I-Giardia duodenum iyigciwane elidala i-giardiasis, isifo samathumbu esivame kakhulu ezinganeni ezincane ezinezimpawu zomtholampilo zohudo.Siye sabika ngaphambilini ukuthi i-extracellular G. duodenalis iqala ukusebenza kwe-intracellular oligomerization-like receptor 3 (NLRP3) ebopha ama-nucleotide futhi ilawula izimpendulo ezivuthayo zokusingatha ngokusebenzisa ukukhishwa kwe-extracellular vesicle (EV).Kodwa-ke, amaphethini aqondile we-molecular we-pathogen-associated duodenococcal EV (GEV) ehilelekile kule nqubo kanye nendima ye-NLRP3 inflammasome ku-giardiasis kusazocaciswa.
I-recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 kanye ne-alpha-7.3 giardins ku-GEV akhiwe, adluliselwa kuma-macrophages ayinhloko we-mouse peritoneal, futhi atholwa ngokukala i-molecule ehlosiwe yokuvuvukala i-caspase-1.Izinga lenkulumo ye-p20 liye lahlolwa..I-G. duodenalis alpha-2 kanye ne-alpha-7.3 giardines ekuqaleni zazikhonjwe ngokulinganisa i-NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 kanye ne-caspase-1 p20), i-IL secretion.Amazinga we-1β, amazinga we-apoptotic spotted protein (ASC) oligomerization, kanye ne-immunofluorescent localization ye-NLRP3 ne-ASC.Indima ye-NLRP3 inflammasome ku-pathogenicity ye-G. duodenalis yabe ihlolwa kusetshenziswa amagundane lapho ukusebenza kwe-NLRP3 kuvinjiwe (amagundane avinjiwe e-NLRP3) kanye nezinguquko ze-pathological isisindo somzimba, umthwalo we-duodenal parasitic, kanye nezicubu ze-duodenal.Ngaphezu kwalokho, siphenye ukuthi i-hiardines alpha-2 kanye ne-alpha-7.3 yenza i-IL-1β secretion in vivo nge-NLRP3 inflammasome futhi sanquma indima yala ma-molecule ku-pathogenicity ye-G. duodenalis kumagundane.
I-Alpha-2 ne-alpha-7.3 giardines yenza kusebenze i-NLRP3 inflammasome in vitro.Lokhu kuholele ekusebenzeni kwe-p20 caspase-1, ukwanda kwamazinga we-NLRP3, i-pro-IL-1β, ne-pro-caspase-1 amaprotheni, ukwanda okuphawulekayo kwe-IL-1β secretion, ukwakheka kwamabala e-ASA endaweni. i-cytoplasm, kanye nokungeniswa kwe-ASA oligomerization.Ukuvuvukala kwe-NLRP3 Ukulahlekelwa kwepenile kwandisa i-pathogenicity ye-G. duodenalis kumagundane.Amagundane aphathwe ngama-cysts nge-gavage evela ku-NLRP3-evinjiwe amagundane abonise inani elikhulayo lama-trophozoite kanye nomonakalo omkhulu ku-duodenal villi, ebonakala nge-necrotic crypts ene-shrunken kanye ne-branching.Ukuhlolwa kwe-vivo kubonise ukuthi i-giardines alpha-2 ne-alpha-7.3 ingabangela ukukhishwa kwe-IL-1β nge-NLRP3 inflammasome, futhi ukugonywa nge-giardines alpha-2 ne-alpha-7.3 kunciphisa i-pathogenicity ye-G. duodenalis kumagundane.
Ihlanganiswe ndawonye, ​​imiphumela yalolu cwaningo iphakamisa ukuthi i-giardia alpha-2 kanye ne-alpha-7.3 ibangela ukukhushulwa kwe-host NLRP3 ukuvuvukala nokunciphisa ukutheleleka kwe-G. duodenalis kumagundane, okuyizinjongo ezithembisayo zokuvimbela i-giardiasis.
I-Giardia duodenum iyi-extracellular protozoan parasite ehlala emathunjini amancane futhi ibangela izehlakalo eziyizigidi ezingama-280 ze-giardiasis enohudo minyaka yonke, ikakhulukazi ezinganeni ezisencane emazweni asathuthuka [1].Abantu bangenwa igciwane ngokuphuza amanzi noma ukudla okungcoliswe ama-M. duodenum cysts, abe esengena esiswini bese ephuma ejusi yesisu.I-Giardia duodenum trophozoites inamathela ku-epithelium ye-duodenal, ibangele isicanucanu, ukuhlanza, isifo sohudo, ubuhlungu besisu, nokuncipha kwesisindo.Abantu abane-immunodeficiency kanye ne-cystic fibrosis bangangenwa izifo.Ukutheleleka kungenzeka futhi ngocansi lomlomo nolwendunu [2].Izidakamizwa ezifana ne-metronidazole, i-tinidazole, ne-nitazoxanide yizindlela ezikhethwayo zokwelapha izifo ze-duodenal [3].Kodwa-ke, le mithi ye-chemotherapy idala imiphumela emibi efana nesicanucanu, umdlavuza, kanye ne-genotoxicity [4].Ngakho-ke, amasu asebenza ngempumelelo kakhulu kudingeka athuthukiswe ukuze kuvinjelwe ukutheleleka kwe-G. duodenalis.
Ama-inflammasomes ayikilasi lama-protein e-cytosolic ayingxenye yempendulo yokuzivikela engaphakathi, esiza ukuvikela ekuhlaselweni kwe-pathogen kanye nokulamula izimpendulo zokuvuvukala [5].Phakathi kwalawa ma-inflammasomes, i-nucleotide-binding oligomerization (NOD) receptor 3 (NLRP3) i-nucleotide-binding oligomerization (NLRP3) i-nucleotide-binding-like inflammasome iye yacwaningwa kabanzi ngoba ingatholwa ngamaphethini ahlukahlukene we-pathogen/umonakalo-ahambisana nama-molecular (PAMP). I-DAMP), ibona, yenza amasosha omzimba azalwa nawo asebenze.futhi ilawula i-homeostasis yamathumbu ezifweni eziningi ezivuthayo [6,7,8].Iqukethe i-pattern recognition receptor (PRR) NLRP3, i-adapter apoptotic spotted protein (ASC), kanye ne-effector procaspase-1 noma i-procaspase-11.I-NLRP3 inflammasome isebenza njengosokhaya ngokumelene nokuhlasela kwe-pathogen, njengoba kuphawulwe ku-Neospora caninum [9], Paracoccidioides brasiliensis [10], kanye nezifundo ze-Leishmania.[11], kodwa futhi kuye kwabikwa ukuthi ukusebenza kwe-NLRP3 inflammasome kunciphisa izimpendulo zamasosha omzimba futhi kukhulisa ukuqhubekela phambili kwesifo, isibonelo, ezibungwini [12].Ngokusekelwe ekutholeni kwethu kwangaphambilini, sibike ukuthi i-extracellular G. duodenalis iqala ukusebenza kwe-intracellular yokuvuvukala kwe-NLRP3 futhi iguqule izimpendulo ezivuthayo zokusingatha ngokufihla ama-extracellular vesicles (EVs) [13].Nokho, indima ye-NLRP3 inflammasome ku-G. duodenalis infection in vivo isazonqunywa.
I-Giardins ekuqaleni yayichazwa njengezingxenye zesakhiwo se-G. duodenalis cytoskeleton futhi idlala indima ebalulekile ekuxhumekeni kwe-trophozoite nokunamathiselwe kweseli ye-epithelial emathunjini amancane.Ukuze uvumelane kangcono nemvelo futhi ukwandise i-pathogenicity yabo, i-G. duodenalis trophozoites yakha isakhiwo esiyingqayizivele se-cytoskeletal esihlanganisa i-8 flagella, i-1 body body, kanye ne-1 ventral disc [14].Ama-trophozoite e-Giardia duodenum asebenzisa i-cytoskeleton yawo ukungena emathunjini amancane angaphezulu, ikakhulukazi i-duodenum, futhi anamathele kuma-enterocyte.Zihlala zifuduka futhi zinamathele kumaseli e-epithelial zisebenzisa i-cell metabolism.Ngakho-ke, kukhona ubudlelwano obuseduze phakathi kwe-cytoskeleton yabo kanye ne-virulence.Ama-giardines aqondene ne-Giardia duodenum ayizingxenye zesakhiwo se-cytoskeleton [15] futhi ahlukaniswe amakilasi amane: α-, β-, γ-, kanye ne-δ-giardines.Kunamalungu angama-21 omndeni we-α-giardin, wonke anekhono elincike ku-calcium lokubopha ama-phospholipids [16].Baphinde baxhuma i-cytoskeleton kulwelwesi lwamaseli.Kubantu abanesifo sohudo esibangelwa i-G. duodenalis, i-α-giardins ivezwa kakhulu futhi i-immunoreactive ngesikhathi sokutheleleka [17].Imithi yokugoma ye-Heterologous esekelwe ku-Giardia alfa-1 evikelwe ngokumelene ne-giardiasis kumagundane futhi ingaba ama-antigens angaba khona okuthuthukiswa komgomo [18].I-Alpha-8 giardin, etholakala ku-membrane ye-plasma kanye ne-flagella, kodwa hhayi ku-ventral disc, ithuthukisa ukuhamba nokukhula kwezinga le-trophozoite ku-G. duodenalis [19].I-Alpha-14 giardin inamathela ezakhiweni ze-microtubule ku-flagella futhi ithinta ukusebenza kwe-G. duodenalis [20].I-Alpha-11 giardine ikhona ngobuningi kuwo wonke umjikelezo wokuphila, futhi ukucindezeleka ngokweqile kwe-alpha-11 giardine kulimaza i-G. duodenalis ngokwayo [21].Kodwa-ke, akucaci ukuthi i-alpha-2 giardine ne-alpha-7.3 giardine zivikela ukutheleleka kwe-G. duodenalis kanye nezindlela zazo ezingaphansi.
Kulolu cwaningo, i-recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardine kanye ne-pcDNA3.1(+)-alpha-7.3 giardine zidluliselwe kumamacrophage e-mouse primary peritoneal ukuze kusebenze umsingathi i-NLRP3.Kwabe sekuhlolwa okuhlosiwe okuhlosiwe.Siphinde sahlola indima ye-NLRP3 inflammasome ku-pathogenicity ye-G. duodenalis, saphenya ukuthi i-alpha-2 ne-alpha-7,3 giardines yenza kusebenze i-NLRP3 inflammasome in vivo, futhi sanquma ukuthi lezi zindima ezimbili ze-giardines ku-pathogenicity G. duodenalis.Umgomo wethu ofanayo kwakuwukuthuthukisa imigomo ethembisayo yokuvimbela ukutheleleka kwe-G. duodenalis.
Uhlobo lwasendle (WT) C57BL/6 amagundane esifazane anamaviki angu-5–8 athengwe e-Liaoning Changsheng Experimental Animal Center (Liaoning, China).Amagundane ayekwazi ukuthola amanzi mahhala, athola ukudla okuvalwa inzalo futhi agcinwa emjikelezweni wokukhanya/wobumnyama ongamahora angu-12/12.Ngaphambi kokutheleleka, amagundane athola ama-antibiotic ad libitum emanzini okuphuza ahlanganiswe ne-ampicillin (1 mg/mL), i-vancomycin (1 mg/mL), ne-neomycin (1.4 mg/mL) (konke okuthengwe e-Shanghai, e-China, izinto eziphilayo zokwenziwa) [22] ].].Amagundane alahlekelwe amandla okudla nokuphuza amahora angu-> 24 futhi alahlekelwa ≥ 20% wesisindo somzimba agunyazwe ngokobuntu ngokugudluka komlomo wesibeletho.
I-WB G. duodenalis trophozoites (iqoqo le-American Type Culture, e-Manassas, e-USA) yengezwe nge-serum ye-fetal bovine engu-12.5% ​​(FBS; Every Green, Zhejiang, China) kanye no-0.1% we-bovine bile (Sigma-Aldrich, St. Missouri, USA ).USA) ngaphansi kwezimo ze-microaerobic.Ama-trophozoite ahlanganayo aqoqwe eqhweni futhi adluliswa ngesilinganiso esingu-1:4 ukuze aphinde akhiqizwe.
I-Giardia duodenum cysts yenziwa njengoba kuchazwe ngaphambilini [23], ama-trophozoite avunwa esigabeni se-logarithmic bese ehlanjululwa nge-encapsulation inducing medium, i-pH 7.1 (elungisiwe i-TYI-S-33) ekugxilweni kokugcina kwe-1 × 106 trophozoites/mL.i-bile concentration 0.05% medium).Ama-Trophozoite akhuliswe ngaphansi kwezimo ze-anaerobic ku-37°C kuze kube yisigaba sokukhula se-logarithmic.Shintsha i-medium kuya ku-cyst inducing medium (pH 7.8; i-TYI-S-33 medium eshintshiwe ne-1% ye-bile concentration) kanye nesiko G. duodenalis ku-37 ° C amahora angu-48-96, lapho ama-cysts okwakheka abonwa ngaphansi kwe-microscope.Ngemva kokuba iningi lama-trophozoite seliyengwe ukwenza ama-cysts, ingxube yesiko yavunwa futhi yaphinde yamiswa emanzini angenalutho ayinyumba ukuze kufakwe ama-trophozoite asele.Ama-cyst abalwa futhi agcinwa ku-4°C ukuze ahlaziywe ngokulandelayo ngeshubhu lesisu kumagundane.
I-Giardia extracellular vesicles (GEVs) yacetshiswa njengoba kuchazwe ngaphambilini [13].Ama-Trophozoite esigabeni sokukhula kwe-logarithmic aphinde amiswa endaweni eguquliwe ye-TYI-S-33 elungiselelwe nge-FBS ephelelwe yi-exosome (Biological Industries, Beit-Haemek, Israel) ukuze ifinyelele ekuhlanganiseni kokugcina kwe-1 × 106 parasites/mL futhi yafakwa amahora angu-12.bahlukaniswa ne-supernatant yesiko nge-centrifugation ku-2000 g imizuzu eyi-10, i-10,000 ama-45 amaminithi, kanye ne-100,000 ama-60 amaminithi.Imvula yancibilika ku-phosphate buffered saline (PBS), yalinganiswa kusetshenziswa i-BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) futhi yagcinwa ku -80° C. noma yasetshenziswa ngokuqondile ukuze kuhlaziywe okwengeziwe.
Ama-macrophages e-peritoneal eyinhloko yegundane alungiswa njengoba kuchazwe ngaphambilini [24].Kafushane, amagundane (amaviki angu-6-8 ubudala) ajovwe (intraperitoneally [ip]) ngo-2.5 ​​ml we-2.98% Difco liquid thioglycol medium (BD, Franklin Lakes, NJ, USA) futhi adliswa ama-palates angu-3-4.Ukumiswa kwama-macrophages kwaqoqwa emgodini wesisu wamagundane ngemva kwe-euthanasia kanye ne-centrifuged izikhathi ezi-3 ku-1000 g imizuzu engu-10.Amaseli avuniwe atholwe nge-flow cytometry kusetshenziswa umaka we-CD11b kuze kube yilapho ukuhlanzeka kweseli kwaba>>98%, kwase kwengezwa kumapuleti e-cellculture cell-cell angu-6 (4.5 x 106 cells/well) futhi afakwe ku-10% FBS (Bioindustry) ku-37°C.kanye ne-5% CO2.
I-RNA ikhishwe ku-1 × 107 trophozoites ku-1 ml ye-TRIzol reagent (Vazyme, Nanjing, China), i-genomic DNA yakhishwa kungqikithi ye-G. duodenalis RNA kusetshenziswa i-MonScript dsDNase (i-Monad, i-Wuhan, i-China) futhi i-DNA ehambisanayo (cDNA) yahlanganiswa. usebenzisa i-MonScript RTIIII Super Mix (Monad) ngokwemiyalelo yomkhiqizi.
Imininingwane yokulandelana kwe-CDS yofuzo oluhlosiwe lwe-G. duodenalis lutholwe ku-NCBI GenBank.Sebenzisa i-Primer 5.0 ukuze udizayine iziqalo zokuhlanganisa ezingenamthungo zofuzo ngalunye oluqondiwe.I-primer eya phambili (5′-3′) iqukethe izingxenye ezintathu: ukulandelana okugqagqene okunevekhtha yomugqa i-pcDNA3.1(+) EcoRV (TGGTGGAATTCTGCAGAT) futhi iqale amakhodoni i-ATG ne-GNN (uma isisekelo sokuqala singeyona i-G).Lokhu kwenzelwa ukuthuthukisa ukusebenza kahle kwenkulumo.Ngaphezu kwalokho, okungenani izisekelo ezihlanganisiwe ze-bp ezingu-16 (okuqukethwe kwe-GC 40–60%/Tm okulinganiselwa ku-55 °C).I-primer ehlehlayo (5′-3′) iqukethe izingxenye ezimbili, ukulandelana okweqanayo nge-EcoRV-linearized vector pcDNA3.1(+) (GCCGCCACTGTGCTGGAT) kanye nesisekelo esihlanganisiwe okungenani esingu-16 bp.(ngaphandle kwezitobhi ezimbili zokugcina).izisekelo) i-codon efana ne-AA noma i-GA ukuvumela ama-plasmid ahlanganisiwe ukuthi aveze amaprotheni awo anelebuli).Ama-primer alandelanayo abhalwe kuThebula 1 futhi ahlanganiswa yi-Kangmet Biotechnology Co., Ltd. (Changchun, China).
Okuhlosiwe kwakhuliswa kusetshenziswa i-Pfu DNA polymerase (Tiangen, Beijing, China) noma i-Ex-taq (Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, China) kusetshenziswa i-G. duodenalis cDNA elungisiwe njengesifanekiso.Ivector ye-eukaryotic expression plasmid pcDNA3.1(+) yahlanganiswa ne-enzyme evimbela i-EcoRV futhi i-dephosphorylated kusetshenziswa i-Fast AP (Thermo Fisher Scientific).Izingcezu ze-pcDNA3.1(+) ezinomugqa nezingcezu zofuzo ezihlosiwe ezithuthukisiwe zahlanzwa kusetshenziswa ikhithi yokuhlanza ijeli ye-DNA (Tiangen) futhi kwalinganiswa kusetshenziswa i-Nanodrop ND-2000 (Thermo Fisher Scientific).Ucezu lwe-pcDNA3.1(+) kanye nocezu ngalunye lofuzo oluqondiwe kwaphinde kwahlanganiswa kusetshenziswa i-MonClone single assembly cloning mix (Monad Biotech Co., Ltd., Suzhou, China) futhi kwaqinisekiswa ukulandelana kwe-DNA kusetshenziswa i-Comate Bioscience Company Limited (Changchun, China) ..
Ama-plasmids angenayo i-Endotoxin i-pcDNA3.1(+)-alpha-2 kanye ne-pcDNA3.1(+)-alpha-7.3 akhiqizwe kusetshenziswa i-SanPrep Endotoxin-free Plasmid Mini Kit (Sangon Biotech).Ukugxilisa ingqondo kuye kwagcinwa ngaphezu kuka-500 ng/µl ukuze kuqinisekiswe ukuthi i-EDTA ku-elution buffer ayiphazamisani nokuhlolwa kokudlulisela.Ama-macrophage e-primary mouse peritoneal akhuliswa kumapuleti angu-6 anemithombo ephelele ye-RPMI 1640 (Biological Industries) amahora angu-12, bese amaseli ahlanjululwa izikhathi ezingu-3 ku-PBS efudumele ukuze kukhishwe i-penicillin ne-streptomycin, bese phakathi nendawo yengezwe nge-medium ephelele.Ama-plasmids angenayo i-Endotoxin i-pcDNA3.1(+)-alpha-2 kanye ne-pcDNA3.1(+)-alpha-7.3 (2.5 μg) ahlanjululwe ngo-125 μl we-Opti-MEM encishisiwe serum medium (Gibco, Thermo Fisher Scientific) ..Kwabe sekuthi u-5 µl we-Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific) yahlanjululwa ngo-125 µl we-serum ephansi ye-Opti-MEM medium.Lungiselela i-liposome-DNA complexes ngokuxuba i-plasmid engena-endotoxin ehlanjululwe ne-Lipofectamine 2000 futhi uvumele ingxube ukuthi ime endaweni yokushisa yasekamelweni imizuzu emi-5.Dlulisa ama-complexes ngokwehlukana kumaseli emthonjeni ngamunye bese uxuba kancane.Ngemuva kwamahora ama-4, indawo yokusebenzela yamaseli yathathelwa indawo u-2 ml we-RPMI 1640 medium ephelele futhi isiko saqhubeka amahora angama-24.I-fresh cell culture medium yengezwe kumaseli futhi yafukanyelwa izikhathi ezahlukahlukene ngokuya ngedizayini yokuhlola.
Amasampula amaprotheni avela kuma-supernatants nama-cell lysates alungiswa njengoba kuchazwe ngaphambilini [25].Imingcele yokudlulisa i-Membrane ye-pro-IL-1β, i-pro-caspase-1, i-caspase-1 p20, i-NLRP3, i-β-actin, ne-His-tag yayingu-200 mA/90 min.Okwe-interleukin 1β (IL-1β; R&D Systems, Minneapolis, Minnesota, USA), caspase-1 (p20) (Adipogen, Switzerland) kanye ne-NLRP3 (Adipogen SA, Epalinges, Switzerland) kanye no-1:5000 eqondise ithegi Yakhe ( Amylet Scientific, Wuhan, China) kanye ne-β-actin (Proteintech, Wuhan, China).
Ukuxhumanisa nge-disuccinimide subrate (DSS) kwenziwa njengoba kuchazwe ngaphambilini [26].Amaseli ahlanzwa izikhathi ezi-3 nge-PBS ebandayo futhi alaliswa ngokuphelele ngenaliti yegeji engu-27 ku-50 µl ASC reaction buffer (pH 8.0) equkethe 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES kanye ne-125 mM NaHCO3.Ingxube ifakwe i-centrifuged ku-5000 g imizuzu engu-3 futhi i-pellet yahlanganiswa ne-10 µl DSS (25 mM ku-DMSO) kanye ne-40 µl ASC yokusabela isibhafa imizuzu engu-30 ku-37°C.Ngemuva kokufakwa phakathi kwe-centrifugation ku-5000 g imizuzu eyi-10, i-pellet yancibilika kusixazululo esingu-40 µl se-ASC reaction buffer kanye no-10 µl we-6x protein loading buffer (TransGen, Beijing, China), bese isixazululo sacinywa ekamelweni lokushisa 15. imiz., Bese ubilise imizuzu eyi-10.Amasampula ephrotheni abe esengaphansi kokucishwa kwaseNtshonalanga kusetshenziswa amasosha omzimba ayinhloko e-ASC (Wanleibio, Shenyang, China) ngesilinganiso sokuhlanjululwa esingu-1:500.
Ukulandela inqubo echazwe ngaphambilini [13], ama-cell culture supernatants avunwa futhi ukukhishwa kwe-cytokine e-pro-inflammatory IL-1β kwanqunywa kusetshenziswa ikhithi ye-IL-1 Beta ELISA (Invitrogen, Thermo Fisher Scientific).Guqula amanani e-OD450nm abe ukugxila kwamaprotheni usebenzisa ijika elijwayelekile le-IL-1β.
Amaseli ambozwe kuma-coverlips ahlanzwa ngobumnene izikhathi ezingu-3 ku-PBS efudumele, efakwe ku-tissue cell fixative (Biosharp, Beijing, China) imizuzu engu-10 ekamelweni lokushisa (RT), ku-0.1% i-Triton X-Permeabilize ku-100 (ihlanjululwe ku-PBS; i-Biosharp ) imizuzu engu-20 ekamelweni lokushisa futhi uvimbele ku-5% serum serum albumin (ku-PBS) amahora angu-2 ekamelweni lokushisa.Amaseli abe esefukanyelwa ngobusuku obungu-4°C namasosha omzimba ayisisekelo amelene ne-ASC (i-1:100 dilution) noma i-NLRP3 (i-1:100 dilution), ngokulandelana, kanye nembuzi ebhalwe ukuthi i-Cy3 emelene nogwaja IgG(H+L) (1:400; EarthOx , San Francisco, CA, USA) noma i-FITC-conjugated goat anti-mouse IgG (1:400; Earthox) ngobusuku obungu-37°C ebumnyameni ihora elingu-1.Ama-nuclei ayegcotshwe nge-Hoechst 33258 (10 μg/ml; UE, Suzhou, China) imizuzu emi-5 futhi abonwa ngaphansi kwesibonakhulu se-fluorescence (Olympus Corporation, Tokyo, Japan).
Amagundane ahlukaniswe ngamaqembu amane (n = 7 eqenjini ngalinye): (i) Iqembu lokulawula elingalungile eliphathwe nge-PBS (i-PBS kuphela; i-gavage 100 µl/igundane i-PBS elandelwa umjovo wansuku zonke we-intraperitoneal 100 µl/mouse PBS 3 amahora kamuva) ., ngokuqhubekayo izinsuku ezingu-7);(ii) iqembu elilawulayo elingalungile eliphathwe nge-MCC950 inhibitor [27] (100 µl/mouse nge-PBS gavage, amahora angu-3 kamuva, isisindo somzimba esingu-10 mg/kg [BW] MCC950 [ku-PBS] saphathwa nge-intraperitoneally nsuku zonke, ubude bezinsuku ezingu-7);(iii) Iqembu le-G. duodenalis cyst infections (1.5 x 106 cysts/mouse by gavage, 3 amahora kamuva, 100 μl/mouse PBS intraperitoneally elawulwa nsuku zonke izinsuku ezingu-7);(iv) Iqembu le-G. duodenalis cyst elihlangene lokutheleleka kwe-MCC950 inhibitor group treatment (1.5×106 cysts/mouse via gavage, 10mg/kg isisindo somzimba MCC950 intraperitoneally nsuku zonke izinsuku ezingu-7 ku-3h).Isisindo somzimba segundane ngalinye sasigadwa nsuku zonke futhi wonke amagundane akhishwa inyumbazane ngosuku lwesi-7.I-duodenum evuniwe (ubude obungu-3 cm) yasikwa yaba izingcezu ezincane ku-1 ml PBS, ama-cysts abhujiswa ubusuku bonke ku-PBS ku-4 ° C, kanye ne-G. duodenalis trophozoites.I-duodenum entsha (ubude obungu-1 cm) yahlukaniselwa ukubola kwe-hematoxylin kanye ne-eosin (H&E).
Amagundane ahlukaniswe ngamaqembu amabili: (i) Iqembu lokulawula i-MOCK kanye (ii) neqembu le-MCC950 inhibitor.Kwakukhona ukwelashwa okuyisihlanu eqenjini ngalinye (n = 7/iqembu lokwelapha): (i) Iqembu lokulawula elibi lokwelashwa kwe-PBS (PBS kuphela; 100 µl/mouse PBS, intramuscular (IM) umjovo (tibialis anterior) [28, 29];( ii) pcDNA3.1(+) iqembu lokulawula elingalungile le-plasmid (100 µg/DNA yegundane, ngomjovo we-intramuscularly); iqembu eliphathwe nge-plasmid pcDNA3.1(+)-alpha-2 (100 μg/mouse DNA, ngomjovo we-intramuscular), kanye (v) neqembu eliphathwa nge-plasmid pcDNA3.1(+)-alpha-7.3 (100 µg/mouse I-DNA, ngemva kwamahora angu-12 okudlula, amagundane eqembu le-MCC950 inhibitor athole umjovo we-intraperitoneal wansuku zonke we-MCC950 (10 mg/kg isisindo somzimba) izinsuku ezingu-7, kuyilapho amagundane eqenjini le-MOCK athola umthamo olinganayo wokwelashwa kwe-PBS.Amasampula egazi aye eqoqwe kumagundane e-eyeballs futhi ashiywe ubusuku bonke ku-4 ° C Amasampula e-Serum ahlukaniswa kusetshenziswa i-enzyme-linked immunosorbent assay (ELISA) kanye nezilinganiso zamazinga e-IL-1β.
Amagundane angamashumi amathathu nanhlanu ahlukaniswa ngamaqembu amahlanu (n=7/group).Iqembu le-1 laliyiqembu lokulawula elingalungile eliphathwa nge-PBS: amagundane athola i-100 μl ye-PBS nge-intramuscularly kanye nezinsuku ze-3 kamuva nge-gavage.Iqembu le-2 liyiqembu elilawulayo elihle elitheleleke nge-G. duodenalis cysts: amagundane ajovwe nge-100 μl ye-PBS, futhi ezinsukwini ezingu-3 kamuva i-1.5 x 106 ama-cysts / igundane ajovwa nge-intragastrically.Iqembu lesithathu - ukugonywa kwe-plasmid nge-pcDNA3.1 (+) kuhlangene neqembu lokulawula ukutheleleka kwe-cyst duodenal: amagundane athola i-100 μg ye-plasmid DNA pcDNA3.1 (+) (im) ngomlomo, 1.5 × 106 ama-cysts / igundane 3 eziningana izinsuku.Amaqembu 4 kanye ne-5 ayeyi-recombinant pcDNA3.1 (+) -alpha-2 giardine plasmid noma i-pcDNA3.1 (+) -alpha-7.3 giardine plasmid ehlangene ne-G. duodenalis cyst infection.Iqembu lokuhlola: amagundane athole i-100 µg ye-pcDNA3.I-1 (+) -giardine plasmid DNA (im), bese emva kwezinsuku ezi-3, ama-cysts/igundane angu-1.5 × 106 ajovwe nge-gavage.Isisindo somzimba segundane ngalinye sagadwa ngemva kokwethulwa kwe-cyst G. duodenalis ngeshubhu.I-duodenum entsha yaqoqwa ukuze kulinganiswe umthamo we-parasitic kanye nokuhlaziywa kwe-HE staining.
Izinguquko ze-Histopathological zahlaziywa ngokwenqubo eshicilelwe ngaphambili [30].I-duodenum entsha yalungiswa nge-tissue cell fixative, yashumekwa kupharafini, yasikwa yaba izingxenye ezingu-4 μm, ingcoliswe nge-H&E futhi yahlaziywa ngaphansi kwesibonakhulu esikhanyayo.Izinguquko ezimele ze-pathological ezigabeni eziyisikhombisa zezicubu ezivela kumagundane ayisikhombisa azimele zahlolwa udokotela wezifo ezingazi ngokwelashwa futhi zathathwa ekukhulisweni kwe-200x.Ubude be-villi nokujula kwama-crypts kukalwa ngokuhambisana nezindlela ezichazwe ngaphambilini.
Imiphumela ye-vitro ne-vivo itholwe ngama-triplicate.Amagrafu akhiqizwa kusetshenziswa i-GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, USA).Umehluko phakathi kwamaqembu amabili uhlaziywe ngokuhlolwa kwe-t, kuyilapho umehluko phakathi kwamaqembu angu-≥3 uhlaziywe ngokuhlaziywa kwendlela eyodwa yokuhluka (ANOVA) kusetshenziswa isofthiwe ye-SPSS (inguqulo 22.0; SPSS IBM Corp., Armonk, NY, USA) .Idatha yahlaziywa ukuze kutholakale i-homogeneity yokuhluka kusetshenziswa ukuhlolwa kuka-Levene okwalandelwa ukuhlolwa kwe-post hoc ka-Bonferroni (B).Ukubaluleka kuvezwa njengo-P<0.05, P<0.01, kanye no-P<0.001 (okungabalulekile [ns]) (P>0.05).
Ukuhlaziywa kwethu kwangaphambilini kwe-GEV proteomics ku-Kyoto Encyclopedia of Genes and Genomes (KEGG) kubonise ukuthi izinhloso eziningi zingase zihileleke ekusebenziseni izindlela zokubonisa ukuvuvukala [13].Sikhethe okuhlosiwe okubili okuthembisayo, i-alpha-2 ne-alpha-7.3 giardins, sikhulise la mamolekyuli futhi siwasebenzise ukuze sakhe i-pcDNA3.1(+) i-eukaryotic expression vector.Ngemuva kokulandelanisa, i-recombinant pcDNA3.1(+)-alpha-2 kanye ne-alpha-7.3 giardine expression plasmids yadluliselwa kuma-macrophages e-primary mouse peritoneal, kanye ne-caspase-1 p20 yesiginesha yephrotheni yokuvuvukala (isiqephu se-caspase-1 ecushiwe) njengokucacisa ama-molecule abalulekile angabangela ukuvuvukala.Imiphumela yabonisa ukuthi i-alpha-2 kanye ne-alpha-7.3 giardines ingakwazi ukuveza inkulumo ye-p20 caspase-1 efana ne-GEV.Awukho umthelela ekusebenzeni kwe-caspase-1 okutholwe ekulawuleni okungalungile okungaphenduliwe (i-PBS kuphela) nokulawula i-plasmid pcDNA3.1 (+) (Umfanekiso 1).
Isilinganiso sokwenziwa kusebenze kwe-p20 caspase-1 nge-pcDNA3.1(+)-alpha-2 kanye ne-alpha-7.3 giardins.I-recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 kanye ne-alpha-7.3 giardines (ngenhla komzila ngamunye) adluliselwa kuma-macrophage wegundane eliyinhloko kanye nama-culture supernatants avunwa emahoreni angu-24 kamuva.Ukucisha okuseNtshonalanga kusetshenziswe ukukala amazinga esiginesha we-caspase-1 p20 inflammasome protein.Iqembu lokwelapha le-PBS kuphela (umzila C) kanye neqembu le-pcDNA3.1(+) le-monotherapy (umzila we-pcDNA3.1) asetshenziswe njengokulawula okungalungile, futhi iqembu lokwelapha i-GEV lasetshenziswa njengokulawula okuhle.Ukuvezwa kwephrotheni ehlanganisiwe kwaqinisekiswa ngokuthola ithegi ye-histidine kuphrotheni ngayinye, futhi amabhande amaprotheni ayelindelwe kwakuyi-alpha-2 giardine (38.2 kDa) ne-alpha-7.3 giardine (37.2 kDa).I-GEV, i-Giardia duodenum extracellular vesicles, pcDNA3.1(+), i-EcoRV-linearized vector, SUP, supernatant
Ukuze unqume ukuthi i-alpha-2 giardine ne-alpha-7.3 giardine zenza i-p20 caspase-1 inkulumo futhi zidlale indima ekwenzeni kusebenze umsingathi we-NLRP3 impendulo yokuvuvukala, i-pcDNA3.1(+)-alpha-2 giardine ne-pcDNA3.1(+)-alpha -I-7.3 giardin yadluliselwa kuma-macrophages e-peritoneal yegundane eyinhloko ne-DNA ye-plasmid recombinant, kanye namazinga okuveza, indawo, kanye ne-oligomerization yamaphrotheni ayinhloko okuvuvukala i-NLRP3 anqunywe.Kulesi sivivinyo, i-GEV isetshenziswe njengeqembu lokulawula elihle, futhi iqembu elingenalo ukwelashwa (i-PBS kuphela) noma iqembu le-pcDNA3.1(+) lokwelapha ukudluliswa kwegciwane kwakuyiqembu elingalungile.Imiphumela yabonisa ukuthi, njengeqembu le-GEV, i-DNA ye-plasmid recombinant ye-giardin pcDNA3.1 (+)-alpha-2 kanye ne-giardin pcDNA3.1 (+) -alpha-7.3 ibangele ukulawulwa kwe-NLRP3, i-pro-IL-1β kanye i-procaspase-1 ne-caspase-1 activation (Fig. 2a).Ukwengeza, womabili ama-giardine adala ukugcinwa okubalulekile kwe-IL-1β (pcDNA3.1: ANOVA, F(4, 10) = 1.625, P = 0.1000; alpha-2 giardine: ANOVA, F(4, 10) = 1.625, P = 0.0007 ).;i-alpha-7.3 giardine: ANOVA, F(4, 10) = 1.625, P<0.0001;I-GEV: ANOVA, F(4, 10) = 1.625, P = 0.0047) (Umfanekiso 2b).Amaprotheni amaningi e-ASC ayeyi-monomeric eqenjini elingalashwa noma eqenjini lokwelapha elidluliselwa nge-pcDNA3.1(+) plasmid, ngokungafani ne-pcDNA3.1(+)-alpha-2 noma i-pcDNA3.1(+)-alpha- 7.3 i-giardine.I-oligomerization ye-ASC yenzeke ku-DNA ye-plasmid recombinant yeqembu noma iqembu le-GEV eliqondile, elibonisa ifomu le-oligomeric (Umfanekiso 2c).Le datha yokuqala iphakamisa ukuthi i-alpha-2 giardine ne-alpha-7,3 giardine ingenza kusebenze ukuvuvukala kwe-NLRP3.Ucwaningo olwalandela lwe-immunofluorescent lwendawo ye-ASC ne-NLRP3 lubonise ukuthi eqenjini lokulawula elingalungile, iphrotheni ye-ASC yahlakazeka kuyo yonke i-cytoplasm futhi yabonakala njengesignali yamachashazi ekugqugquzelweni kwe-pcDNA3.1 (+) -alpha-2 nge-giardine noma i-pcDNA3.I-1(+)-alpha-7,3 iqembu le-giardine noma iqembu le-GEV lokulawula elihle (Umfanekiso 2d).Ekulawulweni okungalungile kanye namaqembu e-pcDNA 3.1 aphethwe nge-plasmid, isignali ye-protein ye-NLRP3 ayizange ibonwe, kuyilapho ichashazi lesignali ye-fluorescent ekuphenduleni i-pcDNA3.1 (+) -alpha-2 giardine noma i-pcDNA3.1 (+) -alpha-7.3 kutholiwe..i-giardine itholakala ku-cytoplasm noma ekugqugquzelweni kwe-HEV (Fig. 2e).Le datha iphinde ibonise ukuthi i-G. duodenalis giardin alpha-2 kanye ne-giardin alpha-7.3 zisebenzisa i-NLRP3 inflammasome kuma-macrophages ayinhloko e-peritoneal yegundane.
I-pcDNA3.1(+)-alpha-2 giardin kanye ne-pcDNA3.1(+)-alpha-7.3 giardin yenza kusebenze i-NLRP3 inflammasome kuma-macrophages e-peritoneal yegundane.Guqula i-recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardin kanye ne-pcDNA3.1(+)-alpha-7.3 giardin ibe yi-primary murine peritoneal macrophage namaseli, noma uvune amandla angaphezu kwavamile phakathi kwamahora angu-24 ukuze kuhlaziywe inkulumo, i-oligomerization , imfihlo.kanye nokwenza kwasendaweni kwamaprotheni ayisihluthulelo sokuvuvukala.Iqembu le-PBS kuphela (C) kanye neqembu le-pcDNA3.1 (+) elilodwa lokwelapha lasetshenziswa njengokulawula okungalungile, futhi iqembu lokwelapha i-GEV lasetshenziswa njengeqembu elihle.a Amaprotheni ayisihluthulelo okuvuvukala i-NLRP3, okuhlanganisa i-NLRP3, i-pro-IL-1β, i-pro-caspase-1, ne-p20 caspase-1, atholwe ukuchithwa kwe-Western.b Amazinga okugcinwa kwe-IL-1β kuma-supernatants anqunywe kusetshenziswa i-enzyme-linked immunosorbent assay (ELISA).Umehluko phakathi kwamaqembu okulawula nawokuhlola uhlaziywe ngokuhlaziywa kwendlela eyodwa yokuhluka (ANOVA) kusetshenziswa i-SPSS software version 22.0.Izinkanyezi zibonisa umehluko omkhulu phakathi kwamaqembu **P<0.01 kanye ne-***P<0.001.c Amazinga e-oligomerization ye-ASC kuma-pellets anqunywa ukuhlaziywa kokuxhumanisa kwe-DSS, kuyilapho amazinga e-ASC kuma-cell lysate asetshenziswa njengokulawula ukulayisha.d Ukubona ngeso lengqondo ukwenziwa kwasendaweni kwe-ISC kusetshenziswa i-immunofluorescence.e I-Immunofluorescence isetshenziselwe ukubona ngeso lengqondo ukwenziwa kwasendaweni kwe-NLRP3.I-ASC, iphrotheni efana ne-apoptotic speck;I-IL, i-interleukin;I-NLRP3, i-nucleotide-binding oligomerization-like receptor 3;ns, akubalulekile (P > 0.05)
Kokubili i-G. duodenalis kanye nama-GEV ewakhiphayo enza kusebenze i-NLRP3 inflammasome futhi alawule ukusabela kokuvuvukala kosokhaya ku-vitro.Ngakho, indima ye-NLRP3 inflammasome ku-pathogenicity ye-G. duodenalis ihlala ingacacile.Ukuze siphenye le nkinga, siklame ukuhlola phakathi kwamagundane atheleleke nge-G. duodenalis cyst kanye namagundane atheleleke nge-G. duodenalis cyst + MCC950 inhibitor treatment futhi saqhathanisa ne-NLRP3 expression inflammasome lapho etheleleke nge-G. duodenalis cyst.Uhlelo olunemininingwane yokuhlolwa luboniswa ku-Fig. 3a.Izinguquko zesisindo somzimba wamagundane emaqenjini ahlukene okwelapha aqashwe izinsuku ezingu-7 ngemva kokutheleleka ngama-cysts, futhi imiphumela iboniswa ku-Fig. 3b.Uma kuqhathaniswa neqembu eliphathwa nge-PBS ehlanzekile, imiphumela yabonisa ukuthi (i) isisindo somzimba wamagundane aphethwe yi-G. duodenalis cyst sehla kusukela ngosuku lwe-3 kuya osukwini lwe-7 ngemva kokutheleleka;(ii) ukwelashwa nge-MCC950 inhibitor akuzange kube nomthelela obalulekile esisindweni somzimba samagundane..Uma kuqhathaniswa neqembu elilodwa lokutheleleka, i-BW yeqembu lokutheleleka kwe-duodenal elashwa nge-MCC950 yehle yaba ngamadigri ahlukahlukene (Usuku 1: ANOVA, F(3, 24) = 1.885, P = 0.0148; Usuku 2: ANOVA, F(3, 24) ) = 0.4602, P<0.0001; Usuku 3: ANOVA, F(3, 24) = 0.8360, P = 0.0010; Usuku 4: ANOVA, F(3, 24) = 1.683, P = 0.0052; (3, 24)=0.6497, P=0.0645; Usuku 6: ANOVA, F(3, 24)=5.457, P=0.0175; Usuku 7: ANOVA, F(3, 24) = 2.893, P = 0.0202).Le datha ibonisa ukuthi i-NLRP3 inflammasome ivikela amagundane ekulahlekelweni kwesisindo esibalulekile ezigabeni zokuqala (izinsuku ezingu-2-4) zokutheleleka kwe-duodenal.Sabe sesihlose ukuthola i-G. duodenalis trophozoites ku-duodenal lavage fluid futhi imiphumela ikhonjiswe kuMfanekiso 3c.Uma kuqhathaniswa neqembu le-G. duodenalis cyst infections, inani lama-trophozoites ku-duodenum landa kakhulu ngemva kokuvimbela i-NLRP3 inflammasome (t (12) = 2.902, P = 0.0133).Izicubu ze-Duodenal ezingcoliswe nge-HE zibonisiwe, uma kuqhathaniswa nokulawulwa okungalungile okuphathwe nge-PBS ne-MCC950 kuphela: (i) Ukutheleleka kwe-G. duodenalis cyst kubangele ukulimala kwe-duodenal villi (ANOVA, F(3, 24)=0.4903, P= 0.0488 ) kanye ne-crypt atrophy (ANOVA, F(3, 24) = 0.4716, P = 0.0089);(ii) i-duodenum evela kumagundane atheleleke nge-G. duodenalis cysts futhi yelashwa nge-MCC950 inhibitors.i-duodenal villi yonakalisiwe futhi ifile (ANOVA, F(3, 24) = 0.4903, P = 0.0144) ene-atrophy ne-crypt branching (ANOVA, F(3, 24) = 0.4716, P = 0, 0481) (Fig. 3d- f).Le miphumela iphakamisa ukuthi i-NLRP3 inflammasome idlala indima ekwehliseni i-pathogenicity ye-G. duodenalis.
Indima ye-NLRP3 inflammasome ku-Giardia duodenum infection.Amagundane ahlukunyezwa (iv) ngama-cysts e-duodenococcal abese ephathwa nge-MCC950 (ip) noma ngaphandle kwayo.Amaqembu okwelapha awodwa ane-PBS noma i-MCC950 asetshenziswe njengezilawuli.Iqembu lokuhlola kanye nemithi yokwelapha.b Isisindo somzimba samagundane eqenjini ngalinye lamaqembu okwelapha ahlukene sagadwa izinsuku ezingu-7.Umehluko phakathi kweqembu lokutheleleka kwe-G. duodenalis kanye neqembu lokwelapha ukutheleleka kwe-G. duodenalis + MCC950 wahlaziywa ngokuhlolwa kwe-t kusetshenziswa i-SPSS software version 22.0.Izinkanyezi zibonisa umehluko omkhulu kokuthi *P<0.05, **P<0.01, noma ***P<0.001.c Umthwalo we-parasitic wanqunywa ngokubala inani lama-trophozoite oketshezini lokuhlanzwa kwe-duodenal.Umehluko phakathi kweqembu lokutheleleka kwe-G. duodenalis kanye neqembu lokwelapha ukutheleleka kwe-G. duodenalis + MCC950 wahlaziywa ngokuhlolwa kwe-t kusetshenziswa i-SPSS software version 22.0.Izinkanyezi zibonisa umehluko omkhulu kokuthi *P <0.05.d Imiphumela ye-Hematoxylin ne-eosin (H&E) ye-duodenal histopathology.Imicibisholo ebomvu ibonisa umonakalo ku-villi, imicibisholo eluhlaza ibonisa umonakalo kuma-crypts.Ibha yesikali: 100 µm.e, f Ukuhlaziywa kwezibalo zobude be-duodenal villus nobude be-crypt yegundane.Izinkanyezi zibonisa umehluko omkhulu kokuthi *P<0.05 kanye ne-**P<0.01.Imiphumela ithathwe ekuhlolweni okuzimele okungu-7 kwebhayoloji.BW, isisindo somzimba;ig, umzila wokulethwa kwe-intragastric;ip, umzila wokulethwa kwe-intraperitoneal;ns, akubalulekile (P > 0.05);i-PBS, i-phosphate buffered saline;I-WT, uhlobo lwasendle
Ukugcinwa kwe-IL-1β kuwuphawu lokusebenza kokuvuvukala.Ukuze sinqume ukuthi i-G. duodenalis alpha-2 giardine kanye ne-alpha-7.3 giardine zenza kusebenze umsingathi we-NLRP3 i-inflammasome ku-vivo, sisebenzise amagundane e-WT angalashiwe (iqembu le-sham) kanye ne-NLRP3 evinjiwe i-inflammasome (iqembu lokwelapha elivinjiwe le-MCC950).Uhlelo olunemininingwane yokuhlolwa luboniswa ku-Fig. 4a.Amaqembu okuhlola ahlanganisa amagundane aphathwe nge-PBS, G. duodenalis cyst treatment by gavage, intramuscular injection of pcDNA3.1, kanye nomjovo we-intramuscular we-pcDNA3.1(+) -alpha-2 giardine noma pcDNA3.1-alpha-7.3 giardine.Ngosuku lwe-7 ngemuva kokuphathwa kwe-intramuscular ye-plasmid ephindaphindiwe, i-serum yaqoqwa futhi izinga le-IL-1β eqenjini ngalinye lanqunywa.Njengoba kuboniswe ku-Figure 4b, eqenjini le-MOCK: (i) uma kuqhathaniswa neqembu le-PBS, ukwelashwa kwe-pcDNA3.1 akuzange kube nomphumela ophawulekayo ku-IL-1β secretion (ANOVA, F (4.29) = 4.062, P=0.9998), noma kunjalo, I-IL-β secretion yayiphakanyiswe kakhulu eqenjini le-G. duodenalis cyst (ANOVA, F (4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine kanye ne-pcDNA3.I-1- I-intramuscular injection ye-alpha-7.3 giardine yanda kakhulu amazinga e-serum IL-1β (ANOVA, F (4, 29) = 4.062, P <0.0001);(iii) i-pcDNA3.1-alpha-7,3 i-giardine yenza amazinga aphezulu e-IL -1β secretion eqenjini le-pcDNA3.1-alpha-2 giardine intramuscular injection (ANOVA, F (4, 29) = 4.062, P = 0.0333) .Uma kuqhathaniswa neqembu ngalinye eqenjini lokwelapha le-MCC950 kanye neqembu le-MOCK: (i) amazinga e-IL-1β emfihlo eqenjini lokulawula le-PBS kanye neqembu lokulawula le-pcDNA3.1 lehle ngezinga elithile ngemva kokuvimbela i-MCC950 inhibitor, kodwa umehluko wawungekho okubalulekile (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F(9, 58) = 3.540, P = 0.5949);(ii) ngemuva kokuvimba i-MCC950., Imfihlo ye-IL-1β yehliswe kakhulu eqenjini le-G. duodenalis cyst-infected, iqembu le-pcDNA3.1-alpha-2 giardine, kanye neqembu le-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).Le miphumela iphakamisa ukuthi i-alpha-2 giardine ne-alpha-7.3 giardine ilamula ukusebenza kwe-NLRP3 inflammasome ku-vivo.
i-pcDNA3.1(+)-giardines yenza kusebenze umsingathi we-NLRP3 we-inflammasome ku-vivo.Amagundane agonyiwe (IM) nge-recombinant eukaryotic expression plasmid pcDNA3.1(+)-alpha-2 giardine noma i-pcDNA3.1(+)-alpha-7.3 giardine bese ephathwa nge-MCC950 (ip; MCC950 group) noma cha (iqembu le-dummy ).I-PBS noma i-pcDNA3.1 (+) iqembu lokwelapha i-plasmid lisetshenziswe njengokulawula okungalungile, iqembu lokwelapha i-cyst ye-G. duodenalis lisetshenziswe njengokulawula okuhle.Iqembu lokuhlola kanye nemithi yokwelapha.b Amazinga we-Serum we-IL-1β kumagundane alinganiswa ngosuku lwe-7 ngokuhlolwa kwe-ELISA.Umehluko phakathi kwamaqembu eqenjini le-MOCK wahlaziywa kusetshenziswa i-ANOVA yendlela eyodwa, futhi umehluko phakathi kweqembu le-MOCK neqembu le-MCC950 wahlaziywa kusetshenziswa ukuhlolwa kwe-t kwenguqulo yesofthiwe ye-SPSS engu-22.0.Izinkanyezi zibonisa umehluko omkhulu phakathi kwamaqembu okwelapha eqenjini le-MOCK, *P<0.05 kanye ne-***P<0.001;izimpawu zedola ($) zibonisa umehluko omkhulu phakathi kweqembu ngalinye eqenjini le-MOCK neqembu le-MCC950 ku-P<0.05.Imiphumela yezilingo eziyisikhombisa ezizimele zebhayoloji.i, umjovo we-intramuscular, ns, awubalulekile (P > 0.05)
Ukuphenya umthelela we-alpha-2 ne-alpha-7.3 giardine-mediated activation ye-NLRP3 host inflammasome ku-G. duodenalis infectivity, sisebenzise amagundane e-WT C57BL/6 futhi sajova i-alpha-2 giardine ne-alpha-7.3 giardine.i-plasmid yajovwa nge-intramuscularly, ngemva kwezinsuku ezingu-3 ngethubhu yesisu ye-G. duodenalis cyst, ngemva kwalokho amagundane abonwa izinsuku ezingu-7.Uhlelo olunemininingwane yokuhlolwa luboniswa ku-Fig. 5a.Isisindo somzimba segundane ngalinye sasilinganiswa nsuku zonke, amasampula ezicubu ze-duodenal entsha aqoqwe ngosuku lwe-7 ngemva kokuphathwa ngeshubhu lesisu, inani lama-trophozoite likalwa, futhi kwabonwa izinguquko ze-histopathological.Njengoba kuboniswe kuMfanekiso 5b, ngokukhula kwesikhathi sokudla, i-BW yamagundane eqenjini ngalinye yanda kancane kancane.I-MT yamagundane yaqala ukwehla ngosuku lwe-3 ngemuva kokuphathwa kwe-intragastric ye-G. duodenalis cysts, futhi kancane kancane yanda.Ukwenziwa kusebenze kwe-NLRP3 inflammasome okudalwe umjovo we-intramuscular we-alpha-2 giardine kanye ne-alpha7.3 giardine kwanciphisa kakhulu ukwehla kwesisindo kumagundane (Usuku 1: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 1.399, P = 0 .9754 Usuku 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30)=1.399, P=0.9987 Usuku 2: pcDNA3.1-alpha-2 giardine, ANOVA, F( 4, 30) = 0.3172, P = 0.9979; Usuku 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.3172, P = 0.8409; Usuku 3: pcDNA3.1-alpha-2 giardine, ANOVA, ANOVA 4, 30) = 0.8222, P = 0.0262 Usuku 3: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.8222, P = 0.0083; Usuku 4: pcDNA3.1-alpha-2 ANOVA , F(4, 30) = 0.5620, P = 0.0012, Usuku 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5620, P <0.0001, Usuku 5: pcDNA3.1-alpha - 2 giardine, ANOVA, F(4, 30) = 0.9728, P < 0.0001 Usuku 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Usuku 6. -1 pcDNA alpha-2 giardine, ANOVA, F(4, 30) = 0.7154, P = 0.0012, Usuku 6: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0006;Usuku 7: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.5369, P<0.0001 Usuku 7: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4 , 30) = 0.5369, P <0.0001).Umthwalo we-parasitic wahlolwa ku-duodenum (Fig. 5c).Uma kuqhathaniswa nokulawulwa okuhle okungalashwa kanye neqembu elijovwe nge-pcDNA3.1 vector engenalutho, inani le-G. duodenalis trophozoites lancishiswa kakhulu emaqenjini ajovwe nge-α-2 giardine kanye ne-α-7,3 giardine (pcDNA3.1-alpha -2 i-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).Ngaphezu kwalokho, i-giardine alfa-7.3 yayivikela kakhulu kumagundane kune-giardine alfa-2 (ANOVA, F (3, 24) = 1.209, P = 0.0081).Imiphumela ye-HE staining ikhonjiswe kumfanekiso.5d–f.Amagundane ajovwe nge-alpha-2 giardine kanye ne-alpha-7.3 giardine ayenezilonda ezimbalwa zezicubu ze-duodenal, ezibonakaliswa ukulimala kwe-villus, uma kuqhathaniswa namagundane ajovwe nge-G. duodenalis kanye namagundane ajovwe nge-G. duodenalis ahlanganiswe ne-pcDNA3 vector engenalutho .1 Zoom.(pcDNA3.1-alpha-2 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0035 noma P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0028 noma P = 0.0055) kanye ne-crypt atrophy encishisiwe (pcDNA3.1-alpha-2 giardine: ANOVA, F(3, 24) = 1.470, P = 0.0264 noma P = 0.0158; pcDNA3.1-alpha-7.3 ANOVADINE , F(3, 24) = 1.470, P = 0.0371 noma P = 0.0191).Le miphumela iphakamisa ukuthi i-alpha-2 giardine ne-alpha-7,3 giardine yehlisa ukutheleleka kwe-G. duodenalis ngokwenza kusebenze i-NLRP3 inflammasome ku-vivo.
Indima ye-pcDNA3.1 (+)-giardins ku-G. duodenalis infection.Amagundane agonyiwe (IM) nge-recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardine noma i-pcDNA3.1(+)-alpha-7.3 giardine abese ephonselwa inselelo ngama-G. duodenalis cysts (ig).Iqembu le-PBS kanye neqembu le-pcDNA3.1 (+) + + lokwelashwa kwe-cyst duodenal lisetshenziswe njengamaqembu okulawula okungalungile, futhi iqembu lokwelapha i-cyst duodenal lisetshenziswe njengeqembu lokulawula elihle.Iqembu lokuhlola kanye nemithi yokwelapha.b I-MT yamagundane kuqembu ngalinye lamaqembu okwelapha ahlukahlukene yaqashwa izinsuku eziyi-7 ngemuva kokuphonselwa inselelo.Izinkanyezi zibonisa umehluko omkhulu phakathi kwamaqembu eqembu le-G. duodenalis kanye neqembu le-pcDNA3.1(+)-alpha-2 giardine, *P <0.05, **P <0.01, kanye ne-***P <0.001;uphawu lwedola ($) lubonisa umehluko omkhulu phakathi kweqembu ngalinye le-G. duodenalis neqembu le-pcDNA3.1(+)-alpha-7.3 jardine, $$P<0.01 kanye ne-$$$P<0.001.c Umthwalo we-parasitic wanqunywa ngokubala inani lama-trophozoite ku-1 ml we-duodenal lavage kusuka ku-duodenum (ubude obu-3 cm) futhi uvezwa njengenani lezimuncagazi ngesentimitha ngayinye ye-duodenum.Umehluko phakathi kweqembu le-G. duodenalis infection, pcDNA3.1(+)-alpha-2 giardine group, kanye neqembu le-pcDNA3.1(+)-alpha-7.3 giardine lihlaziywe ngendlela eyodwa i-ANOVA kusetshenziswa i-SPSS software version 22.0.Izinkanyezi zibonisa umehluko omkhulu kokuthi **P<0.01 kanye nokuthi ***P<0.001.d Izinguquko ze-Histopathological ku-duodenum.Imicibisholo ebomvu ibonisa umonakalo ku-villi, imicibisholo eluhlaza ibonisa umonakalo kuma-crypts.Ibha yesikali: 100 µm.e, f Ukuhlaziywa kwezibalo zobude begundane duodenal villus (e) nobude be-crypt (f).Umehluko phakathi kwamaqembu ku-Figure 1d uhlaziywe nge-ANOVA yendlela eyodwa kusetshenziswa i-SPSS software version 22.0.Izinkanyezi zibonisa umehluko omkhulu kokuthi *P<0.05 kanye ne-**P<0.01.Imiphumela yezilingo eziyisikhombisa ezizimele zebhayoloji.ns, akubalulekile (P > 0.05)
I-Giardia duodenum iyi-parasite yamathumbu eyaziwa kakhulu kubantu nezinye izilwane ezincelisayo ezibangela i-giardiasis.Ngo-2004, yafakwa ku-WHO Neglected Disease Initiative ngenxa yokusabalala kwayo okuphezulu eminyakeni eyi-6, ikakhulukazi emiphakathini yesimo esiphansi senhlalo-mnotho [32].Amasosha omzimba azalwa edlala indima ebalulekile ekuphenduleni kokuzivikela komzimba ekuthelelekeni kwe-G. duodenalis.Ama-macrophage egundane kubikwe ukuthi agwinya futhi abulala i-G. duodenalis ngokukhulula izicupho ezingaphandle kweseli [33].Ucwaningo lwethu lwangaphambilini lubonise ukuthi i-G. duodenalis, i-parasite ye-extracellular engeyona invasive, ivuselela i-p38 MAPK, ERK, NF-κB p65, kanye ne-NLRP3 izindlela zokubonisa ukuvuvukala kuma-macrophages wegundane ukuze ulawule izimpendulo ezivuthayo zokusingatha, futhi i-GEV ekhishwe ingase ithuthukise le nqubo.13], 24].Kodwa-ke, ama-PAMP aqondile ahilelekile ku-NLRP3 ukuvuvukala okulawulwa yi-inflammasome ku-GEV kanye nendima ye-NLRP3 inflammasome ku-giardiasis kusazocaciswa.Ukuze sikhanyisele le mibuzo emibili, senze lolu cwaningo.
I-NLRP3 inflammasome itholakala ku-cytoplasm yamangqamuzana omzimba omzimba futhi ingasetshenziswa yizinhlayiya ezihlukahlukene ezifana namakristalu e-uric acid, ubuthi, amabhaktheriya, amagciwane, nama-parasites.Ezifundweni zebhaktheriya, ubuthi buye babonwa njengama-PAMP abalulekile enza kusebenze izinzwa ezivuthayo, okuholela ekuvuvukeni nasekufeni kweseli [34].Obunye ubuthi obuhlukahlukene ngokwesakhiwo, obufana ne-hemolysin obuvela ku-Staphylococcus aureus [35] kanye ne-Escherichia coli [36], i-hemolysin BL (HBL) evela ku-enterotoxin (NHE) [37], yenza kusebenze ukuvuvukala kwe-NLRP3.Ucwaningo lwegciwane lubonise ukuthi amaprotheni ayingozi njenge-SARS-COV-2 envelope (E) protein [38] kanye ne-Zika virus NS5 protein [39] angama-PAMP abalulekile abonwa yi-NLRP3 receptor.Ezifundweni ze-parasite, ama-parasite amaningi kuye kwabikwa ukuthi ahlotshaniswa nokusebenza kwe-inflammasome, njenge-Toxoplasma gondii, i-Trichomonas vaginalis [40], i-Trypanosoma cruzi [41], ne-Leishmania [42].Amaprotheni aminyene we-granule GRA35, GRA42, kanye ne-GR43, ahlotshaniswa ne-virulence ye-Toxoplasma gondii, ayadingeka ukuze kufakwe i-pyroptosis ku-Lewis rat macrophages [43].Ngaphezu kwalokho, ezinye izifundo ze-Leishmania zigxile kuma-molecule ngamanye ahilelekile ku-NLRP3 inflammasome, njenge-parasite membrane lipophosphoglycan [44] noma i-zinc metalloprotease [45].Phakathi komndeni wezakhi zofuzo ezifana ne-alpha-giardin, i-alpha-1 giardin ikhonjiswe ukuthi ingase ibe ikhandidethi lokugoma elihlinzeka ngokuvikeleka ku-G. duodenalis kumodeli yegundane [18].Ocwaningweni lwethu, sikhethe izici ze-G. duodenalis virulence i-alpha-2 kanye ne-alpha-7,3 giardines, ehlukile ku-giardia kodwa ebikiwe kancane.Lezi zakhi zofuzo ezimbili eziqondiwe zenziwe ku-pcDNA3.1(+) i-eukaryotic expression vector ukuze kuhlaziywe ukusebenza kokuvuvukala.
Kumodeli yethu yegundane, izingcezu ze-caspase eziqhekekile zisebenza njengezimpawu zokuvula ukuvuvukala.Lapho ivuselelwa, i-NLRP3 isebenzisana ne-ASC, iqoqa ama-procaspases, futhi ikhiqize ama-caspases asebenzayo ahlukanisa i-pro-IL-1β ne-pro-IL-18 ku-IL-1β ne-IL-18 evuthiwe, ngokulandelana -18.Ama-caspases avuthayo (ama-caspases-1, -4, -5 kanye -11) awumndeni ogciniwe wama-cysteine ​​​​proteases abalulekile ekuzivikeleni kwangaphakathi futhi abandakanyeka ekuvuvukeni nasekufeni kweseli okuhleliwe [46].I-Caspase-1 icushwe yi-canonical inflammasomes [47], kuyilapho i-caspases-4, -5, ne--11 ihlukaniswa ngesikhathi sokwakhiwa kwe-inflammasomes ye-atypical [48].Kulolu cwaningo, sisebenzise ama-macrophage e-mouse peritoneal njengemodeli futhi saphenya i-p20 caspase-1 eklanywe i-caspase-1 njengophawu lwe-host NLRP3 activation activation ezifundweni zokutheleleka kwe-G. duodenalis.Imiphumela yabonisa ukuthi ama-alpha-giardin amaningi anesibopho sokusebenza okuvamile kokuvuvukala, okuhambisana nokutholakala kwama-molecule e-virulence abalulekile ahilelekile kumabhaktheriya namagciwane.Kodwa-ke, ucwaningo lwethu luyisikrini sokuqala kuphela futhi kukhona amanye ama-molecule angenza kusebenze ama-inflammasomes angewona ama-classical, njengoba ucwaningo lwethu lwangaphambilini lwathola kokubili ama-inflammasomes e-classical kanye ne-non-classical ku-G. duodenalis infection [13].Ukuze siqhubeke sinqume ukuthi ingabe i-p20 caspase-1 ekhiqiziwe ihlotshaniswa ne-NLRP3 inflammasome, sidlulisele i-alpha-2 kanye ne-alpha-7.3 giardins kuma-macrophages e-peritoneal yegundane ukuze sinqume amazinga ayinhloko we-molecule we-molecule kanye namazinga e-oligomerization ye-ASC, siqinisekisa ukuthi womabili ama-α-giardin ayasebenza. ukuvuvukala kwe-NLRP3.Imiphumela yethu ihluke kancane kuleyo kaManko-Prykhoda et al., owabika ukuthi ukukhuthazwa kwamaseli e-Caco-2 ane-G. muris noma i-E. coli EPEC strains kuphela kungakhuphula amandla e-fluorescence e-NLRP3, ASC, ne-caspase-1, nakuba kungenjalo kakhulu, kuyilapho i-costimulation ye-G. muris ne-E. coli ikhulise amazinga amaprotheni amathathu [49].Lokhu kuhluka kungase kubangelwe umehluko ekukhethweni kwezinhlobo ze-Giardia, imigqa yamaseli, namaseli ayisisekelo.Siphinde senza izivivinyo ze-vivo sisebenzisa i-MCC950 kumagundane wesifazane we-WT C57BL/6 onamasonto angu-5, asengozini yokuthola i-G. duodenalis.I-MCC950 iyi-molecule encane enamandla futhi ekhethayo ye-NLRP3 inhibitor evimba ukusebenza kwe-canonical kanye ne-non-canonical NLRP3 ekugxilweni kwe-nanomolar.I-MCC950 ivimbela ukusebenza kwe-NLRP3 kodwa akuthinti ukwenziwa kusebenze kwe-AIM2, NLRC4, kanye ne-NLRP1 izindlela zokuvuvukala noma izindlela zokusayina ze-TLR [27].I-MCC950 ivimba ukusebenza kwe-NLRP3 kodwa ayikuvimbeli ukuqaliswa kwe-NLRP3, i-K+ efflux, ukungena kwe-Ca2+, noma ukuxhumana phakathi kwe-NLRP3 ne-ASC;esikhundleni salokho, ivimbela ukusebenza kwe-NLRP3 inflammasome ngokuvimbela i-ASC oligomerization [27].Ngakho-ke, sisebenzise i-MCC950 ocwaningweni lwe-vivo ukuze sinqume indima ye-NLRP3 inflammasome ngemva komjovo we-giardine.I-caspase-1 p10 ecushiwe ihlukanisa ama-cytokines ane-pro-inflammatory pro-IL-1β kanye ne-pro-IL-18 ku-IL-1β evuthiwe kanye ne-IL-18 [50].Kulolu cwaningo, amazinga e-serum IL-1β kumagundane aphethwe yi-giardine ane-MCC950 noma angenayo asetshenziswe njengenkomba yokuthi i-NLRP3 inflammasome yenziwe yasebenza.Njengoba bekulindelekile, ukwelashwa kwe-MCC950 kwehlisa kakhulu amazinga e-serum IL-1β.Le datha ibonisa ngokucacile ukuthi i-G. duodenalis giardin alfa-2 kanye ne-giardin alfa-7.3 bayakwazi ukwenza kusebenze i-NLRP3 mouse inflammasome.
Idatha ebalulekile eqoqwe kule minyaka eyishumi edlule ibonise ukuthi i-IL-17A iyisilawuli esiyinhloko sokuvikeleka ngokumelene ne-G. muris, idala ukusayina kwe-IL-17RA, ikhiqize ama-peptide e-antimicrobial, futhi ilawula ukusebenza kokugcwalisa [51].Kodwa-ke, ukutheleleka kwe-Giardia kwenzeka kaningi kubantu abadala abasebasha, futhi kubikwe ukuthi ukutheleleka kwe-Giardia kumagundane amancane akusebenzi impendulo ye-IL-17A ukuze yenze umphumela wayo wokuvikela [52], okwenza abacwaningi babheke enye i-Giardia ye-immunomodulatory.izindlela zokutheleleka kwe-helminth.Ababhali bocwaningo lwakamuva babike ukuthi i-G. muris ingakwazi ukwenza i-NLRP3 inflammasome yi-E. coli EPEC, ekhuthaza ukukhiqizwa kwama-peptide e-antimicrobial futhi inciphise umthamo wayo wokunamathisela kanye nenani lama-trophozoite emgudwini wamathumbu, ngaleyo ndlela inciphise ubukhulu bekholoni. izifo ezibangelwa i-bacilli [49].I-NLRP3 inflammasome ihileleke ekuthuthukiseni izifo ezihlukahlukene.Ucwaningo luye lwabonisa ukuthi i-Pseudomonas aeruginosa idala i-autophagy kuma-macrophages ukugwema ukufa kweseli, futhi le nqubo incike ekusebenzeni kwe-NLRP3 inflammasome [53].Ku-N. caninum, ukusetshenziswa kwe-oxygen esebenzayo yezinhlobo ze-NLRP3 inflammasome kunciphisa ukuphindaphinda kwayo kumsingathi, okwenza kube okuhloswe ngakho ukwelashwa [9].I-Paracoccidioides brasiliensis itholakale yenza kusebenze i-NLRP3 inflammasome kumaseli e-dendritic atholakala emnkantsha wegundane, okuholela ekukhululweni kwe-cytokine evuthayo i-IL-1β, edlala indima ebalulekile ekuvikelweni kokusingatha [10].Izinhlobo ezimbalwa ze-Leishmania, okuhlanganisa i-L. amazonensis, L. major, L. braziliensis, kanye ne-L. infantum chagasi, zisebenzisa i-NLRP3 kanye ne-ASC-dependent caspase-1 kuma-macrophage, kanye ne-Leishmania infection.Ukuphindaphinda kwe-parasite kuthuthukisiwe kumagundane ashodayo kuhlobo lwe-NLRP3/ASC/caspase-1 [11].Zamboni et al.Ukutheleleka kwe-Leishmania kuye kwabikwa ukuthi kwenze kusebenze i-NLRP3 inflammasome kuma-macrophages, okuvimbela ukuphindaphinda kwe-intracellular parasite.Ngakho, i-Leishmania ingase ivimbele ukusebenzisa i-NLRP3 njengesu lokugwema.Ezifundweni ze-vivo, i-NLRP3 inflammasome ibe negalelo ekuqedeni i-Leishmania, kodwa ayizange ithinte izicubu [54].Ngakolunye uhlangothi, ezifundweni ze-helminthiasis, ukwenziwa kusebenze kwe-NLRP3 inflammasome kucindezela ukuzivikela komphikisi ngokumelene ne-helminthiasis yamathumbu [12].I-Shigella ingelinye lamagciwane abangela isifo sohudo emhlabeni wonke.Lawa mabhaktheriya angakwazi ukukhiqiza i-IL-1β ngokusebenzisa i-P2X7 receptor-mediated K+ efflux, izinhlobo ze-oxygen esebenzayo, i-lysosomal acidification, kanye nokulimala kwe-mitochondrial.I-NLRP3 inflammasome ilawula kabi i-phagocytosis kanye nomsebenzi we-bactericidal wama-macrophages ngokumelene ne-Shigella [55].Ucwaningo lwe-Plasmodium lubonise ukuthi i-AIM2, i-NLRP3 noma i-caspase-1 entula amagundane atheleleke nge-Plasmodium akhiqiza amazinga aphezulu ohlobo lwe-interferon ye-1 futhi amelana kakhulu nokutheleleka kwe-Plasmodium [56].Kodwa-ke, indima ye-alpha-2 giardine ne-alpha-7.3 giardine ekwenzeni ukusebenza kwe-pathogenic ye-NLRP3 ukuvuvukala kumagundane akucaci.
Kulolu cwaningo, ukuvinjelwa kwe-NLRP3 inflammasome nge-MCC950 kunciphise i-BW futhi kwandise inani lama-trophozoite oketshezini lokuhlanza emathunjini kumagundane, okuholela ekushintsheni okubi kakhulu kwe-pathological kuzicubu ze-duodenal.I-Alpha-2 giardine ne-alpha-7.3 giardine yenza kusebenze igundane elingumsingathi i-NLRP3 inflammasome, andise isisindo somzimba wegundane, anciphise inani lama-trophozoite oketshezini lokugezwa kwamathumbu, futhi anciphise izilonda ze-duodenal.Le miphumela iphakamisa ukuthi i-G. duodenalis ingakwazi ukusebenzisa i-NLRP3 host inflammasome nge-alpha-2 giardine ne-alpha-7,3 giardine, inciphise i-pathogenicity ye-G. duodenalis kumagundane.
Ngokuhlangene, imiphumela yethu ibonisa ukuthi i-alpha-2 ne-alpha-7.3 giardines yenza kusebenze i-NLRP3 host inflammasome futhi inciphisa ukutheleleka kwe-G. duodenalis kumagundane.Ngakho-ke, lezi zinhlayiya ziyizinjongo ezithembisayo zokuvimbela i-giardiasis.
       Data supporting the results of this study can be obtained from the respective author at gongpt@jlu.edu.cn.
Liang AKS, Liang AAM, Huang AHC, Sergi KM, Kam JKM.I-Giardiasis: isifinyezo.Muva nje kuvele ukuthi uPat Inflamm akazwani nezidakamizwa.2019;13:134–43.
Escobedo AA, Tsimerman S. Giardiasis: ukubuyekezwa kwe-pharmacotherapy.Umbono Wochwepheshe kasokhemisi.2007;8: 1885-902.
Tian Huafeng, Chen Bin, Wen Jianfeng.I-Giardiasis, ukumelana nezidakamizwa kanye nokutholakala kwezinhloso ezintsha.Ithelela okuhlosiwe kwezidakamizwa ze-Disord.2010;10:295–302.
Wang Z, Zhang X, Xiao Yi, Zhang W, Wu X, Qin T, njll. I-NLRP3 izifo ezivuthayo kanye nezifo ezivuthayo.I-Oxide Med Cell Longev.2020;2020:4063562.
Chen GY, Núñez G. Indima ye-inflammasome ekuvuvukeni kwamathumbu nomdlavuza.I-Gastroenterology.2011;141:1986–99.
Pellegrini C, Antonioli L, Lopez-Castejon G, Blandizzi C, Fornai M. Canonical kanye ne-atypical NLRP3 activation inflammasome ezimpambanweni zokumelana nokuzivikela komzimba kanye nokuvuvukala kwamathumbu.ngaphambi kokuzivikela komzimba.2017;8:36.
Li L, Wang XC, Gong PT, Zhang N, Zhang X, Li S, et al.I-ROS-mediated NLRP3 activation inflammasome activation ihileleke ekuphenduleni ukutheleleka kwe-N. caninum.I-parasite vector.2020;13:449.