Cov qauv kev coj ua ntawm cistanche Showicola polysaccharide thiab nws cov nyhuv tiv thaiv ntawm high glucose-inducts hk -2 kev raug mob cell

1.2.5.8 Nrhiav tau ntawm Mitochondrial membrane muaj peev xwm

HK {-2} pa yog inoculated hauv {1}} cov phaj zoo thiab kev coj noj coj ua. Tom qab sib xyaw ua kom pom kev, nruab nrab hauv nruab nrab hauv {2} Cov phaj zoo} Cov phiaj xwm tswj tau zoo thiab muaj zog tswj hwm tau ntxiv rau cov pab pawg hauv qab "1.2.5.6". Yaug ib zaug nrog PBS, ntxiv 1 ml ntawm cell kev cai nruab nrab tau sib xyaw kom huv si thiab muab tso rau hauv cov kab lis kev cai ntawm tes hauv qhov tsaus ntuj rau 20 min. Lub hlwb tau ntxuav ob zaug nrog JC -1}}}) thiab cov duab tau sau siv cov tsom iav fluorescence.

 

 

 

 

Cov Nyiaj Pab Contanche

 

1.2.5.9 Heechst33258 txoj kev rau kev tshawb pom ntawm tes Apoptosis

Lub cell slides tau muab tso rau hauv 6- Cov phaj zoo, thiab hk} Cov phaj zoo tshaj plaws hauv lub xov tooj ntawm tes ntawm 1 × 105 hlwb / zoo. Tom qab cov tshuaj cuam tshuam cov tshuaj cuam tshuam, lub hoechst33258 staining khoom siv tau siv rau staining. Txoj kev kuaj mob ntawm tes raug kuaj thiab cov duab tau sau siv cov tsom iav fluorescence.

 

1.2.5.1 sab hnub poob blotting los kuaj

Qhov kev qhia ntawm cov protein ntsig txog tlr4 / nf-κb signinging pathwing thiab kho cov hlwb raws li txoj kev nyob rau hauv "1.2.5.6". Tom qab ntawd, tag nrho cov protein hauv lub hlwb tau muab rho tawm raws li cov lus qhia ntawm cov khoom muaj protein cov khoom siv protein extraction. Protein Quam tau ua tiav siv cov xyoob bicinchinic acid (BCA) Proteification qauv, thiab cov protein yog denatured thiab khaws cia}} degree tub yees.
10% Sodium Dodecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulfate polyacrylamide (PVDF) MICRANY SHRORIDES (PEMRANE RAU ​​TOM QAB TOM QAB
Lub hlwb tau raug muab tso rau hauv ib lub tsev pheeb suab hauv qab nrog kaum tsib -20 (tbst) chav thiab thaiv ntawm chav tsev kub. Thawj tshuaj tua kab mob rau TLR4, NF-κB P65, P-NF-κB P65, thiab -Ta tau siv rauTshawb cov nqaij daim tawv(Antibodies tau diluted rau tbst daws ntawm ib lub ntim piv ntawm 1: 2000) ntawm 4 degree thaum hmo ntuj. Tus TBT
Daim nyias nyias tau diluted nrog cov tshuaj tiv thaiv theem nrab (antibody thiab tbst kev daws tau diluted ntawm ib lub suab piv ntawm 1: 2000) thiab tsim kom zoo ntawm chav tsev kub rau 1 h. Tom qab ntxuav daim nyias nyias muaj 3 zaug nrog TBTS, ECL lumnescent reagent tau ntxiv rau kev kuaj pom, thiab cov protein qhia tau pom los ntawm Gel Kev Ntsuas. Duab J software tau siv los tshawb xyuas cov protein grayscale tus nqi, nrog -tacin li sab hauv siv.

1.3 cov ntaub ntawv ua haujlwm

SPPSs 23. {{0 thiab graphpad prism 6.01 tau siv rau cov ntaub ntawv tsom xam. Kev sim Shapiro-Wilk tau siv los sim cov qauv cov qauv, thiab xim av-pleev xim lossis Bartlett txoj kev sim tau siv los sim homogeneity ntawm cov qauv ntaub ntawv. Qhov ntsuas cov ntaub ntawv nrog kev faib tawm ib txwm muaj thiab homogeneity ntawm kev sib txawv tau qhia raws li txhais tau tias ± txheem kev sib txawv. (x ± s) Qhia cov lus piav qhia, ib txoj kev Anova tau siv rau kev sim, thiab Dunnett cov txheej txheem tau siv rau kev sib piv khub yog tias cov lus tseem ceeb yog cov ntsiab lus tseem ceeb.
Rau Post Hoc Pairwise kev sib piv ntawm cov sib txawv ntawm cov kab mob T3, thiab P <0.01 qhia txog qhov sib txawv ntawm ob pawg.

2 cov qhab nia thiab tshuaj ntsuam

2.1 Kev txiav txim siab ntawm tag nrho cov piam thaj, Uronic acid thiab protein cov ntsiab lus ntawm CDPS

Txoj kab sib luag linear ntawm polysaccharide cov ntsiab lus yog: y=5. 7628x {{} {4}}. 999. Qhov kev sim uas tau qhia tau tias thaum nruab nrab ntawm 7 txog 70 μg / mL, muaj txoj kev sib tw zoo ntawm cov qauv zoo nkauj (x) thiab cov nqi nqus dej (y). Cov feem pua ​​suav ntawm polysaccharide cov ntsiab lus hauv cov qauv yog: 81.02%.
Qhov kev sib tham linear ntawm cov qauv ua rau koj cov kua qaub yog: y {= 1. 15715, r} {4}}. 9968. Qhov kev sim qhia tau hais tias thaum nruab nrab ntawm 20 thiab 100 μg / mL, muaj ib txoj kev sib raug zoo ntawm cov qauv uronic acc acid concentration (x) thiab cov nqi nqus dej (y). Cov ntsiab lus ureonic acid nyob hauv cov qauv yog: 4.43%.
Cov txheej txheem tawm ua kab tawm yog: y {= 1. 2671x {2}}. 2145}. 9969. Qhov kev sim qhia tau tias ntawm 20 thiab 1 00}} μg / mL, muaj cov protein zoo ntawm cov txheej txheem ua si (X) thiab cov txiaj ntsig kev nqus tau (y). Cov ntsiab lus protein hauv cov qauv yog: 4.00%.

 

2.2 molecular hnyav faib ntawm cdps

Qhov hnyav molecular ntawm CDPS tau txiav txim siab los ntawm kev ua haujlwm siab gel peelation peelation chomataphy Tandem sab hauv qab nrhiav. Nrog RT li (x) thiab lgmp li (y), sib npaug ntawm LGMP-RT Calibration Curnve tau suav nrog:}. 661, r²=0. 9956; RT yog (x), lgmw yog (y), lub LGMW-RT Calibration nkhaus nkhaus yog:}} RT yog (x), lgmn yog (y), lub LGMN-RT Calibration nkhaus qhov sib npaug yog:}}. 7, r² {19}}. 9955; Raws li kev nkhaus txheem txheem, cov cai suav nrog tau los laij cov molecular qhov hnyav ntawm txhua qhov qauv. Cov qauv molecular pom ntawm cov qauv yog li hauv qab no, thiab cov txiaj ntsig kev ua tiav tau muab faib rau hauv lub rooj 2 Raws li qhia hauv daim duab 1.

 

 

2.3 CDPS Monosaccharide Composition

CDPS tau raug tshuaj xyuas los ntawm ion chromatography, thiab cov chromatogram yog qhia tau tias piv nrog lub sijhawm ntsuas ntawm 16 Monosaccharide Qauv, 5 Monosaccharides hauv CDPS. Los ntawm piv rau cov spectra nrog cov qauv monosaccharide, CDPS tau tsim los ntawm cov piam thaj, galactose, fammose, mannose thiab sucrose. , arabose. Qhov kev sib piv cov hniav nyiaj hniav puas yog {3}}. 734 {}}. {}}. 0502: 0.0255: 0.0178. Los ntawm cov qauv hniav phem, nws tuaj yeem pom tias CDPs yog tsuas yog tsim cov piam thaj, galactose thiab rammames. Ntawm lawv, cov txiaj ntsig zoo rau 73.405%, galactose account rau 5.381%, thiab cov lej nyiaj tau rau 5.02%.

 

Nco tseg: Man.mannose, GLC N.GLUCSIDE, RHA.GALACTOSAMINE ACR, GLC.GALUCUCE, GL.GALACTE, xyl.xylose, ara. arabinose, fuc.fucose.

2.4 CDPS Fourier Fourrared Tsom xam

Lub ncov ze ze 3246 cm -1 yog tshwm sim los ntawm cov pab pawg hydroxyl, qhia txog kev ua kom ncab ntawm lub ncov siab, qhia tias CDPS muaj cov zis Acid, 1374.31 cm -1 Peak: Tshwj xeeb yog nyob rau hauv methyl (-ch {18 {) pawg polysacchar. Qhov nqus tau lub ncov ntawm 1018.38 cm {21}}} Kuv muaj feem cuam tshuam txog kev ncab cov hlua khi, uas yog pom muaj nyob hauv Polysaccharides. Three weak absorption peaks in the range of 1200-1000 cm-1 indicate the presence of pyranose in CDPs. Qhov nqus tau peaks ntawm 870 thiab 930 cm -1}}} nrhiav tau tias hlycosidic bonds yuav muaj nyob hauv CDPS. Saib Daim Duab 3.

 

2.5 Cov teebmeem tshuaj lom los ntawm CDPS ntawm ib txwm hk {2}}} cells

Piv nrog con, ntawm 24 h tom qab lub cell tobility pib txo qis dua tom qab CDPS Concentration tau hu rau 1000 tus txiv neej tau hu ua 1000 tus ug / ml (p<0.05); at 48 h after administration, the cell viability decreased when the CDPs concentration reached 800 μg/mL and did not exceed 80 %; after 1000 µg/mL, the cell viability began to decline significantly (P<0.05); this may be related to the imbalance of cell osmotic pressure and cell membrane damage caused by the high concentration of polysaccharides. There was no significant difference in cell viability when compared with the concentrations of 25, 50, 100, 200, and 400 µg/mL, so the concentrations of CDPs of 100, 200, and 400 µg/mL when cell viability did not decrease significantly were selected as the drug intervention concentrations. See Table 3.

 

3 kev sib tham thiab xaus lus

Polysaccharidesyog high molecular phaus polymers muaj ntau dua 10 tus monosaccharides. Nws tau raug tshaj tawm tias polysaccharides muajimmunomodulatory, antioxidant, anti-inflammatory thiab hypoglycemic cuam tshuam[15]. Raws li ib qho ntawm cov tshuaj tseem ceeb hauv cistanche Showicanca, CDPS muaj cov tshuaj muaj txiaj ntsig zoo. Txoj kev kawm no ua rau pom tus qauv ntawm CDPS thiab pom tias lawv tuaj yeem txhim kho kev puas tsuaj ntawm hk {}} cov hlwb ua los ntawm thaj chaw muaj dej qab zib.

 

Cov qauv nyuaj ntawm polysaccharides yog qhov sib thooj ntawm lawv cov hom thiab cov txheeb ze ntawm polysaccharidide, thiab hom glycosidic bonds muaj cov ntsiab lus [16]. Cov txheeb ze txheeb ze cov molecular loj muab polysaccharides ntau cov chaw nquag thiab tuaj yeem nthuav tawm cov kev ua kom muaj zog.
Txawm li cas los xij, txoj kev ua kom loj dua yuav nce transmembrane tsis kam, tabkaum polysaccharide nqus kev nqus, thiab cuam tshuam rau kev ua si. Conversely, lub cev me polysaccharides kuj tsis tuaj yeem tsim tau cov qauv siv ua haujlwm zoo, ua rau txo cov haujlwm [17]. Thaum tshawb txog kev tshawb fawb roj ntsha ntawm polysaccharides, nws yog qhov tsim nyog yuav tsum tau txiav txim siab txog Monosaccharide Composition, txheeb ze cov khoom sib xyaw ua ke thiab Glycosidic Bond yam [18]. Hauv txoj kev tshawb no, ion chromatography txiav txim siab tias CDPS tau tsim muaj cov piam thaj, galactose, uas muaj raws li daim ntawv tshaj tawm ntawm Xu li al. [19]. Qhov hnyav-nruab nrab molecular hnyav ntawm CDS ntsuas los ntawm kev ua kua siab kua gel chratography yog 3444, 431 kda, feem. Qhov hnyav molecular ntawm peb cistanche polysaccharides los ntawm plaub cistancho-hom tshuaj muaj tshuaj ntsuam los ntawm yang li al. [20] tau zoo sib xws. CDPs tuaj yeem muaj -Txoj Glycosidic Bonds, thiab tej zaum muaj cov-classglucose lossis -mannose. Polysaccharides tsim ntawm -gleClue lossis -mannpe tau yooj yim lees paub los ntawm tus xov tooj-nyiam receptor 4 (TLR4) lossis TLR2 [21].
DN yog ib qho kev sib txawv ntau tshaj plaws ntawm cov ntshav qab zib, thiab nws cov pathogenesis yog ze ze rau oxidative kev nyuaj siab, mitochondrial kawg, thiab lub xov tooj ntawm tes [22]. Txoj kev tshawb no tshawb xyuas cov nyhuv tiv thaiv CDPS ntawm high guz {2} κb signing

Nyob rau hauv cov dej qab zib high ghlucose, hk -2 Cov hlwb nthuav tawm cov oxidative oxidative loj [23]. Cov kev tshawb fawb tau pom tias cov piam thaj hauv qab zib ntau ua ntau cov khoom ntawm intracellular Ros. Qhov ntau npaum li cas ntawm Ros tuaj yeem puas puas kev puas tsuaj ntawm tes cov nab, ua rau muaj kev nce qib ntawm lipid peroxidation khoom mda [24]. Nyob rau tib lub sijhawm, cov haujlwm ntawm sod thiab GSH-PX, qhov tseem ceeb tshuaj tiv thaiv kab mob antioxidant, qhia tias cov tshuaj tiv thaiv kab mob antioxidant ntawm cov hlwb ua rau [25]. Qhov tsis txaus no hauv oxidation-antioxidation system ntxiv txhawb nqa cov lus qhia thiab kev ua haujlwm ntawm tlr4 [26]. Cov kev tshawb fawb yav dhau los tau pom tias qhov kev tawm tsam ntau dhau tuaj yeem lees paub los ntawm TLR4 raws li cov qauv phom sij (damps), tsim cov lus pom zoo Hauv txoj kev tshawb no, CDPS pretreatment txo qis Ros qib, nce cov dej qab zib thiab GSH-PX kev ua ub no, thiab cov ntsiab lus MDA tsawg dua. Qhov no antioxidant nyhuv qhia tias CDPS tuaj yeem tswj cov lus teb oxidative ntawm ntau lub hom phiaj.
Nyob rau hauv tlr4 / nf-κb signaling txoj kev, ua haujlwm rau kev ua haujlwm ntawm Ikk ua haujlwm los ntawm kev ua haujlwm hauv lub cev, uas nyeg ua rau Iκb [28]. Txoj kev kawm tam sim no pom tias cov hmoov ntau ntxiv tau nce siab txog qhov NF-κB P65 los ntawm Cytoplasm mus rau lub nucleus. Cov kev tshawb fawb tau pom tias P65 tsuas yog tsis thab oxidative kev nyuaj siab tab sis kuj cuam tshuam mitochondrial muaj nuj nqi [29].
Nws tau pom los ntawm JC -1}}}}} Kev kho mob siab uas ua rau pom tias muaj kev ua kom zoo dua ntawm cov tshuaj ntsuab Mitochondrial ua tsis tau zoo ntxiv ntxiv rau kev tsim cov Ros, sib sau ua lub voj voog vancycle [30]. CDPS pretricatment tuaj yeem tswj hwm qhov kev ruaj ntseg ntawm Mitochondrial Mitochondrial Mithbrane yuav ua tiav los ntawm kev nthuav qhia ntawm TLR4 thiab ua kom NF-κB.
Mitochondrial tab kaum thiab pheej oxident oxidative kev nyuaj siab kawg ua rau ntawm tes Apoptosis [31]. Hoechst staining qhia tawm tias lub xov tooj ntawm lub xov tooj ntawm cov glucose zoo li pom zoo xws li hws, hws thiab tawg [32]. Cov kev hloov pauv no cuam tshuam nrog Mitochondrial-kho cov kev tso tawm ntawm Cyoptosis G, ua kom tiav ntawm Caspase Cascade, thiab thaum kawg xov tooj ntawm lub cev [33]. Lub nuclear morphology ntawm cov hlwb hauv CDPS pretrectment pab pawg yog cia li tswj mitochondrial muaj nuj nqi thiab cuam tshuam oxidating kev ntxhov siab.
Hauv cov ntsiab lus, qhov kev kawm no siv tshuaj lom zem oxidative (Ros, Sod, Echs}), mit--1
Ua ntej, los ntawm kev txhim kho cov tshuaj tua kab mob antioxidant ntawm cov hlwb, txo qib ntawm Ros thiab txo qhov kev ua kom muaj tl4; Thib ob, los ntawm inhibiting qhia ntawm tlr4 thiab phosphorylation ntawm Iκb, thaiv cov kev sib tsoo nuclear ntawm NF-κB; Thaum kawg, los ntawm kev txhim kho mitochondrial muaj nuj nqi, inhibiting cell Apoptosis. Kev tuag, yog li ua si lub luag haujlwm tiv thaiv DN. Qhov ntau-hom phiaj synergistic effect ua rau CDPS qhia cov kev thov kev pab zoo hauv kev kho DN. Nyob rau hauv kev tshawb fawb tom ntej, cov qauv polysaccharation yuav txiav txim siab los ntawm x-tshav sib txawv resonance spectroscopy, thiab roj roj chromroography, thiab roj chromatography, thiab roj chromatography-huab cua spectrometry. Tsis tas li ntawd, qhov nruab nrab molecular qhov tshwm sim qhia tau tias CDPS yog cov polysaccharidesous polysaccharides. Tom ntej no, peb yuav cais thiab huv homogeneous polysaccharide, nrhiav cov qauv kev ua si thiab kev ua ub no, thiab qhia meej cov qauv-kev sib raug zoo.