Kev Txhim Kho Ntawm Cov Tshuaj Tiv Thaiv-Angiogenic Los Ntawm Delphinidin Thaum Encapsulated Hauv Me Me Extracellular Vesicles Part 1

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

Abstract:(1) Tom qab: Cov anthocyanin delphinidin nthuav tawm cov khoom tiv thaiv angiogenic ob leeg hauv vitro thiab hauv vivo angiogenesis qauv. Txawm li cas los xij, hauv vivo delphinidin yog absorbed tsis zoo, yog li nws cov bioavailability thiab stability txo nws cov teebmeem los tiv thaiv angiogenic. Txoj haujlwm tam sim no siv qhov zoo ntawm cov khoom me me (EV) los txhim kho kev ruaj ntseg thiab kev ua tau zoo ntawm delphinidin. Thaum encapsulated hauv sEVs, delphinidin inhibits cov theem sib txawv ntawm angiogenesis ntawm tib neeg aortic endothelial hlwb (HAoECs). Ib txoj hauv kev raws li UHPLC-HRMS tau siv los ntsuas delphinidin metabolites hauv sEVs. Kev soj ntsuam kev loj hlob, nitric oxide (NO) ntau lawm, thiab Matrigel assay tau soj ntsuam hauv HAoECs.(3) Cov txiaj ntsig: Delphinidine, 3-O- -rutinoside, thiab Peonidin-3-galactoside tau pom ob qho tib si hauv delphinidin thiab delphinidin-loaded sEVs.sEV-loaded delphinidin nce lub zog ntawm dawb delphinidin 2-fold rau endothelial proliferation,10-fold rau endothelial TSIS MUAJ ntau lawm, thiab 100-fold rau capillary zoo li tsim . Yog li, sEV-loaded delphinidin exerts cuam tshuam rau cov theem sib txawv ntawm angiogenesis. (4) Cov ntsiab lus: sEVs yuav raug suav hais tias yog ib txoj hauv kev cog lus kom xa delphinidin rau lub hom phiaj ntawm cov kab mob angiogenesis, suav nrog mob qog noj ntshav thiab cov kab mob cuam tshuam nrog cov vascularization ntau dhau.

Thov nias ntawm no kom paub ntxiv

Ntsiab lus: delphinidin; endothelial hlwb; angiogenesis; me me extracellular vesicles; mob qog noj ntshav; kab mob plawv

1. Taw qhia

Polyphenols feem ntau pom muaj nyob rau hauv cov khoom noj thiab dej haus cov nroj tsuag thiab muab saj thiab xim ntawm cov zaub mov cog. Ntxiv mus, kev tshawb fawb txog kev kis mob tau tshaj tawm txog kev txo qis hauv plawv plawv thiab mob qog noj ntshav cuam tshuam nrog kev noj zaub mov nplua nuj hauv polyphenols [1-3.

Delphinidin (2-(3,4,5-tri-hydroxyphenyl)chromenylium-3,5,7-triol) yog anthocyanin nplua nuj nyob rau hauv pigmented zaub thiab txiv hmab txiv ntoo, tshwj xeeb tshaj yog berries thiab liab grapes. Peb yav dhau los tau tshaj tawm tias delphinidin muaj tib lub chaw muag tshuaj raws li tag nrho cov extract ntawm liab cawu cub polyphenolic tebchaw los txhawb kev nce ntawm intracellular calcium concentration thiab ua kom cov tyrosine kinases [3], ua rau endothelial nitric oxide (NO) ntau lawm tom qab estrogen receptor alpha. (Ero) kev txhawb zog [4]. Tsis tas li ntawd, peb tau tshaj tawm tias delphinidin ntawm ERo ua raws li kev tiv thaiv kab mob thiab tiv thaiv kab mob uas tuaj yeem hloov pauv T lymphocyte proliferation thiab sib txawv hauv cov neeg mob uas muaj kev pheej hmoo ntawm cov hlab plawv [5].

Thaum kawg, peb tau pom tias delphinidin qhia cov khoom siv los tiv thaiv angiogenic, ob qho tib si hauv vitro thiab hauv vivo angiogenesis qauv, thiab txo qis hauv vivo qog loj hlob ntawm melanoma [6-10]. Tseeb tiag, delphinidin inhibits endothelial cell proliferation los ntawm kev koom tes ntawm cyclin D1- thiab A-dependent txoj kev [6,7]. Peb kuj tau tshaj tawm txog kev sib koom ua ke ntawm inhibition ntawm VEGF-induced mitochondrial biogenesis los ntawm Akt txoj kev los ntawm delphinidin thiab nws cov nyhuv anti-angiogenic [8]. Ntxiv mus, delphinidin txo cov qog loj hlob ntawm melanoma qog hlwb hauv vivo los ntawm kev ua tshwj xeeb ntawm endothelial cell proliferation. Cov txheej txheem qhia txog kev sib koom ua ke ntawm inhibition ntawm VEGF-induced proliferation ntawm VEGFR2 signaling, MAPK, PI3K, thiab ntawm qib transcription ntawm CREB / ATF1 yam, thiab inhibition ntawm phopsphodiesterase2 [9]. Qhov zoo tshaj plaws, cov koob tshuaj ntau ntawm delphinidin txo qis neovascularization nyob rau hauv vivo qauv ntawm angiogenesis tshwm sim los ntawm ischemia siv tus qauv nas ntawm femoral artery ligature [10]. Ua ke, cov ntaub ntawv no qhia tau hais tias delphinidin yog ib qho kev cog lus los tiv thaiv cov kab mob cuam tshuam nrog kev mob plawv thiab qog nqaij hlav.

Txawm li cas los xij, delphinidin tsis muaj zog los txhawb cov txiaj ntsig zoo piv rau tag nrho cov cawv liab liab polyphenol rho tawm, tshwj xeeb tshaj yog nyob rau hauv inducing endothelium-dependent TSIS-mediated vasodilatation [11]. Tseeb, delphinidin yog lub teeb-rhiab thiab ruaj khov ntawm pH<3; therefore,="" it="" degrades="" rapidly="" under="" physiological="" conditions.="" moreover,="" delphinidin="" is="" poorly="" absorbed,="" and="" thus="" its="" modest="" bioavailability="" and="" stability="" reduce="" its="" effects="" both="" in="" vitro="" and="" in="" vivo.="" the="" measurement="" of="" delphinidin="" and="" its="" conjugated="" metabolites="" in="" plasma="" indicates="" its="" low="" bioavailability="" [12].="" hence,="" it="" is="" important="" to="" find="" new="" strategies="" to="" enhance="" delphinidin="" bioavailability="" and="">

Ib lub tswv yim los daws cov teeb meem no yog kev siv cov hlwv extracellular (EVs) raws li kev xa tshuaj. Peb nyuam qhuav pom tias EVs, suav nrog EVs loj thiab me (sEVs), yog nanostructures los ntawm cov khoom sib txawv subcellular compartment, kov yeej cov kev txwv ntawm classical nano-formulations. sEVs txo qis instability thiab immunogenicity, txhim kho bioavailability thiab lub hom phiaj xaiv [13]. Qee cov ntawv tshaj tawm tau qis dua qhov kev tiv thaiv ntawm EVs tso tawm los ntawm cov hlwb kho nrog polyphenols. Tseeb, sEVs enriched nrog miR-21 los ntawm cov hlwb kho nrog curcumin txo cov qog cell loj hlob thiab angiogenesis, kho endothelial permeability, thiab txo cov cell viability ntawm cov kab mob qog noj ntshav [14]. Tsis tas li ntawd, miR-16-enriched sEVs los ntawm cov hlwb kho nrog epigallocatechin gallate suppressed qog loj hlob [15].

Cistanche tuaj yeem txhim kho kev tiv thaiv

Hauv kev tshawb fawb tam sim no, peb tau siv qhov zoo ntawm sEV cov khoom los txhim kho kev ruaj ntseg thiab kev ua tau zoo ntawm delphinidin.sEV-loaded delphinidin induced angiogenesis inhibition siv tib neeg aortic endothelial hlwb (HAoECs). Delphinidin cov ntsiab lus nyob rau hauv cov nqe lus ntawm metabolites nyob rau hauv cov EVs kuj tau txiav txim.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1.Cell Culture

HAoECs (Promocell, Heidelberg, Lub teb chaws Yelemees) tau coj mus kuaj ntawm 37 degree thiab 5 feem pua ​​CO2 hauv endothelial cell loj hlob nruab nrab MV2 (Promocell) ntxiv nrog 1 feem pua ​​penicillin/streptomycin (Sigma-Aldrich, St. Quentin Fallavier, Fabkis). Cells tau trypsinized ntawm 70/80 feem pua ​​confluence thiab tau siv ntawm nqe 3 thiab 6 rau txhua qhov kev sim.

JAWS II dendritic cell kab tau yuav los ntawm American Type Culture Collection (CRL-1194; ATCC; Manassas, VA, USA). JAWS II hlwb tau loj hlob ntawm 37 degree thiab 5 feem pua ​​CO, nyob rau hauv ib tug ua tiav kab lis kev cai nruab nrab ntawm alpha yam tseem ceeb nruab nrab (Lonza; Basel, Switzerland) uas muaj ribonucleosides thiab deoxyribonucleosides thiab ntxiv nrog 20 feem pua ​​​​fetal bovine serum (FBS) (Gibco, Life Technologies; Grand Island, NY, USA), 4 mM L-glutamine (Lonza), 1 mM sodium pyruvate (Lonza), 1 feem pua ​​penicillin/-streptomycin (penicillin/-streptomycin, Sigma-Aldrich) thiab 5ng/mL murine GM-CSF (Miltenyi Biotec; San Diego, CA, USA). Cells tau trypsinized ntawm 70/80 feem pua ​​confluence thiab tau siv ntawm nqe 8 thiab 16 rau txhua qhov kev sim.

2.2.sEVIsolation

JAWS II hlwb tau cog rau ntawm qhov ceev ntawm 5 × 10 degree hlwb hauv T175 cell kab lis kev cai ntim hauv qhov nruab nrab loj hlob tiav, thiab lawv tau tshaib plab hauv FBS ua ntej kev sib cais. Cell nruab nrab yog centrifuged ntawm 300 × g thiab 2000 ×g rau 10 min kom tshem tawm cov hlwb thiab cov khib nyiab ntawm tes, raws li. Qhov tshwm sim supernatant tau centrifuged ntawm 20, 000 × g rau 30 min kom tsis suav nrog EVs loj. Lub supernatant tau centrifuged ntawm 200, 000 ×g (Optima MAX-XP ultracentrifuge thiab MLA-50 rotor, Beckman Coulter, Villepinte, Fabkis) rau 2h rau pelletize sEVs. Tom qab ntawd, sEVs tau ntxuav hauv phosphate-buffered saline (PBS) (NaCl 137 mM, KCl 2.7 mM, Na2HPO410 mM, KH2PO41.8 mM, pH=7.4) thiab tsis ntev los no ntawm 200,000 × g rau 2 h. Thaum kawg, sEVpellets raug tshem tawm hauv 1 mL ntawm PBS thiab khaws cia ntawm 4 degree kom txog thaum siv tom qab. Tus nqi ntawm sEVs tau txiav txim siab siv txoj hauv kev Lowry, nrog bovine serum albumin (Sigma-Aldrich) raws li tus qauv.sEVs tau siv ntawm 10 ug / mL.

2.3. Delphinidin Loading

Delphinidin tau npaj rau hauv dej ntawm pH{{0}} nrog {{10}}.1 feem pua ​​DMSO thiaj li ncav cuag qhov concentration ntawm 10 ug/mL. Cov sEVs tau ntxiv (2 mg), thiab cov tshuaj tau nplawm thiab ces vortexed rau 10 min. Tom qab 2 h ntawm ultracentrifugation ntawm 200, 000 × g, cov pellet tau muab rov ua dua hauv 1 mL ntawm 0.1 feem pua ​​​​DMSO lossis PBS. Delphinidin absorbance tau ntsuas ntawm 530 nm, thiab tus qauv nkhaus nrog ntau qhov sib txawv (0.1 txog 10 ug / mL) ntawm dawb delphinidin tau ua. Qhov feem pua ​​​​ntawm qhov ua tau zoo ntawm kev thauj khoom ntawm sEVs yog 9 feem pua, ntawm nws tus kheej ntawm cov concentration ntawm delphinidin siv (cov ntaub ntawv tsis qhia). Yog li, tus nqi ntawm delphinidin tau hloov kho kom tau txais qhov xav tau concentration (0.1 txog 5 ug / mL) hauv 10 ug / mL sEVs. Txhawm rau tshem tawm delphinidin dawb, cov hlwv no tau ntxuav ob zaug.

2.4. Nanoparticle Tracking Analysis (NTA)

sEV cov qauv tau diluted hauv sterile NaCl0.9 feem pua ​​, thiab qhov loj me faib tau txheeb xyuas siv NanoSight NS300 (Malvern Instruments Ltd., Malvern, UK). Cov yeeb yaj kiab raug kaw. NTA software txiav txim qhov loj me me uas siv qhov sib npaug Stokes-Enstein.

2.5. Transmission Electronic Microscopy

sEVs yog thawj zaug kho thaum hmo ntuj ntawm 4 degree nrog 2.5 feem pua ​​glutaraldehyde (LFG Distribution, Lyon, Fabkis) hauv 0.1 M PBS. Tom qab ntawd, sEVs raug ntxuav ob zaug hauv PBS los ntawm 100, 000 × g centrifugation rau 70 min.sEVs tau muab tso rau ntawm daim phiaj tooj liab rau 2 min thiab tsis zoo stained nrog 20 μL ntawm uranyl acetate 5 feem pua ​​(diluted hauv ethanol 50 feem pua. ) rau 30s. Grids tau soj ntsuam nrog Jeol JEM 1400 microscope (Jeol, Croissy sur Seine, Fabkis) ua haujlwm ntawm 120 keV.

2.6. Kev txiav txim siab ntawm Delphinidin Metabolite hauv sEVs

Kev npaj ua piv txwv yog raws li hauv qab no∶250 μL methanol (MeOH) tau ntxiv rau 10ug sEVs reconstituted hauv PBS, thiab kuaj tau raug rau 20 min ultrasonication. Ob puas uL ntawm MeOH tau ntxiv, thiab cov qauv tau centrifuged (10, 000 × g, 10 min, 4 degree ) thiab evaporated hauv miVac duo concentrator (Genevac Ltd., Ipswich, UK). Cov extract qhuav tau rov tsim dua nrog 200 μL LC-MS qib dej uas muaj 1 feem pua ​​​​ formic acid. Qhov sib tov tau raug rau qhov thib ob centrifugation (10,{11}} × g, 5 min, 4 degree) ua ntej ultra-high-performance kua chromatography txuas rau high-resolution mass spectrometry (UHPLC-HRMS) tsom xam txhawm rau txheeb xyuas delphinidin metabolites nrog kev ntsuas qhov tseeb.

Kev sib cais chromatographic tau ua tiav nrog Kinetex@ 1.7 μm XB C18,150 × 2.1 mm kem ua ke nrog cov kab ke SecurityGard C18 (Phenomenex). Cov theem txawb tau muaj HO hauv channel A thiab acetonitrile hauv channel B, ob qho tib si muaj 0.1 feem pua ​​formic acid. Qhov elution gradient (A∶B, o/ø) tau raws li hauv qab no∶ tuav cov xwm txheej pib 95:5 rau 2 min, ua raws li cov kab ncaj nraim ntawm 95:5 txog 0:100 dhau ib lub sij hawm 6 min, tuav ntawm 0: 100 rau 3 min, rov qab mus rau thawj zaug 95: 5 thiab tuav cov xwm txheej no rau 3.5 min. Tus nqi ntws tas li ntawm 0.300 mL / min tau siv; Qhov ntim ntawm txhaj tshuaj yog 10 μL.

Tag nrho scan thiab tsom SIM loj spectra tau txais nyob rau hauv qhov zoo ionization hom, siv kev daws teeb meem 70, 000 Tag Nrho Dav ntawm Ib Nrab Ntau Tshaj (FWHM) nrog kev tswj tsis siv neeg nce (AGC) lub hom phiaj ntawm 3 × 10 degree ions thiab qhov siab tshaj plaws ion txhaj lub sij hawm (IT) ntawm 200 ms. Cov ntaub ntawv-dependent MS / MS thwmsim tau txais hauv 'Top5's cov ntaub ntawv-nyob ntawm hom.

Cov metabolites qhia hauv cov ntaub ntawv [16-18] tau saib xyuas: Delphinidin, alde-hyde, phloroglucinol aldehyde, gallic acid, chalcone, petunidin-3-galactoside, petunidin-3-arabinoside, petunidin {{4} }O-rutinoside, delphinidin-3-arabinoside, delphinidin-3-galactoside, delphinidin 3-O-(6-coumaroylglucoside), delphinidin 3-O- - rutinoside, cyanidin-3-galactoside, cyanidin 3-O- -rutinoside, Peonidin-3-galactoside thiab malvidin-3-galactoside.

Kev ntsuas ntsuas txhua hnub tau ua los ntawm kev tso dej ntawm Pierce LTO Velos ESI zoo / tsis zoo calibration cov khoom siv raws li kev pom zoo los ntawm cov chaw tsim khoom. Xcalibur 2.2 soft-ware (Thermo Fisher Scientific, San Jose, CA, USA) tau siv rau kev nrhiav tau cov ntaub ntawv, thiab TraceFinder 3.0 software (Thermo Fisher Scientific) tau ua haujlwm rau kev ua cov ntaub ntawv.

2.7. Cell Viability Assay

1 × 104 HAoECs tau muab noob rau hauv 96- phaj zoo thiab kab lis kev cai rau 24 teev thiab kho nrog delphinidin (1 txog 10 ug / mL). Tom qab ntawd, 5 ug/mL ntawm 3-(4,5-dime-thylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-({ {15}}sulfophenyl)-2H- tetrazolium (MTS reagent, Promega, WI, USA) tau ntxiv rau hauv txhua qhov dej thiab incubated ntawm 37 degree rau 120 min. Lub absorbance tau ntsuas ntawm CLARIOstar (BMG LABTECH, Ortenberg, Lub teb chaws Yelemees) spectrophotometer ntawm 490 nm.

2.8. Kev ntsuam xyuas kev loj hlob

Kev soj ntsuam kev loj hlob tau ua tiav siv CyQUANT Cell proliferation Assay cov khoom (Invitrogen, Carlsbad, CA, USA) raws li cov chaw tsim khoom cov lus pom zoo. Luv luv, 1.5 × 10 * hlwb tau cog rau hauv 96- lub phaj zoo. Cells tau tshaib plab rau 2 teev thiab tom qab ntawd kho nrog delphinidin, haiv neeg sEVs lossis sEVs thauj nrog delphinidin ntawm ntau qhov sib txawv. Tom qab 24 teev ntawm incubation, hlwb raug ntxuav nrog PBS, thiab ib tug dye-binding tov yog ntxiv. Cells tau incubated ntawm 37 degree rau 30 min. Tus nyeem ntawv fluorescent microplate (CLARIOstar9, BMG LABTECH, Ortenberg, Lub teb chaws Yelemees) nrog cov ntxaij lim dej rau 485 nm excitation thiab 530 nm emission tau siv rau kev ntsuas fluorescence.

2.9.NO Production Assaty

HAoECs tau cog rau ntawm 8-zoo swb (Ibidi, Gräfelfing, Lub teb chaws Yelemees) ntawm tus nqi ntawm 3 × 104 hlwb rau ib qhov dej (ie, 3 × 104 hlwb / cm²) hauv 300 μL ntawm qhov nruab nrab. Ntawm 70-80 feem pua ​​​​ntawm qhov sib txuam, cov hlwb raug txhawb rau 24 teev nrog delphinidin, haiv neeg sEVs lossis sEV-loaded delphinidin. Adenosine triphosphate (ATP) tau siv los ua kev tswj xyuas zoo (10 μM, Sigma-Aldrich) los txhawb kev tsim NO. Tom qab 24 teev, qhov nruab nrab ntawm txhua lub qhov dej raug tshem tawm, thiab diaminofluorescein diacetate (DAF-2 DA) sojntsuam ntxiv (5 μM rau 30 min, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Tom qab ntawd, cov qhov dej tau ntxuav nrog PBS. Cells tau kho nrog paraformaldehyde (4 feem pua ​​, 20 min). Fluorescence tau nyeem los ntawm confocal microscopy (Zeiss, Jena, Lub teb chaws Yelemees, LSM700). Plaub daim duab tau txais, thiab ImageJ software tau siv los ua kom muaj nuj nqis.

2.10.Matrigel Assay

HAoECs tau cog rau hauv qhov dej coated nrog Matrigel (gel ntawm extracellular matrix ntawm murine sarcoma ntawm Engelbreth-Holm-Swarm, Sigma-Aldrich). xaub Angiogenesis phaj (lbidi) thiab tom qab ntawd incubated rau 45 min ntawm 37 degree los tsim ib gel. HAoECs tau noob thiab incubated ntawm 37 degree thiab 5 feem pua ​​CO, rau 45 min ua ntej kev kho mob nrog delphinidin, haiv neeg sEVs lossis sEV-loaded delphinidin , tom qab ntawd los ntawm kev incubation ntawm 12 mus rau 14h ntawm 37 degree thiab 5 feem pua ​​CO. Cov tsim ntawm "capillary zoo li" tau pom nrog ib tug optical microscope (Olympus CK40). Duab Software.

2.11.Kev txheeb xyuas qhov tseeb

Cov txiaj ntsig tau qhia tias txhais tau tias ± SEM. Qhov tseem ceeb ntawm qhov sib txawv ntawm cov pab pawg tau txiav txim siab los ntawm kev txheeb xyuas qhov sib txawv (ANOVA), tom qab ntawd los ntawm Tukey ntau qhov kev sib piv-isons test. p-tus nqi<0.05 were="" considered="">

3. Cov txiaj ntsig

3.1.sEV Tus cwj pwm thiab kev thauj khoom ntawm Delphinidin

Nyob rau hauv kev pom zoo nrog cov ntaub ntawv, delphinidin loaded nyob rau hauv sEVs tsis induce hloov qhov loj ntawm lub vesicles, yog 118.7 ± 2.9 thiab 111.7 ± 1.8nm rau khoob sEVs thiab delphinidin-loaded sEVs, raws li tau txiav txim los ntawm Nanoparticle Tracking Analysis (Daim duab. 1A) thiab lees paub los ntawm electron microscopy tsom xam (Daim duab 1B). Tsis tas li ntawd, ob hom sEVs, haiv neeg thiab cov neeg thauj khoom nrog delphinidin, qhia cov cim exosomal xws li ALIX, CD63, thiab TSG101 ntawm qib zoo sib xws (Daim duab 1C), thaum lawv tsis qhia ß-actin, cov cim ntawm EVs loj.

Daim duab 1. Characterization of sEVs.(A) Size distributions of native sEVs and sEVs loaded with delphinidin based on NTA measurements. (B) Tus Neeg Sawv Cev Sib Txuas Electron Microscopy duab ntawm haiv neeg sEVs thiab sEVs thauj khoom nrog delphinidin. Scale bar=100 nm.(C) Western Blot tsom xam qhia kev qhia ntawm Alix, CD63, TSG101, thiab ß-Actin hauv sEVs thiab sEVs loaded nrog delphinidin.

Kab lus no yog muab rho tawm los ntawm Nutrients 2021, 13, 4378. https://doi.org/10.3390/nu13124378