Giardia duodenum yog kab mob parasitic uas ua rau giardiasis, kab mob hauv plab tshwj xeeb tshaj yog nyob rau hauv cov menyuam yaus uas muaj cov tsos mob raws plab.Peb tau tshaj tawm yav dhau los tias extracellular G. duodenalis ua rau kev ua haujlwm ntawm intracellular oligomerization-zoo li receptor 3 (NLRP3) khi nucleotides thiab tswj hwm tus tswv tsev inflammatory teb los ntawm extracellular vesicle (EV) secretion.Txawm li cas los xij, cov qauv molecular tseeb ntawm cov kab mob sib txuas nrog duodenococcal EV (GEV) koom nrog hauv cov txheej txheem no thiab lub luag haujlwm ntawm NLRP3 inflammasome hauv giardiasis tseem yuav tsum tau piav qhia.
Recombinant eukaryotic qhia plasmids pcDNA3.1(+)-alpha-2 thiab alpha-7.3 giardins hauv GEV tau tsim, hloov mus rau nas thawj peritoneal macrophages, thiab kuaj pom los ntawm kev ntsuas qhov mob lub hom phiaj molecule caspase-1.Qib p20 qhia tau raug tshuaj xyuas..G. duodenalis alpha-2 thiab alpha-7.3 giardines yog thawj zaug txheeb xyuas los ntawm kev ntsuas NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 thiab caspase-1 p20), IL secretion.1β theem, apoptotic spotted protein (ASC) oligomerization theem, thiab immunofluorescent localization ntawm NLRP3 thiab ASC.Lub luag hauj lwm ntawm NLRP3 inflammasome nyob rau hauv pathogenicity ntawm G. duodenalis tau soj ntsuam siv nas nyob rau hauv uas NLRP3 activation raug thaiv (NLRP3 blocked nas) thiab pathological hloov nyob rau hauv lub cev hnyav, duodenal parasitic load, thiab duodenal ntaub so ntswg.Tsis tas li ntawd, peb tau tshawb xyuas seb hiardines alpha-2 thiab alpha-7.3 induce IL-1β secretion hauv vivo ntawm NLRP3 inflammasome thiab txiav txim siab lub luag haujlwm ntawm cov molecules hauv cov kab mob ntawm G. duodenalis hauv cov nas.
Alpha-2 thiab alpha-7.3 giardines induce lub activation ntawm NLRP3 inflammasome hauv vitro.Qhov no tau ua rau kev ua kom muaj p20 caspase-1, nce qib ntawm NLRP3, pro-IL-1β, thiab pro-caspase-1 cov proteins, qhov tseem ceeb nce hauv IL-1β secretion, tsim ntawm ASA me ntsis hauv cytoplasm, thiab induction ntawm ASA oligomerization.NLRP3 o Penile poob exacerbates lub pathogenicity ntawm G. duodenalis nyob rau hauv nas.Cov nas kho nrog cov hlwv los ntawm gavage los ntawm NLRP3-thaiv cov nas pom muaj ntau tus trophozoites thiab kev puas tsuaj loj rau lub duodenal villi, tus cwj pwm los ntawm necrotic crypts nrog shrunken thiab branching.Hauv vivo kev sim tau pom tias giardines alpha-2 thiab alpha-7.3 tuaj yeem ua rau muaj kev zais ntawm IL-1β ntawm NLRP3 inflammasome, thiab kev txhaj tshuaj nrog giardines alpha-2 thiab alpha-7.3 txo cov kab mob ntawm G. duodenalis hauv cov nas.
Ua ke, cov txiaj ntsig ntawm txoj kev tshawb no qhia tias giardia alpha-2 thiab alpha-7.3 ua rau muaj kev cuam tshuam ntawm tus tswv tsev NLRP3 o thiab txo cov kab mob G. duodenalis hauv cov nas, uas yog lub hom phiaj zoo rau kev tiv thaiv giardiasis.
Giardia duodenum yog ib qho kab mob ntxiv ntawm cov kab mob protozoan uas nyob hauv txoj hnyuv me thiab ua rau 280 lab tus neeg mob giardiasis nrog raws plab ib xyoos ib zaug, tshwj xeeb tshaj yog cov menyuam yaus hauv cov tebchaws tsim [1].Cov neeg kis tau los ntawm cov dej haus los yog cov khoom noj uas muaj kab mob M. duodenum cysts, uas tom qab ntawd nkag mus rau hauv plab thiab tawm hauv cov kua txiv hmab txiv ntoo.Giardia duodenum trophozoites txuas rau duodenal epithelium, ua rau xeev siab, ntuav, raws plab, mob plab, thiab poob phaus.Cov tib neeg uas muaj kev tiv thaiv kab mob thiab cystic fibrosis yog qhov kis tau tus kab mob.Kev kis kab mob tuaj yeem tshwm sim los ntawm qhov ncauj thiab qhov quav [2].Cov tshuaj xws li metronidazole, tinidazole, thiab nitazoxanide yog cov kev xaiv kho mob zoo tshaj plaws rau cov kab mob duodenal [3].Txawm li cas los xij, cov tshuaj chemotherapy no ua rau muaj kev phiv tsis zoo xws li xeev siab, carcinogenesis, thiab genotoxicity [4].Yog li ntawd, yuav tsum tau tsim cov tswv yim zoo dua los tiv thaiv G. duodenalis kab mob.
Inflammasomes yog ib chav kawm ntawm cytosolic protein complexes uas yog ib feem ntawm lub cev tiv thaiv kab mob hauv lub cev, pab tiv thaiv kab mob cuam tshuam thiab kho cov lus teb inflammatory [5].Ntawm cov inflammasomes no, nucleotide-binding oligomerization (NOD) receptor 3 (NLRP3) nucleotide-binding oligomerization (NLRP3) nucleotide-binding-like inflammasome tau kawm ntau heev vim tias nws tuaj yeem kuaj tau los ntawm ntau yam kab mob / kev puas tsuaj cuam tshuam nrog molecular qauv (PAMP / PAMP). DAMP), lees paub, ua kom lub cev tiv thaiv kab mob hauv lub cev.thiab tswj txoj hnyuv homeostasis hauv ntau cov kab mob inflammatory [6,7,8].Nws muaj cov qauv lees paub receptor (PRR) NLRP3, ib qho adapter apoptotic spotted protein (ASC), thiab ib qho effector procaspase-1 lossis procaspase-11.NLRP3 inflammasome ua tus tswv cuab tiv thaiv kab mob cuam tshuam, raws li pom hauv Neospora caninum [9], Paracoccidioides brasiliensis [10], thiab Leishmania kev tshawb fawb.[11], tab sis nws kuj tau tshaj tawm tias kev ua haujlwm ntawm NLRP3 inflammasome txwv kev tiv thaiv lub cev tiv thaiv kab mob thiab ua rau muaj kab mob hnyav dua, piv txwv li, hauv kab mob [12].Raws li peb cov kev tshawb pom yav dhau los, peb tau tshaj tawm tias extracellular G. duodenalis ua rau intracellular activation ntawm NLRP3 o thiab modulates host inflammatory teb los ntawm secreting extracellular vesicles (EVs) [13].Txawm li cas los xij, lub luag haujlwm ntawm NLRP3 inflammasome hauv G. duodenalis kab mob hauv vivo tseem yuav raug txiav txim siab.
Giardins yog thawj zaug piav raws li cov qauv ntawm G. duodenalis cytoskeleton thiab ua lub luag haujlwm tseem ceeb hauv trophozoite motility thiab epithelial cell txuas hauv txoj hnyuv me.Txhawm rau hloov mus rau ib puag ncig zoo dua thiab nce lawv cov kab mob, G. duodenalis trophozoites tsim cov qauv cytoskeletal tshwj xeeb uas muaj 8 flagella, 1 nruab nrab lub cev, thiab 1 ventral disc [14].Cov trophozoites ntawm Giardia duodenum siv lawv cov cytoskeleton nkag mus rau hauv cov hnyuv loj, tshwj xeeb tshaj yog cov duodenum, thiab txuas rau enterocytes.Lawv tas li migrate thiab txuas mus rau cov hlwb epithelial siv cell metabolism.Yog li ntawd, muaj kev sib raug zoo ntawm lawv cov cytoskeleton thiab virulence.Giardines tshwj xeeb rau Giardia duodenum yog cov khoom ntawm cov qauv cytoskeleton [15] thiab muab faib ua plaub chav kawm: α-, β-, γ-, thiab δ-giardines.Muaj 21 tus tswv cuab ntawm tsev neeg α-giardin, tag nrho cov uas muaj calcium-dependent peev xwm los khi phospholipids [16].Lawv kuj txuas lub cytoskeleton mus rau lub cell membrane.Hauv cov tib neeg uas muaj mob raws plab los ntawm G. duodenalis, α-giardins tau hais tawm thiab tiv thaiv kab mob thaum kis kab mob [17].Cov tshuaj tiv thaiv kab mob heterologous raws li Giardia alfa-1 tiv thaiv giardiasis hauv nas thiab yog cov muaj peev xwm sib tw antigens rau kev tsim tshuaj tiv thaiv [18].Alpha-8 giardin, localized nyob rau hauv plasma membrane thiab flagella, tab sis tsis nyob rau hauv lub ventral disc, txhim khu lub motility thiab loj hlob tus nqi ntawm trophozoites nyob rau hauv G. duodenalis [19].Alpha-14 giardin txuas rau microtubule cov qauv ntawm flagella thiab cuam tshuam rau kev muaj peev xwm ntawm G. duodenalis [20].Alpha-11 giardine muaj nyob rau hauv ntau thoob plaws lub neej voj voog, thiab overexpression ntawm alpha-11 giardine puas G. duodenalis nws tus kheej [21].Txawm li cas los xij, nws tsis paub meej tias alpha-2 giardine thiab alpha-7.3 giardine tiv thaiv kab mob G. duodenalis thiab lawv cov txheej txheem hauv qab no.
Nyob rau hauv txoj kev tshawb no, recombinant eukaryotic qhia plasmids pcDNA3.1(+)-alpha-2 giardine thiab pcDNA3.1(+)-alpha-7.3 giardine tau kis mus rau hauv nas thawj peritoneal macrophages los qhib party NLRP3.Tom qab ntawd cov hom phiaj mob mob tau raug tshuaj xyuas.Peb kuj tau soj ntsuam lub luag haujlwm ntawm NLRP3 inflammasome hauv cov kab mob ntawm G. duodenalis, tshawb xyuas seb alpha-2 thiab alpha-7,3 giardines induce activation ntawm NLRP3 inflammasome nyob rau hauv vivo, thiab txiav txim siab tias ob lub luag hauj lwm ntawm giardines nyob rau hauv lub pathogenicity ntawm. G. duodenalis.Peb lub hom phiaj tseem ceeb yog los tsim cov phiaj xwm zoo rau kev tiv thaiv kab mob G. duodenalis.
Cov tsiaj qus (WT) C57BL / 6 poj niam nas hnub nyoog 5-8 lub lis piam tau yuav los ntawm Liaoning Changsheng Experimental Animal Center (Liaoning, Suav).Cov nas tau nkag mus rau dej dawb, tau txais cov zaub mov tsis huv thiab khaws cia rau hauv 12/12 teev lub teeb / tsaus ntuj.Ua ntej kis kab mob, nas tau txais cov tshuaj tua kab mob ad libitum hauv cov dej haus ntxiv nrog ampicillin (1 mg / mL), vancomycin (1 mg / mL), thiab neomycin (1.4 mg / mL) (txhua yam yuav los ntawm Shanghai, Tuam Tshoj, cov khoom siv dag zog) [22 ].].Cov nas uas poob tsis muaj peev xwm noj thiab haus rau > 24 teev thiab poob ≥ 20% lub cev qhov hnyav tau humanely euthanized los ntawm lub ncauj tsev menyuam dislocation.
WB G. duodenalis trophozoites (American Type Culture Collection, Manassas, USA) tau supplemented nrog 12.5% ​​​​fetal bovine serum (FBS; Txhua Green, Zhejiang, Tuam Tshoj) thiab 0.1% bovine bile (Sigma-Aldrich, St. Missouri, USA ).USA) nyob rau hauv microaerobic tej yam kev mob.Confluent trophozoites tau sau rau ntawm cov dej khov thiab dhau ntawm qhov sib piv ntawm 1: 4 rau kev tsim tawm ntxiv.
Giardia duodenum cysts raug ntxias raws li tau piav qhia yav dhau los [23], trophozoites tau sau nyob rau hauv logarithmic theem thiab tom qab ntawd diluted nrog encapsulation inducing nruab nrab, pH 7.1 (hloov TYI-S-33) mus rau qhov kawg concentration ntawm 1 × 106 trophozoites / mL.kua tsib concentration 0.05% nruab nrab).Trophozoites tau coj mus rau hauv cov xwm txheej anaerobic ntawm 37 ° C mus txog rau theem kev loj hlob ntawm logarithmic.Hloov qhov nruab nrab mus rau cyst inducing nruab nrab (pH 7.8; hloov kho TYI-S-33 nruab nrab nrog 1% kua tsib concentration) thiab kab lis kev cai G. duodenalis ntawm 37 ° C rau 48-96 teev, thaum lub sij hawm tsim cov hlwv tau pom nyob rau hauv lub tshuab tsom.Tom qab feem ntau ntawm cov trophozoites tau raug ntxias los tsim cov hlwv, cov kab lis kev cai sib xyaw tau raug sau thiab rov muab tso rau hauv cov dej tsis huv deionized kom lyse cov trophozoites ntxiv.Cysts raug suav thiab khaws cia ntawm 4 ° C rau kev soj ntsuam tom qab los ntawm lub plab hnyuv hauv cov nas.
Giardia extracellular vesicles (GEVs) tau enriched raws li tau piav yav tas los [13].Trophozoites nyob rau hauv logarithmic kev loj hlob theem tau resuspended nyob rau hauv hloov kho TYI-S-33 nruab nrab npaj nrog exosome-depleted FBS (Biological Industries, Beit-Haemek, Ixayees) mus rau qhov kawg concentration ntawm 1 × 106 parasites / mL thiab incubated rau 12 teev.tau cais tawm ntawm kab lis kev cai supernatant los ntawm centrifugation ntawm 2000 g rau 10 min, 10,000 g rau 45 min, thiab 100,000 g rau 60 min.Precipitates tau yaj hauv phosphate buffered saline (PBS), suav nrog siv BCA protein kuaj cov khoom (Thermo Fisher Scientific, Waltham, MA, USA) thiab khaws cia ntawm -80 ° C. lossis siv ncaj qha rau kev tshuaj xyuas ntxiv.
Thawj nas ntawm qhov peritoneal macrophages tau npaj raws li tau piav ua ntej [24].Luv luv, nas (hnub nyoog 6-8 lub lis piam) tau txhaj tshuaj (intraperitoneally [ip]) nrog 2.5 ml ntawm 2.98% Difco kua thioglycol nruab nrab (BD, Franklin Lakes, NJ, USA) thiab noj 3-4 palates.Kev ncua ntawm macrophages tau sau los ntawm lub plab kab noj hniav ntawm nas tom qab euthanasia thiab centrifuged 3 zaug ntawm 1000 g rau 10 min.Cov noob qoob loo tau kuaj pom los ntawm kev ntws cytometry siv CD11b cim kom txog thaum cell purity yog> 98%, ces ntxiv rau 6-zoo cell kab lis kev cai daim hlau (4.5 x 106 hlwb / zoo) thiab incubated nrog 10% FBS (Bioindustry) ntawm 37 ° C.thiab 5% CO2.
RNA tau muab rho tawm los ntawm 1 × 107 trophozoites nyob rau hauv 1 ml ntawm TRIzol reagent (Vazyme, Nanjing, Tuam Tshoj), genomic DNA tau muab rho tawm los ntawm tag nrho G. duodenalis RNA siv MonScript dsDNase (Monad, Wuhan, Tuam Tshoj) thiab complementary DNA (cDNA) tau synthesized. siv MonScript RTIIII Super Mix (Monad) raws li cov chaw tsim khoom cov lus qhia.
CDS ib ntus cov ntaub ntawv rau lub hom phiaj G. duodenalis noob tau txais los ntawm NCBI GenBank.Siv Primer 5.0 los tsim tshwj xeeb seamless cloning primers rau txhua hom phiaj.Lub pem hauv ntej primer (5′-3′) muaj peb seem: ib tug sib tshooj sib tshooj nrog ib tug linearized vector pcDNA3.1(+) EcoRV (TGGTGGAATTCTGCAGAT) thiab pib codons ATG thiab GNN (yog tias thawj lub hauv paus tsis yog G).Qhov no yog ua los txhim kho kev ua tau zoo ntawm cov lus qhia.Tsis tas li ntawd, tsawg kawg yog 16 bp ua ke hauv paus (GC cov ntsiab lus 40–60% / Tm kwv yees li 55 ° C).Qhov rov qab primer (5′-3′) muaj ob ntu, sib tshooj sib tshooj nrog EcoRV-linearized vector pcDNA3.1(+) (GCCGCCACTGTGCTGGAT) thiab lub hauv paus ua ke ntawm tsawg kawg 16 bp.(tsis suav nrog ob qhov chaw nres tsheb kawg).bases) ib codon xws li AA los yog GA kom tso cai rau cov plasmids recombinant qhia lawv cov ntawv sau npe proteins).Cov primer sequences tau teev nyob rau hauv Table 1 thiab tau synthesized los ntawm Kangmet Biotechnology Co., Ltd. (Changchun, Tuam Tshoj).
Cov hom phiaj tau nthuav dav siv Pfu DNA polymerase (Tiangen, Beijing, Suav teb) lossis Ex-taq (Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, Tuam Tshoj) siv cov khoom npaj G. duodenalis cDNA ua tus qauv.Lub eukaryotic qhia vector plasmid pcDNA3.1(+) yog linearized nrog txwv enzyme EcoRV thiab dephosphorylated siv Fast AP (Thermo Fisher Scientific).Linearized pcDNA3.1(+) fragments thiab amplified lub hom phiaj gene fragments tau purified siv DNA gel purification cov khoom (Tiangen) thiab quantified siv ib tug Nanodrop ND-2000 (Thermo Fisher Scientific).Cov pcDNA3.1(+) fragment thiab txhua lub hom phiaj gene fragment tau recombined siv MonClone ib tug sib dhos cloning mix (Monad Biotech Co., Ltd., Suzhou, Suav teb) thiab tau lees paub los ntawm DNA sequencing siv Comate Bioscience Company Limited (Changchun, Suav teb)..
Endotoxin-free plasmids pcDNA3.1(+)-alpha-2 thiab pcDNA3.1(+)-alpha-7.3 tau tsim los siv SanPrep Endotoxin-free Plasmid Mini Kit (Sangon Biotech).Cov concentration tau khaws cia siab tshaj 500 ng / µl kom ntseeg tau tias EDTA nyob rau hauv elution buffer tsis cuam tshuam nrog kev hloov pauv.Primary nas peritoneal macrophages tau coj mus rau hauv 6-zoo daim hlau nrog ua tiav RPMI 1640 nruab nrab (Biological Industries) rau 12 teev, ces cov hlwb raug ntxuav 3 zaug hauv PBS sov so kom tshem tau cov penicillin thiab streptomycin, thiab tom qab ntawd hauv nruab nrab ntxiv nrog rau qhov nruab nrab.Endotoxin-free plasmids pcDNA3.1(+)-alpha-2 thiab pcDNA3.1(+)-alpha-7.3 (2.5 μg) tau diluted hauv 125 μl ntawm Opti-MEM txo cov ntshav nruab nrab (Gibco, Thermo Fisher Scientific)..Tom qab ntawd 5 µl ntawm Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific) tau diluted hauv 125 µl ntawm cov ntshav qis Opti-MEM nruab nrab.Npaj liposome-DNA complexes los ntawm kev sib tov cov diluted endotoxin-dawb plasmid nrog Lipofectamine 2000 thiab cia cov sib tov sawv ntawm chav tsev kub li 5 feeb.Hloov cov complexes cais mus rau cov hlwb hauv txhua qhov dej thiab sib tov maj mam.Tom qab 4 teev, cell kab lis kev cai nruab nrab tau hloov nrog 2 ml ntawm tiav RPMI 1640 nruab nrab thiab kab lis kev cai tau txuas ntxiv rau 24 teev.Fresh cell kab lis kev cai nruab nrab tau ntxiv rau lub hlwb thiab incubated rau ntau lub sij hawm cov ntsiab lus nyob ntawm seb qhov kev ntsuam xyuas tsim.
Cov qauv proteins los ntawm supernatants thiab cell lysates tau npaj raws li tau piav qhia yav dhau los [25].Membrane hloov tsis tau rau pro-IL-1β, pro-caspase-1, caspase-1 p20, NLRP3, β-actin, thiab His-tag yog 200 mA / 90 min.Rau interleukin 1β (IL-1β; R&D Systems, Minneapolis, Minnesota, USA), caspase-1 (p20) (Adipogen, Switzerland) thiab NLRP3 (Adipogen SA, Epalinges, Switzerland) thiab 1: 5000 lub hom phiaj Nws tag (Amylet Scientific, Wuhan, Tuam Tshoj) thiab β-actin (Proteintech, Wuhan, Tuam Tshoj).
Kev sib txuas nrog disuccinimide suberate (DSS) tau ua raws li tau piav qhia yav dhau los [26].Cells raug ntxuav 3 zaug nrog txias PBS thiab tag nrho lysed nrog 27 gauge koob hauv 50 µl ASC cov tshuaj tiv thaiv tsis zoo (pH 8.0) uas muaj 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES thiab 125 mM NaHCO3.Qhov sib tov yog centrifuged ntawm 5000 g rau 3 min thiab cov pellet yog sutured nrog 10 µl DSS (25 mM hauv DMSO) thiab 40 µl ASC cov tshuaj tiv thaiv tsis rau 30 min ntawm 37 ° C.Tom qab centrifugation ntawm 5000 g rau 10 min, cov pellet tau yaj nyob rau hauv ib qho kev daws ntawm 40 µl ntawm ASC cov tshuaj tiv thaiv thiab 10 µl ntawm 6x protein loading tsis (TransGen, Beijing, Tuam Tshoj), thiab ces cov tshuaj tau quenched ntawm chav tsev kub rau 15. min., Tom qab ntawd boil 10 feeb.Cov qauv protein tau raug rau Western blotting siv thawj cov tshuaj tiv thaiv ASC (Wanleibio, Shenyang, Tuam Tshoj) ntawm qhov sib piv ntawm 1: 500.
Ua raws li cov txheej txheem tau piav qhia yav dhau los [13], cell culture supernatants tau sau thiab tso tawm ntawm cov pro-inflammatory cytokine IL-1β tau txiav txim siab siv tus nas IL-1 Beta ELISA cov khoom siv (Invitrogen, Thermo Fisher Scientific).Hloov OD450nm qhov tseem ceeb rau cov protein ntau siv IL-1β tus qauv nkhaus.
Cells coated on coverlips tau maj mam ntxuav 3 zaug nyob rau hauv sov PBS, tsau nyob rau hauv cov ntaub so ntswg cell fixative (Biosharp, Beijing, Tuam Tshoj) rau 10 min ntawm chav tsev kub (RT), nyob rau hauv 0.1% Triton X-Permeabilize ntawm 100 ( diluted hauv PBS; Biosharp ) rau 20 feeb ntawm chav tsev kub thiab thaiv hauv 5% bovine serum albumin (hauv PBS) rau 2 teev ntawm chav tsev kub.Cells tau muab tso rau ib hmos ntawm 4 ° C nrog thawj cov tshuaj tiv thaiv ASC (1: 100 dilution) los yog NLRP3 (1: 100 dilution), feem, thiab Cy3 sau cov tshis tiv thaiv luav IgG (H + L) (1: 400; EarthOx , San Francisco, CA, USA) lossis FITC-conjugated tshis anti-nas IgG (1:400; Earthox) hmo ntuj ntawm 37 ° C hauv qhov tsaus ntuj rau 1 teev.Lub nuclei tau stained nrog Hoechst 33258 (10 μg / ml; UE, Suzhou, Tuam Tshoj) rau 5 feeb thiab soj ntsuam nyob rau hauv lub fluorescence microscope (Olympus Corporation, Tokyo, Nyiv).
Cov nas tau muab faib ua plaub pawg (n = 7 hauv txhua pawg): (i) PBS-kho tsis zoo pab pawg tswj hwm (PBS nkaus xwb; gavage 100 µl / nas PBS tom qab txhaj tshuaj intraperitoneal txhua hnub 100 µl / nas PBS 3 teev tom qab)., tsis tu ncua rau 7 hnub);(ii) pawg tswj tsis zoo kho nrog MCC950 inhibitor [27] (100 µl / nas ntawm PBS gavage, 3 teev tom qab, 10 mg / kg lub cev qhov hnyav [BW] MCC950 [hauv PBS] tau muab tshuaj intraperitoneally txhua hnub, ntev 7 hnub);(iii) G. duodenalis cyst kab mob pawg (1.5 x 106 cysts / nas los ntawm gavage, 3 teev tom qab, 100 μl / nas PBS intraperitoneally muab tshuaj txhua hnub rau 7 hnub);(iv) G. duodenalis cyst ua ke kab mob pawg MCC950 inhibitor kev kho mob pawg (1.5 × 106 hlwv / nas ntawm gavage, 10mg / kg lub cev hnyav MCC950 intraperitoneally txhua hnub rau 7 hnub ntawm 3h).Lub cev hnyav ntawm txhua tus nas tau saib xyuas txhua hnub thiab tag nrho cov nas tau euthanized rau hnub 7.Sau duodenum (3 cm ntev) raug txiav rau hauv me me pieces nyob rau hauv 1 ml PBS, cysts raug puas lawm hmo ntuj nyob rau hauv PBS ntawm 4 ° C, thiab G. duodenalis trophozoites.Fresh duodenum (1 cm ntev) raug cais rau hematoxylin thiab eosin (H&E) staining.
Cov nas tau muab faib ua ob pawg: (i) MOCK pawg tswj hwm thiab (ii) MCC950 inhibitor pawg.Muaj tsib txoj kev kho mob hauv txhua pab pawg (n = 7 / pab pawg kho mob): (i) PBS kho tsis zoo pab pawg tswj hwm (PBS nkaus xwb; 100 µl / nas PBS, intramuscular (IM) txhaj tshuaj (tibialis anterior) [28, 29] ;( ii) pcDNA3.1(+) plasmid negative control group (100 µg/nas DNA, via intramuscular injection); pab pawg kho nrog plasmid pcDNA3.1(+)-alpha-2 (100 μg / nas DNA, los ntawm kev txhaj tshuaj intramuscular), thiab (v) ib pawg kho nrog plasmid pcDNA3.1(+)-alpha-7.3 (100 µg / nas. DNA, tom qab 12 teev dhau los, cov nas hauv MCC950 inhibitor pab pawg tau txais kev txhaj tshuaj intraperitoneal txhua hnub ntawm MCC950 (10 mg / kg lub cev hnyav) rau 7 hnub, thaum cov nas hauv MOCK tau txais qhov sib npaug ntawm kev kho PBS. Cov ntshav kuaj tau. sau los ntawm cov nas pob qhov muag thiab sab laug thaum hmo ntuj ntawm 4 ° C cov ntshav kuaj tau raug cais tawm siv enzyme-linked immunosorbent assay (ELISA) rau thiab ntsuas cov qib IL-1β.
Peb caug-tsib nas tau muab faib ua tsib pawg (n=7/pab).Pawg 1 yog pawg tswj tsis zoo uas tau kho nrog PBS: nas tau txais 100 μl ntawm PBS intramuscularly thiab 3 hnub tom qab los ntawm gavage.Pawg 2 yog ib pawg tswj xyuas zoo uas kis tau tus kab mob G. duodenalis cysts: nas tau txhaj nrog 100 μl ntawm PBS, thiab 3 hnub tom qab ntawd 1.5 x 106 cysts / nas tau txhaj tshuaj intragastrically.Pab pawg thib peb - ​​kev txhaj tshuaj plasmid nrog pcDNA3.1(+) ua ke nrog pab pawg tswj xyuas kab mob duodenal cyst: nas tau txais 100 μg ntawm plasmid DNA pcDNA3.1(+)(im) qhov ncauj, 1.5 × 106 cysts / nas 3 rau ntau hnub.Pawg 4 thiab 5 yog recombinant pcDNA3.1(+)-alpha-2 giardine plasmid los yog pcDNA3.1(+)-alpha-7.3 giardine plasmid ua ke nrog G. duodenalis cyst kab mob.Pab pawg sim: nas tau txais 100 µg ntawm pcDNA3.1(+)-giardine plasmid DNA (im), tom qab ntawd 3 hnub tom qab, 1.5 × 106 cysts / nas raug txhaj ntawm gavage.Lub cev hnyav ntawm txhua tus nas raug saib xyuas tom qab kev qhia txog G. duodenalis cyst los ntawm lub raj.Fresh duodenum tau sau rau kev ntsuas kab mob parasitic thiab HE staining tsom xam.
Cov kev hloov pauv histopathological tau txheeb xyuas raws li cov txheej txheem luam tawm yav dhau los [30].Cov duodenum tshiab tau kho nrog cov ntaub so ntswg cell fixative, embedded nyob rau hauv paraffin, txiav mus rau hauv 4 μm seem, stained nrog H&E thiab soj ntsuam nyob rau hauv lub teeb lub teeb.Cov neeg sawv cev ntawm cov kev hloov pauv pathological nyob rau hauv xya cov ntaub so ntswg los ntawm xya tus nas ywj siab tau soj ntsuam los ntawm tus kws kho mob tsis paub txog kev kho mob thiab raug ntes ntawm 200x magnification.Qhov ntev ntawm villi thiab qhov tob ntawm cov crypts raug ntsuas raws li cov txheej txheem tau piav qhia dhau los.
Cov txiaj ntsig hauv vitro thiab hauv vivo tau txais hauv triplicate.Cov duab tau tsim los siv GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, USA).Qhov sib txawv ntawm ob pawg tau txheeb xyuas los ntawm t-test, thaum qhov sib txawv ntawm ≥3 pawg tau txheeb xyuas los ntawm ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) siv SPSS software (version 22.0; SPSS IBM Corp., Armonk, NY, USA).Cov ntaub ntawv raug soj ntsuam rau homogeneity ntawm qhov sib txawv siv Levene qhov kev sim ua raws li Bonferroni's post hoc test (B).Qhov tseem ceeb yog qhia raws li P<0.05, P<0.01, thiab P<0.001 (tsis tseem ceeb [ns]) (P> 0.05).
Peb qhov kev txheeb xyuas yav dhau los ntawm GEV proteomics hauv Kyoto Encyclopedia of Genes thiab Genomes (KEGG) tau qhia tias ntau lub hom phiaj tuaj yeem koom nrog hauv kev ua kom cov kab mob inflammatory [13].Peb tau xaiv ob lub hom phiaj zoo, alpha-2 thiab alpha-7.3 giardins, nthuav cov molecules thiab siv lawv los tsim cov pcDNA3.1(+) eukaryotic qhia vector.Tom qab ua kom tiav, recombinant pcDNA3.1(+)-alpha-2 thiab alpha-7.3 giardine qhia plasmids tau hloov mus rau hauv thawj nas peritoneal macrophages, thiab caspase-1 p20 kos npe protein ntawm o (ib feem ntawm lub tshuab ua kom tau caspase-1) tau txheeb xyuas. raws li elucidating tseem ceeb molecules uas tuaj yeem ua rau mob.Cov txiaj ntsig tau pom tias alpha-2 thiab alpha-7.3 giardines tuaj yeem ua rau p20 caspase-1 qhia zoo ib yam li GEV.Tsis muaj kev cuam tshuam rau caspase-1 ua kom pom muaj nyob rau hauv kev tswj tsis zoo (PBS nkaus xwb) thiab plasmid tswj pcDNA3.1(+) (Daim duab 1).
Kev ntsuas p20 caspase-1 ua kom los ntawm pcDNA3.1(+)-alpha-2 thiab alpha-7.3 giardins.Recombinant eukaryotic qhia plasmids pcDNA3.1(+)-alpha-2 thiab alpha-7.3 giardines (saum toj no txhua txoj kab) tau hloov mus rau hauv thawj nas peritoneal macrophages thiab kab lis kev cai supernatants tau sau 24 teev tom qab.Western blotting tau siv los ntsuas qhov qhia theem ntawm kos npe caspase-1 p20 inflammasome protein.Pab pawg kho mob PBS nkaus xwb (txoj kab C) thiab pcDNA3.1 (+) pawg kho mob monotherapy (pcDNA3.1 txoj kab) tau siv los ua kev tswj tsis zoo, thiab GEV pab pawg kho mob tau siv los tswj qhov zoo.Kev nthuav qhia ntawm cov recombinant protein tau lees paub los ntawm kev kuaj pom cov cim histidine hauv txhua cov protein, thiab cov protein uas xav tau yog alpha-2 giardine (38.2 kDa) thiab alpha-7.3 giardine (37.2 kDa).GEV, Giardia duodenum extracellular vesicles, pcDNA3.1(+), EcoRV-linearized vector, SUP, supernatant
Txhawm rau txiav txim siab seb alpha-2 giardine thiab alpha-7.3 giardine induce p20 caspase-1 qhia thiab ua lub luag haujlwm hauv kev ua kom tus tswv tsev NLRP3 inflammatory teb, pcDNA3.1(+)-alpha-2 giardine thiab pcDNA3.1(+)-alpha -7.3 giardin tau hloov mus rau hauv thawj nas peritoneal macrophages nrog recombinant plasmid DNA, thiab theem ntawm kev qhia, localization, thiab oligomerization ntawm cov tseem ceeb inflammatory proteins NLRP3 tau txiav txim.Hauv qhov kev sim no, GEV tau siv los ua pawg tswj hwm qhov zoo, thiab tsis muaj pab pawg kho mob (PBS nkaus xwb) lossis pcDNA3.1 (+) pab pawg kho mob hloov pauv yog pawg tsis zoo.Cov txiaj ntsig tau pom tias, ib yam li hauv GEV pawg, recombinant plasmid DNA ntawm giardin pcDNA3.1(+)-alpha-2 thiab giardin pcDNA3.1(+)-alpha-7.3 ua rau muaj kev tswj hwm ntawm NLRP3, pro-IL-1β thiab procaspase-1 thiab caspase-1 activation (Fig. 2a).Tsis tas li ntawd, ob qho tib si giardines induced IL-1β secretion tseem ceeb (pcDNA3.1: ANOVA, F(4, 10) = 1.625, P = 0.1000; alpha-2 giardine: ANOVA, F(4, 10) = 1.625, P = 0.0007 ).;alpha-7.3 giardine: ANOVA, F(4, 10) = 1.625, P<0.0001;GEV: ANOVA, F(4, 10) = 1.625, P = 0.0047) (Figure 2b).Feem ntau ASC cov proteins yog monomeric hauv pab pawg tsis kho lossis hauv pab pawg kho mob kis nrog pcDNA3.1(+) plasmid, sib piv rau pcDNA3.1(+)-alpha-2 lossis pcDNA3.1(+)-alpha- 7.3 loj.ASC oligomerization tshwm sim nyob rau hauv recombinant plasmid DNA ntawm GEV zoo tswj pawg los yog pab pawg, uas qhia ib daim ntawv oligomeric (Daim duab 2c).Cov ntaub ntawv ua ntej no qhia tias alpha-2 giardine thiab alpha-7,3 giardine tuaj yeem ua rau NLRP3 ua kom mob.Cov kev tshawb fawb immunofluorescent tom qab ntawm thaj chaw ntawm ASC thiab NLRP3 tau pom tias hauv pawg tswj hwm tsis zoo, ASC cov protein tau tawg thoob plaws hauv cytoplasm thiab tshwm sim raws li cov teeb liab dot thaum stimulation ntawm pcDNA3.1(+)-alpha-2 nrog giardine lossis pcDNA3.1(+)-alpha-7,3 giardine pawg lossis GEV zoo tswj pawg (Daim duab 2d).Hauv kev tswj tsis zoo thiab plasmid-kho pcDNA 3.1 pawg, NLRP3 protein teeb liab tsis tau kuaj pom, thaum lub teeb liab fluorescent dot hauv teb rau pcDNA3.1(+)-alpha-2 giardine lossis pcDNA3.1(+)-alpha-7.3 tau kuaj pom..giardine muaj nyob rau hauv cytoplasm los yog thaum stimulation ntawm HEV (Fig. 2e).Cov ntaub ntawv no qhia ntxiv tias G. duodenalis giardin alpha-2 thiab giardin alpha-7.3 qhib NLRP3 inflammasome hauv nas thawj peritoneal macrophages.
pcDNA3.1(+)-alpha-2 giardin thiab pcDNA3.1(+)-alpha-7.3 giardin qhib NLRP3 inflammasome hauv nas peritoneal macrophages.Hloov cov recombinant eukaryotic qhia plasmids pcDNA3.1(+)-alpha-2 giardin thiab pcDNA3.1(+)-alpha-7.3 giardin rau hauv thawj murine peritoneal macrophages thiab hlwb, los yog sau cov supernatant hauv 24 teev rau kev tsom xam ntawm kev qhia, oligomerization , tso quav tso.thiab localization ntawm qhov tseem ceeb inflammatory proteins.Pab pawg PBS-tsuas yog (C) thiab pcDNA3.1 (+) ib pab pawg kho mob tau siv los ua kev tswj tsis zoo, thiab GEV pawg kho mob tau siv los ua pawg zoo.Ib qho tseem ceeb inflammatory proteins NLRP3, suav nrog NLRP3, pro-IL-1β, pro-caspase-1, thiab p20 caspase-1, tau kuaj pom los ntawm Western blotting.b Cov theem ntawm kev tso tawm ntawm IL-1β nyob rau hauv cov supernatants tau txiav txim siab siv enzyme-linked immunosorbent assay (ELISA).Qhov sib txawv ntawm kev tswj thiab kev sim pab pawg tau soj ntsuam los ntawm ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) siv SPSS software version 22.0.Asterisks qhia qhov sib txawv tseem ceeb ntawm pawg **P<0.01 thiab ***P<0.001.c ASC oligomerization qib hauv cov pellets tau txiav txim siab los ntawm DSS cross-linking tsom xam, thaum ASC qib hauv cell lysates tau siv los tswj kev thauj khoom.d Visualization ntawm ISC localization siv immunofluorescence.e Immunofluorescence tau siv los pom qhov chaw ntawm NLRP3.ASC, apoptotic speck-zoo li protein;IL, interleukin;NLRP3, nucleotide-binding oligomerization-zoo li receptor 3;ns, tsis tseem ceeb (P> 0.05)
Ob leeg G. duodenalis thiab GEVs nws secretes qhib NLRP3 inflammasome thiab tswj tus tswv tsev inflammatory teb nyob rau hauv vitro.Yog li, lub luag haujlwm ntawm NLRP3 inflammasome hauv pathogenicity ntawm G. duodenalis tseem tsis meej.Txhawm rau tshawb xyuas qhov teeb meem no, peb tau tsim ib qho kev sim ntawm cov nas uas muaj kab mob G. duodenalis cyst thiab nas kis nrog G. duodenalis cyst + MCC950 inhibitor kev kho mob thiab muab piv rau NLRP3 inflammasome qhia thaum kis G. duodenalis cyst.Cov txheej txheem ntxaws ntxaws ntawm qhov kev sim tau pom hauv daim duab 3a.Kev hloov pauv ntawm lub cev hnyav ntawm cov nas hauv ntau pawg kho mob tau saib xyuas rau 7 hnub tom qab kis kab mob, thiab cov txiaj ntsig tau pom hauv daim duab 3b.Muab piv rau cov pab pawg kho nrog PBS ntshiab, cov txiaj ntsig tau pom tias (i) lub cev hnyav ntawm cov nas uas kis tus kab mob G. duodenalis cyst poob qis txij hnub 3 mus rau hnub 7 tom qab kis kab mob;(ii) kev kho mob nrog MCC950 inhibitor tsis muaj qhov cuam tshuam loj rau lub cev hnyav ntawm cov nas..Piv rau ib pawg kab mob sib kis, BW ntawm pawg kab mob duodenal kho nrog MCC950 poob mus rau qhov sib txawv (Hnub 1: ANOVA, F(3, 24) = 1.885, P = 0.0148; Hnub 2: ANOVA, F(3, 24) ) = 0.4602, P<0.0001; Hnub 3: ANOVA, F(3, 24) = 0.8360, P = 0.0010; Hnub 4: ANOVA, F(3, 24) = 1.683, P = 0.0052 Hnub 5: A NO (3, 24)=0.6497, P=0.0645; Hnub 6: ANOVA, F(3, 24)=5.457, P=0.0175; Hnub 7: ANOVA, F(3, 24) = 2.893, P = 0.0202).Cov ntaub ntawv no qhia tau hais tias NLRP3 inflammasome tiv thaiv cov nas los ntawm qhov hnyav poob thaum ntxov (2-4 hnub) ntawm tus kab mob duodenal.Peb mam li tsom mus kuaj G. duodenalis trophozoites hauv duodenal lavage kua thiab cov txiaj ntsig tau pom hauv daim duab 3c.Piv nrog rau G. duodenalis cyst kab mob pawg, tus naj npawb ntawm trophozoites nyob rau hauv duodenum ho nce tom qab thaiv cov NLRP3 inflammasome (t(12) = 2.902, P = 0.0133).Cov ntaub so ntswg duodenal stained nrog HE pom, piv rau kev tswj tsis zoo uas kho nrog PBS thiab MCC950 ib leeg: (i) G. duodenalis cyst kab mob ua rau muaj kev puas tsuaj rau duodenal villi (ANOVA, F(3, 24) = 0.4903, P = 0.0488 ) thiab crypt atrophy (ANOVA, F(3, 24) = 0.4716, P = 0.0089);(ii) duodenum los ntawm nas kis kab mob G. duodenalis hlwv thiab kho nrog MCC950 inhibitors.duodenal villi raug puas tsuaj thiab tuag (ANOVA, F(3, 24) = 0.4903, P = 0.0144) nrog atrophy thiab crypt branching (ANOVA, F (3, 24) = 0.4716, P = 0, 0481) (Fig. 3d- f).Cov txiaj ntsig no qhia tias NLRP3 inflammasome plays lub luag haujlwm hauv kev txo cov kab mob ntawm G. duodenalis.
Lub luag haujlwm ntawm NLRP3 inflammasome hauv Giardia duodenum kab mob.Cov nas raug gavaged (iv) nrog duodenococcal cysts thiab tom qab ntawd kho nrog lossis tsis muaj MCC950 (ip).Ib pawg kho mob nrog PBS lossis MCC950 tau siv los ua kev tswj hwm.Kev sim pab pawg thiab kev kho mob.b Lub cev hnyav ntawm cov nas nyob hauv txhua pab pawg kho mob tau saib xyuas rau 7 hnub.Qhov sib txawv ntawm pawg kab mob G. duodenalis thiab G. duodenalis + MCC950 pab pawg kho kab mob tau raug tshuaj xyuas los ntawm t-test siv SPSS software version 22.0.Asterisks qhia qhov sib txawv tseem ceeb ntawm *P<0.05, **P<0.01, lossis ***P<0.001.c Parasitic load tau txiav txim los ntawm suav tus naj npawb ntawm trophozoites hauv duodenal lavage kua.Qhov sib txawv ntawm pawg kab mob G. duodenalis thiab G. duodenalis + MCC950 pab pawg kho kab mob tau raug tshuaj xyuas los ntawm t-test siv SPSS software version 22.0.asterisks qhia qhov sib txawv tseem ceeb ntawm *P <0.05.d Hematoxylin thiab eosin (H&E) staining tshwm sim ntawm duodenal histopathology.Cov xub liab qhia kev puas tsuaj rau lub villi, cov xub ntsuab qhia kev puas tsuaj rau cov crypts.Scale bar: 100 µm.e, f Kev txheeb xyuas ntawm duodenal villus qhov siab thiab nas crypt qhov siab.Asterisks qhia qhov sib txawv tseem ceeb ntawm *P<0.05 thiab **P<0.01.Cov txiaj ntsig tau muab los ntawm 7 qhov kev sim tshuaj lom neeg ywj pheej.BW, lub cev hnyav;ig, intragastric tus me nyuam txoj kev;ip, intraperitoneal txoj kev xa khoom;ns, tsis tseem ceeb (P> 0.05);PBS, phosphate buffered saline;WT, hom tsiaj qus
Kev tso tawm ntawm IL-1β yog ib qho cim ntawm kev ua kom mob.Txhawm rau txiav txim siab seb G. duodenalis alpha-2 giardine thiab alpha-7.3 giardine qhib NLRP3 party inflammasome hauv vivo, peb tau siv cov nas uas tsis kho WT (sham group) thiab NLRP3 inflammasome-blocked nas (MCC950 inhibited treatment group).Cov txheej txheem ntxaws ntxaws ntawm qhov kev sim tau pom hauv daim duab 4a.Cov pab pawg sim muaj xws li nas kho nrog PBS, G. duodenalis cyst kev kho mob los ntawm gavage, intramuscular txhaj ntawm pcDNA3.1, thiab intramuscular txhaj ntawm pcDNA3.1(+)-alpha-2 giardine los yog pcDNA3.1-alpha-7.3 giardine.Hnub 7 tom qab kev tswj hwm intramuscular ntawm recombinant plasmid, ntshav tau sau thiab qib ntawm IL-1β hauv txhua pab pawg tau txiav txim siab.Raws li pom hauv daim duab 4b, hauv pawg MOCK: (i) piv rau pawg PBS, kev kho pcDNA3.1 tsis muaj qhov cuam tshuam loj rau IL-1β secretion (ANOVA, F(4.29) = 4.062, P = 0.9998), txawm li cas los xij, IL-β secretion tau nce siab hauv G. duodenalis cyst pawg (ANOVA, F(4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine thiab pcDNA3.1- Kev txhaj tshuaj intramuscular ntawm alpha-7.3 giardine nce qib IL-1β (ANOVA, F(4, 29) = 4.062, P<0.0001);(iii) pcDNA3.1-alpha-7,3 giardine induced siab ntawm IL -1β secretion hauv pcDNA3.1-alpha-2 giardine intramuscular txhaj pab pawg (ANOVA, F(4, 29) = 4.062, P = 0.0333) .Piv nrog rau txhua pab pawg hauv pab pawg kho mob MCC950 thiab MOCK pawg: (i) IL-1β tso tawm qib hauv pawg tswj hwm PBS thiab pawg tswj hwm pcDNA3.1 tau txo qis rau qee yam tom qab thaiv MCC950 inhibitor, tab sis qhov sib txawv tsis yog. tseem ceeb (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F(9, 58) = 3.540, P = 0.5949);(ii) tom qab thaiv MCC950., IL-1β secretion tau txo qis hauv G. duodenalis cyst-infected pawg, pawg pcDNA3.1-alpha-2 giardine, thiab pawg pcDNA3.1-alpha-7.3 giardine (G. duodenalis: ANOVA, F(9). , 58) = 3.540 , P = 0.0120; ) = 3.540, P = 0.0164).Cov txiaj ntsig no qhia tias alpha-2 giardine thiab alpha-7.3 giardine kho kom haum rau kev ua kom NLRP3 inflammasome hauv vivo.
pcDNA3.1(+)-giardines qhib NLRP3 party inflammasome hauv vivo.Cov nas tau txhaj tshuaj tiv thaiv kab mob (IM) nrog recombinant eukaryotic qhia plasmid pcDNA3.1(+)-alpha-2 giardine lossis pcDNA3.1(+)-alpha-7.3 giardine thiab tom qab ntawd kho nrog MCC950 (ip; MCC950 pawg) lossis tsis yog (dummy pawg ).PBS lossis pcDNA3.1(+) plasmid kho pab pawg tau siv los ua kev tswj tsis zoo, pawg kho G. duodenalis cyst tau siv los ua kev tswj xyuas zoo.Kev sim pab pawg thiab kev kho mob.b Ntshav Qab Zib qib IL-1β hauv cov nas tau ntsuas rau hnub 7 los ntawm ELISA kev soj ntsuam.Qhov sib txawv ntawm cov pab pawg hauv MOCK pawg tau txheeb xyuas siv ib-txoj kev ANOVA, thiab qhov sib txawv ntawm pawg MOCK thiab pawg MCC950 tau txheeb xyuas siv t-test ntawm SPSS software version 22.0.asterisks qhia qhov sib txawv tseem ceeb ntawm pab pawg kho mob hauv MOCK pawg, *P<0.05 thiab ***P<0.001;Cov cim nyiaj daus las ($) qhia qhov sib txawv tseem ceeb ntawm txhua pab pawg hauv MOCK pawg thiab MCC950 pawg ntawm P<0.05.Cov txiaj ntsig ntawm xya qhov kev sim tshuaj lom neeg ywj pheej.i, intramuscular txhaj, ns, tsis tseem ceeb (P> 0.05)
Txhawm rau tshawb xyuas cov txiaj ntsig ntawm alpha-2 thiab alpha-7.3 giardine-mediated activation ntawm NLRP3 host inflammasome ntawm G. duodenalis kab mob, peb siv WT C57BL/6 nas thiab txhaj tshuaj alpha-2 giardine thiab alpha-7.3 giardine.Cov plasmid tau txhaj tshuaj intramuscularly, tom qab 3 hnub dhau los ntawm lub plab hnyuv ntawm G. duodenalis cyst, tom qab ntawd cov nas tau pom 7 hnub.Cov txheej txheem ntxaws ntxaws ntawm qhov kev sim tau pom hauv daim duab 5a.Lub cev hnyav ntawm txhua tus nas raug ntsuas txhua hnub, cov qauv ntawm cov ntaub so ntswg tshiab tau sau rau hnub 7 tom qab kev tswj hwm los ntawm lub plab hnyuv, ntsuas tus naj npawb ntawm trophozoites, thiab cov kev hloov pauv ntawm histopathological tau pom.Raws li pom hauv daim duab 5b, nrog rau lub sijhawm pub mis ntxiv, BW ntawm cov nas nyob hauv txhua pab pawg maj mam nce.MT ntawm cov nas pib txo qis rau hnub thib 3 tom qab kev tswj hwm intragastric ntawm G. duodenalis cysts, thiab tom qab ntawd maj mam nce.Kev ua kom NLRP3 inflammasome tshwm sim los ntawm kev txhaj tshuaj intramuscular ntawm alpha-2 giardine thiab alpha7.3 giardine ua rau muaj kev poob qis hauv cov nas (Hnub 1: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 1.39, = 0.9754 Hnub 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30)=1.399, P=0.9987 Hnub 2: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.3172, P = 0.9979; Hnub 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.3172, P = 0.8409; Hnub 3: pcDNA3.1-alpha-2 giardine, ANOVA, 4, 30) = 0.8222, P = 0.0262 Hnub 3: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4 , 30) = 0.8222, P = 0.0083 Hnub 4: pcDNA3.1-alpha-A2 , F(4, 30) = 0.5620, P = 0.0012, Hnub 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5620, P < 0.0001, Hnub 5: pcDNA3.1-alpha - 2 giardine, ANOVA, F(4, 30) = 0.9728, P < 0.0001 Hnub 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F(4, 30) = 0.9728, P < 0.0001 Hnub 3: 6.1pcDNA alpha-2 giardine, ANOVA, F(4, 30) = 0.7154, P = 0.0012, Hnub 6: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.7154, P = 0.0006;Hnub 7: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.5369, P<0.0001 Hnub 7: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5369, P <0.0001).Cov kab mob cab tau raug soj ntsuam hauv duodenum (Fig. 5c).Piv nrog rau kev tswj tsis tau zoo thiab cov pab pawg tau txhaj nrog cov khoob pcDNA3.1 vector, tus naj npawb ntawm G. duodenalis trophozoites tau txo qis hauv cov pab pawg txhaj tshuaj α-2 giardine thiab α-7,3 giardine (pcDNA3.1-alpha. -2 giardine : ANOVA, F(3, 24) = 1.209, P = 0.0002, pcDNA3.1-alpha-7.3 giardine: ANOVA, F(3, 24) = 1.209, P<0.0001).Tsis tas li ntawd, giardine alfa-7.3 tau tiv thaiv cov nas ntau dua li giardine alfa-2 (ANOVA, F(3, 24) = 1.209, P = 0.0081).Cov txiaj ntsig ntawm HE staining yog qhia hauv daim duab.5d-f ib.Cov nas txhaj tshuaj nrog alpha-2 giardine thiab alpha-7.3 giardine muaj tsawg dua cov ntaub so ntswg duodenal, tshwm sim los ntawm villus puas, piv rau cov nas txhaj nrog G. duodenalis thiab cov nas txhaj nrog G. duodenalis ua ke nrog ib qho khoob pcDNA3 vector .1 Zoom.(pcDNA3.1-alpha-2 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0035 los yog P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0028 los yog P = 0.0055) thiab txo crypt atrophy (pcDNA3.1-alpha-2 giardine: ANOVA, F(3, 24) = 1.470, P = 0.0264 los yog P = 0.0158; pcDNA3.1-alpha-7.3 , F(3, 24) = 1.470, P = 0.0371 or P = 0.0191).Cov txiaj ntsig no qhia tias alpha-2 giardine thiab alpha-7,3 giardine txo cov kab mob G. duodenalis los ntawm kev ua kom NLRP3 inflammasome hauv vivo.
Lub luag haujlwm ntawm pcDNA3.1(+)-giardins hauv G. duodenalis kab mob.Cov nas tau txhaj tshuaj tiv thaiv kab mob (IM) nrog recombinant eukaryotic qhia plasmids pcDNA3.1(+)-alpha-2 giardine los yog pcDNA3.1(+)-alpha-7.3 giardine thiab ces sib tw nrog G. duodenalis cysts (ig).Pab pawg PBS thiab pcDNA3.1(+) + pab pawg kho mob duodenal cyst tau siv los ua pawg tswj tsis zoo, thiab pab pawg kho mob duodenal cyst tau siv los ua pawg tswj hwm zoo.Kev sim pab pawg thiab kev kho mob.b Cov MT ntawm cov nas hauv txhua pab pawg kho mob tau saib xyuas rau 7 hnub tom qab kev sib tw.Asterisks qhia qhov sib txawv tseem ceeb ntawm pawg hauv G. duodenalis pawg thiab pcDNA3.1(+)-alpha-2 giardine pawg, *P <0.05, **P <0.01, thiab ***P <0.001;tus lej kos npe ($) qhia qhov sib txawv tseem ceeb ntawm txhua pawg G. duodenalis thiab pcDNA3.1(+)-alpha-7.3 jardine pawg, $$P<0.01 thiab $$$P<0.001.C Parasitic load tau txiav txim los ntawm kev suav tus naj npawb ntawm trophozoites hauv 1 ml ntawm duodenal lavage los ntawm duodenum (3 cm ntev) thiab qhia raws li tus naj npawb ntawm cov cab ib cm ntawm duodenum.Qhov sib txawv ntawm pawg kab mob G. duodenalis, pawg pcDNA3.1(+)-alpha-2 giardine, thiab pawg pcDNA3.1(+)-alpha-7.3 giardine tau soj ntsuam los ntawm ib-txoj kev ANOVA siv SPSS software version 22.0.Asterisks qhia qhov sib txawv tseem ceeb ntawm **P<0.01 thiab ***P<0.001.d Histopathological hloov nyob rau hauv duodenum.Cov xub liab qhia kev puas tsuaj rau lub villi, cov xub ntsuab qhia kev puas tsuaj rau cov crypts.Scale bar: 100 µm.e, f Kev txheeb xyuas ntawm nas duodenal villus qhov siab (e) thiab crypt qhov siab (f).Qhov sib txawv ntawm pawg hauv daim duab 1d tau txheeb xyuas los ntawm ib txoj kev ANOVA siv SPSS software version 22.0.Asterisks qhia qhov sib txawv tseem ceeb ntawm *P<0.05 thiab **P<0.01.Cov txiaj ntsig ntawm xya qhov kev sim tshuaj lom neeg ywj pheej.ns, tsis tseem ceeb (P> 0.05)
Giardia duodenum yog cov kab mob hauv plab uas paub zoo ntawm tib neeg thiab lwm yam tsiaj txhu uas ua rau giardiasis.Hauv xyoo 2004, nws tau suav nrog WHO Cov Kab Mob Tsis Pom Zoo vim tias nws muaj ntau tshaj li 6 xyoo, tshwj xeeb tshaj yog nyob rau hauv cov zej zog uas muaj kev noj qab haus huv tsawg [32].Lub cev tiv thaiv kab mob hauv lub cev ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob rau G. duodenalis kab mob.Nas macrophages tau raug tshaj tawm rau engulf thiab tua G. duodenalis los ntawm kev tso cov ntxiab extracellular [33].Peb cov kev tshawb fawb yav dhau los tau pom tias G. duodenalis, cov kab mob tsis muaj kab mob ntxiv, ua rau p38 MAPK, ERK, NF-κB p65, thiab NLRP3 inflammatory signaling pathways in mouse macrophages to control host inflammatory responses, and release GEV may improve this process.13], ib., 24.Txawm li cas los xij, qhov tseeb PAMPs koom nrog NLRP3 inflammasome-regulated o nyob rau hauv GEV thiab lub luag hauj lwm ntawm NLRP3 inflammasome nyob rau hauv giardiasis tseem yuav elucidated.Txhawm rau ua kom pom tseeb ntawm ob lo lus nug no, peb tau ua qhov kev tshawb fawb no.
NLRP3 inflammasome nyob rau hauv cytoplasm ntawm lub cev tiv thaiv kab mob thiab tuaj yeem ua rau muaj ntau yam xws li uric acid crystals, co toxins, kab mob, kab mob, thiab kab mob cab.Hauv kev tshawb fawb kab mob, co toxins tau raug txheeb xyuas tias yog PAMPs tseem ceeb uas ua rau lub cev muaj zog, ua rau mob thiab tuag ntawm tes [34].Qee cov kab mob sib txawv, xws li hemolysin los ntawm Staphylococcus aureus [35] thiab Escherichia coli [36], hemolysin BL (HBL) los ntawm enterotoxin (NHE) [37], induce activation of NLRP3 o.Cov kev tshawb fawb txog kab mob tau pom tias cov kab mob muaj kab mob xws li SARS-COV-2 lub hnab ntawv (E) protein [38] thiab Zika tus kab mob NS5 protein [39] yog qhov tseem ceeb PAMPs lees paub los ntawm NLRP3 receptor.Hauv kev tshawb fawb kab mob, ntau tus kab mob tau raug tshaj tawm tias muaj feem cuam tshuam nrog kev ua kom mob plab, xws li Toxoplasma gondii, Trichomonas vaginalis [40], Trypanosoma cruzi [41], thiab Leishmania [42].Cov tshuaj granule tuab gra35, GRA42, thiab GRA42, cuam tshuam nrog tus virulence toxoPlasmaTsis tas li ntawd, qee qhov kev tshawb fawb Leishmania tau tsom mus rau ib tus neeg molecules koom nrog hauv NLRP3 inflammasome, xws li kab mob cab lipophosphoglycan [44] lossis zinc metalloprotease [45].Ntawm cov annexin-zoo li alpha-giardin tsev neeg ntawm cov noob, alpha-1 giardin tau pom tias yog ib tus neeg muaj peev xwm txhaj tshuaj tiv thaiv kev tiv thaiv G. duodenalis hauv tus qauv nas [18].Hauv peb txoj kev tshawb fawb, peb tau xaiv G. duodenalis virulence yam tseem ceeb alpha-2 thiab alpha-7,3 giardines, uas yog tshwj xeeb rau giardia tab sis kuj qhia tsawg dua.Ob lub hom phiaj no tau cloned rau hauv pcDNA3.1(+) eukaryotic expression system vector rau kev tsom xam ntawm kev ua kom mob.
Hauv peb cov qauv nas, cleaved caspase fragments ua cov cim ntawm kev ua kom mob.Thaum stimulation, NLRP3 cuam tshuam nrog ASC, recruits procaspases, thiab generates active caspases uas cleave pro-IL-1β thiab pro-IL-18 mus rau mature IL-1β thiab IL-18, raws li -18.Inflammatory caspases (caspases-1, -4, -5 thiab -11) yog ib tsev neeg khaws cia ntawm cysteine ​​​​proteases uas yog ib qho tseem ceeb rau kev tiv thaiv innate thiab koom nrog o thiab programmed cell tuag [46].Caspase-1 yog qhib los ntawm canonical inflammasomes [47], thaum caspases-4, -5, thiab -11 yog cleaved thaum lub sij hawm tsim ntawm atypical inflammasomes [48].Hauv txoj kev tshawb no, peb siv nas peritoneal macrophages ua tus qauv thiab tshawb xyuas p20 caspase-1 cleaved caspase-1 ua tus cim ntawm tus tswv tsev NLRP3 o ua kom ua rau hauv kev tshawb fawb ntawm G. duodenalis kab mob.Cov txiaj ntsig tau pom tias ntau alpha-giardins yog lub luag haujlwm rau kev ua haujlwm ntawm qhov mob, uas zoo ib yam nrog kev tshawb pom ntawm cov kab mob tseem ceeb ntawm cov kab mob cuam tshuam nrog cov kab mob thiab cov kab mob.Txawm li cas los xij, peb txoj kev tshawb fawb tsuas yog ib qho kev tshuaj ntsuam ua ntej thiab muaj lwm cov molecules uas tuaj yeem ua rau cov kab mob uas tsis yog classical inflammasomes, raws li peb txoj kev tshawb fawb yav dhau los pom ob qho tib si classical thiab non-classical inflammasomes hauv G. duodenalis kab mob [13].Txhawm rau txiav txim siab ntxiv seb qhov tsim tawm p20 caspase-1 puas cuam tshuam nrog NLRP3 inflammasome, peb tau hloov pauv alpha-2 thiab alpha-7.3 giardins rau hauv nas peritoneal macrophages los txiav txim siab cov ntsiab lus tseem ceeb ntawm cov protein qhia theem thiab ASC oligomerization theem, lees paub tias ob qho tib si α-giardins qhib. Inflammasome NLRP3.Peb cov txiaj ntsig tau txawv me ntsis los ntawm Manko-Prykhoda li al., uas tau tshaj tawm tias kev txhawb nqa ntawm Caco-2 hlwb nrog G. muris lossis E. coli EPEC hom kab mob ib leeg tuaj yeem ua kom cov fluorescence siv NLRP3, ASC, thiab caspase-1, Txawm hais tias tsis tseem ceeb, thaum yuav ua li cas costimulation ntawm G. muris thiab E. coli nce qib ntawm peb cov proteins [49].Qhov sib txawv no yuav yog vim qhov sib txawv ntawm kev xaiv ntawm Giardia hom, kab xov tooj ntawm tes, thiab cov hlwb thawj.Peb kuj tau ua nyob rau hauv vivo kev soj ntsuam siv MCC950 hauv 5-lub lim tiam poj niam WT C57BL/6 nas, uas muaj ntau dua rau G. duodenalis.MCC950 yog ib qho muaj zog thiab xaiv me me molecule NLRP3 inhibitor uas thaiv cov kev ua haujlwm canonical thiab non-canonical NLRP3 ntawm nanomolar concentrations.MCC950 inhibits NLRP3 activation tab sis tsis cuam tshuam rau kev ua haujlwm ntawm AIM2, NLRC4, thiab NLRP1 inflammatory pathways lossis TLR signaling pathways [27].MCC950 thaiv NLRP3 ua kom tab sis tsis txwv NLRP3 pib, K+ efflux, Ca2+ influx, lossis kev cuam tshuam ntawm NLRP3 thiab ASC;Hloov chaw, nws inhibits NLRP3 inflammasome activation los ntawm thaiv ASC oligomerization [27].Yog li, peb siv MCC950 hauv kev tshawb fawb hauv vivo los txiav txim lub luag haujlwm ntawm NLRP3 inflammasome tom qab txhaj tshuaj giardine.Activated caspase-1 p10 cleaves pro-inflammatory cytokines pro-IL-1β thiab pro-IL-18 rau mature IL-1β thiab IL-18 [50].Hauv txoj kev tshawb no, cov qib IL-1β hauv cov nas uas kho giardine nrog lossis tsis muaj MCC950 tau siv los ua qhov taw qhia ntawm seb NLRP3 inflammasome tau qhib.Raws li kev cia siab, MCC950 kev kho mob txo qis ntshav IL-1β qib.Cov ntaub ntawv no qhia meej meej tias G. duodenalis giardin alfa-2 thiab giardin alfa-7.3 muaj peev xwm ua kom NLRP3 nas inflammasome.
Cov ntaub ntawv tseem ceeb tau sau ntau xyoo dhau los tau pom tias IL-17A yog tus tswv tswj hwm kev tiv thaiv kab mob G. muris, ua rau IL-17RA signaling, tsim cov tshuaj tua kab mob peptides, thiab tswj kev ua kom muaj zog ntxiv [51].Txawm li cas los xij, Giardia kab mob tshwm sim ntau zaus hauv cov tub ntxhais hluas, thiab nws tau tshaj tawm tias Giardia kab mob hauv cov nas me tsis ua rau cov lus teb IL-17A los ua kom nws cov txiaj ntsig tiv thaiv [52], ua rau cov neeg tshawb nrhiav nrhiav lwm yam immunomodulatory Giardia.mechanisms ntawm tus kab mob helminth.Cov kws sau ntawv ntawm kev tshawb fawb tsis ntev los no tau tshaj tawm tias G. muris tuaj yeem qhib NLRP3 inflammasome los ntawm E. coli EPEC, uas txhawb kev tsim cov tshuaj tiv thaiv kab mob peptides thiab txo nws cov peev xwm txuas thiab tus naj npawb ntawm trophozoites hauv txoj hnyuv, yog li txo qhov hnyav ntawm txoj hnyuv. kab mob tshwm sim los ntawm kab mob [49].NLRP3 inflammasome koom nrog hauv kev txhim kho ntau yam kab mob.Cov kev tshawb fawb tau pom tias Pseudomonas aeruginosa ua rau autophagy hauv macrophages kom tsis txhob tuag ntawm tes, thiab cov txheej txheem no nyob ntawm qhov ua kom NLRP3 inflammasome [53].Rau N. caninum, reactive oxygen hom-mediated activation ntawm NLRP3 inflammasome txwv nws replication nyob rau hauv tus tswv tsev, ua rau nws muaj peev xwm kho lub hom phiaj [9].Paracoccidioides brasiliensis tau pom los ua kom muaj kev ua haujlwm ntawm NLRP3 inflammasome hauv nas pob txha pob txha-derived dendritic hlwb, uas ua rau tso tawm cov inflammatory cytokine IL-1β, uas ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv tus tswv [10].Ntau hom Leishmania, suav nrog L. amazonensis, L. loj, L. braziliensis, thiab L. infantum chagasi, qhib NLRP3 thiab ASC-dependent caspase-1 hauv macrophages, nrog rau Leishmania kab mob.Parasite replication yog txhim kho nyob rau hauv nas tsis muaj nyob rau hauv NLRP3/ASC/caspase-1 noob [11].Zamboni et al.Tus kab mob Leishmania tau tshaj tawm tias yuav ua rau muaj kev ua kom NLRP3 inflammasome hauv macrophages, uas txwv tsis pub cov kab mob sib kis hauv lub cev.Yog li, Leishmania tuaj yeem cuam tshuam NLRP3 ua kom zoo li kev zam.Hauv kev tshawb fawb vivo, NLRP3 inflammasome pab txhawb kev tshem tawm ntawm Leishmania, tab sis tsis cuam tshuam rau cov ntaub so ntswg [54].Hloov pauv, hauv kev tshawb fawb helminthiasis, ua kom NLRP3 inflammasome suppressed tus tswv tsev tiv thaiv kev tiv thaiv kab mob plab helminthiasis [12].Shigella yog ib qho ntawm cov kab mob tseem ceeb ua rau raws plab thoob ntiaj teb.Cov kab mob no tuaj yeem ua rau IL-1β ntau lawm los ntawm P2X7 receptor-mediated K + efflux, reactive oxygen hom, lysosomal acidification, thiab mitochondrial puas.NLRP3 inflammasome tsis zoo tswj phagocytosis thiab bactericidal kev ua ntawm macrophages tawm tsam Shigella [55].Cov kev tshawb fawb Plasmodium tau pom tias AIM2, NLRP3 lossis caspase-1 cov nas tsis muaj kab mob Plasmodium ua rau muaj qib siab ntawm hom 1 interferon thiab tiv taus cov kab mob Plasmodium ntau dua [56].Txawm li cas los xij, lub luag haujlwm ntawm alpha-2 giardine thiab alpha-7.3 giardine hauv inducing pathogenic activation ntawm NLRP3 o nyob rau hauv nas yog tsis meej.
Nyob rau hauv txoj kev tshawb no, inhibition ntawm NLRP3 inflammasome los ntawm MCC950 txo BW thiab nce tus naj npawb ntawm trophozoites nyob rau hauv plab hnyuv lavage kua nyob rau hauv nas, uas ua rau ntau heev pathological hloov nyob rau hauv duodenal ntaub so ntswg.Alpha-2 giardine thiab alpha-7.3 giardine qhib lub party nas NLRP3 inflammasome, nce nas lub cev hnyav, txo tus naj npawb ntawm trophozoites hauv plab hnyuv lavage kua, thiab alleviate pathological duodenal lesions.Cov txiaj ntsig no qhia tias G. duodenalis tuaj yeem qhib NLRP3 tus tswv tsev inflammasome ntawm alpha-2 giardine thiab alpha-7,3 giardine, txo cov kab mob ntawm G. duodenalis hauv cov nas.
Ua ke, peb cov txiaj ntsig tau pom tias alpha-2 thiab alpha-7.3 giardines ua rau muaj kev ua kom NLRP3 tus tswv tsev inflammasome thiab txo cov kab mob G. duodenalis hauv cov nas.Yog li ntawd, cov molecules yog lub hom phiaj cog lus rau kev tiv thaiv ntawm giardiasis.
       Data supporting the results of this study can be obtained from the respective author at gongpt@jlu.edu.cn.
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