Ntu 1: Kev Sib Txuas Network Pharmacology Thiab Zebrafish Qauv Los Tshawb Fawb Cov Txheej Txheem Ua Haujlwm Ntawm Cistanche Tubulosa Rau Kev Kho Mob Osteoporosis Thiab Alzheimer's Disease
Mar 30, 2022
Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791
Ying-Qi Li 1, Yi Chen 1, Jia-Yi Fang 1, Si-Qi Jiang 1, Ping Li 1, * thiab Fei Li 1, 2, *
TSAB NTAWV
Ethnopharmacological qhov tseeb:Osteoporosis (OP) thiab Alzheimer's disease (AD) yog cov kab mob geriatric concurrent, thiab ntau cov kev tshawb fawb qhia txog kev sib txuas ntawm lawv cov kab mob. Cistanche tubulosa (Schenk) Wight (CT) yog ib hom tshuaj suav siv dav siv thiab tau siv dav los kho OP thiab AD, raws li. Txawm li cas los xij, cov khoom xyaw nquag rau ob qho tib si cov kab mob ib txhij thiab cov txheej txheem hauv qab yog txwv.
Lub hom phiaj ntawm kev kawm:Qhov kev ua haujlwm no yog tsim los tsim kom muaj txiaj ntsig zoo thiab txhim khu kev qha kev tshuaj ntsuam xyuas lub network kom pom cov txiaj ntsig zoo sib xws hauv CT uas tuaj yeem tiv thaiv AD thiab OP ib txhij. Thiab nws yuav muab cov kev xav tshiab ntawm kev sib txuas ntawm OP thiab AD ntawm molecular mechanisms.
Khoom siv thiab txoj kev:Qhov cuam tshuam ob ntawm CT tau raug tshuaj xyuas los ntawm kev sib koom ua ke ntau cov ntaub ntawv thiab kev tshuaj xyuas dav dav ntawm qib kws tshuaj hauv lub network. Lub hom phiaj kev sib koom ua yeeb yaj kiab tau tsim los qhia qhov sib txawv ntawm qhov cuam tshuam ntawm ntau hom kev sib txuas. Txhawm rau ua pov thawj qhov ua tau zoo ntawm lub network no, qee qhov sib txuas tau raug xaiv los txheeb xyuas hauv Pre-induced OP model thiab AlCl3-induced AD model of zebrafish raws li topological parameters.
Ntsiab lus:Network Pharmacology; Osteoporosis;Alzheimer tus kab mob; Tubulosa; Zebrafish.
Cistanche muaj cov haujlwm tiv thaiv Alzheimer.
1. Taw qhia
Lub hnub nyoog nyiam ntawm cov pej xeem ua rau muaj kev nce ntxiv ntawm osteoporosis (OP) thiab Alzheimer's disease (AD) ntau xyoo (Parnetti li al., 2019; Rizzoli, 2018). Tam sim no ob qho tib si OP thiab AD tau lees paub tias yog qhov kev hem thawj loj rau pej xeem kev noj qab haus huv vim lawv tuaj yeem ua tiav yam tsis muaj tsos mob thiab ua rau muaj kev cuam tshuam loj hauv zej zog (Erkkinen li al., 2018; Jonsson li al., 2018). Raws li cov kab mob uas muaj hnub nyoog, lawv feem ntau pom muaj tshwm sim hauv kev kho mob nrog kev xiam oob qhab thiab kev tuag (Dengler-Crish thiab Elefteriou, 2019). OP yog ib qho kab mob ntawm cov pob txha uas muaj cov pob txha loj thiab cov pob txha tsis zoo ntawm cov pob txha, nrog kev txhim kho cov pob txha tawg thiab muaj kev pheej hmoo ntawm pob txha (Rizzoli, 2018). AD yog lub hlwb degeneration uas tshwm sim los ntawm kev poob ntawm kev nco thiab lwm yam kev txawj ntse (Bondi li al., 2017). Txog rau tam sim no, tseem tsis muaj kev xaiv muaj zog txaus los tiv thaiv lossis kho ob kab mob ntawm lawv txoj kev etiology thiab pathogenesis. Thiab qhov phem tshaj yog tias tus neeg mob feem ntau muaj cov kev mob tshwm sim (Brown, 2017; Huang and Mucke, 2012).
Raws li theoretical lub hauv paus ntawm kev sib txawv ntawm cov tsos mob (Sucher, 2013), cov tshuaj suav tshuaj suav tshuaj (TCMs) nrog ntau lub ntsiab lus tsom ntau tshaj li ib qho kev kho mob pathophysiological muaj qhov tshwj xeeb zoo tshaj hauv kev kho mob ntawm qee yam kab mob uas paub txog. Raws li tshuaj ntsuab raum tonic, Cistanche tubulosa (Schenk) Wight (CT) yog dav siv los tiv thaiv kev nco thiab atrophic deility ntawm cov pob txha los ntawm cov kws kho mob suav tshuaj (Fu li al., 2018). Dhau li ntawm nws cov kev ua tau zoo ib txwm muaj, kev tshawb fawb pom tau hais tias nws muaj ntau yam kev ua haujlwm tshuaj, xws li ameliorating kev paub thiab kev coj cwj pwm tsis zoo (Wu et al., 2014), anti-inflammatory (Fu et al., 2018), txhawb cov pob txha tsim, thiab suppressing pob txha. resorption (Li et al., 2013; Li et al., 2012), as well as antioxidant act (Zhang et al., 2016). Tab sis qhov nyuaj ntawm nws cov khoom xyaw nquag thiab cov txheej txheem metabolic tau teeb meem hauv ob qho tib si tshawb nrhiav lub hom phiaj hauv tib neeg cov ntaub so ntswg thiab nthuav tawm cov txheej txheem ntawm kev ua.
Qhov tseem ceeb tshaj, kev sib tham txhim khu kev qha ntawm cov hom phiaj kho mob ntawm OP / AD yog qhov tsim nyog. Tshwj xeeb tshaj yog raws li kev xav ntawm "kev kho tib yam rau cov kab mob sib txawv" hauv TCMs, thiab cov txheej txheem ntawm "cov khoom siv kho mob ntau yam, kev siv tshuaj lom neeg" hauv network pharmacology, kev sim nrhiav cov hom phiaj tseem ceeb ntawm ob qho tib si kab mob yuav muaj qhov tshwj xeeb tshaj yog siv tau. lub ntsiab lus nyob rau hauv muab kev taw qhia rau kev tiv thaiv thiab kev tswj ntawm OP thiab AD. Zebrafish muaj qhov zoo sib xws hauv lawv cov pob txha physiology rau cov tsiaj nyeg thiab cov noob homologous heev rau tib neeg. Nws yog ib qho muaj thiab ntxim nyiam tsiaj qauv hauv vivo rau OP thiab AD kev tshawb fawb (Bergen li al., 2019; Newman li al., 2014). Yog li, qhov kev tshawb fawb tshuaj hauv lub network no tau tsom mus rau kev tshuaj xyuas ob qhov cuam tshuam rau cov neeg sawv cev ntawm CT, tshawb nrhiav cov hom phiaj muaj peev xwm ntawm OP thiab AD, nrog rau kev nthuav tawm qhov muaj peev xwm ntawm kev siv CT los kho ob kab mob ib txhij. Hauv txoj kev tshawb no, cov hom phiaj cov ntaub ntawv ntawm CT cov khoom xyaw nquag, cov genomes, thiab cov ntaub ntawv proteomics ntawm OP thiab AD tau raug sau los ntawm cov ntaub ntawv sib txawv los tsim kom muaj kev sib tham sib network, thiab kev txheeb xyuas ntxiv tau tsim los nrhiav kev sib koom ua ke ntawm CT rau kev kho OP thiab AD. . Tsis tas li ntawd, peb tau ntxias OP thiab AD cov qauv zebrafish raws li, thiab ntes cov ntsiab lus tseem ceeb hauv lub network los txheeb xyuas lawv cov txiaj ntsig thiab lub hom phiaj rau ob kab mob.
Cistanche txhawb kev loj hlob ntawm sclerotin.
2. Cov ntaub ntawv thiab cov txheej txheem
2.1 Reagents
Cov qauv tshuaj ntawm genistein (GE), quercetin (QU), abietic acid (AA), -sitosterol (BSS) (purity Ntau dua lossis sib npaug li 98 feem pua) tau yuav los ntawm Chroma Biotechnology Co., Ltd. (Chengdu, Suav). Kab lis kev cai daim hlau tau txais los ntawm Wuxi NEST Biotechnology Co., Ltd. (Wuxi, Suav). Prednisolone (Pre), Etidronate Disodium (Ed), Donepezil HCL (DPZ), AlCl3·6H2O tau yuav los ntawm Aladdin Reagent Inc. Zebrafish alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), acetylcholinesterase (AChE), thiab acetyltransferase (ChAT) enzyme-linked immunosorbent assay (ELISA) cov khoom siv tau muab los ntawm Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, Suav). Trizol tau txais los ntawm Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, Suav teb). Transtar Top Green qPCR SuperMix tau yuav los ntawm TransGen Biotech. Co., Ltd. (Beijing, Suav).
cistanche extract
2.2 Tsiaj
Cov neeg laus zebrafish (AB hom, 4 lub hlis) tau khaws cia ntawm 28 degree nyob rau hauv 14/10 h lub teeb / tsaus voj voog nyob rau hauv ib lub thoob dej yug ntses nrog cov dej tshiab pauv. Lawv tau khaws cia ob zaug hauv ib hnub nrog kev noj zaub mov tshiab-hatched brine cw (Chen li al., 2018). Feem ntau fertilized embryos raug xaiv thiab coj mus rau 3 hnub tom qab fertilization (dpf) nyob rau hauv lub teeb loj hlob incubator. Txhua qhov kev sim tsiaj tau ua raws li cov lus qhia ntawm lub koom haum thiab tsoomfwv rau kev sim tsiaj.
2.3 Network Pharmacological txheej txheem
2.3.1 Chemical Space Calculation and Candidate Compounds Screening
Tag nrho cov ntaub ntawv tshuaj lom neeg ntawm CT tau sau los ntawm Traditional Chinese Medicine Systems Pharmacology Database thiab Analysis Platform. TCMSP yog lub tshuab pharmacology platform ntawm Suav tshuaj ntsuab tshuaj ntsuab uas ntes cov kev sib raug zoo ntawm cov tshuaj, hom phiaj, thiab kab mob (Ru li al., 2014). Lub caij no, plaub qhov tseem ceeb ntawm cov tshuaj muaj feem cuam tshuam hauv TCMSP tau txais rau qhov kev ntsuam xyuas tseem ceeb (PCA), suav nrog qhov hnyav molecular (MW), kev faib coefficient ntawm octanol thiab dej (AlogP), hydrogen pub suav (Hdon), thiab hydrogen txais suav ( Hacc). Kev faib tshuaj lom neeg ntawm CT tau tsim nrog cov ntsuas saum toj no siv SIMCA software (Version 14.1, Umetrics). Thiab cov tshuaj me me uas tau pom zoo rau AD/OP los ntawm DrugBank tau ua tiav hauv tib chav kawm. Cov khoom xyaw los ntawm CT tau lim los ntawm kev siv tshuaj zoo li (DL). DL yog ib lub tswv yim zoo siv hauv kev tsim tshuaj rau kev kwv yees li cas "zoo li yeeb tshuaj" lub zeem muag txuas ntxiv yog. Cov cuab yeej tseem ceeb no yog siv los ua ib qho kev xaiv rau cov "tshuaj-zoo li" cov tshuaj nyob rau hauv cov tsoos suav tshuaj ntsuab thiab nws pab kom optimize pharmacokinetics thiab kws tshuaj (Tao li al., 2013). Raws li cov lus qhia hauv cov ntaub ntawv thiab TCMSP, peb xaiv DL Ntau dua lossis sib npaug rau 0.18 raws li kev lim dej. Cov khoom xyaw ua raws li qhov xwm txheej tau raug xa tawm mus rau kev tshuaj ntsuam tom ntej. Lawv 2-dimensional (2D) thiab 3D qauv tau pleev xim los ntawm ChemDraw Professional 16.0 software. Thiab cov qauv 3D tau ua kom zoo dua los ntawm kev ua haujlwm ntawm kev txo qis zog hauv Chem3D 16.0.
2.3.2 OP thiab AD Associated Targets Collection
Txhua lub koom haum noob rau OP thiab AD tau sau los ntawm DisGeNET thiab GeneCards. Lub DisGeNET database yog ib qho ntawm cov kev tshawb pom loj tshaj plaws ntawm cov noob caj noob ces thiab kev sib txawv cuam tshuam nrog tib neeg cov kab mob (Pinero li al., 2017). GeneCards yog ib qho kev sib koom ua ke ntawm tib neeg cov noob uas suav nrog genomic, transcriptomic, proteomic, genetic, kho mob, thiab cov ntaub ntawv ua haujlwm (Safran li al., 2010).
2.3.3 Compound Targets Collection thiab Inverse Docking Prediction
GeneCards database thiab Swiss TargetPrediction database tau ua ke los kwv yees cov hom phiaj tseem ceeb ntawm cov khoom xyaw nquag hauv CT. GeneCards qhia txog lub hom phiaj tseem ceeb ntawm cov tebchaw, thaum lub Swiss TargetPrediction database tuaj yeem kwv yees qhov feem ntau cov hom phiaj macromolecular ntawm me me molecules (Daina li al., 2019). Ua ntej kev twv, cov qauv ntawm cov neeg sib tw molecules tau hloov los ntawm mol2 hom rau SMILES hom los ntawm kev siv Qhib Babel (Version 2.4.1).
2.3.4 Protein-protein Interaction Analysis
STRING database tau siv los tshawb pom kev sib cuam tshuam ntawm ib pawg ntawm CT-OP-AD cov hom phiaj sib koom. Tom qab tshawb nrhiav nyob rau hauv tus qauv ntawm "Homo sapiens", qhov kev sib cuam tshuam ntawm cov protein-protein (PPI) network tau pom los ntawm Cytoscape (Version 3.7.2) software. STRING yog cov ntaub ntawv uas paub thiab kwv yees PPI, muab los ntawm genomic cov ntsiab lus kwv yees, kev sim siab dhau los ntawm kev sim, (tso tseg) kev sib tham, kev siv cov ntawv sau cia, thiab kev paub dhau los hauv cov ntaub ntawv (Szklarczyk li al., 2017).
Cistanche yog qhov zoo rau Alzheimer's kab mob.
2.3.5 GO thiab KEGG Pathway Enrichment Analysis
Gene Ontology (GO) txhim kho kev txheeb xyuas tau ua tiav ntawm Funrich software. FunRich (Version 3.1.3) yog software ua haujlwm siv feem ntau rau kev txheeb xyuas cov noob thiab cov protein (Pathan li al., 2015). ConsensusPathDB-tib neeg database tau ua haujlwm los ua txoj hauv kev txhim kho kev txheeb xyuas. Cov npe noob tau raug xa mus rau hauv cov ntaub ntawv nrog qhov teeb tsa "p-value cutoff" li 0.01. Tom qab tau txais cov txiaj ntsig, tsuas yog txoj hauv kev uas cuam tshuam nrog OP thiab / lossis AD raug xaiv thiab muab faib ua pawg raws li KEGG pawg ua haujlwm. CPDB yog ib qho kev sib txuas lus tsis sib haum xeeb uas muaj binary thiab complex protein-protein, noob caj noob ces, metabolic, signaling, gene regulatory, thiab yeeb-tshuaj kev sib cuam tshuam, nrog rau txoj kev biochemical hauv Homo sapiens (Kamburov li al., 2013).
2.4 Cov txheej txheem sim rau OP Zebrafish Model
2.4.1 Qauv Grouping
Zebrafish larvae tau muab faib ua ob peb pawg: pawg tswj, pawg qauv, qauv ntxiv rau Ed pawg, qauv ntxiv pawg sib txuas, txhua tus muaj 30 larvae. Cov pab pawg tswj hwm tau khaws cia hauv nruab nrab nrog 0.2 feem pua DMSO. Pawg qauv tau kho nrog Pre (25 µM) los ntawm 3 dpf mus rau 8 dpf. Cov qauv ntxiv rau Ed pawg tau koom ua ke nrog Pre thiab Ed (30 µg / mL).
2.4.2 Alizarin Liab Staining
Tom qab 5 hnub ntawm kab lis kev cai nrog sib txawv tebchaw, larvae raug tshuaj loog, tsau, thiab bleached nyob rau hauv lem. Alizarin liab tau siv los ua cov pob txha taub hau ntawm zebrafish. 1 feem pua KOH thiab glycerol (qhov sib piv ntawm 3: 1, 1: 1 thiab 1: 3) tau siv los ua kom tshem tawm ntau dhau. Thaum kawg, lub stereomicroscope (Olympus SZX16) tau ua haujlwm los ntes cov duab zebrafish staining hauv qab txoj haujlwm. Staining area and integrated optical density (IOD) raug xam los ntawm Image-Pro Plus 6.0.
2.4.3 ALP thiab TRAP Activity Determination
Zebrafish larvae ntawm 3 dpf tau subcultured nyob rau hauv 6-zoo daim hlau thiab incubated nrog Pre nyob rau hauv lub xub ntiag los yog tsis muaj cov tebchaw rau 5 hnub. Tom qab ntawd lawv tau sau los ntsuas ALP cov haujlwm. ALP kev ua haujlwm tau txiav txim siab los ntawm kev siv cov khoom siv zebrafish ALP ELISA raws li cov chaw tsim khoom raws tu qauv. Txoj hauv kev los ntsuas TRAP kev ua haujlwm yog tib yam li ALP.
2.5 Cov txheej txheem sim rau AD Zebrafish Model
2.5.1 Qauv Grouping
Zebrafish larvae tau muab faib ua ob peb pawg: pawg tswj, pawg qauv, qauv ntxiv rau DPZ pawg, cov qauv ntxiv rau pawg, txhua tus muaj 30 larvae. Cov pab pawg tswj hwm tau khaws cia hauv nruab nrab nrog 0.2 feem pua DMSO. Pawg qauv tau kho nrog AlCl3 (150 µM, pH 5.8) los ntawm 3 dpf mus rau 6 dpf. Cov qauv ntxiv rau DPZ pab pawg tau raug kho nrog AlCl3 thiab DPZ (8 µM).
2.5.2 Kev Ntsuas Tus Cwj Pwm
Tom qab 3 hnub ntawm kev xa tshuaj, larvae txav tau kaw los ntawm ViewPoint tus cwj pwm ntsuas (Zebralab 2018, ViewPoint Life Sciences Co., Ltd.). Txhua qhov kev sim tau ua tiav hauv 60 min ntawm 28˚C, muaj 3 lub voj voog ntawm lub teeb / tsaus ntuj (10 feeb txhua rau lub teeb thiab tsaus). Raws li cov txheej txheem hauv tsab ntawv tshaj tawm dhau los, qhov nruab nrab ceev (AS), kev hloov pauv ceev (∆S), dyskinesia rov qab tus nqi (DRR, kab zauv a), thiab kev ua haujlwm ntawm cov lus teb (RE, kab zauv b) raug xaiv raws li kev siv tshuaj tiv thaiv AD. Kev ntsuas ntsuas hauv cov qauv no (Pan et al., 2019; Wenhai li al., 2016).
AS (mm/sec)=D / T, DRR (%)=100 × (ASDrug - ASModel) / (ASControl - ASModel), a
qhov twg D yog qhov txav deb ntawm zebrafish thaum 60 min, T yog lub sijhawm sim.
RE (%)=100 × (∆SDrug - ∆SModel) / (∆SControl - ∆SModel), b
qhov twg ∆S yog qhov hloov ceev ntawm zebrafish nyob rau hauv lub teeb / tsaus voj voog.
2.5.3 AChE thiab ChaT Kev Ntsuas Kev Ua Si
Zebrafish larvae ntawm 3 dpf tau subcultured nyob rau hauv 6-zoo daim hlau thiab incubated nrog AlCl3 nyob rau hauv lub xub ntiag los yog tsis muaj cov tebchaw rau 3 hnub. Tom qab ntawd lawv tau sau rau AChE cov haujlwm. Kev ua haujlwm AChE tau kuaj pom los ntawm kev siv zebrafish AChE ELISA cov khoom siv raws li cov chaw tsim khoom raws tu qauv. Txoj hauv kev los ntsuas qhov kev ua CHAT yog tib yam li AChE.
2.6 Quantitative Real-Time PCR Analysis
Zebrafish larvae tau muab faib ua OP thiab AD pawg. Cov pab pawg OP muaj xws li pawg tswj hwm, pab pawg Pre, thiab Pre plus pawg pawg, kab lis kev cai los ntawm 3 dpf txog 8 dpf. AD pawg suav nrog pawg tswj hwm, AlCl3 pawg, thiab AlCl3 ntxiv rau pawg sib txuas, kab lis kev cai los ntawm 3 dpf txog 5 dpf. Txhua pawg muaj 30 larvae. Tag nrho RNA raug muab rho tawm los ntawm cov qauv zebrafish larvae los ntawm Trizol. Cov qauv RNA tau thim rov qab mus rau cDNA raws li cov chaw tsim khoom qhia ntawm cov khoom siv cDNA. mRNA qhia theem tau kuaj pom los ntawm lub sijhawm tiag tiag fluorescent quantitative PCR (Roche LightCycler96). Cov txiaj ntsig tau normalized rau -actin qhia thiab ntsuas los ntawm kev sib piv (2−∆∆Ct). Forward thiab thim rov qab primers siv tau los ntawm Sangon Biotech (Shanghai, Tuam Tshoj) thiab teev nyob rau hauv Ntxiv Table S1.
cistanche thiab Tongkat ali hmoovpab txhim kho kev tiv thaiv.
Yog xav paub ntxiv, thov nyem qhov no.
2.7 Kev Tshawb Fawb Txog Kev Tshawb Fawb
GraphPad Prism version 8.00 tau siv los piav qhia txog kev txheeb cais. Cov ntaub ntawv tau nthuav tawm raws li qhov txhais tau tias ± SD thiab tshuaj xyuas los ntawm ANOVA txoj hauv kev los ntsuas qhov sib txawv ntawm txhua qhov kev sim txiav txim siab P Tsawg dua lossis sib npaug rau 0.05 yog qhov tseem ceeb.
