FOXO tswj kev nthuav tawm ntawm Antimicrobial Peptides Thiab Txhawb phagocytosis ntawm Hemocytes hauv Cwj Pwm Antibacterial Immunity
Aug 31, 2023
Abstract
Invertebrates vam khom lub cev tiv thaiv kab mob, suav nrog kev tiv thaiv kab mob humoral thiab cellular, tiv thaiv kab mob pathogenic. Cov kev tshawb fawb yav dhau los tau qhia tias forkhead box transcription factor O (FOXO) koom nrog cov tshuaj tiv thaiv kab mob ntawm cov tsiaj txhu thiab lub plab humoral tiv thaiv kab mob ntawm cov pob txha. Txawm li cas los xij, seb FOXO puas koom nrog hauv kev tswj hwm lub cev thiab kev tiv thaiv kab mob hauv lub cev tsis paub meej. Hauv txoj kev tshawb fawb tam sim no, peb tau txheeb xyuas FOXO los ntawm cws (Marsupenaeus japonicus) thiab pom tias nws tau nthuav tawm ntawm cov theem qis hauv cov cw ib txwm, tab sis tau hloov kho ntau heev hauv cws sib tw los ntawm Vibrio anguillarum. FOXO ua lub luag haujlwm tseem ceeb hauv kev tswj hwm hemolymph thiab plab hnyuv microbiota homeostasis los ntawm kev txhawb nqa kev qhia ntawm Relish, qhov kev hloov pauv ntawm lub cev tsis muaj zog (IMD) txoj hauv kev rau kev qhia txog tshuaj tiv thaiv kab mob peptides (AMPs) hauv cw. Peb kuj pom tau tias cov kab mob kis tau ua rau FOXO thiab ua rau nws txoj kev hloov pauv hauv nuclear los ntawm kev txo serine / threonine kinase AKT kev ua. Nyob rau hauv lub nucleus, activated FOXO ncaj qha tswj kev qhia ntawm nws lub hom phiaj Amp thiab Relish noob tiv thaiv kab mob. Tsis tas li ntawd, FOXO tau txheeb xyuas tias tau koom nrog hauv kev tiv thaiv ntawm tes los ntawm kev txhawb nqa phagocytosis ntawm hemocytes los ntawm kev txhawb nqa kev qhia ntawm phagocytotic receptor scavenger receptor C (Src), thiab ob qho me me GTPases, Rab5 thiab Rab7, uas muaj feem xyuam rau phagosome trafficking rau lysosome. hauv cytoplasm. Muab ua ke, peb cov txiaj ntsig tau qhia tias FOXO siv nws cov teebmeem ntawm homeostasis ntawm hemolymph thiab enteric microbiota los ntawm kev ua kom txoj hauv kev IMD hauv cov cw ib txwm, thiab ncaj qha lossis ncaj qha txhawb AMP kev qhia thiab txhim kho phagocytosis ntawm hemocytes tawm tsam cov kab mob hauv cov kab mob cw. Txoj kev tshawb no tau nthuav tawm cov haujlwm sib txawv ntawm FOXO hauv mucosal (hauv zos) thiab kev tiv thaiv kab mob tiv thaiv kab mob ntawm cov pob txha.
cistanche ntxiv cov txiaj ntsig-yuav ua li cas ntxiv dag zog rau lub cev
Nyem qhov no mus saib Cistanche Enhance Immunity khoom
【Nug ntxiv】 Email: cindy.xue@wecistanche.com / Whats App: 0086 18599088692 / Wechat: 18599088692
Taw qhia
Lub forkhead box transcription factor O tsev neeg cov proteins (FOXOs) koom nrog ntau yam tseem ceeb ntawm cov kab mob lom neeg, nrog rau kev tswj xyuas lub voj voog ntawm tes, kho DNA puas, tiv thaiv kab mob qog noj ntshav, thiab kev tswj lub neej [1,2]. Cov tsiaj nyeg muaj plaub yam FOXO proteins (FOXO1, FOXO3, FOXO4, thiab FOXO6); Txawm li cas los xij, invertebrates tsuas muaj ib qho FOXO, uas tseem hu ua Daf-16 hauv Caenorhabditis elegans [3]. Kev ua haujlwm ntawm FOXO yog tswj hwm los ntawm ntau hom kev hloov pauv tom qab kev txhais lus, xws li phosphorylation, acetylation, methylation, thiab ubiquitination [4]. Cov kev hloov kho no cuam tshuam rau FOXO nuclear translocation lossis tawm ntawm lub hauv paus, thiab nws cov kev cuam tshuam nrog co-repressors thiab co-activators, uas tuaj yeem txhawb lossis txo FOXO kev ua haujlwm thiab kho nws txoj haujlwm lom neeg sib txawv [5,6]. Tom qab ua kom tiav, FOXO koom nrog ntau lub zog ntawm lub cev los ntawm kev tswj hwm kev hloov pauv ntawm ntau hom phiaj hom phiaj [7]. FOXOs yog qhib los ntawm ntau yam stimuli extracellular, nrog rau kev loj hlob yam, cytokines, thiab cov tshuaj hormones [8]. Cov cim kev loj hlob, xws li insulin lossis insulin-zoo li kev loj hlob yam 1 (IGF-1), cuam tshuam nrog cov receptors ntawm txoj kev insulin thiab ua kom phosphatidylinositol 3-kinase (PI3K) signaling, ua rau phosphorylation ntawm FOXO los ntawm cov serine/threonine kinase, protein kinase B (AKT) [9]. Phosphorylated FOXO translocates los ntawm lub nucleus mus rau cytosol, los yog tiv thaiv nws translocation ntawm cytosol mus rau lub nucleus, qhov twg nws yuav ubiquitinated, ua rau nws degradation los ntawm cov proteasome [10]. Los ntawm qhov sib txawv, thaum tsis muaj cov cim qhia kev loj hlob sab nraud, PI3K-AKT teeb liab txoj hauv kev tsis ua haujlwm, thiab tsis muaj phosphorylated FOXO hloov mus rau hauv lub nucleus los txhawb cov hom phiaj hloov pauv [11]. FOXO kuj koom nrog hauv cov tshuaj tiv thaiv kab mob thiab tshuaj tiv thaiv kab mob hauv lub cev tiv thaiv kab mob ntawm cov leeg tsis muaj zog [12,13]. Kev ua kom ntev ntev ntawm FOXO nyob rau hauv cov hnyuv laus ntawm Drosophila represses qhov kev qhia ntawm peptidoglycan paub protein SC2 (ib tug tsis zoo regulator ntawm lub cev tsis muaj zog (IMD) txoj kev) thiab lov plab hnyuv kab mob homeostasis [14]. FOXO ncaj qha tswj cov tshuaj tiv thaiv kab mob peptide (AMP) cov noob hauv cov ntaub so ntswg epithelial ntawm Drosophila uas tsis muaj kab mob hauv cov lus teb rau kev tshaib kev nqhis, uas yog ywj pheej ntawm cov kab mob hauv lub cev tiv thaiv kab mob [15]. Txawm li cas los xij, cov kab mob kuj tuaj yeem ua rau enterocyte FOXO nkag mus rau hauv lub nucleus los ntawm cytoplasm ncaj qha rau hauv cov hnyuv Drosophila [12]. FOXO-raws li kev cai ntawm AMPs kuj tshwm sim nyob rau hauv cov ntaub so ntswg mammalian epithelial, qhia tias qhov no yog evolutionarily khaws cia rau hauv cov tsiaj [16]. Ntau qhov kev tshawb fawb pom tau tias FOXOs tau qhib los ntawm cov kab mob lossis cytokine stimulation hauv mucosal tiv thaiv kab mob [17]. Lawv txoj kev hloov mus rau lub nucleus thiab khi rau cov neeg txhawb nqa ntawm lawv cov hom phiaj yog txhawb nqa los ntawm mitogen-activated protein (MAP) kinase txoj kev thiab inhibited los ntawm PI3K / AKT txoj kev. Lub hom phiaj ntawm FOXOs muaj xws li pro-inflammatory signaling molecules, adhesion molecules, thiab chemokine receptors [17]. Txawm hais tias FOXOs koom nrog hauv lub cev tiv thaiv kab mob tiv thaiv kab mob tseem tsis paub meej. Txhawb nqa qhov kev qhia ntawm AMPs tawm tsam cov kab mob yog ib qho tseem ceeb ntawm kev humoral innate immunity hauv invertebrates [18]. Hauv Drosophila innate kev tiv thaiv, Txoj Kev Hu Xov Tooj thiab IMD txoj hauv kev tau koom nrog kev tswj hwm ntawm AMP qhia, thiab AMPs ua lub luag haujlwm tseem ceeb hauv kev tshem tawm cov kab mob nkag mus [19–21]. Zoo ib yam li kab tiv thaiv kab mob, Tus Xov Tooj, IMD, thiab Janus kinase (JAK) / lub teeb liab transducer thiab activator ntawm transcription (STAT) txoj hauv kev tau koom nrog AMP kev tswj hwm hauv crustaceans [22,23]. Txawm hais tias FOXO tuaj yeem tswj hwm qhov kev qhia ntawm AMPs ywj pheej ntawm Tus Xov Tooj, IMD, thiab JAK / STAT txoj hauv kev tiv thaiv kab mob kab mob hauv crustaceans xav tau kev qhia meej. Ntxiv nrog rau kev tiv thaiv kab mob humoral, invertebrates tseem vam khom lub cev tiv thaiv kab mob los ua haujlwm hauv lub cev tiv thaiv kab mob tiv thaiv kab mob. Phagocytosis los ntawm hemocytes yog ib qho ntawm cov txheej txheem tseem ceeb rau kev tiv thaiv cellular tiv thaiv kab mob [24]. Cov kev tshawb fawb yav dhau los tau pom tias FOXO1-kev kho mob autophagy yog xav tau rau kev tsim kho lub cev (NK) cell [25]. Ntxiv mus, FOXO koom nrog txhawb cov kab mob phagocytosis los ntawm neutrophils [26]. Txawm hais tias tsis muaj lub cev tiv thaiv kab mob tshwj xeeb hauv crustaceans, xws li NK hlwb lossis neutrophils, cw hemocytes tuaj yeem ua rau lub cev tiv thaiv kab mob tiv thaiv kab mob los ntawm phagocytosis [27]. Txawm li cas los xij, seb FOXO koom nrog cov kab mob phagocytosis ntawm hemocytes hauv cw yog tsis paub meej. Cw aquaculture yog ib qho ntawm lub ntiaj teb kev lag luam loj hlob sai tshaj plaws rau kev tsim cov tsiaj protein thiab tau ua rau muaj txiaj ntsig zoo rau kev ua kom tau raws li qhov xav tau ntawm cov tsiaj protein ntau ntxiv. Tam sim no, thoob ntiaj teb kev tsim cov cw yog kwv yees li 4.88 lab tons nrog tus nqi ntawm ib ncig ntawm 39 billion USD [28]. Txawm li cas los xij, kev yug tsiaj ntau ua ke ua rau muaj kev ntxhov siab ntau rau tsiaj, uas pab txhawb cov kab mob, nrog rau cov kab mob thiab cov kab mob, kev sib faib thiab kev kho mob, uas ua rau muaj kev lag luam loj rau kev lag luam [29]. Kev tshawb fawb txog kev tiv thaiv kab mob cw tuaj yeem muab cov tswv yim tshiab rau kev tiv thaiv kab mob thiab kev tswj hwm [30]. Hauv txoj kev tshawb fawb tam sim no, peb tau txheeb xyuas FOXO hauv kuruma cw (Marsupenaeus japonicus). Tom qab knockdown Foxo qhia, ua raws li kev sib tw Vibrio anguillarum, peb pom tias muaj peev xwm tshem tawm cov kab mob thiab cov cw ciaj sia tau txo qis. Kev tshawb fawb ntxiv tau qhia tias FOXO txhawb kev qhia ntawm AMPs ncaj qha lossis tsis ncaj, thiab txhim kho phagocytosis ntawm cov kab mob hauv cov cw. Cov txheej txheem ua tau ntawm FOXO cov teebmeem tiv thaiv kab mob hauv cw tau txheeb xyuas.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Cov txiaj ntsig
FOXO yog qhia nyob rau hauv basal theem nyob rau hauv unchallenged cws thiab yog upregulated nyob rau hauv cws sib tw nrog V. anguillarum
Los ntawm hemocyte transcriptome sequencing ntawm M. japonicus, peb tau txais tag nrho-ntev cDNA ib ntus ntawm Foxo (GenBank accession naj npawb: MW080526). Kev sib piv cov npe tau pom tias qhov kev kwv yees FOXO protein muaj Forkhead (FH) domain, uas zoo ib yam li Homosapiens FOXOs suav nrog FOXO1, FOXO3, FOXO4, thiab FOXO6, thiab dais zoo li AKT phosphorylation (S1A Fig). Cov txiaj ntsig ntawm phylogenetic tsom xam pom tias FOXOs tau muab faib ua ob pawg suav nrog cov pob txha caj qaum thiab cov leeg tsis muaj zog FOXOs, thiab cov cw FOXO tau koom ua ke hauv ceg invertebrate hauv tsob ntoo phylogenetic (S1B Fig). Zoo ib yam li tib neeg FOXOs [31], cw FOXO muaj extracellular regulated kinase (ERK) phosphorylation hloov chaw thiab CREB Binding Protein (CBP) acetylation hloov chaw, tab sis tsis muaj sir tuin 1 (SIRT1) deacetylation hloov chaw thiab c-JUN N-terminal kinase (JNK) phosphorylation hloov chaw (S2 Fig). Polyclonal antibodies paub cw FOXO tau npaj siv lub recombinant FH domain ntawm FOXO qhia hauv Escherichia coli (Fig 1A). Kev soj ntsuam ntawm cov ntaub so ntswg faib ntawm FOXO ntawm mRNA thiab qib protein tau qhia tias nws tau nthuav tawm hauv txhua cov ntaub so ntswg (Fig 1B). Lub sijhawm kawm ntawm FOXO qhia nyob rau hauv cov hemocytes thiab cov hnyuv ntawm cw sib tw los ntawm V. anguillarum tau soj ntsuam siv qPCR thiab western blotting. Cov txiaj ntsig tau pom tias FOXO tau tswj hwm hauv cov hemocytes thiab cov hnyuv ntawm mRNA qib (Daim duab 1C thiab 1D) thiab qib protein (Fig 1E thiab 1F). Cov txiaj ntsig no tau qhia tias FOXO koom nrog cov lus teb rau V. anguillarum kab mob thiab ua rau peb tshawb nrhiav kev ua haujlwm ntawm FOXO hauv kev tiv thaiv cw.
FOXO koom nrog hauv kev tswj hwm homeostasis ntawm hemolymph thiab plab hnyuv microflora hauv cov cw noj qab haus huv
Txhawm rau tshawb nrhiav kev ua haujlwm ntawm FOXO hauv hemolymph thiab plab hnyuv microflora homeostasis hauv cw, RNA cuam tshuam tau ua thiab kuaj cov kab mob. Cov txiaj ntsig tau pom tias qhov kev qhia ntawm Foxo txo qis hauv Foxo-RNAi cw (Fig 2A thiab 2B). Cov txiaj ntsig tawm ntawm Foxo RNAi kuj tau txheeb xyuas los ntawm kev txheeb xyuas qhov kev qhia ntawm lwm hom Fox tsev neeg (xws li Foxk2 thiab Foxn3). Cov txiaj ntsig tau pom tias qhov kev poob qis ntawm Foxo tsis txo qis kev qhia theem ntawm Foxk2 lossis Foxn3 (S3A-S3C Fig). Siv western blotting thiab fluorescent immunocytochemistry kev soj ntsuam, peb ntxiv soj ntsuam cov subcellular faib ntawm FOXO nyob rau hauv cov hnyuv thiab hemocytes ntawm cw nyuaj los ntawm cov kab mob tom qab lub silencing ntawm Foxo. Cov txiaj ntsig tau pom tias piv nrog dsGfp pawg, FOXO qib hauv cov nucleus poob qis heev hauv Foxo-RNAi cw, txawm tias cov cw tau kis los ntawm V. anguillarum (S3D–S4E 'Figs). Tag nrho cov txiaj ntsig saum toj no tau qhia tias peb qhov kev ntsuam xyuas Foxo RNAi tuaj yeem ua rau Foxo ntsiag to hauv hemocytes thiab hnyuv (xws li hauv cytoplasm thiab nucleus). Tom ntej no, peb txheeb xyuas cov kab mob hauv cov hemolymph thiab cov hnyuv tom qab knockdown ntawm Foxo hauv cov cw hauv ib txwm muaj (tsis muaj kab mob sib tw). Cov txiaj ntsig tau pom tias tus naj npawb ntawm cov kab mob hauv hemolymph thiab cov hnyuv tau nce zuj zus tom qab Foxo qhia tau ua tiav lawm (Daim duab 2C thiab 2D). Peb txuas ntxiv txheeb xyuas qhov ciaj sia taus ntawm Foxo-RNAi cw yam tsis muaj kab mob sib tw, thiab cov txiaj ntsig tau pom tias qhov ciaj sia taus ntawm Foxo-knockdown cws poob qis dua piv nrog cov kev tswj hwm (Daim duab 2E). Txhawm rau txheeb xyuas ntxiv cov txiaj ntsig no, peb tau txheeb xyuas qhov muaj sia nyob ntawm Foxo-RNAi cw kho nrog tshuaj tua kab mob thiab cov txiaj ntsig tau pom tias piv nrog pawg dsGfp, qhov ciaj sia taus ntawm cov kab mob tsis muaj cw tsis tau txo qis (Daim duab 2F). Tag nrho cov txiaj ntsig tau qhia tias FOXO ua lub luag haujlwm hauv homeostasis ntawm lub hauv vivo microbiota (xws li hemolymph thiab enteric microbiota) hauv cw nyob rau hauv ib txwm muaj.
cistanche ntxiv cov txiaj ntsig-nce kev tiv thaiv
FOXO koom nrog kev tswj hwm ntawm hemolymph thiab plab hnyuv microflora homeostasis los ntawm kev txhawb nqa Relish thiab Amp qhia hauv cov cw noj qab haus huv
Cov kev tshawb fawb yav dhau los qhia tau hais tias cov cw noj qab haus huv muaj cov kab mob tsawg tab sis ruaj khov hauv cov kab mob hemolymph, thiab cov kab mob siab tas li ntawm cov kab mob hauv plab hnyuv, thiab cov tshuaj tiv thaiv kab mob, xws li tshuaj tiv thaiv kab mob peptides (AMPs) tswj los ntawm cov khoom siv nuclear kappa B (NF. -κB) txoj hauv kev thiab cov pa oxygen reactive (ROS) tswj los ntawm ob txoj kev oxidase (DUOX), ua lub luag haujlwm tseem ceeb hauv homeostasis hemolymph thiab plab hnyuv microbiota [32–34]. Txhawm rau tshawb nrhiav cov txheej txheem ntawm FOXO txoj kev koom tes hauv kev tswj hwm ntawm hemolymph thiab plab hnyuv microflora homeostasis nyob rau hauv ib txwm muaj, peb thawj zaug kuaj pom cov subcellular faib ntawm FOXO hauv hemocytes thiab cov hnyuv nyob rau hauv ib txwm muaj. Cov txiaj ntsig tau pom cov teeb liab tsis muaj zog ntawm FOXO nyob rau hauv lub nuclei ntawm cw hemocytes thiab plab hnyuv hlwb nyob rau hauv ib txwm muaj mob (Daim duab 3A thiab 3B), qhia tias me ntsis FOXO tau qhib los ntawm lub hauv vivo microbiota hauv cws hauv ib txwm muaj. AMP kev qhia yog tswj hwm los ntawm IMD txoj hauv kev hauv cov hnyuv ntawm Drosophila [35]. Yog li ntawd, peb xav tias qhov kev hloov pauv hloov Relish yuav yog lub hom phiaj ntawm FOXO. Peb tau kuaj pom mRNA qhia ntawm Relish, qhov kev hloov pauv ntawm IMD txoj hauv kev, hauv cw hemocytes thiab hnyuv tom qab lub khob ntawm Foxo. Cov txiaj ntsig tau pom tias Relish qhia tau txo qis hauv Foxo-knockdown cw (Fig 3C thiab 3D), thiab cov txiaj ntsig zoo sib xws tau txais ntawm qib protein (Fig 3E thiab 3E'). Cov txiaj ntsig tau qhia tias FOXO tuaj yeem txhawb nqa kev qhia ntawm Relish thiab qhib IMD txoj hauv kev. Txhawm rau kom paub meej tias FOXO koom nrog hemo lymph thiab gastrointestinal tract homeostasis los ntawm IMD signaling, cov kab mob tsis muaj kab mob tau txais (Fig 3F) thiab RELISH activation thiab AMP qhia tau pom. Piv nrog rau qhov ntawd hauv kev tswj hwm, tus nqi ntawm FOXO thiab RELISH nyob rau hauv lub nuclei ntawm plab hnyuv hlwb thiab hemocytes txo qis (Daim duab 3G thiab 3H). Lub caij no, cov lus qhia ntawm AMPs, suav nrog Crustins (CrusI-2 rau 5) thiab Anti-lipopolysaccharide yam (Alf A1, B1, C1, D1 thiab E1), tejzaum nws tswj los ntawm FOXO thiab / lossis RELISH tau tshuaj xyuas hauv cov kab mob. -dawb cw, thiab cov txiaj ntsig tau pom tias kev qhia ntawm CrusI-3, Alf-B1 thiab Alf-C1 poob qis hauv hemo cytes thiab cov hnyuv ntawm cw (Daim duab 3I thiab 3J). Muab hais tias qhov kev qhia ntawm Alf-B1 thiab Alf C1 tau tswj hwm los ntawm IMD txoj hauv kev [36], cov txiaj ntsig no tau qhia tias FOXO koom nrog kev tswj hwm homeostasis ntawm hemolymph thiab plab hnyuv ib txwm muaj los ntawm IMD txoj hauv kev los txhawb kev qhia ntawm AMPs.
Daim duab 1. FOXO tau tswj hwm hauv cov cw sib tw los ntawm V. anguillarum. (A) Recombinant qhia ntawm FH domain ntawm FOXO nyob rau hauv E. coli thiab western blotting kom ntes FOXO siv polyclonal antibodies tawm tsam FOXO npaj rau hauv luav. Txoj kab 1, tag nrho cov proteins ntawm E. coli nrog Foxo-pGEX4T-1. Txoj kab 2, tag nrho cov proteins ntawm cov kab mob tom qab IPTG induction. Txoj kab 3, purified recombinant FOXO. Txoj kab 4, FOXO nyob rau hauv cov hnyuv ntawm cov cw tsis kho tau raug kuaj pom siv western blotting. M, Protein molecular mass markers. (B) Cov ntaub so ntswg faib tawm ntawm Foxo ntawm mRNA (saum vaj huam sib luag) thiab cov protein (cov vaj huam sib luag qis) tau kuaj pom los ntawm RT-PCR thiab sab hnub poob blotting, feem. ACTB yog lub npe luv ntawm -actin. (C, D) Cov qauv qhia mRNA ntawm Foxo hauv hemocytes (C) thiab plab hnyuv (D) tau soj ntsuam los ntawm qPCR siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 ua cov noob tswj endogenous. (E, F) Cov qauv qhia protein ntawm FOXO hauv hemocytes (E) thiab plab hnyuv (F) tau kuaj pom los ntawm sab hnub poob blotting. Cov txiaj ntsig ntawm kev txheeb xyuas kev txheeb xyuas ntawm peb qhov kev hloov pauv tau pom nyob rau hauv qab panels. Cov bands nyob rau sab hnub poob blot tau digitalized siv ImageJ software los ntawm kev txheeb xyuas cov bands los ntawm peb qhov kev ywj pheej rov ua dua. Cov txheeb ze qhia theem ntawm FOXO / -actin tau qhia raws li qhov nruab nrab ± SD, thiab tus nqi ntawm cov cw tswj tau teeb tsa ua ib qho. Cov kab yuam kev hauv daim duab qhia tias SDs (peb qhov rov ua dua).
FOXO tau qhib rau hauv cw los ntawm V. anguillarum kev sib tw thiab koom nrog cov kab mob tshem tawm ntawm hemolymph thiab hnyuv.
Nws tau tshaj tawm tias FOXO tuaj yeem qhib los ntawm cov kab mob los yog cytokine stimulation thiab hloov mus rau lub nucleus los tswj cov kev qhia ntawm qee lub hom phiaj hauv cov tsiaj txhu [17]. Txhawm rau txheeb xyuas seb FOXO puas tau qhib rau hauv cws sib tw los ntawm cov kab mob, cov subcellular faib ntawm FOXO nyob rau hauv cov hnyuv ntawm cov cw raug tshuaj xyuas siv western blotting. Cov txiaj ntsig tau pom tias tus nqi ntawm FOXO nyob rau hauv cytoplasm ntawm cov kab mob hauv plab hnyuv tau txo qis los ntawm 2 mus rau 4 h tom qab V. anguillarum kev sib tw (Fig 4A). Nyob rau hauv sib piv, tus nqi ntawm FOXO nyob rau hauv lub nucleus ntawm txoj hnyuv hlwb tau nce ntau heev tom qab kis tau tus kab mob V. anguillarum (Fig 4B). Txhawm rau txheeb xyuas lub luag haujlwm ntawm FOXO hauv kev tiv thaiv kab mob, kev tshuaj ntsuam fluorescent immunocytochemical tau siv los soj ntsuam cov kev hloov pauv ntawm FOXO hauv hemocytes ntawm cw 2 h post V. txhua xyoo larum stimulation. Peb pom tias cov kab mob sib tw ua rau FOXO hloov mus rau hauv lub nucleus tau nce zuj zus tab sis tsis muaj kev hloov pauv hauv PBS-kev sib tw cw (Daim duab 4C thiab 4C'). Txhawm rau tshawb xyuas seb lub nuclear translocated FOXO puas koom nrog kev tshem tawm cov kab mob, peb tau txheeb xyuas cov kab mob hauv cov hemolymph thiab cov hnyuv hauv Foxo-knockdown cw thaum 6 teev tom qab kev sib tw kab mob. Peb pom tias tom qab lub khob ntawm Foxo, cov kab mob thauj khoom hauv hemolymph thiab cov hnyuv tau nce ntau dua piv nrog rau hauv pawg tswj hwm (Daim duab 4D). Ntxiv mus, qhov ciaj sia taus ntawm Foxo-knockdown cw kis tus kab mob V. anguillarum poob qis dua piv nrog cov kev tswj hwm (Daim duab 4E). Cov txiaj ntsig no tau qhia tias FOXO tau qhib rau hauv cov hlwb enteric thiab hemocytes, thiab qhia tias FOXO koom nrog hauv zos thiab lub cev tiv thaiv kab mob tiv thaiv kab mob.
Fig 2. FOXO koom nrog hauv plab hnyuv (hauv zos) thiab cov kab mob tiv thaiv kab mob tiv thaiv kab mob. (A, B) Kev ua tau zoo ntawm Foxo-RNAi hauv hemocytes (A) thiab plab hnyuv (B) ntawm cw, raws li kev txiav txim siab siv qPCR (A) thiab western blotting (B). QPCR hnub tsom xam siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 raws li endogenous tswj noob. Cov kab yuam kev qhia SDs. (C) Kev txheeb xyuas cov kab mob hemolymph ntawm Foxo-knockdown cw los ntawm cov kab lis kev cai LB khoom (peb rov ua dua); dsGfp txhaj tshuaj tau siv los ua tus tswj. Qhov ratio ntawm dilution yog 1:10. (D). Tus naj npawb ntawm cov kab mob hauv hemolymph thiab cov hnyuv hauv Foxo knockdown cw thiab dsGfp-txhaj tshuaj cw yam tsis muaj kab mob sib tw. (E). Qhov ciaj sia taus ntawm Foxo-RNAi cw yam tsis muaj kab mob sib tw. dsGfp txhaj tshuaj tau siv los ua tus tswj. (F). Qhov ciaj sia taus ntawm Foxo-RNAi cw kho nrog tshuaj tua kab mob. dsGfp txhaj tshuaj tau siv los tswj. Ib txwm: hom cw qus tsis kho nrog tshuaj tua kab mob.
Fig 3. Ib qho basal npaum li cas ntawm FOXO translocates mus rau hauv lub nuclei ntawm hemocytes thiab cov hnyuv hlwb txhawb kev Relish qhia thiab tom qab ntawd activates IMD txoj kev tswj hemolymph thiab plab hnyuv microbiota homeostasis nyob rau hauv ib txwm tej yam kev mob. (A) Immunocytochemistry tau siv los txheeb xyuas cov subcellular faib ntawm FOXO hauv cov hemocytes ntawm cov cw ib txwm. Scale bar=20 μm. Cytoplasm; Nug: Nucleus. (B) Western blotting tau siv los txheeb xyuas cov subcellular faib ntawm FOXO nyob rau hauv cov hnyuv hlwb los ntawm ib txwm cws thiab V. anguillarum-mob cw. (C) Qhov kev qhia ntawm Relish ntawm mRNA qib tau kuaj pom hauv hemocytes ntawm cw tom qab knockdown ntawm Foxo los ntawm qPCR siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 raws li endogenous tswj noob. (D) Cov lus qhia ntawm Relish ntawm mRNA qib hauv cov hnyuv ntawm cw tom qab lub khob ntawm Foxo tau kuaj pom los ntawm qPCR siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 ua cov noob tswj endogenous. (E) Cov qib RELISH tau kuaj pom siv western blotting hauv cw tom qab lub khob ntawm Foxo. (E') Peb qhov kev hloov pauv ntawm vaj huam sib luag E tau digitalized siv ImageJ software. (F) Cov kab mob hnyav ntawm hemolymph thiab cov hnyuv hauv cw 24 teev tom qab kho tshuaj tua kab mob. (G) FOXO nyob rau hauv lub nucleus tau kuaj pom siv western blotting hauv hemocytes thiab cov hnyuv ntawm cw 24 h tom qab kho nrog tshuaj tua kab mob. (H) RELISH nyob rau hauv lub nucleus tau kuaj pom siv western blotting nyob rau hauv cw hemocytes thiab hnyuv tom qab 24 teev tom qab kho nrog tshuaj tua kab mob. (I, J) Qhov kev qhia ntawm Amps hauv hemocytes (I) thiab txoj hnyuv (J) ntawm cov kab mob tsis muaj kab mob tau txiav txim siab los ntawm qPCR siv -actin thiab Ef-1 raws li cov noob siv sab hauv. Cov cw li qub (H2O txhaj) tau siv los ua kev tswj hwm.
Cov kev tshawb fawb yav dhau los tau qhia tias FOXO transcriptional kev ua haujlwm yog tswj hwm los ntawm kev hloov kho phosphorylation thiab tias serine / threonine kinase AKT tswj cov haujlwm ntawm Forkhead transcription yam los ntawm phosphorylating lawv thiab inhibiting lawv tsis meej translocation [9,37]. Txhawm rau tshawb xyuas seb qhov nce ntawm FOXO nyob rau hauv lub nucleus tom qab cov kab mob pathogenic cuam tshuam los ntawm AKT, peb thawj zaug kuaj pom cov ntsiab lus ntawm FOXO hauv cytoplasm thiab nucleus los ntawm sab hnub poob blotting tom qab lub khob ntawm Akt ntawm cw. Cov txiaj ntsig tau pom tias tus nqi ntawm FOXO nyob rau hauv lub nucleus ntawm hemocytes thiab plab hnyuv hlwb tau nce ntau tom qab knockdown ntawm Akt qhia (Fig 5A thiab 5B) hauv cw 2 h tom qab kis kab mob nrog V. anguillarum (Fig 5C thiab 5C'). Txhawm rau txheeb xyuas cov txiaj ntsig kev sim, peb kuj tau siv qhov kev ntsuam xyuas immunocytochemistry los soj ntsuam cov subcellular faib ntawm FOXO nyob rau hauv lub hemocytes ntawm cw tom qab knockdown ntawm Akt. Cov txiaj ntsig tau pom tias FOXO tau nce ntxiv hauv cov nucleus ntawm hemocytes thaum Akt tau tsoo 2 h tom qab kis kab mob nrog V. anguillarum (Fig 5D thiab 5D'). Txhawm rau tshawb xyuas seb cov kab mob puas cuam tshuam rau AKT kev ua haujlwm, peb tau tshawb pom qhov hloov pauv phosphorylation ntawm AKT tom qab cov kab mob kis tau siv tib neeg p-AKT (Ser473) antibody (Phosphorylation site ntawm cw AKT yog Ser486) thiab pom tias theem ntawm phosphorylated AKT poob qis hauv hemocytes thiab cov hnyuv ntawm cw tom qab 30 min ntawm V. anguillarum kab mob (Daim duab 5E thiab 5F). Cov txiaj ntsig no tau qhia tias cov kab mob kis tau tuaj yeem txo cov qib ntawm phosphorylated AKT, uas inhibits cov enzyme kev ua si thiab txo lub peev xwm ntawm AKT rau phosphorylate FOXO. Cov tsis-phosphorylated FOXO nkag mus rau hauv lub nucleus thiab induces kev qhia ntawm nws lub hom phiaj genes. daim duab qhia tias SDs.
cistanche extract
Cov kab mob ua rau txo qis kev ua haujlwm ntawm AKT thiab ua rau FOXO nuclear translocation
FOXO exerts tiv thaiv kab mob tiv thaiv invading kab mob los ntawm kev tswj cov kev qhia ntawm antimicrobial peptides nyob rau hauv cws kis los ntawm cov kab mob.
Txhawm rau tshawb xyuas cov txheej txheem uas FOXO cuam tshuam cov lus teb tawm tsam cov kab mob hauv cw, peb thawj zaug txheeb xyuas cov tshuaj tiv thaiv kab mob qog noj ntshav tswj hwm los ntawm FOXO. Tom qab kev vam meej knockdown ntawm Foxo nyob rau hauv hemocytes thiab cov hnyuv ntawm cw nrog los yog tsis muaj V. anguillarum kev sib tw, cov kev qhia theem ntawm CrusI-3, Alf-B1, Alf-C1, thiab Alf-E1 txo qis heev nyob rau hauv lub silenced cws piv. nrog cov neeg hauv pawg tswj hwm (Daim duab 6A thiab 6B). Txhawm rau txheeb xyuas cov txiaj ntsig ntxiv, peb kuj tau kuaj pom cov lus qhia ntawm plaub AMPs tom qab knockdown ntawm Akt. Cov txiaj ntsig tau pom tias qhov kev qhia ntawm plaub AMPs tau nce ntau ntxiv 6 h tom qab V. anguil larum kab mob piv nrog rau hauv pawg tswj hwm (Daim duab 6C thiab 6D). Piv nrog rau kev tswj hwm AMPs (xws li Alf-B1, thiab Alf-C1) nyob rau hauv ib txwm muaj (Daim duab 3I thiab 3J), cov noob ntxiv ntawm AMPs (CrusI-3, Alf-E1) kuj tau tswj hwm hauv cov cw kis los ntawm V. . anguillarum (Fig 6A thiab 6B). Peb txoj kev tshawb fawb yav dhau los tau qhia tias RELISH tau txhawb nqa qhov nthuav tawm ntawm Alf-B1 thiab Alf-C1 hauv cov cw [36]. Cov txiaj ntsig no qhia tau tias FOXO tuaj yeem tswj hwm AMP kev qhia ncaj qha, xws li CrusI-3 thiab Alf-E1, ywj siab ntawm IMD txoj kev taw qhia. Txhawm rau kom paub meej qhov kev xav, qhov kev tshuaj ntsuam CIP tau ua los xyuas seb FOXO tuaj yeem khi rau tus txhawb nqa ntawm Alf-E1. Qhov kev txhawb nqa qib ntawm Alf-E1 tau txais (S4 Fig) thiab FOXO khi qhov chaw tau kwv yees (Daim duab 6E). Cov txiaj ntsig ChiP tau pom tias FOXO tuaj yeem khi rau Alf-E1 tus txhawb nqa (Daim duab 6F). Cov txiaj ntsig no tau qhia tias FOXO kuj koom nrog kev tiv thaiv kab mob los ntawm kev tswj hwm kev qhia ntawm AMPs hauv cw.
Fig 4. FOXO translocated mus rau hauv lub nucleus hauv cw sib tw los ntawm V. anguillarum. (A, B) Cov qauv FOXO faib nyob rau hauv cytoplasm (A) thiab nucleus (B) ntawm cov hnyuv hlwb ntawm cws sib tw los ntawm cov kab mob, raws li kev soj ntsuam siv western blotting (qis panels ntawm A thiab B). Sab hnub poob blotting bands tau digitalized siv ImageJ software los ntawm kev luam theej duab los ntawm peb qhov rov ua dua. Tus txheeb ze qhia theem ntawm FOXO / -actin lossis FOXO / Histone 3 tau qhia raws li qhov nruab nrab ± SD, thiab tus nqi ntawm cov cw tswj tau raug teeb tsa ua ib qho. (C) Kev hloov pauv nuclear ntawm FOXO hauv hemocytes ntawm cw 2 h tom qab V. Kev sib tw anguillarum tau kuaj pom siv cov tshuaj fluorescent immunocytochemical. PBS txhaj tshuaj yog siv los tswj. Scale bar=20 μm. (C') Kev txheeb xyuas ntawm vaj huam sib luag C Lub colocalization ntawm FOXO thiab DAPI-stained nuclei hauv hemocytes tau soj ntsuam los ntawm WCIF ImageJ software. (D) Tus naj npawb ntawm cov kab mob hauv hemolymph thiab cov hnyuv ntawm Foxo-knockdown cw ua raws li V. anguillarum txhaj; dsGfp-txhaj tshuaj nyob rau hauv cws tom qab txhaj tshuaj yog siv los tswj. (E) Qhov ciaj sia taus ntawm Foxo-RNAi cw kis los ntawm V. anguillarum. dsGfp txhaj tshuaj tau siv los ua tus tswj.
FOXO txhawb nqa phagocytosis ntawm hemocytes tawm tsam cov kab mob los ntawm kev txhawb nqa SRC kev qhia
Cov kev tshawb fawb yav dhau los tau tshaj tawm tias FOXO cuam tshuam ncaj qha nrog cov cheeb tsam txhawb nqa ntawm CXCR2 thiab CD11b los tswj cov phagocytosis ntawm neutrophils hauv cov tsiaj txhu [26]. Txhawm rau kuaj seb FOXO puas cuam tshuam nrog hemocyte phagocytosis hauv cws cov kab mob tiv thaiv kab mob, peb tau tshuaj xyuas tus nqi phagocytic tom qab cuam tshuam nrog Foxo siv ntau txoj kev. Tom qab knockdown ntawm Foxo, phagocytic tus nqi thiab phagocytic Performance index ntawm Foxo-knockdown pawg tau qis dua li ntawm pawg tswj hwm (dsGfp) (Daim duab 7A thiab 7A'). Peb kuj tau siv cov cytometry ntws los ntsuas qhov phagocytic tus nqi ntawm hemocytes tom qab kev ntsiag to ntawm Foxo. Cov txiaj ntsig tau pom tias phage cytosis tus nqi ntawm pawg Foxo-RNAi kuj txo qis dua piv nrog cov pab pawg dsGfp (Fig 7B thiab 7B'). Txhawm rau kom ua tiav cov txiaj ntsig no ntxiv, peb kuj tau tshuaj xyuas tus nqi phagocytic tom qab knockdown ntawm Akt. Cov txiaj ntsig tau pom tias piv nrog cov pab pawg tswj hwm, phagocytic tus nqi thiab phagocytic Performance index nce ntau (Daim duab 7C thiab 7C'). Cov txiaj ntsig no tau qhia tias FOXO txhawb nqa phagocytosis ntawm hemocytes hauv cw. Txhawm rau tshawb xyuas seb FOXO txhim kho li cas hemocyte phagocytosis hauv cw, peb tau tshawb pom qhov kev qhia ntawm cov caj ces yav dhau los koom nrog hauv phagocytosis ntawm cw. Scavenger receptor class B (SRB) thiab chav C (SRC) tau tshaj tawm tias muaj kev koom tes hauv hemocyte phagocytosis [38,39]. Peb pom tias mRNA qhia theem ntawm Src, es tsis yog Srb, txo qis heev tom qab ntsiag to ntawm Foxo nyob rau hauv hemocytes thiab hnyuv (Daim duab 7D thiab 7E), whereas qhov kev qhia ntawm Src nce ntau heev tom qab knockdown ntawm Akt nyob rau hauv cw (Fig 7F). Cov txiaj ntsig zoo sib xws tau txais ntawm qib protein (Daim duab 7G thiab 7G'). Cov txiaj ntsig no tau qhia tias FOXO txhawb nqa hemocyte phagocytosis ntawm cov kab mob hauv cw los ntawm kev txhawb nqa SRC kev qhia.
FOXO txhawb kev qhia ntawm RAB5 thiab RAB7 thaum lub sijhawm phagocytosis hauv cw
Cov kev tshawb fawb yav dhau los tau pom tias cov GTPases me me, RAB5 thiab RAB7, yog cov tswj hwm tseem ceeb ntawm kev thauj cov hlwv tshiab endocytosed mus rau thaum ntxov thiab lig endosomes [40]. RAB5 yog tus tswj hwm qhov tseem ceeb ntawm cov txheej xwm fusion thawj zaug thiab yog tus cim ntawm endosomes thaum ntxov [41,42]. RAB7 yog xav tau rau phagosome-lysosome fusion lig thiab yog ib qho cim ntawm lig endosomes [43]. Txhawm rau tshawb xyuas seb RAB5 thiab RAB7 puas koom nrog kev tshem tawm cov kab mob hauv cw, peb thawj zaug txheeb xyuas cov qauv qhia ntawm Rab5 thiab Rab7 thiab pom tias ob lub GTPases me me tau raug tswj hwm hauv cov cw sib tw nrog cov kab mob (S5A-S5D Fig). Peb tau soj ntsuam ntxiv txog qhov muaj sia nyob ntawm Rab5-RNAi thiab Rab7-RNAi cw tom qab kev sib tw ntawm cov kab mob, thiab cov txiaj ntsig tau pom tias qhov ciaj sia taus ntawm Rab5 thiab Rab7 knockdown cw poob qis dua piv nrog cov tswj ( S5E thiab S5F daim duab). Cov txiaj ntsig no tau qhia tias RAB5 thiab RAB7 ua haujlwm tseem ceeb hauv kev tiv thaiv kab mob kab mob. Txhawm rau tshawb xyuas seb RAB5 thiab RAB7 puas koom nrog FOXO-mediated phagocytosis, peb tau tshuaj xyuas mRNA qhia theem ntawm Rab5 thiab Rab7 tom qab cuam tshuam nrog Foxo qhia hauv cw. Cov txiaj ntsig tau pom tias qib kev qhia ntawm Rab5 thiab Rab7 txo qis hauv hemocytes thiab cov hnyuv (Figs 8A thiab S6A). whereas qhov kev qhia ntawm Rab5 thiab Rab7 tau nce ntau tom qab lub khob ntawm Akt hauv cw (Fig 8B). Txhawm rau kom paub meej ntxiv cov txiaj ntsig saum toj no, qib protein ntawm RAB5 thiab RAB7 hauv Foxo lossis Akt knockdown cws kuj tau kuaj pom. Piv nrog rau pawg dsGfp txhaj tshuaj, RAB5 thiab RAB7 qib kuj tau txo qis hauv cov hemocytes thiab cov hnyuv hauv Foxo-knockdown cw tab sis tau nce ntau hauv Akt-knockdown cw ntawm 2 h tom qab stimulation los ntawm V. anguillarum (Figs 8C–8D '; S6B. thiab S6B'). Cov txiaj ntsig no tau qhia tias RAB5 thiab RAB7 koom nrog FOXO-mediated phage cytosis ntawm hemocytes hauv cw. Peb tau tshawb pom ntxiv tias RAB5 thiab RAB7 tau sib koom ua ke nrog V. anguillarum hauv cw hemocytes (Fig 9). Ua ke, cov txiaj ntsig tau pom tias RAB5 thiab RAB7 koom nrog FOXO-mediated phagocytosis hauv cw.
Fig 5. Cov kab mob kis tau txo qis AKT kev ua, uas txhawb nqa FOXO nuclear translocation hauv hemocytes thiab enteric hlwb ntawm cw. (A, B) Kev ua tau zoo ntawm Akt-RNAi hauv hemocytes thiab cov hnyuv ntawm cw, raws li kev txiav txim los ntawm qPCR siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 raws li endogenous tswj noob (A) thiab western blotting (B). (C) Tus nqi ntawm FOXO nyob rau hauv lub cytoplasm thiab nucleus ntawm txoj hnyuv los ntawm Akt-knockdown cw tau soj ntsuam los ntawm sab hnub poob blotting. dsGfp tau siv los ua kev tswj hwm. (C') Kev txheeb xyuas ntawm vaj huam sib luag (C). ImageJ software tau siv los ua digitalize bands los ntawm kev luam theej duab peb rov ua dua. (D) Kev hloov pauv nuclear ntawm FOXO hauv hemocytes ntawm Akt-knockdown cw tau kuaj pom 2 h tom qab V. anguillarum kev sib tw siv cov tshuaj fluorescent immunocytochemical. dsGfp tau siv los ua kev tswj hwm. Scale bar=20 μm. (D') Kev txheeb xyuas ntawm (D). WCIF ImageJ software tau siv los txheeb xyuas qhov sib koom ua ke los ntawm fluorescence siv piv ntawm anti-FOXO (ntsuab) thiab DAPI-stained nucleus (xiav) hauv hemocytes tom qab kho tus nqi raug. (E, F) Cov ntsiab lus hloov pauv ntawm phosphorylated AKT (p-AKT) hauv hemocytes (E) thiab plab hnyuv (F) ntawm cw sib tw los ntawm V. anguillarum. Cov vaj huam sib luag hauv qab yog cov duab digitalized ntawm (E) thiab (F) raws li cov qhab nia ntawm sab hnub poob blotting thiab kev txheeb xyuas txheeb cais, feem.
Cistanche tshuaj yej
Kev sib tham
FOXO, raws li qhov tseem ceeb tshaj plaws ntawm kev hloov pauv, koom nrog ntau lub zog ntawm lub cev thiab metabolic [44,45]. Hauv kev tshawb fawb tam sim no, peb tau txheeb xyuas FOXO los ntawm cw (M. japonicus) thiab pom tias nws koom nrog kev tswj hwm homeostasis ntawm hemolymph thiab enteric microbiota los ntawm kev tswj hwm AMP kev qhia nyob rau hauv ib txwm muaj. FOXO tuaj yeem qhib tau los ntawm cov kab mob kab mob hauv cw thiab hloov mus rau hauv lub nucleus, qhov twg nws tswj hwm ncaj qha thiab tsis ncaj (los ntawm txoj kev IMD) cov lus qhia ntawm AMPs. FOXO upregulated qhia ntawm phagocytosis ntsig txog cov noob, xws li SRC thiab me GTPases, RAB5 thiab RAB7, los txhawb hemocyte phagocytosis tawm tsam cov kab mob. Yog li ntawd, cw FOXO plays lub luag haujlwm sib txawv hauv lub cev thiab hauv zos (enteric) lub cev tiv thaiv kab mob tiv thaiv kab mob (Daim duab 10). Piv nrog rau tib neeg FOXOs, cw FOXO muaj kev txuag cov amino acid ib ntus thiab AKT phosphorylation qhov chaw; Txawm li cas los xij, qhov thib ob AKT phosphorylation site ntawm cw FOXO yog threonine es tsis txhob serine. AKT phosphorylation ntawm FOXO tswj lub nuclear thiab cytoplasmic thauj ntawm FOXO [9]. Hauv txoj kev tshawb no, peb pom tias AKT muaj feem cuam tshuam nrog kev tswj hwm ntawm FOXO qhov kev nkag mus rau nuclear. Tom qab tus kab mob V. anguillarum, theem ntawm phosphorylated AKT tau txo qis, uas qhia tau hais tias AKT kev ua tau txo qis hauv cov kab mob-gen-infected cw, yog li ua kom muaj kev tuav pov hwm nuclear lossis nuclear translocation ntawm FOXO, thiab tom qab ntawd inducing kev qhia ntawm lub hom phiaj noob, xws li Relish thiab Amp, tiv thaiv kab mob. Cov kev tshawb fawb yav dhau los tau pom tias FOXO koom nrog hauv homeostasis ntawm Drosophila txoj hnyuv nal microflora los ntawm kev tawm tsam ntawm peptidoglycan lees paub protein SC2 (tus tswj tsis zoo ntawm IMD txoj hauv kev) los ua txoj hauv kev rau AMP qhia [14]. RELISH tau tshaj tawm tias yog lub hom phiaj tseem ceeb ntawm FOXO hauv kev tswj hwm ntawm hypoxia kam rau hauv Drosophila [46]. Zoo li lwm yam tsiaj, cw lub plab zom mov muaj qhov siab thiab tsis tu ncua microbiota. Cov pov thawj ntxiv kuj qhia tau hais tias hemolymph ntawm qee cov dej noj qab haus huv invertebrates, suav nrog cws kuj muaj cov kab mob ruaj khov [47]. Peb txoj kev tshawb fawb yav dhau los tau pom tias C-hom lectin koom nrog hauv kev tswj hwm homeostasis ntawm hemolymph microbiota los ntawm kev tswj hwm cov lus qhia ntawm AMPs [34], tab sis peb tsis paub tias txoj kev teeb liab twg koom nrog hauv kev cai ntawm AMP kev qhia. Ntawm no, peb cov txiaj ntsig tau qhia tias FOXO tau koom nrog hauv kev tswj hwm ntawm hemolymph thiab enteric microflora homeostasis los ntawm IMD signaling nyob rau hauv ib txwm muaj mob. Hauv cov cw tsis muaj kab mob, qhov kev tshem tawm ntawm Foxo ua rau txo qis kev nthuav qhia ntawm Relish, thiab cov cw ciaj sia tau txo qis, txawm tias tsis muaj kab mob. Peb kuj pom ib tug me me ntawm FOXO nyob rau hauv lub nucleus ntawm ib txwm cws hlwb. Yog li ntawd, peb xav tias FOXO koom nrog hauv kev tswj hwm homeostasis ntawm hemolymph thiab enteric flora nyob rau hauv ib txwm muaj los ntawm kev txhawb nqa Relish qhia thiab tom qab ntawd txhim kho AMP qhia los ntawm IMD txoj hauv kev. Cov txiaj ntsig no tau qhia tias FOXO koom nrog hauv zej zog thiab kev tiv thaiv kab mob los txhawb microbiota homeostasis ntawm hemolymph thiab gastrointestinal ib ntsuj av hauv cw. Kev tiv thaiv kab mob humoral plays lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob kab mob hauv cov nqi rov qab los ntawm kev tsim cov roj teeb ntawm AMPs [21,48]. Cov lus teb humoral muaj xws li inducible synthesis thiab secretion ntawm AMPs, uas yog tso rau hauv hemolymph raws li ib tug sys-temic teb, los yog tso tawm mus rau lub epidermal nto los yog kab noj hniav ntawm lub plab hnyuv ib ntsuj av tau teb [19]. AMP kev qhia feem ntau yog tswj hwm los ntawm Tus Xov Tooj / Dif thiab IMD / Relish signaling txoj hauv kev hauv cov txiv hmab txiv ntoo yoov [49]. Tam sim no, ntau tsev neeg ntawm AMPs tau txheeb xyuas hauv cw, suav nrog crustins, anti-lipopolysaccharide yam (Alfs), thiab penaeidins [50]. Txoj kev tshawb fawb tam sim no tau qhia tias FOXO tuaj yeem ncaj qha lossis ncaj qha (los ntawm IMD / Relish signaling) tswj kev qhia ntawm AMPs, suav nrog CrusI-3, Alf-B1, Alf-C1, thiab Alf-E1. Peb xav tias kev ua kom qis ntawm FOXO hauv cov hemocytes thiab cov hnyuv ntawm cw nyob rau hauv cov xwm txheej zoo tuaj yeem tswj hwm lub hom phiaj tseem ceeb ntawm Foxo, piv txwv li, Relish, kom tswj tau qhov homeostasis ntawm vivo microbiota. Thaum lub sij hawm kis kab mob, cov kab mob invading ua rau FOXO, feem ntau yog translocated rau hauv lub nucleus, uas nws tswj ntau lub hom phiaj, nrog rau qee cov AMPs, xws li Alf-E1 thiab CrusI-3, ntxiv rau Relish. Noj ua ke, FOXO koom nrog kev tswj hwm ntawm hemolymph thiab plab hnyuv microbiota homeostasis hauv cov cw noj qab haus huv los ntawm kev ua kom theem pib ntawm IMD signaling hauv hemolymph thiab gastrointestinal ib ntsuj av. Cov kab mob kis tau ua rau FOXO los ntawm kev txo qis AKT kev ua haujlwm thiab txhawb nqa kev qhia ntawm AMPs ncaj qha thiab tsis ncaj rau hauv kev tiv thaiv kab mob thiab hauv zos (hauv plab hnyuv) kev tiv thaiv. Phagocytosis yog ib qho receptor-mediated, actin thiab ATP-dependent, qhov tshwm sim uas tshwm sim los ntawm kev khi ntawm cov khoom los yog cov kab mob rau cov cytomembrane receptors tshwj xeeb, thiab sawv cev rau cov txheej txheem tiv thaiv kab mob tseem ceeb uas tus tswv tsev tuaj yeem tiv thaiv nws tus kheej los ntawm cov kab mob [24]. Cov GTPases me, xws li RAB5 thiab RAB7, tau koom nrog hauv kev hloov kho daim nyias nyias txuas ntxiv thiab vesicle khiav hauv phagocytosis. Peb txoj kev tshawb fawb yav dhau los tau tshaj tawm tias scavenger receptors SRB thiab SRC koom nrog hemocyte phagocytosis ua cov receptors hauv cw [38,39]. Hauv kev tshawb fawb tam sim no, peb pom tias tom qab lub khob ntawm Foxo, Src qhia tau txo qis, thiab hemocyte phagocytosis tus nqi txo qis, qhia tias Src yog lub hom phiaj ntawm FOXO thiab FOXO koom nrog SRC-mediated phagocytosis ntawm cov kab mob pathogenic. FOXO kuj tseem tuaj yeem txhawb nqa kev qhia ntawm ob lub GTPases me, RAB5 thiab RAB7. Cov GTPases me me no koom nrog hauv kev thauj mus los ntawm thiab yog qhov tseem ceeb ntawm kev txiav txim siab ntawm, thaum ntxov thiab lig phagosomes [51]. Phagosomes migrate los ntawm periphery ntawm lub cell mus rau thaj tsam intracellular, nrog RAB5 raug hloov los ntawm RAB7 thaum thauj, thiab cov khoom kawg yog thauj mus rau lysosome rau degradation [52,53]. Peb kuj pom tias RAB5 thiab RAB7 tuaj yeem ua ke nrog cov kab mob hauv hemocytes, uas qhia tau hais tias cov kab mob tau phagocytized los ntawm hemocytes hauv cw los ntawm kev dhau los ntawm phagosomes ntxov thiab lig ua ntej thaum kawg tau degraded hauv lysosomes. Yog li, FOXO koom nrog hauv kev tiv thaiv kab mob ntawm tes los ntawm kev txhawb nqa phagocytosis ntawm hemocytes. Hauv kev xaus, peb cov txiaj ntsig tau qhia tias FOXO tsis tsuas yog koom nrog kev tswj hwm microbiota homeostasis nyob rau hauv cov xwm txheej ib txwm muaj tab sis tseem nyob rau hauv kev tshem tawm cov kab mob pathogenic hauv cov cw kab mob los ntawm kev txhawb nqa kev qhia ntawm AMPs thiab phagocytosis ntawm hemocytes.
Fig 6. FOXO ua nws lub luag haujlwm tiv thaiv kab mob hauv cw los ntawm kev tswj cov kev qhia ntawm AMPs. (A, B) Qhov kev qhia ntawm Amps nyob rau hauv hemocytes (A) thiab txoj hnyuv (B) ntawm Foxo-knockdown cw, raws li txiav txim los ntawm qPCR siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 raws li endogenous tswj noob; dsGfp txhaj rau hauv cw yog siv los tswj. Cov ntaub ntawv tau raug txheeb xyuas los ntawm kev siv Mann-Whiteny U test. (C, D) Cov lus qhia ntawm Amps hauv hemocytes (C) thiab txoj hnyuv (D) ntawm Akt knockdown cw tau tshuaj xyuas los ntawm qPCR siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 raws li cov noob tswj endogenous; dsGfp txhaj rau hauv cw yog siv los tswj. Qhov sib txawv tseem ceeb tau txheeb xyuas siv ib txoj kev ANOVA. (E) Schematic daim duab ntawm qhov kwv yees khi qhov chaw ntawm FOXO ntawm Alf-E1 txhawb nqa. (F) Chip thiab RT-PCR tau siv los txheeb xyuas FOXO khi rau Alf-E1 cov txheej txheem txhawb nqa siv primers (Alf-E1-F thiab Alf-E1-R, Table 1). RT-PCR kuj tau siv los ua kom nthuav dav encoding cheeb tsam ntawm Alf-E1 nrog RT-PCR primers (Alf-E1-RT-F thiab Alf-E1-RT-R. Table 1) siv CIP tau DNA ua template. IgG antibody tau siv los tswj, thiab ib txwm genomic DNA amplified fragment tau siv los lees paub lub hom phiaj band.
Daim duab 7. FOXO txhim kho hemocyte phagocytosis ntawm cov kab mob hauv cw los ntawm kev txhawb nqa SRC kev qhia. (A) Hemocyte phagocytosis ntawm V. anguillarum tau soj ntsuam siv fluorescent immunocytochemical assay nyob rau hauv lub fluorescence microscope. V. anguillarum tau sau nrog FITC (ntsuab) thiab cell nuclei raug stained nrog DAPI (xiav). Cov xub liab qhia txog phagocytic hemocytes. Scale bar=20 μm. (A') Kev txheeb xyuas ntawm phagocytic tus nqi thiab phagocytic Performance index hauv Foxo-knockdown cw. Cov pab pawg txhaj tshuaj dsGfp tau siv los ua tus tswj. Tsib puas hemocytes raug suav nrog hauv fluorescence microscope hauv txhua qhov kev sim. Cov ntaub ntawv tau raug txheeb xyuas los ntawm kev siv Cov Tub Ntxhais Kawm T-test. (B) Cov kab mob phagocytosis los ntawm hemocytes txheeb xyuas siv cov cytometry ntws. Cov phagocytosed hemocytes uas muaj cov kab mob (R2) tau raug cais los ntawm cov uas tsis yog-phagocytosed hemocytes (R3) raws li cov teeb liab fluorescence ntawm cov kab mob uas sau npe rau hauv hemocytes siv IDEAS Application v6.0 software. (B') Tus nqi phagocytic ntawm hemocytes raws li cov ntaub ntawv ntws cytometry. Peb txhiab hemocytes raug suav rau txhua qhov kev tshuaj ntsuam thiab peb rov ua dua. Pawg dsGfp tau siv los ua tus tswj. (C) Hemocyte phagocytosis ntawm V. anguillarum nyob rau hauv Akt knockdown cw saib nyob rau hauv lub fluorescence microscope. Scale bar=10 μm. (C') Kev txheeb xyuas ntawm phagocytic tus nqi thiab phagocytic index raws li cov ntaub ntawv hauv vaj huam sib luag (C). (D) mRNA qhia theem ntawm Srb thiab Src hauv hemocytes los ntawm Foxo knockdown cw soj ntsuam los ntawm qPCR siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 raws li endogenous tswj noob. dsGfp txhaj tshuaj tau siv los ua tus tswj. (E) mRNA qhia ntawm Srb thiab Src nyob rau hauv cov hnyuv ntawm Foxo-RNAi cw soj ntsuam los ntawm qPCR siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 raws li endogenous tswj noob. (F) mRNA qhia ntawm Src nyob rau hauv hemocytes ntawm Akt-RNAi cw soj ntsuam los ntawm qPCR siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 raws li endogenous tswj noob. (G) Cov qib protein ntawm SRC hauv hemocytes ntawm Foxo-RNAi los yog Akt-RNAi cw, raws li kev soj ntsuam siv western blotting. dsGfp txhaj tshuaj siv los tswj. (G') Kev txheeb cais raws li cov ntaub ntawv ntawm peb lub vaj huam sib luag replicates (G).
Cov ntaub ntawv thiab cov txheej txheem
Ethics nqe lus
Txhua qhov kev sim tsiaj tau ua raws li Tuam Tshoj Cov Lus Qhia Hauv Tebchaws (GB 14922.2-2011 thiab GB 14925-2010). Cov kev sim luav rau kev npaj tshuaj tiv thaiv kab mob hauv txoj kev tshawb fawb tau ua raws li cov txheej txheem pom zoo los ntawm Tsiaj Saib Xyuas & Welfare Commit tee ntawm Shandong University School of Life Sciences (SYDWLL-2021-54). Kev pom zoo raws li cov cai ntawm kev siv cov cw hauv peb txoj kev tshawb fawb tsis yog qhov tsim nyog vim tias peb cov kev tshawb fawb tau siv qee cov cws uas nquag siv los ntawm cov zej zog thoob plaws.
Tsiaj
Cov cw noj qab nyob zoo (Marsupenaeus japonicus), hnyav li 6-10 g txhua, tau txais los ntawm cov khoom lag luam hauv dej hauv Qingdao, xeev Shandong, Suav. Ua ntej qhov kev sim, cw tau coj mus kuaj yam tsawg kawg 1 hnub nyob rau hauv lub cw kab lis kev cai system nrog aerated seawater ntawm 23-25˚C rau acclimation rau qhov chaw tshiab.
Daim duab 8. RAB5 thiab RAB7 koom nrog FOXO-mediated phagocytosis ntawm hemocytes hauv cw. (A, B) qPCR tau siv los txheeb xyuas mRNA qhia theem ntawm Rab5 thiab Rab7 hauv hemocytes ntawm Foxo-knockdown cw (A) thiab Akt-knockdown cw (B) nrog thiab tsis muaj kab mob. QPCR siv geometric txhais tau tias qhia ntawm -actin thiab Ef-1 ua cov noob tswj endogenous. (C) Cov qib protein ntawm RAB5 thiab RAB7 hauv hemocytes ntawm Foxo-knockdown cw ntawm 2 h tom qab V. anguillarum kev sib tw txiav txim siab siv western blotting. (C') Kev txheeb xyuas cov ntaub ntawv raws li cov ntaub ntawv ntawm peb lub vaj huam sib luag replicates (C). (D) Cov qib protein ntawm RAB5 thiab RAB7 hauv hemocytes ntawm Akt-knockdown cw ntawm 2 h tom qab V. anguillarum kev sib tw txiav txim siab siv western blotting. (D') Kev txheeb xyuas cov ntaub ntawv raws li cov ntaub ntawv ntawm peb lub vaj huam sib luag replicates (D).
Bioinformatic tsom xam
Tag nrho-ntev cDNA ib ntus ntawm Foxo tau txais los ntawm hemocyte thiab plab hnyuv transcrip tome sequencing ntawm M. japonicus (BGI, Shenzhen, Suav). Qhov qhib kev nyeem ntawv (ORF) ntawm Foxo tau nthuav dav siv ib khub ntawm primers (Foxo-EX-F thiab Foxo-EX-R; Table 1) rau kev pom zoo ua ntu zus. Cov cDNA sib raug zoo tau muab txhais ua lus kom tau txais cov protein txiav tawm los ntawm kev siv ExPASy-Translation tool (http://cn.expasy.org/). Kev txheeb xyuas qhov sib xws tau ua tiav siv BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi/), thiab qhov kev kwv yees sau npe tau ua tiav siv SMART (http://smart.emblheidelberg.de). Ib tsob ntoo phylogenetic ntawm FOXOs los ntawm ntau hom tau tsim los siv MEGA 6.0 pro gram [54].
Fig 9. RAB5 thiab RAB7 co-localize nrog V. anguillarum. Kev sib koom ua ke ntawm RAB5 thiab RAB7 nrog FITC sau npe-V. anguillarum nyob rau hauv cw hemocytes, raws li kev soj ntsuam siv fluorescent immunocytochemical assay. Cov kab mob tau sau nrog FITC (ntsuab) thiab txhaj rau hauv cw. Hemocytes tau sau los ntawm tsib cw ntawm 30 min thiab 1 h tom qab txhaj tshuaj kab mob thiab incubated nrog anti-RAB5 los yog anti RAB7 antibodies. Cov tshuaj tiv thaiv kab mob thib ob yog tshuaj tua kab mob IgG Alexa-546 (liab). Nuclei tau stained nrog DAPI (xiav). Co-localization hauv hemocytes yog qhia los ntawm cov xub dawb. Scale bar=10 μm.
Daim duab 10. Schematic sawv cev ntawm FOXO kev koom tes nyob rau hauv lub zos thiab systemic innate tiv thaiv nyob rau hauv cw. (A) Hauv cov xwm txheej tsis muaj kab mob, FOXO tswj hwm homeostasis ntawm hemolymph thiab plab hnyuv microbiota los ntawm kev txhawb nqa Relish thiab tom qab AMP qhia. (B) Hauv cov kab mob, AKT phosphorylation txo qis los ntawm cov kab mob pathogen, uas ua rau cov ntsiab lus FOXO nce ntxiv hauv cov nucleus. FOXO ncaj qha txhawb kev nthuav qhia ntawm Relish thiab AMPs kom tshem tawm cov kab mob nkag mus; Ntawm qhov tod tes, FOXO txhawb cov phagocytosis ntawm cov kab mob los ntawm txoj kev SRC-mediated phagocytosis.
Cov kab mob sib tw thiab cov qauv sau
Vibrio anguillarum (ATCC 43305) tau txais los ntawm Marine College, Shandong University (Wei hai, Tuam Tshoj) thiab tam sim no khaws cia hauv peb chav kuaj. V. anguillarum tau coj mus rau hmo ntuj ntawm 37˚C hauv LB nruab nrab thiab rov ua dua rau 4 teev ntawm 1:100 inoculation rau hnub ob. Cov kab mob hauv cov theem kev loj hlob ntawm logarithmic tau sau los ntawm centrifugation ntawm 5000 × g rau 5 min ntawm 4˚C thiab ntxuav ob zaug nrog phosphate-buffered saline (PBS: 10 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, thiab 1.8 mM KH2PO4. pH 7.4). V. anguillarum resuspended hauv phosphate buffered saline rau kev sib tw cw. Kwv yees li 2 × 107 colony forming units (CFU) / cw tau txhaj rau hauv plab ntawm txhua tus cw siv lub microsyringe. Pawg tswj hwm tau txhaj nrog tib lub ntim ntawm PBS. Cov hemocytes, lub plawv, hepatopancreas, gill, plab, thiab cov hnyuv tau sau los ntawm tsawg kawg peb cws rau RNA thiab protein ntau ntxiv. Rau hemocyte sau, hemolymph tau muab rho tawm los ntawm lub qhov ntswg qhov ntswg siv lub koob txhaj tshuaj preloaded nrog 0.8 ml ntawm anticoagulant tsis (10% sodium citrate), thiab ces sai centrifuged ntawm 700 × g rau 8 min ntawm 4˚C rau hemocyte cais.
RNA thiab protein extraction, thiab cDNA synthesis
Tag Nrho RNA tau muab rho tawm los ntawm cov hemocytes thiab cov kabmob sib txawv ntawm cov cw siv TRIzol (ET101, Transgen, Beijing, Suav). Cov proteins los ntawm cov kab mob sib txawv tau muab rho tawm siv Radioimmunoprecipitation assay (RIPA) Lysis Buffer (P0013B, Beyotime, Jiangsu, Suav). Thawj strand cDNA synthesis tau ua los ntawm cDNA Synthesis Kit (5x All-in-One RT MasterMix; Applied Biological Materials-abm, Vancouver, Canada), ua raws li manufac turer cov lus qhia.
Table 1. Cov txheej txheem ntawm primers siv nyob rau hauv txoj kev tshawb no.
MasterMix; Applied Biological Materials-abm, Vancouver, Canada), ua raws li manufac turer cov lus qhia.
Recombinant qhia thiab kev npaj tshuaj tiv thaiv kab mob
Cov cDNA ib ntus ntawm Foxo tau nthuav dav los ntawm kev rov qab hloov pauv cov tshuaj tiv thaiv polymerase saw (RT-PCR) siv cov primers Foxo-EX-F thiab Foxo-EX-R (Table 1). Cov khoom siv PCR huv si tom qab ntawd tau zom nrog kev txwv enzymes thiab ligated rau hauv plasmid pGEX-4T-1 (GE Healthcare, Piscataway, NJ, USA). thiab cov plasmids tsim tau hloov mus rau hauv Escher ichia coli Rosseta (DE3) hlwb. Protein qhia tau raug ntxias siv 1 mM isopropyl - - D-thio galactopyranoside ntawm 37˚C. Cov recombinant proteins tau raug affinity chromatography siv glutathione (GST) resin (C600031, BBI, Shanghai, Suav teb), ua raws li cov chaw tsim tshuaj paus cov lus qhia. Luav antisera tiv thaiv FOXO tau npaj raws li txoj kev piav qhia yav dhau los [55].
Western blotting
Cov proteins uas muab rho tawm los ntawm cov kab mob sib txawv los yog cov hemocytes tau muab cais siv 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Cov proteins nyob rau hauv SDS-PAGE gels tau pauv mus rau cellulose nitrate membrane siv hloov tsis tau (25 mM Tris-HCl, 20 mM glycine, 0.037% mM SDS, thiab 20% ethanol) thiab incubated nyob rau hauv 5% skim mis nyuj los yog 3% bovine serum albumin (BSA) nyob rau hauv Tris-buffered saline (TBS: 150 mM NaCl, 10 mM Tris HCl, pH 8.0) rau 1 h nrog maj mam co ntawm chav tsev kub. Cov membranes ces incubated nrog thawj antiserum: Anti-FOXO ntawm ib tug 1:100 dilution, anti-RAB5 ntawm 1:25, anti Rab7 ntawm 1:25, anti-beta actin (ACTB) ntawm 1:250 (npaj nyob rau hauv peb kuaj. ); Histone-3 poly clonal antibodies (A2348, ABclonal, Wuhan, Tuam Tshoj, 1:2500); anti-phosphorylated (p)-AKT polyclonal antibodies (WLP001a, Wanleibio, Shenyang, Tuam Tshoj, 1: 500) thiab co maj mam thaum hmo ntuj ntawm 4˚C. Tom qab ntxuav peb zaug nrog TBST (0.1% Tween-20 ntxiv rau TBS), cov mem branes tau incubated nrog lwm yam tshuaj tiv thaiv kab mob (ZB2308 ZSGB-Bio, Beijing, Tuam Tshoj, 1:5,000) los yog ( ZB2301 ZSGB-Bio, Beijing, Tuam Tshoj, 1: 5, 000) rau 3 teev ntawm chav tsev kub nrog maj mam shake ing. Cov tshuaj tiv thaiv kab mob tiv thaiv kab mob tau tsim los siv cov tshuaj nitrotetrazolium xiav chloride (A610379, BBI) thiab P-toluidine ntsev (A610072, BBI) kev daws teeb meem nyob rau hauv qhov tsaus ntuj lossis siv cov tshuaj kho kom zoo nkauj (ECL). -actin lossis Histone-3 tau siv sivs.
Cov ntaub so ntswg faib thiab nthuav qhia cov qauv hauv cov cw sib tw los ntawm cov kab mob
Cov ntaub so ntswg faib ntawm Foxo / FOXO nyob rau hauv hemocytes, lub plawv, gill, hnoos qeev, plab, thiab hnyuv tau soj ntsuam siv RT-PCR nrog primers Foxo-F thiab Foxo-R (Table 1) thiab los ntawm sab hnub poob blotting nrog anti-FOXO cov tshuaj tiv thaiv, raws. Cov txheej txheem PCR muaj qhov pib incubation ntawm 94˚C rau 3 min; ua raws li 35 cycles ntawm 94˚C rau 30 s, 50˚C rau 30 s, thiab 72˚C rau 30 s; ua raws li 72˚C rau 10 feeb. Cov khoom PCR tau txheeb xyuas siv agarose gel electrophoresis (1.5% agarose). Cov -actin gene (-actin-F thiab -actin-R; Table 1) tau siv los ua kev tswj hwm sab hauv. Lub sij hawm qhia cov qauv ntawm Foxo / FOXO ntawm RNA thiab qib protein nyob hauv cov cw sib tw nrog V. anguillarum tau txheeb xyuas siv ntau npaum li cas ntawm PCR (qPCR) hauv lub thermal cycler (qTOWER3, ANALYTIK JENA AG, Jena, Germany) thiab los ntawm sab hnub poob ern blotting, ntsig txog. Cov txheej txheem qPCR suav nrog: 95˚C rau 10 min; 40 cycles ntawm 95˚C rau 10 s thiab 60˚C rau 50 s; thiab tom qab ntawd lub sijhawm melting ntawm 65˚C txog 95˚C. Cov lus qhia pro cov ntaub ntawv ntawm Foxo tau tsim los siv 2-ΔΔCT txoj kev [56] siv lub geometric txhais tau tias ntawm ob lub hauv paus tswj cov noob ntawm -actin thiab Ef-1 rau normalization. Qhov kev ua tau zoo ntawm primer khub hauv qPCR tau txheeb xyuas raws li MIQE txoj kev [57] nrog rau 10-fold logarithmic dilution ntawm cDNA sib tov los tsim cov qauv kab nkhaus.
Kev cais tawm ntawm cov protein thiab cytoplasmic
Cov ntaub so ntswg tau muab tshem tawm los ntawm cw thiab ntxuav hauv PBS peb zaug. Protein extrac tion tau ua los ntawm kev siv cov khoom siv nuclear thiab cytoplasmic protein extraction (R0050, Solarbio, Beijing, Tuam Tshoj) ua raws li cov chaw tsim khoom cov lus qhia. Yam tsawg kawg peb cws tau siv rau kev tshem tawm cov protein kom tshem tawm qhov sib txawv ntawm tus kheej.
RNA cuam tshuam kev soj ntsuam
Txhawm rau txheeb xyuas cov noob muaj nuj nqi, peb tau ua RNA cuam tshuam (RNAi) kev soj ntsuam. Ob peb khub ntawm cov primers uas muaj T7 cov txheej txheem txhawb nqa (Foxo-RI-F thiab Foxo-RI-R; Rab5-RI-F thiab Rab5 -}RI-R; Rab7-RI- F thiab Rab7 -RI-R; Akt-RI-F thiab Akt-RI-R. Table 1) tau tsim los nthuav cov qauv rau dsRNA synthesis. Siv T7 RNA polymerase (EP0111, Thermo Fisher Scientific, Waltham, MA, USA) thiab cDNA template, peb muab cov dsRNA fragment. Ob-strand Green fluorescent protein dsGfp RNA tau txais kev tswj hwm, uas tau nthuav dav siv Gfp-RI-F thiab Gfp -RI-R (Table 1) ua primers rau dsGfp synthesis. Tom qab ntawd, cov dsRNA fragments (50 ug / txhua) tau txhaj rau hauv lub plab ntu ntawm M. japonicus siv microsyringe thiab txhaj tshuaj thib ob tau ua 12 teev tom qab siv tib koob tshuaj. RNA cuam tshuam kev ua haujlwm tau raug tshuaj xyuas los ntawm kev siv ntau lub sijhawm rov qab hloov pauv PCR (qRT-PCR, suav nrog rov qab hloov pauv ntawm RNA los ua cDNA, uas yog tom qab ntawd siv los ua tus qauv hauv qPCR tshuaj ntsuam) lossis sab hnub poob blotting 24 h tom qab txhaj tshuaj thib ob. -actin tau siv los ua kev siv sab hauv.
Cov ntaub ntawv
1. Calnan DR, Brunet A. The FoxO code. Oncogene. 2008; 27(16):2276–2288. https://doi.org/10.1038/ onc.2008.21 PMID: 18391970 ib.
2. Tran H, Brunet A, Grenier JM, Datta SR, Fornace AJ, DiStefano PS, et al. DNA kho txoj kev txhawb nqa los ntawm lub forkhead transcription factor FOXO3a los ntawm Gadd45 protein. Kev tshawb fawb. 2002; 296 (5567): 530–534. https://doi.org/10.1126/science.1068712 PMID: 11964479 ib.
3. Martins R, Lithgow GJ, Link W. Long live FOXO: unraveling lub luag hauj lwm ntawm FOXO proteins nyob rau hauv kev laus thiab ntev gevity. Kev laus Cell. 2016; 15(2):196–207 : kuv. https://doi.org/10.1111/acel.12427 PMID: 26643314 ib.
4. Vogt PK, Jiang H, Aoki M. Triple txheej tswj-phosphorylation, acetylation thiab ubiquitination ntawm FOXO proteins. Cell Cycle. 2005; 4(7):908–913. https://doi.org/10.4161/cc.4.7.1796 PMID: 15917664. 5. Daitoku H, Sakamaki J, Fukamizu A. Regulation of FoxO transcription factors by acetylation and pro tein-protein interactions. Bba-Mol Cell Res. 2011; 1813 (11): 1954–1960. https://doi.org/10.1016/j. bbamcr.2011.03.001 PMID: 21396404.
6. Wang Y, Zhou YM, Graves DT. FOXO transcription yam tseem ceeb: lawv qhov tseem ceeb thiab kev tswj hwm. Biomed Res Int. 2014; 2014:1–13. https://doi.org/10.1155/2014/925350 PMID: 24864265 ib.
7. Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, et al. Cov noob uas ua raws cov dej ntws ntawm DAF-16 los cuam tshuam lub neej ntawm Caenorhabditis elegans. Xwm. 2003; 424(6946):277– 284. https://doi.org/10.1038/nature01789 PMID: 12845331. 8. Sergi C, Shen F, Liu SM. Insulin/IGF-1R, SIRT1, thiab FOXOs pathways-ib qho kev sib cuam tshuam plat daim ntawv rau pob txha thiab osteosarcoma. Pem hauv ntej Endocrinol (Lausanne). 2019; 10:93–103. https://doi.org/10. 3389/fendo.2019.00093 PMID: 30881341 PAB.
9. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al. Akt txhawb nqa txoj sia nyob ntawm tes los ntawm phosphorylat ing thiab inhibiting lub forkhead transcription factor. Cell. Xyoo 1999; 96(6):857–{5}} ib. https://doi.org/10.1016/ s0092-8674(00)80595-4 PMID: 10102273.
10. Huang H, Regan KM, Wang F, Wang DP, Smith DI, van Deursen JMA, et al. Skp2 inhibits FOX01 hauv cov qog nqaij hlav los ntawm ubiquitin-mediated degradation. P Natl Acad Sci USA. 2005; 102(5):1649–1654. https://doi.org/10.1073/pnas.0406789102 PMID: 15668399.
11. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, et al. Lub Fork head transcription factor DAF-16 transduces insulin-zoo li metabolic thiab lub neej ntev teeb liab hauv C. elegans. Xwm. Xyoo 1997; 389 (6654): 994–999. https://doi.org/10.1038/40194 PMID: 9353126 ib.
12. Fink C, Hoffmann J, Knop M, Li Y, Isermann K, Roeder T. plab hnyuv FoxO signaling yuav tsum muaj sia nyob rau qhov ncauj kab mob hauv Drosophila. Mucosal Immunol. 2016; 9(4):927–936. https://doi.org/10.1038/mi. PIB: 26627459, 2015.
13. Spellberg MJ, Marr MT. FOXO tswj RNA cuam tshuam hauv Drosophila thiab tiv thaiv tus kab mob RNA. P Natl Acad Sci USA. 2015; 112 (47): 14587–14592. https://doi.org/10.1073/pnas. 1517124112 PIB: 26553999.
14. Guo LL, Karpac J, Tran SL, Jasper H. PGRP-SC2 txhawb nqa lub plab Immune homeostasis kom txwv tsis pub commen sal dysbiosis thiab ncua lub neej. Cell. 2014; 156(1–2): 109–122. https://doi.org/10.1016/j.cell.2013.12. 018 PIB: 24439372.
15. Becker T, Loch G, Beyer M, Zinke I, Aschenbrenner AC, Carrera P, thiab al. FOXO-dependent kev cai ntawm innate immune homeostasis. Xwm. 2010; 463 (7279): 369–373. https://doi.org/10.1038/ nature08698 PMID: 20090753.
16. Seiler F, Hellberg J, Lepper PM, Kamyschnikow A, Herr C, Bischoff M, et al. FOXO transcription yam tseem ceeb tswj lub cev tiv thaiv kab mob hauv lub cev ua pa epithelial. J Immunol. 2013; 190(4):1603–1613. https://doi.org/10.4049/jimmunol.1200596 PMID: 23315071.
17. Graves DT, Milovanova TN. Mucosal tiv thaiv kab mob thiab FOXO1 transcription yam. Pem hauv ntej Immunol. 2019; 10:2530–2541 IB. https://doi.org/10.3389/fimmu.2019.02530 PMID: 31849924 ib.
18. Bulet P, Stocklin R, Menin L. Anti-microbial peptides: los ntawm invertebrates mus rau vertebrates. Immunol Rev. 2004; 198:169–184. https://doi.org/10.1111/j.0105-2896.2004.0124.x PMID: 15199962.
19. Buchon N, Silverman N, Cherry S. Kev tiv thaiv kab mob hauv Drosophila melanogaster—los ntawm kev paub txog microbial mus rau tag nrho cov kab mob physiology. Nat Rev Immunol. 2014; 14(12): 796–810. https://doi.org/10.1038/nri3763 PMID: 25421701 ib.
20. Hoffmann JA, Reichhart JM. Drosophila innate immunity: ib qho kev xav ntawm evolutionary. Nat Immunol. 2002; 3(2):121–126. https://doi.org/10.1038/ni0202-121 PMID: 11812988.
21. Hultmark D. Drosophila tiv thaiv kab mob: txoj hauv kev thiab cov qauv. Curr Opin Immunol. 2003; 15(1):12–19 : kuv. https://doi.org/10.1016/s0952-7915(02)00005-5 PMID: 12495727.
22. Li FH, Xiang JH. Tsis ntev los no kev nce qib hauv kev tshawb fawb txog kev tiv thaiv kab mob hauv lub cev ntawm cw hauv Suav teb. Dev Comp Immunol. 2013; 39(1–2:11–26). https://doi.org/10.1016/j.dci.2012.03.016 PMID: 22484214 ib.
23. Sun JJ, Lan JF, Zhao XF, Vasta GR, Wang JX. Kev khi ntawm C-type lectin's coiled-coil domain rau Domeless receptor ncaj qha ua rau JAK / STAT txoj hauv kev hauv lub cev tiv thaiv kab mob rau cov kab mob bacte rial. PLoS Pathog. 2017; 13(9):e1006626. https://doi.org/10.1371/journal.ppat.1006626 PMID: 28931061 ib.
24. Gordon S. Phagocytosis: ib qho txheej txheem immunobiologic. Kev tiv thaiv. 2016; 44(3):463–475. https://doi.org/ 10.1016/j.immuni.2016.02.026 PMID: 26982354 ib.
25. Wang S, Xia PY, Huang GL, Zhu PP, Liu J, Ye BQ, et al. FoxO1-kev kho mob autophagy yog xav tau rau NK cell kev loj hlob thiab kev tiv thaiv hauv lub cev. Nat Commun. 2016; 7: 11023–11037. https://doi.org/10.1038/ ncomms11023 PMID: 27010363.
26. Dong G, Song L, Tian C, Wang Y, Miao F, Zheng J, et al. FOXO1 tswj cov kab mob-induced neutrophil kev ua haujlwm. Pem hauv ntej Immunol. 2017; 8: 1088–1102. https://doi.org/10.3389/fimmu.2017.01088 PMID: 28928749 ib.
27. Wang XW, Zhao XF, Wang JX. C-hom lectin khi rau beta-Integrin los txhawb hemocytic phagocytosis nyob rau hauv ib qho invertebrate. J Biol Chem. 2014; 289(4): 2405–2414 : kuv. https://doi.org/10.1074/jbc.M113.528885 PMID: 24324258 ib.
28. Norouzitallab P, Baruah K, Vanrompay D, Bossier P. Qhia cws tus kheej tiv thaiv kab mob. Trends Biotechnol. 2019; 37(1):16–19 : kuv. https://doi.org/10.1016/j.tibtech.2018.05.007 PMID: 29914649.
29. Flegel TW. Keeb kwm tshwm sim, cuam tshuam thiab tam sim no cov xwm txheej ntawm cws kab mob hauv Asia. J Invertebr Pathol. 2012; 110(2): 166–173. https://doi.org/10.1016/j.jip.2012.03.004 PMID: 22429834 ib.
30. Wang PH, Huang TZ, Zhang XB, He JG. Antiviral tiv thaiv hauv cw: Los ntawm lub cev tiv thaiv kab mob mus rau kab mob intionfec. Antivir Res. 2014; 108:129–141. https://doi.org/10.1016/j.antiviral.2014.05.013 PMID: 24886688.
31. Obsil T, Obsilova V. Structure/function relationships underlying regulation of FOXO transcription fac tors. Oncogene. 2008; 27(16):2263–2275. https://doi.org/10.1038/onc.2008.20 PMID: 18391969.
