Estrogenic Activity Ntawm Glycosides Los Ntawm Cistanche Deserticola Raws li Estrogen Receptors Adjuvant hauv Vitro
Mar 05, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
Hui Song, Wen-Lan Li, Xiang-Ming Sun, Yang Hu, Jing-Xin Ding, Yu-Bin Ji, Jing-Ya Wang
Taw qhia
Cistanche deserticola("Rou Cong Rong" hauv Suav), ib hom tshuaj suav tshuaj ntsuab uas tau sau thawj zaug hauv ShenNongBenCaoJing, tau siv rau kev kho mob raum tsis muaj zog, impotence, senile cem quav, thiab ntshav tsis txaus rau ntau txhiab xyoo.[1] Nws yog feem ntau faib rau hauv thaj chaw suab puam ntawm Northwestern Tuam Tshoj, xws li Gansu thiab Xinjiang.[2] Kev tshawb fawb tshuaj niaj hnub no pom tau tias C. deserticola tuaj yeem ua haujlwm dav dav, xws li kev tswj hwm cov tshuaj hormones, immunomodulatory, antioxidative, neuroprotective, anti-inflammatory, thiab estrogenic kev ua haujlwm.[3,4] Glycosides ntawm Cistanches (GCs) raug suav hais tias yog ib qho ntawm cov tshuaj. lub ntsiab active Cheebtsam nrog ntau yam teebmeem lom.[5,6]
Cov tshuaj estrogen receptors- (ERs- ) thiab ER yog cov subtypes ntawm ERs uas tuaj yeem tswj hwm kev ua haujlwm ntawm lub cev.[7] Cov proteins no tuaj yeem tswj hwm qhov kev hloov pauv ntawm cov hom phiaj los ntawm kev khi rau DNA tswj cov kab ke hauv lub cell nucleus. Ob leeg ntawm cov subtypes tau qhia pom zoo nyob rau hauv cov hlab plawv thiab lub hauv nruab nrab lub paj hlwb.[8,9] ER muaj nyob rau hauv lub caj pas mammary, uterus, zes qe menyuam, pob txha, txiv neej lub cev xeeb tub, thiab prostate. ER yog tam sim no tsuas yog nyob rau hauv prostate, zes qe menyuam, thiab zais zis.[10,11] Ntxiv mus, muaj ib co kev physiological luag hauj lwm rau ob subtypes, xws li nyob rau hauv kev loj hlob thiab kev ua hauj lwm ntawm lub zes qe menyuam thiab kev tiv thaiv ntawm lub plawv system. [12,13] Kev tshawb fawb yav dhau los hauv peb pab pawg tau nthuav tawm cov tshuaj estrogenic-zoo li cov txheej txheem ntawm GCs los ntawm cov txheej txheem ntawm metabolomic tsom xam.[14] Hauv kev kawm tas mus li, peb tau txheeb xyuas cov txheej txheem ntawm kev ua estrogenic ntawm GCs ntawm MCF-7 cell kab ntsig txog ERs.
Cov khoom siv thiab cov txheej txheem
Cov cuab yeej
ECO-170P-230 incubator yog los ntawm New Brunswick Scientific (China) Co., Ltd., Bio-Rad 680 microplate nyeem ntawv, thiab electrophoresis apparatus yog los ntawm Bio-Rad Laboratories, Inc., CX21 microscope yog los ntawm Olympus Co., Ltd. , GIS-2019 gel imaging system yog los ntawm Tanon Science thiab Technology Co., Ltd., lub tiaj hauv qab phaj yog los ntawm Corning Inc., EPICS-XL ntws cytometry yog los ntawm Beckman-Coulter Inc., thiab StepOnePlus Real-Time polymerase saw cov tshuaj tiv thaiv (PCR) system yog los ntawm Thermo Fisher Scientific.
Tshuaj thiab reagents
GCs tau npaj rau hauv peb lub chaw kuaj mob raws li txoj kev tau tshaj tawm yav dhau los, [14] thiab qhov purity ntawm GCs tau txiav txim siab yog 52 feem pua (acteoside tau siv los ua tus cim los txiav txim siab GCs) los ntawm ultraviolet spectrophotometric. Estradiol (E2) tau yuav los ntawm Yuanye Biotechnology Co., Ltd. (Shanghai, Suav). Fetal bovine serum (FBS) thiab RPMI-1640 yog los ntawm Gibco Co., Ltd., 96-zoo ca hauv qab phaj yog los ntawm Corning Inc., thiab dimethyl sulfoxide (DMSO) thiab 3-[4,5-dimethyl-2-thiazolyl ]-2,5-diphenyltetrazolium bromide (MTT) tau yuav los ntawm Sigma-Aldrich Corporation. TRIzol reagent, Reverse Transcription (RT) Kit, thiab TB Green™ RT-PCR Kit tau yuav los ntawm TaKaRa Co., Ltd., Dalian, Suav. Anti-ER Rabbit pAb, anti-ER Rabbit pAb, thiab -actin antibodies tau yuav los ntawm Wanlei Biotechnology Co., Ltd. Tag nrho lwm cov tshuaj tau muag los ntawm cov neeg muag khoom txheem.
Kev npaj ua qauv
Kev npaj ntawm glycosides ntawm Cistanches Tshuag tov
GCs extract tau yaj hauv DMSO los ua cov tshuaj hauv Tshuag nrog cov concentration ntawm 0.1051 g / ml thiab khaws cia ntawm 4 degree . Phenol liab-dawb RPMI-1640 tau siv los dilute cov tshuaj hauv Tshuag rau ntau qhov ntau ua ntej siv.
Kev npaj ntawm estradiol Tshuag tov
E2 extract tau yaj nyob rau hauv DMSO los ua cov tshuaj hauv Tshuag nrog cov concentration ntawm 0.27 mg / ml thiab khaws cia ntawm 4 degree . Phenol liab-dawb RPMI-1640 tau siv los dilute cov tshuaj hauv Tshuag rau ntau qhov ntau ua ntej siv.
Kev npaj ntawm charcoal dextran kho-fetal bovine serum
Pua pua milliliters ntawm FBS tau tov nrog 250 mg ntawm activated charcoal hmoov thiab 25 mg dextran. Tom qab incubating rau 45 min ntawm 55 degree, qhov sib tov yog centrifuged ntawm 4 degree, 1000 rpm rau 15 min. Cov supernatant tau lim siv Millipore Express PES membrane kom tau cov charcoal dextran-treated (CDT)-FBS thiab khaws cia ntawm −20 degree.
Cell kab lis kev cai
Tib neeg mob qog noj ntshav MCF-7 cell kab tau txais los ntawm American hom kab lis kev cai sau (ATCC). Tsis tas li ntawd, cov hlwb raug coj los siv nrog RPMI-1640 nruab nrab uas muaj 10 feem pua FBS thiab 1 feem pua penicillin-streptomycin ntawm 37 degree nyob rau hauv ib qho av humidified 5 feem pua CO2 cua. Cells tau coj mus rau hauv cov phenol liab-dawb RPMI-1640 nruab nrab uas muaj 5 feem pua CDT-FBS 4 hnub ua ntej MTT kev soj ntsuam kom cov tshuaj estrogen hauv cov hlwb yuav tsum tau tshem tawm.
Kev tsom xam ntawm cov cell proliferation ntawm estradiol ntawm MCF -7 kab ntawm tes
Kev loj hlob ntawm tes raug ntsuas los ntawm kev ntsuas MTT rau MCF-7 hlwb. }}.1, 1, 10, 100, thiab 1000 nM. MCF-7 cov kab ntawm tes tau raug coj los ua phenol liab-dawb RPMI-1640 nruab nrab rau 4 hnub. Tom qab ntxuav los ntawm phosphate-buffered saline (PBS) peb zaug, 100 µL ntawm FBS-free RPMI-1640 nruab nrab tau ntxiv rau hauv 96-zoo daim hlau ntawm 1.5 × 104 rau ib qhov dej thiab incubated ntawm 37 degree rau 24 teev. Tom qab ntawd, cov hlwb raug kho nrog ntau qhov ntau ntawm cov tshuaj E2 rau 72 teev. Tom qab ntawd, 100 µL ntawm MTT tov (0.5 mg / mL) tau ntxiv rau txhua qhov dej. Cells tau incubated rau 4 h. Thaum kawg, 150 µL ntawm DMSO tau ntxiv kom yaj cov formazan crystals. Qhov nqus tau raug ntsuas ntawm 570 nm los ntawm tus nyeem ntawv microplate, thiab qhov kev nthuav dav (PR) raug xam.
Kev txheeb xyuas cov cell loj hlob ntawm glycosides ntawm Cistanches ntawm MCF-7 kab ntawm tes
Kev loj hlob ntawm tes raug ntsuas los ntawm kev ntsuas MTT rau MCF-7 hlwb. GCs tau yaj hauv RPMI-1640 kab lis kev cai xov xwm uas muaj 0.1 feem pua DMSO ntawm qhov kawg ntawm 0.0175, 0.175, 1.75, 17.5, thiab 175 µg /ml. MCF-7 cov kab ntawm tes tau raug coj los ua hauv phenol liab-dawb RPMI-1640 nruab nrab uas muaj 5 feem pua CDT-FBS rau 4 hnub. Tom qab ntxuav los ntawm PBS peb zaug, 100 µL ntawm FBS-free RPMI-1640 nruab nrab tau ntxiv rau hauv 96-zoo daim hlau ntawm 1.5 × 104 rau ib qhov dej thiab incubated ntawm 37 degree rau 24 teev. Tom qab ntawd, cov hlwb raug kho nrog ntau qhov ntau ntawm cov tshuaj GCs rau 24, 48, thiab 72 h, feem. Tom qab ntawd, 100 µL ntawm MTT tov (0.5 mg / mL) tau ntxiv rau txhua qhov dej. Cells tau incubated rau 4 h. Thaum kawg, 150 µL ntawm DMSO tau ntxiv kom yaj cov formazan crystals. Qhov nqus tau ntsuas ntawm 570 nm los ntawm tus nyeem ntawv microplate, thiab PR tau suav. Phenol liab-dawb RPMI-1640 thiab nruab nrab uas muaj E2 (2.725 × 10-3 µg / ml) yog cov pab pawg tsis zoo thiab zoo, feem.
Kev tsom xam ntawm cell proliferation ntawm glycosides ntawm Cistanches nrog fulvestrant ntawm MCF -7 kab ntawm tes
Kev loj hlob ntawm tes raug ntsuas los ntawm kev ntsuas MTT rau MCF-7 hlwb. GCs tau yaj hauv RPMI-1640 kab lis kev cai xov xwm uas muaj 0.1 feem pua DMSO ntawm qhov kawg ntawm 1.75, 17.5, thiab 175 µg/ml. MCF-7 cov kab ntawm tes tau raug coj los ua hauv phenol liab-dawb RPMI-1640 nruab nrab uas muaj 5 feem pua CDT-FBS rau 4 hnub. Tom qab ntxuav los ntawm PBS peb zaug, 100 µL ntawm FBS-free RPMI-1640 nruab nrab tau ntxiv rau hauv 96-zoo daim hlau ntawm 1.5 × 104 rau ib qhov dej thiab incubated ntawm 37 degree rau 24 teev. Tom qab ntawd, cov hlwb raug kho nrog ntau qhov ntau ntawm cov tshuaj GCs thiab incubated nrog fulvestrant (6.06 × 10-5 µg / ml) rau 48 h. Tom qab ntawd, 100 µL ntawm MTT tov (0.5 mg / mL) tau ntxiv rau txhua qhov dej. Cells tau incubated rau 4 h. Thaum kawg, 150 µL ntawm DMSO tau ntxiv kom yaj cov formazan crystals. Qhov nqus tau ntsuas ntawm 570 nm los ntawm tus nyeem ntawv microplate, thiab PR tau suav. Phenol liab-dawb RPMI-1640 thiab nruab nrab uas muaj E2 (2.725 × 10-3 µg / ml) yog cov pab pawg tsis zoo thiab zoo raws li.
Kev ntsuam xyuas ntawm tes
GCs tau yaj hauv RPMI-1640 kab lis kev cai xov xwm uas muaj 0.1 feem pua DMSO ntawm qhov kawg ntawm 1.75, 17.5, thiab 175 µg/ml. MCF-7-cell kab tau coj mus rau hauv phenol liab-dawb RPMI-1640 nruab nrab uas muaj 5 feem pua CDT-FBS rau 4 hnub. Tom qab ntxuav los ntawm PBS peb zaug, 1 mL ntawm FBS-dawb RPMI-1640 nruab nrab tau ntxiv rau hauv 6-zoo daim hlau ntawm 2.5 × 105 rau ib qhov dej thiab incubated ntawm 37 degree rau 24 teev. Tom qab ntawd, cov hlwb raug kho nrog ntau qhov ntau ntawm cov tshuaj GCs thiab incubated nrog fulvestrant (6.06 × 10-5 µg / ml) rau 48 h. Tom qab ntawd, cov hlwb raug sau thiab ntxuav nrog PBS peb zaug, centrifuged ntawm 4 degree , 1000 rpm rau 10 min, thiab kho nrog 70 feem pua ethanol ntawm −20 degree, thaum hmo ntuj. Tom qab ntxuav nrog PBS ob zaug, cov hlwb raug stained nrog PI tov (50 mg / mL) uas muaj 1 mg / mL ntawm RNase A thiab 0.1 feem pua Triton X-100 rau 30 min hauv qhov tsaus ntuj ntawm 4 degree. Lub voj voog ntawm tes tau kuaj pom siv cov cytometry ntws, thiab qhov ntsuas kev loj hlob (PI) tau suav raws li hauv qab no. Phenol liab-dawb RPMI-1640 thiab nruab nrab uas muaj E2 (2.725 × 10-3 µg / ml) yog cov pab pawg tsis zoo thiab zoo, feem. PI=(S ntxiv rau G2 M)/(G0 / G1 ntxiv S ntxiv rau G2 M) × 100 feem pua
RNA cais tawm thiab lub sij hawm ntawm lub sij hawm ntau npaum li cas polymerase saw cov tshuaj tiv thaiv
Txhawm rau ntsuas qib ntawm mRNA, kev ntsuas RT-quantitative PCR (qPCR) tau siv. Tag nrho cov cellular RNA tau muab rho tawm nrog TRIzol reagent. Rau qPCR, RNA tau thim rov qab rau cDNA los ntawm 4 µg ntawm tag nrho RNA siv cov khoom siv RT. Kev tshuaj xyuas RT-PCR tau ua los ntawm TB Green™ RT-PCR Kit. Tag nrho cov txheej txheem tau ua raws li cov neeg tsim khoom cov lus qhia. Cov primer khub siv rau kev nthuav dav ntawm ER yog 5'-GGGAAGTATGGCTATGGAATCTG-3' (rau pem hauv ntej) thiab 5'-TGGCTGGACACATATAGTCGTT-3' (rov qab); cov primer khub siv rau amplification ntawm ER yog 5'-AGTGCCGCTTTGGAGAGCTG-3' (forward) thiab 5'-CCTGGGTCGCTGTGACCAGA-3' (rov qab). PCR cov xwm txheej rau ER thiab ER yog 94 degree rau 3 min ua raws li 37 thiab 40 cycles, feem, ntawm 94 degree rau 30 s, 58 degree rau 2 min, thiab 72 degree rau 2 min. Cov khub primer siv rau kev nthuav dav ntawm GAPDH yog 5'-GGACGCGAGATCCCCAAAAAT-3' (rau pem hauv ntej) thiab 5'-GGCTGTTGTCATACTTCTCATGG-3' (rov qab). PCR cov xwm txheej rau GAPDH yog 94 degree rau 3 min ua raws li 30 lub voj voog ntawm 94 degree rau 30 s, 58 degree rau 2 min, thiab 72 degree rau 2 min. Txhua lub sijhawm RT-qPCR tau rov ua dua ntau dua ob zaug. Rau qhov no, GAPDH tau siv los ua kev tswj hwm sab hauv. Qhov txheeb ze ntawm cov noob qhia ntawm ER / ER ntawm cov tshuaj tiv thaiv kab mob sib txuas nrog cov hlwb tau suav nrog: 2−(∆∆Ct).
Western blotting
Kev nthuav qhia ntawm ER thiab ER cov proteins tau kuaj pom los ntawm kev tsom xam sab hnub poob raws li cov txheej txheem tshaj tawm nrog kev hloov kho me me. Luv luv, MCF-7 hlwb tau loj hlob hauv 6-zoo daim hlau ntawm 2.5 × 105 hlwb ntawm ib qhov dej thiab kho nrog GCs ntawm qhov kawg ntawm 1.75, 17.5, thiab 175 µg / ml rau 48 h, raws li. Tom qab incubation, tag nrho-cell rho tawm tau npaj siv RIPA tsis muaj 1 mM PMSF. Proteins nyob rau hauv tag nrho-cell extracts raug cais los ntawm 12 feem pua sodium dodecyl sulfate-polyacrylamide gel electrophoresis thiab ces pauv mus rau polyvinylidene difluoride daim nyias nyias. Lub membrane tau thaiv nrog 5 feem pua (w/v) skim mis yaj nyob rau hauv TBST tsis thiab incubated nrog thawj thiab theem nrab cov tshuaj tiv thaiv nyob rau hauv lem. Cov tshuaj tiv thaiv kab mob tau raug soj ntsuam.
Kev txheeb cais
Cov txiaj ntsig tau raug nthuav tawm raws li kev txhais tau tias thiab tus qauv sib txawv, thiab qhov tseem ceeb ntawm cov ntaub ntawv tau ua los ntawm Tub Ntxhais Kawm T-test nrog SPSS 21.0 software (IBM Co., Ltd. America). P<0.05 was="" considered="" statistically="">
TSEEM CEEB
Tom qab 24 teev ntawm E2 kev kho mob, qhov kev loj hlob ntawm MCF-7 hlwb tau pom tseeb txhim kho [Daim duab 1a]. 10nM ntawm E2 cov tshuaj tuaj yeem txhawb kev loj hlob ntawm cov hlwb pom tseeb (123.89 feem pua). Txawm li cas los xij, nrog qhov nce ntawm E2 concentration, lub peev xwm loj hlob ntawm tes tsis muaj zog. Li no, 10nM yog qhov zoo tshaj plaws concentration ntawm E2 hauv qhov kev sim no.
GCs pawg ntawm cov concentrations ntawm 1.75, 17.5, thiab 175 µg / mg tuaj yeem txhim kho kev loj hlob ntawm MCF-7 cell kab nrog lub sijhawm thiab ntau npaum li cas thiab nthuav tawm qhov sib txawv tseem ceeb nrog pab pawg tsis zoo [Daim duab 1b]. Piv nrog rau pawg tsis zoo, pawg E2 tau nthuav tawm pom tseeb kev loj hlob (120.06 feem pua).
Cov tshuaj sib xyaw ua ke (CMG) (fulvestrant nrog E2 lossis GCs) hauv CDT-FBS nruab nrab tau incubated nrog MCF-4 hlwb. Tom qab 72 teev ntawm incubation, CMG tuaj yeem ua rau muaj kev cuam tshuam ntawm PR piv nrog rau ib pawg tshuaj (IMG) (P < 0.01)="" [daim="" duab="">
Flow cytometry tsom xam pom tias GCs tuaj yeem nce PI tus nqi me ntsis uas qhia tias GCs tuaj yeem nce qib G{{{0}}}/G1 theem cell mus rau S thiab G2/M theem [Daim duab 2 thiab Table 1] thiab txhawb nqa cell DNA. synthesis. Qhov tshwm sim qhia tau hais tias cov txheej txheem ntawm GCs ntawm MCF-7 zoo ib yam li E2. Hauv CMG (fulvestrant nrog GCs), PI tus nqi ntawm tes PI tau poob qis rau ntawm IMG me ntsis (P <>
Kev tshuaj xyuas RT-PCR tau qhia tias cov kab lus ntawm ER thiab ER mRNA tau nce ntxiv piv nrog rau pawg tswj hwm (P < 0.01) nyob rau hauv ib koob tshuaj tom qab raug kho los ntawm qhov sib txawv ntawm GCs rau 48 h [Daim duab 3a thiab b].
Western blot tshwm sim qhia tau hais tias tom qab kev kho mob nrog ntau qhov ntau ntawm GCs rau 48 h, cov ntsiab lus ntawm ER thiab ER proteins hauv MCF-7 nce nrog GCs nce raws li kev noj ntau npaum li cas [Daim duab 3c-e]
XAIV
Hauv kev tshawb fawb no, cov txiaj ntsig ntawm GCs ntawm kev loj hlob thiab lub voj voog ntawm tes ntawm MCF-7 tau soj ntsuam los ntawm txoj kev ntawm MTT thiab ntws cytometry, feem. GCs tuaj yeem ua rau muaj kev loj hlob ntawm MCF-7 hlwb ntawm qhov concentration ntawm 1.75, 17.5, thiab 175 µg / ml tom qab incubated rau 72 teev. Txawm li cas los xij, qhov nce yuav qeeb qeeb thaum CCs thiab fulvestrant tau siv ua ke. GCs tuaj yeem nce PI tus nqi ntawm MCF-7 hlwb, txo cov cell ntawm G1 theem, thiab nce cell ntawm S thiab G2 theem.
Fulvestrant yog ib qho ER-tshwj xeeb antagonist, uas thaiv cov nuclear localization ntawm ER los ntawm impairing receptor dimerization thiab lub zog-dependent nuclear thauj.[17] Hauv kev tshawb fawb no, qhov kev txiav txim siab tau lees paub tias fulvestrant tuaj yeem ua rau tsis muaj zog ntawm kev loj hlob ntawm GCs ntawm MCF-7 hlwb, thiab nws tuaj yeem cuam tshuam rau kev hloov pauv ntawm tes. Li no, txoj kev tshawb no qhia txog cov kev ua estrogenic ntawm GCs, thiab kuj, ER yog lub hom phiaj ntawm GCs. Hauv kev sim RT-PCR thiab sab hnub poob blot, mRNA thiab protein qhia ntawm ER thiab ER hauv MCF-7 hlwb tau nce nrog qhov nce ntawm GCs concentration. Yog li, GCs tuaj yeem ua lub luag haujlwm hauv kev ua estrogenic raws li kev tswj hwm mRNA thiab cov proteins ntawm ER thiab ER.
