Beta-glucan Enhanced Immune Response To Newcastle Disease Vaccine Thiab Hloov MRNA Kev Qhia Txog Spleen hauv Qaib

TSAB NTAWV

Txoj kev tshawb no tau ua los tshawb xyuas cov txiaj ntsig ntawm kev tswj hwm qhov ncauj ntawm b-glucan (G70), ib yam khoom tau los ntawm cov phab ntsa ntawm tes ntawm cov poov xab, ntawm Newcastle tus kab mob (NDV)-specific hemagglutination inhibition (HI) titers, lymphocyte proliferation, thiab Lub luag haujlwm ntawm T lymphocyte subpopulations hauv qaib tau kho nrog cov tshuaj tiv thaiv NDV. Tsis tas li ntawd, kev cuam tshuam ntawm b-glucan ntawm splenic gene qhia tau tshawb xyuas los ntawm kev sau cov ntawv sau ua ke. Cov txiaj ntsig tau qhia tias kev ntxiv ntawm b-glucan txhawb nqa cov ntshav titer NDV HI nce NDV stimulation Performance index ntawm lymphocytes hauv cov ntshav peripheral thiab plab hnyuv, thiab txhawb kev sib txawv ntawm T lymphocytes rau hauv CD4+ T hlwb. Kev soj ntsuam RNA sequencing (RNA-seq) pom tau hais tias G70 upregulated lub mRNA kab lus hais txog G-protein coupled receptor thiab MHC chav kawm I polypeptide, thiab downregulated cov mRNA kab lus hais txog cathelicidin thiab betadefensin. Cov nyhuv immunomodulatory ntawm G70 tuaj yeem ua haujlwm los ntawm mitogen-activated protein kinase qhia txoj hauv kev. Txhawm rau suav nrog, G70 tuaj yeem txhawb kev tiv thaiv kab mob ntawm cov tshuaj tiv thaiv NDV nyob hauv cov qaib thiab tuaj yeem siv los ua tus neeg sib tw muaj peev xwm hauv kev lag luam nqaij qaib.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Hot Tags: tshuaj tiv thaiv kab mob Newcastle, beta-glucan, adjuvant, dub-pob txha qaib, RNA-seq

Taw qhia

Poov xab cell phab ntsa polysaccharides yog ncaj qha riginateted los ntawm lub cell phab ntsa ntawm Saccharomyces cerevisiae thiab yog dav siv raws li kev loj hlob txhawb, antimicrobial agents, los yog immunomodulators nyob rau hauv cov nqaij qaib los txhim kho kev tsim khoom thiab kev noj qab haus huv (Schiavone li al., 2017; Hasted et al., 2017). Yav dhau los, ib yam khoom hu ua PW220, uas muaj cov poov xab cell phab ntsa polysaccharides, tau pom los txhawb lub cev tiv thaiv kab mob los ntawm Newcastle tus kab mob (NDV) thiab hloov cov microbial zej zog ntawm cecum ntawm qaib los ntawm kev tswj qhov ncauj (Bi li al., 2020 ). Poov xab cell phab ntsa polysaccharides feem ntau yog ua los ntawm b-glucan thiab mannan-oligosaccharides (Wang li al., 2018). Hauv kev tshawb fawb tam sim no, b-glucan tau tshawb xyuas rau nws cov txiaj ntsig ntawm kev tiv thaiv kab mob rau cov tshuaj tiv thaiv NDV hauv qaib. Tus po yog ib qho tseem ceeb ntawm lub cev los pib lub cev tiv thaiv kab mob. Ib txoj kev tshawb fawb tsis ntev los no tau qhia tias PW220, uas yog cov khoom lag luam ntawm cov phab ntsa ntawm cov poov xab, txhim kho mRNA qhia ntawm TGF-b, IL-6, TLR5, GATA-3, thiab T-bet nyob rau hauv tus po ntawm qaib ( Ib., 2022). Txawm li cas los xij, cov txheej txheem tshwj xeeb yuav tsum tau piav qhia. RNA sequencing (RNA-seq) yog ib qho ntawm cov thev naus laus zis siab tshaj plaws uas tuaj yeem siv rau kev soj ntsuam ntau cov noob qhia thiab muab kev tshuaj xyuas ntawm cov lus qhia sib txawv ntawm qib ntawm tag nrho cov ntawv sau tseg (Zhao li al., 2018). Nrog rau qhov zoo ntawm qhov siab thiab tus nqi qis, RNA-seq tau siv dav hauv kev tshawb fawb tsiaj txhu thiab nqaij qaib (Teixeira li al., 2019; Yuan et al., 2020). Yog li, hauv txoj kev tshawb no, cov txiaj ntsig ntawm kev tswj hwm b-glucan ntawm NDV-specific hemagglutination inhibition (HI) titers, kev loj hlob ntawm lymphocytes, thiab T-lymphocyte subpopulations hauv qaib qhov ncauj tau txhaj tshuaj tiv thaiv NDV. Tsis tas li ntawd, transcriptome tsom xam tau siv los ntsuas qhov cuam tshuam ntawm b-glucan ntawm cov noob qhia thiab cov teeb liab hauv qab txoj hauv kev hauv cov poov xab ntawm qaib.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Nyem qhov no mus saib Cistanche Enhance Immunity khoom

【Nug ntxiv】 Email: cindy.xue@wecistanche.com / Whats App: 0086 18599088692 / Wechat: 18599088692

Tsiaj txhu

Ib hnub qub lag luam dub-pob txha qaib (txiv neej) tau txais los ntawm Guizhou Yushun Poultry Co., Ltd. (Anshun, Suav). Cov qaib tau muab faib ua cov hlau tawb tso cai pub pub dawb thiab dej. Nyob rau hauv thawj 7 d, chav tsev kub yog khaws cia ntawm 33 ° C mus rau 35 degree Cand ces maj mam txo los ntawm 1 ° C rau txhua txhua 2 d mus txog rau 27 ° C. Tag nrho cov qaib tau ua tiav los ntawm cov qauv tsim los ntawm Southwest University Animal Care thiab Siv Pawg Neeg Saib Xyuas Kev Ncaj Ncees (Ethics Certificate Number: IAC-2021-0057). Tag nrho cov noog sim tau euthanized nrog CO2 thaum kawg ntawm txoj kev tshawb no.

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

Tshuaj tiv thaiv

Cov tshuaj tiv thaiv nyob ntawm NDV (Strain La Sota) tau muab los ntawm Qingdao YEBIO Bioengineering Co., Ltd. (Qingdao, Suav).

Reagents 

Poov xab cell phab ntsa khoom (YP) G70 tau los ntawm cov poov xab cell (Saccharomyces cerevisiae) phab ntsa, uas muaj b-glucan (Ntau tshaj los yog sib npaug rau 70%) (AngelG70, QB/T4572, Angel Poov xab, Yichang, Suav teb). Ob leeg antigen thiab zoo tswj sera rau ntsuas NDV-specific HI titers tau yuav los ntawm Qingdao Regen Diagnostics Development Center (Qingdao, Suav). Anti-chicken CD3-APC (C2818-T9580), CD4-FITC (D0117-WA78E), thiab CD8- PE (L{{14} }}TH49T) hauv nas tau muab los ntawm Nanjing Zeweil Biological Technology Co., Ltd. (Nanjing, Suav).

Kev sim tsim

Plaub caug-yim dub-pob txha qaib thaum muaj hnub nyoog 5 hnub tau muab faib ua 3 pawg (Table 1) thiab tau muab cov khoom noj basal (Table 2) lossis cov zaub mov basal nrog 0.1% G7{{10} } (b-glucan, 0.7 g / kg) ntxiv rau lub sijhawm ntawm kev tshawb fawb tam sim no. Pawg H (G70 + Tshuaj Tiv Thaiv) thiab C (Vaccine) tau txhaj tshuaj tiv thaiv qhov ncauj nrog NDV tshuaj tiv thaiv ntawm 14 thiab 28 d, raws li. Pab pawg S (Sailne) tau kho nrog qhov sib npaug ntawm cov kua qaub. Cov ntshav kuaj tau raug sau los ntawm tis venipuncture thaum muaj hnub nyoog 5, 7, 14, 21, 28, 35, thiab 42 d los kuaj HI titer. Xya hnub tom qab txhaj tshuaj tiv thaiv kab mob, 8 tus qaib nyob rau hauv txhua kem tau random khaws thiab txi los ntawm asphyxiation siv CO2. Lymphocytes tau muab cais los ntawm ob qho tib si peripheral ntshav thiab jejunum los ua cov lymphocyte proliferation thiab ntws cytometry tsom xam. Nyob rau hauv cov ntaub ntawv ntawm jejunal sampling, lub duodenal suspensory ligament yog qhov pib ntawm lub jejunum thiab ib tug 5 cm ntev seem tau sau los ntawm qhov pib taw tes ntawm lub jejunum. Cov poov xab tau khov sai sai hauv cov kua nitrogen thiab tom qab ntawd khaws cia ntawm -80 degree rau RT-qPCR thiab kev sib txuas ua ke.

Rooj 1. Kev sim tsim

Table 2. 1 Cov ntsiab lus thiab cov ntsiab lus ntawm cov khoom noj broiler basal kev sim.

HI Kev Kawm

Qhov kev ntsuam xyuas rau NDV-tshwj xeeb HI titers hauv cov ntshav tau ua raws li cov lus piav qhia dhau los (Ball li al., 2019). Hauv luv luv, cov ntshav tau diluted nrog phosphate-buffered saline (PBS) ntawm 1: 2 txog 1: 1,024 hauv V-hauv qab 96-zoo microtiter phaj. Tom qab ntawd, 25 mL ntawm NDV dilution tau ntxiv rau ib qhov dej thiab incubated ntawm 37 degree rau 30 min. Tom qab ntawd, 25 mL ntawm 1% rooster erythrocyte raug ncua rau txhua qhov dej thiab incubated ntawm 37 degree rau 30 min. Tag nrho cov qauv raug kuaj ob zaug thiab ob qho tib si zoo thiab tsis zoo tswj tau suav nrog ntawm txhua lub phaj. HI titers tau ua raws li qhov loj tshaj dilution uas ua rau muaj kev cuam tshuam tag nrho ntawm hemagglutination. Qhov nruab nrab HI titer thiab tus qauv yuam kev (SE) raug ntsuas rau ib pawg.

Kev rho tawm ntawm Lymphocytes los ntawm cov ntshav Peripheral

Lymphocytes raug cais thiab sau los ntawm cov ntshav lymphocytes peripheral siv cov Lymphocyte Separation Medium Kit (Tianjin Haoyang Biological Manufacture Co. Ltd., Tianjin, Suav). Tom qab ntawd, cov lymphocytes tau muab tso rau hauv kev ncua nrog RPMI 1640 nruab nrab (Solarbio, Beijing, Tuam Tshoj) suav nrog 5% fetal calf serum thiab 25 mM HEPES (pH 7.0).

Kev rho tawm ntawm Lymphocytes Los ntawm Jejunum

Cov txheej txheem ua haujlwm tau ua tiav raws li cov lus piav qhia dhau los, thiab nrog kev hloov kho me ntsis (Yuan li al., 2020). Hauv luv luv, siv 70 hli ntawm tes lim los faib cov hnyuv rau hauv 4 mL ntawm PBS txias. Tom qab ntawd, kev ncua ntawm cov qauv hlwb tau centrifuged ntawm 4,500 rpm rau 12 min nrog kev tshem tawm ntawm supernatant. Tom qab ntawd, rov tshem tawm cov plab hnyuv mus rau qhov nruab nrab (Solarbio Co., Beijing, Tuam Tshoj) suav nrog 5% fetal bovine serum (FBS) hauv RPMI 1640 (Sijiqing Co., Hangzhou, Suav). Thaum kawg, cov khoom siv qaib lymphocyte cais tawm (Tianjin Haoyang Biological Manufacture Co. Ltd.) cais cov lymphocytes. cov ntaub so ntswg hauv plab tau muab faib ua 4 mL ntawm PBS txias siv 70 mm cell lim. Tom ntej no, kev ncua ntawm cov qauv hlwb tau centrifuged ntawm 4,500 rpm rau 12 min thiab cov supernatant raug muab pov tseg. Tom qab ntawd, cov kab mob hauv plab tau rov qab mus rau hauv RPMI 1640 (Solarbio Co.) uas muaj 5% FBS (Sijiqing Co.). Thaum kawg, cov lymphocytes raug cais siv cov khoom siv qaib lymphocyte cais tawm (Tianjin Haoyang Biological Manufacture Co. Ltd.).

Kev loj hlob ntawm Lymphocytes 

Cov noob tau cog rau hauv 96- cov phiaj zoo (5 £ 105 ib qhov dej), txhawb nqa los ntawm cov tshuaj tiv thaiv ntawm inactivated NDV rau 4 hemagglutination units los yog qhov sib npaug ntawm cov ntsev ntsev. Txhua tus qauv tau sim nyob rau hauv 3 replicates. Cov hlwb thiab cov tshuaj tiv thaiv tau incubated rau 44 h ntawm 37 degree hauv 5% CO2 thiab tom qab ntawd 50 mL methyl thiazolyl tetrazolium (MTT) (2 mg / mL) ntxiv rau txhua qhov dej thiab incubated rau lwm 4 h. Cov qauv raug centrifuged ntawm 1,200 £ g rau 8 min ntawm qhov kub thiab txias. Tom qab ntawd, tus supernatant tau ceev faj tshem tawm thiab 100 mL ntawm DMSO tau ntxiv rau txhua qhov dej. Cov phiajcim tau shaken rau 8 min kom solubilize cov muaju. Thaum kawg, qhov nruab nrab qhov muag pom qhov ntom ntom (OD) tau kaw ntawm 570 nm. Qhov stimulation Performance index (SI) tau txais los ntawm kev sib npaug: OD qhov tseem ceeb ntawm cov qhov dej muaj zog / OD qhov tseem ceeb ntawm cov qhov dej tsis muaj zog (Cui li al., 2020).

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

Flow Cytometry Analysis ntawm T-Lymphocyte Subpopulations

Lymphoid cell suspensions raug cais thiab ntxuav ob zaug nrog PBS. Lub cell concentration tau hloov mus rau 106 / mL thiab tom qab ntawd khaws cia rau hauv dej khov. Txhua tus qauv raug stained nrog 2 mL ntawm nas los tiv thaiv nqaij qaib CD3-APC, CD4- FITC, thiab nas los tiv thaiv qaib CD8-PE nyob rau hauv lub teeb-pov thawj rau 30 min, ua raws los ntawm 2 ntxuav nrog PBS. Cov hlwb raug soj ntsuam los ntawm kev ntsuas cytometry ntawm Flow cytometer (BD Bioscience, San Jose, CA).

RT-qPCR Kev Ntsuam Xyuas

Tag nrho RNA tau muab los ntawm tus po siv TRIzol reagent (Takara, Shiga, Nyiv) raws li cov chaw tsim khoom cov lus qhia thiab tom qab ntawd hloov mus rau cDNA siv PrimeScript RT Master Mix (Takara, Dalian, Tuam Tshoj) ntawm T100 thermal cycler (Bio-Rad, Hercules, CA). Cov nqaij qaib beta-actin tau ua haujlwm raws li kev tswj cov noob caj noob ces. RT-qPCR ntawm cov noob xaiv tau ua tiav ntawm Multiple Real-Time PCR System (AB, Carlsbad, CA) nrog SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara). Txoj kev sib txheeb ntau npaum li cas (244CT) tau ua los ntsuas qhov ntau qhov sib txawv (Bi li al., 2022). Cov txheej txheem primer tau teev tseg hauv Table 3.

RNA Extraction

Txhua tus ntawm 3 tus qauv spleen los ntawm Pawg H (G70 +Cov tshuaj tiv thaiv) thiab Pawg C (Vaccine) tau siv rau RNA-seq nrog TRIzol reagent (Takara). Tom qab ntawd, RNA raug kuaj rau kev sib kis thiab degradation nrog 1% agarose gel thiab RNA purity raug tshuaj xyuas siv NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA). Qhov siab ntawm RNA tau txiav txim siab ua haujlwm Qubit RNA Assay Kit hauv Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA). RNA kev ncaj ncees raug ntsuas siv RNA Nano 6000 Assay Kit ntawm Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA). Cov txiaj ntsig tau pom tias RNA ua tiav thiab tsis muaj DNA paug.

Transscriptome Analysis

Transcriptome sequencing, sequence assembling, thiab cov ntaub ntawv tsom xam yog muab los ntawm Novogene Bioinformatics Technology Co. Ltd. (Beijing, Tuam Tshoj). Cov txheej txheem tseem ceeb rau kev sau ntawv tau teev tseg raws li hauv qab no: 1) Kev ua kom huv ntawm mRNA los ntawm tag nrho RNA tau ua los ntawm poly-T oligo-txuas sib nqus hlaws. Fragmentation tau ua nrog divalent cations hauv NEB Next First Strand Synthesis Reaction Buffer (5X) ntawm qhov kub siab. 2) Thawj-strand cDNA yog synthe sized siv random hexamer primer thiab Moloney Murine Leukemia Virus (M-MuLV) Rov qab. Thib ob strand cDNA synthesis yog ces synthesized siv DNA Polymerase I thiab RNase H. 3) seem overhangs tau hloov mus rau blunt kawg los ntawm cov exonuclease/polymerase kev ua ub no. Ib tug hairpin voj qauv NEB Tom ntej Adapter yog ces ligated los npaj rau hybridization tom qab adenylation ntawm 3' kawg ntawm DNA tawg. 4) Txog 250 txog 300 bp cDNA fragments tau txais thiab lub tsev qiv ntawv tau huv si siv Beckman Coulter's AMPure XP system (Beckman Coulter, Beverly, MA). Tom qab ntawd 3 mL NEBU SER Enzyme (Thermo Fisher, Hillsboro, OR) tau siv nrog qhov loj-xaiv, adaptor-ligated cDNA ntawm 37 degree C rau 15 min, thiab ua raws li 5 min ntawm 95 degree. PCR amplification tau ua tiav nrog Phusion High-Fidelity DNA polymerase. Thaum kawg, PCR cov khoom raug purified (AMPure XP system) thiab cov tsev qiv ntawv zoo tau kwv yees siv Agilent Bioanalyzer 2100 system. Kev sib koom ua ke ntawm cov qauv ntsuas ntsuas tau ua tiav ntawm TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA). Tom qab ntawd, qhov sib txuas tau ua tiav ntawm Illumina Novaseq platform thiab 150 bp sib txuas-kawg nyeem tau tsim. Cov ntaub ntawv hais txog genome thiab gene qauv annotation tau rub tawm los ntawm genome lub vev xaib (ftp://ftp.ensembl.org/pub/release- 98/fasta/gallus_gallus/ thiab ftp://ftp. ensembl.org/ pub/re-lease-98/gtf/gallus_gallus/). Ib qho Performance index ntawm cov ntaub ntawv siv genome tau tsim los siv HISAT2 (v2.0.5) thiab ua ke-kawg huv nyeem tau ua raws li cov ntaub ntawv siv genome. Tus naj npawb ntawm kev nyeem daim ntawv qhia rau txhua tus noob thiab Fragments Ib Kilobase ib lab (FPKM) rau txhua tus noob raws li qhov ntev ntawm cov noob thiab cov naj npawb ntawm cov ntawv nyeem tau muab rau cov noob tau suav nrog siv Feature Counts v1.5.{{38} }p3 ib. Kev qhia sib txawv ntawm pawg H (G70 + Tshuaj Tiv Thaiv) thiab pawg C (Cov Tshuaj Tiv Thaiv) tau muab los ntawm kev siv DESeq2 R pob (1.16.1). Genes nrog P <0.05 thiab |log2 (fold hloov)| > 1 tau txhais tias yog hom kab mob sib txawv (DEGs).

Table 3. Sequences of primers for RT-qPCR.

Real-Time Quantitative PCR Validation 

Ob qhov kev tswj hwm DEG thiab 4 qhov kev tswj hwm tsis zoo hauv kev sib piv ntawm pawg H (G70 + Tshuaj Tiv Thaiv) vs. Pawg C (Cov Tshuaj Tiv Thaiv) tau raug xaiv los lees paub cov txiaj ntsig ntawm cov ntawv sau los ntawm RT-qPCR. RNA tau hloov mus rau cDNA siv PrimeScript RT Master Mix (Takara) ntawm T100 thermal cycler (Bio-Rad). Cov txheej txheem primer tau teev nyob rau hauv Table 3. Nqaij qaib b-actin tau siv los ua cov noob tswj sab hauv. RT-qPCR ntawm cov noob xaiv tau ua tiav ntawm Ntau Lub Sijhawm PCR System (Applied Biosystems, Carlsbad, CA) nrog SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara). Ib txoj hauv kev sib txheeb ntau npaum li cas (244CT) tau siv los ntsuas qhov sib txawv ntawm qhov ntsuas. Tag nrho cov qauv tau ua nyob rau hauv triplicate rau kev soj ntsuam.

Kev txheeb cais

Ib txoj kev ANOVA nrog Duncan post hoc xeem tau siv rau ntau qhov kev sib piv ntawm pab pawg siv SPSS software (version 21.0, SPSS Inc., Chicago, IL). Cov ntaub ntawv raug nthuav tawm raws li qhov txhais tau tias § SE. Tus nqi ntawm P < 0.05 tau suav tias yog qhov tseem ceeb.

TAM SIM NO Cov nyhuv ntawm b-Glucan ntawm Serum Antibody Titers

Raws li pom hauv daim duab 1, NDV-tshwj xeeb HI titers tau txo qis hauv txhua pab pawg ua ntej txhaj tshuaj tiv thaiv thiab nce hauv pawg H (G70+Vaccine) thiab C (Vaccine) tom qab txhaj tshuaj. Interestingly, siab dua HI titers tau pom nyob rau hauv pawg H (G70+Cov tshuaj tiv thaiv) ntawm 21 (P > 0.05), 28 (P < 0.{{ 14}}5), 35 (P> 0.05), thiab 42 (P> 0.05) d hnub nyoog tshaj hauv pawg C (Vaccine).

Cov nyhuv ntawm b-Glucan ntawm Lymphocyte Proliferation

Cov nyhuv ntawm G70 ntawm kev loj hlob ntawm peripheral ntshav lymphocytes yog piav qhia hauv daim duab 2A. SI hauv cov ntshav lymphocytes tau txhim kho nyob rau hauv cov noog ntxiv nrog G70 (G70 + Tshuaj Tiv Thaiv) ntawm 7 d (P <0.05) cov tshuaj tiv thaiv kab mob, thaum piv nrog pawg tshuaj tiv thaiv. Tsis tas li ntawd, SI hauv jejunal lymphocytes tau nce siab ntawm 7 d (P < 0.05) tom qab txhaj tshuaj tiv thaiv kab mob thaum piv nrog pawg tshuaj tiv thaiv (Daim duab 2B).

Cov nyhuv ntawm b-Glucan ntawm qhov sib piv ntawm CD4 + / CD8 + T Cell hauv Peripheral Ntshav 

Cov duab 3A thiab 3B tau qhia txog qhov gating ntawm lymphocytes thiab CD3 + hlwb thiab daim duab 3C rau 3E pom qhov piv ntawm CD4 + /CD8 + T hlwb hauv G70, tshuaj tiv thaiv, thiab pab pawg saline, ntsig txog. Raws li pom hauv daim duab 3F, tsis muaj qhov sib txawv ntawm pawg tshuaj tiv thaiv thiab pawg Saline tom qab txhaj tshuaj tiv thaiv kab mob (P > 0.05), qhov feem ntawm CD4 + /CD{{12} } T cell tau pom tseeb siab dua hauv G70 (G70 + Cov Tshuaj Tiv Thaiv) pawg dua li hauv pawg tshuaj tiv thaiv (P <0.05).

Daim duab 1. Cov txiaj ntsig ntawm kev tswj hwm qhov ncauj ntawm b-glucan ntawm cov tshuaj tiv thaiv kab mob ntshav rau cov tshuaj tiv thaiv NDV. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias § SE.

Hais txog Gene Expression

Raws li tau piav qhia hauv daim duab 4, nce mRNA kev nthuav qhia ntawm TGF-b (P < 0 05), IL-6 (P < 0 0. }5), thiab TLR5 (P < 0). Tsis muaj qhov sib txawv tseem ceeb hauv mRNA qhia ntawm IFN-g (P> 0.05), TLR4 (P> 0.05), thiab TLR3 (P> 0.05) hauv tus po ntawm G70 (G70 + Tshuaj Tiv Thaiv) thiab Tshuaj Tiv Thaiv pab pawg.

RNA Sequencing Data Analysis 

Sequencing ntawm RNA los ntawm 8 lub tsev qiv ntawv spleen yielded 24.1 G ntawm raw cov ntaub ntawv raws li qhia hauv Table S1 ntawm cov khoom ntxiv. CN txhais tau tias pawg tshuaj tiv thaiv, thiab HN txhais tau tias pawg G70 + Tshuaj tiv thaiv. Lub tsev qiv ntawv C1, C2, C3, C4, H1, H2, H3, thiab H4 feem, suav nrog 45,591,492, 47,424,782, 46,193,492, 54,403,376, 47,118,476,458,45,45,45,476 438 original nyeem. Ua raws li aptamer, qhov tsis meej ntawm cov kab lus tsis meej thiab qhov tsis zoo tau raug tshem tawm, tawm hauv 44,050,198, 45,449,884, 44,261,112, 52,097,846, 45,542,404, 44,566,41,29, 44,564,404, 44,564,29. nyeem. Ntau tshaj 96% ntawm cov ntawv nyeem huv yog nyob ntawm cov ntawv nyeem thawj, thiab 8 lub tsev qiv ntawv tau sib koom ua ke siv HISAT2 rau kev sib txuas cov ntawv nyeem tawm tsam cov ntaub ntawv siv los ntawm Gallus genome. Tsis tas li ntawd, cov ntaub ntawv huv huv tau teeb tsa rau cov ntaub ntawv no hauv ntau dua 95% ntawm cov neeg mob. Tshwj xeeb rau cov tsev qiv ntawv los ntawm C1, C2, C3, C4, H1, H2, H3, thiab H4 yog 40,921,384 (92.9%), 41,302,100 (90.87%), 40,771,075 (92.11%), 478,43% (92.11%), 478,93% .2 %), 40,213,444 (90.23%), 40,922,895 (92.77%), thiab 40,971,972 (93.06%) nyeem tau muab tshwj xeeb rau cov ntaub ntawv siv, feem.

Differentially nthuav qhia Genes

Raws li tau hais hauv daim duab 5A, tag nrho ntawm 198 qhov sib txawv qhia cov noob, suav nrog 47 cov noob tau tswj hwm thiab 151 cov noob tswj tsis tau raug txheeb xyuas. Cov DEGs tau piav qhia meej hauv cov khoom siv ntxiv Table S2. Daim duab 5B qhia tias DEGs muaj kev kho kom rov zoo dua qub.

Kev txheeb xyuas ntawm Gene Ontology Classification thiab Kyoto Encyclopedia of Genes thiab Genomes Enrichment

Cov noob uas muaj qhov sib txawv ntawm cov noob tau muab faib ua 3 pawg ua haujlwm tseem ceeb raws li Gene Ontology (GO) kev faib tawm: txheej txheem lom neeg, cov khoom siv ntawm tes, thiab kev ua haujlwm molecular. Sab saum toj 10 GO cov ntsiab lus nyob rau hauv 3 pawg tau nthuav tawm hauv daim duab 6. Muaj ib qho tseem ceeb ntawm cov noob muaj feem xyuam rau "kev teb rau kev tiv thaiv kab mob," "chemokine teb," " teb rau cov tshuaj tua kab mob humoral," "tiv thaiv cov kab mob, "" tiv thaiv kab mob rau cov tshuaj tua kab mob lom neeg kho mob los ntawm cov tshuaj tua kab mob peptides," "tiv thaiv teb tiv thaiv kab mob Gram-tsis zoo," thiab "tiv thaiv teb rau cov kab mob Gram-zoo" hauv qeb ntawm cov txheej txheem lom neeg. Tsis tas li ntawd, nyob rau hauv qeb ntawm molecular muaj nuj nqi, qhov zoo kawg nkaus piv ntawm cov noob muaj feem xyuam rau "CC motif chemokine receptor (CCR) chemokine receptor binding," "chemokine receptor binding," thiab "lipopolysaccharide binding." Tsis tas li ntawd, "mitochondrial respiratory chain complex I," "NADH dehydrogenase complex," thiab "chaw ua pa" yog cov ntsiab lus tseem ceeb tshaj plaws hauv cov khoom ntawm tes. Kyoto Encyclopedia ntawm Genes thiab Genomes txoj kev tsom xam kuj tau ua tiav ntawm cov noob caj noob ces sib txawv. Cov txiaj ntsig tau qhia tias cov noob tau muab faib ua 7 txoj hauv kev suav nrog "Neuroactive ligand-receptor kev cuam tshuam," "Gap junction," "mitogen-activated protein kinase (MAPK) signaling pathway," "ABC transporters," "Biosynthesis of amino acids," "Drug metabolism," thiab "Protein export."

Daim duab 2. Qhov cuam tshuam ntawm kev tswj qhov ncauj ntawm b-glucan ntawm lymphocyte stimulating index (SI). (A) Peripheral ntshav lymphocytes; (B) plab hnyuv lymphocytes. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias § SE.

Daim duab 3. Qhov cuam tshuam ntawm kev tswj qhov ncauj ntawm b-glucan ntawm CD4+/CD8+ cell ratio ntawm peripheral ntshav. (A) Rooj vag ntawm lymphocytes; (B) rooj vag ntawm CD3+ T cells; (C) zaus ntawm CD3+ CD4 + CD8 + T hlwb hauv G70+ pab pawg tshuaj tiv thaiv; (D) zaus ntawm CD3+ CD4 + CD8 + T hlwb hauv pawg tshuaj tiv thaiv; (E) zaus ntawm CD3+ CD4 + CD8 + T cells hauv Saline pawg; (F) bardiagram sawv cev rau CD4+/CD{19}} piv. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias § SE.

Daim duab 4. Cov txiaj ntsig ntawm kev tswj qhov ncauj ntawm b-glucan ntawm mRNA qhia hauv nqaij qaib spleen. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias § SE.

Daim duab 5. Cov ntsiab lus ntawm RNA-Seq cov ntaub ntawv. (A) Daim ntawv teev cov noob caj noob ces sib txawv, (B) daim ntawv qhia ua ke ntawm DEGs.

Kev txheeb xyuas cov noob qhia txawv ntawm RT-qPCR

6 DEGs uas tau nce- lossis qis-tswj tau raug lees paub los ntawm PCR lub sijhawm tiag tiag. Qhov tshwm sim tau qhia tias RT-qPCR cov ntaub ntawv feem ntau tau ua raws li cov ntaub ntawv RNA-seq feem ntau, qhia txog qhov ua tau zoo sib xws (Daim duab 7). Kev nthuav qhia ntawm mRNA ntawm MAPK Pathway Associated Genes Raws li pom hauv daim duab 8, txo qis mRNA qhia ntawm FAS (P < 0 05) thiab CACNA2 (P < 0.05) yog kuaj pom nyob rau hauv tus po ntawm cov qaib hauv pawg G70 (G70 + Tshuaj Tiv Thaiv) thaum piv nrog rau pawg tshuaj tiv thaiv. Tsis tas li ntawd, cov naj npawb nce mRNA qhia ntawm DUSP5 thiab DUSP4, thiab cov lej txo qis mRNA qhia ntawm p38, JNK, thiab ERK tau kuaj pom hauv pawg G70 (G70 + Cov Tshuaj Tiv Thaiv) thaum piv nrog pawg tshuaj tiv thaiv (P> 0.05) .

Kev sib tham

Cov tshuaj tiv thaiv nyob ND tau nthuav dav hauv cov qaib ua liaj ua teb thiab tseem tseem muaj cov kab mob Newcastle tshwm sim nyob rau hauv cov tsiaj txhu tiv thaiv kab mob (Zhang li al., 2022). Cov tshuaj tiv thaiv zoo tshaj plaws tau txhais los txhawb kev tiv thaiv kab mob ntawm tes thiab mucosal nrog rau kev tiv thaiv kab mob zoo (Shan li al., 2019). Yog li ntawd, ntau thiab ntau cov kev saib xyuas tau raug coj mus rau adjuvants uas tuaj yeem ua rau mucosal nrog rau cov lus teb ntawm lub cev tiv thaiv kab mob (Chen li al., 2014; Yu et al., 2015). Piv nrog rau txoj kev txhaj tshuaj, kev tswj xyuas qhov ncauj yog ib qho yooj yim dua uas txo cov nqi thiab ua rau muaj kev ntxhov siab tsawg rau cov qaib broiler (Boyaka li al., 2003; Zhang et al., 2008). Cov txiaj ntsig ntawm txoj kev tshawb fawb no tau pom tias kev noj zaub mov ntxiv nrog b-glucan tau nce qib ntawm NDV tshwj xeeb HI titers hauv cov ntshav ntawm qaib, uas zoo ib yam nrog cov ntaub ntawv dhau los (Horst et al., 2019). NDV-cov tshuaj tiv thaiv tshwj xeeb yog qhov tseem ceeb rau kev txhim kho kev tiv thaiv kab mob humoral los tiv thaiv qaib los ntawm NDV kab mob (Martinez li al., 2018; Li et al., 2020). B lymphocytes tsim cov tshuaj tiv thaiv thiab T lymphocytes nthuav dav hauv cov lus teb rau mitogen (Bohacova li al., 2021). Nyob rau hauv txoj kev tshawb no, qhov stimulation Performance index ntawm nqaij qaib peripheral ntshav lymphocytes rau NDV ntawm pawg G70 yog pom tseeb ntau dua li cov tshuaj tiv thaiv pawg, qhia tias ntau T lymphocytes tau qhib. Cov subpopulations uas tseem ceeb ntawm T lymphocytes yog CD4 + thiab CD{15}} T hlwb (Cui li al., 2020). CD4 + T hlwb feem ntau tsim cytokines thiab txhawb B cell kom loj hlob, whereas CD{18}} T hlwb yog txuam nrog tua cov phiaj hlwb (Zhang li al., 2021b). Hauv qhov kev tshawb fawb no, qhov piv ntawm CD4 +/CD{21}} T hlwb kuaj pom hauv G70 pab pawg tau nce ntxiv, qhia tias G70 tuaj yeem qhib CD ntau dua 4 + T hlwb los ntawm cov ntshav peripheral. Tsis tas li ntawd, peb tau pom qhov ua rau lub plab zom mov ntau dua li cov qog ntshav qog ntshav hauv cov pab pawg G70 ntau dua li hauv pawg Tshuaj Tiv Thaiv, uas tau lees paub tias b-glucan tuaj yeem ua rau mob plab hnyuv lymphocytes. Peb cov kev sim yav dhau los tau lees paub tias cov poov xab cell phab ntsa extract PW220 nce cov hnyuv tshwj xeeb sIgA thiab IgA + hlwb (Bi li al., 2020). Qhov ntawd yog hais tias, ob qho tib si b-glucan G70 thiab PW220 muaj txiaj ntsig zoo los txhawb txoj hnyuv mucosal.

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

Kev txhim kho kev tiv thaiv ntawm cov tsiaj los ntawm kev tswj hwm ntawm b-glucan tau tshaj tawm yav dhau los. Guo et al. (2003) tau lees paub tias kev tswj hwm ntawm b-glucan txhim kho qhov ntsuas ntawm bursa thiab NDV cov tshuaj tiv thaiv tshwj xeeb hauv qaib. Levine et al. (2018) tau qhia tias b-glucan supplementation tuaj yeem txhim kho plab hnyuv MHC Ⅱ thiab nce cov CD45 + hlwb kom txo tau txoj hnyuv.

Li et al. (2005) tau tshaj tawm tias kev noj zaub mov b-glucan tuaj yeem txhim kho mRNA qhia qib ntawm IL-8 thiab TGF-b hauv plab hnyuv ntawm npua. Cov txheej txheem ntawm cov nyhuv immunomodulatory ntawm b-glucan tsis meej. Kim et al. (2010) tau qhia tias b-glucan txhawb kev loj hlob ntawm cov hlwb dendritic los ntawm kev tswj cov kev qhia ntawm CD40, CD80, thiab CD86. Tsis tas li ntawd, Lee and Kim (2014) and Su et al. (2020) tau tshaj tawm tias b-glucan activated pattern recognition receptors xws li Dectin{14}} receptor, Hu-like receptor, thiab CR3 (Baert li al., 2015). Tus po yog ib lub cev tseem ceeb rau kev pib tiv thaiv kab mob thiab ua lub luag haujlwm tseem ceeb hauv ob qho tib si hauv lub cev thiab kev tiv thaiv kab mob (Bronte thiab Pittet, 2013). Txawm li cas los xij, tsuas yog muaj kev tshawb fawb me ntsis raws li RNA-seq tsom xam ntawm mRNA qhia hauv tus po tom qab qhov ncauj tswj hwm ntawm poov xab polysaccharides. Hauv kev tshawb fawb tam sim no, Kyoto Encyclopedia ntawm Genes thiab Genomes txoj kev tsom xam pom tias cov noob no tau muab faib ua 2 txoj hauv kev, suav nrog "Neuroactive ligand-receptor kev sib cuam tshuam" thiab "MAPK signaling pathway." Cov receptors rau cov qauv kev lees paub lub hom phiaj b-glucan tau ua kom muaj txiaj ntsig zoo ntawm lub cev tiv thaiv kab mob (Goodridge li al., 2009). Tom qab b-glucan lees paub, tyrosine kinase thiab nuclear factor kappaB (NF-kappaB) tau txhawb nqa los ntawm cov receptors, uas ua rau muaj kev cuam tshuam ntawm pro-inflammatory cytokine secretion thiab ua kom lub cev tiv thaiv kab mob (Kankkunen li al., 2010; Masuda etal. ., 2012; Xu et al., 2016; Bode et al., 2019). Lub MAPK, ua ib tsev neeg ntawm serine-threonine protein kinases, plays lub luag haujlwm tseem ceeb hauv kev mob thiab cytokine ntau lawm hauv qaib. Byun et al. (2016) pom tau hais tias b-glucan nce kev tsim tawm ntawm interferon-g thiab interleukin -2 thiab ua rau muaj kev cuam tshuam ntau dua ntawm phosphorylation ntawm MAPK p38 hauv peritoneal macrophage. Wang et al. (2014) tau tshaj tawm tias b-glucan txo qis cov lus teb inflammatory hauv THP-1 hlwb los ntawm inhibiting p38 MAPK activation. Hauv qhov kev tshawb fawb no, 13 DEGs suav nrog DUSP4, CACNA2D1, PDGFD, PTPRR, ENSGALG00000006351, FAS, MAP2K3, MYC, DUSP5, MAX, SRF, PRKCB, thiab EGFR tau qhia hauv MAPK immune cov lus teb uas yuav tswj tau txoj hauv kev, ntawm spleen ntawm MAPK hauv qaib. MAPK yog ib chav kawm tshwj xeeb ntawm Ser / Thr kinases hauv cov hlwb uas feem ntau koom nrog hauv cov lus teb ntawm tes xws li kev tiv thaiv kab mob, apoptosis, thiab kev loj hlob. Tsev neeg MAPK suav nrog tsawg kawg 3 lub tsev neeg: c-Jun N-terminal kinase (JNK), p38 MAPK, thiab extracellular signal-regulated kinase (ERK) (Hong et al., 2020; Zhang et al., 2021a). DUSP4 thiab DUSP5 yog dual-specificity phosphatase uas tshwj xeeb inhibit kev ua haujlwm ntawm MAPK tsev neeg xws li p38, JNK, thiab ERK, ua si lub luag haujlwm tseem ceeb hauv kev tiv thaiv inflammatory overreaction thiab txhawb lub regression ntawm o ntawm lub cev (Imasato li al. ., 2002; Talreja et al., 2021). Tsis tas li ntawd, ib qho me me uas tsis yog-coding RNA npe hu ua gga-miR-200a-3p tau raug tshaj tawm los tawm tsam qhov kev qhia ntawm cov kab mob pro-inflammatory thiab yog li tswj hwm tus tswv tsev autoimmune teb los ntawm kev tswj tsis zoo ntawm MAPK txoj hauv kev. thiab nws downstream signaling molecules (Pham li al., 2017). Hauv kev tshawb fawb tam sim no, RT-qPCR tsom xam pom tias mRNA qhia ntawm P38, JNK, thiab ERK txo qis thiab mRNA qhia ntawm DUSP4 thiab DUSP5 nce hauv qaib noj b-glucan (G70). Cov txiaj ntsig no qhia tias kev tiv thaiv kev tiv thaiv kab mob ntawm b-glucan (G70) ntawm cov tshuaj tiv thaiv kab mob Newcastle tuaj yeem cuam tshuam nrog cov kev tawm tswv yim tsis zoo ntawm cov tshuaj tiv thaiv kab mob sib kho los ntawm MAPK. Tsis tas li ntawd, FAS yog ib yam khoom tseem ceeb uas kho apoptosis thiab tseem ceeb heev rau lub cev tiv thaiv kab mob (Krueger li al., 2003; Guegan and Legembre, 2018). Lub caij no, CACNA2 yog ib qho hluav taws xob-nyob ntawm calcium channel subunit, uas muaj kev txhawb nqa ntawm cov cell proliferation, tsiv teb tsaws, thiab ntxeem tau (Sun li al., 2020). Hauv txoj kev tshawb no, FAS thiab CACNA2 tau txo qis hauv cov qaib ntxiv nrog b-glucan (G70), qhia tias b-glucan (G70) ua rau lub cev tiv thaiv kab mob feem ntau los ntawm inhibiting immune cell apoptosis thiab ntsuas autoimmunity. Cov kev tshawb pom no muab cov lus qhia theoretical rau kev siv b-glucan (G70) raws li kev tiv thaiv kab mob hauv cov chaw kho mob. Txawm li cas los xij, cov txheej txheem ntawm b-glucan txhim kho lub cev tiv thaiv kab mob los ntawm kev tawm tswv yim tsis zoo ntawm MAPK txoj hauv kev xav tau pov thawj ntxiv. Nws yog qhov nthuav kom nco ntsoov tias cov noob encoding cathelicidin suav nrog CATH3, CATH1, CATH2, thiab cov noob encoding b-defensin suav nrog AvBD6, AvBD7, AvBD1, thiab AvBD4 tau poob qis-tswj hauv qhov sib piv H (Pab G70 + Tshuaj tiv thaiv) vs. Tswj (Cov Tshuaj Tiv Thaiv Pab Pawg). Host Defense Peptides (HDPs) yog cov tshuaj molecules uas muaj lub luag haujlwm tseem ceeb hauv lub cev tsis muaj zog (Cuperus li al., 2013). Cov peptides no tau pom nyob rau hauv ntau hom tsiaj los ntawm tsiaj txhu mus rau noog. Muaj 4 cathelicidins hauv cov menyuam qaib, suav nrog CATH1, CATH2, CATH3, thiab CTHB1, uas tua tau ntau yam kab mob (Hamad et al., 2017). Avian beta-defensins (AvBDs), hloov npe hu ua gallinacean, yog cov cationic peptides me me nrog 3 cysteine ​​disulfide bonds ntawm lawv cov cysteine ​​residues thiab ua lub luag haujlwm tseem ceeb hauv lub cev tsis muaj zog (Yoshimura, 2015). Kev txo qis hauv kev qhia ntawm cathelicidins thiab beta-defensin tau piav qhia los ntawm cov lus qhia tawm tswv yim, qhov twg cov kab mob thiab cov kab mob parabacterial raug txo los ntawm cov poov xab cell phab ntsa polysaccharides (Bi li al., 2020). Cov txiaj ntsig zoo sib xws tau tshwm sim hauv lwm cov kev tshawb fawb. Shao et al. (2016) tau qhia tias kev ntxiv cov poov xab b-glucan hauv broiler qaib tua cov kab mob Salmonella thiab txo qis kev qhia ntawm HDPs los ntawm kev txheeb xyuas PCR ntau. Hauv txoj kev tshawb no, cov khoom lag luam G70 feem ntau tsim los ntawm b-glucan txhim kho qib ntawm NDV-cov tshuaj tiv thaiv tshwj xeeb hauv cov ntshav, nce NDV stimulation Performance index ntawm peripheral ntshav lymphocytes thiab plab hnyuv lymphocytes, thiab txhawb kev sib txawv ntawm peripheral ntshav T lymphocytes rau hauv CD{{ 123}} T cells. Tsis tas li ntawd, RNA-seq tsom xam pom tias G70 tau tswj hwm mRNA cov lus hais txog G-protein txuas nrog serotonin receptor thiab MHC chav kawm I polypeptide thiab txo qis mRNA cov lus hais txog cathelicidin thiab beta-defensin. Xav txog cov txiaj ntsig zoo ntawm G70 ntawm cov tshuaj tiv thaiv ND hauv nqaij qaib, cov nyhuv ntawm G70 ntawm lwm cov tshuaj tiv thaiv qaib xws li cov tshuaj tiv thaiv kab mob khaub thuas avian tuaj yeem kawm ntxiv.

Daim duab 6. KEGG pathway analysis.

Daim duab 7. RT-qPCR kev lees paub ntawm cov neeg xaiv DEG xaiv. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias § SE.

Daim duab 8. txheeb ze mRNA qhia hauv tus po. Tag nrho RNA tau muab rho tawm los ntawm tus po. Cov nqaij qaib b-actin tau txais kev pabcuam raws li kev tswj hwm sab hauv. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias § SE.

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