Anti-aging Effects Of Ribes Meyeri Anthocyanins On Neural Stem Cells And Aging Mice Part 2

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

Nar cawm senescent phenotypes ntawm 23M-NSCs

R. Meyer anthocyanins yog nplua nuj nyob rau hauv ntau hom flavonoids thiab bioactive Cheebtsam [1,3-5].Los txiav txim seb lub Cheebtsam ntawm R. Meyer anthocyanins plays ib tug tseem ceeb luag hauj lwm nyob rau hauv anti-aging, peb ua ib tug metabolomic tsom xam ntawm R. Meyer anthocyanins. Peb tau nthuav tawm tias Nar yuav yog ib qho tseem ceeb ntawm flavonoid metabolite vim tias txoj hauv kev flavonoid biosynthetic tau nplua nuj nyob hauv R. Meyer anthocyanins (Table 1) ntxiv. Txhawm rau txiav txim siab qhov cuam tshuam los ntawm Nar, ntau qhov concentrations (0, 1.7,3.4,6.8,13.6, thiab 27.2 ug/mL) tau siv rau 23M-NSCs thiab CCK-8 kev soj ntsuam Raws li Daim Duab Ntxiv 3 qhia tau hais tias, 6.8 ug/mL Nar kev kho mob nce cell viability los ntawm 150 feem pua ​​​​. Txhawm rau tshawb nrhiav ntxiv los tiv thaiv kev cuam tshuam ntawm Nar, 23M-NSCs tau kho nrog Nar, thiab cov cim qhia txog kev laus raug kuaj pom. .hesperidin sivHauv 23M-NSCs kho nrog 6.8 ug / mL Nar, p16nk4a mRNA qhia tau txo qis (Daim duab 3A). Nar kuj tseem txuas ntxiv cov telomeres ntawm kab lis kev cai mNSCs thiab nce DNA synthesis (Daim duab 3B,3C). Txhawm rau tshawb fawb ntxiv txog qhov cuam tshuam ntawm Nar ntawm kev loj hlob ntawm tes, peb tau ua cov tshuaj tiv thaiv kab mob hauv lub cev ntawm Ki67, uas yog cov tshuaj tiv thaiv kab mob ntawm cov mitotic hlwb uas tau nthuav tawm thoob plaws hauv lub voj voog ntawm tes, tshwj tsis yog hauv G0 [19,27]. Kev kho mob nrog Nar txhim kho cell proliferation, raws li kev ntsuas los ntawm cov xov tooj ntawm Ki67- cov cell zoo (Daim duab 3D, 3E). Tom ntej no, peb kuj tau kuaj pom qhov sib txawv ntawm NSCs rau hauv cov neurons. Tseeb tiag, 6.8ug/mL Nar kev kho mob kuj tau nce tus naj npawb ntawm MAP2-cov hlwb zoo (Daim duab 3F,3G). Cov txiaj ntsig zoo li no qhia tau hais tias Nar tuaj yeem thim rov qab 23M-NSC senescence.

Daim duab 2. Ribes Meyer anthocyanins txhim khu nas nco thiab kev kawm muaj peev xwm los ntawm kev nce neural stem cells (NSCs). (A) Lub sijhawm yog siv los nrhiav lub platform. (B) Lub sijhawm raug coj los ua ntej mus txog lub platform. (C) txhais tau tias nrawm los nrhiav lub platform. (D) Nyob deb ua luam dej ua ntej nrhiav lub platform. (E. F) Kev kho mob nrog R.poob teb chaws Ottoman cistanchetxhua tus anthocyanin nce neurosphere tus lej thiab qhov loj me piv nrog kev tswj hwm. (G) Neurospheres ot 50-100 um qhov loj me tau nce hauv tus lej los ntawm R. Meyer anthocyanin kev kho mob hauv nas. Cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab ± SD ntawm peb qhov kev sim ywj pheej. *P<><0.01, and=""><0.0001 compared="" with="" untreated="" cells."n"="" indicates="" the="" number="" of="" animals="" in="" each="" experimental="">

Daim duab 3. Qhov cuam tshuam ntawm naringenin (Nar) ntawm kev nce qib ntawm nas neural qia hlwb (MNCs). (A) p16k4a mRNA qhia tau ntsuas los ntawm qRT-PCR hauv 23M-NSCs nrog thiab tsis kho ntawm 6.8 ug / mL Nar rau 48 teev. (B) Cell cycle phase distributions of 23M-NSCs kho nrog Nar rau 48 h thiab tswj cov hlwb. (C) Cov txheeb ze telomere ntev ntawm 23M-NSCs tau nce ntau nrog Nar kho, (D) Immunofluorescence Ki67 staining ntawm cov leeg kho nrog Nar rau 48 h, nrog DAPI nuclear labeling, (E) Quantification ntawm (D), (F) Tus Neeg Sawv Cev teb ntawm MAP2 thiab GFAPimmunofluorescence staining ntawm kab lis kev cai mNSCs tom qab tswj thiab Nar kho. (G) Quantification ntawm (F). Cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab ± SD ntawm peb qhov kev sim ywj pheej. *P<>< 0.01,="" and="">< 0.0001="" compared="" with="" untreated="">

Thov nias ntawm no kom paub ntxiv

Nar txhim kho kev nco thiab kev paub hauv cov nas laus

Cov qib kev txawj ntse poob qis nrog kev laus, thiab kev kho kev laus tuaj yeem txhim kho kev txawj ntse [11, 28]. Txhawm rau txheeb xyuas qhov kev tiv thaiv kev laus ntawm Nar hauv Vivo, peb txhaj 20 mg / kg Nar rau hauv cov laus C57BL / 6 nas (12 lub hlis) los ntawm tus Tsov tus tw leeg. Qhov kev xeem MWM tau siv los tshuaj xyuas qhov chaw nco thiab kev txawj ntse ntawm cov nas.micronized purified flavonoid feem 1000 mg sivTshaj li 5 hnub ntawm kev rov ua dua tshiab, lub sijhawm siv rau Nar-kho cov nas laus kom pom lub platform maj mam txo qis piv nrog pawg tswj hwm (Daim duab 4A). Cov txiaj ntsig kev xeem tau qhia tias lub sijhawm ua ntej mus txog lub platform raug txo qis (Daim duab 4B), thaum lub sijhawm siv hauv thaj chaw platform tau nce (Daim duab 4C). Tsis tas li ntawd, Nar-kho cov nas laus tau pom nyob rau hauv thaj chaw platform ntawm ntau zaus tshaj li pawg tswj hwm (Daim duab 4D). Ua ke, cov txiaj ntsig no qhia tias Nar tuaj yeem txhim kho kev nco qab thiab kev txawj ntse hauv cov nas laus.

RNA sequencing tsom xam ntawm Nar-kho ntshav los ntawm cov nas laus

Txhawm rau tshawb xyuas cov Nar-specific anti-aging mechanisms, peb tau soj ntsuam cov noob qhia profile ntawm Nar-kho ntshav los ntawm cov nas laus siv RNA sequencing (RNA-seq). Cov txiaj ntsig tau nthuav tawm 1,961 qhov sib txawv qhia cov noob (DEGs). Feem ntau, Nar downregulated gene qhia, thiab tsuas yog ib tug me me ntawm cov noob tau up-regulated piv nrog cov pab pawg neeg tswj tsis kho (Daim duab 5A, 5B). Tsis tas li ntawd, cov kev taw qhia tseem ceeb ntawm cov noob caj noob ces cuam tshuam txog kev laus raug txo qis, tshwj xeeb tshaj yog cov nyob hauv TNF signaling pathway (Daim duab 5C).oteflavonoidCov txiaj ntsig no txhais tau tias Nar cuam tshuam rau TNF signaling txoj hauv kev, yog li cuam tshuam rau kev ua haujlwm ntawm TNF-a, uas cuam tshuam rau kev laus txog kev paub tsis meej [29,30]. Yog li ntawd, peb tau soj ntsuam TNF- qib hauv cov ntshav ntawm cov nas. Cov txiaj ntsig tau los ntawm d immunosorbent assays (ELISA) enzyme-txuas

Daim duab 4. Naringenin (Nar) txhim khu kev nco thiab kev kawm muaj peev xwm hauv cov nas laus. (A) Kev khiav tawm latency ntawm Nar-kho laus nas yog luv dua piv nrog pawg tswj hwm. (B) Lub sij hawm coj mus rau thawj zaug mus txog lub platform yog luv dua nyob rau hauv Nar-kho cov laus nas tshaj nyob rau hauv pawg tswj. (C) Lub sijhawm siv sijhawm nyob hauv thaj chaw platform. (D) Ntau zaus tau pom nyob rau hauv thaj chaw platform rau Nar-kho cov laus nas piv nrog pawg tswj hwm. Cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab ± SD ntawm peb qhov kev sim ywj pheej.*P<>< 0.01,=""><0.0001 compared="" with="" the="" control="" group."n"="" indicates="" the="" number="" of="" animals="" used="" for="" each="" experimental="" group.="" revealed="" that="" nar="" treatment="" led="" to="" the="" downregulation="" of="" plasma="" tnf-a="" levels="" (figure="" 5d).="" we="" also="" examined="" pl6ink4agene="" expression="" in="" the="" hippocampus="" of="" mice="" and="" demonstrated="" that="" nar="" treatment="" downregulates="" pl6ink4amrna="" expression(figure="">

Cistanche tuaj yeem tiv thaiv kev laus

Kev sib tham

Kev laus yog txuam nrog kev pheej hmoo siab ntawm kev paub txog kev poob qis nrog rau kev poob ntawm kev kawm thiab kev nco muaj peev xwm [22,23]. Neuronal poob thiab impaired neurogenesis muaj feem cuam tshuam nrog lub hnub nyoog cuam tshuam lub hlwb tsis ua haujlwm [11-13]. Yog li, nce tus naj npawb ntawm cov neurons thiab txhim kho neurogenesis yog qhov tseem ceeb rau kev tiv thaiv kev laus. R. Meyer, tsob nroj nplua nuj nyob rau hauv flavonoids thiab lwm yam bioactive tshuaj [1, 3-6], muaj ntau lub zog uas tseem tsis tau piav qhia. Hauv txoj kev tshawb fawb tam sim no, R. Meyer anthocyanins txhawb NSC kev loj hlob thiab nce kev tsim cov neurospheres nrog cov qauv morphology. R. Meyer anthocyanin kev kho mob kuj tau txhim kho cov cellular aging phenotype, nrog txo qis ROS thiab aging-related P16nk4 noob qhia, nce DNA synthesis theem, thiab lengthened telomeres.puritans vitamin cNtxiv mus, immunofluorescence staining tshwm sim tau qhia tias R Meyer anthocyanins txhawb kev tsim cov neurons. Cov txiaj ntsig no qhia tau hais tias R. Meyer anthocyanins muaj kev tiv thaiv kev laus ntawm mNSCs, thiab tseem tuaj yeem txhawb kev tsim cov neurons hauv vitro ncaj qha. Tsis tas li ntawd, kev paub txog kev poob qis yog cuam tshuam nrog kev laus [22, 23]. Peb cov txiaj ntsig MWM tau pom tias kev tswj hwm intragastric ntawm R. Meyer anthocyanins txhim kho kev nco thiab kev kawm muaj peev xwm hauv cov nas laus, thiab kuj nce ntxiv.

Figure 5. Tumor necrosis factor(TNF)-a plays an essential role in naringenin (Nar)-mediated anti-aging effects. (A) Summary of gene expressions in mouse blood, as determined by RNA-seq. (B)Heatmap showing differentially expressed genes (DEGs) in the blood between control and Nar-treated mice. DEGs were identified by a fold change of >2. 0 or<0.5. (c)bar="" graph="" showing="" the="" major="" correlated="" signaling="" pathways.="" (d)="" heatmap="" showing="" degs="" in="" the="" tnf="" signaling="" pathway="" (e)="" plasma="" tnf-a="" levels="" determined="" by="" enzyme-linked="" immunosorbent="" assay.="" (e)="" p16hka="" gene="" expression="" levels,="" as="" determined="" by="" qrt-pcr.="" data="" are="" presented="" as="" the="" mean="" ±="" sd="" of="" three="" independent=""><><><0.0001 compared="" with="" the="" control="" group.="" nsc="" and="" neuronal="" numbers="" in="" the="" hippocampus="" of="" aging="" mice.="" these="" results="" suggest="" that="" r.="" meyer="" anthocyanins="" may="" enhance="" cognitive="" function="" by="" increasing="" nsc="" proliferation="" and="">

Kev siv cov tshuaj metabolomic los tshawb xyuas cov ntsiab lus tshwj xeeb ntawm R. Meyer anthocyanins tau qhia tias Nar yuav yog ib qho tseem ceeb ntawm flavonoid metabolite. Nar tau tshaj tawm yav dhau los rau ameliorate myocardial cell senescence [31], txhim kho lub peev xwm metabolic ntawm txoj hnyuv [32], thiab siv tshuaj tiv thaiv kab mob [33] thiab tiv thaiv mob qog noj ntshav [34]. Txawm li cas los xij, qhov cuam tshuam ntawm Nar ntawm NSCs thaum laus tseem tsis paub. Txhawm rau tshawb xyuas qhov cuam tshuam los tiv thaiv kev laus ntawm R. Meyer anthocyanins, peb tau tshawb fawb ntxiv siv Nar. Kev kho mob nrog 6.8 ug / mL Nar nce cell viability, txo P16ink4a noob qhia, lengthened telomeres, thiab txhawb NSC sib txawv rau hauv neurons hauv vitro. Txhawm rau ntsuas qhov cuam tshuam ntawm Nar ntawm kev loj hlob ntawm tes, peb tau ua cov kev tshawb fawb immunofluorescence. Cov txiaj ntsig tau qhia tias Nar kev kho mob tau nce ob tus lej ntawm Ki67-cov hlwb zoo thiab feem ntawm MAP{12}} cov hlwb zoo, qhia tias Nar tuaj yeem txhawb nqa neurogenesis. Tsis tas li ntawd, qhov cuam tshuam ntawm Nar ntawm kev kawm thiab kev nco kuj raug soj ntsuam hauv cov nas laus. Qhov kev xeem MWM tau pom tau hais tias kev kho mob Nar txhim kho spatial leaming hauv cov nas laus. Interestingly, RNA-seq tsom xam tau qhia tias Nar tuaj yeem cuam tshuam kev laus ntawm TNF txoj kev taw qhia, tshwj xeeb tshaj yog los ntawm kev txo qis TNF-ib qho kev qhia hauv cov ntshav ntawm cov nas laus. ELISA kev soj ntsuam kuj tau qhia tias kev kho mob Nar txo cov ntshav TNF-ib theem piv nrog rau kev tswj cov nas laus. TNF-a yog ib qho tseem ceeb hauv TNF signaling pathway thiab yog ze ze rau kev paub txog kev laus. Nws lub luag haujlwm suav nrog kev txhawb nqa kev hloov pauv pathological hauv hippocampal synapses [29] thiab inhibition ntawm precursor cell proliferation [35]. Hloov TNF qib yog txuam nrog kev paub tsis meej hauv kev nyuaj siab, schizophrenia, kev puas siab puas ntsws bipolar, thiab Alzheimer's kab mob [30,36,37]. Tshwj xeeb tshaj yog, TNF-a yog upregulated rau cov neeg mob Alzheimer's kab mob [38].

Hauv cov ntsiab lus, peb txoj kev tshawb fawb pom tau tias R. Meyer anthocyanins txhim kho cov teebmeem ntawm kev laus hauv NSCs ntawm Nar, uas txo qis TNF-ib theem hauv vivo thiab txhim kho kev paub txog kev laus nas. Sib sau ua ke, peb cov kev tshawb pom muab ib lub tswv yim tshiab rau kev txhim kho kev kho mob, tsom mus rau kev paub ntau dua ntawm cov nqi tshuaj ntawm R.Meyer anthocyanins.

Cov khoom siv thiab cov txheej txheem

Kev sim tsiaj

Txoj kev tshawb no tau pom zoo los ntawm Tongji University Animal Care Committee thiab ua raws li cov txheej txheem hauv tsev kawm ntawv. R. Meyer anthocyanins tau muab tshuaj intragastrically rau 2 lub hlis rau 12-hli cov nas uas muaj hnub nyoog khaws cia ntawm C57BL/6 keeb kwm yav dhau. Nar tau txhaj rau hauv cov hlab ntsha caudal rau 1-hli mus rau 12- cov nas muaj hnub nyoog khaws cia ntawm C57BL/6 keeb kwm yav dhau. Cov nas tswj tau muab tib lub ntim ntawm phosphate-buffered saline (PBS).

Kev npaj ntawm R. Meyer anthocyanins thiab Nar

R. Meyer txiv hmab txiv ntoo (500 g) tau homogenized siv cov ntaub so ntswg homogenizer thiab tshem tawm hauv 1 L methanol uas muaj 0.1 feem pua ​​​​hvdrochloric acid. Ultrasonic extraction tau ua peb zaug rau 30 min ntawm chav tsev kub. Cov extracts tau muab tso ua ke, thiab cov methanol tau concentrated nyob rau hauv kev txo qis kom tau txais cov rho tawm ntawm R Meyer. Cov extract tau ntxiv purified nrog AB -8 macroporous resin kom nce cov ntsiab lus anthocyanin.

Naringenin(C15H1205; MW: 272.25; purity: Ntau dua lossis sib npaug li 98 feem pua; HPLC qib) tau yuav los ntawm Chengdu Push Biotechnology Co. Ltd.(Sichuan, Tuam Tshoj) thiab khaws cia ntawm 2 degree {{6 }} degree nyob rau hauv ib puag ncig qhuav tsis muaj teeb. Qhov concentration ntawm cov tshuaj hauv Tshuag yog 100 mM hauv dimethyl sulfoxide (DMSO). Qhov kawg DMSO concentration tsis tshaj 0.1 feem pua ​​​​hauv cov kab lis kev cai nruab nrab.

Neurosphere thiab kev ntsuam xyuas sib txawv

NSCs tau muab los ntawm tus nas SVZ. Cov nas tau muab xyeem tom qab tshuaj loog thiab SVZ tau ua tib zoo txiav thiab sib cais rau hauv ib lub hlwb nrog 0.4 U/mL papain(Imogen, Alexandria, VA, USA; Cat No: LS003126). NSCs tau plated hauv 10 cm lauj kaub tais diav uas muaj 10 mL NSC Basal Medium nrog 10 ng / mL EGF (PeproTech, Cranbury, NJ, USA; Cat No: AF-100-15) thiab 10 ng / mL FGF (PeproTech, Cranbury, NJ , USA; Cat No:AF-100-18B). Neurospheres tau kis txhua 5 hnub. NSCs los ntawm qhov kev tsis sib haum xeeb tshiab -2 neurospheres tau noob rau ntawm laminin (Gibco, Gaithersburg, MD, USA; Cat No: 23017015) thiab poly-L-ornithine hydrobromide (Poly-L; Sigma Aldrich, St Louis, MO, USA ; Cat No: P3655)-coated iav slides hauv NSCBasalMedium yam tsis muaj kev sib txawv ntxiv. Cov hlwb sib txawv tau tiv thaiv kab mob rau Tu1 (beta-tubulin II; Thermo Fisher Scientific, Waltham, MA, USA; Cat No: 480011), MAP2 (Thermo Fisher Scientific, Cat No: MA5-12826), thiab glial fibrillary acidic protein (GFAP; Thermo Fisher Scientific, Cat No. PA5-16291), thiab tom qab ntawd suav nrog 4'6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Cat No: D1306). Slides raug tshuaj xyuas siv Olympus BX53 microscope (Olympus, Madison, WI, USA). Quantification ntawm TuJ1- thiab MAP2- cov hlwb zoo tau ua los ntawm kev suav cov cim npe los ntawm peb qhov kev sim ywj pheej. Kev txheeb xyuas txheeb cais tau ua los ntawm kev sib tw t-test.

Kab lus no yog muab rho tawm los ntawm www.aging-us.com AGING 2020, Vol. 12, nr 17