yutian Prefecture, Xinjiang Uygur Autonomous Region, Tuam Tshoj. Voucher cov qauv tau muab tso rau hauv Lub Tsev Haujlwm Tseem Ceeb ntawm Cov Tshuaj Kho Mob Siab, Thib Peb Cov Tub Rog Kho Mob University (Chongqing, Tuam Tshoj) thiab tau lees paub los ntawm Yi Zhang los ntawm Chengdu University of Traditional Chinese Medicine (Chengdu, Suav).

3.3. Extraction thiab rho tawm cov khoom cog

Cua qhuav C. tubulosa (0.6 kg) stems tau hmoov thiab muab rho tawm nrog 50% ethanol rau 2 h ntawm 70 degree . Tom qab tshem tawm cov kuab tshuaj nyob rau hauv lub siab txo, ethanolic extract (212.5 g) tau yaj thiab muab tshem tawm hauv dej (2 L) thiab muab rho tawm nrog n-butanol (2 L × 3). N-butanol extract (110 g) tau chromatographed ntawm ib kab ntawv polyamide thiab eluted nrog ethanol / dej (0:100, 5:95, 15:85, 30:70, 50:50) ua tsib feem (A-E) . Fraction B (65 g) tau muab faib rau ntawm kab ODS; elution nrog ethanol: dej (1:9) sib cais compound 1 (3 g). Fraction D (10.5 g) yog fractionated ntawm ib kem ODS; elution nrog ethanol / dej (1:4) sib cais compound 2 (1 g).

NMR spectra tau kaw rau ntawm Bruker Avance 600 spectrometer hauv CD3OD nrog Tetramethylsilane (TMS) raws li tus qauv sab hauv. Cov spectrum loj tau nqa tawm ntawm BioTOF-Q huab hwm coj spectrometer.

3.4. Cell Culture

EA.hy926 cell kab tau txais los ntawm American Type Culture Collection (Manassas, VA, USA). Cells tau coj mus kuaj hauv RPMI 1640 nruab nrab, ntxiv nrog 10% (v/v) fetal bovine serum thiab 1% (v/v) penicillin-streptomycin nyob rau hauv ib puag ncig humidified nrog 5% CO2 ntawm 37 degree.

3.5. Cell Viability

Cell viability raug soj ntsuam siv MTT thiab CCK{{0}} kev soj ntsuam. Cov tshuaj 1 thiab 2, thiab lawv cov aglycones, caffeic acid (3) thiab 3, 4-dihydroxylphenylethanol (4) tau yaj hauv DMSO mus rau qhov kawg ntawm DMSO concentration <0.1% (v/v) hauv kab lis kev cai xov xwm.

EA.hy926 hlwb tau cog rau ntawm 96-cov phiaj zoo ntawm 4 × 103 hlwb/zoo. Tom qab incubation rau 24 teev, cov hlwb raug kho nrog 6.25-50 μM compound 1, 2, 3, lossis 4 rau 24 teev. Cov kab lis kev cai nruab nrab raug tshem tawm thiab cov hlwb raug ntxuav ob zaug nrog phosphate-buffered saline (PBS). Aliquots (10 μL) ntawm Tshuag MTT tov (5 mg·mL−1) tau ntxiv rau txhua qhov dej uas muaj 100 μL ntawm nruab nrab thiab incubated nrog hlwb rau 4 h. Tom qab incubation, qhov nruab nrab raug tshem tawm thiab 150 μL aliquots ntawm DMSO tau ntxiv rau txhua lub qhov dej kom solubilize lub formazan crystals. Absorbance tau ntsuas ntawm 490 nm siv lub microplate nyeem ntawv (Bio-Tek Synergy HT, Winooski, VT, USA). Cell viability tau qhia raws li feem pua ​​​​ntawm MTT txo, muab 100% tus nqi rau kev nqus ntawm cov tswj hlwb. Txhua qhov kev sim tau ua peb zaug thiab nthuav tawm raws li qhov txhais tau tias ± tus qauv sib txawv (SD).

Kev ntsuam xyuas CCK-8 tau ua raws li cov ntaub ntawv nrog kev hloov kho me ntsis [23]. Tshwj xeeb, cov hlwb raug kho nrog 6.25–50 μM cov tshuaj 1, 2, 3, lossis 4 rau 24 teev thiab ntxuav ob zaug nrog PBS ua ntej ntxiv 10 μL CCK-8 tov thiab 100 μL ntawm kab lis kev cai xov xwm. Tom qab incubating lub microplate ntawm 37 degree rau 2 h, absorbance yog ntsuas ntawm 450 nm siv tus nyeem ntawv microplate. Cell viability tau qhia raws li feem pua ​​​​ntawm cov hlwb uas muaj feem cuam tshuam rau kev tswj cov hlwb (100%). Txhua qhov kev sim tau ua tiav peb zaug thiab qhia tias txhais tau tias ± SD.

3.6. Kev ntsuam xyuas Apoptosis los ntawm Hoechst 33342 Staining

Apoptotic morphological hloov pauv tau pom los ntawm Hoechst 33342 staining. Luv luv, EA.hy926 hlwb tau cog rau ntawm 48- cov phiaj zoo (2 × 104 hlwb / zoo) thiab kho nrog 50 μM compound 1, 2, 3, lossis 4 rau 48 h ntawm 37 degree, ntxuav ob zaug nrog PBS, thiab kho nrog 4% (v/v) paraformaldehyde rau 15 min ntawm chav tsev kub. Tom qab yaug ob zaug nrog PBS, cov hlwb raug stained nrog Hoechst 33342 (10 ug·mL−1) rau 5 min ntawm chav tsev kub thiab ntxuav ob zaug nrog PBS. Hoechst-stained nuclei tau pom los ntawm kev siv lub tshuab fluorescence (Olympus, Tokyo, Nyiv).

3.7. Apoptosis Analysis los ntawm Flow Cytometry

Apoptotic hlwb tau kuaj pom los ntawm annexin V-FITC / PI ob-staining thiab flow cytometry tsom xam. Luv luv, tom qab kev kho mob nrog 50 μM compound 1, 2, 3, lossis 4 rau 48 h, cov hlwb raug sau thiab rov ua dua hauv PBS ntawm 1 × 105 hlwb·mL−1. Tom qab centrifugation ntawm 500 × g rau 5 min ntawm 25 degree, 195 μL ntawm annexin V-FITC binding tsis thiab 5 μL ntawm annexin V-FITC tau ntxiv. Tom qab maj mam vortex-sib tov, qhov sib tov yog incubated rau 10 min ntawm chav tsev kub nyob rau hauv qhov tsaus ntuj nti. Tom qab centrifugation ntawm 500 × g rau 5 min ntawm 25 degree, 190 μL ntawm FITC-conjugated annexin V binding tsis thiab 10 μL PI tau ntxiv. Tom qab maj mam sib xyaw vortex, cov qauv raug tshuaj xyuas siv FACSCalibur Flow Cytometer (Becton Dickinson, San Jose, CA, USA) hauv 30 min.

3.8. Kev txheeb cais

Tag nrho cov ntaub ntawv sim tau qhia tias txhais tau tias ± SD. Qhov tseem ceeb ntawm qhov sib txawv ntawm cov qauv kev sim thiab kev tswj xyuas tau raug soj ntsuam los ntawm ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) siv SPSS software (version 11.5). Kev txheeb xyuas qhov tseem ceeb tau teeb tsa ntawm p < 0.05.

4. Cov lus xaus

Peb cov txiaj ntsig tau qhia tias qhov kev kwv yees ntau dhau ntawm cov hlwb siv tau pom hauv MTT qhov kev ntsuam xyuas tuaj yeem npog cov cytotoxicity ntawm qee cov tebchaw. Yog li ntawd, thaum lub sijhawm tshuaj ntsuam xyuas cov txheej txheem, peb pom zoo kom siv ntau txoj hauv kev los txiav txim siab cov txiaj ntsig ntawm cov ntsiab lus tshwj xeeb ntawm kev muaj peev xwm ntawm tes (xws li CCK-8 thiab 3 H-TdR kev soj ntsuam) kom tsis txhob tsim cov txiaj ntsig tsis raug.

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