Immunostimulatory Activity Ntawm Dej-extractable Polysaccharides Los ntawm Cistanche Deserticola Ua Ib tsob nroj Adjuvant hauv Vitro Thiab hauv Vivo
Apr 08, 2024
Taw qhia
Cov tshuaj tiv thaiv tseem ceeb heev rau kev tswj lossis tiv thaiv kab mob. Cov tshuaj tiv thaiv uas tsim tawm tshiab thiab tam sim no tsim muaj cov tshuaj tiv thaiv kab mob sib xyaw ua kom zoo dua qub nrog kev nyab xeeb dua, tab sis cov tshuaj dawb huv tsis tuaj yeem txhawb lub cev tiv thaiv kab mob zoo dua piv nrog cov tshuaj tiv thaiv kab mob ua kom tsis muaj zog lossis tsis ua haujlwm. Nrog kev pab ntawm adjuvants, cov tshuaj tiv thaiv tuaj yeem ua rau lub cev tiv thaiv kab mob tsis tu ncua [1,2]. Cov tshuaj muaj zog tshiab tshiab muaj kev txhawj xeeb hauv ob qho tshuaj tiv thaivkev loj hlob thiab tib neeg kev noj qab haus huv. Txog niaj hnub no, ntau yam adjuvants tau raug txheeb xyuas thiab tau kawm ntau. Cov tshuaj tiv thaiv kab mob no muaj ntau yam zoo, suav nrog txo qhov xav tau ntawm cov tshuaj tiv thaiv kab mob, txo qis cov tshuaj tiv thaiv kab mob uas xav tau rau cov tshuaj tiv thaiv kab mob ib txwm muaj, thiab ua rau muaj kev cuam tshuam sai dua, dav dua, thiab muaj zog tiv thaiv kab mob teb [3–5]. Txawm hais tias muaj ntau lub zog adjuvants tau tsim, xws li lipopolysaccharide (LPS) thiab Freund's tiav adjuvant (FCA), lawv tsis tau siv dav vim lawv cov tshuaj lom. Li no, tsuas yog ob peb yam ntxiv, xws li Alum, tau tso cai rau kev siv tshuaj kho mob [6]. Zuag qhia tag nrho, cov tshuaj tiv thaiv zoo dua thiab muaj kev nyab xeeb dua yuav tsum tau tsim los txhawb kev tiv thaiv kev tiv thaiv zoo dua thiab kho cov tshuaj tiv thaiv kab mob sib kis thiab tsis muaj kab mob.
NATURAL CISTANCHE TUBULOSA FOR ENHANCING Immunity PHGS75% ECH 30% ACT 12%
Kev nyab xeeb yog qhov tseem ceeb hauv kev txhim kho cov adjuvants. Suav herbaceous constituents muaj xws li cov as-ham thiab tseem ceeb active composites, xws li phenolic tebchaw thiab polysaccharides, uas yuav ua tau raws li haib immunostimulants [7-9]. Ntawm lawv, polysaccharides los ntawm cov tshuaj suav tshuaj suav tshuaj tau nthuav dav vim lawv cov haujlwm immunostimulatory thiab tsis muaj tshuaj lom. Piv txwv li, inulin yog ib qho tshuaj tiv thaiv kab mob tsis muaj tshuaj lom ntawm lwm cov tshuaj ntxiv xws li FCA. AdvaxTM delta inulin adjuvant kuj tau ua tiav qhov kev tiv thaiv kab mob ntawm cov tshuaj tiv thaiv [10,11]. Astragalus, tseem hu ua Huangqi hauv Suav thiab Radix Astragali hauv Latin, tau nthuav dav thoob plaws ntiaj teb. Polysaccharide yog ib qho ntawm nws cov khoom xyaw tseem ceeb ua lub luag haujlwm rau kev ua haujlwm immunomodulatory. Cov kev tshawb fawb soj ntsuam tau pom tias Astragalus polysaccharide muaj zog immunomodulatory teebmeem ob leeg hauv vitro thiab hauv vivo [12, 13]. Lycium barbarum polysaccharides raws li cov tshuaj tiv thaiv adjuvant kuj tau pom tias muaj kev txhim kho zoo thiab txhawb cov teebmeem [14, 15]. Cov kev tshawb fawb no qhia tias Suav tshuaj ntsuab polysaccharides yog cov neeg sib tw zoo tshaj plaws rau kev txhim kho ntxiv.
Cistanche deserticolaYC Ma (CD, "Rou Cong Rong" hauv Suav) yog cov tshuaj suav tshuaj muaj txiaj ntsig zoo thiab feem ntau suav tias yog "Ginseng ntawm cov suab puam" vim tias nws cov txiaj ntsig zoo tshaj plaws. Nws tau muab faib rau hauv cheeb tsam arid lossis semi-arid hauv Xinjiang. Hauv Suav teb, nws cov qia qhuav tau siv los ua cov khoom noj tonic rau ntau pua xyoo [16, 17]. Tau ntau xyoo, boiled CD tau siv los ua cov khoom noj tonic los kho kev puas tsuaj ntau dhau, qhia tias nws muaj kev nyab xeeb rau kev tswj qhov ncauj. Cov nroj tsuag no dav suav hais tias yog vim nws txoj haujlwm dav dav. Cov kev tshawb fawb phytochemical thiab pharmacological tsis ntev los no tau pom tias polysaccharides yog cov khoom siv roj ntsha tseem ceeb thiab muaj ntau yam teebmeem lom, suav nrog.immunomodulatory kev ua ub no, antioxidant nyhuv, thiab anti-inflammatory teebmeem[18–21]. Txawm li cas los xij, kev tiv thaiv kev tiv thaiv kev ua haujlwm ntawm dej-extractable polysaccharides los ntawm C. deserticola hauv Xinjiang tsis tshua muaj qhia.
Nws paub zoo tias DCs yog ib qho tseem ceeb ntawm cov kab mob antigen-presenting cells (APCs) muab cov cim qhia xav tau rau kev pib lub cev tiv thaiv kab mob thiab hloov kho cov hlwb innate thiab yoog raws. Qee cov adjuvants txhim kho antigen uptake los ntawm DCs tuaj yeem nce co-stimulatory lossis MHC molecules thiabtxhim khu kev tiv thaiv. Thaum DCs maturation pib, ntau co-stimulatory molecules thiab cytokines raug tsim thiab DCs qhia txawv phenotypes [22, 23].
Hauv txoj kev tshawb no, peb thawj zaug siv seb WPCD tuaj yeem txhawb kev ua haujlwm ntawm DCs ntawm TLR4 kev taw qhia txoj hauv kev hauv vivo. Txij li thaum DCs yog qhov sib txuas ntawm lub cev thiab lub cev tiv thaiv kab mob, peb xav tias DCs ua kom muaj zog los ntawm WPCD yuav cuam tshuam cov txiaj ntsig ntawm kev tiv thaiv kab mob. Peb tau tshuaj xyuas cov tshuaj tiv thaiv kab mob tshwj xeeb rau ovalbumin (OVA) hauv cov nas uas kho nrog WPCD, suav nrog titers ntawm IgG, IgG1, thiab IgG2a subclass, T- thiab B- proliferation, thiab kev tsim cov cytokine. Peb tau tshawb xyuas seb WPCD kev kho mob puas yuav ua rau muaj kev ua haujlwm ntawm DCs thiab Treg hlwb hauv tus po ntawm cov nas no. Peb qhov kev tshawb pom tau ua pov thawj tias muaj peev xwm ua haujlwm immunomodulatory ntawm ntuj polysaccharides los ntawm C. deserticola thiab nthuav dav nws daim ntawv thov.
Cov ntaub ntawv thiab cov txheej txheem
Tsiaj txhu
Yim mus rau kaum-lub lim tiam-laus C57BL/6, BALB/c, lossis ICR poj niam nas tau yuav los ntawm Thawj Tsev Kho Mob ntawm Xinjiang Medical University (Urimuqi, Xinjiang, Suav). Cov nas tau nyob hauv cov kab mob tsis muaj kab mob raws li cov lus qhia ntawm Pawg Saib Xyuas Tsiaj thiab Siv Tshuaj (ACUC) ntawm Xinjiang University for Animal Health thiab Wellbeing. Cov txheej txheem kev sim tsiaj tau pom zoo los ntawm AUCC ntawm Xinjiang University.
Extraction ntawm aqueous extracts
C. deserticola yog ib tsob nroj hauv Xinjiang Xeev hauv Suav teb. Lub crude polysaccharide tau txais los ntawm dej extraction thiab ethanol nag lossis daus. Luv luv, 100 g ntawm qhuav C. deserticola yog av rau hauv hmoov thiab ces lim. Cov hmoov tau refluxed nrog roj av ether ntawm chav tsev kub dua thiab tom qab ntawd refluxed nrog anhydrous ethanol rau 1 h kom tshem tawm cov khoom xyaw xim thiab lipids. Cov seem tau muab rho tawm nrog dej kub rau peb zaug thiab cov khoom rho tawm tau muab sib xyaw ua ke, centrifuged ntawm 4000 rpm rau 10 min, thiab refluxed ntawm 60˚C rau 4 h. Plaub zaug ntawm qhov ntim ntawm 95% ethanol tau ntxiv rau hauv cov kev daws rau precipitate crude polysaccharides. Cov polysaccharides nyoos tau rov ua dua hauv dej distilled thiab kho nrog Sevage reagent kom tshem tawm cov protein. Extracts tau yaj nyob rau hauv PBS thiab sterilized los ntawm ib tug 0.22-μm lim. Thaum kawg, tag nrho cov ntsiab lus qab zib ntawm WPCD yog 59.58%, raws li qhia los ntawm phenol-sulfuric acid tsom xam [24].
Generation ntawm DCs
BM-DCs tau tsim tawm raws li txoj kev piav qhia yav dhau los nrog kev hloov kho me ntsis [25]. Cov pob txha pob txha DCs los ntawm C57BL/6 nas tau tawm nrog RPMI-1640 ua tiav nruab nrab (Gibco) ntxiv nrog 10% fetal calf serum (Hyclone). Cov hlwb uas tau sau tseg tau rov raug ncua hauv RPMI-1640 nruab nrab nrog 50 μM -mercaptoethanol (Sigma, St Louis, MO) thiab 20 ng/mL murine GM-CSF (Peprotech, Rocky Hill, NY) thiab incubated ntawm 37˚ C hauv 5% CO2 cua. Ib nrab ntawm cov kab lis kev cai nruab nrab tau hloov los ntawm qhov nruab nrab tshiab uas muaj GM-CSF txhua 1-2 hnub. Cells tau sau rau hnub 6, kho nrog ntau koob tshuaj ntawm WPCD, thiab LPS (100 ng / mL) (Sigma, St Louis, MO) rau 24 teev, thiab tom qab ntawd soj ntsuam los ntawm flow cytometry.
Kev tshawb pom ntawm maturation ntawm DCs thiab T cell ua rau hauv vitro
Cov txiaj ntsig hauv vitro maturation ntawm WPCD ntawm BM-DCs tau soj ntsuam raws li cov txiaj ntsig ntawm phenotypic tsom xam los ntawm FACs. Hnub 7, BM-DCs los ntawm C57BL/6 raug ntxias nyob rau hauv lub xub ntiag ntawm GM-CSF thiab tom qab ntawd kho nrog ntau qhov sib txawv ntawm WPCD (0.01, 0.{{ 11}}2, 0.05, 0.1, thiab 0.2 mg/mL) rau 12 teev hauv triplicate. LPS (100 ng / mL) tau siv los ua kev tswj xyuas zoo. Tom qab BM-DCs tau ntxuav hauv PBS thiab Fc Block (BD Biosciences, CA) tau siv los tiv thaiv cov tshuaj tiv thaiv kab mob tsis tshwj xeeb, cov hlwb tau stained hauv PBS los ntawm kev siv FITC-, PE-, lossis APC-conjugated CD40, CD11c, CD{ {23}}, CD80, thiab MHC-II cov tshuaj tiv thaiv kab mob (BD) rau 20 feeb ntawm 37˚C. Cov hlwb stained tau kuaj pom los ntawm FACs Calibur (BD). Cov ntaub ntawv tsom xam tau ua nrog FlowJo software (Tree Star).
Nyob rau hauv DCs maturation xyaum ua tej yam nyob rau hauv vitro, cell culture supernatants kuj tau sau los soj ntsuam IL-12 thiab TNF- los ntawm kev siv cytokine assay kits (Boster, Wuhan, Suav teb) raws li cov chaw tsim tshuaj paus cov lus qhia. Lub absorbance ntawm 450 nm tau ntsuas nrog ELISA phaj nyeem ntawv (Bio-Rad, USA). Cytokine ntau nyob rau hauv cov qauv raug xam nrog tus qauv nkhaus ntawm recombinant cytokines raws li txoj kev regression linear.
Txhawm rau kuaj lawv cov allogenic stimulatory kev ua, qhov kev sim ntawm kev sib xyaw lymphocyte cov tshuaj tiv thaiv (MLR) tau ua raws li txoj kev dhau los [26] los ntawm MTT (Sigma, St Louis, MO) assay. Luv luv, BM-DCs los ntawm C57BL / 6 nas siv los ua cov hlwb stimulator tau zoo nyob rau Hnub 7 hauv RPMI-1640 nruab nrab ntxiv rau GM-CSF thiab kho nrog ntau qhov ntau ntawm WPCD rau 12 teev ntawm 37˚C. Cells raug ntxuav thiab rov muab ncua rau hauv RPMI -1640 nruab nrab ua ntej plating rau hauv 24- lub phaj zoo. Singleplenocyte suspension responseer cells raug cais tawm ntawm cov nas BALB/c. BM-DCs tau tov nrog splenocytes raws li qhov sib piv ntawm 1: 5 thiab 1: 10. Cells raug coj los ntawm 37˚C hauv 5% CO2 cua rau peb hnub hauv triplicate. Kev kho LPS tau siv los ua kev tswj xyuas zoo. Cov kab lis kev cai tau ntxiv tom qab nrog 20 μL ntawm MTT rau qhov kawg 4-h cog qoob loo. Cov absorbances ntawm kev ntsuas wavelength (490 nm) thiab siv wavelength (650 nm) tau ntsuas rau T cell proliferation tsom xam. Tag nrho cov qauv raug ntsuas los ntawm kev tswj hwm keeb kwm yav dhau.
NATURAL CISTANCHE TUBULOSA rau kev txhim kho koj lub cev tiv thaiv kab mob PHGS75% ECH 30% ACT 12%
Kev kho mob los ntawm TLR4 inhibitor hauv vitro
BM-DCs tau kho ua ntej nrog 5 μM TAK-242 (inhibitor ntawm TLR4, Medchemexpress Inc. USA) rau 1 h thiab tom qab ntawd co-cultured nrog ntau qhov ntau ntawm WPCD (100 thiab 200 ug / mL) rau 12 teev nyob rau hauv lub xub ntiag los yog tsis muaj Golgi Nres hauv triplicate. Hauv kev tswj hwm zoo, LPS tau ntxiv. Cells thiab supernatants tau kuaj pom los ntawm FACs rau kev txheeb xyuas cov cim saum npoo CD86 thiab CD40 thiab los ntawm ELISA rau kev txheeb xyuas cytokines TNF-a thiab IL-12, raws li.
Kev txhaj tshuaj tiv thaiv kab mob
Kev kuaj mob toxicity
Nyob rau hauv qhov ncauj mob toxicity test nyob rau hauv txoj kev tshawb no, 40 noj qab nyob zoo cov neeg laus ICR nas. ICR nas tau yoo mov (zaub mov tab sis tsis dej thaum hmo ntuj) ua ntej noj. WPCD tau muab tshuaj rau qhov ncauj raws li qhov koob tshuaj 0, 50, 500, lossis 5000 mg / kg lub cev hnyav rau txhua tus tsiaj rau 7 hnub. Saline-kho thiab alum-kho nas tau suav nrog hauv pawg tswj hwm. Cov tsiaj tau pom txhua hnub rau 14 hnub sib law liag. Cov nas tau pom ib tus zuj zus los sau txog kev tuag thiab cov tsos mob tshwm sim. Tsis tas li ntawd, lub cev hnyav thiab khoom noj thiab dej haus tau raug ntsuas thoob plaws hauv qhov kev sim. Hnub 14, tus po los yog thymus Performance index raug xam raws li (spleen lossis thymus hnyav / bodyweight) × 10.
Kev kuaj pom ntawm WPCD immunomodulatory kev ua haujlwm hauv vivo.
Txhawm rau tshawb xyuas seb WPCD puas muaj cov tshuaj tiv thaiv kab mob, ovalbumin (OVA) (Sigma) tau siv los ua tus qauv antigen, thiab poj niam ICR nas tau muab cais ua 7 pawg ( pawg tswj tsis zoo (0.9% NaCl thiab WPCD 400 ug). ) thiab kev tswj zoo (Alum 200 ug nrog OVA)). Cov nas tau muab tshuaj subcutaneously ob zaug nrog 10 ug OVA ib leeg lossis WPCD nrog OVA raws li koob tshuaj ntawm 20, 100, lossis 400 ug nrog lub sijhawm 2-lub lim tiam. Cov ntshav kuaj thiab tus po tau txais tom qab txhaj tshuaj tiv thaiv kab mob txhawm rau txhawm rau kuaj IgG titers, IgG subclasses, splenocyte proliferation thiab cytokine, DCs nto markers, thiab Treg zaus.
Kev kuaj pom ntawm OVA cov tshuaj tiv thaiv tshwj xeeb
OVA-specific IgG titers thiab subclasses raug tshuaj xyuas los ntawm ELISA raws li txoj kev dhau los [27]. ELISA daim hlau (Nunc, Thermo Fisher Scientific) tau coated thaum hmo ntuj thiab tom qab ntawd thaiv. Cov qauv ntshav tau diluted serially rau IgG titer tsom xam lossis kuaj pom ntawm IgG1 thiab IgG2a (Southern Biotech, Inc.). Cov phiajcim tau muab tso rau hauv PBST uas muaj HRP-conjugated anti-nas IgG, IgG1, thiab IgG2a rau 1 h ntawm 37˚C. Cov tshuaj tiv thaiv colorimetric tau tsim nrog tetramethylbenzidine (TMB) thiab tom qab ntawd cov absorbances ntawm 450 nm / 655 nm raug ntsuas thiab qhia tias qhov muag pom qhov ntom ntom (OD) units.
Kev kuaj pom ntawm splenocyte proliferation thiab cytokine
Splenocyte proliferation tau txiav txim los ntawm MTT kev soj ntsuam. Hnub 21 tom qab txhaj tshuaj tiv thaiv thawj zaug, tau txais ib qho kev ncua ntawm splenocyte. Cells tau coj mus kuaj hauv RPMI-1640 nruab nrab hauv 96- cov phiaj zoo raws li qhov concentration ntawm 1 × 106 hlwb / zoo hauv triplicate. Cov kab lis kev cai tau txhawb nqa nrog OVA (qhov kawg concentration 10 ug / mL), ConA (kawg concentration 5 ug / mL) (Sigma, St Louis, MO), thiab LPS (qhov kawg concentration 100 ng / mL) rau 48 h ntawm 37˚C. . Cells raug coj mus kuaj rau 48 h thiab 20 μL (5 mg / mL) ntawm MTT (Sigma, St Louis, MO) tau ntxiv rau hauv txhua qhov dej thiab incubated rau lwm 4 h. Tom qab ntxiv 50 μL ntawm DMSO rau hauv txhua lub qhov dej kom tsis txhob muaj xim tsim (Sigma), daim hlau tau nyeem ntawm 570 nm los ntawm microtiter phaj nyeem (BioRad, CA, USA). Splenocyte proliferation tau nthuav tawm raws li stimulation Performance index (SI), uas yog OD570 nm piv ntawm ib tug stimulated zoo rau ib tug unstimulated zoo.
Cov txiaj ntsig ntawm IL-4 thiab IFN- nyob rau hauv T hlwb tau ntsuas los ntawm intracellular cytokine staining raws li cov txheej txheem luam tawm nrog kev hloov kho me me [27, 28]. Kev ncua ib leeg splenocyte tau npaj rau Hnub 21 tom qab txhaj tshuaj tiv thaiv thawj zaug. Tom qab cov ntshav liab lysis, lub splenocyte (2 × 106 hlwb / mL) tau incubated nrog OVA (10 ug / mL) rau 4-h stimulation thiab Golgi nres (BD) tau ntxiv rau 12 h incubation, PMA zoo tswj. Cells tau sau, ntxuav nrog PBS, stained nrog CD4-FITC/CD{11}}FITC, thiab tsau thiab permeabilized nrog Cytofix/Cytoperm kit (BD) raws li cov chaw tsim tshuaj paus cov lus qhia. Intracellular cytokine staining tau ua nrog qhov tsim nyog ntawm IL-4-PE lossis IFN- -APC cov tshuaj tiv thaiv ntawm 4˚C rau 20 min. Stained cells raug kuaj pom los ntawm FACs. Kev txheeb xyuas cov ntaub ntawv tau ua tiav hauv FlowJo.
Kev ntsuam xyuas ntawm DCs maturation thiab Treg hlwb hauv tus po
Rau kev soj ntsuam ntawm DC maturation los ntawm splenocytes hauv nas, cell nto staining tau ua nrog CD11c-PE, CD40-FITC, thiab CD80-APC antibodies. Hnub 3 tom qab txhaj tshuaj tiv thaiv thawj zaug, ib qho kev tshem tawm splenocyte (1 × 106 hlwb / mL) tau txais thiab cov hlwb raug rau ob npaug. Lub zog fluorescent tau ntsuas los ntawm FACs Calibur thiab cov ntaub ntawv ntsuas tau ntsuas los ntawm FlowJo.
Txhawm rau saib seb WPCD puas tuaj yeem txo qis- tswj cov zaus ntawm CD4+ CD25+ Foxp3+ Treg hlwb. Kev ncua ib leeg splenocyte tau npaj rau Hnub 7 tom qab txhaj tshuaj thib ob. Lub splenocyte (2 × 106 hlwb / mL) tau raug rau ntawm lub xov tooj ntawm tes staining nrog CD4-APC antibody, tom qab los ntawm nuclear cytokine staining nrog CD tsim nyog25-FITC thiab Foxp3-PE antibodies nrog cov nas tswj T-cell staining kit (eBiosciences) raws li cov chaw tsim khoom cov lus qhia. Qhov zaus ntawm CD4+ CD25+ Foxp3+ Treg hlwb raug kuaj ntawm FACs. Kev txheeb xyuas cov ntaub ntawv tau ua tiav hauv FlowJo.
Kev txheeb cais
Ib-txoj kev tsom xam ntawm qhov sib txawv (ANOVA) kev xeem (Tukey's Multiple-Comparison Test) tau ua los tshuaj xyuas qhov sib txawv ntawm ntau pawg kev sim. Tag nrho cov txiaj ntsig tau qhia tias txhais tau tias ± SD. P< 0.05 is believed to be statistically significant. The statistical analyses were performed using Prism 5.0 software.
Cov txiaj ntsig WPCD txhawb kev loj hlob thiab kev ua haujlwm ntawm BM-DCs hauv vitro
Adaptive kev tiv thaiv yog txiav txim los ntawm kev ua kom DCs, nrog rau cov hom co-stimulatory molecules thiab cytokines [29, 30]. Thaum xub thawj, peb tau tshawb xyuas seb WPCD puas tuaj yeem txhawb kev nthuav qhia ntawm co-stimulatory molecules. Raws li cov koob tshuaj sib txawv ntawm WPCD, BM-DCs
los ntawm C57BL / 6 tau tswj hwm. Kev qhia theem ntawm CD11c, CD86, CD80, CD40, thiab MHC-II hauv hlwb raug tshuaj xyuas (Daim duab 1). Cov hlwb gated nrog SSC thiab FSC tau pom tias kev kho mob nrog cov koob tshuaj sib txawv ntawm WPCD tsis hloov pauv morphology ntawm BM-DCs (cov ntaub ntawv tsis qhia). Cov kev qhia theem ntawm CD86, CD80, thiab CD40 tau nce siab-tswj raws li koob tshuaj piv rau cov pab pawg tsis tau kho thiab mus txog toj siab hauv koob tshuaj ntawm 20 ug / mL ntawm WPCD, tab sis qhov txawv yog tsis tseem ceeb piv nrog pawg LPS (Fig 1A–1C). Kev qhia theem ntawm MHC-II tau nce ntxiv rau nws qhov siab tshaj plaws nyob rau hauv koob tshuaj 50 ug / mL (Daim duab 1D). Cov txiaj ntsig ntawm kev soj ntsuam ntawm cov cim ntawm tes tau pom tias DCs kho nrog LPS lossis WPCD tau pom muaj kev qhia ntau ntxiv ntawm CD86, CD80, CD40, thiab MHC-II thiab txhawb nqa phenotypic maturation.
Qee cov adjuvants tuaj yeem ua kom cov co-stimulatory molecules ntawm DCs los yog ncaj qha induce lub secretion ntawm cytokines. IL-12 thiab TNF- yog cov cytokines loj rau kev ua kom lub cev tiv thaiv kab mob Th1. Peb txheeb xyuas cov txiaj ntsig ntawm cytokine hauv BM-DCs raws li kev kho WPCD. Tom qab BM-DCs tau kho nrog cov ntsiab lus sib txawv ntawm WPCD rau 12 teev, cov supernatant tau sau los kuaj xyuas cov ntsiab lus ntawm IL-12 thiab TNF- nrog cov khoom siv ELISA. WPCD tuaj yeem txhaj tshuaj raws li kev tsim tawm ntawm IL-12 (Daim duab 2A) thiab TNF- (Fig 2B). Cov txiaj ntsig tau pom tias WPCD tuaj yeem ua rau kev ua haujlwm loj ntawm DCs.
Hauv kev tiv thaiv kab mob, nws yog ib qho tsim nyog yuav tsum ua kom DCs ua haujlwm. Tom qab ntawd, qib siab dua ntawm allogeneic T-cell proliferation yog induced los ntawm tag nrho mature DCs. Yog li, cov lus teb ua haujlwm ntawm DCs rau WPCD tau tshawb xyuas los ntawm MLR. Lub peev xwm allostimulatory ntawm WPCD-induced DCs, zoo ib yam li LPS, tau txhim kho zoo heev nyob rau hauv ib koob tshuaj raws li qhov piv txwv (Daim duab 2C thiab 2D). Cov txiaj ntsig tau qhia tias WPCD txhim kho kev nthuav qhia antigen thiab ua kom zoo dua T-cell teb los ntawm MLR hauv vitro.
WPCD txhawb kev loj hlob ntawm DCs ntawm TLR4 txoj hauv kev
Nws tuaj yeem raug txiav txim siab los ntawm cov txiaj ntsig saum toj no uas BM-DCs qhib los ntawm WPCD qhia zoo sib xws DCs nto molecule kab lus rau LPS (a TLR4 ligand). Yog li peb xav tias BM-DCs tau qhib los ntawm WPCD ntawm txoj kev TLR4. Tom qab BM-DCs tau pretreated nrog TAK-242 (TLR4 inhibitor) thiab co-cultured nrog WPCD thiab LPS rau 12 h, kev qhia ntawm CD40, CD86, TNF-a, los yog IL{{12 }} tau kuaj pom los ntawm FACs lossis ELISA. Raws li pom nyob rau hauv daim duab 3A thiab 3B, kev kho TAK-242 tau ua rau muaj kev cuam tshuam tsis zoo ntawm CD40 thiab CD{18}} ntxias los ntawm LPS lossis WPCD, thiab cytokine ntau lawm ntawm IL-12 lossis TNF-a kuj tseem txo qis (Daim duab 3C thiab 3D). Cov txiaj ntsig tau qhia tias WPCD tuaj yeem ua rau DC maturation los ntawm txoj kev TLR4.
WPCD txhim kho kev lom zem thiab kev tiv thaiv ntawm tes
Rau kev sib piv, peb tau soj ntsuam qhov cuam tshuam ntawm WPCD ntawm kev tshem tawm cov tshuaj tiv thaiv kab mob humoral hauv OVA-tiv thaiv nas. Ua ntej txhaj tshuaj tiv thaiv thiab hnub tim 14, 21,
35, thiab 49 tom qab txhaj tshuaj tiv thaiv thawj zaug, cov ntshav tau sau los kuaj xyuas IgG titer thiab IgG subclasses serum los ntawm ELISA. IgG cov tshuaj tiv thaiv kab mob rau OVA tau nce ntxiv nrog kev nce hauv koob tshuaj ntawm WPCD kev tswj hwm (100 thiab 400 ug) nyob rau hauv koob tshuaj (Daim duab 4). Qhov siab tshaj plaws yog 100 ug / mL ntawm WPCD thiab nce IgG cov lus teb los ntawm ob zaug piv rau OVA ib leeg rau Hnub 21, 35 thiab 49 (Fig 4A). Kev nce ntxiv hauv OVA tshwj xeeb IgG1 thiab IgG2a cov tshuaj tiv thaiv kab mob qis dua 20 thiab 100 ug ntawm WPCD kev tswj hwm tau muab piv nrog OVA pab pawg hauv ntshav rau hnub 35 (Fig 4B). Lub caij no, Alum txhawb nqa tsuas yog qib kev qhia ntawm OVA-specific IgG thiab IgG1 cov tshuaj tiv thaiv hauv cov nas uas tau txhaj tshuaj. WPCD ib leeg tsis ua rau OVA cov tshuaj tiv thaiv tshwj xeeb. Cov txiaj ntsig saum toj no tau qhia tias WPCD tsim cov tshuaj tiv thaiv kab mob siab dua thiab sib npaug Th1 / Th2 cov lus teb ntau dua li Alum.
Splenocyte proliferation yog lwm qhov qhia txog kev tiv thaiv cellular. ConA txhawb nqa T-cells thiab LPS txhawb nqa B-cell proliferation. Hnub 21 tom qab txhaj tshuaj tiv thaiv thawj zaug, ib qho kev tshem tawm ntawm splenocyte tau txais thiab ua raws li kev txhawb nqa nrog OVA (10 ug / mL), OVA323-339 (10 ug / mL) peptide, ConA (5 ug / mL), thiab LPS ( 5 ug / ml) rau 48 h. Tom qab ntawd qhov kev loj hlob ntawm T hlwb raug ntsuas los ntawm MTT txoj kev. WPCD tuaj yeem txhawb nqa OVA-antigen, Con A-mitogen, thiab LPS mitogen-stimulated splenocyte proliferation hauv cov nas uas tau txhaj tshuaj OVA thiab WPCD (Fig 5) thiab qhov kev pom zoo ntawm WPCD yog 100 ug / mL. Cov ntaub ntawv no tau qhia tias WPCD ua cov tshuaj tiv thaiv kab mob hauv OVA-tiv thaiv nas tuaj yeem ua kom muaj txiaj ntsig zoo ntawm T-cells thiab B hlwb dua li Alum.
Txhua qhov kev sim WCPD adjuvants tsim nrog cov tshuaj tiv thaiv OVA tsim muaj zog sib npaug thiab kev tiv thaiv ntawm tes (Figs 4 thiab 5). Raws li cov ntaub ntawv no, txhawm rau ntsuas WPCD cuam tshuam ntxiv ntawm OVA-tshwj xeeb T pab (Th) cov lus teb ntawm tes, peb tau txheeb xyuas cov kab lus cytokine ntxiv hauv CD8+ thiab CD4+ T hlwb los ntawm Flow cytometry (Fig 6) . Hnub 21 tom qab txhaj tshuaj tiv thaiv thawj zaug, ib qho kab mob ntawm cov nas tau raug cais tawm thiab tom qab ntawd koom nrog OVA (10 ug / mL). IL-4 tawm los hauv CD4+ T hlwb hauv pawg tswj hwm nrog 100 ug OVA / WPCD tau ntau dua li hauv OVA / Alum pawg thiab OVA pawg thiab zoo ib yam li hauv PMA (Fig 6A). Ntxiv mus, IFN- tawm los hauv CD8+ thiab CD{15}} T hlwb kuj tseem muaj txiaj ntsig zoo hauv cov nas uas tswj hwm OVA / WPCD (20, 100, thiab 400 ug) (Fig 6B thiab 6C). Piv nrog Alum adjuvant, WPCD tau pom muaj peev xwm zoo dua ntawm inducing IL-4 thiab IFN- secretions los ntawm T hlwb.
WPCD-stimulated maturation ntawm DCs thiab txo Treg zaus hauv vivo DCs tau lees paub tias yog APCs muaj zog tshaj plaws koom nrog hauv kev pib cov lus teb hauv lub cev. Yog li, Hnub 3 tom qab txhaj tshuaj tiv thaiv thawj zaug, peb kuj tau tshawb xyuas qhov cuam tshuam ntawm WPCD ntawm CD40 thiab CD80 ntawm DCs ntawm tus po nyob rau hauv cov nas (Daim duab 7A thiab 7B). Cov nas nyob rau hauv 100-ug OVA / WPCD pab pawg ua tau ntau dua ntawm CD40 thiab CD80 ntawm DCs dua li cov hauv pawg OVA / Alum. Cov ntaub ntawv no tau qhia tias WPCD tuaj yeem qhib DCs thiab ua rau DC maturation hauv nas. Treg hlwb tuaj yeem sib npaug ntawm kev kam rau siab thiab kev tiv thaiv kab mob. Txhawm rau tshawb xyuas ntxiv li cas WPCD hloov kho lub cev tiv thaiv kab mob, Treg hlwb nyob rau hauv tus po nyob rau hauv nas tau stained nrog nas tswj T cell staining kit. Hnub 21 tom qab txhaj tshuaj thawj zaug, qhov tsawg zaus ntawm CD25+ Foxp3+ Treg hlwb tau pom nyob rau hauv tag nrho CD4+ T cells (Fig 7C). Piv nrog OVA thiab OVA / Alum pawg, WPCD pawg tau pom qhov txo qis Treg zaus.
Kev soj ntsuam kev nyab xeeb ntawm WPCD hauv nas
Txhawm rau kwv yees qhov ncauj mob toxicity, peb tau ua qhov kev kuaj mob hnyav. Cov nas tsuag hauv qhov ncauj nrog 5000 mg / kg lub cev qhov hnyav tsis pom muaj tus cwj pwm txawv txav lossis cov kev mob tshwm sim thiab tsis muaj kev tuag nyob hauv qhov kev ntsuas tshuaj lom. Tsis muaj qhov sib txawv ntawm lub cev qhov hnyav nce, Thymus Performance index, thiab Spleen Performance index tau sau tseg ntawm ntau pawg ntawm cov nas uas tau tswj hwm nrog cov koob tshuaj sib txawv ntawm WPCD thiab tsis muaj qhov sib txawv tseem ceeb (Table 1). Tsis pom kev tuag tom qab 14 hnub. Yog li, LD50 tus nqi ntawm WPCD yog ntau tshaj 5000 mg ib kg lub cev hnyav.
Txhawm rau kuaj seb WPCD puas muaj kev cuam tshuam tsis zoo rau kev loj hlob ntawm cov nas, ua ntej thiab tom qab txhaj tshuaj tiv thaiv subcutaneous, lub cev hnyav tau txiav txim siab rau txhua tus nas (Table 2). Hauv kev soj ntsuam tom ntej ntawm cov nas, cov kev mob tshwm sim lossis kev coj cwj pwm txawv txawv tsis tau pom. Tsis tas li ntawd, lub cev hnyav ntawm cov nas uas tau tswj hwm nrog WPCD thiab cov nas uas siv cov kua ntsev los yog OVA / Alum tsis pom qhov txawv txav. Cov txiaj ntsig kev soj ntsuam no tau pom tias kev tswj hwm ntawm WPCD muaj kev nyab xeeb.
Kev sib tham
Adjuvants yog cov khoom tseem ceeb hauv cov tshuaj tiv thaiv [31]. Vim muaj kev nce qib hauv genomics thiab proteomics, ntau thiab ntau cov recombinants thiab hluavtaws tshuaj tiv thaiv molecules raug txheeb xyuas. Yog li ntawd, yuav tsum tau ntau adjuvants thiab formulations. Qee qhov muaj zog adjuvants feem ntau muaj feem cuam tshuam rau kev muaj tshuaj lom ntxiv, piv txwv li, FCA. Yog li ntawd, nws yog ib qho tsim nyog yuav tau nrhiav ib qho kev nyab xeeb uas muaj cov khoom sib txawv sib txawv thaum kawg ua rau lub cev xav tau.Polysaccharides los ntawm Suav tshuaj ntsuab yog nontoxicthiab qhia tsis muaj kev phiv loj [32,28]. Yuav tsum muaj ib qho tshuaj tiv thaiv zoo rau qee yam tshuaj tiv thaiv.
Cistanche deserticola yog ib qho tseem ceeb tshuaj ntsuab thiab dav muaj nyob rau hauv arid av thiab sov suab puam nyob rau sab qaum teb hnub poob ntawm Tuam Tshoj. Cistanche deserticola muaj kev txhawj xeeb dav vim nws cov bioactivities xws li immunomodulatory, antioxidative, antibacterial, thiab antitumor teebmeem [20,21]. Qee qhov kev nce qib tau ua tiav hauv cov qauv kev ua haujlwm thiab kev tiv thaiv kev tiv thaiv ntawm Cistanche deserticola, uas yog tus neeg sib tw zoo rau kev tsim cov adjuvants. Peb tau sim soj ntsuam cov teebmeem adjuvant thiab mechanism ntawm WPCD hauv vitro thiab hauv vivo. Kev tswj hwm subcutaneous ntawm WPCD tau txhawb nqa kev lom zem thiab lub cev tiv thaiv kab mob los ntawm kev ua kom cov tshuaj tiv thaiv kab mob hauv cov ntshav thiab lymphocyte proliferation, txhim kho cov kev qhia ntawm cytokines, nce-regulating DC maturation, thiab down-regulating zaus ntawm CD4+ CD{{5} } Foxp3+ Treg cells.
DCs yog APCs tseem ceeb rau priming naive T cells. Kev ua kom DCs yog qhov tseem ceeb rau cov neeg ua haujlwm. DCs, tshwj xeeb tshaj yog murine marrow-derived DCs, feem ntau yog siv los ntsuas cov tshuaj tiv thaiv thiab tshuaj tiv thaiv. Flow cytometry tuaj yeem paub qhov txawv ntawm cov hlwb uas tau ua haujlwm tom qab ntes cov antigen los ntawm cov hlwb nyob ib puag ncig. Hauv FCM tsom xam, qib ntawm kev sib sau ua ke ntawm stimulated
hlwb yog qhov qhia tsis ncaj ntawm kev ntsuas kev nyab xeeb ntawm immunomodulators [3]. Yog li, WPCD adjuvant cov ntaub ntawv ua haujlwm hauv DCs hauv vitro tuaj yeem muab cov ntaub ntawv tseem ceeb rau cov qauv tsiaj. Raws li tus qauv BM-DC, cov kev qhia theem ntawm MHC-II, CD86, CD80, thiab CD40, cytokine ntau lawm, thiab allogenic T cell proliferation tau kuaj pom nyob rau hauv qhov zoo tshaj WPCD concentration ntau. Cov theem qhia ntawm MHC-II, CD86, CD80, thiab CD40 tau nce-tswj hauv BM-DCs thiab cov txiaj ntsig ntawm TNF-a thiab IL-12 tau nce ntxiv raws li kev siv tshuaj. Allogeneic T cell proliferation tau pom. Lub morphology ntawm BM-DCs tsis tau hloov. Los ntawm inducing BM-DCs activation thiab lub secretion ntawm inflammatory cytokines nyob rau hauv vitro, WPCD adjuvant yuav ho nce tus nqi ntawm antigen thiab tus naj npawb ntawm APCs thiab tsub kom T hlwb kom secrete IFN-, uas pab txhawb rau kev txhim kho ntawm immunomodulatory kev ua si.
Ntau yamSuav herbaceous polysaccharidesmuaj peev xwm ua kom lub cev tsis muaj zog thiab muaj peev xwm ua tau zoo heev los ntawm kev txhawb nqa DC kev loj hlob los ntawm txoj kev TLR4 [33,28]. Yog li, peb xav tias TLR4 koom nrog txoj hauv kev taw qhia ntawm WPCD-induced DCs maturation. Raws li kev cia siab, TLR4 inhibitor kev kho mob ua rau txo qis hauv TNF-a thiab IL-12. Tsis tas li ntawd, qhov inhibition ntawm TLR4 txoj hauv kev kuj cuam tshuam qhov kev qhia ntawm CD40 thiab CD80 ntawm DCs, qhia tias kev loj hlob ntawm DCs yog nyob ntawm TLR4. Yog li ntawd, nws yog qhov pom tseeb tias TLR4 koom nrog WPCD-induced DCs maturation.
NATURAL CISTANCHE TUBULOSA rau kev txhim kho kev tiv thaiv kab mob PHGS75% ECH 30% ACT 12%
Qhov koob tshuaj zoo tshaj plaws ntawm cov tshuaj tiv thaiv kab mob thiab tshuaj tiv thaiv yog txiav txim siab pom hauv ntau qhov kev sim. Txhawm rau tshawb nrhiav kev sib raug zoo ntawm koob tshuaj ntawm WPCD adjuvant thiab OVA antigens hauv cov nas, peb tau xaiv cov koob tshuaj WPCD sib txawv raws li cov ntaub ntawv qhia dhau los ntawm BM-DCs hauv vitro rau subcutaneously tswj ICR nas ob zaug. Peb pom tias WPCD tau nce cov txiaj ntsig ntawm OVA cov tshuaj tiv thaiv tshwj xeeb thiab ua rau muaj kev sib npaug Th1 / Th2 lub cev tiv thaiv kab mob nrog kev txhim kho ntawm IgG1 thiab IgG2a qib. Tshwj xeeb tshaj yog, qib IgG2a siab dua qhov uas tau kho nrog Alum hauv cov koob tshuaj zoo tshaj. Yog li ntawd, WPCD ua rau muaj ntau dua ntawm cov tshuaj tiv thaiv tshwj xeeb thiab kev ua tau zoo dua nrog kev txhaj tshuaj qis dua.
Cov tshuaj tiv thaiv tshiab yuav tsum muaj qhov cuam tshuam ntawm cov lus teb ntawm tes muaj zog, uas muaj cov tshuaj tiv thaiv kab mob, T pab (Th) hlwb, thiab cytotoxic T lymphocytes (CTLs). Cov tshuaj tiv thaiv kab mob txhawm rau ua kom muaj zog T cell teb. Qhov zoo tshaj plaws adjuvant txhim kho qhov muaj zog ntawm cov tshuaj tiv thaiv thiab txhawb nqa kev tiv thaiv kab mob ntawm tes tsis ua rau muaj tshuaj lom [34]. Ntawm cov kev soj ntsuam hauv cov qauv txhaj tshuaj tiv thaiv nas, WPCD tau ua rau muaj kev nce ntxiv hauv OVA tshwj xeeb thiab tsis tshwj xeeb splenocyte proliferation piv nrog Alum. Qee qhov adjuvants up-regulate cytokines thiab lub cev tiv thaiv kab mob. Piv txwv li, saponins tuaj yeem txhawb nqa lub cev tiv thaiv kab mob hauv lub cev rau cov tshuaj tiv thaiv uas ib txwm ua rau tsuas yog cov tshuaj tiv thaiv kab mob. Peb tau xaiv IL-4 thiab IFN- ua cov ntsuas ntsuas ntsuas qhov ntsuas tsis ncaj rau hauv cov nas. WPCD tuaj yeem ntxias ntau IL-4 thiab IFN- secretions tshaj Alum. Yog li, cov lus teb muaj zog T-cell thiab cytokine secretions tau pom los ntawm kev txhaj tshuaj tiv thaiv WPCD, qhia tias WPCD tuaj yeem ua kom muaj zog lymphocytes kom zais cov Th1-hom cytokine thiab Th2- hom cytokine ntau dua. Alum.
Adjuvants influencing antigen nthuav qhia tuaj yeem cuam tshuam cov txheej txheem tiv thaiv kab mob. Treg hlwb tuaj yeem hloov kho Th1 thiab Th2 cov lus teb [35]. Ib koob tshuaj uas tsim nyog ntawm WPCD tuaj yeem txhawb kev loj hlob ntawm DCs hauv cov nas los ntawm kev nce qib ntawm CD80 thiab CD40 thiab ua kom muaj peev xwm ntawm OVA antigen kev nthuav qhia hauv lub cev tiv thaiv kab mob thaum ntxov. Cov txiaj ntsig ntawm kev sim ntawm DCs ua kom muaj zog thiab Treg zaus pom tau tias WPCD ua rau kev loj hlob ntawm DCs thiab txo qis Treg zaus hauv tus po hauv cov nas. Cov txiaj ntsig no tau ua pov thawj tias WPCD tau txhim kho qhov ua tau zoo ntawm cov tshuaj tiv thaiv OVA los ntawm kev ua kom lub hom phiaj ntawm cov tshuaj tiv thaiv kab mob tam sim no thiab txhawb kev ua kom T-cell tshwj xeeb.
Nyob rau hauv txoj kev loj hlob ntawm adjuvants, nws yog ib qho nyuaj rau xaiv induce lub cev tiv thaiv kab mob uas tsim nyog tiv thaiv tus kab mob coj. Adjuvant tsim nyog yuav tsum muaj kev phiv tsawg thiab toxicity rau tib neeg los yog tsiaj txhu. Yog li, WPCD kev nyab xeeb yuav tsum raug txiav txim siab, suav nrog cov kev mob tshwm sim tam sim ntawd thiab mus sij hawm ntev. Hauv txoj kev tshawb no, peb tshawb nrhiav qhov mob toxicity ntawm WPCD thiab qhov tsis zoo ntawm WPCD ntawm kev loj hlob ntawm nas rau 110 hnub. Cov txiaj ntsig ntawm kev mob toxicity ntawm WPCD pom tias lub cev qhov hnyav, thymus Performance index, lossis Spleen Performance index tsis muaj qhov sib txawv loj. Tus nqi LD50 ntawm WPCD yog ntau tshaj 5000 mg ib kg lub cev hnyav. Thoob plaws hauv qhov kev sim tom qab kev soj ntsuam ntawm cov nas, tsis muaj kev phiv los yog kev coj cwj pwm txawv txav hauv cov nas. Cov txiaj ntsig no tau qhia tias WPCD muaj kev nyab xeeb.
Hauv kev xaus, hauv qhov kev tshawb fawb no, WPCD, cov khoom siv raw polysaccharide rho tawm los ntawm C. deserticola los ntawm Xinjiang, tau nthuav tawm qee yam khoom ntawm adjuvants. Piv txwv li, WPCD tau txhim kho Th1 thiab Th2 cov lus teb los ntawm kev ua kom DCs, nce cov tshuaj tiv thaiv kab mob, thiab txhim kho cytokine ntau lawm. Tsis tas li ntawd, peb tau tshawb nrhiav cov txheej txheem ntawm WPCD kev ua tau zoo los ntawm kev txheeb xyuas DCs kev loj hlob thiab kev ua haujlwm ntawm TLR4 txoj hauv kev hauv vitro. Hauv cov nas tsuas yog kho nrog WPCD, tsis pom muaj kev phiv tshwm sim. Yog li, WPCD muaj ntau dua immunostimulatory kev ua haujlwm hauv cellular thiab humoral tiv thaiv kab mob. Adjuvants yog qhov sib xyaw ntawm ntau lub tebchaw. Kev sib xyaw ntawm ntau cov khoom siv raw tuaj yeem muaj txiaj ntsig zoo dua li ib tsob nroj extract. Nws yog ib qho tsim nyog yuav tsum tshawb nrhiav WPCD cov ntaub ntawv rho tawm, sim lawv cov txiaj ntsig thiab kev nyab xeeb, thiab nthuav tawm cov txheej txheem ntawm lawv cov teebmeem.
NATURAL CISTANCHE TUBULOSA rau kev txhim kho kev tiv thaiv kab mob PHGS75% ECH 30% ACT 12%
