Giardia duodenum bụ parasitic organism nke na-akpata giardiasis, ọrịa eriri afọ nke na-adịkarị na ụmụaka nwere ihe mgbaàmà nke afọ ọsịsa.Anyị na-akọ na mbụ na extracellular G. duodenalis na-akpalite ọrụ nke intracellular oligomerization-dị ka receptor 3 (NLRP3) na-ejikọta nucleotides ma na-achịkwa nzaghachi mkpali nke ndị ọbịa site na nzuzo nzuzo nke extracellular vesicle (EV).Otú ọ dị, kpọmkwem usoro mkpụrụ ndụ ihe nketa nke pathogen-ejikọta duodenococcal EV (GEV) na-etinye aka na usoro a na ọrụ NLRP3 inflammasome na giardiasis ka na-anọgide na-akọwapụta.
Ewubere okwu eukaryotic recombinant plasmids pcDNA3.1 (+) -alpha-2 na alpha-7.3 giardins na GEV, gbanwere n'ime mouse primary peritoneal macrophages, wee chọpụta site na ịlele mbufụt lekwasịrị molekul caspase-1.A na-enyocha ọkwa p20..G. duodenalis alpha-2 na alpha-7.3 giardines bụ nke a chọpụtara na mbụ site na ịlele NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 na caspase-1 p20), IL secretion.1β ọkwa, protein apoptotic spotted (ASC) oligomerization ọkwa, na immunofluorescent localization nke NLRP3 na ASC.A na-enyocha ọrụ nke NLRP3 inflammasome na pathogenicity nke G. duodenalis site na iji ụmụ oke na-egbochi NLRP3 activation (NLRP3 blocked ụmụ oke) na mgbanwe pathological na arọ ahụ, ibu parasitic duodenal, na anụ ahụ duodenal.Tụkwasị na nke ahụ, anyị nyochara ma hiardines alpha-2 na alpha-7.3 na-ebute IL-1β nzuzo na vivo site na NLRP3 inflammasome ma kpebie ọrụ nke ụmụ irighiri ihe ndị a na pathogenicity nke G. duodenalis na ụmụ oke.
Alfa-2 na alfa-7.3 giardines na-ebute mmalite nke NLRP3 inflammasome in vitro.Nke a mere ka ọ rụọ ọrụ nke p20 caspase-1, mmụba na ọkwa nkwupụta nke NLRP3, pro-IL-1β, na pro-caspase-1 protein, mmụba dị ịrịba ama na nzuzo IL-1β, nhazi nke ntụpọ ASA n'ime ahụ. cytoplasm, na ntinye nke ASA oligomerization.NLRP3 mbufụt Penile ọnwụ na-eme ka pathogenicity nke G. duodenalis na ụmụ oke.Ụmụ oke ejiri cysts na-emeso ya site na ụmụ oke NLRP3 egbochiri egosipụtara ọnụ ọgụgụ trophozoites na-arịwanye elu na mmebi dị ukwuu na duodenal villi, nke e ji necrotic crypts na shrunken na alaka.Nnwale na vivo egosila na giardines alpha-2 na alpha-7.3 nwere ike ime ka nzuzo nke IL-1β site na NLRP3 inflammasome, na ịgba ọgwụ mgbochi ọrịa na giardines alpha-2 na alpha-7.3 belatara pathogenicity nke G. duodenalis na ụmụ oke.
Ejikọtara ọnụ, nsonaazụ nke ọmụmụ a na-atụ aro na giardia alpha-2 na alpha-7.3 na-eme ka mmụba nke onye ọbịa NLRP3 mbufụt ma belata infectivity nke G. duodenalis na ụmụ oke, bụ ndị na-ekwe nkwa ebumnuche maka igbochi giardiasis.
Giardia duodenum bụ parasaiti protozoan extracellular nke bi na eriri afọ ma na-ebute ọrịa giardiasis nde 280 na afọ ọsịsa kwa afọ, ọkachasị n'etiti ụmụaka na mba ndị ka na-emepe emepe [1].Ndị mmadụ na-ebute ọrịa site na mmiri ọñụñụ ma ọ bụ ihe oriri nke nwere M. duodenum cysts, nke na-abanye n'ime afọ wee wepụ ya na mmiri gastric.Giardia duodenum trophozoites na-etinye aka na epithelium duodenal, na-akpata ọgbụgbọ, ọgbụgbọ, afọ ọsịsa, mgbu afọ, na mbelata ibu.Ndị nwere immunodeficiency na cystic fibrosis nwere ike ibute ọrịa.Ọrịa nwekwara ike ime site na ọnụ na gbasara ike mmekọahụ [2].Ọgwụ dị ka metronidazole, tinidazole, na nitazoxanide bụ nhọrọ ọgwụgwọ kachasị mma maka ọrịa duodenal [3].Otú ọ dị, ọgwụ ndị a chemotherapy na-akpata mmetụta ọjọọ dị ka ọgbụgbọ, carcinogenesis, na genotoxicity [4].Ya mere, ọ dị mkpa ka e mepụta atụmatụ dị irè karị iji gbochie ọrịa G. duodenalis.
Inflammasomes bụ klaasị nke ogige protein cytosolic bụ akụkụ nke nzaghachi mgbochi ebumpụta ụwa, na-enyere aka ịgbachitere mbuso agha nje na mezie nzaghachi mkpali [5].N'ime ihe ndị a na-egbuke egbuke, nucleotide-binding oligomerization (NOD) 3 (NLRP3) nucleotide-binding oligomerization (NLRP3) nucleotide-binding-like inflammasome ka amụchara nke ọma n'ihi na enwere ike ịhụ ya site n'ụdị dị iche iche nke nje pathogen / mmebi-metụtara (PAMP/). DAMP), na-amata, na-arụ ọrụ usoro ahụ ji alụso ọrịa ọgụ.ma na-achịkwa homeostasis intestinal n'ọtụtụ ọrịa mkpali [6,7,8].Ọ na-agụnye ihe nnabata nnabata (PRR) NLRP3, ihe nkwụnye ihe nkwụnye ntụpọ apoptotic (ASC), yana procaspase-1 ma ọ bụ procaspase-11.NLRP3 inflammasome na-eme dị ka onye ọbịa megide mbuso agha pathogen, dị ka a hụrụ na Neospora caninum [9], Paracoccidioides brasiliensis [10], na ọmụmụ Leishmania.[11], ma a kọwokwa na ịgbalite NLRP3 inflammasome na-egbochi nzaghachi mgbochi nchebe ma na-eme ka ọganihu ọrịa dịkwuo njọ, dịka ọmụmaatụ, na ikpuru [12].Dabere na nchoputa anyị gara aga, anyị kọrọ na extracellular G. duodenalis na-akpalite intracellular activation nke NLRP3 mbufụt na modulates host mkpali nzaghachi site secreting extracellular vesicles (EVs) [13].Otú ọ dị, ọrụ nke NLRP3 inflammasome na G. duodenalis ọrịa na vivo ka ga-ekpebi.
A kọwara Giardins na mbụ dị ka ihe nhazi nke G. duodenalis cytoskeleton ma na-arụ ọrụ dị mkpa na motility trophozoite na ntinye sel epithelial na eriri afọ.Iji mee ka ọ dịkwuo mma na gburugburu ebe obibi ma mee ka ọrịa ha dịkwuo elu, G. duodenalis trophozoites mepụtara usoro cytoskeletal pụrụ iche nke nwere 8 flagella, 1 n'etiti ahụ, na 1 ventral disc [14].Ndị trophozoites nke Giardia duodenum na-eji cytoskeleton ha banye n'ime eriri afọ nke elu, karịsịa duodenum, ma tinye ya na enterocytes.Ha na-akwaga mgbe niile ma na-etinye aka na sel epithelial site na iji cell metabolism.Ya mere, enwere mmekọrịta chiri anya n'etiti cytoskeleton ha na virulence.Giardine ndị akọwapụtara maka Giardia duodenum bụ akụkụ nke nhazi cytoskeleton [15] ma kewara ya na klas anọ: α-, β-, γ-, na δ-giardines.Enwere ndị otu 21 nke ezinụlọ α-giardin, ha niile nwere ikike dabere na calcium iji kekọta phospholipids [16].Ha na-ejikọta cytoskeleton na akpụkpọ ahụ cell.N'ime ndị nwere afọ ọsịsa nke G. duodenalis kpatara, α-giardins na-egosipụta nke ọma na immunoreactive n'oge ọrịa [17].Ọgwụ mgbochi dị iche iche dabere na Giardia alfa-1 chebere megide giardiasis na ụmụ oke ma bụrụ ndị nwere ike ịbụ antigens maka mmepe ịgba ọgwụ mgbochi [18].Alfa-8 giardin, nke dị na akpụkpọ ahụ plasma na flagella, ma ọ bụghị na diski ventral, na-eme ka motility na uto nke trophozoites na G. duodenalis [19].Alfa-14 giardin na-ejikọta na microtubule owuwu na flagella ma na-emetụta viability nke G. duodenalis [20].Alfa-11 giardine dị n'ụba n'oge ndụ ya niile, na nkwuwa okwu nke alfa-11 giardine na-emebi G. duodenalis n'onwe ya [21].Otú ọ dị, amabeghị ma alpha-2 giardine na alpha-7.3 giardine na-echebe megide ọrịa G. duodenalis na usoro ha dị n'okpuru.
N'ime ọmụmụ ihe a, recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine na pcDNA3.1 (+) -alpha-7.3 giardine gbanwere n'ime mouse primary peritoneal macrophages iji rụọ ọrụ NLRP3.A na-enyocha ihe ndị na-ere ọkụ.Anyị na-enyochakwa ọrụ nke NLRP3 inflammasome na pathogenicity nke G. duodenalis, nyochara ma alpha-2 na alpha-7,3 giardines ịkpalite activation nke NLRP3 inflammasome na vivo, na kpebisiri ike na abụọ ndị a ọrụ nke giardines na pathogenicity nke. G. duodenal.Ebumnobi anyị na-emekarị bụ ịzụlite ebumnuche ndị na-ekwe nkwa maka igbochi ọrịa G. duodenalis.
Ụdị anụ ọhịa (WT) C57BL/6 ụmụ oke nwanyị gbara izu 5–8 ka zụtara n'aka Liaoning Changsheng Experimental Animal Center (Liaoning, China).Ụmụ oke nwere ohere ịnweta mmiri n'efu, nata nri gbasiri ike ma debe ya na ọkụ elekere 12/12 / ọchịchịrị.Tupu ọrịa ahụ, ụmụ oke natara ọgwụ nje ad libitum n'ime mmiri ọ drinkingụ drinkingụ agbakwunyere na ampicillin (1 mg / mL), vancomycin (1 mg / mL), na neomycin (1.4 mg / mL) (nke a zụtara na Shanghai, China, ihe ndị na-emepụta ihe) [22] ].].Ụmụ oke ndị enweghị ike iri ihe na ịṅụ ihe ọṅụṅụ ruo> awa 24 ma tụfuo ≥ 20% arọ ahụ bụ ihe na-eme ka mmadụ ghara ịdị n'otu site na mgbapụ cervical.
WB G. duodenalis trophozoites (Ụdị Ụdị Omenala America, Manassas, USA) ka ejiri 12.5% ​​serum bovine bovine (FBS; Green ọ bụla, Zhejiang, China) na 0.1% bovine bile (Sigma-Aldrich, St. Missouri, USA) gbakwunyere. ).USA) n'okpuru ọnọdụ microaerobic.A na-anakọta trophozoites confluent na ice wee gafere na nha nke 1: 4 maka mmụba ọzọ.
A kpalitere cysts duodenum giardia dị ka akọwara na mbụ [23], a na-egbute trophozoites na oge logarithmic wee gwakọta ya na encapsulation inducing medium, pH 7.1 (gbanwetụrụ TYI-S-33) na ntinye ikpeazụ nke 1 × 106 trophozoites/mL.ntinye uche bile 0.05% ọkara).Azụlitere Trophozoites n'okpuru ọnọdụ anaerobic na 37 Celsius ruo oge uto logarithmic.Gbanwee usoro ka ọ bụrụ ihe na-eme ka ọkpụkpụ na-eme ka ọkpụkpụ (pH 7.8; gbanwee TYI-S-33 na 1% bile focus) na omenala G. duodenalis na 37 ° C maka awa 48-96, n'oge nke a na-ahụ cysts nguzobe n'okpuru microscope.Mgbe ọtụtụ n'ime trophozoites a kpalitere ka ha ghọọ cysts, a na-egbute ngwakọta omenala ahụ ma tinyeghachi ya na mmiri na-adịghị mma iji lyse trophozoites fọdụrụnụ.A na-agụta cysts ma chekwaa ya na 4 Celsius C maka nyocha ndị na-esote site na tube gastric na ụmụ oke.
Giardia extracellular vesicles (GEVs) ka emelitere dị ka akọwara na mbụ [13].Trophozoites na oge uto logarithmic ka eweghachiri na mgbanwe TYI-S-33 akwadoro na FBS exosome depleted (Industries Biological, Beit-Haemek, Israel) na ntinye ikpeazụ nke 1 × 106 parasites / mL wee tinye ya maka awa 12.e kewapụrụ ya na ndị na-emepụta omenala site na centrifugation na 2000 g maka 10 min, 10,000 g maka 45 min, na 100,000 g maka 60 min.A gbazere ebili mmiri na phosphate buffered saline (PBS), nke agbaziri site na iji ngwa nyocha protein BCA (Thermo Fisher Scientific, Waltham, MA, USA) wee chekwaa ya na -80° C. ma ọ bụ jiri ya mee nyocha ọzọ.
A kwadebere macrophages peritoneal òké mbụ dị ka akọwara na mbụ [24].Na nkenke, a gbara ụmụ oke (ndị dị afọ 6-8 izu) (intrapereritoneally [ip]) na 2.5 ml nke 2.98% Difco liquid thioglycol medium (BD, Franklin Lakes, NJ, USA) wee nye 3-4 palates.A na-anakọta nkwusioru nke macrophages site na oghere afọ nke ụmụ oke mgbe euthanasia na centrifuged ugboro 3 na 1000 g maka 10 min.Achọpụtara mkpụrụ ndụ ewepụtara site na cytometry na-asọpụta site na iji akara CD11b ruo mgbe ịdị ọcha cell dị> 98%, wee tinye ya na 6-nke ọma cell plate plate (sel 4.5 x 106 / ọma) wee tinye ya na 10% FBS (Bioindustry) na 37°C.na 5% CO2.
A na-ewepụta RNA site na 1 × 107 trophozoites na 1 ml nke TRIzol reagent (Vazyme, Nanjing, China), DNA nke genomic bụ site na mkpokọta G. duodenalis RNA site na iji MonScript dsDNase (Monad, Wuhan, China) na DNA nkwado (cDNA) na-arụkọ ọrụ. iji MonScript RTIIII Super Mix (Monad) dịka ntuziaka onye nrụpụta siri dị.
E nwetara ozi usoro CDS maka ebumnuche G. duodenalis site na NCBI GenBank.Jiri Primer 5.0 chepụta primers na-enweghị ntụpọ maka mkpụrụ ndụ ihe nketa ọ bụla.The n'ihu primer (5'-3′) nwere akụkụ atọ: usoro na-agafe agafe na vector pcDNA3.1 (+) EcoRV (TGGTGGAATTCTGCAGAT) wee malite codons ATG na GNN (ọ bụrụ na ntọala mbụ abụghị G).A na-eme nke a iji melite arụmọrụ nke okwu ahụ.Na mgbakwunye, ọ dịkarịa ala 16 bp jikọtara ntọala (ọdịnaya GC 40-60% / Tm ihe ruru 55 Celsius C).The reverse primer (5′-3′) nwere akụkụ abụọ, usoro na-agafe agafe nwere vector pcDNA3.1 (+) (GCCGCCACTGTGCTGAT) yana ntọala jikọtara ma ọ dịkarịa ala 16 bp.(ewezuga nkwụsị abụọ ikpeazụ).bases) codon dị ka AA ma ọ bụ GA iji kwe ka plasmids recombinant kwupụta protein ha akpọrọ).Edepụtara usoro izizi na Tebụl 1 ma bụrụ nke Kangmet Biotechnology Co., Ltd. (Changchun, China) mebere ya.
A na-eme ka ebumnuche dị elu site na iji Pfu DNA polymerase (Tiangen, Beijing, China) ma ọ bụ Ex-taq (Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, China) site na iji G. duodenalis cDNA a kwadebere dị ka ndebiri.Eukaryotic okwu vector plasmid pcDNA3.1(+) ka ejiri mmachi enzyme EcoRV na dephosphorylated jiri ngwa ngwa AP (Thermo Fisher Scientific).A na-asachapụ iberibe pcDNA3.1 (+) linearized na mpịkọ mkpụrụ ndụ ihe nketa agbakwunyere site na iji DNA gel purification kit (Tiangen) wee tụọ ya site na iji Nanodrop ND-2000 (Thermo Fisher Scientific).A na-ejikọta iberi pcDNA3.1 (+) na mpempe mkpụrụ ndụ nke ọ bụla ezubere iche site na iji MonClone otu cloning mix (Monad Biotech Co., Ltd., Suzhou, China) ma kwadoro usoro DNA site na iji Comate Bioscience Company Limited (Changchun, China) ..
pcDNA3.1(+) -alpha-2 na pcDNA3.1(+) -alpha-7.3 bụ nke emere site na iji Plasmid Mini Kit (Sangon Biotech) na-enweghị SanPrep Endotoxin.A na-edobe ntinye uche n'elu 500 ng/µl iji hụ na EDTA na ebe nchekwa elution egbochighị nyocha ntụgharị.A na-emepụta macrophages nke mbụ nke peritoneal mouse na efere 6-nke nwere ọkara RPMI 1640 zuru ezu (Industries Biological) maka awa 12, mgbe ahụ, a na-asacha mkpụrụ ndụ ugboro 3 na PBS na-ekpo ọkụ iji wepụ penicillin na streptomycin, wee na ọkara gbakwunyere na ọkara zuru ezu.plasmids na-enweghị Endotoxin pcDNA3.1 (+) -alpha-2 na pcDNA3.1 (+) -alpha-7.3 (2.5 μg) na 125 μl nke Opti-MEM belatara ọbara ọbara (Gibco, Thermo Fisher Scientific) ..Mgbe ahụ, 5 µl nke Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific) ka agbazere na 125 µl nke obere ọbara Opti-MEM.Kwadebe ogige liposome-DNA site na ịgwakọta plasmid na-enweghị endotoxin na Lipofectamine 2000 na ikwe ka ngwakọta guzoro n'ime ụlọ maka nkeji ise.Nyefee ogige ndị ahụ iche na sel n'ime olulu mmiri ọ bụla wee gwakọta nwayọọ nwayọọ.Mgbe awa 4 gachara, a na-eji 2 ml nke RPMI 1640 zuru ezu dochie usoro omenala cell na omenala na-aga n'ihu ruo awa 24.Agbakwunyere usoro omenala ọhụrụ n'ime mkpụrụ ndụ ma tinye ya maka oge dị iche iche dabere na nhazi nyocha.
A kwadebere ihe nlele protein sitere na supernatants na cell lysates dị ka akọwara na mbụ [25].Usoro mbufe membrane maka pro-IL-1β, pro-caspase-1, caspase-1 p20, NLRP3, β-actin, na mkpado Ya bụ 200 mA/90 min.Maka interleukin 1β (IL-1β; R & D Systems, Minneapolis, Minnesota, USA), caspase-1 (p20) (Adipogen, Switzerland) na NLRP3 (Adipogen SA, Epalinges, Switzerland) na 1: 5000 na-ezubere mkpado Ya (Amylet Scientific, Wuhan, China) na β-actin (Proteintech, Wuhan, China).
Cross-linking na disuccinimide suberate (DSS) mere dị ka akọwara na mbụ [26].A na-asacha mkpụrụ ndụ ugboro 3 na PBS oyi ma jiri ntụ ntụ 27 mee ya kpamkpam na 50 µl ASC mmeghachi omume mmeghachi omume (pH 8.0) nwere 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES na 125 mM NaHCO3.A na-etinye ngwakọta ahụ na 5000 g maka 3 min ma jiri 10 µl DSS (25 mM na DMSO) kpuchie pellet ahụ na 40 µl ASC nzaghachi mmeghachi omume maka 30 min na 37 ° C.Mgbe centrifugation na 5000 g maka 10 min, a gbazere pellet na ngwọta nke 40 µl nke ASC mmeghachi omume buffer na 10 µl nke 6x protein loading buffer (TransGen, Beijing, China), na mgbe ahụ, a kwụsịrị ngwọta na ụlọ okpomọkụ maka 15. min., Mgbe ahụ, sie minit 10.A na-edobe ihe nlele protein n'ọdịdị ọdịda anyanwụ site na iji ọgwụ mgbochi ASC bụ isi (Wanleibio, Shenyang, China) na nha nke 1:500.
N'ịgbaso usoro akọwara na mbụ [13], a na-egbute ndị na-ahụ maka omenala cell na nzuzo nke cytokine pro-inflammatory IL-1β kpebiri site na iji ngwa IL-1 Beta ELISA (Invitrogen, Thermo Fisher Scientific).Tụgharịa ụkpụrụ OD450nm ka ọ bụrụ mkpokọta protein site na iji usoro ọkọlọtọ IL-1β.
A na-asacha mkpụrụ ndụ ndị a na-ekpuchi na mkpuchi na-eji nwayọọ 3 ugboro na PBS na-ekpo ọkụ, na-edozi ya na cell fixative (Biosharp, Beijing, China) maka 10 min na ụlọ okpomọkụ (RT), na 0.1% Triton X-Permeabilize na 100 (diluted na PBS; Biosharp). ) maka nkeji 20 n'ime ụlọ okpomọkụ ma gbochie 5% bovine serum albumin (na PBS) maka awa 2 na ụlọ okpomọkụ.A na-etinye mkpụrụ ndụ n'otu abalị na 4 Celsius C na ọgwụ mgbochi mbụ megide ASC (1: 100 dilution) ma ọ bụ NLRP3 (1: 100 dilution), n'otu n'otu, na Cy3 akpọrọ ewu mgbochi rabbit IgG (H + L) (1:400; EarthOx). , San Francisco, CA, USA) ma ọ bụ FITC-conjugated ewu mgbochi òké IgG (1:400; Earthox) n'abali na 37 Celsius C n'ọchịchịrị maka 1 hour.Ejiri Hoechst 33258 (10 μg/ml; UE, Suzhou, China) nwere ntụ maka nkeji 5 wee hụ n'okpuru microscope fluorescence (Olympus Corporation, Tokyo, Japan).
E kewara oke oke n'ime otu anọ (n = 7 na otu ọ bụla): (i) PBS-emeso ndị na-adịghị mma nchịkwa (PBS naanị; gavage 100 µl / òké PBS sochiri kwa ụbọchị intraperitoneal injection 100 µl / òké PBS 3 awa mgbe e mesịrị) ., na-aga n'ihu maka ụbọchị 7);(ii) otu njikwa na-adịghị mma na MCC950 inhibitor [27] (100 µl / òké site na PBS gavage, 3 awa mgbe e mesịrị, 10 mg / kg arọ ahụ [BW] MCC950 [na PBS] na-enye intraperitoneally kwa ụbọchị, oge 7 ụbọchị);(iii) G. duodenalis cyst ọrịa otu (1.5 x 106 cysts / òké site gavage, 3 awa mgbe e mesịrị, 100 μl / òké PBS intraperitoneally ndinọ kwa ụbọchị maka 7 ụbọchị);(iv) G. duodenalis cyst jikọtara ọrịa otu MCC950 inhibitor ọgwụgwọ otu (1.5 × 106 cysts / òké via gavage, 10mg / kg ahu arọ MCC950 intraperitoneally kwa ụbọchị maka 7 ụbọchị na 3h).A na-enyocha ịdị arọ nke òké ọ bụla kwa ụbọchị, a na-ewepụkwa ụmụ oke niile n'ụbọchị nke asaa.A na-egbutu duodenum (3 cm ogologo) n'ime obere iberibe na 1 ml PBS, a na-ebibi cysts n'otu abalị na PBS na 4 Celsius C, na G. duodenalis trophozoites.Duodenum ọhụrụ (ogologo 1 cm) dịpụrụ adịpụ maka hematoxylin na eosin (H&E).
E kewara oke oke ụzọ abụọ: (i) otu njikwa MOCK na (ii) MCC950 inhibitor otu.Enwere ọgwụgwọ ise n'ime otu ọ bụla (n = 7 / otu ọgwụgwọ): (i) PBS ọgwụgwọ ọjọọ nchịkwa (PBS naanị; 100 µl / òké PBS, intramuscular (IM) injection (tibialis anterior) [28, 29];( ii) pcDNA3.1 (+) plasmid ọjọọ akara otu (100 µg / òké DNA, site intramuscular injection); Otu a na-emeso ya na plasmid pcDNA3.1 (+) -alpha-2 (100 μg/mouse DNA, site na intramuscular injections), na (v) otu a na-eji plasmid pcDNA3.1 (+) -alpha-7.3 (100 μg/mouse) DNA, mgbe awa iri na abụọ gafechara, ụmụ oke na MCC950 inhibitor otu natara ọgwụ intraperitoneal kwa ụbọchị nke MCC950 (10 mg / kg arọ ahụ) maka ụbọchị 7, ebe ụmụ oke na MOCK natara nha nha nke ọgwụgwọ PBS. A na-enyocha ọbara ọbara. anakọtara site na ụmụ oke anya wee hapụ otu abalị na 4 Celsius C, a na-ekewapụta ihe nlele ọbara site na iji immunosorbent assay (ELISA) jikọtara ọnụ maka na nha ọkwa IL-1β.
E kewara ụmụ oke iri atọ na ise ka ọ bụrụ otu ise (n=7/otu).Otu 1 bụ otu njikwa na-adịghị mma ejiri PBS mesoo: ụmụ oke natara 100 μl PBS intramuscularly na ụbọchị 3 ka e mesịrị site na gavage.Otu 2 bụ otu njikwa dị mma na-ebute G. duodenalis cysts: a na-agbanye ụmụ oke na 100 μl nke PBS, na ụbọchị 3 ka e mesịrị 1.5 x 106 cysts / òké na-agbanye n'ime intragastrically.Otu nke atọ - ọgwụ mgbochi plasmid na pcDNA3.1 (+) jikọtara ya na otu njikwa maka ọrịa duodenal cyst ọrịa: ụmụ oke natara 100 μg nke plasmid DNA pcDNA3.1 (+) (im) ọnụ, 1.5 × 106 cysts / òké 3 maka ọtụtụ. ụbọchị.Otu 4 na 5 bụ recombinant pcDNA3.1 (+) -alpha-2 giardine plasmid ma ọ bụ pcDNA3.1 (+) -alpha-7.3 giardine plasmid na ngwakọta G. duodenalis cyst ọrịa.Otu nnwale: ụmụ oke natara 100 μg nke pcDNA3.1 (+) -giardine plasmid DNA (im), mgbe ụbọchị 3 gachara, a na-agbanye 1.5 × 106 cysts / òké site na gavage.A na-enyocha ịdị arọ nke òké ọ bụla mgbe ntinye nke G. duodenalis cyst site na tube.A nakọtara duodenum ọhụrụ maka nha ibu parasitic yana nyocha ntụpọ HE.
A na-enyocha mgbanwe akụkọ ihe mere eme dịka usoro e bipụtara na mbụ [30].A na-edozi duodenum ọhụrụ ya na cell fixative, agbakwunyere na paraffin, bee n'ime akụkụ 4 μm, tinye ya na H&E wee nyochaa n'okpuru microscope ọkụ.A na-enyocha mgbanwe mgbanwe ndị na-anọchi anya na akụkụ anụ ahụ asaa sitere na ụmụ oke asaa nọọrọ onwe ha site n'aka onye na-ahụ maka ọrịa na-amaghị ọgwụgwọ ahụ ma jide ya na 200x magnification.A na-atụle ogologo villi na omimi nke crypts dị ka usoro ndị a kọwara na mbụ.
E nwetara nsonaazụ in vitro na in vivo na ugboro atọ.Emepụtara eserese site na iji GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, USA).A na-enyocha ọdịiche dị n'etiti otu abụọ site na t-ule, ebe a na-enyocha ọdịiche dị n'etiti ≥3 otu site na nyocha nke otu ụzọ (ANOVA) site na iji software SPSS (mbipute 22.0; SPSS IBM Corp., Armonk, NY, USA).A na-enyocha data maka ịdị n'otu nke ọdịiche site na iji ule Levene nke Bonferroni's post hoc test (B) sochiri.A na-akọwapụta mkpa dị ka P <0.05, P<0.01, na P<0.001 (adịghị mkpa [ns]) (P> 0.05).
Nyocha anyị gara aga nke proteomics GEV na Kyoto Encyclopedia of Genes and Genomes (KEGG) gosiri na ọtụtụ ebumnuche nwere ike itinye aka na ntinye nke ụzọ mgbaàmà ọkụ ọkụ [13].Anyị họrọ ebumnuche abụọ na-ekwe nkwa, alfa-2 na alfa-7.3 giardins, kwalite ụmụ irighiri ihe ndị a wee jiri ha wuo pcDNA3.1(+) eukaryotic expression vector.Mgbe usoro, recombinant pcDNA3.1 (+) -alpha-2 na alpha-7.3 giardine okwu plasmids gbanwere n'ime isi mouse peritoneal macrophages, na caspase-1 p20 mbinye aka protein nke mbufụt (iberibe nke arụ ọrụ caspase-1) amata. dị ka elucidating isi ụmụ irighiri ihe nwere ike ịkpalite mbufụt.Nsonaazụ gosiri na alfa-2 na alfa-7.3 giardines nwere ike ime ka p20 caspase-1 okwu yiri GEV.Enweghị mmetụta na ịgbalite caspase-1 na njikwa adịghị mma na-adịghị edozi (PBS naanị) na pcDNA3.1 (+) plasmid (Nkeji 1).
Ntụle nke p20 caspase-1 ịgbalite site na pcDNA3.1(+) -alpha-2 na alfa-7.3 giardins.Ebufere okwu eukaryotic recombinant plasmids pcDNA3.1 (+) -alpha-2 na alpha-7.3 giardines (n'elu ụzọ nke ọ bụla) n'ime macrophages bụ isi nke òké peritoneal macrophages nke omenala bụ ihe a na-egbute mgbe awa 24 gachara.A na-eji blọgụ Western iji tụọ ọkwa ngosipụta nke protein caspase-1 p20 na-ere ọkụ.A na-eji PBS-naanị ọgwụgwọ otu (lane C) na pcDNA3.1 (+) monotherapy otu (pcDNA3.1 lane) dị ka njikwa na-adịghị mma, a na-ejikwa otu ọgwụgwọ GEV mee ihe dị ka njikwa dị mma.A kwadoro nkwupụta nke protein recombinant site na ịchọta mkpado histidine na protein ọ bụla, na eriri protein a na-atụ anya ya bụ alpha-2 giardine (38.2 kDa) na alpha-7.3 giardine (37.2 kDa).GEV, Giardia duodenum extracellular vesicles, pcDNA3.1 (+), EcoRV-linearized vector, SUP, supernatant
Iji chọpụta ma alpha-2 giardine na alpha-7.3 giardine na-ebute p20 caspase-1 okwu ma na-ekere òkè n'ịgbalite nzaghachi mkpali NLRP3 onye ọbịa, pcDNA3.1 (+) -alpha-2 giardine na pcDNA3.1 (+) -alpha -7.3 giardin gbanwere n'ime macrophages peritoneal macrophages bụ isi na plasmid DNA recombinant, na ọkwa nke okwu, nhazi, na oligomerization nke protein protein NLRP3 bụ isi.N'ime nnwale a, a na-eji GEV dị ka ndị na-ahụ maka njikwa dị mma, na enweghị otu ọgwụgwọ (PBS naanị) ma ọ bụ pcDNA3.1 (+) ọgwụgwọ ntụgharị bụ otu ọjọọ.Nsonaazụ gosiri na, dị ka ọ dị na GEV otu, recombinant plasmid DNA nke giardin pcDNA3.1 (+) -alpha-2 na giardin pcDNA3.1 (+) -alpha-7.3 rụpụtara n'elu NLRP3, pro-IL-1β na procaspase-1 na caspase-1 ịgbalite (Fig. 2a).Tụkwasị na nke ahụ, ma giardines abụọ kpalitere ihe nzuzo IL-1β dị ịrịba ama (pcDNA3.1: ANOVA, F (4, 10) = 1.625, P = 0.1000; alpha-2 giardine: ANOVA, F (4, 10) = 1.625, P = 0.0007 ).;alfa-7.3 giardine: ANOVA, F (4, 10) = 1.625, P<0.0001;GEV: ANOVA, F (4, 10) = 1.625, P = 0.0047) (Foto 2b).Ọtụtụ ndị na-edozi ASC bụ monomeric n'ime ndị na-adịghị agwọ ọrịa ma ọ bụ n'ime usoro ọgwụgwọ a gbanwere na pcDNA3.1 (+) plasmid, n'ụzọ dị iche na pcDNA3.1 (+) -alpha-2 ma ọ bụ pcDNA3.1 (+) -alpha- 7.3 giardine.ASC oligomerization mere na recombinant plasmid DNA nke GEV nti njikwa ma ọ bụ otu, na-egosi ụdị oligomeric (Nyocha 2c).Ihe omuma nke mbu a na-egosi na alpha-2 giardine na alpha-7,3 giardine nwere ike ime ka NLRP3 rụọ ọrụ mbufụt.Ọmụmụ ihe ọmụmụ immunofluorescent sochirinụ nke mpaghara ASC na NLRP3 gosiri na n'ime otu njikwa na-adịghị mma, protein ASC gbasasịa na cytoplasm ma pụta dị ka akara ntụpọ n'elu mkpali nke pcDNA3.1 (+) -alpha-2 na giardine ma ọ bụ pcDNA3.1 (+) -alpha-7,3 giardine otu ma ọ bụ GEV mma akara otu (Nyocha 2d).Na njikwa na-adịghị mma na plasmid-emeso pcDNA 3.1 otu, achọpụtaghị akara protein NLRP3, ebe akara ngosi fluorescent na nzaghachi na pcDNA3.1 (+) -alpha-2 giardine ma ọ bụ pcDNA3.1 (+) - alpha-7.3 achọpụtara..A na-ahụ giardine na cytoplasm ma ọ bụ na mkpali nke HEV (Fig 2e).Ihe data ndị a na-egosikwa na G. duodenalis giardin alpha-2 na giardin alpha-7.3 na-arụ ọrụ NLRP3 inflammasome na mouse primary peritoneal macrophages.
pcDNA3.1 (+) -alpha-2 giardin na pcDNA3.1 (+) -alpha-7.3 giardin na-arụ ọrụ NLRP3 inflammasome na mouse peritoneal macrophages.Nyefee okwu eukaryotic recombinant plasmids pcDNA3.1 (+) -alpha-2 giardin na pcDNA3.1 (+) -alpha-7.3 giardin n'ime murine peritoneal macrophages na sel, ma ọ bụ na-ewe ihe dị elu n'ime 24 h maka nyocha nke okwu, oligomerization. , nzuzo.na localization nke isi mkpali protein.A na-eji otu PBS-naanị (C) na pcDNA3.1 (+) otu usoro ọgwụgwọ dị ka njikwa na-adịghị mma, a na-ejikwa otu GEV ọgwụgwọ dịka otu dị mma.a Key mkpali proteins NLRP3, gụnyere NLRP3, pro-IL-1β, pro-caspase-1, na p20 caspase-1, achọpụtara site Western blotting.b A na-ekpebi ọkwa nke nzuzo nke IL-1β n'ime ihe ndị dị elu site na iji enzyme-linked immunosorbent assay (ELISA).A tụlere ọdịiche dị n'etiti njikwa na otu nnwale site na nyocha otu ụzọ nke iche iche (ANOVA) site na iji ụdị ngwanrọ SPSS 22.0.Asterisks na-egosi ọdịiche dị ukwuu n'etiti otu **P<0.01 na ***P <0.001.c ASC oligomerization ọkwa na pellets kpebisiri ike site na nyocha njikọ njikọta nke DSS, ebe a na-eji ọkwa ASC na cell lysates dị ka njikwa nbudata.d Nleta anya nke mpaghara ISC site na iji immunofluorescence.e ji Immunofluorescence mee ihe iji were anya nke uche hụ nhazi nke NLRP3.ASC, protein apoptotic speck;IL, interleukin;NLRP3, nucleotide-binding oligomerization-like receptor 3;ns, ọ bụghị ihe dị mkpa (P> 0.05)
Ma G. duodenalis na GEVs ọ na-ezoro ezo na-arụ ọrụ NLRP3 inflammasome ma na-achịkwa nzaghachi mkpali ndị ọbịa na vitro.Ya mere, ọrụ nke NLRP3 inflammasome na pathogenicity nke G. duodenalis ka edoghị anya.Iji nyochaa okwu a, anyị mere nnwale n'etiti ụmụ oke na-ebute G. duodenalis cyst na ụmụ oke na-ebute G. duodenalis cyst + MCC950 inhibitor ọgwụgwọ ma tụnyere NLRP3 inflammasome okwu mgbe ọrịa G. duodenalis cyst.E gosipụtara atụmatụ zuru ezu nke nnwale ahụ na Fig. 3a.A na-enyocha mgbanwe na oke ahụ nke ụmụ oke na otu ọgwụgwọ dị iche iche maka ụbọchị 7 mgbe ọrịa na cysts gasịrị, a na-egosipụtakwa nsonaazụ ya na Fig. 3b.E jiri ya tụnyere otu a na-emeso ya na PBS dị ọcha, nsonaazụ gosiri na (i) arọ ahụ nke ụmụ oke na-ebute G. duodenalis cyst belatara site na ụbọchị 3 ruo ụbọchị 7 mgbe ọrịa gasịrị;(ii) ọgwụgwọ na MCC950 inhibitor enweghị mmetụta dị ukwuu n'ịdị arọ ahụ nke ụmụ oke..E jiri ya tụnyere otu ọrịa na-efe efe, BW nke otu ọrịa duodenal nke a na-emeso ya na MCC950 belatara ruo ogo dị iche iche (Ụbọchị 1: ANOVA, F (3, 24) = 1.885, P = 0.0148; Day 2: ANOVA, F (3, 24) ) = 0.4602, P <0.0001; Ụbọchị 3: ANOVA, F (3, 24) = 0.8360, P = 0.0010; Ụbọchị 4: ANOVA, F (3, 24) = 1.683, P = 0.0052; F. 5: A (3, 24) = 0.6497, P=0.0645; Ụbọchị 6: ANOVA, F (3, 24)=5.457, P=0.0175; Ụbọchị 7: ANOVA, F(3, 24) = 2.893, P = 0.0202).Data ndị a na-egosi na NLRP3 inflammasome na-echebe ụmụ oke site na oke ibu dị arọ na mmalite mmalite (2-4 ụbọchị) nke ọrịa duodenal.Mgbe ahụ, anyị na-achọ ịchọpụta G. duodenalis trophozoites na duodenal lavage fluid na nsonaazụ na-egosi na Figure 3c.E jiri ya tụnyere G. duodenalis cyst ọrịa otu, ọnụ ọgụgụ nke trophozoites na duodenum abawanyela nke ukwuu mgbe igbochi NLRP3 inflammasome (t (12) = 2.902, P = 0.0133).Anụ ahụ duodenal nwere ntụpọ HE gosiri, ma e jiri ya tụnyere njikwa na-adịghị mma na PBS na MCC950 naanị: (i) G. duodenalis cyst ọrịa butere mmebi nke duodenal villi (ANOVA, F(3, 24) = 0.4903, P= 0.0488 ) na crypt atrophy (ANOVA, F (3, 24) = 0.4716, P = 0.0089);(ii) duodenum sitere na ụmụ oke butere G. duodenalis cysts wee mesoo ya na ihe mgbochi MCC950.duodenal villi mebiri ma nwụọ (ANOVA, F (3, 24) = 0.4903, P = 0.0144) na atrophy na crypt branching (ANOVA, F (3, 24) = 0.4716, P = 0, 0481) (Fig. 3d- f) .Nsonaazụ ndị a na-egosi na NLRP3 inflammasome na-ekere òkè n'ibelata pathogenicity nke G. duodenalis.
Ọrụ NLRP3 inflammasome na ọrịa Giardia duodenum.A na-eji cysts duodenococcal kpuchie ụmụ oke (iv) wee mesoo ya ma ọ bụ na-enweghị MCC950 (ip).A na-eji otu ọgwụgwọ otu nwere PBS ma ọ bụ MCC950 dịka njikwa.Otu nnwale na usoro ọgwụgwọ.b A na-enyocha oke ahụ nke ụmụ oke na nke ọ bụla n'ime usoro ọgwụgwọ dị iche iche maka ụbọchị 7.A na-enyocha ihe dị iche n'etiti G. duodenalis ọrịa otu na G. duodenalis + MCC950 ọrịa ọgwụgwọ otu site t-ule iji SPSS software version 22.0.Asterisks na-egosi nnukwu ọdịiche na * P <0.05, ** P <0.01, ma ọ bụ *** P <0.001.c Ekpebiri ibu parasitic site n'ịgụ ọnụ ọgụgụ trophozoites na mmiri mmiri duodenal lavage.A na-enyocha ihe dị iche n'etiti G. duodenalis ọrịa otu na G. duodenalis + MCC950 ọrịa ọgwụgwọ otu site t-ule iji SPSS software version 22.0.Asterisks na-egosi nnukwu ọdịiche na * P <0.05.d Hematoxylin na eosin (H&E) nsonaazụ ntụpọ nke duodenal histopathology.Akụ na-acha uhie uhie na-egosi mmebi nke villi, akụ ndụ ndụ na-egosi mmebi nke crypts.Ogwe ọnụ ọgụgụ: 100µm.e, f Statistical analysis of duodenal villus elu na òké crypt elu.Asterisks na-egosi nnukwu ọdịiche na * P <0.05 na ** P <0.01.A na-ewepụta nsonaazụ ya site na nnwale 7 nọọrọ onwe ya.BW, ibu ahụ;ig, ụzọ nnyefe intragastric;ip, ụzọ nnyefe intraperitoneal;ns, ọ bụghị ihe dị ịrịba ama (P> 0.05);PBS, saline buffered phosphate;WT, ụdị anụ ọhịa
Ihe nzuzo nke IL-1β bụ akara ngosi nke ịgbalite ọkụ.Iji chọpụta ma G. duodenalis alpha-2 giardine na alpha-7.3 giardine na-arụ ọrụ NLRP3 host inflammasome na vivo, anyị na-eji WT ụmụ oke na-adịghị agwọ ọrịa (sham group) na NLRP3 inflammasome-blocked ụmụ oke (MCC950 na-egbochi ọgwụgwọ otu).E gosipụtara atụmatụ zuru ezu nke nnwale ahụ na Fig. 4a.Otu nnwale gụnyere ụmụ oke na PBS, G. duodenalis cyst ọgwụgwọ site gavage, intramuscular injection nke pcDNA3.1, na intramuscular injection nke pcDNA3.1 (+) -alpha-2 giardine ma ọ bụ pcDNA3.1-alpha-7.3 giardine.N'ụbọchị nke asaa mgbe nchịkwa intramuscular nke plasmid recombinant gasịrị, a na-anakọta serum na ọkwa IL-1β n'ime otu ọ bụla.Dị ka e gosiri na Figure 4b, na MOCK otu: (i) tụnyere PBS otu, pcDNA3.1 ọgwụgwọ enweghị mmetụta dị ịrịba ama na IL-1β secretion (ANOVA, F (4.29) = 4.062, P = 0.9998), Otú ọ dị, A na-ebuli ihe nzuzo IL-β dị elu na G. duodenalis cyst group (ANOVA, F (4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine na pcDNA3.1- Intramuscular injection nke alpha-7.3 giardine budata ụbara ọbara IL-1β ọkwa (ANOVA, F (4, 29) = 4.062, P <0.0001);(iii) pcDNA3.1-alpha-7,3 giardine butere ọkwa dị elu nke IL -1β secretion na pcDNA3.1-alpha-2 giardine intramuscular injection group (ANOVA, F (4, 29) = 4.062, P = 0.0333) .E jiri ya tụnyere otu ọ bụla n'ime usoro ọgwụgwọ MCC950 na otu MOCK: (i) ọkwa nzuzo nke IL-1β na otu njikwa PBS na otu njikwa pcDNA3.1 belatara ruo n'ókè ụfọdụ mgbe igbochi MCC950 inhibitor, mana ọdịiche ahụ abụghị. dị ịrịba ama (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F (9, 58) = 3.540, P = 0.5949);(ii) ka emechiri MCC950., A na-ebelata ihe nzuzo nzuzo IL-1β na G. duodenalis cyst-ọrịa, otu pcDNA3.1-alpha-2 giardine, na pcDNA3.1-alpha-7.3 giardine (G. duodenalis: ANOVA, F (9) , 58) = 3.540, P = 0.0120; pcDNA3.1-alpha-2 giardine: ANOVA, F (9, 58) = 3.540, P = 0.0447; pcDNA3.1-alpha-7.3 giardine: ANOVA, F ) = 3.540, P = 0.0164).Nsonaazụ ndị a na-atụ aro na alpha-2 giardine na alpha-7.3 giardine na-eme ka ọ rụọ ọrụ nke NLRP3 inflammasome na vivo.
pcDNA3.1 (+) -giardine na-arụ ọrụ NLRP3 host inflammasome na vivo.A na-enye ụmụ oke ọgwụ mgbochi (IM) na recombinant eukaryotic expression plasmid pcDNA3.1 (+) -alpha-2 giardine ma ọ bụ pcDNA3.1 (+) -alpha-7.3 giardine wee mesoo ya na MCC950 (ip; MCC950 otu) ma ọ bụ (otu dummy). ).A na-eji PBS ma ọ bụ pcDNA3.1 (+) ọgwụgwọ plasmid mee ihe dị ka nchịkwa na-adịghị mma, a na-eji G. duodenalis cyst ọgwụgwọ otu njikwa dị mma.Otu nnwale na usoro ọgwụgwọ.b Serum ọkwa nke IL-1β na ụmụ oke ka a tụrụ na ụbọchị 7 site na nyocha ELISA.A na-enyocha ọdịiche dị n'etiti otu dị na MOCK site na iji ANOVA otu ụzọ, na ọdịiche dị n'etiti MOCK otu na otu MCC950 ka a na-enyocha site na iji t-ule nke ụdị software SPSS 22.0.Asterisks na-egosi ọdịiche dị ukwuu n'etiti otu ọgwụgwọ na otu MOCK, * P <0.05 na *** P <0.001;akara dollar ($) na-egosi nnukwu ọdịiche dị n'etiti otu ọ bụla n'ime MOCK na otu MCC950 na P<0.05.Nsonaazụ nke nnwale ndu ndu asaa kwụụrụ onwe ya.i, ogwu ogwu intramuscular, ns, abaghị uru (P> 0.05)
Iji nyochaa mmetụta nke alpha-2 na alpha-7.3 giardine-mediated activation nke NLRP3 host inflammasome na G. duodenalis infectivity, anyị na-eji WT C57BL / 6 ụmụ oke ma tinye alpha-2 giardine na alpha-7.3 giardine.A na-agbanye plasmid ahụ intramuscularly, mgbe ụbọchị 3 gasịrị site na tube gastric nke G. duodenalis cyst, mgbe nke ahụ gasịrị, a na-ahụ ụmụ oke maka ụbọchị 7.E gosipụtara atụmatụ zuru ezu nke nnwale ahụ na Fig. 5a.A na-atụle ịdị arọ nke òké ọ bụla kwa ụbọchị, a na-anakọta ihe atụ nke anụ ahụ duodenal ọhụrụ n'ụbọchị nke asaa mgbe nchịkwa gasịrị site na tube gastric, a na-atụle ọnụ ọgụgụ nke trophozoites, na mgbanwe histopathological.Dị ka egosiri na eserese 5b, na-abawanye oge nri, BW ụmụ oke na otu ọ bụla ji nwayọọ nwayọọ na-abawanye.MT nke ụmụ oke malitere ibelata na ụbọchị 3 mgbe nchịkwa intragastric nke G. duodenalis cysts, wee jiri nwayọọ nwayọọ mụbaa.Ịrụ ọrụ nke NLRP3 inflammasome kpaliri site na intramuscular injection nke alpha-2 giardine na alpha7.3 giardine budata mbelata ibu arọ na ụmụ oke (Ụbọchị 1: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 1.399, P = 0 .9754 Ụbọchị 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 1.399, P=0.9987 Ụbọchị 2: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 0.3172, P = 0.9979; Ụbọchị 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.3172, P = 0.8409; Ụbọchị 3: pcDNA3.1-alpha-2, F 4, 30) = 0.8222, P = 0.0262 Day 3: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.8222, P = 0.0083; Ụbọchị 4: pcDNA3.1-alpha-2 giardine , F (4, 30) = 0.5620, P = 0.0012, Ụbọchị 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.5620, P <0.0001, Ụbọchị 5: pcDNA3.1-alpha - 2 giardine, ANOVA, F (4, 30) = 0.9728, P <0.0001 Ụbọchị 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Ụbọchị 6: pcDNA3 . alpha-2 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0012, Ụbọchị 6: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0006;Ụbọchị 7: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 0.5369, P<0.0001 Ụbọchị 7: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.5369, P <0.0001).A na-enyocha ibu parasitic na duodenum (Fig 5c).E jiri ya tụnyere njikwa dị mma na-adịghị edozi ya na otu a na-agbanye na pcDNA3.1 vector efu, ọnụ ọgụgụ G. duodenalis trophozoites belatara nke ukwuu n'ime ìgwè ndị a na-agbanye na α-2 giardine na 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).Na mgbakwunye, giardine alfa-7.3 na-echebe karịa na ụmụ oke karịa giardine alfa-2 (ANOVA, F (3, 24) = 1.209, P = 0.0081).A na-egosi nsonaazụ HE na fig.5d–f.Ụmụ oke agbanyere alpha-2 giardine na alpha-7.3 giardine nwere ọnya anụ ahụ duodenal pere mpe, gosipụtara site na mmebi villus, ma e jiri ya tụnyere ụmụ oke etinyere na G. duodenalis na ụmụ oke etinyere na G. duodenalis na ngwakọta na pcDNA3 vector .1 mbugharị efu.(pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 2.466, P = 0.0035 ma ọ bụ P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F (3, 24) = 2.466, P = 0.0028 ma ọ bụ P = 0.0055) na ibelata atrophy crypt (pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 1.470, P = 0.0264 ma ọ bụ P = 0.0158; pcDNA3.1-alpha: 7.3 giardine , F (3, 24) = 1.470, P = 0.0371 ma ọ bụ P = 0.0191).Nsonaazụ ndị a na-egosi na alpha-2 giardine na alpha-7,3 giardine na-ebelata ọrịa nke G. duodenalis site n'ịkwalite NLRP3 inflammasome na vivo.
Ọrụ nke pcDNA3.1 (+) -giardins na ọrịa G. duodenalis.A na-enye ụmụ oke ọgwụ mgbochi (IM) na recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine ma ọ bụ pcDNA3.1 (+) - alpha-7.3 giardine wee gbaa aka na G. duodenalis cysts (ig).A na-eji otu PBS na pcDNA3.1 (+) + duodenal cyst treatment group mee ihe dị ka ndị na-ahụ maka nchịkwa na-adịghị mma, a na-ejikwa òtù ọgwụgwọ cystitis duodenal mee ihe dị ka otu nchịkwa dị mma.Otu nnwale na usoro ọgwụgwọ.b A na-enyocha MT nke ụmụ oke na nke ọ bụla n'ime otu ọgwụgwọ dị iche iche maka ụbọchị 7 mgbe ihe ịma aka gasịrị.Asterisks na-egosi ọdịiche dị ịrịba ama n'etiti otu G. duodenalis na otu pcDNA3.1 (+) -alpha-2 giardine group, * P <0.05, ** P <0.01, na ***P <0.001;akara dollar ($) na-egosi nnukwu ọdịiche dị n'etiti otu G. duodenalis na pcDNA3.1 (+) - alpha-7.3 jardine group, $$P <0.01 na $$$P <0.001.c Ekpebiri ibu parasitic site n'ịgụ ọnụ ọgụgụ nke trophozoites na 1 ml nke duodenal lavage sitere na duodenum (ogologo 3 cm) ma gosipụta ya dị ka ọnụọgụ parasites kwa cm nke duodenum.Ọdịiche dị n'etiti G. duodenalis ọrịa otu, pcDNA3.1 (+) -alpha-2 giardine otu, na pcDNA3.1 (+) -alpha-7.3 giardine otu e nyochaa otu ụzọ ANOVA iji SPSS software version 22.0.Asterisks na-egosi nnukwu ọdịiche na **P <0.01 na ***P <0.001.d mgbanwe histopathological na duodenum.Akụ na-acha uhie uhie na-egosi mmebi nke villi, akụ ndụ ndụ na-egosi mmebi nke crypts.Ogwe ọnụ ọgụgụ: 100µm.e, f Statistical analysis of òké duodenal villus elu (e) na crypt elu (f).ANOVA nwere otu ụzọ nyochara ndịiche dị n'etiti otu dị na eserese 1d site na iji ụdị ngwanrọ SPSS 22.0.Asterisks na-egosi nnukwu ọdịiche na * P <0.05 na ** P <0.01.Nsonaazụ nke nnwale ndu ndu asaa kwụụrụ onwe ya.ns, ọ bụghị ihe dị mkpa (P> 0.05)
Giardia duodenum bụ nje nje eriri afọ a ma ama nke mmadụ na anụmanụ ndị ọzọ na-ebute giardiasis.N'afọ 2004, etinyere ya na mmemme WHO leghaara ọrịa anya n'ihi oke ya karịrị afọ 6, ọkachasị n'obodo ndị nwere ọnọdụ akụ na ụba dị ala [32].Usoro ahụ ji alụso ọrịa ọgụ na-arụ ọrụ dị oke mkpa na nzaghachi mgbochi ọrịa G. duodenalis.A kọwo macrophages òké ka ọ na-ekpuchi ma gbuo G. duodenalis site na ịhapụ ọnyà extracellular [33].Ọmụmụ ihe anyị gara aga egosila na G. duodenalis, parasite extracellular na-adịghị emerụ ahụ, na-arụ ọrụ p38 MAPK, ERK, NF-κB p65, na NLRP3 ụzọ mgbaàmà na-egbuke egbuke na mouse macrophages iji na-achịkwa nzaghachi mkpali ndị ọbịa, ma wepụta GEV nwere ike ịkwalite usoro a.13], 24].Otú ọ dị, kpọmkwem PAMPs na-etinye aka na NLRP3 inflammasome-regulated inflammation na GEV na ọrụ NLRP3 inflammasome na giardiasis ka na-anọgide na-akọwapụta.Iji mee ka a ghọtakwuo ajụjụ abụọ a, anyị duziri ọmụmụ ihe a.
NLRP3 inflammasome dị na cytoplasm nke mkpụrụ ndụ na-alụso ọrịa ọgụ ma nwee ike rụọ ọrụ site na ihe dị iche iche dị ka kristal uric acid, nsị, nje bacteria, nje na nje nje.N'ime ọmụmụ ihe nje bacteria, a chọpụtala nsị dị ka isi PAMPs nke na-eme ka ihe mmetụta na-egbuke egbuke, na-eduga na mbufụt na ọnwụ cell [34].Ụfọdụ nsí dị iche iche dị iche iche, dị ka hemolysin sitere na Staphylococcus aureus [35] na Escherichia coli [36], hemolysin BL (HBL) sitere na enterotoxin (NHE) [37], na-ebute mbufụt nke NLRP3.Ọmụmụ ihe gbasara nje egosila na protein virulence dị ka SARS-COV-2 envelopu (E) protein [38] na nje Zika NS5 protein [39] bụ ihe PAMP dị mkpa nke onye nnabata NLRP3 ghọtara.N'ime ọmụmụ ihe parasaiti, a kọwawo ọtụtụ nje nje na-ejikọta ya na ntinye ọkụ ọkụ, dị ka Toxoplasma gondii, Trichomonas vaginalis [40], Trypanosoma cruzi [41], na Leishmania [42].A chọrọ protein granul granule GRA35, GRA42, na GRA43, jikọtara ya na virulence nke Toxoplasma gondii, maka ntinye nke pyroptosis na Lewis rat macrophages [43].Na mgbakwunye, ụfọdụ ọmụmụ Leishmania lekwasịrị anya na ụmụ irighiri ihe ọ bụla na-etinye aka na NLRP3 inflammasome, dị ka parasite membrane lipophosphoglycan [44] ma ọ bụ zinc metalloprotease [45].N'ime annexin-dị ka alfa-giardin ezinụlọ nke mkpụrụ ndụ ihe nketa, alpha-1 giardin egosila na ọ bụ onye nwere ike ịgba ọgwụ mgbochi na-enye nchebe megide G. duodenalis na ụdị òké [18].N'ime ọmụmụ ihe anyị, anyị họrọ G. duodenalis virulence factor alpha-2 na alpha-7,3 giardines, bụ ndị pụrụ iche na giardia ma ọ dị ntakịrị kọrọ.Edebere mkpụrụ ndụ ihe nketa abụọ a n'ime pcDNA3.1 (+) eukaryotic expression system vector maka nyocha nke ịgbalite mbufụt.
N'ụdị òké anyị, iberisị kaspase gbawara agbawa na-eje ozi dị ka ihe nrịbama nke ịgbalite mkpasu iwe.N'elu mkpali, NLRP3 na ASC na-emekọrịta ihe, na-ewebata procaspases, ma na-emepụta caspases na-arụ ọrụ nke na-ekewa pro-IL-1β na pro-IL-18 n'ime IL-1β na IL-18 tozuru okè, n'otu n'otu -18.Caspases na-egbuke egbuke (caspases-1, -4, -5 na -11) bụ ezinụlọ echekwara nke cysteine ​​​​proteases nke dị oke mkpa maka nchebe ebumpụta ụwa ma na-etinye aka na mbufụt na ọnwụ cell .Caspase-1 na-arụ ọrụ site na canonical inflammasomes [47], ebe caspases-4, -5, na -11 na-agbajie n'oge e guzobere inflammasomes atypical [48].N'ime ọmụmụ ihe a, anyị na-eji mouse peritoneal macrophages dị ka ihe nlereanya ma nyochaa p20 caspase-1 cleaved caspase-1 dị ka akara nke onye ọbịa NLRP3 mbufụt activation na ọmụmụ nke G. duodenalis ọrịa.Nsonaazụ gosiri na ọtụtụ alfa-giardins bụ ndị na-ahụ maka mmegharị ahụ nke mbufụt, bụ nke kwekọrọ na nchọpụta nke mkpụrụ ndụ nje na-akpata isi na-etinye aka na nje bacteria na nje.Agbanyeghị, ọmụmụ ihe anyị bụ naanị ihuenyo mbido yana enwere ụmụ irighiri ihe ndị ọzọ nwere ike ịgbalite inflammasomes na-abụghị nke oge gboo, dịka ọmụmụ anyị gara aga chọtara ma oge gboo na nke na-abụghị nke oge gboo na ọrịa G. duodenalis [13].Iji chọpụta ma p20 caspase-1 emepụtara na-ejikọta ya na NLRP3 inflammasome, anyị gbanwere alpha-2 na alpha-7.3 giardins n'ime macrophages peritoneal macrophages iji chọpụta ọkwa okwu protein molecule isi na ọkwa ASC oligomerization, na-ekwenye na ma α-giardins na-arụ ọrụ. NLRP3 na-egbu egbu.Nsonaazụ anyị dịtụ iche na nke Manko-Prykhoda et al., bụ ndị kọrọ na mkpali nke mkpụrụ ndụ Caco-2 na G. muris ma ọ bụ E. coli EPEC strains naanị nwere ike ime ka fluorescence siri ike nke NLRP3, ASC, na caspase-1, ọ bụ ezie na ọ bụghị nke ukwuu, ebe ka ọnụ ahịa G. muris na E. coli si gbasaa ọkwa nke protein atọ [49].Ọdịiche a nwere ike ịbụ n'ihi ọdịiche dị na nhọrọ nke ụdị Giardia, ahịrị sel, na sel ndị mbụ.Anyị mekwara nyocha vivo site na iji MCC950 n'ime ụmụ nwanyị WT C57BL/6 dị izu 5, nke nwere ike ibute G. duodenalis.MCC950 bụ onye na-emechi molekul NLRP3 dị ike ma na-ahọrọ nke na-egbochi ọrụ NLRP3 nke canonical na nke na-abụghị nke canonical na nchịkọta nanomolar.MCC950 na-egbochi ọrụ NLRP3 mana ọ naghị emetụta ntinye nke AIM2, NLRC4, na NLRP1 ụzọ mkpali ma ọ bụ ụzọ akara TLR [27].MCC950 na-egbochi ọrụ NLRP3 mana ọ naghị egbochi mmalite NLRP3, K+ efflux, Ca2+ influx, ma ọ bụ mmekọrịta dị n'etiti NLRP3 na ASC;kama, ọ na-egbochi NLRP3 inflammasome activation site na igbochi ASC oligomerization [27].Ya mere, anyị na-eji MCC950 na nyocha in vivo iji chọpụta ọrụ nke NLRP3 inflammasome mgbe mgbatị giardine gasịrị.Caspase-1 p10 arụ ọrụ na-ekewa cytokines pro-inflammatory pro-IL-1β na pro-IL-18 n'ime IL-1β tozuru okè na IL-18 [50].N'ime ọmụmụ ihe a, a na-eji ọkwa serum IL-1β n'ime ụmụ oke giardine na-agwọ ma ọ bụ na-enweghị MCC950 dị ka ihe na-egosi ma NLRP3 inflammasome agbanyere.Dị ka a tụrụ anya ya, ọgwụgwọ MCC950 belatara ọkwa serum IL-1β nke ukwuu.Ihe data ndị a na-egosi n'ụzọ doro anya na G. duodenalis giardin alfa-2 na giardin alfa-7.3 nwere ike ime ka NLRP3 òké na-egbuke egbuke.
Data dị ịrịba ama chịkọbara n'ime afọ iri gara aga egosila na IL-17A bụ onye nchịkwa nchịkwa nke mgbochi megide G. muris, na-eme ka akara ngosi IL-17RA, na-emepụta peptides antimicrobial, na ịhazi ọrụ mmeju [51].Otú ọ dị, ọrịa Giardia na-apụta ugboro ugboro na ndị na-eto eto, a kọwakwara na ọrịa Giardia na ụmụ oke ụmụ oke anaghị eme ka nzaghachi IL-17A na-arụ ọrụ nchebe ya [52], na-akpali ndị nchọpụta ịchọ Giardia immunomodulatory ọzọ.usoro nke ọrịa helminth.Ndị na-ede akwụkwọ na-adịbeghị anya kwuru na G. muris nwere ike ime ka NLRP3 inflammasome site na E. coli EPEC, nke na-akwalite mmepụta nke peptides antimicrobial ma na-ebelata ikike ntinye ya na ọnụ ọgụgụ nke trophozoites na eriri afọ, si otú ahụ na-ebelata ịdị njọ nke colon. ọrịa ndị sitere na bacilli [49].NLRP3 inflammasome na-etinye aka na mmepe nke ọrịa dị iche iche.Nnyocha egosiwo na Pseudomonas aeruginosa na-ebute autophagy na macrophages iji zere ọnwụ cell, na usoro a dabere na ntinye nke NLRP3 inflammasome [53].Maka N. caninum, ụdị ikuku oxygen na-emeghachi omume na-arụ ọrụ nke NLRP3 inflammasome na-amachi mmegharị ya na onye ọbịa, na-eme ka ọ bụrụ ebumnuche ọgwụgwọ [9].Achọpụtala Paracoccidioides brasiliensis ka ọ na-akpalite NLRP3 inflammasome na mkpụrụ ndụ dendritic na-enweta ụmị ọkpụkpụ, na-ebute ntọhapụ nke cytokine na-egbuke egbuke IL-1β, nke na-arụ ọrụ dị oke mkpa na nchekwa ndị ọbịa [10].Ọtụtụ ụdị Leishmania, gụnyere L. amazonensis, L. major, L. braziliensis, na L. infantum chagasi, na-arụ ọrụ NLRP3 na ASC-based caspase-1 na macrophages, yana ọrịa Leishmania.A na-akwalite mmegharị njegharị n'ime ụmụ oke na-erughị eru na NLRP3/ASC/caspase-1 gene [11].Zamboni et al.A kọwo na ọrịa Leishmania na-ebute mkpali nke NLRP3 inflammasome na macrophages, nke na-egbochi mmegharị nje nje intracellular.Ya mere, Leishmania nwere ike igbochi NLRP3 ịgbalite dịka atụmatụ ịzere.Na ọmụmụ vivo, NLRP3 inflammasome nyere aka na mkpochapụ Leishmania, ma ọ dịghị emetụta anụ ahụ [54].N'aka nke ọzọ, na ọmụmụ helminthiasis, ịgbalite NLRP3 inflammasome na-egbochi nchebe nchebe nke onye ọbịa megide helminthiasis eriri afọ [12].Shigella bụ otu n'ime nje bacteria na-ebute afọ ọsịsa n'ụwa niile.Nje bacteria ndị a nwere ike ịkpata mmepụta IL-1β site na P2X7 onye na-anabata K + efflux, ụdị oxygen na-emeghachi omume, lysosomal acidification, na mmebi mitochondrial.NLRP3 inflammasome na-achịkwa phagocytosis na-adịghị mma na ọrụ bactericidal nke macrophages megide Shigella [55].Ọmụmụ Plasmodium egosila na AIM2, NLRP3 ma ọ bụ caspase-1 ụmụ oke na-arịa ọrịa Plasmodium na-emepụta ọkwa dị elu nke ụdị 1 interferon ma na-eguzogide ọgwụ Plasmodium [56].Otú ọ dị, ọrụ nke alpha-2 giardine na alpha-7.3 giardine na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-akpata ọrịa NLRP3 mbufụt na ụmụ oke adịghị edozi.
N'ime ọmụmụ ihe a, mgbochi nke NLRP3 inflammasome site na MCC950 belatara BW ma mee ka ọnụ ọgụgụ nke trophozoites dị n'ime eriri afọ nke eriri afọ na ụmụ oke, na-ebute mgbanwe mgbanwe dị ukwuu nke pathological na anụ ahụ duodenal.Alfa-2 giardine na alpha-7.3 giardine na-eme ka òké NLRP3 na-egbu egbu, na-abawanye arọ ahụ, belata ọnụ ọgụgụ nke trophozoites na mmiri mmiri nke eriri afọ, ma na-ebelata ọnya duodenal pathological.Nsonaazụ ndị a na-egosi na G. duodenalis nwere ike ime ka NLRP3 host inflammasome rụọ ọrụ site na alpha-2 giardine na alpha-7,3 giardine, na-ebelata pathogenicity nke G. duodenalis na ụmụ oke.
Na mkpokọta, nsonaazụ anyị na-egosi na alpha-2 na alpha-7.3 giardines na-eme ka arụ ọrụ nke NLRP3 host inflammasome ma belata ọrịa nke G. duodenalis na ụmụ oke.Ya mere, ụmụ irighiri ihe ndị a bụ ebumnuche na-ekwe nkwa maka igbochi giardiasis.
       Data supporting the results of this study can be obtained from the respective author at gongpt@jlu.edu.cn.
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