PART 1 Cistanche Deserticola Polysaccharide Inhibits OVX-vim cov pob txha poob hauv nas thiab RANKL-induced Osteoclastogenesis

1. Taw qhia

Osteoporosis-ib yam kab mob uas tshwm sim los ntawm cov pob txha poob qis, qhov txawv txav ntawm cov pob txha microstructure, nce pob txha fragility, thiab pob txha tawg (De Martinis, Di Benedetto, Mengoli, & Ginaldi, 2006) - tam sim no cuam tshuam rau ntau tshaj 200 lab tus tib neeg thoob ntiaj teb thiab nthuav tawm kev kho mob thiab Socioeconomic nra rau lub neej niaj hnub (Strom et al., 2011). Kev kho mob osteoporosis feem ntau tsom rau kev hloov tshuaj hormones, bisphosphonate, lossis denosumab therapy. Cov txheej txheem no muaj txiaj ntsig zoo tab sis ua rau muaj kev phiv ntev ntev xws li kev pheej hmoo ntawm mob qog noj ntshav mis thiab atypical femur fractures (Black, Bauer, Schwartz, Cummings, & Rosen, 2012; Rachner, Khosla, & Hof- Bauer, 2011). Yog li ntawd, nws yog ib qho tsim nyog yuav tsum tau tshawb xyuas cov tshuaj uas tsis tuaj yeem cuam tshuam xwbosteoporosistab sis kuj muaj tsawg dua cov kev mob tshwm sim tsis zoo.

Cistanche deserticola(CD), cov khoom noj khoom haus tonic thiab tshuaj siv dav hauv Suav teb, lub npe hu ua "desert ginseng", yog lub qia qhuav succulent nrog nplai nplooj ntawmC. deserticolaYC Ma (Gu, Yang, & Huang, 2016) thiab muaj ntau yam thiab siv tau zootshuajkev ua si, xws li kev tiv thaiv kab mob, tiv thaiv oxidation, thiab tiv thaivosteoporosiskhoom (Hu et al., 2020; Li et al., 2012; Zhang et al., 2014). Cov kev tshawb fawb yav dhau los ntawm cov tshuaj nquag ntawm CD ntawm kev kho pob txha pob txha feem ntau tsom rauphenylethanoidglycosides(Li, Jiang, & Gu, 2018; Xu, Zhang, Wang, Yao, & Ma, 2017). Txawm li cas los xij, CD tseem muaj lwm cov khoom siv zoo xws li iridoids, lignans,polysaccharides(Wang, Zhang, & Xie, 2012). Kev kho mob ntawm cov tshuaj suav tshuaj (TCM) feem ntau yog dej decoction.Polysaccharidesyog cov dej-soluble Cheebtsam thiab feem ntau yuav yog lub ntsiab pharmacodynamic Cheebtsam ntawm TCM. Polysaccharides muab rho tawm los ntawm TCM tau tshaj tawm tias muaj kev tiv thaiv.osteoporosiscov nyhuv thiab ob peb yam kev mob tshwm sim, uas feem ntau tau siv hauv tsev kho mob (xws li, polysaccharides muab rho tawm los ntawm Epimedium brevicornum (Zheng, He, Wu, Cai, & Wei, 2020), Achyranthes bidentata (Zhang, Zhang, Zhang, Wang, & Yan, 2018), thiab Morinda officinalis (Yan li al., 2019)). Yog li ntawd, peb hypothesized tiasCistanche deserticolapolysaccharide(CDP) muab rho tawm los ntawm CD tej zaum yuav yog ib qho tshuaj muaj txiaj ntsig zoo rau kev kho pob txha.

Feem ntau, cov pob txha resorption thiab cov pob txha tsim kom muaj qhov sib npaug ntawm cov pob txha hloov kho hauv kev sib koom tes nrog ntau hom hlwb, suav nrog osteoclasts, osteoblasts, pob txha hauv cov hlwb, thiab osteocytes (Kular, Tickner, Chim, & Xu, 2012). Kev tawg ntawm qhov sib npaug no tuaj yeem ua rau ntau yam kab mob ntawm cov pob txha metabolic, xws liosteoporosis(Zhu et al., 2018) thiab osteosclerosis (Ihde et al., 2011). Osteoclasts, raws li qhov kawg-cov hlwb sib txawv los ntawm cov kab mob mononuclear / macrophage, yog tib lub hlwb uas muaj cov pob txha resorption muaj nuj nqi (Teitelbaum, 2000). Muaj ob lub cytokines tseem ceeb uas koom nrog kev sib txawv ntawm osteoclast thiab maturation (Koga li al., 2004): macrophage colony-stimulating factor (M-CSF) thiab receptor activator ntawm nuclear factor κB (NF-κB) ligand (RANKL). M-CSF kho qhov sib txawv ntawm hematopoietic qia hlwb rau hauv osteoclast progenitors thiab txhawb kev sib txawv ntawm osteoclasts los ntawm upregulating cov lus qhia ntawm RANK receptor (Takayanagi, 2007). Kev sib khi ntawm RANKL thiab RANK ntawm osteoclast cell nto ua rau hauv qab no: (1) recruitment ntawm signaling adapter molecules xws li TNF receptor-associated factor 6 (TRAF6); (2) ua kom muaj ntau lub hom phiaj nqes hav, suav nrog NF- κB thiab mitogen-activated protein kinases (MAPKs); thiab (3) upregulation ntawm kev qhia theem ntawm nuclear factor ntawm activated T-hlwb, cytoplasmic 1 (NFATc1) (Liu li al., 2019; Yamashita li al., 2007). Kev ua kom cov kev taw qhia no ncaj qha tswj cov kev qhia ntawm osteoclast noob, suav nrog acid phosphatase 5 (Acp5) [encoding tartrate-resistant acid phosphatase (TRAcP)], matrix metalloproteinase 9 (MMP9), thiab cathepsin K (CTSK) (Boyle, Simone). , & Lacey, 2003). Yog li ntawd, qhov inhibition ntawm RANKL-induced osteoclast sib txawv-txog kev taw qhia txoj hauv kev yog ib txoj hauv kev kho mob rau osteoporosis.

Hauv txoj kev tshawb no, peb tsom los txiav txim siab qhov cuam tshuam ntawm CDP kev kho mob ntawm ovariectomized (OVX) vim yogosteoporosisnas qauv hauv vivo thiab ntawm RANKL-induced osteoclast kev ua haujlwm hauv vitro, nrog rau kev tsom mus rau kev qhia ntawm osteoclast-specific genes thiab ua kom NFATc1 thiab NF-κB thiab MAPKs qhia txoj hauv kev. Peb qhov kev tshawb pom tuaj yeem muab kev nkag siab tshiab rau hauv lub peev xwm ntawm CDP raws li cov tshuaj muaj kev nyab xeeb thiab zoo rau kev kho pob txha.

Yog xav paub ntxiv thov hu rau:Joanna.jia@wecistanche.com

Polysaccharide Cistanche deserticola muaj ntau yam cuam tshuam rau pob txha pob txha, nyem qhov no kom paub ntau ntxiv

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Cov tshuaj thiab cov qauv sau

CD (no. 180801) tau yuav los ntawm Anhui Jishun Traditional Chinese Medicine Co., Ltd. (Anhui, Tuam Tshoj) thiab txheeb xyuas los ntawm xibfwb Haibo Huang los ntawm Tsev Kawm Ntawv Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, Suav. Estradiol valerate ntsiav tshuaj tau yuav los ntawm Bayer (Leverkusen, North-Rhine-Westphalia, Lub teb chaws Yelemees). Alpha-hloov yam tsawg kawg nkaus qhov tseem ceeb nruab nrab

(-MEM) thiab fetal bovine serum (FBS) tau txais los ntawm Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Penicillin / streptomycin thiab TRAcP staining kit tau txais los ntawm Solarbio (Beijing, Suav). Recombinant nas M-CSF thiab recombinant nas RANKL tau raug kho los ntawm R&D Systems (Minneapolis, MN, USA). Hydroxyapatite-coated daim hlau tau yuav los ntawm Corning Life Sciences (St. Lowell, MA, USA). Thawj cov tshuaj tiv thaiv rau NFATc1 thiab CTSK (Santa Cruz Biotechnology, Santa Cruz, CA, USA); IκB-, p65, P-p65, p38, P-p38, ERK1/2, P-ERK1/2, JNK, thiab P-JNK (Cell Signaling Technology, Dan-vers, MA, USA); thiab -actin (CWBIO, Beijing, Tuam Tshoj) kuj tau. Lwm cov reagents siv nyob rau hauv txoj kev tshawb no yog ntawm analytical qib, thiab cov dej tau purified los ntawm Milli-Q dej purification system (Millipore, Bedford, MA, USA).

2.2. CDP rho tawm

Lub CD yog av rau hmoov zoo thiab tov nrog roj av ether thrice kom kov yeej nws. Cov khoom seem tau sau los ntawm kev pom thiab tom qab ntawd qhuav ntawm chav tsev kub. Covpolysaccharidestau muab rho tawm los ntawm cov qauv kho ua ntej peb zaug nrog dej rau 2 h, concentrated, precipitated los ntawm kev ntxiv ntawm anhydrous ethanol mus rau qhov kawg concentration ntawm 80 feem pua ​​(v / v), thiab khaws cia ntawm 4 ◦C rau 24 teev. Cov precipitates tau sau los ntawm centrifugation ntawm 3000 rpm rau 20 min, yaj hauv dej distilled, thiab purified (tshem tawm cov proteins dawb) siv txoj kev Sevag (Zhao li al., 2019). Cov rov zoopolysaccharidestau dialyzed, qhuav, thiab khaws cia kom txog thaum siv ntxiv. Tsis tas li ntawd, cov tshuaj muaj pes tsawg leeg tau pom tias tag nrho cov suab thaj, uronic acid, sulfate, thiab cov ntsiab lus protein ntawm CDP yog 67.63 0.98 feem pua ​​, 21.05 0.49 feem pua ​​, 1.{{7 }}.24 feem pua ​​, thiab 8.81 0.36 feem pua ​​.

Cov monosaccharide muaj pes tsawg leeg thiab Fourier transform infrared tsom xam ntawm CDP tau pom nyob rau hauv Ntxiv daim duab 1 thiab 2.

2.3. OVX-induced osteoporosis nas qauv

Txhua qhov kev sim tsiaj tau pom zoo los ntawm Pawg Saib Xyuas Kev Ncaj Ncees Tsiaj ntawm Guangzhou University of Chinese Medicine (pom zoo SYXK 2019-0202). Peb caug 6-cov poj niam hnub nyoog muaj hnub nyoog C57BL/6 nas tau muab los ntawm Kev Tshawb Fawb Tsiaj Chaw ntawm Guangzhou University of Chinese Medicine. Tom qab acclimatization rau 1 lub lis piam, ib pawg nas tau txais kev ua haujlwm sham (Sham pab pawg), thaum cov nas ntxiv tau raug rau kev ua haujlwm ovariectomy (OVX pawg). Tom qab kev phais lawm, cov nas raug tso cai rov zoo tau 1-lub lim tiam. Tom qab ntawd, cov nas ua tau zoo ua qauv tau muab faib ua 5 pawg, nrog rau 6 nas rau ib pawg:

(1) Sham pawg: kho nrog dej distilled, (2) OVX pawg: kho nrog dej distilled, (3) OVX E2 pawg (E2): kho nrog 0.13 mg/kg estradiol valerate ntsiav tshuaj, (4 ) OVX CDP low dose group (CDP-L): kho nrog 300 mg/kg ntawm CDP, (5) OVX CDP high dose pawg (CDP-H): kho nrog 600 mg/kg ntawm CDP. Cov dej Distilled, E2, los yog CDP tau muab los ntawm gavage ib hnub twg. Tag nrho cov nas tau txi tom qab lub sijhawm kho mob 12- lub lim tiam (Fig. 1A). Lub tsev menyuam tau sau thiab hnyav, thiab sab laug tibias tau sau rau kev kuaj tom ntej. Tag nrho cov ntshav kuaj tau sau thiab centrifuged ntawm 5000 rpm rau

15 feeb ntawm 4 ◦C. Cov tshuaj serum supernatants tau aspirated thiab khaws cia ntawm

-80 ◦C kom txog rau thaum kev soj ntsuam ntxiv.

2.4. Micro-computed tomography (micro-CT) tsom xam thiab pob txha histomorphometry

Cov tibias sab laug tau kho nrog 4 feem pua ​​​​ntawm paraformaldehyde rau 48 h thiab tom qab ntawd muab tso rau hauv cov raj centrifuge uas muaj cov ntsev ib txwm. Cov qauv ruaj khov tau raug tshuaj xyuas nrog Skyscan 1172 micro-CT ntsuas (Bruker micro-CT, Skyscan, Kontich, Belgium) siv cov chaw hauv qab no: voltage, 80 kV; qhov tam sim no, 100 μA; Al, 0.5 hli lim; pixel

loj 9,76m;. thiab cov kauj ruam tig, 0.6◦. Ob peb parameters

Cov pob txha trabecular, suav nrog pob txha pob txha pob txha ntom ntom (BMD), pob txha ntim feem (BV / TV), pob txha pob txha ntawm tag nrho ntim (BS / TV), trabecular tooj (Tb. N), thiab trabecular spacing (Tb. sp) tau ntsuas siv CT-Analyser® software (Bruker micro-CT, Skyscan). Cov duab peb-dimensional tau tsim los siv CTVol software (Bruker micro-CT. Skyscan).

Tom qab micro-CT tsom xam, sab laug tibias tau decalcified nyob rau hauv 14 feem pua ​​​​EDTA tov thiab embedded rau hauv paraffin rau sectioning. Cov qauv tau muab hlais rau hauv 5 µm ntu uas siv microtome ua ntej hematoxylin thiab eosin (H&E) thiab TRAcP staining thwmsim. Cov seem stained raug tshuaj xyuas thiab sau tseg siv Olympus CX 31 micro-scope (Olympus Optical Co., Ltd, Tokyo, Nyiv).

Cistanchetuaj yeem txhim kho pob txha ceev thiab txo cov pob txha

2.5. Kev tshuaj ntsuam xyuas ntshav

Cov calcium (Ca) thiab phosphorus (P) concentrations tau txiav txim siab raws li cov lus qhia cov khoom siv tsim los ntawm Nanjing Jiancheng Bioengineering Institute (Nanjing, Suav). TRAcP-5b thiab RANKL qib hauv cov ntshav tau tshuaj xyuas siv TRAcP-5b ELISA cov khoom siv (CUSABIO, Wuhan, Tuam Tshoj) thiab RANKL ELISA cov khoom siv (Cloud-CloneCorp., Wuhan, Tuam Tshoj), feem.

2.6. Kev ntsuam xyuas hauv vitro osteoclastogenesis

BMMs raug cais tawm ntawm tibia thiab femur ntawm 6- poj niam hnub nyoog C57BL/6 nas. Cov hlwb raug cais tawm hauv -MEM nruab nrab uas muaj 25 ng / mL M-CSF, 10 feem pua ​​FBS, thiab 1 feem pua ​​​​penicillin / streptomycin. Thaum ncav cuag qhov sib txuam, cov BMMs (1 104 cov hlwb / lub qhov dej) tau cog rau hauv 96- cov phiaj xwm zoo thiab kho nrog CDP nyob rau hauv muaj 50 ng / mL RANKL. Qhov nruab nrab thiab CDP raug hloov txhua 2 hnub. Tom qab 7 hnub, cov hlwb raug kho nrog 4 feem pua ​​​​ntawm paraformaldehyde thiab stained rau qhov muaj TRAcP. Tus naj npawb ntawm osteoclasts, txhais tau tias yog TRAcP- cov hlwb zoo nrog peb lossis ntau dua nuclei, raug suav thiab sau tseg siv lub tshuab ntsuas lub teeb.

2.7. Kev ntsuam xyuas cell proliferation

Cov BMMs (1 × 104 lub hlwb / lub qhov dej) tau muab noob rau hauv 96- lub phaj zoo thiab incubated thaum hmo ntuj. Tom qab qhov no, cov hlwb raug kho nrog ntau qhov sib txawv ntawm CDP (0, 0.625, 1.25, 2.5, 5, 10, 20, thiab 40 µg / mL) rau 48 h. Lub cell confluence tau kuaj pom thiab tshuaj xyuas siv IncuCyte ZOOM® (Essen BioScience, Ann Arbor, MI, USA), lub sijhawm ntev, lub sijhawm tiag tiag ntawm cov duab nyob ntawm tes.

2.8. Hydroxyapatite resorption xeem

Cov BMMs ({{0}}} hlwb/zoo) tau noob nyob rau hauv ib lub phaj hydroxyapatite-coated, kho nrog CDP ntawm qhov qhia tau hais tias concentrations (0, 5, thiab 10 µg/mL), thiab kab lis kev cai ua kom tiav -MEM muaj 25 ng / mL M-CSF thiab 50 ng / mL RANKL. Tom qab 7 hnub, cov hlwb raug ntxuav nrog 10 feem pua ​​​​ntawm cov tshuaj bleach kom tshem tawm cov khoom ntawm tes. Cov duab ntawm hydroxyapatite resorption thaj chaw raug ntes siv IncuCyte ZOOM® thiab tshuaj xyuas siv ImageJ software (NIH, Bethesda, MD, USA) (Abramoff, Magelhaes, & Ram, 2003).

2.9. Kev cais RNA thiab lub sijhawm rov qab rov qab-quantitative PCR (RT-qPCR)

Cov BMMs ({{0}}} hlwb/zoo) tau noob nyob rau hauv ib tug 6-zoo phaj thiab stimulated nrog RANKL thiab M-CSF nyob rau hauv lub xub ntiag ntawm CDP ntawm txawv concentrations (0, 5, thiab 10 µg / ml) rau 5 hnub. Tag nrho RNA raug cais tawm ntawm cov hlwb lossis cov pob txha ntawm cov nas siv TRIzol reagent (Sigma- Aldrich). Complementary DNA tau tsim los ntawm 2 µg tag nrho RNA siv cov khoom siv rov qab transcriptase (TransGen Biotech, Beijing, Suav). Cov tshuaj tiv thaiv RT-qPCR tau npaj siv PerfectStart™ Green qPCR SuperMix (TransGen Biotech) thiab kuaj pom los ntawm ABI 7500 system (Applied

Biosystems, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cov kev caij tsheb kauj vab rau PCR tau teeb tsa raws li hauv qab no: 95 ◦C rau 5 feeb, ua raws li 40 lub voj voog ntawm 95 ◦C rau 15 s, thiab 60 ◦C rau 30 s. Cov primers tshwj xeeb siv tau pom nyob rau hauv Table 1, thiab qhov ntau ntawm txhua lub hom phiaj gene yog normalized rau GAPDH (internal tswj).

2.10. Western blot tsom xam

Cov BMMs ({{0}} cell/well) tau noob nyob rau hauv 6-zoo phaj. Rau lub sijhawm luv luv, cov hlwb tau ua ntej incubated nrog qhov sib txawv ntawm CDP (0, 5, thiab 10 µg / mL) rau 1 h, thiab tom qab ntawd kho nrog 50 ng / mL RANKL rau 30 min. . Nyob rau lub sijhawm ntev, cov hlwb tau txhawb nqa nrog 50 ng / mL RANKL hnub 3, 5, thiab 7 nyob rau hauv muaj CDP (0, 5, thiab 10 µg / mL). Tag nrho cov proteins tau muab rho tawm siv RIPA lysis buffer (CWBIO). Proteins raug cais los ntawm 10 feem pua ​​SDS-PAGE thiab pauv mus rau PVDF daim nyias nyias. Cov daim nyias nyias tau thaiv nrog 5 feem pua ​​​​skim mis nyuj nyob rau hauv chav tsev kub rau 2 h, incubated nrog thawj.

cov tshuaj tiv thaiv ib hmos ntawm 4 ◦C, thiab tom qab ntawd nrog rau qhov sib thooj

cov tshuaj tiv thaiv rau 1.5 h. Cov membranes tau tsim los siv ECL reagents (Millipore Corp., Billerica, MA, USA), thiab cov duab tau coj los siv Tanon 5200 Chemiluminescence Imaging System (Tanon Science thiab Technology, Shanghai, Suav).

Cistanchetuaj yeem txhim kho pob txha ceev

2.11. Kev kuaj pom ntawm NFATc1 translocation siv immunofluorescence test

Cov BMMs tau cog rau hauv {{{0}}} cov ntawv npog zoo, txhawb nqa nrog RANKL thiab M-CSF, thiab kho nrog 10 µg/mL CDP rau 5 hnub. Cov hlwb raug kho nrog 4 feem pua ​​​​ntawm paraformaldehyde rau 15 feeb, ntxuav peb zaug nrog PBS, thiab permeabilized nrog 0.1 feem pua ​​Triton X-100 rau 10 min. Cov hlwb tau muab sib xyaw nrog 10 feem pua ​​​​tshis ntshav (CWBIO) thiab incubated rau 2 teev. Tom qab ntawd, cov hlwb raug tsim nrog thawj cov tshuaj tiv thaiv thaum hmo ntuj ntawm 4 ◦C thiab tom qab ntawd nrog Alexa Fluor 488 tshis los tiv thaiv nas thib ob antibody (Abcam, Cambridge, MA, USA) hauv qhov tsaus ntuj rau 1 teev. Cov npog npog tau ntxuav nrog PBS, mounted hauv Prolong Gold Antifade Reagent nrog 4′, 6-diamidino-2-phenyl in-dole (DAPI) (Solar), thiab

Kev tshuaj xyuas siv Zeiss LSM 800 nrog Airyscan confocal microscope (Carl Zeiss, Oberkochen, Lub teb chaws Yelemees).

2.12. Kev txheeb cais

Tag nrho cov ntaub ntawv sim tau raug tshuaj xyuas siv SPSS 25.0 statistical software (IBM Corporation, Armonk, NY, USA). Cov ntaub ntawv rau kev tshawb fawb tsiaj thiab cell yog qhia raws li txhais tau tias ± SEM thiab txhais tau tias ± SD, feem. Cov txiaj ntsig tau yog siv ib txoj kev tsom xam ntawm qhov sib txawv lossis Cov Tub Ntxhais Kawm T-test. Tus nqi ntawm p < 0.05="" tau="" suav="" tias="" yog="" qhov="" tseem="">

Cistanchetuaj yeem txoapoptosisthiab txhim khu pob txhaceev