I-Giardia duodenum sisifunxi-gazi esibangela i-giardiasis, usulelo lwamathumbu oluxhaphake ngakumbi kubantwana abancinci abaneempawu zekliniki zorhudo.Siye sabika ngaphambili ukuba i-extracellular G. duodenalis ibangela ukuba kusebenze i-intracellular oligomerization-like receptor 3 (NLRP3) ebopha i-nucleotides kwaye ilawula iimpendulo ezivuthayo ezithintekayo ngokusebenzisa i-extracellular vesicle (EV) secretion.Nangona kunjalo, iipatheni ezichanekileyo ze-molecular ye-pathogen-associated duodenococcal EV (GEV) echaphazelekayo kule nkqubo kunye nendima ye-NLRP3 inflammasome kwi-giardiasis ihlala icaciswa.
I-recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 kunye ne-alpha-7.3 giardins kwi-GEV zakhiwa, zatshintshelwa kwi-mouse primary peritoneal macrophages, kwaye zichongiwe ngokulinganisa imolekyuli ekujoliswe kuyo ukudumba caspase-1.Inqanaba lokubonisa i-p20 lihlolwe..I-G. duodenalis alpha-2 kunye ne-alpha-7.3 giardines zachongwa ekuqaleni ngokulinganisa i-NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 kunye ne-caspase-1 p20), i-IL secretion.Amanqanaba e-1β, i-apoptotic spotted protein (ASC) amanqanaba e-oligomerization, kunye ne-immunofluorescent localization ye-NLRP3 kunye ne-ASC.Indima ye-NLRP3 inflammasome kwi-pathogenicity ye-G. duodenalis emva koko ihlolwe kusetyenziswa iigundane apho i-NLRP3 isebenze ivaliwe (i-NLRP3 ivinjiwe iigundane) kunye neenguqu ze-pathological kubunzima bomzimba, umthwalo we-duodenal parasitic, kunye nezicubu ze-duodenal zibekwe esweni.Ukongezelela, siye saphanda ukuba ngaba i-hiardines alpha-2 kunye ne-alpha-7.3 yenza i-IL-1β secretion in vivo ngokusebenzisa i-NLRP3 inflammasome kwaye inqume indima yale molekyuli kwi-pathogenicity ye-G. duodenalis kwiigundane.
I-Alpha-2 kunye ne-alpha-7.3 giardines yenza ukuba kusebenze i-NLRP3 inflammasome in vitro.Oku kwakhokelela ekusebenzeni kwe-p20 caspase-1, ukunyuka kwamanqanaba okubonakaliswa kwe-NLRP3, i-pro-IL-1β, kunye neprotheyini ye-pro-caspase-1, ukwanda okukhulu kwe-IL-1β secretion, ukubunjwa kwamabala e-ASA kwindawo. icytoplasm, kunye nokungeniswa kwe-ASA oligomerization.Ukuvuvukala kwe-NLRP3 Ukulahleka kwePenile kwandisa i-pathogenicity ye-G. duodenalis kwiigundane.Iigundane eziphathwe ngama-cysts nge-gavage ukusuka kwi-NLRP3-evaliweyo iigundane zibonise inani elongezelelweyo le-trophozoites kunye nomonakalo omkhulu kwi-duodenal villi, ebonakaliswa yi-necrotic crypts kunye ne-shrunken kunye ne-branching.Uvavanyo lwe-vivo lubonise ukuba i-giardines alpha-2 kunye ne-alpha-7.3 inokubangela ukukhutshwa kwe-IL-1β nge-NLRP3 inflammasome, kunye nokugonywa nge-giardines alpha-2 kunye ne-alpha-7.3 yanciphisa i-pathogenicity ye-G. duodenalis kwiigundane.
Kuthatyathwe kunye, iziphumo zolu phononongo zibonisa ukuba i-giardia alpha-2 kunye ne-alpha-7.3 ibangela ukunyuswa kwe-host NLRP3 ukuvuvukala kunye nokunciphisa ukusuleleka kwe-G. duodenalis kwiigundane, ezijolise ekujoliswe kuyo ekukhuseleni i-giardiasis.
I-Giardia duodenum yi-extracellular protozoan parasite ehlala emathunjini amancinci kwaye ibangela iimeko ze-280 yezigidi ze-giardiasis kunye nohudo ngonyaka, ngakumbi kubantwana abancinci kumazwe asakhasayo [1].Abantu bosulelwa ngamanzi okusela okanye ukutya okungcoliswe yi-M. duodenum cysts, ethi emva koko ingene esiswini kwaye ikhutshwe kwijusi yesisu.I-Giardia duodenum trophozoites inamathele kwi-epithelium ye-duodenal, ibangela isicaphucaphu, ukuhlanza, isifo sohudo, intlungu yesisu, kunye nokunciphisa umzimba.Abantu abane-immunodeficiency kunye ne-cystic fibrosis basengozini yosulelo.Usulelo lunokwenzeka nangokwabelana ngesondo ngomlomo nangempundu [2].Amachiza afana ne-metronidazole, i-tinidazole, kunye ne-nitazoxanide zezona zikhethwayo zonyango losulelo lwe-duodenal [3].Nangona kunjalo, la machiza e-chemotherapy abangela iziphumo ezibi ezifana nesicaphucaphu, i-carcinogenesis, kunye ne-genotoxicity [4].Ngoko ke, izicwangciso ezisebenzayo ngakumbi kufuneka zenziwe ukuthintela usulelo lwe-G. duodenalis.
I-Inflammasomes yiklasi ye-cytosolic protein complexes eyingxenye yempendulo ye-immune ye-innate, inceda ukukhusela ekuhlaselweni kwe-pathogen kunye nokudibanisa iimpendulo ezivuthayo [5].Phakathi kwezi inflammasomes, i-nucleotide-binding oligomerization (NOD) receptor 3 (NLRP3) i-nucleotide-binding oligomerization (NLRP3) i-nucleotide-binding-like inflammasome ifundwe ngokubanzi ngenxa yokuba inokubonwa ngeendlela ezahlukeneyo ze-pathogen / umonakalo-ehambelana neepatheni ze-molecular (PAMP). I-DAMP), iyayibona, ivule inkqubo yokhuselo lomzimba.kwaye ilawula i-homeostasis yamathumbu kwizifo ezininzi ezivuthayo [6,7,8].Iqukethe i-pattern recognition receptor (PRR) NLRP3, i-adapter apoptotic spotted protein (ASC), kunye neprothector procaspase-1 okanye iprocaspase-11.I-NLRP3 inflammasome isebenza njengomkhosi ngokuchasene nokuhlasela kwe-pathogen, njengoko kubonwa kwi-Neospora caninum [9], i-Paracoccidioides brasiliensis [10], kunye nezifundo ze-Leishmania.[11], kodwa kuye kwaxelwa ukuba ukusetyenziswa kwe-NLRP3 i-inflammasome imida yokukhusela iimpendulo zokuzivikela kunye nokwandisa ukuqhubela phambili kwesifo, umzekelo, kwiimbungu [12].Ngokusekelwe kwiziphumo zethu zangaphambili, siye sabika ukuba i-extracellular G. duodenalis ibangela ukusebenza kwe-intracellular ye-NLRP3 ukuvuvukala kwaye imodareyitha iimpendulo ezivuthayo ngokukhupha i-extracellular vesicles (EVs) [13].Nangona kunjalo, indima ye-NLRP3 i-inflammasome kwi-G. duodenalis usulelo kwi-vivo ihlala izimisele.
I-Giardins ekuqaleni yayichazwa njengenxalenye yesakhiwo se-G. duodenalis cytoskeleton kwaye idlala indima ebalulekileyo kwi-trophozoite motility kunye ne-epithelial cell attachment kumathumbu amancinci.Ukuze ulungelelanise kakuhle indawo kunye nokwandisa i-pathogenicity yabo, i-G. duodenalis trophozoites yavelisa isakhiwo esiyingqayizivele se-cytoskeletal esiquka i-8 flagella, i-1 yomzimba ophakathi, kunye ne-1 ventral disc [14].I-trophozoites ye-Giardia duodenum isebenzisa i-cytoskeleton ukuze ingene kumathumbu amancinci aphezulu, ngakumbi i-duodenum, kwaye ifake kwi-enterocytes.Bahlala befuduka kwaye banamathele kwiiseli ze-epithelial usebenzisa i-cell metabolism.Ngoko ke, kukho ubudlelwane obusondeleyo phakathi kwe-cytoskeleton kunye ne-virulence.Iigiardines ezikhethekileyo kwi-Giardia duodenum ziyinxalenye yesakhiwo se-cytoskeleton [15] kwaye zihlulwe zibe ziiklasi ezine: α-, β-, γ-, kunye ne-δ-giardines.Kukho amalungu e-21 yentsapho ye-α-giardin, yonke into enekhono elixhomekeke kwi-calcium ukubopha i-phospholipids [16].Bakwadibanisa i-cytoskeleton kwi-membrane yeseli.Kubantu abanesifo sohudo esibangelwa yi-G. duodenalis, i-α-giardins ibonakaliswe kakhulu kwaye i-immunoreactive ngexesha losulelo [17].Ugonyo lwe-Heterologous olusekwe kwi-Giardia alfa-1 ekhuselweyo kwi-giardiasis kwiimpuku kwaye zingama-antigens anokubakho kuphuhliso lwesitofu [18].I-Alpha-8 giardin, efumaneka kwi-membrane ye-plasma kunye ne-flagella, kodwa kungekhona kwi-disc ye-ventral, iphakamisa i-motility kunye nesantya sokukhula kwe-trophozoites kwi-G. duodenalis [19].I-Alpha-14 giardin inamathele kwizakhiwo ze-microtubule kwi-flagella kwaye ichaphazela ukusebenza kwe-G. duodenalis [20].I-Alpha-11 giardine ikhona ngobuninzi kumjikelezo wobomi, kwaye ukugqithisa kwe-alpha-11 giardine yonakalisa i-G. duodenalis ngokwayo [21].Nangona kunjalo, akucaci ukuba i-alpha-2 giardine kunye ne-alpha-7.3 giardine zikhusela ukusuleleka kwi-G. duodenalis kunye neendlela zabo eziphantsi.
Kolu phononongo, i-recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine kunye ne-pcDNA3.1 (+) -alpha-7.3 giardine zatshintshelwa kwimouse primary peritoneal macrophages ukuze kusebenze i-NLRP3 yomkhosi.Iithagethi ezivuthayo zaye zahlolwa.Siphinde savavanya indima ye-NLRP3 inflammasome kwi-pathogenicity ye-G. duodenalis, uphando malunga nokuba i-alpha-2 kunye ne-alpha-7,3 giardines yenza ukuba kusebenze i-NLRP3 inflammasome kwi-vivo, kwaye yagqiba ukuba ezi zimbini iindima ze-giardines kwi-pathogenicity G. duodenalis.Injongo yethu efanayo yayikukuphuhlisa iinjongo ezithembisayo zokuthintela usulelo lwe-G. duodenalis.
Uhlobo lwasendle (WT) C57BL/6 iimpuku ezibhinqileyo ezineminyaka eyi-5–8 yeeveki zathengwa kwiZiko loMvavanyo leZilwanyana laseLiaoning Changsheng (Liaoning, China).Iigundane zazinokufikelela simahla emanzini, zifumana ukutya okwenziwa inzala kwaye zigcinwe kumjikelo weyure we-12/12 wokukhanya/ubumnyama.Ngaphambi kokusuleleka, iigundane zafumana i-antibiotics ad libitum emanzini okusela agalelwe i-ampicillin (1 mg / mL), i-vancomycin (1 mg / mL), kunye neomycin (1.4 mg / mL) (zonke ezithengwa eShanghai, eChina, izinto eziphilayo) [22] ].].Iigundane eziye zalahlekelwa amandla okutya kunye nokusela > iiyure ze-24 kwaye zalahlekelwa ≥ 20% ubunzima bomzimba zaye zaxhaswa ngobuntu ngokukhutshwa komlomo wesibeleko.
I-WB G. duodenalis trophozoites (iNgqokelela yeNkcubeko yoHlobo lwaseMelika, iManassas, eU.SA) yongezwa nge-12.5% ​​ye-fetal bovine serum (FBS; Yonke iGreen, iZhejiang, eChina) kunye ne-0.1% ye-bovine bile (Sigma-Aldrich, St. Missouri, USA) ).USA) phantsi kweemeko ze-microaerobic.Iitrophozoite ezidibeneyo zaqokelelwa emkhenkceni kwaye zagqithiswa kumlinganiselo we-1: 4 ukwenzela ukuveliswa kwakhona.
I-Giardia duodenum cysts yenziwe njengoko ichazwe ngaphambili [23], i-trophozoites yavunwa kwisigaba se-logarithmic kwaye yahlanjululwa nge-encapsulation inducing medium, i-pH 7.1 (i-TYI-S-33 elungisiweyo) ukuya kwi-concentration yokugqibela ye-1 × 106 trophozoites / mL.i-bile concentration 0.05% medium).I-Trophozoites yakhuliswa phantsi kweemeko ze-anaerobic kwi-37 ° C kude kube yinqanaba lokukhula kwe-logarithmic.Guqula i-medium kwi-cyst inducing medium (pH 7.8; i-TYI-S-33 elungisiweyo ephakathi kunye ne-1% ye-bile concentration) kunye nenkcubeko G. duodenalis kwi-37 ° C kwiiyure ze-48-96, apho i-cysts yokwakheka yabonwa phantsi kwe-microscope.Emva kokuba uninzi lwe-trophozoite luye lwanyanzelwa ukuba lwenze ama-cysts, umxube wenkcubeko wavunwa kwaye waphinda wamiswa emanzini angenazintsholongwane ukuze adibanise i-trophozoites eseleyo.I-Cysts yayibalwe kwaye igcinwe kwi-4 ° C ukwenzela uhlalutyo olulandelayo nge-tube yesisu kwiigundane.
I-Giardia extracellular vesicles (GEVs) yatyetyiswa njengoko kuchaziwe ngaphambili [13].I-Trophozoites kwisigaba sokukhula kwe-logarithmic yaphinda yamiswa kwi-TYI-S-33 medium modified elungiselelwe nge-exosome-depleted FBS (Biological Industries, Beit-Haemek, Israel) ukuya kugxininiso lokugqibela lwe-1 × 106 parasites / mL kwaye ifakwe kwiiyure ze-12.zahlulwe kwinkcubeko supernatant nge centrifugation kwi 2000 g for 10 min, 10,000 g for 45 min, kunye 100,000 g for 60 min.I-precipitates yachithwa kwi-phosphate buffered saline (PBS), ukulinganisa usebenzisa i-BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) kwaye igcinwe kwi -80 ° C. okanye isetyenziswe ngokuthe ngqo ukuhlalutya okungaphezulu.
I-macrophages ye-peritoneal ye-primary mouse yalungiswa njengoko kuchaziwe ngaphambili [24].Ngokufutshane, iigundane (ezineminyaka eyi-6-8 iiveki) zifakwe (intraperitoneally [ip]) kunye ne-2.5 ml ye-2.98% ye-Difco liquid thioglycol medium (BD, Franklin Lakes, NJ, USA) kwaye zondliwe i-3-4 palates.Ukumiswa kwe-macrophages kwaqokelelwa kwisigxina sesisu seegundane emva kwe-euthanasia kunye ne-centrifuged ngamaxesha e-3 kwi-1000 g ye-10 min.Iiseli ezivuniweyo zifunyenwe nge-cytometry yokuhamba kusetyenziswa i-CD11b marker de i-cell purity i> 98%, emva koko yongezwa kwiiplate ze-cell culture cell ze-6 (4.5 x 106 iiseli / kakuhle) kwaye zifakwe kwi-10% FBS (Bioindustry) kwi-37 ° C.kunye ne-5% CO2.
I-RNA ikhutshwe kwi-1 × i-107 trophozoites kwi-1 ml ye-reagent ye-TRIzol (Vazyme, Nanjing, China), i-DNA ye-genomic ikhutshwe kwi-G. duodenalis RNA iyonke isebenzisa i-MonScript dsDNase (i-Monad, i-Wuhan, i-China) kunye ne-DNA eyongezelelweyo (cDNA) yenziwe. usebenzisa iMonScript RTIIII Super Mix (Monad) ngokwemiyalelo yomenzi.
Ulwazi lokulandelelana kweCDS kwithagethi yeG. duodenalis gene yafunyanwa kwi-NCBI GenBank.Sebenzisa i-Primer 5.0 ukuyila iiprimer ze-cloning ezingenamthungo kwijini nganye ekujoliswe kuyo.I-primer yangaphambili (5′-3′) iqulathe amacandelo amathathu: ulandelelwano olugqitheneyo kunye ne-layini yevektha pcDNA3.1(+) EcoRV (TGGTGGAATTCTGCAGAT) kwaye iqale iicodons ATG kunye ne-GNN (ukuba isiseko sokuqala asiyiyo i-G).Oku kwenziwa ukuphucula ukusebenza kakuhle kwentetho.Ukongezelela, ubuncinane i-16 bp iziseko ezidibeneyo (i-GC umxholo we-40-60% / Tm malunga ne-55 ° C).I-primer ebuyela umva (5′-3′) iqulathe iinxalenye ezimbini, ukulandelelana okugqitheneyo kunye ne-EcoRV-linearized vector pcDNA3.1 (+) (GCCGCCACTGTGCTGGAT) kunye nesiseko esidibeneyo se-16 bp.(ngaphandle kokumisa ezibini zokugqibela).iziseko) i-codon efana ne-AA okanye i-GA ukuvumela ii-plasmids eziphinda-phindeneyo ukuba ziveze iiproteni zazo ezibhalwe).Ukulandelelana kwe-primer zidweliswe kwiThebhile 1 kwaye yenziwe yi-Kangmet Biotechnology Co., Ltd. (Changchun, China).
Iithagethi zandiswa kusetyenziswa i-Pfu DNA polymerase (i-Tiangen, i-Beijing, i-China) okanye i-Ex-taq (i-Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, China) isebenzisa i-G. duodenalis cDNA elungiselelwe njenge template.Ivector ye-eukaryotic expression plasmid pcDNA3.1(+) yalungelelaniswa ne-ecoRV ye-restriction enzyme kunye ne-dephosphorylated usebenzisa i-Fast AP (i-Thermo Fisher Scientific).I-linearized pcDNA3.1 (+) iziqwenga kunye neziqhekeza ezijoliswe kuzo ezijoliswe kuzo zahlanjululwa kusetyenziswa ikiti yokucoca ijeli ye-DNA (i-Tiangen) kwaye ilinganiswe ngokusebenzisa i-Nanodrop ND-2000 (i-Thermo Fisher Scientific).Iqhekeza le-pcDNA3.1 (+) kunye neqhekeza ngalinye lofuzo ekujoliswe kulo liphinde ladityaniswa kusetyenziswa i-MonClone enye indibano ye-cloning mix (i-Monad Biotech Co., Ltd., eSuzhou, e-China) kwaye yaqinisekiswa ngokulandelelana kwe-DNA kusetyenziswa i-Comate Bioscience Company Limited (Changchun, China) ..
I-Endotoxin-free plasmids pcDNA3.1 (+)-alpha-2 kunye ne-pcDNA3.1 (+)-alpha-7.3 zenziwe kusetyenziswa i-SanPrep Endotoxin-free Plasmid Mini Kit (Sangon Biotech).Ugxininiso lwagcinwa ngaphezu kwe-500 ng/µl ukuqinisekisa ukuba i-EDTA kwi-elution buffer ayizange iphazamise uvavanyo lokudluliselwa.I-macrophages ye-mouse ye-peritoneal yeprayimari yakhuliswa kwiipleyiti ze-6 ezipheleleyo kunye ne-RPMI 1640 epheleleyo (i-Biological Industries) kwiiyure ze-12, emva koko iiseli zihlanjwe ngamaxesha e-3 kwi-PBS efudumeleyo ukususa i-penicillin kunye ne-streptomycin, kwaye ke phakathi kongezwa kunye ne-medium epheleleyo.I-Endotoxin-free plasmids pcDNA3.1 (+)-alpha-2 kunye ne-pcDNA3.1 (+)-alpha-7.3 (2.5 μg) yaxutywa kwi-125 μl ye-Opti-MEM eyanciphisa i-serum medium (Gibco, Thermo Fisher Scientific) ..Emva koko i-5 µl ye-Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific) yaxutywa kwi-125 µl ye-serum ephantsi ye-Opti-MEM medium.Lungiselela i-liposome-DNA complexes ngokuxuba i-plasmid e-diluted endotoxin-free kunye ne-Lipofectamine 2000 kwaye uvumele umxube ukuba ume kwindawo yokushisa kwemizuzu emi-5.Dlulisa ii-complexes ngokwahlukeneyo kwiiseli kwiqula ngalinye kwaye udibanise ngokukhawuleza.Emva kweeyure ze-4, i-cellculture medium yatshintshwa nge-2 ml ye-RPMI 1640 epheleleyo kunye nenkcubeko yaqhubeka iiyure ze-24.Iseli entsha inkcubeko medium yongezwa kwiiseli kwaye incubated ngamaxesha ahlukeneyo kuxhomekeke kuyilo assay.
Iisampulu zeprotheyini ezivela kwi-supernatants kunye ne-cell lysates zalungiswa njengoko kuchaziwe ngaphambili [25].Iiparamitha zokudlulisa i-Membrane ye-pro-IL-1β, i-pro-caspase-1, i-caspase-1 p20, i-NLRP3, i-β-actin, kunye ne-His-tag yayiyi-200 mA / 90 min.I-interleukin 1β (IL-1β; iR & D Systems, Minneapolis, Minnesota, USA), caspase-1 (p20) (Adipogen, Switzerland) kunye ne-NLRP3 (Adipogen SA, Epalinges, Switzerland) kunye ne-1: 5000 ejolise kwithegi yakhe (Amylet Scientific, Wuhan, China) kunye β-actin (Proteintech, Wuhan, China).
I-Cross-linking kunye ne-disuccinimide subrate (DSS) yenziwa njengoko kuchazwe ngaphambili [26].Iiseli zahlanjwa amaxesha e-3 nge-PBS ebandayo kwaye yahlanjululwa ngokupheleleyo ngenaliti ye-27 ye-gauge kwi-50 µl ASC reaction buffer (pH 8.0) equlethe i-25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES kunye ne-125 mM NaHCO3.Umxube wawufakwe kwi-centrifuged kwi-5000 g nge-3 min kwaye i-pellet yathanjiswa nge-10 µl DSS (25 mM kwi-DMSO) kunye ne-40 µl ye-ASC yokuphendula i-buffer ye-30 min kwi-37°C.Emva kwe-centrifugation kwi-5000 g ye-10 min, i-pellet yachithwa kwisisombululo se-40 µl ye-ASC reaction buffer kunye ne-10 µl ye-6x ye-protein yokulayisha i-buffer (i-TransGen, e-Beijing, e-China), kwaye isisombululo sacinywa kwiqondo lokushisa kwe-15. imiz., Emva koko ubilise imizuzu eyi-10.Iisampulu zeprotheyini zaye ziphantsi kwe-Western blotting usebenzisa i-anti-ASC eziphambili (i-Wanleibio, i-Shenyang, i-China) kwi-dilution ratio ye-1: 500.
Ukulandela inkqubo echazwe ngaphambili [13], i-cell culture supernatants yavunwa kwaye imfihlo ye-cytokine ye-pro-inflammatory IL-1β yamiselwa ngokusebenzisa i-mouse IL-1 Beta ELISA kit (Invitrogen, Thermo Fisher Scientific).Guqula ixabiso le-OD450nm libe kukugxininiswa kweeprotheyini usebenzisa i-IL-1β ijika eliqhelekileyo.
Iiseli ezifakwe kwii-coverlips zihlanjwe ngobumnene ngamaxesha e-3 kwi-PBS efudumeleyo, egxininiswe kwi-tissue cell fixative (i-Biosharp, eBeijing, eChina) kwi-10 min kwiqondo lokushisa lokushisa (RT), kwi-0.1% i-Triton X-Permeabilize kwi-100 (ihlanjululwe kwi-PBS; i-Biosharp ) imizuzu engama-20 kwindawo yokushisa kunye nebhloko kwi-5% ye-bovine serum albumin (kwi-PBS) kwiiyure ezingama-2 kwiqondo lokushisa.Iiseli zaye zafakwa ngobusuku kwi-4 ° C kunye nezithinteli eziphambili ezichasene ne-ASC (1: 100 dilution) okanye i-NLRP3 (1: 100 dilution), ngokulandelanayo, kunye ne-Cy3 ebhalwe ibhokhwe echasene nomvundla IgG (H + L) (1: 400; EarthOx , San Francisco, CA, USA) okanye i-FITC-conjugated ibhokhwe ye-anti-mouse IgG (1: 400; Earthox) ngobusuku kwi-37 ° C ebumnyameni ngeyure eli-1.I-nuclei yachithwa nge-Hoechst 33258 (10 μg / ml; UE, Suzhou, China) imizuzu emi-5 kwaye yabonwa phantsi kwe-microscope ye-fluorescence (i-Olympus Corporation, eTokyo, eJapan).
Iigundane zahlulwe zangamaqela amane (n = 7 kwiqela ngalinye): (i) I-PBS-iphathwe nge-PBS yeqela elingalunganga lokulawula (i-PBS kuphela; i-gavage 100 µl / i-PBS ye-mouse ilandelwa yi-injection ye-intraperitoneal yemihla ngemihla 100 µl / i-mouse PBS kwiiyure ze-3 kamva)., ngokuqhubekayo iintsuku ezi-7);(ii) iqela lokulawula elibi eliphathwa nge-MCC950 inhibitor [27] (100 µl / mouse nge-PBS gavage, iiyure ze-3 kamva, i-10 mg / kg ubunzima bomzimba [BW] MCC950 [kwi-PBS] ilawulwa nge-intraperitoneally yonke imihla, ubude beentsuku ze-7);(iii) Iqela le-G. duodenalis cyst infections (1.5 x 106 cysts / mouse by gavage, iiyure ezi-3 kamva, i-100 μl / impuku ye-PBS ilawulwa nge-intraperitoneally yonke imihla kwiintsuku ze-7);(iv) I-G. duodenalis cyst edibeneyo yosulelo lweqela le-MCC950 inhibitor unyango lweqela (1.5 × 106 cysts / mouse nge-gavage, 10mg / kg ubunzima bomzimba we-MCC950 intraperitoneally yonke imihla ngeentsuku ze-7 kwi-3h).Ubunzima bomzimba wemouse nganye bujongwa imihla ngemihla kwaye zonke iigundane zaye zaxhaswa ngosuku lwe-7th.I-duodenum evuniweyo (i-3 cm ubude) yanqunyulwa kwiincinci ezincinci kwi-1 ml ye-PBS, i-cysts yatshatyalaliswa ngobusuku kwi-PBS kwi-4 ° C, kunye ne-G. duodenalis trophozoites.I-duodenum entsha (i-1 cm ubude) yabekwa yodwa ngenxa ye-hematoxylin kunye ne-eosin (H&E) ukungcolisa.
Iigundane zahlulwe ngamaqela amabini: (i) Iqela lokulawula i-MOCK kunye (ii) neqela le-MCC950 inhibitor.Kwakukho unyango oluhlanu kwiqela ngalinye (n = 7 / iqela lonyango): (i) unyango lwe-PBS iqela lokulawula elibi (PBS kuphela; 100 µl / mouse PBS, intramuscular (IM) injection (tibialis anterior) [28, 29];( ii) pcDNA3.1(+) iqela lolawulo olungalunganga lweplasmid (100 µg/mouse DNA, ngesitofu se-intramuscular) (iii) G. duodenalis cyst group positive control (1.5 x 106 cysts/mouse, via gavage) (iv) a iqela eliphathwe ngeplasmid pcDNA3.1 (+)-alpha-2 (100 μg/mouse DNA, ngesitofu se-intramuscular), kunye (v) neqela eliphathwa nge-plasmid pcDNA3.1 (+)-alpha-7.3 (100 µg/mouse I-DNA, emva kweeyure ze-12 zokuhamba, iigundane kwiqela le-MCC950 inhibitor zifumana inaliti ye-intraperitoneal ye-MCC950 yonke imihla (10 mg / kg ubunzima bomzimba) kwiintsuku ze-7, ngelixa iigundane kwiqela le-MOCK zifumana umthamo olinganayo wonyango lwe-PBS. eqokelelwe kwiigundane ze-eyeballs kwaye zashiya ubusuku bonke kwi-4 ° C Iisampulu ze-Serum zahlukaniswa zisebenzisa i-enzyme-linked immunosorbent assay (ELISA) kunye nemilinganiselo ye-IL-1β.
Iimpuku ezingamashumi amathathu anesihlanu zahlulwa zangamaqela amahlanu (n=7/group).Iqela le-1 yayiliqela elibi lolawulo eliphathwayo nge-PBS: iigundane zafumana i-100 μl ye-PBS nge-intramuscularly kunye neentsuku ze-3 kamva nge-gavage.Iqela le-2 liqela lokulawula elilungileyo elisuleleke kwi-G. duodenalis cysts: iigundane zafakwa nge-100 μl ye-PBS, kunye neentsuku ze-3 kamva i-1.5 x 106 i-cysts / i-mouse ifakwe intragastically.Iqela lesithathu - ugonyo lweplasmid kunye ne-pcDNA3.1 (+) ngokudibanisa neqela lolawulo losulelo lwe-cyst duodenal: iimpuku zafumana i-100 μg ye-plasmid DNA pcDNA3.1 (+) (im) ngomlomo, 1.5 × 106 cysts / mouse 3 eziliqela iintsuku.Amaqela e-4 kunye ne-5 ayeyi-recombinant pcDNA3.1 (+) -alpha-2 giardine plasmid okanye i-pcDNA3.1 (+) -alpha-7.3 giardine plasmid ngokudibanisa ne-G. duodenalis cyst infection.Iqela lovavanyo: iimpuku zifumene i-100 µg ye-pcDNA3.I-1 (+) -giardine plasmid DNA (im), emva kweentsuku ze-3, i-1.5 × 106 i-cysts / i-mouse ifakwe nge-gavage.Ubunzima bomzimba wemouse nganye bubekwe iliso emva kokuqaliswa kwe-cyst ye-G. duodenalis ngokusebenzisa ityhubhu.I-duodenum entsha yaqokelelwa imilinganiselo yomthwalo we-parasitic kunye nohlalutyo lwe-HE staining.
Utshintsho lwe-Histopathological lwahlalutywa ngokwenkqubo epapashwe ngaphambili [30].I-duodenum entsha yalungiswa kunye ne-tissue cell fixative, ifakwe kwiparafini, inqunywe kumacandelo e-4 μm, ifakwe i-H & E kwaye ihlalutye phantsi kwe-microscope yokukhanya.Utshintsho olumeleyo lwe-pathological kumacandelo asixhenxe e-tissue ukusuka kwiigundane ezisixhenxe ezizimeleyo zavavanywa yi-pathologist engazi ngonyango kwaye yabanjwa kwi-200x yokukhulisa.Ubude be-villi kunye nobunzulu be-crypts bulinganiswa ngokuhambelana neendlela ezichazwe ngaphambili.
Iziphumo ze-vitro kunye ne-vivo zifunyenwe ngokuphindwe kathathu.Iigrafu zenziwa kusetyenziswa iGraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, USA).Ukwahluka phakathi kwamaqela amabini kwahlalutywa ngovavanyo lwe-t, ngelixa ukuhlukana phakathi kwamaqela e-≥3 kwahlalutywa ngokuhlalutya kwendlela enye yokuhluka (ANOVA) usebenzisa isofthiwe ye-SPSS (inguqulo 22.0; SPSS IBM Corp., Armonk, NY, USA).Idatha yahlaziywa i-homogeneity of variance kusetyenziswa uvavanyo lukaLevene olulandelwa luvavanyo lwe-post hoc lukaBonferroni (B).Ukubaluleka kubonakaliswa njenge-P <0.05, P <0.01, kunye ne-P <0.001 (ingabalulekanga [ns]) (P> 0.05).
Uhlalutyo lwethu lwangaphambili lwe-GEV proteomics kwi-Kyoto Encyclopedia yeGenes kunye neGenomes (KEGG) lubonise ukuba ezininzi iithagethi zinokubandakanyeka ekusebenzeni kweendlela zokubonisa ukuvuvukala [13].Sikhethe iithagethi ezimbini ezithembisayo, i-alpha-2 kunye ne-alpha-7.3 giardins, ukwandisa ezi molekyuli kwaye zisebenzise ukwakha i-pcDNA3.1 (+) i-eukaryotic expression vector.Emva kolandelelwano, i-recombinant pcDNA3.1 (+)-alpha-2 kunye ne-alpha-7.3 giardine expression plasmids yatshintshelwa kwi-primary mouse peritoneal macrophages, kunye ne-caspase-1 p20 yesiginesha yeprotheni yokudumba (iqhekeza le-caspase-1 esebenzayo) ichongiwe. njengokucacisa iimolekyuli eziphambili ezinokubangela ukuvuvukala.Iziphumo zibonise ukuba i-alpha-2 kunye ne-alpha-7.3 giardines inokubangela i-p20 caspase-1 ibonakaliso efana ne-GEV.Akukho mpembelelo kwi-activation ye-caspase-1 ifunyenwe kulawulo olubi olungaphendulwanga (PBS kuphela) kunye nolawulo lwe-plasmid pcDNA3.1 (+) (Umfanekiso 1).
Umlinganiselo we-p20 caspase-1 isebenze nge-pcDNA3.1 (+) -alpha-2 kunye ne-alpha-7.3 giardins.I-Recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 kunye ne-alpha-7.3 giardines (ngasentla kwendlela nganye) zatshintshelwa kwiprimary mouse peritoneal macrophages kunye nenkcubeko supernatants zavunwa kwiiyure ezingama-24 kamva.I-Western blotting yayisetyenziselwa ukulinganisa amanqanaba okubonakaliswa kwe-signature caspase-1 p20 iprotheni evuthayo.Iqela lonyango lwe-PBS kuphela (umzila C) kunye ne-pcDNA3.1 (+) iqela le-monotherapy (umzila we-pcDNA3.1) wasetyenziswa njengolawulo olubi, kwaye iqela lonyango lwe-GEV lisetyenziswe njengolawulo oluhle.Ukubonakaliswa kweprotheni edibeneyo kuqinisekiswe ngokufumanisa ithegi ye-histidine kwiprotheni nganye, kwaye i-protein bands elindelekileyo yayiyi-alpha-2 giardine (38.2 kDa) kunye ne-alpha-7.3 giardine (37.2 kDa).I-GEV, i-Giardia duodenum extracellular vesicles, i-pcDNA3.1(+), i-EcoRV-linearized vector, SUP, supernatant
Ukugqiba ukuba ngaba i-alpha-2 giardine kunye ne-alpha-7.3 giardine yenza i-p20 caspase-1 intetho kwaye idlale indima ekusebenziseni i-host NLRP3 impendulo yokuvuvukala, i-pcDNA3.1 (+) -alpha-2 giardine kunye ne-pcDNA3.1 (+) -alpha -I-7.3 i-giardin idluliselwe kwi-macrophages ye-mouse ephambili ye-peritoneal macrophages kunye ne-DNA ye-plasmid edibeneyo, kunye namanqanaba okuvakalisa, indawo, kunye ne-oligomerization yeeprotheni eziphambili ezivuthayo ze-NLRP3 zinqunywe.Kulo vavanyo, i-GEV isetyenziswe njengeqela lokulawula elilungileyo, kwaye akukho qela lonyango (PBS kuphela) okanye i-pcDNA3.1 (+) iqela lonyango lokudluliselwa kwaba liqela elibi.Iziphumo zibonise ukuba, njengeqela le-GEV, i-plasmid DNA ye-giardin pcDNA3.1 (+) -alpha-2 kunye ne-giardin pcDNA3.1 (+) -alpha-7.3 ibangele ukulawulwa kwe-NLRP3, pro-IL-1β kunye iprocaspase-1 kunye ne-caspase-1 activation (Umfanekiso 2a).Ukongezelela, zombini i-giardines yenze i-IL-1β secretion ebalulekileyo (pcDNA3.1: ANOVA, F (4, 10) = 1.625, P = 0.1000; alpha-2 giardine: ANOVA, F (4, 10) = 1.625, P = 0.0007 ).;alpha-7.3 giardine: ANOVA, F (4, 10) = 1.625, P <0.0001;I-GEV: ANOVA, F (4, 10) = 1.625, P = 0.0047) (Umfanekiso 2b).Uninzi lweeprotheyini ze-ASC zaziyi-monomeric kwiqela elingenalo unyango okanye kwiqela lonyango elidluliselwe kwi-pcDNA3.1 (+) i-plasmid, ngokuchasene ne-pcDNA3.1 (+) -alpha-2 okanye i-pcDNA3.1 (+) -alpha- 7.3 igiardine.I-ASC i-oligomerization yenzeke kwi-DNA ye-plasmid edibeneyo ye-GEV yeqela elilawulayo okanye iqela, elibonisa ifom ye-oligomeric (Umfanekiso 2c).Ezi datha zangaphambili zibonisa ukuba i-alpha-2 giardine kunye ne-alpha-7,3 giardine inokwenza i-NLRP3 isebenze ukuvuvukala.Izifundo ezilandelayo ze-immunofluorescent zendawo ye-ASC kunye ne-NLRP3 zibonise ukuba kwiqela lokulawula elibi, iprotheni ye-ASC yasasazeka kwi-cytoplasm kwaye yabonakala njengomqondiso wechaphaza ekukhuthazeni i-pcDNA3.1 (+) -alpha-2 kunye ne-giardine okanye i-pcDNA3.I-1 (+)-alpha-7,3 iqela le-giardine okanye iqela le-GEV elilungileyo lokulawula (Umfanekiso 2d).Kulawulo olubi kunye namaqela e-pcDNA ephathwa nge-plasmid, i-protein ye-NLRP3 ayizange ibonwe, ngelixa i-dot ye-fluorescent ye-fluorescent ekuphenduleni i-pcDNA3.1 (+) -alpha-2 giardine okanye i-pcDNA3.1 (+) -alpha-7.3 yabhaqwa..i-giardine ifumaneka kwi-cytoplasm okanye ekukhuthazeni i-HEV (Umfanekiso 2e).Ezi datha zibonisa ngakumbi ukuba i-G. duodenalis giardin alpha-2 kunye ne-giardin alpha-7.3 isebenze i-NLRP3 inflammasome kwi-mouse primary peritoneal macrophages.
pcDNA3.1 (+) -alpha-2 giardin kunye pcDNA3.1 (+) -alpha-7.3 giardin isebenze NLRP3 inflammasome in mouse peritoneal macrophages.Guqula i-recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardin kunye ne-pcDNA3.1(+)-alpha-7.3 giardin kwiprimary murine peritoneal macrophages kunye neeseli, okanye uvune i-supernatant ngaphakathi kwe-24 h ukuhlalutya intetho, i-oligomerization , imfihlo.kunye nokufumaneka kwendawo yeeprotheni eziphambili ezivuthayo.Iqela le-PBS kuphela (C) kunye ne-pcDNA3.1 (+) iqela elilodwa lonyango lisetyenziswe njengolawulo olubi, kwaye iqela lonyango lwe-GEV lisetyenziswe njengeqela elihle.a Iiprotheyini eziphambili ezivuthayo ze-NLRP3, ezibandakanya i-NLRP3, i-pro-IL-1β, i-pro-caspase-1, kunye ne-p20 caspase-1, zifunyenwe yi-Western blotting.b Amanqanaba emfihlo ye-IL-1β kwi-supernatants anqunywe ngokusebenzisa i-enzyme-linked immunosorbent assay (ELISA).Umahluko phakathi kwamaqela olawulo kunye novavanyo lwahlalutywa ngohlalutyo lwendlela enye yokwahluka (ANOVA) usebenzisa i-SPSS software version 22.0.Iinkwenkwezi zibonisa umahluko omkhulu phakathi kwamaqela ** P <0.01 kunye ne-***P <0.001.c Amanqanaba e-Oligomerization ye-ASC kwiipellets aye amiselwa luhlalutyo lwe-DSS olunqamlezayo, ngelixa amanqanaba e-ASC kwiiseli zeseli asetyenziswa njengolawulo lokulayisha.d Ukubonwa kwe-ISC yendawo kusetyenziswa i-immunofluorescence.e I-Immunofluorescence isetyenziselwe ukubona umfanekiso wendawo ye-NLRP3.I-ASC, iprotheni efana ne-apoptotic speck;I-IL, i-interleukin;I-NLRP3, i-nucleotide-binding oligomerization-like receptor 3;ns, ayibalulekanga (P > 0.05)
Zombini i-G. duodenalis kunye ne-GEVs eyifihlayo isebenze i-NLRP3 inflammasome kwaye ilawule iimpendulo ezivuthayo zomkhosi kwi-vitro.Ngaloo ndlela, indima ye-NLRP3 inflammasome kwi-pathogenicity ye-G. duodenalis ihlala ingacacanga.Ukuphanda lo mbandela, senze umfuniselo phakathi kweegundane ezisuleleke nge-G. duodenalis cyst kunye neempuku ezosulelwe yiG.Isicwangciso esineenkcukacha zovavanyo luboniswe kumfanekiso 3a.Utshintsho kubunzima bomzimba weegundane kumaqela onyango ahlukeneyo abekwe iliso kwiintsuku ze-7 emva kokusuleleka kwi-cysts, kwaye iziphumo ziboniswe kwi-Fig 3b.Xa kuthelekiswa neqela eliphathwe nge-PBS ecocekileyo, iziphumo zibonise ukuba (i) ubunzima bomzimba weegundane ezinesifo se-G. duodenalis cyst yancipha ukusuka kwi-3 ukuya kwi-7 emva kokusuleleka;(ii) unyango nge-MCC950 inhibitor aluzange lube nempembelelo ebalulekileyo kubunzima bomzimba weempuku..Xa kuthelekiswa neqela elilodwa losulelo, i-BW yeqela losulelo lwe-duodenal eliphathwa nge-MCC950 liyancipha ukuya kwii-degrees ezihlukeneyo (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, F = 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).Ezi nkcukacha zibonisa ukuba i-NLRP3 inflammasome ikhusela iigundane ekulahlekeni kwesisindo esibalulekileyo kwizigaba zokuqala (iintsuku ze-2-4) ze-duodenal infection.Emva koko sijonge ukufumanisa i-G. duodenalis trophozoites kwi-duodenal lavage fluid kwaye iziphumo ziboniswe kuMzobo 3c.Xa kuthelekiswa neqela le-G. duodenalis cyst infections, inani le-trophozoites kwi-duodenum landa kakhulu emva kokuthintela i-NLRP3 inflammasome (t (12) = 2.902, P = 0.0133).Izicubu ze-Duodenal ezingcoliswe yi-HE zibonise, xa kuthelekiswa nolawulo olubi oluphathwa nge-PBS kunye ne-MCC950 yodwa: (i) I-G. duodenalis usulelo lwe-cyst lubangele umonakalo kwi-duodenal villi (ANOVA, F (3, 24) = 0.4903, P = 0.0488 ) kunye ne-crypt atrophy (ANOVA, F (3, 24) = 0.4716, P = 0.0089);(ii) i-duodenum evela kwiigundane ezosulelwe yi-G. duodenalis cysts kwaye iphathwe nge-MCC950 inhibitors.i-duodenal villi yonakaliswe kwaye ifile (ANOVA, F (3, 24) = 0.4903, P = 0.0144) kunye ne-atrophy kunye ne-crypt branching (ANOVA, F (3, 24) = 0.4716, P = 0, 0481) (Umfanekiso 3d- f).Ezi ziphumo zibonisa ukuba i-NLRP3 inflammasome idlala indima ekunciphiseni i-pathogenicity ye-G. duodenalis.
Indima ye-NLRP3 inflammasome kwi-Giardia duodenum infection.Iimpuku zahluthwa (iv) nge-duodenococcal cysts emva koko zaphathwa nge-MCC950 okanye ngaphandle kwayo (ip).Amaqela onyango olunye kunye ne-PBS okanye i-MCC950 yasetyenziswa njengolawulo.Iqela lovavanyo kunye nerejimeni yonyango.b Ubunzima bomzimba weempuku kwiqela ngalinye lamaqela onyango ahlukeneyo abekwe esweni iintsuku ezisi-7.Umahluko phakathi kweqela losulelo lwe-G. duodenalis kunye ne-G. duodenalis + MCC950 iqela lonyango losulelo lwahlalutywa ngovavanyo lwe-t usebenzisa i-SPSS software version 22.0.Iinkwenkwezi zibonisa umahluko omkhulu kwi- * P <0.05, ** P <0.01, okanye *** P <0.001.c Umthwalo weParasitic wamiselwa ngokubala inani le-trophozoites kulwelo lokuhlanjululwa kwe-duodenal.Umahluko phakathi kweqela losulelo lwe-G. duodenalis kunye ne-G. duodenalis + MCC950 iqela lonyango losulelo lwahlalutywa ngovavanyo lwe-t usebenzisa i-SPSS software version 22.0.Iinkwenkwezi zibonisa umahluko omkhulu kwi * P <0.05.d I-Hematoxylin kunye ne-eosin (H&E) iziphumo ezingcolisayo ze-duodenal histopathology.Iintolo ezibomvu zibonisa umonakalo kwi-villi, iintolo eziluhlaza zibonisa umonakalo kwi-crypts.Isikali sebar: 100 µm.e, f Uhlalutyo lwamanani lwe-duodenal villus ukuphakama kunye nokuphakama kwe-crypt ye mouse.Iinkwenkwezi zibonisa umahluko omkhulu kwi- * P <0.05 kunye ne ** P <0.01.Iziphumo zithathwe kuvavanyo oluzimeleyo lwebhayoloji esi-7.BW, ubunzima bomzimba;ig, indlela yokuhanjiswa kwe-intragastric;ip, indlela yokuhanjiswa kwe-intraperitoneal;ns, ayibalulekanga (P > 0.05);i-PBS, i-phosphate buffered saline;WT, uhlobo lwasendle
Imfihlo ye-IL-1β yinto ephawulekayo yokusebenza kokuvuvukala.Ukufumanisa ukuba i-G. duodenalis alpha-2 giardine kunye ne-alpha-7.3 i-giardine isebenze i-NLRP3 i-host inflammasome kwi-vivo, sasebenzisa iigundane ze-WT ezingaphendulwanga (iqela le-sham) kunye ne-NLRP3 i-inflammasome-blocked mice (MCC950 inhibited treatment group).Isicwangciso esineenkcukacha zovavanyo luboniswe kumfanekiso we-4a.Amaqela ovavanyo aquka iigundane eziphathwe nge-PBS, unyango lwe-G. duodenalis cyst nge-gavage, inaliti ye-intramuscular ye-pcDNA3.1, kunye ne-intramuscular injection ye-pcDNA3.1 (+) -alpha-2 giardine okanye i-pcDNA3.1-alpha-7.3 giardine.Ngomhla we-7 emva kokulawulwa kwe-intramuscular of recombinant plasmid, i-serum yaqokelelwa kwaye inqanaba le-IL-1β kwiqela ngalinye linqunywe.Njengoko kuboniswe kuMfanekiso 4b, kwiqela le-MOCK: (i) xa kuthelekiswa neqela le-PBS, unyango lwe-pcDNA3.1 lwalungenayo impembelelo ebalulekileyo kwi-IL-1β secretion (ANOVA, F (4.29) = 4.062, P = 0.9998), nangona kunjalo, I-IL-β secretion yaphakanyiswa kakhulu kwiqela le-G. duodenalis cyst (ANOVA, F (4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine kunye ne-pcDNA3.I-1- I-injection ye-intramuscular ye-alpha-7.3 giardine yandisa kakhulu i-serum IL-1β amanqanaba (ANOVA, F (4, 29) = 4.062, P <0.0001);(iii) i-pcDNA3.1-alpha-7,3 i-giardine yenza amanqanaba aphezulu e-IL -1β secretion kwi-pcDNA3.1-alpha-2 giardine intramuscular injection group (ANOVA, F (4, 29) = 4.062, P = 0.0333) .Xa kuthelekiswa neqela ngalinye kwiqela le-MCC950 lonyango kunye neqela le-MOCK: (i) i-IL-1β amanqanaba e-secretion kwiqela lokulawula i-PBS kunye neqela lokulawula i-pcDNA3.1 lehla ukuya kwinqanaba elithile emva kokuthintela i-MCC950 inhibitor, kodwa umehluko wawungekho. okubalulekileyo (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F (9, 58) = 3.540, P = 0.5949);(ii) emva kokuvala i-MCC950., I-IL-1β secretion yancitshiswa kakhulu kwiqela le-G. duodenalis cyst-infected, iqela le-pcDNA3.1-alpha-2 giardine, kunye ne-pcDNA3.1-alpha-7.3 iqela le-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).Ezi ziphumo zibonisa ukuba i-alpha-2 giardine kunye ne-alpha-7.3 giardine idibanisa ukusebenza kwe-NLRP3 inflammasome kwi-vivo.
I-pcDNA3.1 (+) -giardines yenza ukuba i-NLRP3 ibambe i-inflammasome kwi-vivo.Iimpuku ziye zagonywa (IM) kunye ne-recombinant eukaryotic expression plasmid pcDNA3.1 (+)-alpha-2 giardine okanye i-pcDNA3.1 (+) -alpha-7.3 giardine emva koko yaphathwa nge-MCC950 (ip; MCC950 iqela) okanye hayi (iqela le-dummy ).I-PBS okanye i-pcDNA3.1 (+) iqela lonyango lwe-plasmid lisetyenziswe njengolawulo olubi, iqela lonyango lwe-G. duodenalis cyst lisetyenziswe njengolawulo oluhle.Iqela lovavanyo kunye nerejimeni yonyango.b Amanqanaba e-Serum ye-IL-1β kwiigundane alinganiswa ngosuku lwe-7 ngovavanyo lwe-ELISA.Ukwahluka phakathi kwamaqela kwiqela le-MOCK kwahlalutywa kusetyenziswa i-ANOVA yendlela enye, kwaye iyantlukwano phakathi kweqela le-MOCK kunye neqela le-MCC950 yahlalutywa kusetyenziswa uvavanyo lwe-t-test ye-SPSS software version 22.0.I-asterisks ibonisa ukungafani okuphawulekayo phakathi kwamaqela onyango kwiqela le-MOCK, * P <0.05 kunye ne-*** P <0.001;iimpawu zeedola ($) zibonisa ukungafani okukhulu phakathi kweqela ngalinye kwiqela le-MOCK kunye neqela le-MCC950 kwi-P <0.05.Iziphumo zemifuniselo yebhayoloji ezimeleyo ezisixhenxe.i, inaliti ye-intramuscular, ns, ayibalulekanga (P > 0.05)
Ukuphanda umphumo we-alpha-2 kunye ne-alpha-7.3 ye-giardine-mediated activation ye-NLRP3 host inflammasome kwi-G. duodenalis infectivity, sasebenzisa iigundane ze-WT C57BL / 6 kunye ne-alpha-2 giardine kunye ne-alpha-7.3 giardine.i-plasmid ifakwe i-intramuscularly, emva kweentsuku ze-3 nge-tube yesisu ye-G. duodenalis cyst, emva koko iigundane zabonwa ngeentsuku ze-7.Isicwangciso esineenkcukacha zovavanyo luboniswe kuMzobo 5a.Ubunzima bomzimba wemouse nganye bulinganiswe imihla ngemihla, iisampulu zezicubu ze-duodenal ezitsha zaqokelelwa ngosuku lwe-7 emva kokulawulwa nge-tube yesisu, inani le-trophozoites lilinganiswe, kwaye utshintsho lwe-histopathological lwabonwa.Njengoko kubonisiwe kuMfanekiso 5b, ngokunyuka kwexesha lokutya, i-BW yeempuku kwiqela ngalinye yanda ngokuthe ngcembe.I-MT yeegundane yaqala ukunciphisa ngosuku lwe-3 emva kokulawulwa kwe-intragastric ye-G. duodenalis cysts, kwaye yanda ngokuthe ngcembe.Ukusebenza kwe-NLRP3 i-inflammasome eyenziwa yi-injection ye-intramuscular ye-alpha-2 giardine kunye ne-alpha7.3 giardine iyancipha kakhulu ukulahleka kwesisindo kwiigundane (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, F 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 lwe-5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F (4, 30) = 0.9728, P <0.0001 Usuku 6. -pcDNA3 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 uhlolwe kwi-duodenum (Umfanekiso we-5c).Xa kuthelekiswa nokulawulwa okungahambi kakuhle kunye neqela elifakwe kwi-pcDNA3.1 vector engenanto, inani le-G. duodenalis trophozoites lancitshiswa kakhulu kumaqela afakwe nge-α-2 giardine kunye ne-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).Ukongezelela, i-giardine alfa-7.3 yayikhusela ngakumbi kwiigundane kune-giardine alfa-2 (ANOVA, F (3, 24) = 1.209, P = 0.0081).Iziphumo ze-HE staining ziboniswe kwifig.5d–f.Iigundane ezifakwe kwi-alpha-2 giardine kunye ne-alpha-7.3 giardine zinezilonda ezincinci zezicubu ze-duodenal, ezibonakaliswa ngumonakalo we-villus, xa kuthelekiswa neegundane ezifakwe kwi-G. duodenalis kunye neegundane ezifakwe kwi-G. duodenalis ngokudibanisa ne-pcDNA3 vector engenanto .1 Zoom.(pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 2.466, P = 0.0035 okanye P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F (3, 24) = 2.466, P = 0.0028 okanye P = 0.0055) kunye nokunciphisa i-crypt atrophy (pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 1.470, P = 0.0264 okanye P = 0.0158; pcDNA3.1-alpha-7.3 ANOVADINE , F (3, 24) = 1.470, P = 0.0371 okanye P = 0.0191).Ezi ziphumo zibonisa ukuba i-alpha-2 giardine kunye ne-alpha-7,3 i-giardine inciphisa ukusuleleka kwe-G. duodenalis ngokuvula i-NLRP3 inflammasome kwi-vivo.
Indima ye-pcDNA3.1 (+)-giardins kwi-G. duodenalis usulelo.Iigundane ziye zagonywa (IM) kunye ne-recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine okanye i-pcDNA3.1 (+) -alpha-7.3 giardine kwaye emva koko icela umngeni kwi-G. duodenalis cysts (ig).Iqela le-PBS kunye ne-pcDNA3.1 (+) + iqela lonyango lwe-cyst duodenal lisetyenziswe njengamaqela olawulo olubi, kwaye iqela lonyango lwe-cyst duodenal lisetyenziswe njengeqela lokulawula okulungileyo.Iqela lovavanyo kunye nerejimeni yonyango.b I-MT yeempuku kwiqela ngalinye lamaqela ahlukeneyo onyango yayibekwe iliso kwiintsuku ze-7 emva komngeni.I-asterisks ibonisa ukungafani okukhulu phakathi kwamaqela kwiqela le-G. duodenalis kunye ne-pcDNA3.1 (+) -alpha-2 giardine iqela, * P <0.05, ** P <0.01, kunye ne-***P <0.001;uphawu lwedola ($) lubonisa umahluko omkhulu phakathi kweqela ngalinye le-G. duodenalis kunye ne-pcDNA3.1 (+)-alpha-7.3 jardine iqela, $ $ P <0.01 kunye ne-$ $ $ P <0.001.c Umthwalo weParasitic unqunywe ngokubala inani le-trophozoites kwi-1 ml ye-duodenal lavage ukusuka kwi-duodenum (i-3 cm ubude) kwaye ibonakaliswe njengenani lee-parasites kwi-cm ye-duodenum.Ukwahluka phakathi kweqela le-G. duodenalis losulelo, i-pcDNA3.1 (+) -alpha-2 iqela le-giardine, kunye ne-pcDNA3.1 (+) -alpha-7.3 iqela le-giardine lihlalutywe yindlela enye ye-ANOVA usebenzisa i-SPSS software version 22.0.Iinkwenkwezi zibonisa umahluko omkhulu ku-*P <0.01 kunye ne-***P <0.001.d Utshintsho lwe-Histopathological kwi-duodenum.Iintolo ezibomvu zibonisa umonakalo kwi-villi, iintolo eziluhlaza zibonisa umonakalo kwi-crypts.Isikali sebar: 100 µm.e, f Uhlalutyo lwamanani lobude bemouse duodenal villus (e) kunye nobude bekripthi (f).Umahluko phakathi kwamaqela kuMfanekiso 1d uhlalutywe yindlela enye ye-ANOVA usebenzisa i-SPSS software version 22.0.Iinkwenkwezi zibonisa umahluko omkhulu kwi- * P <0.05 kunye ne ** P <0.01.Iziphumo zemifuniselo yebhayoloji ezimeleyo ezisixhenxe.ns, ayibalulekanga (P > 0.05)
I-Giardia duodenum sisifunxi-gazi esaziwayo samathumbu ebantwini kunye nezinye izilwanyana ezanyisayo ezibangela igiardiasis.Kwi-2004, ifakwe kwi-WHO engahoywayo yeZifo ngenxa yokuxhaphaka kwayo kwiminyaka eyi-6, ngakumbi kwiindawo zokuhlala eziphantsi kwemeko yentlalo-qoqosho [32].I-immune system yemvelo idlala indima ebalulekileyo ekuphenduleni komzimba kwi-G. duodenalis usulelo.Iimacrophage zempuku ziye zabikwa ukuba ziginya kwaye zibulale i-G. duodenalis ngokukhulula imigibe engaphandle [33].Uphononongo lwethu lwangaphambili lubonise ukuba i-G. duodenalis, i-parasite ye-extracellular engena-invasive, ivuselela i-p38 MAPK, i-ERK, i-NF-κB p65, kunye ne-NLRP3 yeendlela zokubonisa ukuvuvukala kwi-macrophages ye-mouse ukulawula iimpendulo ezivuthayo zomkhosi, kwaye i-GEV ekhutshweyo inokuphucula le nkqubo.13], 24].Nangona kunjalo, ii-PAMP ezichanekileyo ezibandakanyekayo kwi-NLRP3 i-inflammasome-regulated inflammation kwi-GEV kunye nendima ye-NLRP3 inflammasome kwi-giardiasis ihlala icaciswa.Ukuze sikhanyisele le mibuzo mibini, siye saqhuba esi sifundo.
I-NLRP3 i-inflammasome ifumaneka kwi-cytoplasm yeeseli ze-immune kwaye inokuthi isebenze ngamasuntswana ahlukeneyo afana ne-uric acid crystals, i-toxins, ibhaktheriya, i-virus kunye ne-parasites.Kwizifundo zebhaktheriya, i-toxins ichongiwe njenge-PAMP eziphambili ezenza izinzwa ezivuthayo, ezikhokelela ekudumbeni nasekufeni kweseli [34].Ezinye i-toxins ezihlukeneyo zesakhiwo, ezifana ne-hemolysin esuka kwi-Staphylococcus aureus [35] kunye ne-Escherichia coli [36], i-hemolysin BL (HBL) esuka kwi-enterotoxin (NHE) [37], yenza ukuba kusebenze ukuvutha kwe-NLRP3.Uphononongo lwentsholongwane lubonise ukuba iiprotheyini zentsholongwane ezifana ne-SARS-COV-2 imvulophu (E) protein [38] kunye ne-Zika virus NS5 protein [39] zibalulekile iiPAMPs eziqatshelwe yi-NLRP3 receptor.Kwizifundo ze-parasite, ezininzi ii-parasites ziye zaxelwa ukuba zidibene ne-host inflammasome activation, njenge-Toxoplasma gondii, i-Trichomonas vaginalis [40], i-Trypanosoma cruzi [41], kunye ne-Leishmania [42].Iiprotheni ezixineneyo ze-granule GRA35, GRA42, kunye ne-GR43, ezinxulumene ne-virulence ye-Toxoplasma gondii, ziyafuneka ukuze kufakwe i-pyroptosis kwi-Lewis rat macrophages [43].Ukongezelela, ezinye izifundo zeLeishmania zijolise kwiimolekyuli ezizimeleyo ezibandakanyekayo kwi-NLRP3 inflammasome, njenge-parasite membrane lipophosphoglycan [44] okanye i-zinc metalloprotease [45].Phakathi kwentsapho ye-alpha-giardin efana ne-alpha-giardin, i-alpha-1 giardin ibonakaliswe ukuba ingaba ngumviwa wokugonya obonelela ngokhuseleko kwi-G. duodenalis kwimodeli yegundane [18].Kuphononongo lwethu, sikhethe i-G. duodenalis virulence factor alpha-2 kunye ne-alpha-7,3 giardines, ezikhethekileyo kwi-giardia kodwa zichazwe kancinci.Ezi zimbini zofuzo ezijoliswe kuzo zaye zahlanganiswa kwi-pcDNA3.1 (+) i-eukaryotic expression expression vector ukuze kuhlalutywe ukuvuvukala kusebenze.
Kwimodeli yethu yemouse, amaqhekeza e-caspase acandekileyo asebenza njengeempawu zokuvula ukuvuvukala.Emva kokuvuselela, i-NLRP3 isebenzisana ne-ASC, ifuna i-procaspases, kwaye ivelise i-caspases esebenzayo eqhekeza i-pro-IL-1β kunye ne-pro-IL-18 kwi-IL-1β evuthiweyo kunye ne-IL-18, ngokulandelanayo -18.I-Caspases evuthayo (i-caspases-1, -4, -5 kunye -11) yintsapho egciniweyo ye-cysteine ​​​​proteases ebaluleke kakhulu ekukhuseleni okungaphakathi kwaye ibandakanyeka kukuvuvukala kunye nokufa kwe-cell program [46].I-Caspase-1 iqhutywe yi-canonical inflammasomes [47], ngelixa i-caspases-4, -5, kunye ne-11 zicatshulwe ngexesha lokuqulunqwa kwe-inflammasomes ye-atypical [48].Kule sifundo, sisebenzise i-mouse peritoneal macrophages njengomzekelo kwaye siphanda i-p20 caspase-1 ecatshulwe i-caspase-1 njengophawu lwe-host NLRP3 ukuvuvukala kusebenze kwizifundo ze-G. duodenalis usulelo.Iziphumo zibonise ukuba ezininzi ze-alpha-giardins zijongene nokusebenza okuqhelekileyo kokuvuvukala, okuhambelana nokufunyanwa kwee-molecule ze-virulence eziphambili ezibandakanyeka kwiibhaktheriya kunye neentsholongwane.Nangona kunjalo, uphononongo lwethu luyisikrini sokuqala kuphela kwaye kukho ezinye iimolekyuli ezinokuthi zisebenze i-inflammasomes ezingekho zakudala, njengoko uphando lwethu lwangaphambili lufumene i-inflammasomes ye-classical kunye ne-non-classical inflammasomes kwi-G. duodenalis infection [13].Ukugqiba ukuba ngaba i-p20 caspase-1 eveliswayo inxulumene ne-NLRP3 inflammasome, satshintshela i-alpha-2 kunye ne-alpha-7.3 giardins kwi-macrophages ye-mouse ye-peritoneal ukugqiba amanqanaba eprotheni ye-molecule kunye namanqanaba e-oligomerization ye-ASC, eqinisekisa ukuba zombini i-α-giardins iyasebenza. i-NLRP3 evuthayo.Iziphumo zethu zihluke kancinane kunezo zikaManko-Prykhoda et al., Oye wabika ukuba ukukhuthazwa kweeseli zeCaco-2 kunye ne-G. muris okanye i-E. coli I-EPEC i-strains yodwa inokunyusa i-fluorescence intensity ye-NLRP3, i-ASC, kunye ne-caspase-1, nangona kungenakubaluleka, ngelixa i-costimulation ye-G. muris kunye ne-E. coli yandisa amanqanaba eeprotheni ezintathu [49].Oku kungafani kusenokuba ngenxa yokungafani kokukhethwa kweentlobo ze-Giardia, imigca yeeseli, kunye neeseli eziphambili.Senze kwakhona kwii-assays ze-vivo sisebenzisa i-MCC950 kwi-5-iveki-ubudala yabasetyhini i-WT C57BL/6 iimpuku, ezinokuthi zichaphazeleke ngakumbi kwi-G. duodenalis.I-MCC950 inamandla kwaye ikhetha i-molecule encinci ye-NLRP3 inhibitor evimba i-canonical kunye ne-non-canonical activation ye-NLRP3 kwi-nanomolar concentrations.I-MCC950 inqanda ukusebenza kwe-NLRP3 kodwa ayichaphazeli ukusebenza kwe-AIM2, i-NLRC4, kunye ne-NLRP1 iindlela zokuvuvukala okanye iindlela zokubonisa i-TLR [27].I-MCC950 ivimbela ukusebenza kwe-NLRP3 kodwa ayikuthinteli ukuqaliswa kwe-NLRP3, i-K + efflux, i-Ca2 + ukungena, okanye ukusebenzisana phakathi kwe-NLRP3 kunye ne-ASC;endaweni yoko, inqanda i-NLRP3 inflammasome activation ngokuthintela i-ASC oligomerization [27].Ke ngoko, sasebenzisa i-MCC950 kwisifundo se-vivo ukufumanisa indima ye-NLRP3 inflammasome emva kwesitofu se-giardine.I-caspase esebenzayo-1 p10 iqhekeza i-cytokines e-pro-inflammatory pro-IL-1β kunye ne-pro-IL-18 ibe yi-IL-1β evuthiweyo kunye ne-IL-18 [50].Kulo cwaningo, amanqanaba e-serum IL-1β kwiigundane eziphathwe nge-giardine kunye okanye ngaphandle kwe-MCC950 zisetyenziswe njengesalathisi sokuba i-NLRP3 inflammasome yenziwe.Njengoko bekulindelekile, unyango lwe-MCC950 lunciphise kakhulu amanqanaba e-serum IL-1β.Ezi datha zibonisa ngokucacileyo ukuba i-G. duodenalis giardin alfa-2 kunye ne-giardin alfa-7.3 iyakwazi ukwenza i-NLRP3 i-mouse inflammasome.
Idatha ebalulekileyo eqokelelwe kwiminyaka elishumi edlulileyo ibonise ukuba i-IL-17A iyi-master regulator ye-immunity ngokumelene ne-G. muris, eyenza i-IL-17RA ibonakaliso, ivelise i-peptides ye-antimicrobial, kunye nokulawula ukusebenza kokuncedisa [51].Nangona kunjalo, usulelo lwe-Giardia lwenzeka rhoqo kubantu abadala abancinci, kwaye kuye kwaxelwa ukuba usulelo lwe-Giardia kwiimpuku eziselula aluyisebenzisi impendulo ye-IL-17A ukuba yenze umphumo wayo wokukhusela [52], okwenza abaphandi bajonge enye i-immunomodulatory Giardia.iindlela zosulelo lwe-helminth.Ababhali bophando olutshanje baxela ukuba i-G.muris inokwenza i-NLRP3 inflammasome yi-E. coli EPEC, ekhuthaza ukuveliswa kwe-peptides ye-antimicrobial kunye nokunciphisa amandla ayo okunamathisela kunye nenani le-trophozoites kwi-intestinal tract, ngaloo ndlela inciphisa ubunzima bekholoni. izifo ezibangelwa yi-bacilli [49].I-NLRP3 inflammasome ibandakanyeka ekuphuhliseni izifo ezahlukahlukeneyo.Ucwaningo luye lwabonisa ukuba i-Pseudomonas aeruginosa ibangela i-autophagy kwi-macrophages ukuphepha ukufa kweeseli, kwaye le nkqubo ixhomekeke ekusebenzeni kwe-NLRP3 inflammasome [53].Kwi-N. caninum, i-oxygen esebenzayo-i-activation ye-oxygen-mediated activation ye-NLRP3 i-inflammasome ikhawulela ukuphindaphinda kwayo kwi-host host, okwenza kube yinto ekujoliswe kuyo yonyango [9].I-Paracoccidioides brasiliensis ifunyenwe ukuba ibangele ukusebenza kwe-NLRP3 inflammasome kwi-mouse ye-mouse ephuma kwiiseli ze-dendritic, ezibangele ukukhululwa kwe-cytokine evuthayo ye-IL-1β, edlala indima ebalulekileyo ekukhuseleni umkhosi [10].Iintlobo ezininzi ze-Leishmania, kuquka i-L. amazonensis, i-L. enkulu, i-L. braziliensis, kunye ne-L. infantum chagasi, yenza i-NLRP3 kunye ne-ASC exhomekeke kwi-caspase-1 kwi-macrophages, kunye nosulelo lwe-Leishmania.Ukuphindaphinda kweParasite kuphuculwe kwiigundane ezisweleyo kwi-NLRP3/ASC/caspase-1 gene [11].Zamboni et al.Ukusuleleka kweLeishmania kuye kwabikwa ukuba kusebenze i-NLRP3 inflammasome kwi-macrophages, ethintela ukuphindaphinda kwe-intracellular parasite.Ke, iLeishmania inokuthintela ukusebenza kwe-NLRP3 njengeqhinga lokuphepha.Kwizifundo ze-vivo, i-NLRP3 inflammasome ibe negalelo ekupheliseni i-Leishmania, kodwa ayizange ichaphazele izicubu [54].Ngokwahlukileyo koko, kwizifundo ze-helminthiasis, ukusebenza kwe-NLRP3 inflammasome kucinezele ukhuselo lomkhosi ngokuchasene ne-helminthiasis yesisu [12].IShigella yenye yeebhaktheriya eziphambili ezibangela urhudo kwihlabathi jikelele.Ezi bhaktheriya zinokubangela ukuveliswa kwe-IL-1β nge-P2X7 i-receptor-mediated K + efflux, iintlobo ze-oksijini ezisebenzayo, i-lysosomal acidification, kunye nomonakalo we-mitochondrial.I-NLRP3 inflammasome ilawula kakubi i-phagocytosis kunye nomsebenzi we-bactericidal we-macrophages ngokuchasene ne-Shigella [55].Izifundo ze-Plasmodium zibonise ukuba i-AIM2, i-NLRP3 okanye i-caspase-1 enqongopheleyo yeegundane ezisuleleke ngePlasmodium zivelisa amanqanaba aphezulu ohlobo lwe-interferon ye-1 kwaye zixhathisa ngakumbi kwi-Plasmodium infection [56].Nangona kunjalo, indima ye-alpha-2 giardine kunye ne-alpha-7.3 giardine ekuqaliseni ukusebenza kwe-pathogenic ye-NLRP3 ukuvuvukala kwiigundane akucaci.
Kolu phononongo, inhibition ye-NLRP3 inflammasome yi-MCC950 yanciphisa i-BW kwaye yandisa inani le-trophozoites kwi-intestinal lavage fluid kwiimpuku, okukhokelela kutshintsho olubi kakhulu lwe-pathological kwizicubu ze-duodenal.I-Alpha-2 giardine kunye ne-alpha-7.3 giardine ivule i-mouse ye-NLRP3 inflammasome, ukwandisa ubunzima bomzimba we-mouse, ukunciphisa inani le-trophozoites kwi-intestinal lavage fluid, kunye nokunciphisa izilonda ze-duodenal pathological.Ezi ziphumo zibonisa ukuba i-G. duodenalis inokuvula i-NLRP3 ibamba i-inflammasome nge-alpha-2 giardine kunye ne-alpha-7,3 giardine, ukunciphisa i-pathogenicity ye-G. duodenalis kwiigundane.
Ngokudibeneyo, iziphumo zethu zibonisa ukuba i-alpha-2 kunye ne-alpha-7.3 giardines yenza ukuba kusebenze i-NLRP3 yokusingatha i-inflammasome kunye nokunciphisa ukusuleleka kwe-G. duodenalis kwiigundane.Ngoko ke, ezi molekyuli zithembisa iinjongo zokuthintela i-giardiasis.
       Data supporting the results of this study can be obtained from the respective author at gongpt@jlu.edu.cn.
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