Aqueous Extracts Los ntawm Cultivated Cistanche Deserticola YC Ma As Polysaccharide Adjuvant Txhawb Kev tiv thaiv kab mob

Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791

Shuangshuang Feng, 1, Xiumei Yang a, Xiang Weng a, 1, Bin Wang b, Ailian Zhang, *

TSAB NTAWV

Ethnopharmacological qhov tseeb:Herbal polysaccharides tau nthuav tawm zoo heevtiv thaiv kab mob- Txhim khu muaj peev xwm. Adjuvants yog cov cuab yeej tseem ceeb rau kev tsim cov tshuaj tiv thaiv zoo. Hauv peb txoj kev tshawb fawb yav dhau los, cov dej-soluble polysaccharide muab rho tawm los ntawm cov tsiaj qusCistanche deserticola YC Mapom muaj zog immunostimulatory kev ua.

Lub hom phiaj ntawm kev kawm:Hauv qhov kev tshawb fawb no, lubtiv thaiv kab mobprofiles thiab kev ua tau zoo ntawm aqueous extracts ntawm cultivatedCistanche deserticola YC Ma(AECCD) ntawm ICR nas tiv thaiv ovalbumin (OVA) tau tshawb xyuas. Hauv kev sim hauv vitro, qhov ua tau DC ua kom lub cev muaj zog los ntawm AECCD raug soj ntsuam.

Cov ntaub ntawv thiab cov txheej txheem: AECCDtau muab rho tawm siv dej kub tom qab uas cov crude polysaccharides tau precipitated los ntawm ethanol. Cov nas yog thawj zaug txhaj tshuaj subcutaneously nrog OVA (10 ug ib tus nas) ib leeg lossis OVA (10 ug ib tus nas) feem ntau muaj cov koob tshuaj sib txawv ntawm AECCD (200, 400, thiab 800 ug ib tus nas) nyob rau Hnub 1 thiab 14 thiab qhov loj thiab kinetics ntawm Cov tshuaj tiv thaiv kab mob thiab cov lus teb ntawm tes-mediated tau raug soj ntsuam.

Cov txiaj ntsig: AECCDelicited vigorous thiab ntev IgG cov lus teb nrog cov lus teb Th1 / Th2 sib xyaw thiab nce qib ntawm Th-txuas cytokines (CD4 ntxiv rau IL-4, CD4 ntxiv rau IFN- , thiab CD8 ntxiv rau IFN- ). Ntxiv mus, AECCD induced lub zog cellulartiv thaiv kab mobcov lus teb tshwm sim los ntawm kev nce splenocyte proliferation nrog rau cov lus teb ua haujlwm T cell. Qhov tseem ceeb, AECCD tau txhim kho qhov kev loj hlob ntawm cov hlwb dendritic (DCs) thiab inhibited Tregs. Hauv kev sim vitro, kev sim ua ntej tau qhia tias AECCD ua rau DC ua kom muaj zog los ntawm kev txhawb nqa phenotypic maturation, cytokine seem, thiab allostimulatory kev ua. Tus xov tooj hu-zoo li receptor 4 (TLR4) yog ib qho tseem ceeb receptor rau DCs ncaj qha khi AECCD. Cov inhibitors ntawm NF-κB txo qis kev qhia qib ntawm CD40, CD80, CD86, thiab MHC-II thiab tsim tawm IFN- , TNF- , thiab IL-6 los ntawm DCs.

Cov lus xaus:Thaum kawg, cov kev tshawb pom no pom tau tiasAECCDtuaj yeem ua kom muaj zog thiab ruaj khov antigen tshwj xeebtiv thaiv kab mobcov lus teb los ntawm DC ua kom, uas tau koom nrog hauv kev tswj hwm ntawm cov cim kev loj hlob thiab cytokine qhia ntawm TLR4-txog NF-κB txoj hauv kev. Txoj kev tshawb no qhia tias AECCD yog qhov muaj peev xwm immunomodulator.

Cistanche tshuaj ntsuabtxhim khotiv thaiv kab mob.

1. Taw qhia

Ntau qhov kev tshawb fawb tau qhia tias cov tshuaj tiv thaiv tuaj yeem txhim kho kev ua tau zoo ntawm cov tshuaj tiv thaiv kab mob sib kis xws li HIV, COVID-19, mob npaws, thiab kab mob ko taw thiab qhov ncauj (Cao li al., 2020; Giuseppe li al., 2018 ). Adjuvants tuaj yeem ua rau muaj ntau hom thiab qhov loj ntawm lub cev tiv thaiv kab mob, yog li kev xaiv adjuvant kom tsim nyog tuaj yeem ua rautiv thaiv kab mobteb yuav tsum tau rau ib tug kab mob los yog antigen (Reed li al., 2013). Ntuj herbaceous extracts tau ntev tau dav siv nyob rau hauv lub neej txhua hnub los yog kev kho mob. Hauv ntau qhov xwm txheej, cov tshuaj suav tshuaj (TCM) thiab nws cov khoom siv, tshwj xeeb tshaj yog ntau yam TCM polysaccharides-derived adjuvants, tau txhim kho cov tshuaj tiv thaiv zoo. TCM polysaccharides (Advax™, Astragalus polysaccharide, Ginseng polysaccharide, Lycium barbarum polysaccharide, thiab Ganoderma lucidum polysaccharide) muaj ntau yam zoo dua lwm cov adjuvants, xws li kev nyab xeeb zoo dua, biocompatibility, muaj zogtiv thaiv kab mobtxhim kho, thiab qis reactogenicity (Li thiab Wang, 2015; Schijns li al., 2020). TCM polysaccharide-based compounds thiab formulations, yog li ntawd, muaj peev xwm ua tau raws li cov neeg sib tw.

Cistanche deserticola YC Ma(Roucongrong hauv Suav) yog cov tshuaj suav tshuaj sau tseg hauv Suav Pharmacopeia thiab tau siv los ua cov khoom noj tonic rau ntau pua xyoo hauv Suav teb (Suav Pharmacopoeia Commission, 1992). Cov kev tshawb fawb tshuaj niaj hnub no tau qhia nws ntau yam kev siv tshuaj thiab tshuaj tiv thaiv kab mob (Dong li al., 2007; Fu li al., 2017). Hauv peb txoj kev tshawb fawb dhau los, cov polysaccharides crude tau los ntawm cov qus Cistanche deserticola YC Ma txhawb cov antigen tshwj xeeb humoral thiab cellular teb thiab tswj kev loj hlob ntawm dendritic hlwb (DCs) (Zhang li al., 2018; Zhao li al., 2019). Txawm li cas los xij, cov tsiaj qus Cistanche deserticola YC Ma tau tshawb nrhiav ntau dhau thiab teev tias yog hom kab mob uas muaj kev puas tsuaj.Cistanche deserticola YC Matau cog rau hauv qhov loj thiab tau siv dav hauv kev txhim kho thiab siv cov peev txheej TCM. Txawm li cas los xij, lub peev xwm adjuvant ntawm cultivated Cistanche deserticola YC Ma tseem tsis tau meej.

DCs yog cov tswj hwm tseem ceeb tiv thaiv kab mob sib kis. Tom qab DCs tau qhib, cov molecules saum npoo thiab cov cytokines pro-inflammatory cytokines ntawm DCs cuam tshuam nrog cov neeg txais kev sib raug zoo los qhib T hlwb thiab ua rau cov lus teb tshwj xeeb antigen thiab tsim cov cytokines directing adaptive.tiv thaiv kab mobcov lus teb (Banchereau thiab Steinman, 1998). Nws tau raug tshaj tawm tias tus xov tooj hu ua receptor 4 (TLR4) ntawm DCs ua haujlwm ua ib qho tseem ceeb polysaccharide receptor (Park li al., 2014; Qi li al., 2016). Qhov tseeb, ntau cov polysaccharides tuaj yeem cuam tshuam ncaj qha lossis tsis ncaj nraim nrog TLR4 ntawm DCs txhawm rau ua kom lub zog qis qis-kappa B (NF-κB), uas tuaj yeem ua rau muaj kev cuam tshuam ntau yam ntawm cov noob caj noob ces xws li IFN-, TNF-, thiab IL{{11} }} thiab cov lus teb inflammatory los tshem tawm cov kab mob (Tian li al., 2019; Zhou et al., 2017; Zhu et al., 2013). Yog li ntawd, DCs thiab NF-κB tau kawm ntau yam raws li lub hom phiaj rau kev kho kab mob.

Hauv txoj kev tshawb no, nrog rau hauv vivo (ICR nas) lub tswv yim, peb thawj zaug soj ntsuam cov teebmeem immunostimulatory ntawm aqueous extracts ntawm cultivatedCistanche deserticola YC Ma(AECCD) nrog cov koob tshuaj sib txawv (qis, nruab nrab, thiab siab) txhawm rau txhawm rau tiv thaiv lub cev tiv thaiv OVA thiab piav qhia txog txoj hauv kev hauv vitro kom qhib DCs thiab cov kev cuam tshuam txog cov receptors. Nrog tus qauv nas, peb tau qhia tias AECCD txhawb nqa cov tshuaj tiv thaiv kab mob ntev ntev nrog kev nce IgG titers thiab cellular.tiv thaiv kab mobteb. Qhov tseem ceeb, AECCD induced Th-associated cytokines, tshwj xeeb tshaj yog IFN- ntau lawm, uas coj mus rau Th1 teb tiv thaiv kab mob. Cov nyhuv induction tau zoo sib xws nrog kev txhim kho DC kev loj hlob thiab txo qis Tregs. Mechanistically,AECCDtxhawb nqa DC ua kom dhau ntawm TLR4-txog NF-κB txoj hauv kev. Hauv cov ntsiab lus, txoj kev tshawb fawb tau lees paub qhov muaj zog adjuvant kev ua haujlwm ntawm AECCD hauv kev txhawb nqa cov lus teb hloov pauv. Txoj kev tshawb no tau tsim lub hauv paus rau kev txhim kho AECCD polysaccharide adjuvants.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Reagents

Ovalbumin (OVA), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), polymyxin B (PMB), concanavalin A (ConA), lipopolysaccharide (LPS), mitomycin C thiab 3,3′,5,5′-Tetramethylbenzidine (TMB) tau txais los ntawm Sigma (USA). Fetal bovine serum (FBS) tau yuav los ntawm Biological Industries. Cell Counting Kit-8 (CCK-8) tau yuav los ntawm Biosharp (Tuam Tshoj). Tshis los tiv thaiv nas IgG/IgG1/IgG2a-HRP conjugates yog los ntawm Southern Biotech (USA). Txhaj Alum yog los ntawm Thermo Scientific Pierce (USA). GM-CSF yog los ntawm Peprotech (USA). Anti-CD{20}}PE, anti-CD4-APC, anti-CD8a-FITC, anti-CD44-PE, anti-CD62L-PE, anti-MHCII -FITC/Percp-cy5.5, anti-IL-4-PE, anti-IFN- -PE, anti-CD86-APC, anti-CD40-FITC/PE, anti- CD11c-PE/FITC, anti-CD80-APC), Golgistop, Cytofix/Cytoperm, thiab Perm/Wash buffer yog tag nrho los ntawm BD Bioscience (USA). Cov khoom siv tswj T-cell staining yog los ntawm eBiosciences (USA). TAK-242 yog los ntawm MedchemExpress (Shanghai, Suav). PDTC yog los ntawm Beyotime Biotechnology (Tuam Tshoj). Multi-analyte flow assay kit yog los ntawm Biolegend (USA).

2.2. Kev npaj ntawm AECCD

C. deserticola (Ib daim ntawv pov thawj No. CD10041602) tau txheeb xyuas los ntawm xibfwb Jiang He.AECCDtau txais los ntawm kev rho tawm dej, ethanol nag lossis daus, thiab txoj kev deproteinization nrog qee qhov kev hloov kho me me (Zhang li al., 2018). Luv luv, cultivated C. deserticola yog thawj zaug muab rho tawm nrog dej ntau zaus thiab tom qab ntawd crude polysaccharides tau pw hmo ntuj los ntawm kev ntxiv ethanol raws li qhov sib piv ntawm 1: 4. Tom qab ntawd cov tau txais crude polysaccharides tau deproteinized los ntawm txoj kev Sevag (Liu li al., 2012). Tag nrho cov ntsiab lus ntawm qab zib tau kuaj pom tias yog 76.82 feem pua ​​​​ntawm txoj kev anthrone-sulfuric acid (Dubois li al., 1951).

cistanche txiv neej cov txiaj ntsighauvtiv thaiv kab mob

2.3. Infrared spectroscopic tsom xam

Rau lub hom phiaj ntawm kev txheeb xyuas cov pab pawg ua haujlwm organic,AECCDnrog KBr raug nias rau hauv pellets rau qhov ntsuas infrared (IR) spectra. IR spectra ntawm cov qauv tau txiav txim siab nyob rau hauv 4000-400 cm-1.

2.4. Hauv vivo kev sim

2.4.1. Tsiaj txhaj tshuaj

Poj niam C57BL/6 nas thiab ICR nas (6 lub lis piam) los ntawm Xinjiang Medical University (Urumqi, Tuam Tshoj) tau acclimatized rau 3 hnub. Tag nrho cov txheej txheem sim tau ua raws li cov kev cai ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Tsiaj Kev sim ntawm Xinjiang Key Laboratory of Biological Resources thiab Genetic Engineering hauv Xinjiang University (BRGE-AE001).

Cov nas tau txhaj tshuaj subcutaneously nrog OVA ib leeg lossis OVA nrog qhov sib txawv ntawm AECCD thiab txhawb nqa rau Hnub 14 raws li pawg sib txawv raws li hauv qab no (6 nas hauv txhua pab pawg): Pab pawg tswj (0.9 feem pua ​​NaCl), OVA pawg ( 10 UG OVA),AECCD-H pawg (AECCD 800 ug), OVA/ AECCD-L pawg (10 ug OVA, AECCD 200 ug), OVA/ACCD-M pawg (10 ug OVA, AECCD 400 ug), OVA/ACCD -H pawg (10 ug OVA, AECCD 800 ug), thiab OVA / Alum pawg (10 ug OVA, Alum100 ug). Cov tshuaj tiv thaiv hauv 100 μL ntawm 0.9 feem pua ​​​​NaCl tau txhaj rau ntawm ob qhov chaw sib txawv (50 μL ntawm txhua qhov chaw) hauv daim tawv nqaij tom qab ntawm txhua tus nas. Tom qab txhaj tshuaj, cov ntshav tau sau los ntawm retro-orbital plexus, thiab sera tau txais thiab khaws cia ntawm - 80 ◦C rau kev tshuaj xyuas ntxiv ntawm IgG nrhiav pom. Splenocyte proliferation thiab nrhiav pom ntawm cytokine, T cell subsets, DCs nto cim thiab Tregs tau ua tom qab tib txoj kev txhaj tshuaj tiv thaiv.

2.4.2. Kev tsom xam Antibody

IgG titres hauv cov ntshav tawm tsam OVA tau txiav txim los ntawm ELISA Hnub 14, 28, 42 thiab 56 tom qab txhaj tshuaj tiv thaiv thawj zaug (Zhang li al., 2018). IgG1 thiab IgG2a tau ntsuas rau Hnub 21. Qhov nqus ntawm 450 nm / 630 nm tau txais los ntawm kev siv tus nyeem ntawv (Bio-Rad, USA). Cov txiaj ntsig ntawm cov tshuaj tiv thaiv kab mob yog qhia raws li IgG titers thiab IgG1 thiab IgG2a isotypes tau qhia tias yog cov absorbances sib thooj.

2.4.3. Kev ntsuam xyuas Splenocyte proliferation

Splenocyte proliferation assay hauv nas tau ua nrog CCK-8 cov khoom kuaj raws li cov chaw tsim khoom cov lus qhia. Hnub 21 tom qab txhaj tshuaj thawj zaug, cov splenocytes raug cais tawm ntawm tus po ntawm cov nas uas tau txhaj tshuaj tiv thaiv kab mob thiab cov erythrocytes tau lysed tom qab sib tsoo. Splenocytes (1 × 106 cell / zoo) tau txhawb nqa nrog OVA (10 ug / mL), OVA 323-339 (10 ug / mL), ConA (10 ug / mL), thiab LPS (10 ug / mL) rau ob hnub thiab cov hlwb dawb paug tau siv los tswj. Cov kev ua haujlwm loj hlob tau nthuav tawm raws li stimulation Performance index (SI), uas yog xam qhov absorbance piv ntawm ib tug stimulated cell mus rau ib tug unstimulated cell nyob rau hauv lub wavelength ntawm 570/630 nm.

Fig. 1. FTIR spectra of AECCD.

2.5. Kev sim hauv vitro

2.5.1. Tsim cov DCs, cell viability, thiab endotoxin qib

DCs muab tau los ntawm cov pob txha pob txha ntawm C57BL/6 nas tau txais raws li txoj kev tau piav qhia yav dhau los (Inaba li al., 1992). Luv luv, cov hlwb raug coj los ntawm RPMI-1640 ua tiav nruab nrab (20 ng/mL GM-CSF thiab 5 ng/mL IL-4). Tom qab rau hnub, tag nrho cov hlwb raug tshem tawm tau sau ua DCs tsis paub qab hau rau kev soj ntsuam tom ntej. Qhov feem pua ​​​​ntawm CD11c ntxiv rau DCs hauv cov hlwb tsis ua raws li tau txiav txim siab yog 94 feem pua ​​​​ntawm cov cytometry ntws.

Lub cell viability ntawm DCs thiab splenocytes los ntawm cov nas thaum AECCD tau kuaj pom nrog CCK-8 cov khoom siv raws li cov chaw tsim khoom cov lus qhia. Cov hlwb los ntawm DCS thiab splenocytes tau txais thiab tom qab ntawd txhawb nrog ntau qhov sib txawv ntawmAECCD(0, 10, 100, 400, 800, thiab 1600 ug/mL). Lub cell viability tau qhia raws li qhov piv ntawm absorbance ntawm pab pawg kho mob rau absorbance ntawm pawg tswj) × 100 feem pua.

Rau lub hom phiaj ntawm kev tsis suav cov txiaj ntsig ntawm endotoxin hauv AECCD, tom qab AECCD (100, 200 thiab 400 ug / mL) thiab LPS (100 ng / mL) raug kho nrog PMB (100 ug / mL) rau 60 min, DCs (5 × 105 hlwb / mL) tau incubated nrog pretreated AECCD (100, 200 thiab 400 ug / mL) thiab LPS rau 12 teev. Tom qab kev kho mob saum toj no, cov qib CD40, CD86, CD80, thiab MHCII raug soj ntsuam.

2.5.2. Analysis ntawm DC phenotype

Cov theem ntawm costimulatory molecules tau txiav txim siab los ntsuas DC maturation los ntawm kev sim hauv vitro. Hnub rau, DCs tau kho nrogAECCD(20, 100, 200 thiab 400 ug / mL) lossis LPS (100 ng / mL) rau 12 teev. Tom qab ntawd, DCs tau stained nrog cov molecules saum npoo thiab soj ntsuam los ntawm kev ntws cytometry.

2.5.3. Cytokine ntau lawm los ntawm DCs hauv vitro

Lub cell kab lis kev cai supernatant tau sau los ntsuas qib ntawm cytokines los ntawm kev siv cov khoom siv ELISA lossis cov khoom siv tshuaj ntsuam xyuas ntau yam nrog CBA txoj kev. Txhua kauj ruam kev sim tau ua raws li cov neeg tsim khoom cov lus qhia.

2.5.4 ib. Allogeneic mixed lymphocyte reaction (MLR)

Splenocytes los ntawm BALB/c nas tau aseptically tau nyob rau hauv RPMI -1640 nruab nrab (Zhang li al., 2018). Mature C57BL/6 DCs (matured nyob rau hauv lub xub ntiag ntawm txawv concentrationsAECCDlos yog LPS) raug kho nrog 50 ug/mL Mitomycin C rau 60 feeb ntawm 37 ◦C. Tom qab ntxuav, DCs tau tov nrog splenocytes raws li qhov sib piv ntawm 1: 5 lossis 1: 10 rau 2 hnub. Cell proliferation tau txiav txim los ntawm MTT kev soj ntsuam.

Fig. 2. OVA-specific antibody titers thiab IgG isotypes hauv cov ntshav. Cov qauv ntshav tau sau tom qab thawj zaug thiab tshuaj tiv thaiv kab mob rau cov tshuaj tiv thaiv ELISA

2.6. Kev txheeb cais

Cov ntaub ntawv raug qhia raws li txhais tau tias ± SD. Cov theem tseem ceeb ntawm qhov sib txawv ntawm kev txheeb cais tau txiav txim los ntawm One-way ANOVA thiab Tukey qhov kev sib piv ntau yam. P < 0.05="" raug="" suav="" hais="" tias="" yog="" qhov="" tseem="">

3. Cov txiaj ntsig

3.1. FTIR spectroscopy

Lub ntsiab nqus peaks ntawmAECCDNyob rau hauv daim duab 1 yog cov yam ntxwv nqus peaks ntawm cov qauv glycosidic sib haum rau O-H stretching vibration ntawm 3342.95 cm- 1. Band ntawm 2933.54 cm- 1 muaj cov yam ntxwv tsis muaj zog nqus ntawm C-H stretching vibrations. Lub ncov ntawm 1657.39 cm-1 yog ntaus nqi rau O-H flexural vibration. Qhov nqus ntawm 1411.00 cm− 1 yog ascribed rau O–H lossis C–H thiab C–O stretching vibrations. Lub ncov ntawm 1018.38 cm-1 kuj qhia CO thiab C-O-C glycosidic band vibrations.

3.2. OVA-specific serum antibody teb

Sera los ntawm cov nas txhaj tshuaj tau raug kuaj xyuas seb muaj cov tshuaj tiv thaiv tsim tawm (IgG, IgG1, thiab IgG2a) tawm tsam OVA. Raws li pom nyob rau hauv daim duab 2A, IgG titers nyob rau hauv nas txhaj tshuaj nrog OVA /AECCD-M elicited higher total IgG (titers: 220,000, 250,000 and 250,000) relative to those of OVA group (titers: 100,000, 100,000 and 120,000), and was close to Alum group (titers: 250,000, 250,000 and 270,000) on Days 28, 42 and 56 after primary vaccination. OVA/AECCD-M/H significantly enhanced IgG1 levels compared to the OVA group (P < 0.05) and the difference between OVA/AECCD-M/H group and Alum group was not significant (P>{{0}}.05). OVA/AECCD-M/H kuj tau txhim kho IgG2a qib piv rau OVA pawg thiab Alum pawg (Fig. 2B thiab C) (P <0.05). cov="" qib="" igg2a="" txhim="" kho="" tuaj="" yeem="" tiv="" thaiv="" kab="" mob="" uas="" xav="" tau="">tiv thaiv kab mobteb.

3.3. Cellular tiv thaiv kab mob

Spleen lymphocyte proliferation tau tshawb xyuas los ntawm MTT kev soj ntsuam raws li kev txhawb nqa nrog OVA lossis OVA323-339. Kev soj ntsuam kev loj hlob tau qhia tias OVA /AECCD-M ob qho tib si tau nce splenocyte proliferation tom qab kis OVA, OVA323-339, Con A lossis LPS txheeb ze rau OVA pawg. OVA thiab OVA323-339 tau txhim kho qhov kev loj hlob ntawm cov lus teb ntau dua li ntawm pawg Alum (Fig. 3) (P <>

Daim duab 3. Splenocyte proliferation thaum kho AECCD hauv vivo

3.4. Ua kom T cell subsets hauv cov kua dej ntws tawm thiab tus po

Lymph nodes thiab spleen los ntawm cov nas txhaj tshuaj tau raug sau los kuaj xyuas T cell subsets nyob rau hnub 21 tom qab txhaj tshuaj tiv thaiv thawj zaug. Qhov feem pua ​​​​ntawm T cell subsets (CD4 ntxiv, CD8 ntxiv, CD44 ntxiv rau T hlwb) los ntawm cov qog ntshav hauv OVA / AECCD-M pawg tau ntau dua li ntawm OVA pawg thiab Alum pawg (Fig. 4A–D) (P. < 0="" 05).="" cov="" feem="" pua="" ​​​​ntawm="" t="" cell="" subsets="" (cd4="" ntxiv="" ,="" cd8="" ntxiv="" ,="" cd44="" ntxiv="" ,="" thiab="" cd62l="" ntxiv="" rau="" t="" cells)="" nyob="" rau="" hauv="" tus="" po="" tau="" ntau="" dua="" li="" cov="" pab="" pawg="" ova="" (fig.="" 4e–j)="" (p=""><0.05). cov="" kev="" tshawb="" pom="" no="" qhia="" tau="">AECCDtxhawb cell-mediatedtiv thaiv kab mobteb.

3.5. Cytokines secreted los ntawm restimulated splenocytes hauv vivo

Th1/Th2 cytokines los ntawm tus po ntawm cov nas uas txhaj tshuaj tiv thaiv kab mob tau txiav txim siab los ntawm cov kab mob hauv lub cev rau hnub 21 tom qab thawj zaug txhaj tshuaj. Cov theem ntawm CD4 ntxiv rau IL-4, CD4 ntxiv rau IFN-, thiab CD8 ntxiv rau IFN- hauv pawg OVA/AECCD-M tau ntau dua li ntawm OVA pawg thiab Alum pawg (P< 0.05,="" fig.="" 5).="" the="" differences="" indicated="" that="">AECCDtuaj yeem txhawb Th1 / Th2 cov lus teb, tshwj xeeb tshaj yog T-cell mediatedtiv thaiv kab mobteb.

3.6. DC maturation thiab Tregs hauv vivo

Kev ua kom DC tuaj yeem pib hloov pauv kev tiv thaiv. Cov teebmeem ntawmAECCDntawm DC maturation nyob rau hauv cov nas txhaj tshuaj tau raug tshuaj xyuas rau hnub 3 tom qab txhaj tshuaj thawj zaug. OVA/AECCD-M induced qib siab tshaj ntawm CD40, CD80, CD86, thiab MHCII ntawm DCs txheeb ze rau OVA pawg thiab qhov sib txawv ntawm OVA/AECCD-M pawg thiab Alum pawg tseem ceeb (Fig. 6A–D) (P <0.05). qhov="" zaus="" ntawm="" cd4="" ntxiv="" rau="" cd25="" ntxiv="" rau="" foxp3="" ntxiv="" tregs="" hauv="" ova="" aeccd-m="" pawg="" tau="" qis="" dua="" li="" hauv="" ova="" pawg="" (fig.="" 6e)="" (p=""><0.05). cov="" ntaub="" ntawv="" no="" tau="" qhia="" tias="" aeccd="" tuaj="" yeem="" qhib="" dcs="" thiab="" qis="" dua="" treg="">

3.7. Kev soj ntsuam ntawm cytotoxicity thiab endotoxin paug

Tom qab kev kho mob nrog txawv concentrations ntawmAECCDrau 2 hnub, cov hlwb los ntawm DCs thiab splenocytes ntawm nas tau siv los txiav txim qhov muaj peev xwm cytotoxicity nrog CCK-8 cov khoom siv. Tsis muaj qhov tseem ceeb inhibited proliferation teb nyob rau hauv lub cell viability ntawm DCs thiab splenocytes piv rau cov tswj pawg (Fig. 7A–B) (P > 0.05). Cov txiaj ntsig no tau pom tias AECCD ntawm qhov concentration ntawm 10–1600 ug/mL tsis pom muaj cytotoxicity tseem ceeb rau DCs thiab splenocytes piv rau kev tswj hwm (P> 0.05). Yog li, qhov concentration ntawm AECCD hauv cov kev sim tom ntej tau teeb tsa kom qis dua 1600 ug / mL. Txhawm rau tshem tawm qhov cuam tshuam ntawm endotoxin paug hauv cov teebmeem ntawm AECCD ntawm DC ua kom, PMB, tshuaj tua kab mob dav pom vim nws cov LPS-neutralizing nyhuv, tau siv los tshem tawm cov kab mob endotoxin (Morrison thiab Jacobs, 1976). Raws li tau piav qhia hauv daim duab 7C–F, CD40, CD80, CD86 lossis MHC-II qhia theem hauv AECCD-kho DCs nyob rau hauv lub xub ntiag ntawm PMB tsis pom qhov hloov pauv loj (P> 0.05). Txawm li cas los xij, qhov poob qis ntawm CD40, CD80 CD86, thiab MHC-II qhia qib tau pom hauv LPS-kho DCs nyob rau hauv muaj PMB (P <0.001). cov="" txiaj="" ntsig="" no="" tau="" pom="" tias="" aeccd="" tsis="" muaj="" cov="" kab="" mob="">

3.8. Phenotypic maturation thiab kev ua haujlwm ntawm DCs hauv vitro

Raws li kev sim tshuaj tiv thaiv saum toj no, phenotypic maturation ntawm DCs tau tshawb nrhiav ntxiv tom qab kev kho mob nrog AECCD hauv vitro. Hnub 6, DCs tsis paub qab hau tau txhawb nqa nrog qhov muab siab ntawm AECCD lossis LPS los txheeb xyuas cov molecules saum npoo.AECCDinduced cell maturation characterized by a majority in the percentage of CD40, CD80 CD86, and MHC II in a dose-dependent yam (P <0.001, fig.="" 8a–d).="" ib="" qho="" zoo="" sib="" xws="" phenotypic="" maturation="" ntawm="" dcs="" kuj="" tau="" pom="" tom="" qab="" stimulation="" nrog="" 100="" ng="" ml="" lps.="" cov="" ntaub="" ntawv="" txheeb="" xyuas="" tau="" qhia="" tias="" aeccd="" txhim="" kho="" dc="" kev="" loj="" hlob.="" los="" ntawm="" cov="" txiaj="" ntsig="" saum="" toj="" no,="" aeccd="" txhim="" kho="" phenotypic="" maturation="" ntawm="" dcs.="" txhawm="" rau="" soj="" ntsuam="" cov="" txiaj="" ntsig="" ntawm="" aeccd="" ntawm="" dc="" kev="" ua="" haujlwm="" hauv="" vitro,="" lub="" peev="" xwm="" ntawm="" aeccd-kho="" dcs="" los="" txhawb="" cov="" allogeneic="" t="" cell="" proliferation="" hauv="" mlr="" ntawm="" dc-splenocyte="" piv="" ntawm="" 1:="" 5="" lossis="" 1:="" 10="" tau="" tshawb="" xyuas.="" dcs="" kho="" nrog="" ntau="" qhov="" sib="" txawv="" ntawm="" aeccd="" ua="" rau="" t="" cell="" proliferation="" tseem="" ceeb="" piv="" rau="" cov="" tsis="" kho-dcs="" (fig.="" 8e–f)="">< 0.001).="" dc="" maturation="" could="" induce="" the="" production="" of="" cytokines.="" next,="" the="" supernatants="" of="" dcs="" treated="" with="" aeccd="" or="" lps="" were="" detected="" by="" elisa="" kit.="" the="" levels="" of="" il-12="" and="" tnf-α="" in="" the="" supernatants="" of="" dcs="" were="" dramatically="" increased="" in="" a="" dose-dependent="" manner="" (p="" <="" 0.001,="" fig.="">

3.9. TLR4, receptor rau AECCD ntawm DCs

DCs tuaj yeem pib lub xov tooj ntawm tes los ntawm daim nyias nyias receptors. Txhawm rau txiav txim siab lub luag haujlwm ntawm TLR4 hauv kev koom nrog hauv AECCD-kho DC maturation, tom qab kev kho ua ntej nrog TLR4 inhibitor TAK-242, DCs tau txhawb nqa nrog cov concentration ntawmAECCD,thiab tom qab ntawd cov xwm txheej ua kom DCs tau tshuaj xyuas los ntawm flow cytometer. Cov kev qhia theem ntawm cytokines nyob rau hauv kab lis kev cai supernatants tau soj ntsuam los ntawm ib tug multi-analyte flow assay kit. Kev tswj hwm ntawm CD40, CD80, CD86, thiab MHC-II raug ntxias los ntawm AECCD tau cuam tshuam loj heev los ntawm kev thaiv TLR4 txoj hauv kev (P< 0.01,="" fig.="" 9a–d).="" moreover,="" compared="" to="" untreated="" control,="" blocking="" tlr4="" with="" tak-242="" had="" a="" more="" significant="" inhibition="" effect="" on="" the="" production="" of="" ifn-γ,="" tnf-α,="" and="" il-6="" (p="" <="" 0.01,="" fig.="">

3.10. Kev ua kom DC los ntawm AECCD ntawm TLR4-txog NF-κB txoj hauv kev

Txhawm rau tshawb xyuas seb cov kab lus ntawm cov molecules saum npoo thiab cytokines ntawm AECCD-kho DCs tau tswj los ntawm NF-κB tom qab DCs tsis paub qab hau pretreated nrog NF-κB inhibitor PDTC tau txhawb nrog AECCD rau 12 h, kev qhia theem ntawm costimulatory molecules thiab cytokines tau ntsuas los ntawm flow cytometer. Cov theem ntawm CD40, CD80 CD86, thiab MHC-II induced los ntawmAECCDtau txo qis dua piv rau kev tswj tsis tau los ntawm kev thaiv NF-κB txoj hauv kev nrog cov inhibitors sib raug (P < 0.001,="" fig.="" 10a–d).="" tib="" lub="" sijhawm,="" kev="" qhia="" ntawm="" ifn-="" ,="" tnf-="" ,="" thiab="" il-6="" kuj="" tseem="" cuam="" tshuam="" los="" ntawm="" pdtc="" (p="">< 0.05,="" fig.="">

4. Kev sib tham

Tam sim no, ntau yam polysaccharides los ntawm TCM tau tshawb xyuas ua cov tshuaj tiv thaiv tib neeg thiab tsiaj txhu (Sun li al., 2018; Rey-Ladino li al., 2011). Hauv qhov kev tshawb fawb no, lubtiv thaiv kab mob-Modulating teebmeem ntawm AECCD thiab thawj qhov kev ua kom lub cev ntawm DCs tau tshawb xyuas hauv vivo thiab hauv vitro los ntawm kev sim ntau yam txhawm rau txhawm rau ntsuas qhov hloov pauv lub cev tiv thaiv kab mob tiv thaiv OVA thiab kev ua haujlwm ntawm DCs. Hauv vivo cov kev sim qhia tias AECCD zoo kawg li txhawb kev tsim cov OVA tshwj xeeb IgG thiab IgG isotypes, txhim kho splenocyte proliferation, thiab induced T cell activation thiab cytokines (IL-4}/IFN- ). Tsis tas li ntawd,AECCDtxhim kho DC maturation thiab nthuav tawm txo qis Tregs. Cov kev tshawb pom no tau qhia tias kev ua haujlwm ntawm AECCD ua ib qho kev ua haujlwm zoo tau pom los ntawm kev txhawb nqa DC maturation thiab inhibiting Tregs. Kev sim hauv vitro tau qhia tias AECCD txhawb nqa phenotypic maturation ntawm DCs, cytokine seem, thiab allostimulatory kev ua ntawm TLR4-txog NF-κB txoj hauv kev. Cov txiaj ntsig kev sim no tau qhia tias AECCD muaj peev xwm tseem ceeb ua lub zog tiv thaiv kab mob.

Txhua qhov adjuvant muaj qhov tshwj xeebtiv thaiv kab mobCov yam ntxwv tawm tsam sib txawv antigens, qhia tias adjuvants muaj peev xwm siv tau hauv cov kab mob sib txawv. Ua ntej, cov khoom siv ntawm kev sib koom ua ke ntawm AECCD nrog OVA tau raug kuaj los ntawm kev txiav txim siab lawv cov teebmeem ntawm kev hloov lub cev tiv thaiv kab mob hauv nas. Ntau cov kev tshawb fawb pom tau hais tias kev tiv thaiv kev tiv thaiv kab mob tau ncaj qha ncaj qha rau cov koob tshuaj nyob rau hauv ib qho chaw (Li et al., 2020; Reed et al., 2020; Zhang et al., 2017). Ib yam li ntawd, hauv txoj kev tshawb no, kev tsom xam ntawm cov tshuaj tiv thaiv kab mob thiab isotype tau qhia tias AECCD nyob rau hauv ib qho kev txhaj tshuaj tau txhim kho OVA-specific IgG, IgG1, thiab IgG2a cov lus teb thiab tias qhov nruab nrab koob ntawm AECCD yog cov koob tshuaj zoo. Hauv cov kev sim tom ntej, peb tsuas yog tshawb xyuas qhov stimulatory nyhuv ntawm AECCD ntawm qhov pom zoo nruab nrab koob. Qhov nruab nrab koob ntawmAECCDua rau muaj qhov nce ntxiv ntawm cov kab mob splenocyte proliferation hauv cov nas txhaj tshuaj tiv thaiv kab mob, qhia tias AECCD txhawb nqa cellular.tiv thaiv kab mobcov lus teb tawm tsam OVA. Cov txiaj ntsig no tau qhia tias AECCD txhim kho ob qho tib si humoral thiab cellular tiv thaiv kab mob, uas cuam tshuam nrog cov peev xwm ntxiv.

cistanchetshuaj xyuastxogtiv thaiv kab mob

Kev ua haujlwm ntawm T-cell ntau heev tuaj yeem ua rau muaj kev tiv thaiv kab mob. Thaum ibtiv thaiv kab mobcov lus teb, activated T hlwb tuaj yeem ncav cuag cov lymphoid cov ntaub so ntswg, nrog rau cov poov xab thiab cov qog nqaij hlav (Steinman, 2012). Qhov nruab nrab koob ntawmAECCDCov tshuaj tiv thaiv tau zoo dua qub vim nws tuaj yeem txhim kho CD4, CD8, CD44, thiab CD62L T cell cov lus teb hauv cov qog ntshav thiab tus po. Ua kom CD8 T hlwb kho cov kab mob tshem tawm los ntawm kev zais IFN- . Proliferated CD4 T cells tau muab faib ua Th1 hlwb thiab Th1 hlwb. Th1 hlwb secreted Th1 cytokine los txhawb cov lymphocyte proliferation thiab Th2 hlwb secreted Th2 cytokine los txhim kho antibody ntau lawm (Seder li al., 2008). Hauv kev tshawb fawb, qib CD4 ntxiv rau IL-4, CD4 ntxiv rau IFN- , thiab CD8 ntxiv rau IFN- tau nce los ntawm AECCD ntawm qhov nruab nrab koob. AECCD induced ib tug balanced Th1/Th2 teb thiab coj tustiv thaiv kab mobteb los ntawm kev hloov cov cytokine network.

Ib qho adjuvant yuav tsum ua kom muaj kev tiv thaiv tshwj xeeb thiab tswj cov txiaj ntsig ntawm kev tiv thaiv kab mob los ntawm kev tswj hwm DCs xwm txheej ntawm ntau cov ntsiab lus (Carrera Silva li al., 2013). Qhov kev qhia ntawm qhov chaw molecules ntawm DCs yog ib qho tseem ceeb qhia rau kev ntsuam xyuas DC maturation thiab feem ntau correlated nrog T lymphocyte sib txawv (Steinman, 2012). Hauv txoj kev tshawb no, AECCD tau txhim kho qhov kev qhia ntawm costimulatory molecules thiab pab txhawb DC kev loj hlob thaum nws tau siv los ua ib qho kev tiv thaiv OVA. Qhov no tej zaum yuav yog ib txoj hauv kev uas AECCD txhawb kev humoral thiab cellulartiv thaiv kab mobteb. Lub caij no,AECCDkuj txo qis zaus ntawm CD4 ntxiv rau CD25 ntxiv rau Foxp3 ntxiv rau Tregs, uas muab kev tiv thaiv kab mob sib npaug tom qab txhaj tshuaj. Peb cov txiaj ntsig tau txhawb kev tshawb pom tsis ntev los no tias ntau yam polysaccharides los ntawm TCM yog tus tswj hwm ntawm DC maturation (Bo li al., 2019; Li et al., 2015; Zhu et al., 2016).

Activation ntawm adaptivetiv thaiv kab mobcov lus teb los ntawm polysaccharide adjuvants yog kho feem ntau ntawm lawv qhov kev lees paub los ntawm cov receptors tshwj xeeb ntawm DCs (Akira, 2004). DCs tau qhib los ntawm TLR4-txoj kev cuam tshuam tuaj yeem zais ntau yam immunomodulators raws li tus kws kho mob tseem ceeb tiv thaiv kab mob (Akira, 2007). Yog li ntawd, hauv cov kev sim tom ntej, peb tau tshawb nrhiav qhov molecular mechanism ntawm stimulatory nyhuv ntawm AECCD ntawm DCs hauv vitro. Txhawm rau soj ntsuam cytotoxicity ntawm AECCD, lub cell viability ntawm DCs thiab splenocytes los ntawm nas yog thawj zaug txiav txim siab. AECCD ntawm 10-1600 ug / mL pom tsis muaj cytotoxicity rau DCs thiab splenocytes. Txhawm rau txiav txim siab LPS paug, peb tau kho AECCD ua ntej nrog PMB. Cov kev qhia theem ntawm CD40, CD80 CD86, thiab MHC-II hauv AECCD-kho DCs tsis tau txo qis, tab sis PMB cuam tshuam qhov kev qhia ntawm costimulatory molecules hauv LPS-kho DCs. Qhov no tau pom tias AECCD tsis muaj cov kab mob endotoxin. Tom qab ntawd, cov xwm txheej ua kom DCs tau tshawb nrhiav ntxiv. Zoo ib yam li LPS-kho DCs, nyob rau hauv ib koob tshuaj,AECCD-kho DCs tau nce qib ntawm CD40, CD80 CD86, thiab MHC II nrog rau kev tsim cov pro-inflammatory cytokine, uas yog ib qho kev ua haujlwm ntawm kev loj hlob ntawm DCs. Hauv MLR, qhov muaj peev xwm nthuav dav ntawm allogeneic T hlwb raug soj ntsuam hauv AECCD-kho DCs. AECCD tuaj yeem ua tus thawj thiab ua kom muaj cov kab mob allogeneic. DCs tau raug kho ua ntej nrog Mitomycin C thiab tom qab ntawd ntxuav kom huv si kom tshem tawm cov reagent no, yog li cov teebmeem ntawm AECCD ntawm DCs tsis zoo li yuav raug ntaus nqi rau kev ua haujlwm mitogenic. Cov ntaub ntawv sim tau qhia tias DCs phenotypic maturation thiab kev ua haujlwm ua haujlwm yuav raug ntaus nqi los ntawm kev txhawb nqa los ntawm AECCD.

Ib tug xov tooj ntawm TCM polysaccharides yuav txhawb DCs maturation ntawm TLR4. NF-κB raws li ib tug tswv cuab ntawm TLR4-txog txoj kev downstream ua lub luag haujlwm tseem ceeb hauv kev tswj cov phenotypic thiab kev ua haujlwm ntawm DCs thiab inducing qhia txog ntau yam noob caj noob ces koom nrog.tiv thaiv kab mobcov lus teb (Qi et al., 2016; Re and Strominger, 2001; Wei et al., 2016; Zhu et al., 2013). Los ntawm kev sim hauv vitro, peb pom tias AECCD txhawb nqa DC kev loj hlob thiab qhov ntawd TAK-242 (TLR4 inhibitor) thiab PDTC (NF-κB inhibitor) txo qis kev qhia qib ntawm CD40, CD80, CD86, thiab MHC -II thiab qib secretion ntawm IFN- , TNF- , thiab IL-6 ntawm AECCD-kho DCs, Cov kev tshawb pom no tau qhia tias TLR4 thiab NF-κB tau koom nrog hauv AECCD-induced DC activation thiab cytokine secretion. Cov txiaj ntsig tau zoo ib yam nrog cov kev tshawb fawb yav dhau los tau tshaj tawm vim tiasAECCDraws li ib tug adjuvant txhim kho antigen lub hom phiaj mus rau DCs.

Hauv cov ntsiab lus, peb cov txiaj ntsig tau pom tias AECCD induced check thiab Th1/Th2 teb, tshwj xeeb tshaj yog T-cell mediatedtiv thaiv kab mobteb tawm tsam OVA. AECCD tau qhib DCs los ntawm TLR4-txoj kev sib kho NF-κB, yog li txhim kho kev tsim cov immunomodulators thiab allogeneic T cell proliferation. AECCD tuaj yeem siv los ua cov tshuaj tiv thaiv zoo hauv kev tsim tshuaj tiv thaiv. Txawm li cas los xij, ib qho kev txheeb xyuas qhov tseeb yuav raug ua los txiav txim seb qhov twg nyob hauvAECCDactivated DCs. Nrog rau kev tsom mus rau cov txheej txheem hais txog yuav ua li cas AECCD boosts immunogenicity, nrog rau kev ua kom zoo ntawm adjuvant formulations thiab txoj hauv kev ntawm kev tswj hwm tshuaj tiv thaiv, peb yuav txuas ntxiv tshawb nrhiav cov tshuaj tiv thaiv kab mob thiab cov khoom tshwj xeeb ntawm AECCD rau lub hom phiaj ntawm kev tsim cov tshuaj tiv thaiv zoo tiv thaiv kab mob sib kis.

cistanchetxhim khotiv thaiv kab mob