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Kev paub

Autofluorescence Raws li Cov Khoom Siv Tsis Siv Neeg Ntawm Senescence Thiab Advanced Glycation End Cov Khoom hauv Cistanche Tubulosa

Apr 11, 2023

Kev sib tham

Yav dhau los, autofluorescence tau siv los saib xyuas lipofuscin, lub hnub nyoog xim, raws li biomarker ntawm senescence.4,13. Txawm tias peb pom liabfluorescence qhia txog lipofuscin raws li pom hauv cov kev tshawb fawb yav dhau los9, xiavfluorescence tau kuaj pom nyob rau theem ua ntej ntawm lub neej thaum cov kab mob tau kuaj xyuas siv M165 FCfluorescence stereomicroscope (Leica Microsystems, Tokyo, Nyiv) (cov ntaub ntawv tsis qhia). Lub hom phiaj ntawm txoj kev tshawb fawb tam sim no yog los tshuaj xyuas seb qhov xiav autofluorescence puas tuaj yeem ua tus cim rhiab dua rau kev taug qab cov kab mob ntawm tus kab mob.

anti- senescence cistanche function

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Covfluorescence ntsuas nyob rau hauv ib tug neeg worms nce nrog lub hnub nyoog; cov worms tsawg duafluoresced, ntev lawv lub neej expectancy. Yog li ntawd, xiavfluorescence tuaj yeem muab lwm biomarker rau tracing senescence. Txawm li cas los xij, Pincus et al. qhia tias cov kab mob uas tom qab tuag (hauv 24 teev)fluoresced vim biosynthesis ntawm kynurenine; cov kws tshawb fawb hu ua lub teeb xiav no"tuagfluorescence". Tshaj tawm, tuagflua reflects emission los ntawm glycosylated daim ntawv ntawm anthranilic acid ua los ntawm txoj kev kynurenine; lub autoflCov khoom siv uorescing raug kuaj pom hauv lysosome zoo li lub plab granules31. Lub teeb xiav no tuaj yeem yog tus cim kev tuag tib yam uas Coburn li al. pom hauvC. elegansrau ob peb teev ua ntej thiab tom qab tuag, thiab hais tias tau pom nyob rau hauv ob qho tib si worms hluas raug mob tuag taus thiab worms tuag ib txwm muaj hnub nyoog. Tseeb tiag, peb tau pom tias covzoo -1mutant, uas tsis muaj kev ua kynureninase, yog autofluorescent tsawg dua li hom tsiaj qus. Txawm li cas los xij, peb cov txiaj ntsig tau qhia tias qee feem ntawm xiavfluorescence yog txuam nrog kev laus dua li kev tuag. Tshwj xeeb, raws li qhia hauv Fig.3b, worms aging nthuav dav ntxiv xiavfluorescence txawm tias tsis suav nrog cov ntaub ntawv tau txais hauv 2 hnub ua ntej kev tuag. Tsis tas li ntawd, worms tseemfluoresced ntau nrog aging, irrespective ntawm lub xeev ntawm lubkynu-1noob. Txofluorescence hauvkynu-1mutant yuav tsum yog li no vim poobntawm acceleration ntawm AGE synthesis los ntawm kynurenines es tsis muaj kev tuagfluorescence32.


Qee cov AGE compounds yogfluorescent27. Peb inferred tias xiavfluorescence tej zaum yuav suav nrog emissions los ntawm cov AGEs, cais los ntawm qhov uas tshwm sim rau kev tuagfluorescence. Thaum tshem tawm cov kab mob protein tau incubated nrog suab thaj hauv vitro, lubfluorescence siv thiab qib ntawm AGEs tau nce ntxiv raws sijhawm. Kab laum kab lis kev cai ntawm qhov nruab nrab uas muaj ribosefluoresced ntau tshaj li tswj tsiaj yog zus nyob rau hauv qhov tsis muaj supplemental ribose, txawm thaumkynu-1tau mutated. Nyob rau hauv sib piv, cov kab mob kab mob tshwm sim nyob rau hauv lub xub ntiag ntawm rifampicin, ib tug paub inhibitor ntawm AGEs ntau lawm, nthuav tawm txo qis ntawm xiav.fluorescence. Tseeb tiag, fluorescence microscopy qhia tau hais tias 13- cov kab mob hnub nyoog muaj hnub nyoog tshaj tawm lub teeb xiav ntau dua li 3- cov tub ntxhais hluas hnub nyoog hnub nyoog. Immunostaining nrog anti-CML los yog anti-pentosidine antibodies ua tsis tau tejyam los qhia qhov muaj diffusely kis AGEs nyob rau hauv ib tug tis qauv zoo li xiav.fluorescence. Covfluorescence tuaj yeem tshwm sim los ntawm lwm qhov ntaufluorescent AGEs xws li vespertine A, LM-1, thiab argpyrimidine33,34.


Txhawm rau muab yolk rau oocysts,C. eleganshermaphrodites noj lawv cov hnyuv biomass, uas ua rau plab hnyuv atrophy thiab ectopic yolk deposition nyob rau hauv lub neej tom ntej. Vitellogenins (yolk proteins) muaj nyob rau ntawm ob-fold ntau dua hauv cov kab qub (piv rau cov tsiaj yau), raws li qhia ua ntej35, thiab nthuav tawm rau ntau duaflureescence tom qab glycation hauv vitro. Cov ntaub ntawv no qhia tias vitellogenins lawv tus kheej yuav tsim tau 12-fold ncefluorescence nyob rau hauv cov laus worms piv rau cov hluas cov hluas theoretically. Western blotting pom tau hais tias lub ntsiab bands reacting nrog anti-CML antibody yog cov yolk proteins. Qhov kev tshawb pom no sib tw nrog cov ntaub ntawv dhau los ntawm Nakamura li al., uas tau kuaj pom glycation hnyav ntawm vitellogenin.36, thiab Golegaonkar li al., uas kuaj pom qhov txo qis glycation hauv vitellogenin-2, vitellogenin-6, thiab elongation factor 1 alpha hauv rifampicin-kho nematodes30. Tshaj tawm, vitellogenins ua raws li antioxidants thiab pab txhawb rau lub neej ntev ntawm zib muv huab tais37. HauvC. elegans, vitellogenins ua lub luag haujlwm tseem ceeb hauv kev ntxhov siab 38. Txij li cov tshuaj tiv thaiv antioxidants tuaj yeem oxidized yooj yim rau lawv tus kheej, tsub zuj zuj ntawm glycated vitellogenin tuaj yeem cuam tshuam kev tiv thaiv los ntawm oxidative kev puas tsuaj, uas ua rau kev sib raug zoo ntawm lub neej expectancy thiab kev siv xiav.fluorescence pom nyob rau hauv txoj kev tshawb no. Txawm li cas los xij, Sornda et al. Tsis ntev los no tau tshaj tawm tias kev ua neej tsis muaj feem cuam tshuam rau oxidative kev nyuaj siab kho mob los ntawm YP115/YP88 (vitellogenin-6)39. Cov kws tshawb fawb tau hais tias kev sib sau ntawm vitellogenin YP170, muab los ntawm vitellogenins 1.–5, ua rau plab hnyuv atrophy thiab txo lub neej, thaum lub sij hawm tsub zuj zuj ntawm YP115 / YP88 yuav retard plab hnyuv atrophy thiab ntev lifespan. Glycation ntawm vitellogenin -6 thiab qhov tshwm sim tsis ua haujlwm yog li ntawd tuaj yeem ua rau muaj qhov tsis zoo. Txawm hais tias elongation yamfluoresced peb-fold ntau ntxiv tom qab nyob rau hauv vitro glycation, tus nqi ntawm cov proteins no zoo sib xws ntawm cov hluas thiab cov laus cab. Yog li ntawd, qhov kev koom tes ntawm qhov tseem ceeb no rau kev txhim kho autofluorescence yuav tsawg dua qhov ua rau vitellogenins.

anti- senescence cistanche function


Cistanchetau siv feem ntau ua qauv raukawmsenescence thiab anti-senescence cuam tshuam. Peb tau thov kom siv cov cab los ua ib qho qauv los tshawb xyuas qhov cuam tshuam rau lub neej ntevntawm cov kab mob lactic acid8. Muab qhov ua qauv qhia (hauv txoj kev tshawb no) uas xiavfluorescence nyob rau hauv nematodes yog ib qho ua tau qhia ntawm AGEs tsub zuj zuj, peb hais tiasCistanche hermaphrodites tuaj yeem ua tus qauv rau kev tshawb fawb txog kev siv hluav taws xob los tiv thaiv kev cuam tshuam uas ua los ntawm kev tawm tsam ntawm AGEs ntau lawm. Hauv qhov sib piv, autofluorescence tsis zoo li yog biomarker ntawm senescence rau txiv neej worms vim vitellogenins yuav tsum tsis tshua muaj.

anti- senescence cistanche function

Fig. 5 Blue fluorescence los ntawm kynu -1 mutants, uas yuav tsum tsis txhob emit tuag fluorescence. ibFluorescence siv (ex 340/em 430) ntawm 7-hnub nyoog thiab 13-hnub qubkynu-1mutants (n = 37 ib.); cov worms laus tseem tawm ntau duafluorescence tshaj cov hluas worms, txawm tias ntawmkynu-1kev hloov pauv. Txawm li cas los, tsis muaj signififi uaCant sib txawv tau kuaj pom nyob rau hauv cov nqi autofluorescence los ntawm Mann–WhitneyU xeem.b Ib tug 7-hnub-laus worm tuav nrog ribose emitted ntau xiavfltxawm tias ntawm uerescencezoo -1kev hloov pauv. Xiavfluorescence ntawmC. elegansloj hlob nrog ribose (n = 24) los yog sorbitol (n = 18) tau muab piv nrog cov kab mob tswj hwm tsis muaj suab thaj (n = 20). Cov txiaj ntsig ntawm autofluorescence tau muab piv nrog cov nonparametric Steel–Txoj kev Dwass (**p < 0.01). c Ciaj sia taus nkhaus ntawmC. elegansloj hlob nrog ribose (n = 62) los yog sorbitol (n = 84) tau muab piv nrog cov tswj kab mob uas tsis muaj suab thaj (n = 64). Cov kab mob muaj 3 hnub nyob rau hnub 0. Nematode ciaj sia taus yog xam los ntawm Kaplan–Meier txoj kev, thiab qhov sib txawv ntawm kev ciaj sia tau raug sim rau qhov tseem ceeb los ntawm kev siv lub cav-qib xeem (**p < 0.01).


Txoj kev

NematodesCistanche Bristol strain N2 thiab nws cov derivative mutant hom tau ua siab zoo muab los ntawm Caenorhabditis Genetics Center, University of Minnesota. Cov kev hloov pauv siv hauv txoj kev tshawb no yog CB1370daf-2 (e1370)thiab CB1003kynu-1 (e1003). Nematodes tau khaws cia thiab nthuav tawm ntawm NGM raws li cov txheej txheem txheej txheem 40. Cov qe qe tau zoo los ntawm cov neeg laus cov kab mob tom qab raug cov tshuaj sodium hypochlorite/sodium hydroxide raws li tau piav qhia dhau los 41. Cov qe suspensions tau incubated thaum hmo ntuj ntawm 25 degree kom tso cai rau hatching, thiab ncua kev kawm ntawv. ntawm L1-stage worms tau centrifuged ntawm 156 ×g rau 1 min. Cov supernatant raug tshem tawm, thiab cov larvae ntxiv tau raug xa mus rau cov tshiab peptone tsis hloov pauv NGM (mNGM) daim hlau npog nrogE. coliHom OP50 (OP50). Cov kab mob tau cog rau ntawm mNGM agar daim hlau ntawm 25 degree tshwj tsis yog raudaf -2strain, uas tau loj hlob ntawm 20 degree mus txog rau theem L4. Tag nrho cov kev ntsuam xyuas tau pib nrog cov kab mob me me uas pib qe-ntev ntawm 25 degree. Tsis muaj kev pom zoo rau kev ncaj ncees rau nematodes.Cov kab mob kab mobOP50 tau siv los ua cov zaub mov tsim thoob ntiaj teb ntawm nematodes. Tryptone soya agar (Nissui Pharmaceutical, Tokyo, Nyiv) tau siv rau kab lis kev cai OP50. Cov kab mob (100 mg ntub hnyav) raug tshem tawm hauv 0.5 mL ntawm M9 tsis; ib 50-µL aliquot ntawm cov kab mob ncua sij hawm ces kis tau rau ntawm mNGM nyob rau hauv 5.5-cm-diameter daim hlau tshwj tsis yog hais tias lwm yam.


anti- senescence cistanche function

Multivariate tsom xam ntawm autofluorescence

Cov pej xeem ntawm 100 tus neeg laus txhua tus tau zoo thaum muaj hnub nyoog 3 thiab 17 hnub,ntxuav peb zaug, concentrated hauv 3 µL ntawm M9 tsis, thiab khaws cia khovntawm–80 degree kom txog thaum siv. Ua ntej rho tawm, 3 µL ntawm lysis tsis muaj (suav nrog50 mM Tris-HCl tsis kam (pH 7.5), 150 mM NaCl, 0.1 feem pua ​​sodium dodecyl sulfate(SDS), 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride (PMSF), thiab
2 feem pua ​​2-mercaptoethanol) thiab 4 µL ntawm 15 feem pua ​​SDS tau muab faib rau txhua lub raj. Cov qauv ces raug raufi uathiab khov-thaw cycles kom tso cov protein. Photoluminescent spectra tau sau nrog afluorescencespectrophotometer (Hitachi FL {{0}}) nrog cov ntaub ntawv txhais software (FL Solutions ver. 2.0). Fluorescence zog ntawm nematode suspensions yog

soj ntsuam siv excitation-emission matrix (EEM)flUorescence spectroscopy. Multivariate tsom xam (ib-nqaj excitation nrog ntauwavelength emission, thiab synchronous-scanningfluorometry) yielded EEM cov phiaj xwm muaj xws li ib leeg-scan excitation, thiab synchronousfluorescencespectra ntawm txhua series tau kos.



Fluorescence spectra ntawm aging worms

Cov kab mob (3–13 hnub) tau rov qab los ntawm kev loj hlob nruab nrab uas muaj 2'-deoxy-5-fluorouridine (Tokyo Chemical Industry, Tokyo, Nyiv). Qhov nocompound blocks lub embryonic txoj kev loj hlob ntawm progeny, yog li precluding kis kab mob los ntawm cov xeeb ntxwv. Ib tug neeg laus worms tau sau rau hauvcev, yuvtsiblub sij hawm, concentrated nyob rau hauv 3 µL ntawm M9 tsis, thiab khaws cia khov ntawm–80 degree kom txog thaum siv. Ua ntej extraction, 100 µL ntawm lysis tsis(muaj 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 10 mM dithiothreitol (DTT), 1 mM PMSF, thiab 1 µL protease inhibitor cocktail (Sigma, St. Louis, MO, USA)) tau ntxiv rau cwb cev. Cov qauv tom qab ntawd tau hauv av siv Mini Cordless Grinder (Funakoshi, Tokyo, Nyiv) tso tawm
protein ntau. Cov ntsiab lus ntawm cov protein tau ntsuas ntau yam siv Pierce™ 660nm Protein Assay Reagent (Thermo Fisher Scientific Meridian,Waltham, MA, USA) thiab NanoDrop OneC ntsuas (Thermo Fisher Scientific). Covfluorescencespectrum tau txiav txim siab rau txhua tus qauv {{0}µL (muaj 1.5μg ntawm tag nrho cov protein) siv lub multimode grating

microplate nyeem ntawv qauv SH-9000Lab nrog SF6 Data Treatment Software(Corona Electric, Ibaraki, Nyiv). Txhua qhov kev ntsuas tau ua peb zaug.


image

Kev ntsuas ntawm autofluorescence ntawm tus kab mob nyob ib leeg (wrap-drop method)

Randomly xaiv worms raug ntxuav nrog M9 tsis, thiab tom qab ntawdib tus kab mob tau muab tso rau hauv 1.0 µL ntawm M9 tsis nyob ntawm Saran Wrap clingfi ualm (Asahi Kasei, Tokyo, Nyiv) tau ncav cuag 384- lub phaj dub zoo(STEM, Tokyo, Nyiv). Cov hollow me me tau tsim ntawm txhua qhov dej siv areplicator (Watson, Kobe, Nyiv). Qhov kev hloov kho no yog txhawm rau rov qab cov kab mob kom zoo tom qab kev ntsuas kom lub hnub nyoog hloov pauv ntawm autofluorescence ntawm txhua tus cab tuaj yeem taug qab. Qhov clingfi ualm tau xaiv vim nws pib-fluoresced tsawg dua lwm yam lag luam muajfi ualms ua.

Txawm li cas los xij, cov ntaub ntawv dawb paug (M9 tsis tsuas yog) tau kuaj peb zaug rautxhua qhov dej, xav txog qhovkev hloov pauvlos ntawm qhov zoo mus rau qhov zoo. Cov kev txwv tsawg kawg nkaus thiab qhov txwv kom muaj nuj nqis tau txiav txim siab raws li cov ntaub ntawv tsis muaj nyob rau txhua hnub raws liμ (lub ntsiab lus ntawm qhov blank)ntxiv3.29σ (standard deviation) thiabμntxiv√2 × 10σ, raws. Lub autofluorescence nyob rau hauv lub cev ntawm txhua tus cab

tau ntes nrog multimode grating microplate nyeem ntawv. Tom qabKev ntsuas, txhua tus cab tau khaws cia ntawm tus kheej ntawm 4.0-cmTxoj kab uas hla phaj npog nrog OP50 (2 mg / 10μL M9 tsis) ntawm 25 degree. Txhuakev soj ntsuam tau ua tiav ntawm qhov tsawg kawg nkaus ntawm 20 worms thiab rov ua dua ob zaug.


Kev ntsuas lub neej expectancy

Cov kab mob tau khaws cia rau ntawm mNGM daim hlau npog nrog OP50 ntawm 25 degree txog 13 hnub. Tom qab kev ntsuam xyuas los ntawmfluorescenceassay, 30 worms txhua tustsiv mus rau cais mNGM daim hlau tshiab uas npog nrog OP50. Cov phiajcim tau muab tso rau ntawm 25 degree, thiab cov kab mob nyob thiab tuag tau qhab nia txhua 24 teev. Tus kab mob tau suav hais tias tuag thaum nws tsis tuaj yeem teb rau qhov kev sib tw nrog tus cab cab.

anti- senescence cistanche function


AGEs tom qab hauv vitro glycation

Kev rho tawm cov protein tau ua raws li tau piav qhia dhau los. Peb-hnub worms tau siv rau kev tsim glycation. Cov qauv protein (2 mg / mL)500 mM qabzib, 100 mM ribose, lossis 500 mM fructose(fi uaconcentration). Tag nrho cov qauv raug incubated ntawm 37 degree rau 0–4 lis. Aliquot (50μL) tau muab xa mus rau lub qhov dej ntawm lub phaj polystyrene
(Sumitomo Bakelite, Tokyo, Nyiv) thiab incubated rau 2 teev ntawm 37 degree . Tom qabtshem tawm cov qauv kev daws teeb meem, lub phaj tau ntxuav nrog PBS-T(phosphate-buffered saline (PBS), 0.05 feem pua ​​Tween 20); 250μL ntawm 3% skim mis nyujtau ntxiv rau txhua qhov dej, thiab lub phaj yog incubated rau 2 h ntawm 37 degree. Txhuazoo ces tau ntxuav nrog PBS-T, thiab 100μL ntawm anti-AGE (anti-CML)
monoclonal antibody (Clone No. 6D12, Trans Genic, Fukuoka, Nyiv; 75 ng /mL) tau muab faib rau txhua qhov dej; lub phaj ces yog incubated nyob rau hauv chav tsevkub rau 1 h. Tom qab ntxuav nrog PBS-T, 100μL ntawm peroxidase-conjugatedtshis anti-nas IgG antibody (Rockland Immunochemicals, Inc., Limerick, PA, USA; 1:150,000) tau muab faib rau txhua qhov dej, thiab
lub phaj yog incubated nyob rau hauv chav tsev kub rau 1 h. Tom qab ntxuav nrog PBS-T, 100μL ntawm cov tshuaj ua haujlwm (tetramethylbenzidine (TMB) staining solution: peroxide tov= 1:1; ELISA POD substrate TMB cov khoom siv, Nrov,Nacalai Tesque, Kyoto, Nyiv) tau muab xa mus rau txhua qhov dej, thiab lub phaj tau khaws cia ntawm chav sov hauv qhov tsaus ntuj kom txog thaum quenching los ntawm

ntxiv ntawm 100μL ntawm 1 mol / L sulfuric acid rau ib qhov dej. Lub absorbance ntawm txhuazoo ntawm 450 nm (sub: 650 nm) ces tau ntsuas tam sim nrog tus nyeem ntawv microplate. ELISA tau ua peb zaug rau txhua qhov kev xeem.


Western blotting rau aging worms

Cov qauv raug kho nrog 4 × Bolt™ LDS Sample Buffer (Thermo Fisher Scientififi uac) thiab 10 × Bolt™ Tus neeg saib xyuas txo qis (Thermo Fisher Scientififi uac), thiab txhua tus qauv muaj 1.5μg ntawm tag nrho cov protein tau khiav ntawm Bolt™ 4–12% Bis-Tris Plus Gel (Thermo Fisher Scientififi uac). Tom qab ntawd cov proteins tau pauv los ntawm gel mus rau polyvinylidenefluoride membrane (ATTO, Tokyo, Nyiv) thiab thaiv nrog 5% skim mis nyuj hauv PBS- 0.1 feem pua ​​Tween 20 rau 1 teev. Lub membrane tau incubated hmo ntuj ntawm 4 degree nrog anti-AGE (anti-CML) monoclonal antibody (Clone No. 6D12 ntawm 1:1000), ntxuav nrog PBS-T, thiab ces incubated nyob rau hauv chav tsev kub rau 1. h nrog peroxidase-conjugated tshis los tiv thaiv nas IgG antibody (1:25,000). Tom qab ntxuav nrog PBS-T, daim nyias nyias tau tsim nrog ECL prime western blotting detection reagent (GE Healthcare UK, Little Chalfont, England) thiab luam theej duab siv ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) lossis ImageQuant LAS 500 (GE Healthcare UK). Membranes ces tau ntxuav nrog PBS-T, incubated nrog Western BLoT Stripping Buffer (Takara, Shiga, Nyiv) rau 30 min ntawm chav tsev kub, thiab ntxuav dua nrog PBS-T. Lub daim nyias nyias tau rov soj ntsuam los ntawm cov tshuaj tiv thaiv vitellogenin antibodies YP115 thiab YP170 (txhua 1:10,000) ntawm 4 degree thaum hmo ntuj42, ntxuav nrog PBS-T, thiab incubated nrog tshis anti-rat IgG antibody conjugated nrog peroxidase (1:2000) (Proteintech, Rosemont, IL, USA) ntawm chav tsev kub rau 2 h. Rau kev thauj khoom ntawm cov tswj, daim nyias nyias tau rov soj ntsuam siv cov tshuaj tiv thaiv anti-actin (Clone No. C4; Merck KGaA, Darmstadt, Lub teb chaws Yelemees) thiab peroxidase-conjugated anti-nas antibody (GE Healthcare UK). Qhov ntom ntawm vitellogenin (YP170 thiab YP115) thiab CML bands nyob rau hauv txhua txoj kab tau normalized tiv thaiv cov actin bands nyob rau hauv txoj kab. Band densities tau txheeb xyuas siv ImageQuant TL software (GE Healthcare). Txhua qhov kev ntsuam xyuas tau ua ob zaug.


Cov teebmeem ntawm ribose thiab rifampicin ntawm AGEs hauv vivo

Peb-hnub worms tau coj los ntawm mNGM uas muaj 400 mM riboselos yog 400 mM sorbitol kom phim lub osmolarity ntawm siab ribose; worm zaub mov tau muab raws li cua sov (100 degree, 10 min)-tua OP50 los tiv thaiv cov kab mob los ntawm fermenting qab zib. Txhawm rau tiv thaiv kev kis kab mob los ntawm cov menyuam yaus, cov kab mob tau hloov mus rau cov phiaj tshiab txhua hnub rau 4 hnub kom txog thaum lawv muaj tag nrho.

tiavqe-tso (thaum 7 hnub). Txhawm rau ntsuas qhov cuam tshuam ntawm ribose, peb tau ua qhov kev ntsuam xyuas ciaj sia thiab ntsuas autofluorescence. Ib tug worm yogxav tias tuag thaum nws tsis teb rau ib tug mos mos kov nrog ib tug cab picker. Cov kab mob uas tuag los ntawm kev ua raws li phab ntsa ntawm lub phaj tsis suav nrog hauv kev tshuaj xyuas thiab raug censored. Hauv kev sim cais,MGM muaj 50 µM (kawgconcentration) rifampicin (dissolved hauvDMSO) tau siv. Txhua qhov kev ntsuas tau rov ua dua ob zaug.


anti- senescence cistanche function

Daim duab 6 Nws pib-fluorescence ntawm vitellogenin (Vit) thiab elongation factor (EF). ibSpectrophotometric (ex 325/em 350–600) ntawm vitellogenin thiabelongation yam ua ntej thiab tom qab glycation hauv vitro los ntawm ribose rau 14 hnub: gly-Vit thiab gly-EF yog glycated vitellogenin thiab glycate elongationfactor, raws. Riboflfl uaavin (Rib) qhia ua tus sawv cevlfluorescent compound. Extract los ntawm 17-hnub qub worms (wild-type N2) yog qhia rau kev sib piv (C. elegans). b Hauv vitro glycation ntawm vitellogenin thiab elongation yam los ntawm ribose yielded nce hauv autofluorescence
dhau sijhawm.c Western blotting tsom xam ntawm AGE thiab vitellogenins hauv cov qauv muab rho tawm los ntawm cov kab mob (tsiaj qus N2) cov neeg muaj hnub nyoog sib txawv. Blots tau muab los ntawm tib gel thiab tau ua tiav nrog txhua cov tshuaj tiv thaiv kab mob.d Quantificationntawm vitellogenins thiab AGEs los ntawm sab hnub poobblotting siv densitometry. Cov kev sim tau rov ua dua ob zaug, thiab cov ntaub ntawv los ntawm tus neeg sawv cev sim tau qhia. Ob kab thiab kab dotted hauv xim liab qhia txog kev hloov pauv ntawm AGEs, thaum cov xim dub qhia txog cov vitellogenins. Kaw lub voj voog thiab hla yog rau cov ntaub ntawv ntawm kev sib tw rau YP170 thiab YP115, ntsig txog


Kev txheeb xyuas cov kab mob qub kab mob tshwj xeeb

Mass spectrometry tsom xam ntawm rho tawm los ntawm 3- thiab 17- hnub qub nematodestau ua ntawm AB SCIEX 5800 LC-MALDI-TOF mass spectrometer (Newark, NJ, USA) nrog Protein Pilot version 4.0. Ob lub matrices taudigested los ntawm trypsin thiab tov nrog ib tug matrix tov (7 mg / L ntawm - cyano-4-hydroxycinnamic acid hauv 0.1 feem pua ​​(v/v) trifluoroacetic acid (TFA), 70 feem pua
(v/v) acetonitrile (ACN)). Covphooj ywgtus nqi siv rau kev sib cais yog 300ML / min,thiab kev sib cais tau ua tiav los ntawm kev siv cov kab hluav taws xob ntawm ob theem txawb ({{0}}}.1 feem pua ​​(v/v) TFA hauv 2 feem pua ​​ACN (cov kuab tshuaj A) thiab 0.1 feem pua ​​(v/v) TFA hauv 80 feem pua ​​ACN(cov kuab tshuaj B)) hauv cov proportions nram qab no: A / B= 100/0–50/50 (60 feeb), A/B= 50/50–0/100 (20 min), A/B= 0/100 (10 feeb). MALDI-TOF Mass system tau siv los muab peptide-massntiv tes. Peptide matching thiabKev tshawb nrhiav protein tau ua tawm tsam Swiss-Prot version 57.0 database.


Extracted proteins tau yaj nyob rau hauv 50 mM ammonium bicarbonate(AmBic) kom tawm tau cov protein ntau ntawm 0.1 thiab 1 µg/µL. Txhawm rau kom ua tiav qhov solubility ntawm cov protein, suav cov ntim dejRapigest (Waters Corporation, Milford, MA, USA) tau ntxiv rau cov txiaj ntsigfi uanal concentrations ntawm 0.1–0.2 feem pua ​​Rapigest nyob rau hauv cov qauv ua ntej digestion. Cov qauv
tau rhuab ntawm 40 degree nrog co rau 10 min; cov ntsiab lus ces yogcentrifuged, thiab qhov tshwm sim pellet raug tshem tawm hauv AmBic uas muaj 10 mM DTT. Cov qauv tau ua kom sov ntawm 80 degree rau 15 min thiab pelleted ntawm chav tsev kub. Ib qho aliquot ntawm 200 mM iodoacetamide (IAM) hauv 50 mMAmBic tau ntxiv rau txhua tus qauv kom ua tiav akawgIAM concentration

20 mM (nyob rau hauv lub xub ntiag ntawm 2 × molar tshaj ntawm DTT); qhov sib tov ces yogincubated nyob rau hauv qhov tsaus ntuj ntawm chav tsev kub rau 30 min kom tso cai rau cov tshuaj tiv thaiv alkylation mus ntxiv. Trypsin hauv 50 mM AmBic tau tov nrog txhua qhov tshuaj ntawm 1:50 (trypsin: protein concentration). Cov qauv tau zom rau yam tsawg kawg 4 teev lossis hmo ntuj ntawm 37 degree nrog co. Ua rawscentrifugation, TFA thiab ACN tau ntxiv rau kev tawm loskawgconcentrations ntawm 0.5–1.0 feem pua ​​TFA thiab 2 feem pua ​​ACN los ntawm ntim. Cov qauv tau shaken rau 2 teev ntawm 60 degree. Tom qab centrifugation ntawm 22,200 ×g rau 5 min, tus supernatant yogpipetted rau hauv lub autosampler vial. Proteins tau zom nrog trypsin ua ntejlos soj ntsuam los ntawm kev rov qab-theem kua chromatography siv qhov kawg 3000RSnanoLC system (Thermo Fisher Scientific) ua ke rau ESI-Q-TOF system (Impact II Bruker Daltonics). Cov poov xab cawv dehydrogenase(ADH1_YEAST) protein tau spiked nyob rau hauv cov qauv raws li tus qauv sab hauv;spiking tau ua kom muaj qhov ntau ntawm kwv yees li 50 fmol ntawmtxheem ntawm kab.


Autoflfluorescence tom qab hauv vitro glycation

Vitellogenin hauv PBS tov ({{0}}.053 µM) tau yuav los ntawm Biosense Laboratories AS (Bergen, NORWAY). Elongation factor solution (1.17 µM) tau yuav los ntawm Abnova Corporation (Taipei City, Taiwan). Cov kev daws teeb meem no tau glycated nrog 0.1 mM ribose rau 23 hnub ntawm 37 degree. Riboflfl uaavin tau yuav los ntawm Sigma (Tokyo, Nyiv) thiab yaj hauv PBS ntawm 0.1 mM. Txhua qhov kev daws teeb meem tau ntsuas los ntawm fluorescence spectrophotometric nyob rau hauv tib yam mob.

Immunofluorescencelos ntawm cov menyuam yaus thiab laus wormsHnub nyoog-synchronized CB1003 pub OP50 tau incubated ntawm 25 degree. Peb-hnub thiab 13- cov neeg laus hnub nyoog muaj hnub nyoog tau permeabilized siv Bouin's tube fixation raws tu qauv43.


anti- senescence cistanche function

Daim duab 7 Fluorescence duab ntawm 3-hnub nyoog thiab 13-hnub qub kynu-1 mutants. ib-cPeb-hnub worms thiabd–f 13-hnub qub cab, nrog cov duab nyoos hauv kab 1. Kom tsis txhob muaj qhov tshwm sim ntawm kev tuagfluorescence uas pib los ntawm 2 hnub ua ntej tus kab mob' demise, tus mutantkynu-1tau siv. Autofluorescence pom nyob rau hauv cov duab hauv kab 2 thiab 3. Cov duab hauv kab 3 tau nthuav dav rom qhov qhia (los ntawm cov duab plaub) ntawm cov duab hauv kab 2. Cov kab mob hnub nyoog (13- hnub nyoog) pibfluorescedkos npefifi uacantly siab dua cov kab menyuam yaus (3-hnub-hnub). Txhua qhov ntsuas bar qhia 50μm. g Quantificationntawm xiavfluorescencesiv ImageJ thiab ImageQuant TL software. Txhua bar sawv cev qhov nruab nrab qhov tseem ceeb ntawmfluorescence siv ib 1 mm2 ntawm cuaj kab. ** qhia tau tias tus lej kos npefifi uatsis tuaj yeem sib txawv ntawm 3-hnub-hnub thiab 13-hnub worms ntawm ap tus nqi < 0.01. Cov kab yuam kev sawv cev rau SE.

Cov qauv tautsauhauv Bouin's khonrog methanol / -mercaptoethanolntawm chav tsev kub rau 30 min. Txhawm rau txhawm rau cuticle, worms tau muab tso rau hauvisopropanol thiab khaws cia ntawm–Ci 80 degrees rau 10 feeb, tom qab ntawd incubated ntawm chav tsev kub rau lwm 30 min. Covkhodaws raug tshem tawm thiabhloov nrog BTB tov (25 mM borate buffer, 0.5 feem pua ​​Triton X-100, 2 feem pua
-mercaptoethanol) ntawm chav tsev kub rau 1 h; incubation hauv BTB yogrov qab rau tag nrho peb zaug. Cov worms ces tau pauv los ntawm BTB mus rau BT (25 mM borate buffer, 0.5 feem pua ​​Triton X-100), incubated nyob rau hauv antibody buffer tov (1 × PBS, 0.5 feem pua Triton X-100, 0.1 mM EDTA, {{10}}}.1% bovine serum albumin (BSA), 0.05% sodium azide, pH 7.2) ntawm chav tsev kub rau

1 h, thiab thaiv nrog cov tshuaj tiv thaiv kab mob tsis muaj tshuaj uas muaj 10 feem pua ​​​​tshuaj ntshav(Cosmo Bio, Tokyo, Nyiv) ntawm 4 degree thaum hmo ntuj. Thawj cov tshuaj tiv thaiv muaj xws li nas monoclonal anti-AGE (anti-CML) antibody (Clone No. 6D12; 1:125) thiab anti-pentosidine antibody (Clone No. PEN-12, Trans Genic; 1:5 0). Cov tshuaj tiv thaiv thib ob muaj xws li Alexa Fluor 555- conjugated tshis anti-nas antibody (Abcam, Cambridge, England; 1:100). Tag nrho cov tshuaj tiv thaiv kab mob tau ua tiav siv cov tshuaj tiv thaiv kab mob uas muaj 0.5 feem pua ​​BSA. Cov qauv tau muab tso rau hauv cov iav slides siv VECTASHIELD antifade mounting nruab nrab (Vector Laboratories, Burlingame, CA, USA).



Fluorescence microscopy

Fluorescence dluab ntawm 3- los yog 13-hnub-laus cov neeg laus cab, mounted rau iav slides raws li tau piav saum toj no, raug kaw siv ib tug invertedfluorescencemicroscope (BZ-X700; Keyence) nrog 10 × lub hom phiaj lens (CFI Plan Fluor DL1 0 ×/0.30). Autoflfl uauorescence ntawm worms ib tug liabfluorescencelos ntawm AlexaFluor 555 tau saib nrog BZ-X DAPI (excitation wavelength (Ex), 360 ± 20 nm; emission wavelength (Em), 460 ± 25 nm) thiab TRITC (Ex, 545 ±12,5 nm; Em, 605 ± 35 nm)lim, raws. Tshooj duab tauntes los ntawm Sectioning hom nrog ib-dimensional slit hom nrog adav 32, thiab Z-pitch pitch 0.7μm rau 40-μm-tuab qauv. Yuav kom suav cov xiavfluorescence, covfluorescenceintensities ntsuas los ntawm Duab
Quant TL software (GE Healthcare) tau normalized rau qhov ntom ntom ntawm 1 hli2 ntawm tus kab mob's projection cheeb tsam. Lub cev loj tau txiav txim siab nrog Adobe Photoshop Elements thiab ImageJ software tsim los ntawm National Institutes of Health. Hauv qhov system no, thaj tsam ntawm tus kab mob's projectiontau kwv yees tau txiav thiab siv los ua qhov ntsuas ntawm lub cev loj.



Kev txheeb cais

Kev sib raug zoo ntawm lub neej expectancy tau suav nrog Spearman's qeb correlation coefficient. Cov qib autofluorescence tom qab hauv vitro glycation tau muab piv nrog ob-factor factorial ANOVA thiab Scheffe's F xeem. CovELISA qhov tseem ceeb tom qab hauv vitro glycation tau muab piv nrog ib qho-qhov ANOVA thiab Dunnett xeem rau ntau qhov sib piv. Nematode ciaj siatau xam los ntawm Kaplan–Meier txoj kev, thiab qhov sib txawv ntawm kev ciaj sia tau raug sim rau qhov tseem ceeb los ntawm kev siv lub log-qib xeem. Cov txiaj ntsig ntawm autofluorescence tom qab kev kho rifampicin thiab kev laus ntawm covdaf -2thiabkynu-1cov mutants tau muab piv rau txhua tus siv Tub Kawm Ntawv's t test, rov ntsuas ob-yam ANOVA, thiab Mann–WhitneyU xeem. autofluorescence muaj nuj nqis tom qab kev kho ribose rau N2 lossis covkynu-1mutant tau muab piv nrog cov nonparametric Steel–Dwass method. Qhov ntom ntawm autofluorescence microscopy tau muab piv nrog Cov Tub Ntxhais Kawm's t-test. Thaum pom qhov tseem ceeb, cov ntaub ntawv yog classififi uaed ua*p < 0.05 and **p < 0.01. All Kev txheeb xyuas txheeb cais tau ua nrog Microsoft Excel ntxiv nroglub add-in softwarentxivStatcel 3 (OMS, Tokyo, Nyiv) thiab JSTAT rau Windows(Nankodo, Tokyo, Nyiv).


Tshaj tawm cov ntsiab lus

Cov ntaub ntawv ntxiv txog kev tshawb fawb tsim muaj nyob rau hauv Kev Tshawb Fawb XwmKev Qhia Summary txuas rau kab lus no.


DATA muaj nyob

Cov ntaub ntawv tsim tawm thaum txoj kev tshawb fawb tam sim no muaj los ntawm kev sib raug zooing sau ntawm qhov kev thov tsim nyog.



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