Cistanche: Echinacoside-induced Nitric Oxide Production hauv Endothelial Cells: Lub luag haujlwm ntawm Androgen Receptor Thiab Txoj Kev PI3K-Akt
Mar 05, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
LI GU, DANHONG LIAN, YIMEI ZHENG, WEI ZHOU, JINLEI GU thiab XIN LIU
Taw qhia
CistancheHoffmg. Et Link yog ib tsob nroj uas muaj kab mob perennial ntawm cov genus ntawm tsev neeg Orobanchaceae. HerbaCistanche, lub qiaCistanche deserticolaY.c. MA thiabCistanche tubulosa(Schenk) R. Nyhav, tau siv los ua tus neeg sawv cev tonic los kho lub raum tsis muaj zog, impotence, morbid leukorrhea thiab senile cem quav (1), uas tej zaum yuav yog vim nws cov androgen-zoo li los yog kev sib deev kev tswj xyuas cov nyhuv (2).Echinacoside(EcH; c35H46O20;
molecular hnyav, 786.73; Daim duab 1), yog ib qho ntawm cov phenylethanoid glycosides (PhGs) cais tawm ntawm cov stems ntawm HerbaCistanche, thiab nthuav tawm ntau yam khoom siv lom neeg, suav nrog cov tshuaj tiv thaiv oxidant, tiv thaiv kev laus, tiv thaiv kab mob, hepatoprotective thiab neuroprotective teebmeem (3). Kev tshawb nrhiav tshuaj niaj hnub niaj hnub tau pom tias muaj ntau yam ntawm HerbaCistanche, suav nrog EcH, acteoside, kankanose, kankanoside F thiab cistanoside F, nthuav tawm cov haujlwm vasorelaxant (4).
Lub endothelium yog ib qho tseem ceeb regulator ntawm vasculature, thiab nitric oxide (NO) yog ib qho tseem ceeb so kom txaus tso tawm los ntawm endothelial hlwb. Los ntawm diffusing mus rau hauv cov nqaij mos hlwb, TSIS MUAJ activates guanylate cyclase, tsub kom cov theem ntawm cyclic guanosine monophosphate (cGMP), thiab ces activates cGMP-dependent protein kinases (PKGs) los txhawb cov leeg du (5). Nws tau pom yav dhau los tias EcH ntawm 350-400 µM ncaj qha ua rau cov leeg nqaij leeg thiab inhibits hypoxia-induced proliferation hauv nas pulmonary artery smooth nqaij hlwb (6), whereas 30-300 µM EcH ua rau mob vasorelaxation hauv endothelium- rings nyob rau hauv ib tug concentration-dependent yam, thiab txhim kho cGMP ntau lawm nyob rau hauv lub corpus cavernosum smooth nqaij ntawm aortic rings cog lus los ntawm phenylephrine (7). Los ntawm kev qhib NO-cGMP-PKG-BKca channels hauv cov leeg nqaij du, 100 lossis 300 µM EcH suppressed noradrenaline-induced contraction nyob rau hauv cov hlab ntsha pulmonary ntawm nas, tshwj xeeb tshaj yog nyob rau hauv endothelium-denuded rings (8). Cov hlwb endothelial yog cov tswj hwm tseem ceeb ntawm cov hlab plawv thiab, nyob rau hauv ntau qhov xwm txheej, nws tau pom tias qhov kev so ntawm endothelial-dependent yog vim muaj cov khoom hloov pauv, xws li NO, tso tawm los ntawm endothelium (9). Txawm li cas los xij, lub tshuab hluav taws xob sab sauv ntawm EcH-induced TSIS tsim nyob rau hauv vascular endothelial hlwb yuav tsum tau tshawb xyuas ntxiv.
Hauv vascular endothelium, TSIS MUAJ tiam neeg feem ntau yog kho los ntawm endothelial NO synthase (eNOS). Ntau pab pawg tau pom tias txoj kev phosphatidylinositol 3-kinase (PI3K) txoj kev ua rau cov serine threonine protein kinase B (Akt), uas ua rau ncaj qha eNOS phosphorylation ntawm serine 1177 (Ser1177) (10). Lub androgen receptor (AR) yog ib tug tswv cuab ntawm nuclear receptor subfamily s3, uas canonically hloov cov noob qhia. AR localiza- tion rau caveolae hauv cell membrane yog koom nrog
cov kev cai uas tsis yog-genomic ntawm endothelial cell muaj nuj nqi los yog gene qhia los ntawm triggering lub c-Src/PI3K/Akt cascade, uas thaum kawg ua rau eNOS phosphorylation thiab TSIS MUAJ ntau lawm (11). Nyob rau hauv tib neeg aortic endothelial hlwb, testos-terone tau raug tshaj tawm kom qhib PI3K / Akt signaling thiab sai induce TSIS MUAJ ntau lawm vim yog kev sib cuam tshuam ncaj qha ntawm AR thiab p85 subunit ntawm PI3K ntawm cov hlab plawv (12). Nws tau tshaj tawm tias EcH ua rau cov tsos mob tsis txaus ntseeg txog cov tshuaj hormones thiab ua rau nws cov teebmeem zoo li androgen vim muaj kev sib tw khi rau AR es tsis txhob siv testosterone (2). Yog li, txoj kev tshawb fawb tam sim no tau pom tias txoj hauv kev PI3K / Akt tuaj yeem koom nrog NO ntau lawm los ntawm AR-dependent eNOS phosphorylation induced los ntawm EcH. Lub hom phiaj ntawm txoj kev tshawb fawb tam sim no yog los soj ntsuam cov teebmeem hauv qab no ntawm EcH: i) Induction ntawm TSIS MUAJ ntau lawm thiab eNOS phosphorylation; ii) kev koom tes ntawm AR hauv eNOS phosphorylation; thiab iii) ua kom txoj hauv kev PI3K/Akt nyob rau hauv tib neeg txoj hlab ntsha endothelial hlwb (HUVEcs), ib tug zoo-paub sim qauv rau kev kawm cov kev cai ntawm endothelial cell functions thiab angiogenesis (13).
Khoom siv thiab txoj kev
Tshuaj thiab reagents.EcH (purity, 92.5 feem pua) tau txais los ntawm National drug Reference Standards in National Institute for the Food and drug control. Antibodies tiv thaiv p-Akt (Ser473; cat. no. ab8805), Akt (cat. no. ab81283), p-eNOS (Ser1177; cat. no. ab184154) thiab eNOS (cat. no. ab76198) tau muas los ntawm Abcam. Cov inhibitors ntawm nilutamide thiab IcI 182780 tau txais los ntawm Sigma-Aldrich; Merck KGA. L-NAME tau yuav los ntawm Adamas-Beta, Ltd., thiab wortmannin tau yuav los ntawm Pribolab.
Cell kab lis kev cai anddrug kev kho mob.HUVEcs tau txais los ntawm Sciencell Research Laboratories Inc. thiab kab lis kev cai hauv endothelial cell nruab nrab (EcM; Sciencell Research Laboratories) nrog 5 feem pua (v/v) fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.) thiab 1 feem pua ntawm cov kab mob endothelial. Kev loj hlob ntxiv (ECGS) ntawm 37˚C (5 feem pua CO2 thiab 95 feem pua cov av) (14). Thaum ncav cuag qhov sib txuam, cov hlwb tau zom nrog trypsin thiab plated hauv EcM nrog 1% FBS thiab 1% EcGS. Rau txhua qhov kev sim, HUVEcs tau plated ntawm qhov concentration ntawm 1x104 / ml thiab loj hlob kom txog thaum ncav cuag qhov sib txuam. Ua ntej kev kho mob nrog EcH los yog lwm yam stimulators, lub hlwb tau incubated nyob rau hauv phenol liab-dawb EcM tsis muaj FBS thiab EcGS rau 6 h rau induce
kev loj hlob raug ntes. Hauv kev sim inhibitory, HUVEcs tau ua ntej incubated nrog ntau yam antagonists lossis inhibitors, suav nrog 10 µM nilutamide, 10 µM IcI 182780, 0.5 mM L-NAME lossis 5 µM wortmannin, rau 30 feeb, nrog lossis tsis muaj ECH, ntawm 37˚C. Hauv txhua pab pawg, suav nrog kev tswj hwm, dMSO tau siv los ua cov kuab tshuaj ntawm qhov sib npaug ntawm 0.001 feem pua.
Kev ntsuas ntawm intracellular NOproduction.
Cov txheeb ze hloov pauv hauv cytosolic NO concentration hauv HUVEcs tau saib xyuas siv fluorescent TSIS sojntsuam DAF-FM (Cayman Chemical Company), raws li tau tshaj tawm yav dhau los (12). Luv luv, cov hlwb tau ntim nrog 5 µM DAF-FM diacetate rau 20 min ntawm 37˚C hauv lub phaj dub microtiter thiab yaug ob peb zaug nrog PBS (pH 7.4). Lub fluorescence tau txiav txim siab ntawm excitation thiab emission wavelengths ntawm 495 thiab 515 nm, ntsig txog, siv lub fluorescent microplate nyeem ntawv (Biotek Synergy H4; BioTek Instruments, Inc.) thiab compact inverted microscope (Nikon eclipse Ts2R; Nikon corporation).
Western blot tsom xam.Raws li yav dhau los tau tshaj tawm (15), cov monolayers ntawm cov hlwb raug ntxuav ob zaug hauv cov dej khov-txias PBS thiab lysed nrog RIPA tsis (P0013d; Beyotime Institute of Biotechnology). Cov protein concentration hauv supernatant tau ntsuas los ntawm kev ntsuas bicinchoninic acid (16). Tom qab ntawd, 30 µg protein raug thauj khoom ib txoj kab, sib cais siv 10 feem pua polyacrylamide gels thiab xa mus rau polyvi- nylidene difluoride daim nyias nyias. Cov daim nyias nyias tau thaiv nrog 5% skimmed mis nyuj rau 1 teev ntawm 25˚C. Tom qab incubation nrog monoclonal antibodies tiv thaiv Akt (1: 1, 000 dilution), p-Akt (1: 500 dilution), eNOS (1: 2, 000 dilution), los yog p-eNOS (1 :1,000 dilution) ntawm 4˚C thaum hmo ntuj, cov ntaub so ntswg tau ntxuav nrog TBST (muaj 0.1 feem pua Tween-20) 4 zaug rau ~ 15 feeb ib zaug ntxuav ntawm 25˚C. Tom qab ntawd, cov membranes tau incubated nrog horseradish peroxidase-conjugated thib ob antibodies (1: 5, 000 dilution; anti-nas antibody, cat. no. A0216, los yog anti-luv antibody, cat. No. A0208, Beyotime Institute) ntawm biotechnology) rau 1 h ntawm 25˚C, thiab kuaj pom siv cov khoom siv kho kom zoo dua qub (cat. no. 32209, Thermo Fisher Scientific, Inc.). Band intensities raug suav nrog siv Image J gel tsom xam software, version 1.8.0_112 (National Institutes of Health).
Me me cuam tshuam (si) RNA kev npaj thiab kev kis kab mob.Qhov ntev ob-stranded RNAs tau tsim los ntawm lub hom phiaj mRNA ntawm AR thiab estrogen receptor (ER) nrog cov kab ke qhia hauv Table I. Cov xwm txheej rau siRNA knockdown koom nrog kev hloov HUVECs ntawm 70 feem pua confluence khaws cia hauv cov tshuaj uas tsis yog tshuaj tua kab mob. kab lis kev cai nruab nrab hauv 60 hli collagen-coated kab lis kev cai tais diav. Kev hloov pauv ntawm 5 nM AR-siRNA lossis 10 nM ER -siRNA nrog Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) tau ua tiav cais, raws li cov chaw tsim khoom cov txheej txheem. Transfection effi-ciency tau soj ntsuam los ntawm qhov rov qab hloov pauv ntau-PcR tsom xam (RT-qPcR), raws li tau tshaj tawm yav dhau los (17). Cov txheej txheem thermo-cycle yog raws li nram no: 10 min ntawm 95˚C; 40 cycles ntawm 95˚C rau 5 sec thiab 60˚C rau 1 min, thiab melting nkhaus ntawm 95˚C rau 15 sec, 60˚C rau 1 min thiab 95˚C rau 15 sec; Peb qhov kev ywj pheej biological replicates tau ua rau txhua tus qauv. Cov kev sim tom ntej tau ua 48 teev tom qab hloov pauv. Primer khub tau tsim los siv Primer Premier v5.0 software (PREMIER Biosoft) nrog cov kab hauv qab no: AR pem hauv ntej, 5'-GGTTAcAccAAAGGGcTAGAA-3' thiab thim rov qab, 5'-GAcTTGTAGAGAGAcAGGGTAGA-3'; ER pem hauv ntej, 5'-ccAGTAccA ATGAcA AGG GAAG -3' thiab rov qab, 5'-TcAcAGGAccAGAcTccATAA-3'; thiab GAPdH rau pem hauv ntej, 5'-cAGGGcTGcTTTTAAcTcTGGTAA-3' thiab rov qab, 5'-GGGTGGAATcATATTGGAAcATGT-3'.
Kev txheeb cais tsom xam.Tag nrho cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab ± tus qauv sib txawv. Kev sib piv ntawm cov pab pawg tau ua tiav siv Kruskal-Wallis xeem lossis ob txoj kev ANOVA nrog Tukey's post hoc test rau ntau qhov sib piv. P<0.05 was="" considered="" to="" indicate="" a="" statistically="" significant="">
Cov txiaj ntsig
ECH induces TSIS MUAJ ntau lawm thiab eNOSphosphorylation.Raws li pom nyob rau hauv daim duab 2A thiab B, 1 µM ECH ho nce intracellular TSIS MUAJ ntau lawm nyob rau hauv HUVEcs piv nrog cov kev tswj tsis zoo hlwb, thaum lub stimulatory nyhuv ntawm EcH tau attenuated mus rau theem tswj los ntawm pretreatment nrog L-NAME. Txhawm rau kom tau txais qhov zoo tshaj plaws concentration, eNOS phosphorylation tau sim ntawm 60 min tom qab kev kho mob nrog EcH ntawm cov concentrations
ntawm {{0}}, 0.01, 0.1, 1 thiab 10 µM. Cov txiaj ntsig tau qhia tias EcH ntawm qhov siab ntawm 0.01-10 µM tuaj yeem ua rau eNOS phosphorylation ntawm Ser 1177 nyob rau hauv ib qho concentration-dependent yam, nrog rau qhov siab tshaj eNOS phosphorylation pom ntawm 10 µM EcH induction (Fig. 2c thiab d). Tsis tas li ntawd, eNOS phosphorylation ntawm Ser1177 tau tshuaj xyuas los ntawm kev txheeb xyuas sab hnub poob ntawm 0, 5, 15, 30, 60 thiab 120 min tom qab incubation nrog 1 µM EcH. Nws tau pom tias eNOS phosphorylation tau nrawm nrawm los ntawm EcH ntawm 5 min, thiab txuas ntxiv mus txog 30 min ntawm incubation (Daim duab 2E thiab F). Tom qab ntawd, txawm tias EcH kho, qhov sib npaug ntawm eNOS phos-phorylation tseem nyob ruaj khov txij li 30 feeb mus rau; Yog li, EcH tsis cuam tshuam tag nrho eNOS kev qhia (Fig. 2c thiab E).
AR kev sib kho ECH-induced TSIS MUAJ ntau lawm thiab eNOScuam tshuam cov teebmeem ntawm AR-siRNA-1 thiab ER -siRNA-2 yog qhov ua tau zoo tshaj plaws hauv kev cuam tshuam cov kev qhia ntawm AR thiab ER hauv HUVEcs hauv txoj kev tshawb no (Fig. 3). Tom qab ntawd, tus antagonist rau AR inhibition-of-function tsom xam thiab siRNA rau AR poob-of-function tsom xam tau siv los ntsuas qhov kev koom tes ntawm AR hauv EcH-induced eNOS ua kom thiab TSIS muaj ntau lawm. Raws li pom nyob rau hauv daim duab 4A, 1 µM ECH ho nce NO ntau lawm hauv HUVEcs (P<0.05). pretreatment="" with="" the="" ar="" antagonist="" nilutamide="" (10="" µm)="" abolished="" ech-induced="" no="" production,="" whereas="" ici182789="" (an="" er="" antagonist)="" did="" not="" exert="" the="" same="" effects.="" furthermore,="" the="" effects="" of="" sirna-mediated="" ar="" knockdown="" on="" no="" production="" were="" examined="" in="" cultured="" cells,="" indicating="" that="" no="" production="" was="" diminished="" by="" transfection="" with="" ar="" sirnas;="" however,="" it="" was="" not="" affected="" by="" er="" sirnas="" or="" control="" random="" sirnas="" (fig.="" 4b).="" representative="" western="" blots="" and="" semi-quantitative="" analysis="" (fig.="" 4c="" and="" d)="" revealed="" that="" the="" phosphorylation="" of="" enos="" induced="" by="" ech="" was="" inhibited="" by="" nilutamide="" and="" ici182789,="" and="" that="" the="" inhibitory="" effect="" of="" nilutamide="" on="" ech-induced="" enos="" activation="" was="" significantly="" higher="" compared="" with="" that="" of="" ici182789,="" which="" indicated="" that="" inhibi-="" tion="" of="" ar="" function="" had="" a="" greater="" impact="" than="" er="" on="" enos="" phosphorylation="" induced="" by="" ech.="" similarly,="" the="" phosphoryla-="" tion="" of="" enos="" induced="" by="" ech="" was="" reduced="" significantly="" in="" cells="" transfected="" with="" ar-sirna="" and="" er-sirna="" compared="" with="" the="" control="" random="" sirna;="" the="" inhibitory="" effect="" of="" ar‑sirna="" on="" ech‑induced="" enos="" activation="" was="" significantly="" stronger="" compared="" with="" that="" of="" er-sirna="" (fig.="" 4e="" and="" f).="" therefore,="" the="" aforementioned="" results="" suggested="" that="" ech="" may="" cause="" ar-dependent="" activation="" of="" enos="" to="" induce="" no="" production="" in="">
ECH qhib tus PI3K/Akt txoj kev.Nyob rau hauv txoj kev tshawb no, Akt phosphorylation ntawm Ser473 tau sim 60 min tom qab EcH incubation ntawm concentrations ntawm 0, 0.01, 0.1 , 1 thiab 10 µM. Cov txiaj ntsig tau qhia tias EcH (0.01-10 µM) tuaj yeem ua rau muaj kev cuam tshuam Akt phosphorylation. Cov txheeb ze qhia theem ntawm p-Akt peaked ntawm 1 thiab 10 µM ntawm EcH kev kho mob (Fig. 5A thiab B). Tsis tas li ntawd, Akt phosphorylation tau kuaj xyuas los ntawm western blot tsom ntawm 0, 5, 15, 30, 60 thiab 120 min tom qab ntxiv 1 µM EcH rau HUVEc kab lis kev cai. Raws li qhia hauv daim duab 5c thiab d, EcH nce Akt phosphoryla-tion sai sai tom qab 5 min ntawm incubation, thiab qhov siab tshaj plaws protein ntau ntawm p-Akt tau pom ntawm 60 min. Los ntawm qhov sib txawv, EcH tsis cuam tshuam rau tag nrho Akt qhia (Fig. 5A thiab c). Ntxiv mus, txhawm rau tshawb xyuas qhov muaj peev xwm ntawm PI3K txoj hauv kev ntawm eNOS phosphorylation, HUVEcs tau kho ua ntej nrog PI3K inhibitor wortmannin ua ntej EcH daim ntawv thov. Wortmannin tau pom los txo EcH-induced TSIS MUAJ ntau lawm mus rau theem pib (Fig. 5E). Ib qho tshwm sim zoo sib xws tau pom siv fluorescence microscopy thaum kuaj EcH-induced DAF-FM fluorescence (Fig. 5F). Tsis tas li ntawd, wortmannin tuaj yeem muaj peev xwm tshem tawm sai eNOS phosphorylation hauv HUVEcs (Fig. 5G thiab H). Cov txiaj ntsig tau hais los saum toj no qhia tias EcH tuaj yeem qhib eNOS phosphorylation thiab TSIS MUAJ ntau lawm ntawm txoj kev PI3K / Akt.
Kev sib tham
EcH yog ib qho kev sib xyaw ntuj cais los ntawm HerbaCistanche, nrog ntau yam pharmacological zog. Nws tau dhau los ua pov thawj tias EcH tau ua kom muaj qhov endothelium-dependent vascular so los ntawm kev qhib NO-cGMP-PKG-BKca raws hauv cov hlab ntsha cov leeg nqaij (7,8). Cov kev tshawb pom tam sim no muab pov thawj tias ECH tau siv AR-dependent activation ntawm eNOS kom tsis muaj kev tsim khoom nrog kev koom tes ntawm PI3K / Akt signaling txoj hauv kev hauv vascular endothelial hlwb.
Raws li cov txheej txheem sab hauv ntawm phab ntsa ntawm cov hlab ntsha, endothelium tuaj yeem nkag siab sai thiab teb rau cov kev hloov pauv hauv cov ntshav ntws, ua rau cov teeb liab xa mus rau cov leeg nqaij hauv qab kom tswj tau cov hlab ntsha (18). Nws tau raug tshaj tawm tias muaj ntau yam endothelial-derived vasodila-tory tebchaw, nrog rau feem ntau prototypical tshuaj.yog NO, tsim los ntawm endothelial isoform ntawm eNOS, uas ua rau phosphorylation (19). Raws li ib txwm muaj, eNOS tseem tsis ua haujlwm thaum khi rau caveolin, thiab ua haujlwm nrog cov xwm txheej hauv qab no hauv endothelial.cells: i) eNOS dissociates los ntawm caveolin-1 thiab cov koom nrog ca2 ntxiv /caM; ii) Heat Shock Protein (HSP)90 txhawb nqa eNOSdimerization thiab nyiam ib tug steric tsim los recruit Akt; thiab iii) calcineurin dephosphorylates Thr495 los txhawb lub xeev ua kom muaj zog (20). Hauv kev tshawb fawb tam sim no, TSIS MUAJ ntau lawm tau nce ntau hauv HUVECs kho nrog 1 µM ECH. Qhov tshwm sim no zoo ib yam li cov txiaj ntsig yav dhau los, qhov twg EcH nce NO tso tawm thiab txhawb nqa cov synthesis ntawm cGMP hauv nas thoracic aortic rings (7). Tsis tas li ntawd, ob qhov chaw phosphorylation, Ser1177 thiab Thr495, zoo li tseem ceeb tshwj xeeb rau kev tswj hwm eNOS kev ua (20), thiab txoj kev tshawb fawb tam sim no tau qhia tias EcH ua rau mob eNOS phosphorylation ntawm Ser1177 nyob rau hauv ib qho concentration-dependent yam. Tsis tas li ntawd, qhov stimuli cuam tshuam nrog cov teebmeem ntawm eNOS cov protein ntau feem ntau txiav txim siab eNOS mRNA stability, thiab yog tias qhov kev txhawb nqa tau khaws cia rau lub sijhawm ntev, eNOS mRNA transcription tshwm sim ntawm mitogen-activated protein kinase thiab nuclear factor κB (20). Muab ua ke, cov kev soj ntsuam no qhia tau hais tias kev ua kom eNOS ntawm Ser1177 yog lub luag haujlwm rau TSIS MUAJ kev sib txuas los ntawm EcH.
Tsis tas li ntawd, kev tshawb fawb ntau ntxiv tau pom tias AR tau nthuav tawm hauv cov hlwb endothelial hauv ntau cov ntaub so ntswg tib neeg, uas qhia txog lub luag haujlwm tseem ceeb rau androgens thiab lawv cov analogues, uas ua los ntawm cov txheej txheem AR-mediated, hauv kev hloov kho ntawm tib neeg endothelial. cell homeostasis (21) . Hauv txoj kev tsis yog classical PI3K / Akt, AR tuaj yeem qhib PI3K los ntawm kev cuam tshuam ncaj qha nrog PI3K kev tswj hwm subunit p85 (22). Txoj kev tshawb fawb tam sim no tau pom tias tus AR antagonist lossis AR siRNA txo qis qhov TSIS TAU thiab eNOS phosphorylation induced los ntawm EcH hauv HUVEcs. Nws tau raug tshaj tawm yav dhau los tias cov tshuaj estrogens induce eNOS phosphorylation thiab txhawb NO ntau lawm ntawm classic ER activation nyob rau hauv endothelial hlwb (23), thiab kev tswj ntawm ECH ho txhim khu kev qha ER nyob rau hauv lub tsev menyuam (24). Txawm li cas los xij, hauv txoj kev tshawb fawb tam sim no, cov txiaj ntsig ntawm AR yog qhov tseem ceeb dua piv nrog ER ntawm EcH-induced eNOS activation thiab TSIS MUAJ ntau lawm. Tsis tas li ntawd, nws tau pom tias EcH ua rau mob NO ntau lawm nyob rau hauv feeb ntawm AR-kev koom tes nrog eNOS ua rau hauv HUVEcs, uas yog raws li qhov tsis yog-genomic ntawm cov lus teb hauv cov hlwb endothelial. AR yog txuam nrog scaffolding proteins, xws li HSP90, HSP70 thiab kinase Src, nyob rau hauv lub cytoplasm, thiab nws tuaj yeem raug thauj mus rau daim nyias nyias los ntawm AR complex hauv 5 min ntawm kev kho testosterone (23). Raws li 'lub hom phiaj nuv ntses' lub tswv yim, HSP90 tau txheeb xyuas raws li PhGs-coupled lub hom phiaj, uas qhia tias EcH tuaj yeem pab txhawb kev sib cais ntawm AR los ntawm scaffolding proteins (25). Lwm txoj kev tshawb fawb qhia tias, hauv hypothalamus, EcH tuaj yeem ua ke nrog AR hnab tshos ntawm cov amino acids Met-894 thiab Val-713 thiab inhibit kev thauj ntawm cytoplasmic AR mus rau lub nucleus (26). Txawm li cas los xij, lub hauv paus txheej txheem los ntawm EcH mediates cytoplasmic AR translocation mus rau daim nyias nyias yuav tsum tau tshawb xyuas ntxiv. Yog li, kev tshawb fawb ntxiv yog yuav tsum tau ua kom paub meej cov txheej txheem los ntawm EcH ua tiav AR-dependent eNOS kev ua haujlwm thiab yuav ua li cas nws yuav cuam tshuam nrog kev khi rau HSP90 hauv vascular endothelial hlwb.
Txoj kev PI3K/Akt yog ib qho tseem ceeb tshaj plaws ntawm kev taw qhia cascades, qhov ua kom muaj zog ntawm kev tsim cov phosphatidylinositol-3,4,5-trisphosphate los khi N-terminal pleckstrin homology ntawm Ser/ Thr kinase Akt. Qhov no pab txhawb Akt recruitment rau plasma membrane (27). Txoj hauv kev PI3K / Akt tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev tswj hwm ntawm NO-dependent so induced los ntawm EcH.
Txoj kev tshawb fawb tam sim no tau qhia tias EcH-induced NO ntau lawm tau txo qis thaum cov hlwb tau tsim nrog PI3K inhibitor wortmannin. Ib tsab ntawv tshaj tawm dhau los tau pom tias 15 mg / kg EcH tau qhib PI3K / Akt signaling txoj hauv kev hauv 5-fluorouracil-suppressed pob txha hlwb hlwb (28). Hauv txoj kev tshawb no, PI3K inhibitors txo qis EcH-induced eNOS phosphorylation ntawm Ser1177. Ntxiv mus, Akt kev ua ub no feem ntau yog tswj hwm los ntawm cov kev cai tswj hwm sab saud, tshwj xeeb tshaj yog PI3K-dependent phosphorylation ntawm Ser473 (20). Nyob rau hauv txoj kev tshawb no, EcH induced Akt phos-phorylation ntawm Ser473 nyob rau hauv ib tug npaum li cas-dependent yam; Ib yam li ntawd, ib qho kev tshawb fawb yav dhau los tau pom tias 5, 10 lossis 20 µM EcH tau siv cov tshuaj tiv thaiv cardioprotective tiv thaiv anoxia / reperfusion kev kho mob raws li koob tshuaj los ntawm kev muaj peev xwm upregulating p-Akt thiab SLc8A3 (29). Cov kev cai hloov pauv ntawm Akt gene tseem tsis paub ntau (30); Yog li ntawd, txoj kev tshawb fawb tam sim no tau tsom mus rau cov kev cai tom qab kev hloov pauv ntawm EcH ntawm Akt. Ua raws li cov lus qhia saum toj no rau hauv kev txiav txim siab, nws tau txiav txim siab tias daim ntawv thov EcH rau HUVEcs tuaj yeem ua rau kev ua haujlwm ntawm PI3K / Akt txoj hauv kev, uas phosphorylates eNOS thiab, tom qab ntawd, nce NO ntau lawm.
Hauv kev xaus, EcH yog ib yam khoom ntuj uas yog cais tawm los ntawm HerbaCistanche.Lub peev xwm txheej txheem hauv qab EcH-vim tsis muaj kev tsim tawm hauv cov hlwb endothelial tuaj yeem suav nrog cov hauv qab no (Fig. 6): i) EcH ua haujlwm raws li kev ua haujlwm ligand ntawm AR uas yog nyob rau hauv lub caveolae hauv cell membrane; ii) PI3K khi rau hydrophobic domain ntawm Akt ntawm Ser473 thiab pab txhawb Akt recruitment rau lub cell membrane; iii) kev nrhiav neeg ua haujlwm ntawm PI3K/Akt cascades ua rau AR-dependent eNOS phosphorylation; thiab iv) tiam ntawm NO yog kho los ntawm eNOS hauv endothelial hlwb. Kev soj ntsuam uas EcH ua rau tsis muaj kev tsim khoom los ntawm AR-dependent phosphorylation ntawm eNOS nrog kev koom tes ntawm PI3K / Akt txoj hauv kev tuaj yeem ua rau muaj kev nkag siab ntxiv txog cov teebmeem vasorelaxant ntawm EcH. Tsis tas li ntawd, EcH tsom mus rau endothelial-derived TSIS tau txoj hauv kev yuav yog vim tsis muaj kev cuam tshuam genomic. Yog li, txoj kev tshawb fawb tam sim no tuaj yeem pab qhia cov txheej txheem los ntawm EcH siv nws cov tshuaj pharmacological los tiv thaiv kab mob plawv.
Kev lees paub
Tsis siv tau.
Nyiaj txiag
Txoj kev tshawb fawb tam sim no tau txais kev txhawb nqa los ntawm National Natural Science Foundation of China (grant no. 81503333).
Muaj ntawm cov ntaub ntawv thiab cov ntaub ntawv
Cov ntaub ntawv tsim tawm thiab tshuaj xyuas thaum lub sijhawm kawm tam sim no yog muaj los ntawm tus kws sau ntawv uas tsim nyog thov.
Cov kws sau ntawv kev pab
LG thiab XL tau tsim thiab tsim cov kev sim. dL thiab YZ ua qhov kev sim. WZ thiab JG txheeb xyuas cov ntaub ntawv. LG tau tsim cov ntawv sau. XL tau tshuaj xyuas thiab kho cov ntawv sau. Txhua tus kws sau ntawv tau nyeem thiab pom zoo cov ntawv sau kawg.
1202 UGthiab al: EcHINAcoSIdE INdUcES NITRIC OXIdE tsim los ntawm AR-dEPendENT PI3K / AKT PATHWAY
Kev ncaj ncees kev pom zoo thiab kev tso cai rau koom
Tsis siv tau.
Tus neeg mob kev tso cai rau luam tawm
Tsis siv tau.
Kev sib tw kev txaus siab
Cov kws sau ntawv tshaj tawm tias lawv tsis muaj kev sib tw nyiam.
Cov ntaub ntawv
1. Suav Pharmacopoeia commission:CistancheHerba. Hauv: Pharmacopoeia ntawm Cov Neeg Sawv Cev ntawm Tuam Tshoj 1. puab tsaig. Med. Sci. Xovxwm, Beijing, pp 135, 2015.
2. Jiang Z, Wang J, Li X, Zhang X: Echinacoside thiabCistanche tubulosa(Schenk)R. wight ameliorate bisphenol A-induced testicular thiab phev puas nyob rau hauv nas los ntawm gonad axis tswj steroidogenic enzymes. J Ethnopharmacol 193: 321-328, 2016.
3. Liu J, Yang L, dong Y, Zhang B thiab Ma X: Echinacoside, ib qho khoom siv tsis zoo hauv kev kho mob paj hlwb thiab lwm yam kab mob. Molecules 23: piiE1213, 2018.
4. Yoshikawa M, Matsuda H, Morikawa T, Xie H, Nakamura S thiab Muraoka O: Phenylethanoid oligoglycosides thiab acylated oligosugars nrog vasorelaxant kev ua los ntawmCistanche tubulosa. Bioorg Med chem 14: 7468-7475, 2006.
5. Arnold WP, Mittal cK, Katsuki S thiab Murad F: Nitric oxide activates guanylate cyclase thiab nce guanosine 3': 5'-cyclic monophosphate theem nyob rau hauv ntau yam ntaub so ntswg npaj. Proc Natl Acad Sci USA 74: 3203-3207, 1977.
6. Gai XY, Tang F, Ma J, Zeng KW, Wang SL, Wang YP, Wuren TN, Lu dX, Zhou Y and Ge RL: Antiproliferative effect of echinaco- side on rat pulmonary artery smooth muscle cells under hypoxia. J Pharmacol Sci 126: 155-163, 2014.
7. He WJ, Fang TH, Ma X, Zhang K, Ma ZZ thiab Tu PF: Echinacoside elicits endothelium-dependent so in rat aortic rings via NO-cGMP pathway. Planta Med 75: 1400-1404, 2009.
8. Gai XY, Wei YH, Zhang W, Wuren TN, Wang YP, Li ZQ, Liu S, Ma L, Lu dX, Zhou Y thiab Ge RL: Echinacoside induces nas pulmonary artery vasorelaxation los ntawm kev qhib NO-cGMP-PKG- BKca channels thiab txo qis intracellular ca2 ntxiv rau qib. Acta Pharmacol Sin 36: 587-596, 2015.
9. Maruhashi T, Kihara Y thiab Higashi Y: Kev ntsuam xyuas ntawm endothe- lium-independent vasodilation: Los ntawm txoj kev mus rau kev soj ntsuam kev pom. J Hypertens 36: 1460-1467, 2018.
10. Ahmad KA, Ze H, chen J, Khan FU, chen X, Xu J thiab derivative on human umbilical vein endothelial cells tiv thaiv oxidative stress-induced raug mob: Kev koom tes ntawm antioxidation thiab PI3k/Akt/eNOS/NOS signaling pathways. Biomed Pharmacother 106: 1734-1741, 2018.
11. Yu J, Akishita M, Eto M, Koizumi H, Hashimoto R, Ogawa S, Tanaka K, Ouchi Y thiab Okabe T: Src kinase-mediates androgen receptor-dependent non-genomic activation of signaling cascade ua rau endothelial nitric oxide synthase . Biochem Bioph Res commun 424: 538-543, 2012.
12. Yu J, Akishita M, Eto M, Ogawa S, Son B, Kato S, Ouchi Y thiab Okabe T: Androgen receptor-dependent activation of endothe-lial nitric oxide synthase in vascular endothelial cells: Lub luag haujlwm ntawm phosphatidylinositol {{3} }kinase/Akt pathway.Endocrinology 151: 1822-1828, 2010.
13. Pittarella P, Squarzanti dF, Molinari c, Invernizzi M, Uberti F thiab Reno F: TSIS TAU-dependent proliferation thiab migration induced by vitamin d in HUVEc. J Steroid Biochem 149: 35-42, 2015.
14. He Y, Luan Z, Fu X thiab Xu X: Overexpression of uncoupling protein 2 inhibits high glucose-induced apoptosis of human umbilical vein endothelial cells. Int J Mol Med 37: 631-638, 2016.
15. Xiao-Hong d, chang-Qin X , Jian-Hua H , Wen-Jiang Z thiab Bing S: Icariin ncua homocysteine-induced endothelial cellular senescence uas muaj feem cuam tshuam ntawm PI3K / AKT-eNOS signaling pathway. Pharm Biol 51: 433-440, 2013.
16. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano Md, Fujimoto EK, Goeke NM, Olson BJ thiab Klenk dc: Kev ntsuas cov protein uas siv bicinchoninic acid. Anal Biochem 150: 76-85, 1985.
17. Gu L, Zhong X, Lian d, Zheng Y, Wang H thiab Liu X: Triterpenoid biosynthesis thiab cov lus teb transcriptional elicited los ntawm nitric oxide nyob rau hauv submerged fermentingGanoderma lucidum. Txheej Txheem Biochem 60: 19-26, 2017.
18. Ellinsworth dc, Sandow SL, Shukla N, Liu Y, Jeremy JY thiab Gutterman dd: Endothelium-derived hyperpolarization thiab coronary vasodilation: muaj ntau haiv neeg thiab kev koom ua ke ntawm epoxye-icosatrienoic acids, hydrogen peroxide, thiab qhov sib txuas. Microcirculation 23: 15-32, 2016.
19. Freed JK thiab Gutterman dd: kev sib txuas lus yog qhov tseem ceeb: Mechanisms of intercellular signaling in vasodilation. J cardiovasc Pharm 69: 264-272, 2017.
20. Quillon A, Fromy B thiab debret R: Endothelium microenvironment sensing ua rau nitric oxide mediated vasodilation: Kev tshuaj xyuas cov paj hlwb thiab biomechanical signals. Nitric Oxide 45: 20-26, 2015.
21. Torres-Estay V, carreno dV, Francisco IF, Sotomayor P, Godoy AS thiab Smith GJ: Androgen receptor in human endothelial cells. J Endocrinol 224: 131-137, 2015.
22. deng Q, Zhang Z, Wu Y, Yu WY, Zhang J, Jiang ZM, Zhang Y, Liang H thiab Gui YT: Non-genomic action of androgens is mediated by fast phosphorylation and regulation of androgen receptor trafficking. Cell Physiol Biochem 43: 223-236,2017.
23. de Oliveira TS, de Oliveira LM, de Oliveira LP, costa RMd, Tostes Rc, Georg Rc, costa EA, Lobato NS, Filgueira FP thiab Ghedini Pc: Ua kom PI3K/Akt txoj kev sib kho los ntawm estrogen receptors tso nyiaj rau estrone-in vascular activation ntawm cGMP signaling. Vascul Pharmacol 110: 42-48, 2018.
24. Li F, Yang X, Yang Y, Guo c, Zhang c, Yang Z thiab Li P: Antiosteoporotic kev ua ntawm echinacoside nyob rau hauv ovariectomized nas. Phytomedicine 20: 549-557, 2013.
25. Zeng KW, Liao LX, Wan YJ, Jiang Y thiab Tu PF: Pharmacological lub hom phiaj kev txheeb xyuas thiab kev ua tau zoo ntawm phenylethanoid glycosides los ntawmCistanchesHerba raws li 'lub hom phiaj nuv ntses' lub tswv yim. chin Tradit Tshuaj ntsuab 49: 173-178, 2018.
26. Jiang Z, Zhou B, Li X, Kirby GM thiab Zhang X: Echinacoside nce phev kom muaj nuj nqis hauv nas los ntawm kev tsom mus rau hypothalamic androgen receptor. Sci Rep 8: 3839-3850, 2018.
27. coffer PJ, Jin J thiab Woodgett JR: Protein kinase B (c-Akt): Ib tug multifunctional mediator ntawm phosphatidylinositol 3-kinase activation. Biochem J 335, 1-13, 1998.
28. Wang S, Zheng G, Tian S, Zhang Y, Shen L, Pak Y, Shen Y thiab Qian J: Echinacoside txhim kho hematopoietic muaj nuj nqi hauv 5-FU-induced myelosuppression nas. Lub Neej Sci 123: 86-92, 2015.
29. chen M, Wang X, Hu B, Zhou J, Wang X, Wei W thiab Zhou H: Tiv thaiv cov teebmeem ntawm echinacoside tiv thaiv anoxia / reperfusion raug mob hauv H9c2 hlwb ntawm kev tswj p-AKT thiab SLc8A3. Biomed Pharmacother 104: 52-59, 2018.
30. Abeyrathna P thiab Su Y: Lub luag haujlwm tseem ceeb ntawm Akt hauv kev ua haujlwm ntawm lub plawv. Vascul Pharmacol 74: 38-48, 2015.
