Abstract.Arbutin yog lub ntuj tsim tawm los ntawm ntau yam nroj tsuag, nrog rau cov nplooj bearberry, uas ua rau muaj kev cuam tshuam ntau yam nrog rau cov tawv nqaij dawb, thiab tiv thaiv inflammatory thiab oxidative kev ntxhov siab tiv thaiv. Txawm li cas los xij, cov teebmeem ntawm arbutin ntawm osteoblasts tseem tsis tau paub. Txoj kev tshawb fawb tam sim no tsom los tshawb xyuas cov haujlwm thiab cov txheej txheem ntawm arbutin ntawm kev loj hlob thiab kev sib txawv ntawm MC3T3-E1 nas osteoblast precursor hlwb hauv vitro. Kev loj hlob ntawm MC3T3-E1 hlwb kho nrog arbutin tau soj ntsuam siv Cell Counting Kit-8 assay and a 5-ethynyl-2'-deoxyuridine labeling assay. Tsis tas li ntawd, lub voj voog ntawm tes thiab apoptosis tau raug tshuaj xyuas los ntawm kev ntsuas cytometry ntws. Cov teebmeem ntawm arbutin ntawm kev sib txawv ntawm osteoblast tau tshawb xyuas siv alkaline phosphatase (ALP) staining thiab los ntawm kev tshuaj xyuas mRNA qhia qib ntawm collagen hom I 1 saw (COL1A1), pob txha-carboxyglutamate protein (BGLAP) thiab Sp7 transcription factor (SP7). Txhawm rau tshawb xyuas cov txheej txheem molecular hauv qab arbutin muaj nuj nqi hauv kev txhawb nqa osteogenesis, mRNA thiab protein qhia theem ntawm runt-related transcription factor 2 (RUNX2) thiab -catenin tau txheeb xyuas los ntawm qhov rov qab hloov pauv-quantitative polymerase saw cov tshuaj tiv thaiv thiab sab hnub poob blotting. Arbutin txhawb nqa MC3T3-E1 cell proliferation thiab nce qhov sib piv ntawm cov hlwb hauv S-theem. Kev kho mob nrog arbutin nce ALP kev ua haujlwm thiab mRNA qhia qib ntawm COL1A1, BGLAP, thiab SP7 hauv MC3T3-E1 hlwb. Tsis tas li ntawd, cov protein thiab mRNA qhia theem ntawm RUNX2 thiab -catenin tau nce ntxiv tom qab kev kho mob nrog arbutin. Ua ke, qhov kev tshawb pom tam sim no tau qhia tias arbutin tuaj yeem txhawb kev loj hlob thiab kev sib txawv ntawm MC3T3-E1 hlwb ntawm Wnt / -catenin signaling pathway.
Raws li kev tshawb fawb cuam tshuam,cistancheyog ib hom tshuaj ntsuab uas hu ua "cov tshuaj ntsuab miracle uas ua rau lub neej ntev". Nws lub ntsiab tivthaiv yogcistanoside, uas muaj ntau yam teebmeem xws liantioxidant, anti-inflammatory, thiab kev tiv thaiv kev ua haujlwm nce qib. Lub mechanism ntawm cistanche thiabtawv nqaij dawbnyob rau hauv cov nyhuv antioxidant ntawm cistanche glycosides. Melanin nyob rau hauv tib neeg daim tawv nqaij yog tsim los ntawm oxidation ntawm tyrosine catalyzed los ntawmtyrosinase, thiab cov tshuaj tiv thaiv oxidation yuav tsum muaj kev koom tes ntawm cov pa, yog li cov pa oxygen dawb radicals hauv lub cev dhau los ua ib qho tseem ceeb cuam tshuam rau kev tsim melanin. Cistanche muaj cistanoside, uas yog ib qho antioxidant thiab tuaj yeem txo qhov tsim ntawm cov dawb radicals hauv lub cev, yog liinhibiting melanin ntau lawm.
Yog xav paub ntxiv:
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Osteoporosis yog ib qho teeb meem kev noj qab haus huv thiab kev noj qab haus huv uas muaj cov pob txha loj thiab cov pob txha tsis zoo, uas ua rau muaj kev pheej hmoo ntawm cov pob txha tawg (1,2). Hauv European Union (EU), qhov muaj feem ntau ntawm cov pob txha pob txha hauv cov tib neeg muaj hnub nyoog Ntau dua lossis sib npaug li 50 xyoo tau kwv yees li ntawm 6.6 thiab 22.1 feem pua rau cov txiv neej thiab poj niam xyoo 2010, raws li. Kev lag luam, tag nrho cov nqi ntawm cov pob txha pob txha rau EU yog ~ € 37.4 billion hauv 2010 (3). Raws li qhov nce hauv lub neej expectancy thiab cov neeg laus zuj zus, cov neeg coob tuaj yeem raug kev puas tsuaj los ntawm pob txha pob txha yav tom ntej (4). Osteoporosis yog tshwm sim los ntawm qhov tsis sib xws ntawm cov pob txha tsim, kho los ntawm osteoblasts, thiab resorption, kho los ntawm osteoclasts (5). Cov kev kho mob osteoporosis muaj xws li tshuaj tiv thaiv resorption (xws li bisphosphonates thiab denosumab), calcitonin, thiab estrogen. Txawm li cas los xij, cov tebchaw no muaj qee qhov kev txwv; tshwj xeeb, lawv txo qis osteogenic turnover tus nqi, thiab los ntawm kev txo cov pob txha-remodeling txheej txheem, lawv ua rau ib tug txo nyob rau hauv cov pob txha tsim (6). Kev kho cov tshuaj estrogen tsis yog qhov zoo tagnrho rau kev kho mob osteoporosis mus sij hawm ntev, vim tias cov tshuaj estrogen siab tuaj yeem ua rau cov uterine los ntshav, mob cancer mis, thiab kab mob plawv (7). Tsis tas li ntawd, cov tshuaj tiv thaiv resorption tsis tuaj yeem kho cov pob txha uas ploj lawm. Txawm li cas los xij, cov tshuaj anabolic tuaj yeem tsim cov pob txha thiab ua kom cov pob txha loj (8). Yog li ntawd, nws yog ib qho tseem ceeb los txheeb xyuas qhov kev nyab xeeb tshiab, thiab cov tshuaj muaj txiaj ntsig zoo tuaj yeem txhawb kev tsim cov pob txha.
Arbutin (4-hydroxyphenyl- ‑D-glucopyranoside; Fig. 1) yog ib qho tshwm sim ntawm hydroquinone derivative (molecular mass 272 Da) muaj nyob rau hauv ntau hom nroj tsuag. Cov qib siab ntawm arbutin tau txheeb xyuas hauv cov nroj tsuag suav nrog, marjoram (Origanum majorana, Lamiaceae) thiab pears (Pyrus communis, Rosaceae) thiab, tshwj xeeb, tsev neeg Ericaceae xws li bearberry nplooj (Arctostaphylos uva-ursi) (9). Arbutin nthuav tawm ntau yam haujlwm lom neeg. Piv txwv li, arbutin yuav siv tau
raws li daim tawv nqaij whitening agent; los ntawm inhibiting tyrosinase kev ua hauv melanosomes, arbutin tau txheeb xyuas los txhawb kev depigmentation (10,11). Ib txoj kev tshawb fawb yav dhau los tau pom tias arbutin tuaj yeem ua haujlwm tiv thaiv los ntawm X-irradiation-induced apoptosis los ntawm kev txo qis hauv cov cell ntawm hydroxyl radicals (12). Tsis tas li ntawd, arbutin tuaj yeem tiv thaiv kev sib txawv ntawm osteoclast los ntawm kev txo qis hauv cov cellular qib ntawm superoxide thiab los ntawm kev txo qis ntawm cov khoom siv hluav taws xob ntawm cov tshuab ua kom T hlwb 1 (13). Txawm li cas los xij, cov teebmeem ntawm arbutin ntawm osteoblast muaj nuj nqi tseem tsis paub. Yog li, txoj kev tshawb fawb tam sim no tsom los tshawb xyuas cov teebmeem thiab cov txheej txheem ntawm arbutin ntawm MC3T3-E1 nas osteoblast precursor cell proliferation thiab sib txawv.
Wnt signaling txoj kev yuav cuam tshuam rau osteoblast thiab osteoclast, ncaj qha thiab tsis ncaj, nce pob txha tsim thiab txo cov pob txha resorption (14). Canonical Wnt signaling tuaj yeem tswj cov kev loj hlob, kev sib txawv, thiab kev ua haujlwm ntawm osteoblastic ntawm ntau qib (15). Hauv cov qauv tsiaj, txo qis kev ua haujlwm ntawm inhibitors ntawm Wnt / -catenin signaling pathway los ntawm kev siv cov tshuaj tiv thaiv kab mob tiv thaiv cov protein uas muaj feem xyuam nrog cov protein 1, sclerostin, thiab dickkopf WNT signaling pathway inhibitor 1 (DKK1), thiab me-molecule inhibitors ntawm glycogen synthase. kinase 3 (GSK-3 ) tuaj yeem ua rau pob txha loj thiab txo qhov kev pheej hmoo ntawm pob txha (16). Yog li ntawd, Wnt signaling pathway sawv cev rau lub hom phiaj kho mob rau kev txhim kho cov tshuaj tshiab los kho pob txha (17). Hauv kev tshawb fawb tam sim no, cov teebmeem ntawm arbutin ntawm MC3T3-E1 cell proliferation thiab sib txawv tau raug tshawb xyuas. Tsis tas li ntawd, cov txheej txheem molecular hauv qab arbutin muaj nuj nqi hauv inducing MC3T3-E1 cell sib txawv tau raug tshuaj xyuas.
Cov ntaub ntawv thiab cov txheej txheem
Tshuaj. Arbutin (purity, Ntau dua lossis sib npaug li 98 feem pua) tau yuav los ntawm Dalian Meilun Biotech Co., Ltd. (Dalian, Tuam Tshoj), yaj hauv dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA, Darmstadt, Lub teb chaws Yelemees), thiab khaws cia ntawm ib concentration ntawm 0.5 M. Recombinant human DKK1 yog yuav los ntawm PeproTech, Inc. (Rocky Hill, NJ, USA; cat. no. 120-30).
Cell kab lis kev cai. MC3T3-E1 nas calvarial pre-osteoblasts tau yuav los ntawm Cell Resource Center ntawm Shanghai Institutes rau Biological Sciences ntawm Tuam Tshoj Academy ntawm Sciences (Shanghai, Tuam Tshoj), thiab tau cultured nyob rau hauv -Minimum Essential Medium (-MEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) ntxiv nrog 10 feem pua fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel), 100 µg/ml streptomycin thiab 100 U/ml penicillin (HyClone; GE Healthcare Life Sciences). Cells tau khaws cia rau hauv lub cell culture incubator nrog 5 feem pua CO2 ntawm 37˚C. Qhov nruab nrab tau hloov txhua hnub. Cells ntawm 80 feem pua ntawm cov confluence tau reseeded rau hauv cov ntaub so ntswg kab lis kev cai flasks tom qab kev kho mob nrog 0.25 feem pua trypsin (HyClone; GE Healthcare Life Sciences) rau 1-2 min ntawm 37˚C. Rau kev sim osteoblastic sib txawv, cov hlwb tau kho nrog cov tshuaj osteogenic ntxiv uas muaj 50 µg / ml L-ascorbic acid (Sigma-Aldrich; Merck KGaA) thiab 10 mM -glycerophosphate disodium ntsev hydrate (Sigma-Aldrich; Merck KGaA) rau 7 9 hnub ntawm 3. ˚C. Rau cov kev tshawb fawb siv tshuab, MC3T3-E1 hlwb raug kho nrog DKK1 (0.5 µg / ml) rau 6 h ntawm 37˚C thiab tom qab ntawd kho nrog arbutin (100 µM) rau 3 hnub ntawm 37˚C.
Cell proliferation. Cov khoom siv suav Cell{{{0}}} kev soj ntsuam (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Nyiv) tau siv los ntsuas qhov cuam tshuam ntawm arbutin ntawm cell proliferation. Cov noob tau cog rau hauv 96- cov phiaj zoo ntawm qhov ntom ntawm 5x103 hlwb / qhov dej rau 24 teev ntawm 37˚C. Tom qab ntawd, cov hlwb raug kho nrog arbutin ntawm ntau qhov ntau (0, 10, 50, thiab 100 µM). Tom qab 24, 48, lossis 72 teev, cov hlwb raug kho nrog 9.1 feem pua CCK-8 cov tshuaj uas muaj 10 µl CCK{18}} thiab 100 µl -MEM rau 1-2 teev ntawm 37˚C. Tus nqi kho qhov muag ntawm txhua qhov dej tau ntsuas los ntawm kev nyeem ntawv microplate (ELx808; BioTek Instruments, Inc., Winooski, VT, USA) ntawm lub wavelength ntawm 450 nm. Kev txheeb ze ntawm cellular viability raug xam raws li qhov piv ntawm qhov nruab nrab absorbance ntawm tus qauv thiab kev tswj.
5-ethynyl-2'-deoxyuridine (EdU) labeling assay. Cov txiaj ntsig ntawm arbutin ntawm kev loj hlob ntawm tes tau ntsuas siv EdU Apollo® 567 hauv vitro Imaging kit (Guangzhou RiboBio Co., Ltd., Guangzhou, Suav). Cells raug inoculated nyob rau hauv ib tug 96-zoo phaj ntawm ib tug ceev ntawm 1x103 hlwb/zoo thiab incubated nyob rau hauv -MEM muaj 10 feem pua FBS nrog 0, 10, 50, los yog 100 µM arbutin. Tom qab 72-h incubation, EdU tau ntxiv rau txhua lub qhov dej ntawm qhov concentration ntawm 50 µM ua ntej incubation ntxiv rau 2 teev ntawm 37˚C. Cells raug ntxuav ob zaug nrog PBS thiab kho nrog PBS uas muaj 4 feem pua paraformaldehyde rau 30 min ntawm chav tsev kub (RT). Tom qab ntxuav nrog glycine (2 mg / ml) thiab PBS, hlwb tau permeabilized nrog Triton X-100 (0.5 feem pua) rau 10 feeb ntawm RT. Cells raug incubated nrog 1X Apollo staining tshuaj tiv thaiv kua ntawm RT rau 30 min nyob rau hauv qhov tsaus ntuj. Cell nuclei raug counterstained nrog 1X Hoechst 33342 rau 30 min ntawm RT. EdU-zoo hlwb raug pom los ntawm fluorescence microscopy (magnification, x200; dab noj hnub Ti; Nikon Corporation, Tokyo, Nyiv) nyob rau hauv tsib qhov chaw xaiv random.
Cell cycle thiab apoptosis tsom xam. MC3T3-E1 hlwb tau muab noob rau hauv 6-zoo daim hlau ntawm qhov ntom ntawm 2x105 hlwb/qhov zoo. Tom qab ib tug 24-h incubation, hlwb raug kho nrog arbutin nyob rau hauv concentrations ntawm 0, 10, 50, thiab 100 µM. Cells raug sau tom qab 3 hnub ntawm RT thiab kho nrog 70 feem pua ethanol rau 12 teev ntawm 4˚C. Cells raug ntxuav peb zaug nrog PBS thiab stained nrog propidium iodide (PI) staining tov (Beyotime Institute of Biotechnology, Haimen, Tuam Tshoj) rau 30 min ntawm 37˚C hauv qhov tsaus ntuj. DNA cov ntsiab lus raug ntsuas siv FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) nrog CellQuest Pro software (Version 5.2; BD Biosciences) thiab ModFit LT software (Version 3.0; Verity Software House, Inc., Topsham, ME, USA). Rau kev soj ntsuam apoptosis, cov hlwb kho nrog arbutin tau sau thiab pleev xim nrog fluorescein isothiocyanate-labeled Annexin-V thiab P I (Dojindo Molecular Technologies, Inc.) hauv qhov tsaus ntuj rau 15 feeb ntawm RT. Lub cell apoptotic tus nqi tau soj ntsuam siv FACSCalibur flow cytometer (BD Biosciences) nrog CellQuest Pro software (Version 5.2; BD Biosciences).
Alkaline phosphatase (ALP) staining assay. Osteoblasts tau cog rau hauv {{0}} cov phiaj zoo ntawm qhov ntom ntawm 5x104 hlwb / zoo incubated nyob rau hauv -MEM uas muaj osteogenic ntxiv thiab kho nrog 0 (tswj), 10, 50, los yog 100 µM arbutin. Tom qab 9 hnub ntawm 37˚C, cov hlwb raug ntxuav peb zaug nrog PBS thiab kho hauv 4 feem pua ntawm paraformaldehyde ntawm RT rau 10 min. Cells tau muab yaug peb zaug nrog dej distilled thiab tom qab stained siv lub 5-bromo-4-chloro-3-indolyl phosphate/nitro xiav tetrazolium chloride ALP xim tsim cov khoom siv (Beyotime Institute of Biotechnology) rau 2 h ntawm RT. Stained cells tau yees duab siv lub teeb pom kev zoo (kev nthuav dav, x40; dab noj hnub Ti; Nikon Corporation).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cov noob tau cog rau hauv 6-cov phiaj zoo ntawm qhov ntom ntawm 2x105 hlwb/qhov zoo. Tom qab kev kho mob nrog arbutin ntawm ntau qhov ntau ntxiv rau 3 hnub ntawm 37˚C, tag nrho RNA tau muab rho tawm los ntawm MC3T3-E1 hlwb siv RNAiso Plus reagent (Takara Biotechnology Co., Ltd., Dalian, Suav). Nyob rau hauv tag nrho, 1 µg RNA tau thim rov qab rau hauv cDNA siv PrimeScript RT reagent cov khoom siv nrog gDNA Eraser (Takara Biotechnology Co., Ltd.), raws li cov chaw tsim khoom cov lus qhia. Cov kev mob tshwm sim yog raws li nram no: 42˚C rau 2 feeb, 37˚C rau 15 feeb, thiab 85˚C rau 5 feeb. qPCR tau ua tiav siv qhov sib npaug ntawm cDNA los ntawm txhua tus qauv hauv qhov ntim tag nrho ntawm 20 µl nrog ABI 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Inc., Waltham, MA, USA) siv SYBR premix Ex Taq II (Takara Biotechnology Co., Ltd.). Cov txheej txheem thermocycling hauv qab no tau siv: Pib denaturation ntawm 95˚C rau 30 sec, ua raws li 40 cycles ntawm 95˚C rau 5 sec thiab 60˚C rau 34 sec. Qhov tshwj xeeb ntawm amplification tau soj ntsuam los ntawm kev txheeb xyuas qhov nkhaus, thiab -actin tau txais kev tswj hwm sab hauv. Cov txheeb ze ntawm cov noob qhia theem tau txheeb xyuas siv 2-ΔΔCq txoj kev (18). Cov txheej txheem ntawm cov primers siv yog cov hauv qab no: Runt-related transcription factor 2 (RUNX2), forward 5'-CCAACCGAGTCATTTAAGGCT-3', thim rov qab 5'-GCTCACGTCCGCTCATCTTG-3'; collagen hom I 1 saw (COL1A1), forward 5'-GCCTCCCAGAACATC ACCTA‑3', thim rov qab 5'-GCAGGGACTTTGAGGTTG-3';pob txha -carboxyglutamate protein (BGLAP), rau pem hauv ntej 5'-CGCTACCTTGGAGCCTTCAGT-3', rov qab 5'-AGGCGGTCTTCAAGCCATAC-3'; Sp7 transcription factor (SP7), rau pem hauv ntej5'-AAGGTTAGGGCAAGGCTTC-3', rov qab 5'-CGTCAGAGGAGTGAACCT-3'; -catenin, pem hauv ntej 5'-ATGGAGCCGGACAGAAAAGC-3', rov qab 5'-CTTTGCCACTCAGGGAAGGA-3'; -actin, forward 5'-GGCTGTATTCCCCTCCATCG-3', rov qab 5'-CCAGTTGTTAACAATGCCATGT-3'.
Western blot tsom xam. MC3T3-E1 hlwb tau muab noob rau hauv 6-cov phiaj zoo ntawm qhov ntom ntawm 2x105 hlwb/qhov zoo. Cov hlwb raug kho nrog arbutin ntawm ntau qhov ntau rau 3 hnub thiab yaug peb zaug nrog dej khov-txias PBS. Tag nrho cov cellular protein tau muab rho tawm los ntawm MC3T3-E1 hlwb siv radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) uas muaj 1 mM phenylmethylsulfonyl fluoride. Proteins raug cais tawm tom qab centrifugation ntawm 13,800 xg rau 15 min ntawm 4˚C. Protein concentration tau txiav txim siab siv cov khoom kuaj bicinchoninic acid protein (Beyotime Institute of Biotechnology). Qhov sib npaug ntawm cov protein (20-30 µg) nyob rau hauv txhua tus qauv raug cais los ntawm 10 feem pua SDS-PAGE rau 2 h ntawm qhov hluav taws xob tas li (110 V) thiab xa mus rau polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). Membranes raug thaiv hauv TBS ntxiv rau Tween-20 (TBST; 20 mM Tris-HCl, 150 mM NaCl pH 7.5 thiab 0.1 feem pua Tween-20) muaj 5 feem pua cov mis nyuj tsis rog ntawm RT rau 2 teev thiab tom qab ntawd incubated hmo ntuj ntawm 4˚C nrog thawj cov tshuaj tiv thaiv tsim nyog. Cov tshuaj tiv thaiv uas siv tau yog cov hauv qab no: Luav monoclonal anti- -catenin (1:5,000; cat. no. ab32572; Abcam, Cambridge, MA, USA), luav polyclonal anti-RUNX2 (1: 1,000; cat. no. ab23981; Abcam) thiab nas polyclonal anti- -actin (1:1,{45}}; cat. no. AF0003; Beyotime Lub koom haum ntawm Biotechnology). Tom qab ntawd, cov ntaub so ntswg tau ntxuav peb zaug nrog TBST, thiab PVDF daim nyias nyias tau muab tso rau ntawm horseradish peroxidase (HRP)-conjugated tshis tiv thaiv luav immunoglobulin G (IgG; 1:10,000; cat. no. ZB{{ 52}}; OriGene Technologies, Inc., Beijing, China) los yog HRP-conjugated tshis anti-nas IgG (1:10,000; cat. no. ZB-2305; OriGene Technologies, Inc.) ntawm RT rau 2 h . Proteins tau pom los ntawm kev siv Enhanced Chemiluminescence reagents (Thermo Fisher Scientific, Inc.) thiab kuaj pom nrog chemiluminescence detection system (Amersham Imager 600; GE Healthcare Life). Proteins tau suav nrog siv ImageJ software (version 1.52; National Institutes of Health, Bethesda, MD, USA). Tom qab normalization, txheeb ze protein qhia qib tau suav nrog -actin raws li kev tswj hwm sab hauv.
Kev txheeb cais. Txhua qhov kev sim tau rov ua nws tus kheej tsawg kawg peb zaug. Kev txheeb xyuas txheeb cais tau ua tiav siv GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab ± tus qauv sib txawv, thiab qhov sib txawv tseem ceeb tau txheeb xyuas los ntawm ib txoj kev tsom xam ntawm qhov sib txawv ua ke nrog Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Cov txiaj ntsig
Arbutin txhawb nqa MC3T3-E1 cell proliferation. Cov teebmeem ntawm arbutin ntawm MC3T3-E1 cell proliferation raug tshuaj xyuas siv CCK-8 assay. Arbutin tau tswj hwm ntawm ntau qhov sib xyaw (0, 10, 50, thiab 100 µM) rau 24, 48, thiab 72 h, thiab CCK-8 kev soj ntsuam tau ua (Fig. 2A). Tom qab 24 teev, tsis muaj qhov sib txawv ntawm cov ntaub ntawv txheeb xyuas hauv osteoblast proliferation piv nrog cov hlwb tsis kho. Txawm li cas los xij, MC3T3-E1 cell proliferation tau nce ntxiv tom qab kev kho mob nrog arbutin ntawm 100 µM tom qab 48 thiab 72 teev. EdU daim ntawv lo tshuaj ntsuam xyuas tau ua tom qab 72 h, thiab cov txiaj ntsig tau pom tias feem pua ntawm EdU-positive MC3T3-E1 hlwb kho nrog arbutin ntawm qhov concentration ntawm 50 thiab 100 µM tau nce ntau dua piv nrog kev tswj tsis tau kho (Daim duab 2B thiab C). .
Arbutin accelerates lub voj voog ntawm tes. Cov teebmeem ntawm arbutin ntawm kev nce ntawm lub voj voog ntawm tes tau raug soj ntsuam los ntawm kev txheeb xyuas lub voj voog ntawm tes (Fig. 3). Kev kho mob nrog arbutin ntawm 50 thiab 100 µM coj mus rau qhov nce hauv feem pua ntawm cov hlwb hauv S-theem (Daim duab 3A thiab C) thiab 100 µM arbutin ua rau txo qis feem pua ntawm cov hlwb hauv G1-theem ( Fig. 3D). Tsis muaj qhov sib txawv ntawm qhov tseem ceeb hauv 10 µM pawg piv nrog pawg tswj hwm. Cov teebmeem ntawm arbutin ntawm MC3T3-E1 apoptosis kuj tau soj ntsuam los ntawm kev siv cytometry ntws (Fig. 3B thiab E). Tus nqi ntawm apoptosis tsis tau hloov pauv ntau tom qab kev kho mob nrog arbutin ntawm 10, 50, thiab 100 µM piv nrog kev tswj hwm.
Cov teebmeem ntawm arbutin ntawm ALP kev ua haujlwm. Cov teebmeem ntawm arbutin ntawm kev sib txawv ntawm osteoblast tau txheeb xyuas los ntawm ALP staining. Tom qab 9 hnub, kev kho mob nrog ntau qhov ntau ntawm arbutin (0, 10, 50, lossis 100 µM) tshwj xeeb tshaj yog nce kev ua haujlwm ALP piv nrog pawg tswj hwm (Daim duab 4A). Cov kev tshawb pom tam sim no qhia tias arbutin tuaj yeem ua rau muaj kev ua haujlwm ntawm ALP hauv MC3T3-E1 hlwb.
Cov teebmeem ntawm arbutin ntawm mRNA qhia theem ntawm COL1A1, -catenin, RUNX2, BGLAP, thiab SP7. mRNA qhia theem ntawm COL1A1, -catenin, RUNX2, BGLAP, thiab SP7 tau soj ntsuam hauv MC3T3-E1 hlwb siv RT-qPCR tom qab kev kho mob nrog arbutin ntawm ntau qhov ntau (0, 10, 50, lossis 100 µM) rau 3 hnub. COL1A1 thiab -catenin qhia qib tau nce hauv osteoblasts kho nrog 10, 50, thiab 100 µM arbutin piv nrog cov hlwb tsis kho (Fig. 4B thiab C). Cov theem qhia ntawm RUNX2, BGLAP, thiab SP7 tau nce ntxiv tom qab kev kho mob nrog arbutin ntawm 50 thiab 100 µM (Fig. 4D-F).
Cov teebmeem ntawm arbutin ntawm cov protein qhia qib ntawm -catenin thiab RUNX2. Txhawm rau tshawb xyuas cov txheej txheem hauv qab ntawm kev sib txawv ntawm arbutin-induced osteoblast, cov protein qhia theem ntawm RUNX2 thiab -catenin raug tshuaj xyuas hauv MC3T3-E1 hlwb los ntawm western blotting (Fig. 5A thiab B). Kev kho mob nrog arbutin ntawm 100 µM nce qib protein ntau ntawm -catenin thiab RUNX2 hauv osteoblasts piv nrog kev tswj hwm (Daim duab 5C thiab D, raws li). Cov txiaj ntsig tam sim no tau qhia tias arbutin tuaj yeem cuam tshuam rau kev sib txawv ntawm osteoblast los ntawm kev tswj hwm cov protein qhia qib ntawm -catenin thiab RUNX2. Txhawm rau tshawb xyuas seb arbutin txhawb kev sib txawv ntawm osteoblastic los ntawm Wnt / -catenin signaling pathway, MC3T3-E1 hlwb raug kho nrog 0.5 µg / ml DKK1 rau 6 teev ua ntej kho nrog 100 µM arbutin.
DKK1 cuam tshuam arbutin-induced RUNX2 protein qhia (Fig. 5E). Cov txiaj ntsig tam sim no tau qhia tias arbutin tuaj yeem cuam tshuam rau kev sib txawv ntawm osteoblast ntawm Wnt / -catenin signaling pathway.
Kev sib tham
Osteoporosis tuaj yeem ua rau muaj pob txha loj thiab kev xiam oob qhab thiab sawv cev rau cov teeb meem muaj hnub nyoog thoob ntiaj teb (19). Cov ntaub ntawv pov thawj pom tau tias qhov tsis sib xws ntawm osteoclasts thiab osteoblasts hauv pob txha tsim thiab resorption tuaj yeem ua rau osteoporosis (20). Qhov tseem ceeb, cov pob txha tsim nyob ntawm osteoblast proliferation thiab sib txawv (21,22).
Muaj ntau yam tshuaj siv los kho pob txha pob txha yog cov tshuaj tiv thaiv kab mob (23); Txawm li cas los xij, cov kev kho mob no tsis tuaj yeem thim rov qab pob txha (24). Cov tshuaj anabolic uas txhawb kev tsim cov pob txha tuaj yeem kho cov pob txha pob txha loj heev thiab poob ntawm pob txha loj (24). Teriparatide yog ib tus neeg sawv cev anabolic uas tau pom los txhawb kev tsim cov pob txha, thiab kev sim tshuaj ntsuam pom tau hais tias kev kho mob nrog teriparatide txo qhov kev pheej hmoo ntawm cov pob txha pob txha tshiab thiab nce pob txha pob txha pob txha hauv lub duav, lumbar qaum, thiab femoral caj dab (25). Tshwj xeeb, teriparatide yog ib qho kev kho mob kim. Yog li ntawd, nws yog ib qho tseem ceeb los tsim cov tshuaj tshiab uas tuaj yeem txhawb kev tsim cov pob txha.
Arbutin yog ib tug cytoprotective tus neeg saib xyuas thiab tsis muaj qhov tseem ceeb cytotoxic cuam tshuam ntawm high concentrations (26). Txawm hais tias cov ntshav concentration ntawm arbutin tsis meej (13), arbutin yog siv los ua cov tshuaj tua kab mob tso zis thiab suav tias yog tus neeg saib xyuas qhov ncauj zoo (27,28). Cov kev tshawb fawb yav dhau los tau pom tias arbutin tuaj yeem nthuav tawm ntau yam kev ua ub no, suav nrog kev ua kom tawv nqaij dawb (10,11), tshuaj tiv thaiv kab mob (29), anticancer (30), thiab kev tawm tsam ntawm osteoclast sib txawv (13). Txawm li cas los xij, kev tshawb fawb ntxiv yuav tsum tau tshawb xyuas qhov cuam tshuam ntawm arbutin ntawm osteoblasts thiab nws lub peev xwm los siv los ua cov khoom siv tshiab rau kev kho mob pob txha. Yog li, txoj kev tshawb fawb tam sim no tau tshawb xyuas qhov cuam tshuam ntawm arbutin ntawm kev loj hlob thiab kev sib txawv ntawm MCET3-E1 hlwb thiab cov txheej txheem hauv qab arbutin muaj nuj nqi hauv vitro.
Cov pob txha tsim muaj feem cuam tshuam nrog tus lej ntawm osteoblasts thiab kev ua haujlwm ntawm ib leeg osteoblasts (31). Tus naj npawb ntawm osteoblasts tuaj yeem nce ntxiv los ntawm kev txhawb nqa ua ntej osteoblast replication lossis sib txawv, lossis los ntawm kev txo cov cell tuag ntawm cov laus osteoblasts (32). Hauv kev tshawb fawb tam sim no, arbutin tau nce kev loj hlob ntawm MC3T3-E1 hlwb tsis cuam tshuam rau tus nqi apoptotic. Tsis tas li ntawd, arbutin nce lub voj voog ntawm tes los ntawm kev hloov cov hlwb ntawm G1-theem mus rau S-theem. Cov txiaj ntsig no tau qhia tias arbutin tuaj yeem ua rau osteoblastic proliferation.
ALP yog qhov cim ntxov ntawm kev sib txawv ntawm osteogenic (33). ALP yog txuam nrog calcification ntawm lub pob txha pob txha thaum tsim pob txha (34). Hauv kev tshawb fawb tam sim no, ALP staining cov txiaj ntsig tau qhia tias osteoblasts kho nrog arbutin ntawm 10, 50, thiab 100 µM, thiab nrog cov tshuaj ntxiv osteogenic rau 9 hnub tau nthuav tawm kev ua haujlwm ntawm ALP, qhia tias arbutin tuaj yeem txhawb kev sib txawv thaum ntxov ntawm osteoblasts.
Osteogenesis tau pom tias yuav tswj hwm los ntawm ntau yam kev hloov pauv, suav nrog RUNX2 thiab SP7, thiab ntau cov pob txha tshwj xeeb matrix proteins, suav nrog ALP,BGLAP, thiab COL1A1 (35). RUNX2 thiab SP7 yog qhov tseem ceebtranscription yam koom nrog hauv kev sib txawv ntawm osteoblastthaum tsim pob txha (36). COL1A1 yog ib qho tshuaj ntxivmatrix protein uas txhawb cov pob txha regeneration thiab osteoblast sib txawv (37) . BGLAP yog ib qho uas tsis yog-collagenouspob txha matrix protein uas tswj cov pob txha turnover thiab pob txhamineralization (38). Cov kev tshawb fawb tam sim no tau pom tias arbutintuaj yeem ua rau mRNA qhia theem tseem ceebosteogenic regulators, suav nrog RUNX2, BGLAP, SP7, thiabCOL1A1. Tsis tas li ntawd, qhov kev qhia theem ntawm -catenin yognce. Cov txiaj ntsig no qhia tias arbutin tuaj yeem txhawb nqaosteoblastic sib txawv los ntawm ib tug mechanism uas muajWnt/ - catenin signaling.
Wnt signaling txoj kev cuam tshuam cov pob txha tsim thaum lub sij hawm kev loj hlob, thiab kev kho pob txha thaum lub sij hawm rov ua dua cov ntaub so ntswg (39). Lub canonical Wnt signaling txoj kev yogtxheeb xyuas tau pib los ntawm Wnt ligand signaling ntawm lub cellnto los ntawm tsawg-density lipoprotein receptor-txogprotein 5 los yog 6 (Lrp5/6) thiab xya-pass transmembraneFrizzled receptor (40). Kev sib cuam tshuam ntawm Wnt ligandsthiab lawv cov receptor yuav inhibit GSK3- hauv cytoplasm,ua rau kev tso tawm ntawm -catenin, cov tshuaj transcriptionalator ntawm canonical Wnt signaling. Tom qab kev tso tawm, -catenintuaj yeem nkag mus rau hauv lub nucleus, yog li tswj cov lus qhiatheem ntawm nws cov hom phiaj (41). RUNX2 belongs rau Runtdomain gene tsev neeg thiab yog ib qho kev hloov pauv uas cuam tshuam nrogosteoblastic sib txawv (42). RUNX2 ua haujlwm tseem ceeblub luag haujlwm hauv kev sib koom tes ntau lub teeb liab txoj hauv kev thaum lub sijhawmosteoblast txawv (43) . Ib tug dab kawm yav dhau loshais tias canonical Wnt signaling yuav ncaj qha tswjRUNX 2. Tshwj xeeb, cov -catenin/HNF1 homeobox Acomplex tuaj yeem qhib qhov qhia theem ntawm RUNX2,yog li txhawb cov pob txha tsim (44). DKK1 muaj zog heevinhibitor ntawm Wnt / -catenin canonical txoj kev, los ntawm kev khirau Lrp5/6 (45,46). Cov txiaj ntsig ntawm kev tshawb fawb tam sim no tau pom zootias kev kho mob nrog arbutin nce cov protein ntauqhia theem ntawm RUNX2 thiab -catenin hauv MC3T3-E1hlwb, thiab DKK1 txo qis cov protein expression theem ntawm RUNX2. Cov txiaj ntsig tam sim no qhia tiasarbutin tuaj yeem txhawb nqa MC3T3-E1 cell sib txawv ntawm canonical Wnt /- catenin txoj kev.
Ua ke, rau qhov zoo tshaj plaws ntawm cov kws sau ntawv paub, qhov kev tshawb fawb tam sim no yog thawj zaug qhia tias arbutin tuaj yeem txhawb kev loj hlob thiab kev sib txawv ntawm MC3T3-E1 hlwb ntawm Wnt / -catenin signaling pathway. Yog li, arbutin tuaj yeem sawv cev rau tus neeg sib tw tshiab rau kev kho pob txha. Txawm li cas los xij, kev tshawb fawb ntxiv yuav tsum txheeb xyuas lub luag haujlwm tshwj xeeb ntawm Wnt / -catenin signaling txoj hauv kev hauv arbutin-induced osteogenesis, suav nrog phosphorylation ntawm -catenin ntawm Ser675 (47); Cov ntaub ntawv ntxiv hais txog lub luag haujlwm ntawm Wnt / -catenin signaling tuaj yeem ua tiav siv Wnt agonists. Yav tom ntej, kev tshawb fawb ntxiv tuaj yeem tshawb xyuas lub peev xwm ntawm arbutin los txhawb kev tsim cov pob txha hauv vivo.
Kev lees paub
Tsis siv tau.
Nyiaj txiag
Txoj haujlwm tam sim no tau txais kev txhawb nqa los ntawm Qhov Kev Pabcuam Loj ntawm National Nature Science Foundation ntawm Tuam Tshoj (daim ntawv pub dawb 81370981) thiab Cov Nyiaj Pab Txhawb Kev Tshawb Fawb ntawm Shengjing Tsev Kho Mob.
Muaj cov ntaub ntawv thiab cov ntaub ntawv
Cov ntaub ntawv siv thiab/lossis tshuaj xyuas thaum lub sijhawm kawm tam sim no muaj los ntawm tus kws sau ntawv raws li qhov kev thov tsim nyog.
Cov neeg sau ntawv pab txhawb
QF, XM, thiab LiY tau xeeb thiab tsim cov kev sim. XM tau ua qhov kev sim thiab sau cov ntawv sau. SL, LiY, LeY, thiab ML tau txheeb xyuas cov ntaub ntawv thiab tshuaj xyuas cov ntawv sau. QF saib xyuas txhua qhov kev tshawb fawb thiab kho cov ntawv sau. Txhua tus kws sau ntawv tau nyeem thiab pom zoo cov ntawv sau kawg.
Kev pom zoo ntawm kev ncaj ncees thiab kev tso cai los koom
Tsis siv tau.
Tus neeg mob tso cai rau kev tshaj tawm
Tsis siv tau.
Kev nyiam sib tw
Cov kws sau ntawv tshaj tawm tias lawv tsis muaj kev nyiam sib tw.
Cov ntaub ntawv
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