Spinach Methanolic Extract Attenuates Retinal Degeneration hauv cov nas ntshav qab zib

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

Abstract:Nws tau raug pom zoo tias spinach methanolic extract (SME) inhibits qhov tsim ntawm qib siab glycation kawg cov khoom (AGEs), uas yog nce thaum lub sij hawm mob ntshav qab zib, yog li nws yog ib qho tseem ceeb kom paub seb SMEs puas muaj txiaj ntsig zoo rau cov ntshav qab zib retina. Hauv txoj kev tshawb no, kev soj ntsuam hauv vitro tau pom tias SME inhibits glycation, carbonyl pawg tsim, thiab txo qis-thiol pab pawg depletion hauv bovine serum albumin incubated los yog txo cov suab thaj lossis methylglyoxal. Cov nyhuv SME hauv cov retinas ntawm streptozotocin-induced diabetic nas (STZ2) kuj tau kawm (n=10) hauv pawg normoglycemic, STZ, STZ nas kho nrog SME, thiab STZ nas kho nrog aminoguanidine (anti-AGEs siv. pab pawg) hauv 12 lub lis piam. Lub retina tau muab faib thiab tiv thaiv kab mob rau Ne-carboxymethyl lysine (CML), receptor RAGE, NADPH-Nox4, inducible nitric oxide synthase (iNOS), 3-nitrotyrosine (NT), nuclear NF-kB, vascular endothelial kev loj hlob yam ( VEGF), glial fibrillary acidic protein (GFAP), S100B protein, thiab TUNEL assay. Lipid peroxidation tau txiav txim siab hauv tag nrho retina los ntawm malondialdehyde (MDA) qib. Cov txiaj ntsig tau pom tias hauv cov ntshav qab zib retina, SME txo qis CML-RAGE co-localization, oxidative stress (NOX4, iNOS, NT, MDA), o (NF-B, VEGF, S10B, GFAP), thiab apoptosis (p.<0.05).therefore, smes="" could="" attenuate="" retinal="" degeneration="" by="" inhibiting="" cml-rage="">

Ntsiab lus:spinach; ntshav qab zib retinopathy; advanced glycation kawg khoom; oxidative kev nyuaj siab; mob; RAGE; carboxymethyl L-Lysine

Thov nias ntawm no kom paub ntxiv

1. Taw qhia

Kev noj zaub mov ntawm cov nroj tsuag tau cuam tshuam nrog kev pheej hmoo tsawg ntawm kev tsim cov kab mob ntev xws li rog rog, ntshav qab zib hom 2, thiab kab mob plawv. Spinach (Spinacia oleracea L.) yog ib qho zaub ntsuab uas siv tau thoob plaws ntiaj teb vim muab cov fibers, vitamins, thiab minerals txaus [1,2]. Ob peb ntawm nws cov phytochemicals tau raug txheeb xyuas, suav nrog chlorophylls, carotenoids (lutein thiab zeaxanthin) [3], thiab phenolic compounds (flavones, flavonoids, coumarins) [4-8] (saib ntxiv 1). Nws tau raug tshaj tawm tias spinach, thaum noj ua zaub mov, dej-soluble extract, los yog nyob rau hauv daim ntawv khov-qhuav, thiab nws cov thylakoids muaj kev tiv thaiv kab mob, tiv thaiv rog, thiab hypoglycemic zog [9]. Cov nyhuv anti-inflammatory ntawm spinach tau pom nyob rau hauv cov tsiaj endotoxemia qauv, cov tsiaj rog rog, thiab cov tib neeg noj qab nyob zoo [9]. Nws cov nyhuv antioxidant tau kawm hauv tib neeg prostate carcinoma hlwb, tsiaj qauv nrog rog rog, UV-irradiated nas, thiab adenocarcinoma ntawm transgenic nas prostate [9,10]. Cov tshuaj tiv thaiv glycation thiab cov tshuaj tiv thaiv kab mob ntawm spinach methanolic extract (SME) tau kawm nyob rau hauv cov qauv ntawm cov piam thaj-induced ntshav qab zib nyob rau hauv zebrafish thiab isoproterenol-induced myocardial necrosis nyob rau hauv nas, feem [11,12]. SME txo qis glycosylated hemoglobin thiab fructosamine, nrog rau cov qib glycated protein, los ntawm kev txo cov kev ua ntawm aldose reductase hauv lub lens ocular [11]. Cov kev tshawb pom saum toj no qhia tias SMEs tuaj yeem muaj kev tiv thaiv kev loj hlob ntawm cov kab mob ntshav qab zib mellitus xws li mob ntshav qab zib retinopathy (DR).

DR nce zuj zus ua rau poob ntawm qhov muag tsis pom kev lossis qhov muag tsis pom kev uas cuam tshuam nrog o, oxidative kev nyuaj siab, thiab tsub zuj zuj ntawm cov khoom kawg glycation (AGEs)[13,14]. Cov AGEs induce protein crosslinking, hloov cov qauv / kev ua haujlwm thiab nws txoj kev hloov pauv / tshem tawm. AGEs tuaj yeem tsim los ntawm cov tshuaj tiv thaiv tsis-enzyme ntawm cov piam thaj nrog cov amino pawg dawb ntawm cov proteins, tsim cov kev hloov pauv hloov pauv xws li Schiff's bases thiab Amadori cov khoom (fructosamine) [15]. Lwm cov txheej txheem rau AGEs tsim muaj xws li "carbonyl stress" txoj hauv kev, qhov twg oxidation ntawm cov suab thaj thiab lipids tsim dicarbonyl inter-mediate compounds xws li glyoxal thiab methylglyoxal (MGO), uas ua rau AGEs tsim raws li N:-carboxymethyl lysine (CML) [ 16]. Kev nce qib CML hauv cov ntshav thiab cov vitreous tuaj yeem ua tus cim biochemical rau ob qho tib si tsos thiab kev loj hlob ntawm DR [17,18]. Nyob rau hauv lub retina, qhov ua rau ntawm receptor RAGE los ntawm AGEs (AGEs-RAGE) induces lub overexpression ntawm glial fibrillary acidic protein (GFAP) thiab pro-inflammatory yam, tswj los ntawm lub nu-clear factor kappa-light-chain-enhancer ntawm activated B. cells(NF-kB)[19]. NF-kB zoo tswj RAGE qhia los ntawm kev ua raws li lub tswv yim zoo [20].cistanche yog dab tsiNtawm qhov tod tes, siab ntau ntawm S100B, calcium-binding protein, kuj tseem tuaj yeem ua rau NF-kB, inducing neuroinflammation thiab glial cell activation [21]. Lub overexpression ntawm S100B nyob rau hauv astrocytes provokes neurotoxic teebmeem tshwm sim los ntawm retinal astrogliosis [22].

Cistanche tuaj yeem tiv thaiv kev laus

Cov tswv yim tau raug sim tsim los tiv thaiv kev puas hlwb. Qee cov phytochemicals ntawm spinach cais los ntawm lwm cov nroj tsuag tau cuam tshuam nrog inhibition ntawm AGEs thiab oxidative stress [8,23-26]. Lutein, astaxanthin, thiab kaempferol tau tiv thaiv tib neeg-derived retinal pigment epithelial hlwb (ARPE-19) los ntawm kev puas tsuaj los ntawm lawv cov anti-AGEs, antioxidant, thiab anti-apoptosis zog [26-28]. Cov txiaj ntsig no qhia tias cov phytochemicals ntawm spinach muaj lub luag haujlwm tseem ceeb hauv kev tiv thaiv ntawm retinal degeneration zoo li DR. Lwm lub tswv yim tau siv aminoguanidine (AG) ua lub zog inhibitor ntawm glycation thiab iNOS kev ua haujlwm, txo qis CML tsub zuj zuj thiab tsim cov reactive di-carbonylic precursors hauv nas [29]. Txawm li cas los xij, nyob rau hauv ib qho kev sim multicentric thiab randomized soj ntsuam nrog 690 cov neeg mob ntshav qab zib, cov txiaj ntsig zoo tsis tau tsim kom meej [30].

Cov kev hloov pauv hauv retinal degeneration nyob rau hauv cov mob hyperglycemic tsis tau kawm. Yog li ntawd, nws yog qhov nthuav kom paub yog tias SME tiv thaiv cov txheej txheej ntawm cov pob txha los ntawm kev puas tsuaj cuam tshuam nrog nws cov tshuaj tiv thaiv AGEs, antioxidant, thiab cov khoom tiv thaiv kab mob hauv retina ntawm streptozotocin-induced mob ntshav qab zib nas.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1.Kev npaj ntawm Methanolic Extract ntawm Spinach (SME)

Cov nplooj tshiab ntawm spinach (S.oleracea L.) tau sau nyob rau lub caij nplooj zeeg-lub caij ntuj no hauv thaj chaw ua liaj ua teb ntawm Puebla Xeev, Mexico, thiab txheeb xyuas los ntawm tus kws kho tsiaj hauv herbarium ntawm National Polytechnic Institute (IPN, Mexico City, Mexico). Daim ntawv pov thawj tus lej 4532 tau muab tso rau hauv herbarium ntawm National School of Biological Sciences ntawm IPN.

Cov nplooj tau dehydrated thiab finely av. Cov nplooj qhuav tau macerated nrog methanol nyob rau hauv chav tsev kub, lim los ntawm cellulose lim (Whatman, Maidstone, UK), thiab qhuav siv lub tshuab nqus tsev vacuum rotatory (BUCHI Rotavapor R{{0}}, Switzerland) ntawm 45 degree rau muab cov pos hniav ntsuab (95.0g / kg nplooj qhuav).Anti aging cistancheSME tau khaws cia hauv qhov tsaus ntuj ntawm 4 degree kom txog thaum siv. Rau txhua qhov kev ntsuam xyuas, cov extract tau rov tsim dua hauv dej distilled [11].

2.2.In Vitro Assays ntawm SME Anti-Glycation Activity

2.2.1. Tsim cov Advanced Glycation End Cov Khoom Tau Los ntawm Bovine Serum Albumin (BSA-AGEs)

Qhov kev ntsuam xyuas BSA-AGEs tau ua tiav, raws li tau piav qhia yav dhau los [31]. Luv luv, 10 mg/mL BSA(fraction V; Sigma-Aldrich, St. Louis, MO, USA) tau ntxiv rau hauv D-glucose 0.5 M, D-fructose {{12 }}.5 M, los yog methylglyoxal 2.5 mM, thiab ua nyob rau hauv ib tug tov 0.1 M PBS (pH 7.4) thiab 0.02 feem pua ​​sodium azide. SME concentrations (5, 10,25, 50, 100, thiab 200 mg / mL) tau ntxiv rau txhua qhov sib xyaw thiab tom qab ntawd incubated ntawm 37 degree rau 4 lub lis piam. Tom qab ntawd, cov suab thaj uas tsis muaj kev cuam tshuam tau raug tshem tawm los ntawm kev lim dej tawm tsam cov dej distilled rau ob hnub ntawm 4 degree. BSA-AGE qib tau txiav txim siab los ntawm fluorescence spectrophotometry (Ex370 / Em 440 nm; qauv LS 30, PerkinElmer LAS Ltd., Buckinghamshire, UK) [32]. Lub glycation ntawm BSA protein los ntawm kev txo cov suab thaj thiab MGO yog ib tug zoo tswj (BSA glycated), thaum incubation ntawm BSA glycated nrog aminoguanidine (1 mg / mL) yog siv raws li kev tswj tsis zoo (BSA glycated-AG). Cov kev ntsuam xyuas tau ua hauv triplicate.

2.2.2. Fructosamine Assay

Qib fructosamine tau txiav txim los ntawm nitro xiav tetrazolium (NBT) assay [33], uas yog raws li lub peev xwm ntawm fructosamine txo NBT, tsim formazan, xim kawg-cov khoom nyob rau hauv alkaline tej yam kev mob. Kaum microliters ntawm ob qho ntawm cov tswj lossis glycated BSA-incubated SME concentrations (BSA-SME) tau ntxiv rau 90 μL ntawm NBT ntawm 2.5 mM, npaj rau hauv carbonate buffer (0.1 M; pH 10.3); Tag nrho cov incubated ntawm 37 ° C rau 30 min. Lub absorbance ntawm 530 nm tau ntsuas. Fructosamine concentrations (nmol / mg) raug xam raws li tus qauv nkhaus ntawm formazan.

2.2.3. Kev txiav txim siab ntawm Carbonyl Groups Tsim thiab Thiol Pab Pawg Depletion hauv BSA-AGEs

Carbonyl pawg concentration tau ntsuas hauv BSA-SME thiab tswj cov qauv, raws li Levine li al. [34]. Ib qho kev daws ntawm 2,4-dinitrophenylhydrazine (DNPH; 10 mM) hauv 400 μL ntawm 2.5 M HCl tau ntxiv rau 100 μL txhua tus qauv thiab incubated nyob rau hauv qhov tsaus ntuj rau 60 min ntawm chav tsev kub.cistanche cov txiaj ntsigTom qab ntawd, 500 μL ntawm trichloroacetic acid (20 feem pua ​​w / v) tau ntxiv thiab khaws cia rau hauv dej khov rau 5 feeb. Cov qauv no tau centrifuged ntawm 4000 rpm rau 15 min, thiab cov protein pellet tau ntxuav peb zaug nrog ethyl acetate / ethanol (1: 1 v / v). Tom qab ntawd, cov qauv raug tshem tawm hauv 250μL guanidine hydrochloride mus rau 6 M. Cov concentration ntawm carbonyl pawg (nmol/mg protein) yog xam los ntawm spectrophotometric ntawm 370 nm absorbance (EON, BioTek Instruments, Inc., Winooski, VT, USA) siv cov absorption coefficient ntawm DNPH (22,000 M-1 cm{13}}).

Raws li Ellman txoj kev, kev txiav txim siab ntawm thiol pawg depletion hauv BSA-SME thiab kev tswj cov qauv tau ua. Luv luv, 10 μL ntawm 5,5'-Disulfanediylbis (2-nitrobenzoic acid) (DNTB; 10 mM, npaj hauv PBS) nrog 50 μL ntawm cov qauv glycated tau incubated rau 15 min ntawm chav tsev kub, Tom qab ntawd qhov absorbance ntawm 410 nm tau ntsuas. Qib ntawm cov pab pawg dawb thiol tau suav nrog siv tus qauv nkhaus ntawm L-cysteine ​​(0.4-11 μM) thiab qhia hauv nmol/mg protein [34].

2.3. Cov txiaj ntsig ntawm SME ntawm Retina ntawm Cov Kab Mob Ntshav Qab Zib 2.3.1.Diabetes Induction hauv cov nas

Wistar txiv neej nas hnyav 250 ± 10 g (N=40) yoo mov rau 8 teev tau siv. Ib koob tshuaj intraperitoneal ntawm streptozotocin (60 mg / kg) hauv citrate buffer (10 mM, pH 4.5) (Sigma-Aldrich, Inc., St. Louis, MO, USA) tau tswj hwm [35]. Peb hnub tom qab, cov piam thaj tau ntsuas (glucometer; ACCU-CHEK nquag; Roche Diagnostics, GmbH, Mannheim, Lub teb chaws Yelemees) los ntawm kev sau ib cov ntshav ntawm tus Tsov tus tw. Cov tsiaj uas muaj glycemic ntau dua 350mg / dL tau raug xaiv rau txoj kev tshawb no. Cov piam thaj hauv cov ntshav tau ntsuas txhua lub lim tiam rau 12 lub lis piam.

2.3.2. Kev sim tsim

Cov streptozotocin-induced diabetic nas (STZ) tau muab faib ua pawg hauv qab no (n =10): STZ kho intragastrically nrog 2 mL ntawm cov dej haus (STZ); STZ kho nrog SME ntawm 400 mg/kg (STZ-SME); normoglycemic nas (NG); thiab STZ kho nrog 50 mg / kg aminoguanidine (AG; Sigma-Aldrich, Inc.)(STZ-AG).STZ-AG tau siv los ua pawg neeg siv tshuaj tiv thaiv AGE. Cov koob tshuaj tau muab tshuaj txhua txhua 24 teev (9:00 teev sawv ntxov) rau 12 lub lis piam. Cov qib glycemic hauv txhua pab pawg tau saib xyuas txhua lub lim tiam. SME ntau npaum li cas ntawm 400 mg / kg yog raws li hauv qab no: 7 g ntawm SME yog tau los ntawm 100g ntawm spinach tshiab (cov ntaub ntawv tsis qhia). Cov nyiaj no yog sib npaug rau kev noj txhua hnub ntawm tus neeg nruab nrab ntawm 70 kg hauv Asmeskas noj [36], uas sib haum rau 100 mg ntawm extract / kg ntawm lub cev hnyav.Cistanche Extract Anti RadiationNtawm qhov tod tes, vim qhov sib txawv ntawm cov metabolism hauv cov nas nrawm, nws raug nquahu kom nce kev noj cov khoom siv ntuj tsim mus txog 6.4 zaug rau kev sib piv nrog tib neeg [37]. Qhov koob tshuaj ntawm 400 mg / kg uas peb tau siv yog raws li cov txiaj ntsig tau txais hauv cov qauv ntawm myocardial necrosis induced nyob rau hauv Wistar nas, uas pom tau tias cov tshuaj tiv thaiv kab mob ntawm SME yog qhov tseem ceeb ntawm 300 mg / kg [12].

2.3.3. Histological thiab Immunohistochemistry Kev Ntsuam Xyuas

Enucleated ob lub qhov muag tau kho nyob rau hauv nruab nrab formalin thiab tom qab ntawd tau lub cev qhuav dej nyob rau hauv cov qib cawv thiab embedded nyob rau hauv paraffin. Histological seem ntawm 2 μm tau teeb tsa rau ntawm cov khoom siv hluav taws xob, dewaxed, thiab rehydrated mus rau cov tshuaj antigen rov qab (K035; 10 × citrate Buffer, pH6; Diagnostic BioSystems, Pleasanton, CA, USA). Ib qho kev tiv thaiv kab mob polymer-based (PolyVuemouse / luav DAB kuaj pom qhov system, Diagnostic BioSystems) tau siv. Cov tshuaj tiv thaiv hauv qab no tau siv: glial fibrillary acidic protein (anti-GFAP; MAB360; Chemicon International, Inc., Temecula, CA, USA); vascular endothelial grown factor (anti-VEGF; sc-7269; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), S100 calcium-binding protein B (anti-S100B; ab52642; Abcam PLC, Cambridge, UK) , thiab nuclear factor kappa-light-chain-enhancer of activated B cells (anti-NF-kB p65; sc-8008).Txhua qhov dilutions yog nyob ntawm 1:200 thiab incubated hmo ntuj ntawm 4 degree . Ib qho tshuaj tiv thaiv kab mob thib ob (Mouse / Rabbit PolyVueTM) tau ntxiv raws li tus neeg muag khoom cov lus qhia.cistanche tshuaj ntsuabCov seem tau stained nrog DAB ntxiv / chromogen substrate thiab hematoxylin. Kev soj ntsuam histological thiab ntes cov duab tau ua nyob rau hauv Carl Zeiss microscope (Carl Zeiss Microscopy GmbH, Jena, Lub teb chaws Yelemees).

Rau immunofluorescence staining, cov slides tau incubated thaum hmo ntuj ntawm 4 degree C nrog cov hauv qab no cov thawj antibodies: carboxymethyl Lysine (anti-CML; ab124145; Abcam PLC, Cambridge, UK), AGE receptor (anti-RAGE; sc-365154), NADPH oxidase 4 (anti-Nox4; ab133303), 3-nitrotyrosine (anti-NT; ab110282), thiab nitric oxide synthase (inducible) (anti-iNOS; A00368-1; Boster Bio, Pleasanton, CA , USA). Tom qab ntawd, cov tshuaj tiv thaiv kab mob hauv qab no tau siv: rau anti-RAGE, tshis anti-nas lgG(FITC)-conjugated(1:200; Santa Cruz, Biotechnology, Inc.); rau anti-CML thiab anti-NT, nas los tiv thaiv luav IgG-PE (SC-3753); thiab rau anti-iNOS thiab anti-Nox4, m-IgGkBP-PE (Sc-516141). Tag nrho cov incubated nyob rau hauv chav tsev kub rau 60 min. Tom qab ntawd, ntu tau muab tso rau hauv qhov nruab nrab uas muaj 4', 6-Diamidine-2'-phenylindole dihydrochloride (DAPI). Anti-CML thiab anti-RAGE cov tshuaj tiv thaiv tau sib xyaw ua ke hauv tib lub swb. FLoidTM Cell Imaging Station (Lub Neej technologies Carlsbad, San Diego, CA, USA) tau siv rau cov duab fluorescence.

2.3.4.Apoptosis Evaluation

Lub apoptosis ntawm retinal hlwb tau kuaj pom raws li phau ntawv piav qhia los ntawm terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) kev soj ntsuam siv In Situ Cell Detection Kit, TMR (tetramethyl-rhodamine{{ 3}}dUTP) liab, version 12 (12156792910; Roche Diagnostics). Luv luv, cov dewaxed slides tau rehydrated thiab yaug ob zaug nrog PBS. Tom qab ntawd, lawv tau muab tso rau hauv TUNEL cov tshuaj tiv thaiv sib xyaw rau hauv qhov chaw ntub dej rau 60 feeb ntawm 37 C. Tom qab ntawd, ntu tau teeb tsa nrog DAPI thiab saib hauv fluorescence microscopy (FLoidTM Cell Imaging Station). IOD rau TUNEL tau suav rau cov txheej txheej ntawm cov retina.

2.3.5. Lipid Peroxidation Assay

Rau kev ntsuam xyuas ntawm malondialdehyde (MDA) qib hauv cov ntaub so ntswg retinal, kwv yees li 3 mg ntawm cov ntaub so ntswg tshiab (n=3) tau homogenated thiab ua tiav raws li yav dhau los re-ported [38]. Cov qib MDA tau raug ntsuas raws li cov khoom siv cov lus qhia (OXItek-TBARS assay kit, Enzo Life Sciences, Farmingdale, NY, USA).

2.4.Kev txheeb xyuas qhov tseeb

Rau kev txheeb cais, tag nrho cov retinal micrographs raug ntes nrog qhov loj ntawm 200 × ntawm 100 μm dhau ntawm thaj chaw kho qhov muag. Kwv yees li 40 daim duab ntawm ib pab tsiaj (n=7) raug tshuaj xyuas. Cov duab tsom xam tau ua nrog software Image-Pro Premier Version 9.0 (Media Cybernetics, Inc., Rockville, MD, USA). Peb siv GraphPad Prism software (La Jolla, CA, USA; version 8.0). Cov duab qhia tau hais tias muaj nuj nqis nyob rau hauv lub thawv-thiab-whisker cov phiaj xwm (nruab nrab, thawj-thib peb quartile, yam tsawg kawg nkaus-tus nqi siab tshaj) rau ganglion cell txheej (GCL), inner nuclear txheej (INL), thiab txheej txheej nuclear (ONL). Qhov nruab nrab thiab tus qauv sib txawv (txhais tau tias ± SD) muaj nyob rau hauv Appendix A (Tables A1-A3). Nyob rau hauv txhua rooj plaub, ib qho kev xeem ANOVA ib-txoj kev ua raws li Tukey qhov kev xeem tau ua. p<0.05 was="" considered="" statistically="">