Giardia duodenum ni ibinyabuzima bya parasitike bitera giardiasis, indwara yo mu mara ikunze kugaragara cyane ku bana bato bafite ibimenyetso by’indwara ya diyare.Twabanje kubamenyesha ko G. duodenalis idasanzwe itera gukora ya oligomerisiyonike imeze nka reseptor 3 (NLRP3) ihuza nucleotide kandi ikagenga ibisubizo byakira binyuze mumyanya ndangagitsina (EV).Nyamara, imiterere nyayo ya molekuline ya virusi itera duodenococcal EV (GEV) igira uruhare muri iki gikorwa ndetse n’uruhare rwa NLRP3 inflammasome muri giardiasis iracyasobanurwa.
Recombinant eukaryotic imvugo plasmide pcDNA3.1 (+) - alpha-2 na alpha-7.3 giardine muri GEV yarubatswe, ihindurwamo imbeba yibanze ya peritoneal macrophage, hanyuma igaragazwa no gupima intego ya molekile caspase-1.Urwego rwo kwerekana imvugo ya p20 rwerekanwe..G. duodenalis alpha-2 na alpha-7.3 giardine yabanje kumenyekana mugupima NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 na caspase-1 p20), isohoka rya IL.1β urwego, apoptotic spotted protein (ASC) urwego rwa oligomerisiyoneri, hamwe na immunofluorescent ya NLRP3 na ASC.Uruhare rwa NLRP3 inflammasome mu gutera indwara ya G. duodenalis rwasuzumwe hifashishijwe imbeba aho ibikorwa bya NLRP3 byahagaritswe (NLRP3 byahagaritse imbeba) kandi impinduka z’indwara ziterwa n'uburemere bw'umubiri, umutwaro wa parasitike duodenal, hamwe na tissue duodenal.Twongeyeho, twakoze iperereza niba hiardines alpha-2 na alpha-7.3 itera IL-1β gusohora muri vivo binyuze muri inflammasome ya NLRP3 maze tumenya uruhare rwa molekile mu gutera indwara ya G. duodenalis mu mbeba.
Alpha-2 na alpha-7.3 giardine itera gukora kwa NLRP3 inflammasome muri vitro.Ibi byatumye ibikorwa bya p20 caspase-1 byiyongera, kwiyongera kurwego rwimvugo ya NLRP3, pro-IL-1β, na poroteyine-ya-caspase-1, kwiyongera cyane mu gusohora kwa IL-1β, gushiraho ibibanza bya ASA muri cytoplasme, hamwe no kwinjiza ASA oligomerisation.NLRP3 inflammation Gutakaza imboro byongera indwara ya G. duodenalis mu mbeba.Imbeba zavuwe na cysts na gavage ziva mu mbeba zafunzwe na NLRP3 zerekanye umubare wiyongereye wa trophozoite no kwangirika cyane kuri villi ya duodenal, irangwa nudusimba twa nekrotike twagabanutse kandi twashami.Mu bushakashatsi bwa vivo bwerekanye ko giardine alpha-2 na alpha-7.3 zishobora gutera ururenda rwa IL-1β binyuze muri NLRP3 inflammasome, kandi gukingirwa hamwe na giardine alpha-2 na alpha-7.3 byagabanije gutera indwara ya G. duodenalis mu mbeba.
Ufatiye hamwe, ibyavuye muri ubu bushakashatsi byerekana ko giardia alpha-2 na alpha-7.3 bitera igenzura ry’umuriro wa NLRP3 kandi bikagabanya kwandura G. duodenalis mu mbeba, bikaba bitanga icyizere cyo gukumira indwara ya giardiyasi.
Giardia duodenum ni parasite idasanzwe ya protozoan iba mu mara mato kandi itera miliyoni 280 zanduye giardiasis hamwe na diyare buri mwaka, cyane cyane mu bana bato bo mu bihugu bikiri mu nzira y'amajyambere [1].Abantu bandura amazi yo kunywa cyangwa ibiryo byandujwe na cyst M. duodenum, hanyuma ikinjira mu gifu igasohoka mumitobe ya gastric.Giardia duodenum trophozoite ifata epitelium ya duodenal, itera isesemi, kuruka, impiswi, kubabara mu nda, no kugabanya ibiro.Abantu bafite immunodeficiency na fibrosis ya cystic barashobora kwandura.Indwara irashobora kandi kubaho binyuze mu mibonano mpuzabitsina mu kanwa no mu kanwa [2].Ibiyobyabwenge nka metronidazole, tinidazole, na nitazoxanide nuburyo bwiza bwo kuvura indwara zifata imyanya ndangagitsina [3].Nyamara, iyi miti ya chimiotherapie itera ingaruka mbi nko kugira isesemi, kanseri, na genotoxicity [4].Niyo mpamvu, ingamba zifatika zigomba gutegurwa kugirango wirinde kwandura G. duodenalis.
Inflammasomes ni icyiciro cya poroteyine za cytosoliki zigize uruhare mu kurwanya ubudahangarwa bw'umubiri, zifasha kwirinda indwara ziterwa na virusi no guhuza ibisubizo by’umuriro [5].Muri izo inflammasomes, nucleotide-binding oligomerisation (NOD) reseptor 3 (NLRP3) nucleotide-binding oligomerisation (NLRP3) nucleotide-binding-nka inflammasome yakozweho ubushakashatsi bwimbitse kuko ishobora gutahurwa nuburyo butandukanye bwa patogene / ibyangiritse bifitanye isano na molekile (PAMP / DAMP), izi, ikora sisitemu yubudahangarwa bw'umubiri.kandi igenga homeostasis yo munda mu ndwara nyinshi zanduza [6,7,8].Igizwe nicyitegererezo cyo kumenyekanisha (PRR) NLRP3, adapt apoptotic spotted proteine ​​(ASC), hamwe na effaspase-1 cyangwa procaspase-11.Indwara ya NLRP3 ikora nk'iyakira kurwanya igitero cya virusi, nk'uko bigaragara muri Neospora caninum [9], Paracoccidioides brasiliensis [10], n'ubushakashatsi bwa Leishmania.]Dufatiye ku byo twabonye mbere, twatangaje ko G. duodenalis idasanzwe itera imbaraga zo mu nda ya NLRP3 kandi ikanahindura ibisubizo byatewe no gusohora imitsi idasanzwe (EVS) [13].Icyakora, uruhare rwa NLRP3 inflammasome mu kwandura G. duodenalis muri vivo ntiruramenyekana.
Giardins yabanje gusobanurwa nkibigize imiterere ya G. duodenalis cytoskeleton kandi igira uruhare runini mukugenda kwa trophozoite no kwifata kwa epiteliyale mu mara mato.Kugira ngo barusheho kumenyera ibidukikije no kongera indwara zabo, G. duodenalis trophozoite yateje imbere imiterere yihariye ya cytoskeletal igizwe na flagella 8, umubiri 1 wo hagati, na disiki 1 ya ventrale [14].Trophozoite ya Giardia duodenum ikoresha cytoskeleton kugirango yinjire mu mara mato yo hejuru, cyane cyane duodenum, hanyuma yomekera kuri enterocytes.Bahora bimuka kandi bagerekaho selile zifata metabolism.Kubwibyo, hari isano ya hafi hagati ya cytoskeleton na virusi.Giardine yihariye ya Giardia duodenum ni ibice bigize imiterere ya cytoskeleton [15] kandi igabanijwemo ibyiciro bine: α-, β-, γ-, na δ-giardine.Hariho abantu 21 bo mu muryango wa α-giardin, bose bafite ubushobozi bushingiye kuri calcium yo guhuza fosifolipide [16].Bahuza kandi cytoskeleton na selile ya selile.Ku bantu barwaye impiswi iterwa na G. duodenalis, α-giardine iragaragazwa cyane kandi ikingira indwara mu gihe cyo kwandura [17].Urukingo rwa Heterologique rushingiye kuri Giardia alfa-1 rwarinze giardiyasi mu mbeba kandi ni antigene zishobora kuba umukandida mu iterambere ry’inkingo [18].Alpha-8 giardin, iherereye muri plasma membrane na flagella, ariko ntabwo iri muri disiki ya ventrale, byongera umuvuduko nubwiyongere bwa trophozoite muri G. duodenalis [19].Alpha-14 giardin ifata imiterere ya microtubule kuri flagella kandi ikagira ingaruka kubuzima bwa G. duodenalis [20].Alpha-11 giardine irahari cyane mubuzima bwose, kandi gukabya gukabije kwa alpha-11 giardine byangiza G. duodenalis ubwayo [21].Icyakora, ntibisobanutse niba alpha-2 giardine na alpha-7.3 giardine birinda kwandura G. duodenalis hamwe nuburyo bukoreshwa.
Muri ubu bushakashatsi, imvugo ya recombinant eukaryotic plasmide pcDNA3.1 (+) - alpha-2 giardine na pcDNA3.1 (+) - alpha-7.3 giardine yahinduwe muri macrophage yibanze ya peritoneal kugirango ikore NLRP3.Intego za Inflammasome noneho zerekanwe.Twasuzumye kandi uruhare rwa NLRP3 inflammasome mu gutera indwara ya G. duodenalis, dukora iperereza niba alpha-2 na alpha-7,3 giardine itera gukora NLRP3 inflammasome muri vivo, maze twemeza ko izo nshingano zombi za giardine mu gutera indwara ya G. duodenalis.Intego yacu twese yari iyo guteza imbere intego zitanga ikizere cyo gukumira indwara ya G. duodenalis.
Ubwoko bw'ishyamba (WT) C57BL / 6 imbeba z'abagore zifite ibyumweru 5-8 byaguzwe mu kigo cy’inyamanswa cya Liaoning Changsheng (Liaoning, Ubushinwa).Imbeba zabonye amazi kubuntu, zakira ibiryo bya sterisile kandi zabitswe mumasaha 12/12 yumucyo / umwijima.Mbere yo kwandura, imbeba zabonye antibiyotike ad libitum mu mazi yo kunywa zongerwamo ampisiline (1 mg / mL), vancomycine (1 mg / mL), na neomycine (1,4 mg / mL) (byose byaguzwe na Shanghai, Ubushinwa, ibinyabuzima byakozwe) [22 ].].Imbeba zatakaje ubushobozi bwo kurya no kunywa amasaha> 24 zigatakaza ≥ 20% ibiro byumubiri byashizwemo ubumuntu na dislocation ya nyababyeyi.
WB G. duodenalis trophozoites (Icyegeranyo cy’umuco w’Abanyamerika, Manassas, Amerika) hiyongereyeho 12.5% ​​ya serumu y’inda (FBS; Icyatsi cyose, Zhejiang, Ubushinwa) na 0.1% bovine (Sigma-Aldrich, St. Missouri, Amerika) ).Amerika) mugihe cya microaerobic.Trophozoite ihuriweho yakusanyirijwe ku rubura kandi inyuzwa ku kigereranyo cya 1: 4 kugirango irusheho kubyara.
Giardia duodenum cysts yatewe nkuko byasobanuwe mbere [23], trophozoite yasaruwe mugice cya logarithmic hanyuma ikavangwa na encapsulation itera imiti, pH 7.1 (yahinduwe TYI-S-33) kugeza kuri 1 × 106 trophozoite / mL.kwibumbira hamwe 0,05% hagati).Trophozoite yarezwe mu bihe bya anaerobic kuri 37 ° C kugeza igihe cyo gukura kwa logarithmic.Hindura uburyo bugera kuri cyst itera (pH 7.8; yahinduwe TYI-S-33 iciriritse hamwe na 1% yibice bya bile) hamwe numuco G. duodenalis kuri 37 ° C mumasaha 48-96, mugihe cysts yabonetse munsi ya microscope.Nyuma yuko benshi muri trophozoite bamaze gushishikarizwa gukora cysts, imvange yumuco yarasaruwe hanyuma ihagarikwa mumazi ya sterile deionised kugirango abone trophozoite isigaye.Cysts yabazwe kandi ibikwa kuri 4 ° C kugirango isesengurwe nyuma ikoresheje umuyoboro wa gastric mu mbeba.
Indwara ya Giardia idasanzwe (GEVs) yakungahajwe nkuko byasobanuwe mbere [13].Trophozoite mu cyiciro cyo gukura kwa logarithmic yahagaritswe mu buryo bwahinduwe bwa TYI-S-33 bwateguwe hamwe na FBS ya exosome-yatakaye (Inganda z’ibinyabuzima, Beit-Haemek, Isiraheli) kugeza ku ntera ya 1 × 106 parasite / mL hanyuma ikorerwa amasaha 12.batandukanijwe n’umuco ndengakamere na centrifugation kuri 2000 g kuminota 10, 10,000 g kuri 45 min, na 100.000 g kuri min 60.Ibicuruzwa byashongeshejwe muri saline ya fosifate (PBS), bigereranywa hifashishijwe ibikoresho bya poroteyine BCA (Thermo Fisher Scientific, Waltham, MA, USA) bikabikwa kuri -80 ° C. cyangwa bigakoreshwa mu buryo butaziguye kugira ngo bisesengurwe.
Imbeba yibanze ya peritoneal macrophage yateguwe nkuko byasobanuwe mbere [24].Muri make, imbeba (zifite ibyumweru 6-8) zatewe (intraperitoneally [ip]) hamwe na ml 2,5 ya 2.98% ya Difco yamazi ya thioglycol (BD, Franklin Lakes, NJ, USA) hanyuma igaburirwa amagage 3-4.Guhagarika macrophage byakusanyirijwe mu cyuho cyinda yimbeba nyuma ya euthanasiya hanyuma igashyiramo inshuro 3 kuri 1000 g kuminota 10.Ingirabuzimafatizo zasaruwe zagaragajwe na cytometrike itemba ikoresheje ikimenyetso cya CD11b kugeza isuku ya selile yari> 98%, hanyuma ikongerwaho ku byapa 6 by’umuco w’akagari (4.5 x 106 selile / iriba) hanyuma igashyiramo 10% FBS (Bioindustry) kuri 37 ° C.na 5% CO2.
RNA yakuwe muri 1 × 107 trophozoite muri ml 1 ya reagent ya TRIzol (Vazyme, Nanjing, Ubushinwa), ADN genomic yakuwe muri G. duodenalis RNA yose ikoresheje MonScript dsDNase (Monad, Wuhan, Ubushinwa) hanyuma ADN yuzuzanya (cDNA). ukoresheje MonScript RTIIII Super Mix (Monad) ukurikije amabwiriza yabakozwe.
Amakuru ya CDS akurikirana intego ya G. duodenalis gene yabonetse muri NCBI GenBank.Koresha Primer 5.0 kugirango ushushanye primers idasanzwe ya clon primers kuri buri gen igenewe.Imbere ya primer (5′-3 ′) igizwe nibice bitatu: urukurikirane rwuzuzanya hamwe na vector umurongo wa pcDNA3.1 (+) EcoRV (TGGTGGAATTCTGCAGAT) hanyuma utangire code ATG na GNN (niba ishingiro ryambere atari G).Ibi bikorwa kugirango tunoze imikorere yimvugo.Mubyongeyeho, byibuze 16 bp ihuriweho hamwe (GC ibirimo 40-60% / Tm hafi 55 ° C).Primer primer (5′-3 ′) igizwe nibice bibiri, urutonde rwuzuzanya hamwe na EcoRV-umurongo wa vector pcDNA3.1 (+) (GCCGCCACTGTGCTGGAT) hamwe nifatizo byibuze 16 bp.(ukuyemo guhagarara kabiri).shingiro) codon nka AA cyangwa GA kugirango yemere plasmide recombinant kwerekana proteine ​​zanditseho).Urutonde rwa primer rwashyizwe ku mbonerahamwe ya 1 kandi rwahujwe na Kangmet Biotechnology Co., Ltd. (Changchun, Ubushinwa).
Intego zongerewe hakoreshejwe polymerase ya Pfu ADN (Tiangen, Beijing, Ubushinwa) cyangwa Ex-taq (Tekara Biomedical Technology [Beijing] Co., Ltd., Beijing, Ubushinwa) ikoresheje G. duodenalis cDNA yateguwe nk'icyitegererezo.Imvugo ya eukaryotic vector plasmid pcDNA3.1 (+) yashyizwe kumurongo hamwe na enzyme yo kubuza EcoRV na dephosifora ikoresheje Byihuta AP (Thermo Fisher Scientific).Ibice bya pcDNA3.1 (+) hamwe nibice bya gene byongeweho byahanaguwe hakoreshejwe ibikoresho byoza ADN (Tiangen) hanyuma bigereranywa hakoreshejwe Nanodrop ND-2000 (Thermo Fisher Scientific).Igice cya pcDNA3.1 (+) na buri gice cyagenewe gene cyongeye guhuzwa hifashishijwe imvange imwe ya MonClone ikomatanya (Monad Biotech Co., Ltd., Suzhou, Ubushinwa) kandi byemejwe na ADN ikurikirana ikoresheje Comate Bioscience Company Limited (Changchun, Ubushinwa)..
Endotoxine idafite plasmide pcDNA3.1 (+) - alpha-2 na pcDNA3.1 (+) - alpha-7.3 byakozwe hakoreshejwe SanPrep Endotoxin idafite Plasmid Mini Kit (Sangon Biotech).Kwibandaho byakomeje hejuru ya 500 ng / µl kugirango barebe ko EDTA muri buffer ya elution itabangamiye ikibazo cyo kwanduza.Imbeba yibanze ya peritoneal macrophage yarezwe mumasahani 6 yuzuye hamwe na RPMI 1640 yuzuye (Biologiya Inganda) mumasaha 12, hanyuma selile zogejwe inshuro 3 muri PBS zishyushye kugirango zikuremo penisiline na streptomycine, hanyuma muburyo bwuzuzanya nuburyo bwuzuye.Endotoxine idafite plasmide pcDNA3.1 (+) - alpha-2 na pcDNA3.1 (+) - alpha-7.3 (2,5 μg) yavomwe muri 125 μl ya Opti-MEM yagabanije serumu (Gibco, Thermo Fisher Scientific)..Noneho 5 µl ya Lipofectamine 2000 yo kwanduza (Invitrogen, Thermo Fisher Scientific) yavomwe muri 125 µl ya serumu nkeya Opti-MEM.Tegura liposome-ADN igizwe no kuvanga plasmid ya endotoxine itavanze na Lipofectamine 2000 hanyuma ukemerera kuvanga guhagarara mubushyuhe bwicyumba muminota 5.Hindura ibice bitandukanye muri selile muri buri riba hanyuma uvange buhoro.Nyuma yamasaha 4, umuco wumuco wakagari wasimbuwe na ml 2 yuzuye ya RPMI 1640 yuzuye kandi umuco wakomeje amasaha 24.Umuco mushya wumuco wongeyeho ingirabuzimafatizo hanyuma ushyirwa mugihe cyigihe gitandukanye ukurikije igishushanyo mbonera.
Ingero za poroteyine ziva mu ndengakamere na lysates zateguwe nk'uko byasobanuwe mbere [25].Ibipimo byo kwimura Membrane kuri pro-IL-1β, pro-caspase-1, caspase-1 p20, NLRP3, β-actin, na tag-ye yari 200 mA / 90 min.Kuri interleukin 1β (IL-1β; Sisitemu ya R&D, Minneapolis, Minnesota, Amerika), caspase-1 (p20) (Adipogen, Ubusuwisi) na NLRP3 (Adipogen SA, Epalinges, Ubusuwisi) na 1: 5000 byibasiye tagi ye (Amylet Scientific, Wuhan, Ubushinwa) na β-actin (Proteintech, Wuhan, Ubushinwa).
Kwambukiranya hamwe na disuccinimide suberate (DSS) byakozwe nkuko byasobanuwe mbere [26].Ingirabuzimafatizo zogejwe inshuro 3 hamwe na PBS ikonje hanyuma zomekwa rwose hamwe nurushinge rwa gipima 27 muri 50 µl ASC reaction reaction (pH 8.0) irimo 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES na 125 mM NaHCO3.Uruvange rwashyizwemo 5000 g muminota 3 hanyuma pellet ishushanywa na 10 µl DSS (25 mM muri DMSO) na 40 µl ASC ya reaction ya min 30 min kuri 37 ° C.Nyuma ya centrifugation kuri 5000 g muminota 10, pellet yashongeshejwe mugisubizo cya 40 µl ya reaction ya ASC na 10 µl ya 6x ya protein yapakira (TransGen, Beijing, Ubushinwa), hanyuma igisubizo kizimya ubushyuhe bwicyumba kuri 15 min., Hanyuma uteke iminota 10.Urugero rwa poroteyine rwakorewe iburengerazuba hifashishijwe antibodiyite zambere zirwanya ASC (Wanleibio, Shenyang, Ubushinwa) ku kigereranyo cya 1: 500.
Nyuma yuburyo bwasobanuwe mbere [13], hasaruwe kandi ndengakamere y’umuco w’akagari maze hasohoka ururenda rwitwa cytokine IL-1β rwifashisha imbeba IL-1 Beta ELISA (Invitrogen, Thermo Fisher Scientific).Hindura OD450nm indangagaciro za protein ukoresheje umurongo wa IL-1β.
Ingirabuzimafatizo zometse ku gipfukisho zogejwe buhoro inshuro 3 muri PBS zishyushye, zishyirwa mu gutunganya ingirabuzimafatizo (Biosharp, Beijing, Ubushinwa) mu minota 10 ku bushyuhe bw’icyumba (RT), muri 0.1% Triton X-Permeabilize kuri 100 (ivanze muri PBS; Biosharp ) muminota 20 mubushyuhe bwicyumba hanyuma uhagarike 5% ya bovine serum albumin (muri PBS) kumasaha 2 mubushyuhe bwicyumba.Ingirabuzimafatizo zashizwemo ijoro ryose kuri 4 ° C hamwe na antibodies zibanze zirwanya ASC (1: 100 dilution) cyangwa NLRP3 (1: 100 dilution), naho Cy3 yanditseho ihene irwanya urukwavu IgG (H + L) (1: 400; EarthOx , San Francisco, CA, Amerika) cyangwa FITC-ihujwe ihene irwanya imbeba IgG (1: 400; Earthox) ijoro ryose kuri 37 ° C mu mwijima isaha 1.Nuclei yandujwe na Hoechst 33258 (10 μg / ml; UE, Suzhou, Ubushinwa) mu minota 5 kandi ikurikiranwa na microscope ya fluorescence (Corporation Olympus, Tokiyo, Ubuyapani).
Imbeba zagabanyijwemo amatsinda ane (n = 7 muri buri tsinda): (i) PBS yavuwe nitsinda rito ryo kugenzura (PBS gusa; gavage 100 µl / imbeba PBS ikurikirwa no guterwa buri munsi intraperitoneal 100 µl / imbeba PBS nyuma yamasaha 3)., ubudahwema iminsi 7);...Uburemere bwumubiri wa buri mbeba bwakurikiranwe burimunsi kandi imbeba zose zashyizwe ahagaragara kumunsi wa 7.Duodenum yasaruwe (cm 3 z'uburebure) yaciwemo uduce duto muri ml 1 PBS, cysts yarasenyutse ijoro ryose muri PBS kuri 4 ° C, na G. duodenalis trophozoite.Dodenum nshya (cm 1 z'uburebure) yitaruye hematoxyline na eosine (H&E).
Imbeba zagabanyijwemo amatsinda abiri: (i) itsinda rishinzwe kugenzura MOCK na (ii) MCC950 itsinda ryabuza.Muri buri tsinda habayeho uburyo butanu bwo kuvura (n = 7 / itsinda ryo kuvura): (i) Itsinda rishinzwe kuvura nabi PBS (PBS gusa; 100 µl / imbeba PBS, inshinge (IM) inshinge (tibialis imbere) [28, 29]; ( ii) pcDNA3.1. itsinda ryavuwe na plasmid pcDNA3.1 (+) - alpha-2 (100 μg / imbeba ADN, ukoresheje inshinge zo mu nda), na (v) itsinda ryavuwe na plasmide pcDNA3.1 (+) - alpha-7.3 (100 µg / imbeba ADN, nyuma yamasaha 12 yo gutambuka, imbeba zo mumatsinda ya inhibitori ya MCC950 zabonye inshinge za intraperitoneal ya buri munsi ya MCC950 (uburemere bwumubiri 10 mg / kg) muminsi 7, mugihe imbeba zo mumatsinda ya MOCK zabonye urugero rumwe rwo kuvura PBS. Ingero zamaraso zari yakusanyirijwe mu mbeba y'amaso hanyuma igasigara ijoro ryose kuri 4 ° C. Ingero za Serumu zashyizwe mu bwigunge hakoreshejwe imisemburo ifitanye isano na immunosorbent (ELISA) no gupima urwego rwa IL-1β.
Imbeba mirongo itatu n'eshanu zagabanyijwemo amatsinda atanu (n = 7 / itsinda).Itsinda rya 1 ryari itsinda rishinzwe kugenzura nabi ryavuwe na PBS: imbeba zakiriye 100 μl za PBS mu buryo butemewe kandi nyuma yiminsi 3 na gavage.Itsinda rya 2 nitsinda ryiza rishinzwe kwandura cysts ya G. duodenalis: imbeba zatewe 100 μl za PBS, hanyuma nyuma yiminsi 3 1.5 x 106 cysts / imbeba zatewe inshinge.Itsinda rya gatatu - gukingira plasmide hamwe na pcDNA3.1 (+) ifatanije nitsinda rishinzwe kugenzura kwandura cysten duodenal: imbeba zakiriye 100 μg za ADN ya plasmide pcDNA3.1 (+) (im) mu kanwa, 1.5 × 106 cysts / imbeba 3 kuri benshi iminsi.Amatsinda ya 4 na 5 yari recombinant pcDNA3.1 (+) - alpha-2 giardine plasmid cyangwa pcDNA3.1 (+) - alpha-7.3 giardine plasmid ifatanije na infection ya G. duodenalis.Itsinda ryubushakashatsi: imbeba zakiriye 100 µg za pcDNA3.1 (+) - giardine plasmid ADN (im), nyuma yiminsi 3, 1.5 × 106 cysts / imbeba yatewe hakoreshejwe gavage.Uburemere bwumubiri wa buri mbeba bwakurikiranwe nyuma yo kwinjiza cyst G. duodenalis ikoresheje umuyoboro.Duodenum nshya yakusanyirijwe kubipima imitwaro ya parasitike hamwe na HE yisesengura.
Impinduka za Histopathologique zasesenguwe hakurikijwe uburyo bwatangajwe mbere [30].Dodenum nshya yashizwe hamwe na tissue selile ikosora, yashyizwe muri paraffine, igabanywamo ibice 4 mm, yandujwe na H&E hanyuma isesengurwa munsi ya microscope yoroheje.Imihindagurikire y’indwara mu bice birindwi byimbeba zivuye ku mbeba ndwi zigenga zasuzumwe n’umuhanga mu bumenyi bw'indwara utazi imiti kandi zafashwe kuri 200x.Uburebure bwa villi hamwe nuburebure bwibisobanuro byapimwe hakurikijwe uburyo bwasobanuwe mbere.
Ibisubizo muri vitro no muri vivo byabonetse muri bitatu.Igishushanyo cyakozwe hakoreshejwe GraphPad Prism 7.00 (GraphPad Software Inc, La Jolla, CA, USA).Itandukaniro riri hagati yitsinda ryombi ryasesenguwe na t-test, mugihe itandukaniro riri hagati yitsinda 3 ryasesenguwe nisesengura ryuburyo bumwe (ANOVA) ukoresheje software ya SPSS (verisiyo 22.0; SPSS IBM Corp., Armonk, NY, USA).Amakuru yasesenguwe kugirango bahuje ibitsina bakoresheje ikizamini cya Levene gikurikirwa na Bonferroni yipimishije (B).Akamaro kagaragazwa nka P <0.05, P <0.01, na P <0.001 (ntabwo ari ngombwa [ns]) (P> 0.05).
Isesengura ryibanze kuri GEV proteomics muri Kyoto Encyclopedia ya Gen na Genomes (KEGG) ryerekanye ko intego nyinshi zishobora kugira uruhare mugukora inzira zerekana ibimenyetso byerekana umuriro [13].Twahisemo intego ebyiri zitanga icyizere, alpha-2 na alpha-7.3 giardins, twongere izo molekile kandi tuyikoreshe mukubaka pcDNA3.1 (+) eukaryotic imvugo ya vector.Nyuma yo gukurikiranwa, recombinant pcDNA3.1 (+) - alpha-2 na alpha-7.3 giardine imvugo ya plasmide yimuriwe muri macrophage yibanze ya imbeba peritoneal, hanyuma caspase-1 p20 proteine ​​yumukono wa inflammation (agace ka caspase-1 ikora) nkugusobanura molekile zingenzi zishobora gutera uburibwe.Ibisubizo byerekanye ko alpha-2 na alpha-7.3 giardine zishobora gutera imvugo p20 caspase-1 imvugo isa na GEV.Nta ngaruka zo gukora caspase-1 zabonetse mugucunga nabi kutavuwe (PBS gusa) no kugenzura plasmid pcDNA3.1 (+) (Ishusho 1).
Gupima p20 caspase-1 gukora na pcDNA3.1 (+) - alpha-2 na alpha-7.3 giardins.Recombinant eukaryotic imvugo plasmide pcDNA3.1 (+) - alpha-2 na alpha-7.3 giardine (hejuru ya buri murongo) byahinduwe muri macrophage yimbeba yibanze kandi ndengakamere yumuco byasaruwe nyuma yamasaha 24.Guhindura iburengerazuba byakoreshejwe mu gupima urwego rwerekana umukono caspase-1 p20 poroteyine.Itsinda ryokuvura gusa PBS (umurongo C) hamwe na pcDNA3.1 (+) itsinda rya monotherapy (pcDNA3.1 lane) ryakoreshejwe nkigenzura ribi, kandi itsinda ryita kuri GEV ryakoreshejwe nkigenzura ryiza.Kugaragaza poroteyine ya recombinant byemejwe no kumenya tagi ya histidine muri buri poroteyine, kandi ibiteganijwe kuri poroteyine byari alpha-2 giardine (38.2 kDa) na alpha-7.3 giardine (37.2 kDa).GEV, Giardia duodenum imitsi idasanzwe, pcDNA3.1 (+), EcoRV-umurongo wa vector, SUP, ndengakamere
Kugirango umenye niba alpha-2 giardine na alpha-7.3 giardine itera imvugo ya p20 caspase-1 kandi ikagira uruhare mukugirango usubize nyiricyubahiro NLRP3, pcDNA3.1 (+) - alpha-2 giardine na pcDNA3.1 (+) - alpha -7.3 giardin yahinduwe muri macrophage yibanze ya macrophage hamwe na ADN ya recombinant plasmid, hanyuma urwego rwo kwerekana, kwimenyekanisha, hamwe na oligomerisiyumu ya proteine ​​zingenzi zitera umuriro NLRP3.Muri ubu bushakashatsi, GEV yakoreshejwe nk'itsinda rishinzwe kugenzura neza, kandi nta tsinda rivura (PBS gusa) cyangwa itsinda rishinzwe kuvura indwara ya pcDNA3.1 (+) ryari itsinda ribi.Ibisubizo byerekanye ko, nko mu itsinda rya GEV, ADN ya recombinant plasmid ya giardin pcDNA3.1 (+) - alpha-2 na giardin pcDNA3.1 (+) - alpha-7.3 byatumye habaho kugenzura NLRP3, pro-IL-1β na procaspase-1 na caspase-1 gukora (Ishusho 2a).Byongeye kandi, giardine zombi zateje ururenda rukomeye rwa 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 ).;alpha-7.3 giardine: ANOVA, F (4, 10) = 1.625, P <0.0001;GEV: ANOVA, F (4, 10) = 1.625, P = 0.0047) (Ishusho 2b).Poroteyine nyinshi za ASC zari monomeric mu itsinda ritavura cyangwa mu itsinda ryo kuvura ryandujwe na plasmide ya pcDNA3.1 (+), bitandukanye na pcDNA3.1 (+) - alpha-2 cyangwa pcDNA3.1 (+) - alpha- 7.3 giardine.ASC oligomerisiyasi yabereye muri recombinant plasmid ADN ya GEV itsinda rishinzwe kugenzura neza cyangwa itsinda, byerekana ifishi ya oligomeri (Ishusho 2c).Aya makuru abanza yerekana ko alpha-2 giardine na alpha-7,3 giardine ishobora gutera NLRP3 gukora.Ubushakashatsi bwakorewe immunofluorescent bwakorewe aho ASC na NLRP3 bwerekanye ko mu itsinda rishinzwe kugenzura nabi, poroteyine ya ASC yakwirakwijwe muri cytoplazme kandi igaragara nk'ikimenyetso cy'akadomo nyuma yo gukurura pcDNA3.1 (+) - alpha-2 hamwe na giardine cyangwa pcDNA3.1 (+) - alpha-7,3 itsinda rya giardine cyangwa itsinda ryiza rya GEV (Ishusho 2d).Mu kugenzura nabi no kuvura plasmide yakozwe na pcDNA 3.1, ikimenyetso cya poroteyine ya NLRP3 nticyagaragaye, mugihe akadomo ka fluorescent gashubije pcDNA3.1 (+) - alpha-2 giardine cyangwa pcDNA3.1 (+) - alpha-7.3 byagaragaye..giardine iboneka muri cytoplazme cyangwa iyo itera HEV (Ishusho 2e).Aya makuru yerekana kandi ko G. duodenalis giardin alpha-2 na giardin alpha-7.3 ikora NLRP3 inflammasome muri macrophage yimbeba yibanze ya peritoneal.
pcDNA3.1 (+) - alpha-2 giardin na pcDNA3.1 (+) - alpha-7.3 giardin ikora NLRP3 inflammasome muri macrophage ya peritoneal.Hindura imvugo ya recombinant eukaryotic plasmide pcDNA3.1 (+) - alpha-2 giardin na pcDNA3.1 (+) - alpha-7.3 giardin muri macrophage na selire primaire ya perine, cyangwa gusarura supernatant muri 24 h kugirango isesengure imvugo, oligomerisation , gusohora.hamwe na poroteyine zingenzi.Itsinda rya PBS gusa (C) hamwe nitsinda rya pcDNA3.1 (+) ryakoreshejwe nkigenzura ribi, naho itsinda rya GEV ryakoreshejwe nkitsinda ryiza.Intungamubiri za poroteyine NLRP3, harimo NLRP3, pro-IL-1β, pro-caspase-1, na p20 caspase-1, zagaragajwe n’iburengerazuba.b Urwego rwo gusohora kwa IL-1β muri ndengakamere rwagenwe hakoreshejwe enzyme ihujwe na immunosorbent assay (ELISA).Itandukaniro riri hagati yubugenzuzi nitsinda ryubushakashatsi ryasesenguwe nuburyo bumwe bwo gusesengura ibintu (ANOVA) ukoresheje verisiyo ya software ya SPSS 22.0.Inyenyeri zerekana itandukaniro rikomeye hagati yitsinda ** P <0.01 na *** P <0.001.c Urwego rwa ASC oligomerisation muri pellet rwagenwe nisesengura rya DSS, mugihe urwego rwa ASC muri selile lysates rwakoreshejwe nkigenzura.d Kubona amashusho ya ISC ukoresheje immunofluorescence.e Immunofluorescence yakoreshejwe mugushushanya aho NLRP3 iherereye.ASC, apoptotique idasanzwe isa na poroteyine;IL, interleukin;NLRP3, nucleotide-ihuza oligomerisation-nkiyakira 3;ns, ntabwo ari ngombwa (P> 0.05)
Byombi G. duodenalis na GEV isohora ikora NLRP3 inflammasome kandi ikanagenzura ibisubizo byakira muri vitro.Rero, uruhare rwa NLRP3 inflammasome mugutera indwara ya G. duodenalis ntirurasobanuka neza.Kugira ngo dukore iperereza kuri iki kibazo, twateguye ubushakashatsi hagati yimbeba zanduye G. duodenalis cyst nimbeba zanduye imiti ya G. duodenalis cyst + MCC950 hanyuma tugereranya imvugo ya NLRP3 iyo yanduye G. duodenalis.Igishushanyo kirambuye cyubushakashatsi cyerekanwe ku gishushanyo cya 3a.Imihindagurikire yuburemere bwimibiri yimbeba mumatsinda atandukanye yo kuvura yakurikiranwe muminsi 7 nyuma yo kwandura cysts, kandi ibisubizo bigaragara kumashusho 3b.Ugereranije n'itsinda rivuwe na PBS itanduye, ibisubizo byagaragaje ko (i) uburemere bw'umubiri w'imbeba zanduye G. duodenalis cyst zagabanutse kuva ku munsi wa 3 kugeza ku munsi wa 7 nyuma yo kwandura;(ii) kuvura hamwe na MCC950 inhibitor nta ngaruka nini byagize ku buremere bw'umubiri w'imbeba..Ugereranije n'itsinda rimwe ryanduye, BW yo mu itsinda ryanduye duodenal yavuwe na MCC950 yagabanutse ku buryo butandukanye (Umunsi wa 1: ANOVA, F (3, 24) = 1.885, P = 0.0148; Umunsi wa 2: ANOVA, F (3, 24 ) = 0.4602, P <0.0001 Umunsi wa 3: ANOVA, F (3, 24) = 0.8360, P = 0.0010 Umunsi wa 4: ANOVA, F (3, 24) = 1.683, P = 0.0052; (3, 24) = 0.6497, P = 0.0645;Aya makuru yerekana ko NLRP3 inflammasome irinda imbeba gutakaza ibiro cyane mugihe cyambere (iminsi 2-4) yanduye.Twahise tugamije kumenya G. duodenalis trophozoite mumazi ya duodenal lavage kandi ibisubizo byerekanwe mubishusho 3c.Ugereranije nitsinda ryanduye rya G. duodenalis, umubare wa trophozoite muri duodenum wiyongereye cyane nyuma yo guhagarika NLRP3 inflammasome (t (12) = 2.902, P = 0.0133).Uturemangingo twa Duodenal twandujwe na HE yerekanye, ugereranije no kugenzura nabi bivurwa na PBS na MCC950 byonyine: (i) G. duodenalis cyst yanduye byangiza villi ya duodenal (ANOVA, F (3, 24) = 0.4903, P = 0.0488 ) no gutaka atrophy (ANOVA, F (3, 24) = 0.4716, P = 0.0089);(ii) duodenum ituruka ku mbeba zanduye G. duodenalis cysts kandi zivurwa na MCC950 inhibitor.villi ya duodenal yangiritse kandi yarapfuye (ANOVA, F (3, 24) = 0.4903, P = 0.0144) hamwe na atrophy hamwe nishami ryihuta (ANOVA, F (3, 24) = 0.4716, P = 0, 0481) (Ishusho 3d- f).Ibisubizo byerekana ko NLRP3 inflammasome igira uruhare mukugabanya indwara ya G. duodenalis.
Uruhare rwa NLRP3 inflammasome muri infection ya Giardia duodenum.Imbeba zashizwemo (iv) hamwe na cyst duodenococcale hanyuma zivurwa hamwe na MCC950 (ip).Amatsinda yo kuvura imwe hamwe na PBS cyangwa MCC950 yakoreshejwe nkigenzura.Itsinda ryubushakashatsi hamwe nuburyo bwo kuvura.b Uburemere bwumubiri wimbeba muri buri tsinda ritandukanye ryakurikiranwe iminsi 7.Itandukaniro riri hagati yitsinda ryanduye rya G. duodenalis nitsinda rya G. duodenalis + MCC950 ryanduye ryasesenguwe na t-test ukoresheje verisiyo ya software ya SPSS 22.0.Asterisks yerekana itandukaniro rikomeye kuri * P <0.05, ** P <0.01, cyangwa *** P <0.001.c Umutwaro wa parasitike wagenwe mukubara umubare wa trophozoite mumazi ya duodenal.Itandukaniro riri hagati yitsinda ryanduye rya G. duodenalis nitsinda rya G. duodenalis + MCC950 ryanduye ryasesenguwe na t-test ukoresheje verisiyo ya software ya SPSS 22.0.Asterisks yerekana itandukaniro rikomeye kuri * P <0.05.d Hematoxyline na eosine (H&E) yerekana ibisubizo bya histopathology duodenal.Imyambi itukura yerekana ibyangiritse kuri villi, imyambi yicyatsi yerekana ibyangiritse.Umwanya munini: 100 µm.e, f Isesengura mibare yuburebure bwa duodenal villus nuburebure bwimbeba.Asterisks yerekana itandukaniro rikomeye kuri * P <0.05 na ** P <0.01.Ibisubizo byakuwe mubushakashatsi 7 bwigenga bwibinyabuzima.BW, uburemere bw'umubiri;ig, inzira yo gutanga intagastric;ip, inzira yo gutanga intraperitoneal;ns, ntabwo ari ngombwa (P> 0.05);PBS, saline ya fosifate;WT, ubwoko bw'ishyamba
Ururenda rwa IL-1β ni ikiranga ibikorwa byo gutwika.Kugirango tumenye niba G. duodenalis alpha-2 giardine na alpha-7.3 giardine ikora NLRP3 yakira inflammasome muri vivo, twakoresheje imbeba za WT zitavuwe (itsinda rya sham) hamwe nimbeba za NLRP3 zifata imbeba (MCC950 yabujije itsinda ryokuvura).Igishushanyo kirambuye cyubushakashatsi cyerekanwe ku gishushanyo cya 4a.Amatsinda yubushakashatsi yari agizwe nimbeba zavuwe na PBS, G. duodenalis cyst ivurwa na gavage, inshinge zo mu nda zatewe na pcDNA3.1, no gutera inshinge za pcDNA3.1 (+) - alpha-2 giardine cyangwa pcDNA3.1-alpha-7.3 giardine.Ku munsi wa 7 nyuma yubuyobozi bwimitsi ya recombinant plasmid, serumu yakusanyijwe kandi urwego rwa IL-1β muri buri tsinda rwaragenwe.Nkuko bigaragara ku gishushanyo cya 4b, mu itsinda rya MOCK: (i) ugereranije nitsinda rya PBS, kuvura pcDNA3.1 nta ngaruka nini byagize ku ibanga rya IL-1β (ANOVA, F (4.29) = 4.062, P = 0.9998), ariko, Isohora rya IL-β ryazamutse cyane mu itsinda rya cyst G. duodenalis (ANOVA, F (4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine na pcDNA3.1- Gutera inshinge ya alpha-7.3 giardine yiyongereye cyane serumu IL-1β (ANOVA, F (4, 29) = 4.062, P <0.0001);(iii) pcDNA3. .Ugereranije na buri tsinda riri mu itsinda ry’ubuvuzi rya MCC950 hamwe nitsinda rya MOCK: (i) IL-1β urwego rwibanga mu itsinda rishinzwe kugenzura PBS hamwe nitsinda rishinzwe kugenzura pcDNA3.1 ryaragabanutse ku rugero runaka nyuma yo guhagarika inhibitori ya MCC950, ariko itandukaniro ntabwo gikomeye (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F (9, 58) = 3.540, P = 0.5949);(ii) nyuma yo guhagarika MCC950.Isohoka rya IL-1β ryaragabanutse cyane mu itsinda ryanduye G. duodenalis cyst, itsinda rya pcDNA3.1-alpha-2 giardine, hamwe nitsinda rya pcDNA3.1-alpha-7.3 (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 (9, 58 ) = 3.540, P = 0.0164).Ibisubizo byerekana ko alpha-2 giardine na alpha-7.3 giardine ihuza ibikorwa bya NLRP3 inflammasome muri vivo.
pcDNA3.1 (+) - giardine ikora NLRP3 yakira inflammasome muri vivo.Imbeba zakingiwe (IM) hamwe na recombinant eukaryotic imvugo plasmid pcDNA3.1 (+) - alpha-2 giardine cyangwa pcDNA3.1 (+) - alpha-7.3 giardine hanyuma ivurwa na MCC950 (ip; itsinda rya MCC950) cyangwa ntabwo (itsinda rya dummy) ).Itsinda rivura PBS cyangwa pcDNA3.1 (+) ryakoreshejwe nkigenzura ribi, itsinda rya G. duodenalis cyst ryakoreshejwe nkigenzura ryiza.Itsinda ryubushakashatsi hamwe nuburyo bwo kuvura.b Urwego rwa serumu ya IL-1β mu mbeba rwapimwe kumunsi wa 7 na ELISA.Itandukaniro riri hagati yitsinda ryitsinda rya MOCK ryasesenguwe hakoreshejwe inzira imwe ya ANOVA, naho itandukaniro riri hagati yitsinda rya MOCK nitsinda rya MCC950 ryasesenguwe hakoreshejwe t-test ya software ya software ya SPSS 22.0.Asterisks yerekana itandukaniro rikomeye hagati yitsinda rivura MOCK, * P <0.05 na *** P <0.001;ibimenyetso by'idolari ($) byerekana itandukaniro rikomeye hagati ya buri tsinda mu itsinda rya MOCK n'itsinda MCC950 kuri P <0.05.Ibisubizo byubushakashatsi burindwi bwigenga.i, inshinge zo mu nda, ns, ntabwo ari ngombwa (P> 0.05)
Kugira ngo dukore iperereza ku ngaruka za alpha-2 na alpha-7.3 giardine-yunganirwa na enterineti ya NLRP3 yakira inflammasome ku ndwara ya G. duodenalis, twakoresheje imbeba za WT C57BL / 6 hanyuma dutera alpha-2 giardine na alpha-7.3 giardine.plasmid yatewe inshinge, nyuma yiminsi 3 ikoresheje umuyoboro wigifu wa cyst G. duodenalis, nyuma yimbeba zimaze iminsi 7 ziboneka.Gahunda irambuye yubushakashatsi irerekanwa mu gishushanyo cya 5a.Uburemere bwumubiri wa buri mbeba bwapimwaga burimunsi, ingero za tissue duodenal nshya zegeranijwe kumunsi wa 7 nyuma yubuyobozi binyuze mumiyoboro ya gastric, hapimwe umubare wa trophozoite, kandi hagaragaye impinduka za histopathologique.Nkuko bigaragara ku gishushanyo cya 5b, hamwe no kongera igihe cyo kugaburira, BW yimbeba muri buri tsinda yiyongereye buhoro buhoro.MT yimbeba zatangiye kugabanuka kumunsi wa 3 nyuma yubuyobozi bwimikorere ya G. duodenalis cysts, hanyuma bwiyongera buhoro buhoro.Gukora kwa NLRP3 inflammasome iterwa no gutera inshinge ya alpha-2 giardine na alpha7.3 giardine byagabanije cyane kugabanya ibiro mu mbeba (Umunsi wa 1: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 1.399, P = 0 .9754 Umunsi wa 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 1.399, P = 0.9987 Umunsi wa 2: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 0.3172, P = 0.9979 Umunsi wa 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.3172, P = 0.8409 Umunsi wa 3: pcDNA3.1-alpha-2 giardine, ANOVA, F (; 4, 30) = 0.8222, P = 0.0262 Umunsi wa 3: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.8222, P = 0.0083 Umunsi wa 4: pcDNA3.1-alpha-2 giardine, ANOVA , F (4, 30) = 0.5620, P = 0.0012, Umunsi wa 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.5620, P <0.0001, Umunsi wa 5: pcDNA3.1-alpha - 2 giardine, ANOVA, F (4, 30) = 0.9728, P <0.0001 Umunsi wa 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F (4, 30) = 0.9728, P <0.0001 Umunsi wa 6: pcDNA3 .1 - alpha-2 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0012, Umunsi wa 6: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0006;Umunsi wa 7: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 0.5369, P <0.0001 Umunsi wa 7: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.5369, P <0.0001).Umutwaro wa parasitike wasuzumwe muri duodenum (Ishusho 5c).Ugereranije no kugenzura kutavuwe neza hamwe nitsinda ryatewe inshinge ya pcDNA3.1 yubusa, umubare wa G. duodenalis trophozoite wagabanutse cyane mumatsinda yatewe na α-2 giardine na α-7,3 giardine (pcDNA3.1-alpha -2 giardine: ANOVA, F (3, 24) = 1.209, P = 0.0002, pcDNA3.1-alpha-7.3 giardine: ANOVA, F (3, 24) = 1.209, P <0.0001).Byongeye kandi, giardine alfa-7.3 yarindaga imbeba kurusha giardine alfa-2 (ANOVA, F (3, 24) = 1.209, P = 0.0081).Ibisubizo bya HE irangi byerekanwe mugitabo.5d - f.Imbeba zatewe na alpha-2 giardine na alpha-7.3 giardine zagize ibikomere bike byumubiri wa duodenal, bigaragazwa no kwangirika kwa villus, ugereranije nimbeba zatewe na G. duodenalis nimbeba zatewe na G. duodenalis zifatanije na vector ya pcDNA3 irimo ubusa .1 Zoom.(pcDNA3. = 0.0028 cyangwa P = 0.0055) no kugabanya atrophy ya crypt (pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 1.470, P = 0.0264 cyangwa P = 0.0158; pcDNA3.1-alpha-7.3 giardine: ANOVA , F (3, 24) = 1.470, P = 0.0371 cyangwa P = 0.0191).Ibisubizo byerekana ko alpha-2 giardine na alpha-7,3 giardine bigabanya kwandura G. duodenalis ukoresheje NLRP3 inflammasome muri vivo.
Uruhare rwa pcDNA3.1 (+) - giardine mu kwandura G. duodenalis.Imbeba zakingiwe (IM) hamwe na recombinant eukaryotic imvugo plasmide pcDNA3.1 (+) - alpha-2 giardine cyangwa pcDNA3.1 (+) - alpha-7.3 giardine hanyuma ihura na cysts ya G. duodenalis (ig).Itsinda rya PBS hamwe na pcDNA3.1 (+) + itsinda ryo kuvura cyst duodenal ryakoreshejwe nkitsinda rishinzwe kugenzura nabi, naho itsinda ryita kumyanya ndangagitsina ryakoreshejwe nk'itsinda rishinzwe kugenzura neza.Itsinda ryubushakashatsi hamwe nuburyo bwo kuvura.b MT yimbeba muri buri tsinda ritandukanye ryakurikiranwe muminsi 7 nyuma yikibazo.Asterisks yerekana itandukaniro rikomeye hagati yitsinda ryo mu itsinda rya G. duodenalis na pcDNA3.1 (+) - itsinda rya alpha-2 giardine, * P <0.05, ** P <0.01, na *** P <0.001;ikimenyetso cy'idolari ($) cyerekana itandukaniro rikomeye hagati ya buri tsinda rya G. duodenalis na pcDNA3.1 (+) - alpha-7.3 itsinda rya jardine, $$ P <0.01 na $$$ P <0.001.c Umutwaro wa parasitike wagenwe no kubara umubare wa trophozoite muri ml 1 ya lavage duodenal kuva kuri duodenum (cm 3 z'uburebure) kandi ugaragazwa nkumubare wa parasite kuri cm ya duodenum.Itandukaniro riri hagati yitsinda ryanduye rya G. duodenalis, pcDNA3.1 (+) - itsinda rya alpha-2 giardine, hamwe na pcDNA3.1 (+) - alpha-7.3 giardine ryasesenguwe ninzira imwe ANOVA ikoresheje verisiyo ya software ya SPSS 22.0.Asterisks yerekana itandukaniro rikomeye kuri ** P <0.01 na *** P <0.001.d Impinduka za Histopathologique muri duodenum.Imyambi itukura yerekana ibyangiritse kuri villi, imyambi yicyatsi yerekana ibyangiritse.Umwanya munini: 100 µm.e, f Isesengura ryibarurishamibare ryimbeba ya duodenal villus (e) nuburebure bwa f (f).Itandukaniro hagati yitsinda mu gishushanyo 1d ryasesenguwe ninzira imwe ANOVA ukoresheje verisiyo ya software ya SPSS 22.0.Asterisks yerekana itandukaniro rikomeye kuri * P <0.05 na ** P <0.01.Ibisubizo byubushakashatsi burindwi bwigenga.ns, ntabwo ari ngombwa (P> 0.05)
Giardia duodenum ni parasite yo munda izwi cyane yabantu nizindi nyamabere zitera giardiasis.Mu 2004, yashyizwe muri gahunda ya OMS yirengagije indwara kubera ko yiganje cyane mu myaka 6, cyane cyane mu baturage bafite imibereho myiza y’ubukungu [32].Ubudahangarwa bw'umubiri bufite uruhare runini mugukingira indwara ya G. duodenalis.Imbeba za macrophage zafashwe kandi zica G. duodenalis mu kurekura imitego idasanzwe [33].Ubushakashatsi bwatubanjirije bwerekanye ko G. duodenalis, parasite idashobora gutera, ikora p38 MAPK, ERK, NF-κB p65, na NLRP3 yerekana ibimenyetso byerekana inzira muri macrophage yimbeba kugirango igenzure ibyakiriwe neza, kandi GEV irekuwe irashobora kongera iyi nzira.13], 24].Nyamara, PAMPs nyayo igira uruhare muri NLRP3 inflammasome-igenzurwa na GEV hamwe nuruhare rwa NLRP3 inflammasome muri giardiasis iracyasobanurwa.Kugira ngo tumenye ibi bibazo byombi, twakoze ubu bushakashatsi.
Indwara ya NLRP3 iherereye muri cytoplazme yingirangingo z'umubiri kandi irashobora gukoreshwa nuduce duto nka kirisiti ya aside irike, uburozi, bagiteri, virusi, na parasite.Mu bushakashatsi bwa bagiteri, uburozi bwamenyekanye nka PAMP zingenzi zikoresha ibyuma bifata ibyuma byangiza, biganisha ku gutwika no gupfa kw ingirabuzimafatizo [34].Uburozi butandukanye butandukanye, nka hemolysine yo muri Staphylococcus aureus [35] na Escherichia coli [36], hemolysine BL (HBL) ikomoka kuri enterotoxine (NHE) [37], itera gukora kwa NLRP3.Ubushakashatsi bwa virusi bwerekanye ko poroteyine za virusi nka poroteyine ya SARS-COV-2 (E) [38] na virusi ya Zika NS5 [39] ari PAMP zingenzi zemewe na reseptor ya NLRP3.Mu bushakashatsi bwakozwe na parasite, byavuzwe ko parasite nyinshi zifitanye isano no gukora inflammasome, nka Toxoplasma gondii, vaginalis ya Trichomonas [40], Trypanosoma cruzi [41], na Leishmania [42].Poroteyine zuzuye za granule GRA35, GRA42, na GRA43, zifitanye isano na virusi ya Toxoplasma gondii, zirakenewe mu kwinjiza pyroptose muri macrophage ya Lewis [43].Byongeye kandi, ubushakashatsi bumwe na bumwe bwa Leishmania bwibanze kuri molekile ku giti cye igira uruhare mu gutwika NLRP3, nka parasite membrane lipophosphoglycan [44] cyangwa zinc metalloprotease [45].Mu muryango wa alne-giardin umeze nka annexine, alpha-1 giardin byagaragaye ko ari umukandida w’inkingo ushobora kurinda G. duodenalis mu buryo bw’imbeba [18].Mu bushakashatsi bwacu, twahisemo virusi ya G. duodenalis alpha-2 na alpha-7,3 giardine, zidasanzwe kuri giardia ariko ugereranije ni bike.Izi genes ebyiri zerekanwe zashyizwe muri pcDNA3.1 (+) eukaryotic ya sisitemu ya sisitemu yo gusesengura imikorere yumuriro.
Muburyo bwimbeba yacu, ibice bya caspase byacitse nkibimenyetso byerekana ibikorwa.Nyuma yo gukanguka, NLRP3 ikorana na ASC, igashakisha procaspase, kandi ikabyara caspase ikora ihuza IL-1β na pro-IL-18 mo IL-1β na IL-18 ikuze -18.Indwara ya caspase (caspase-1, -4, -5 na -11) ni umuryango wabungabunzwe wa poroteyine ya sisitemu ifite akamaro kanini mu kwirwanaho kavukire kandi igira uruhare mu gutwika no gupfa byateguwe na selile [46].Caspase-1 ikorwa na Canonical inflammasomes [47], mugihe caspase-4, -5, na -11 zometseho mugihe cyo gushinga ibintu bidasanzwe [48].Muri ubu bushakashatsi, twakoresheje imbeba peritoneal macrophage nkicyitegererezo kandi dukora iperereza kuri p20 caspase-1 yacaguye caspase-1 nkikimenyetso cyerekana ibikorwa bya NLRP3 byo gutwika mubushakashatsi bwanduye G. duodenalis.Ibisubizo byerekanye ko alpha-giardine nyinshi zifite inshingano zo gukora ibikorwa bisanzwe byo gutwika, ibyo bikaba bihuye no kuvumbura molekile zingenzi za virusi zigira uruhare muri bagiteri na virusi.Nyamara, ubushakashatsi bwacu ni ecran ibanza gusa kandi hariho izindi molekile zishobora gukora inflammasomes itari classique, nkuko ubushakashatsi bwacu bwabanje bwerekanye ko inflammasomes ya classique na non-classique yanduye G. duodenalis [13].Kugirango turusheho kumenya niba p20 caspase yakozwe 1 ifitanye isano na NLRP3 inflammasome, twahinduye alpha-2 na alpha-7.3 giardine muri macrophage yimbeba kugirango tumenye urwego rwingenzi rwa poroteyine zerekana urwego rwa ASC oligomerisation, twemeza ko α-giardine zombi zikora. inflammasome NLRP3.Ibisubizo byacu bitandukanye cyane n’ibya Manko-Prykhoda n'abandi, bavuze ko gukangurira ingirabuzimafatizo za Caco-2 hamwe na G. muris cyangwa E. coli EPEC byonyine bishobora kongera ubukana bwa fluorescence ya NLRP3, ASC, na caspase-1, nubwo bidasobanutse neza, mugihe uburyo kwigana G. muris na E. coli byongereye urwego rwa poroteyine eshatu [49].Uku kunyuranya gushobora guterwa no gutandukanya guhitamo ubwoko bwa Giardia, imirongo ya selile, na selile yibanze.Twakoze kandi muri vivo assays dukoresheje MCC950 mubyumweru 5 byimbeba WT C57BL / 6 yimbeba, zishobora kwibasirwa na G. duodenalis.MCC950 ninzitizi ikomeye kandi ihitamo molekile ntoya ya NLRP3 ibuza gukora Canonical na non-Canonical NLRP3 yibikorwa bya nanomolar.MCC950 ibuza gukora NLRP3 ariko ntabwo igira ingaruka kumikorere ya AIM2, NLRC4, na NLRP1 cyangwa inzira ya TLR yerekana inzira [27].MCC950 ihagarika ibikorwa bya NLRP3 ariko ntibibuza gutangiza NLRP3, K + efflux, Ca2 + kwinjira, cyangwa imikoranire hagati ya NLRP3 na ASC;ahubwo, irabuza gukora NLRP3 inflammasome muguhagarika ASC oligomerisation [27].Kubwibyo, twakoresheje MCC950 mubushakashatsi bwa vivo kugirango tumenye uruhare rwa NLRP3 inflammasome nyuma yo guterwa giardine.Caspase ikora-1 p10 ikora cytokine itera inflammatory IL-1β na pro-IL-18 ikuze IL-1β na IL-18 [50].Muri ubu bushakashatsi, urugero rwa serumu IL-1β mu mbeba zivuwe na giardine cyangwa zidafite MCC950 zakoreshejwe nk'ikimenyetso cyo kumenya niba NLRP3 inflammasome yarakozwe.Nkuko byari byitezwe, kuvura MCC950 byagabanije cyane serumu IL-1β.Aya makuru yerekana neza ko G. duodenalis giardin alfa-2 na giardin alfa-7.3 bashoboye gukora imbeba ya NLRP3 inflammasome.
Amakuru y'ingenzi yakusanyirijwe mu myaka icumi ishize yerekanye ko IL-17A ari yo igenga ubudahangarwa bw'umubiri wa G. muris, itera ibimenyetso bya IL-17RA, ikora peptide ya mikorobe, kandi igenga ibikorwa byuzuzanya [51].Nyamara, kwandura Giardia kugaragara cyane ku rubyiruko rukuze, kandi byavuzwe ko kwandura Giardia mu mbeba zikiri nto bidakora igisubizo cya IL-17A kugira ngo bigire ingaruka zo kubarinda [52], bituma abashakashatsi bashakisha izindi Giardia ikingira indwara.uburyo bwo kwandura helminth.Abanditsi b'ubushakashatsi buherutse gukorwa batangaje ko G. muris ishobora gukora NLRP3 inflammasome na E. coli EPEC, iteza imbere umusaruro wa peptide yica mikorobe kandi ikagabanya ubushobozi bwo kuyifata ndetse n'umubare wa trophozoite mu mara, bityo bikagabanya ubukana bwa colon indwara ziterwa na bacili [49].NLRP3 inflammasome igira uruhare mugutezimbere indwara zitandukanye.Ubushakashatsi bwerekanye ko Pseudomonas aeruginosa itera autofagy muri macrophage kugirango birinde urupfu, kandi iki gikorwa giterwa no gukora kwa NLRP3 inflammasome [53].Kuri N. caninum, ubwoko bwa ogisijeni ikora neza-ihuza ibikorwa bya NLRP3 inflammasome igabanya kwigana kwayo, bityo bikaba intego yo kuvura [9].Paracoccidioides brasiliensis yasanze itera imbaraga za NLRP3 inflammasome mu ngirabuzimafatizo ya dendritic selile yimbeba, bivamo irekurwa rya cytokine IL-1β, igira uruhare runini mu kwirwanaho [10].Ubwoko butandukanye bwa Leishmania, harimo L. amazonensis, L. major, L. braziliensis, na L. infantum chagasi, bukora NLRP3 na ASC iterwa na caspase-1 muri macrophage, ndetse no kwandura Leishmania.Kwigana parasite byongerewe imbaraga mu mbeba zabuze muri NLRP3 / ASC / caspase-1 gene [11].Zamboni n'abandi.Indwara ya Leishmania yavuzwe ko itera gukora NLRP3 inflammasome muri macrophage, igabanya parasite yo mu nda.Rero, Leishmania irashobora guhagarika ibikorwa bya NLRP3 nkingamba zo kwirinda.Mu bushakashatsi bwa vivo, NLRP3 inflammasome yagize uruhare mu kurandura Leishmania, ariko ntiyagize ingaruka ku ngingo.Ku rundi ruhande, mu bushakashatsi bwa helminthiasis, gukora kwa NLRP3 inflammasome byahagaritse ubudahangarwa bw'umubiri bwakingira indwara ya gastrointestinal helminthiasis [12].Shigella ni imwe muri bagiteri nyamukuru zitera impiswi ku isi.Izi bagiteri zirashobora gutuma IL-1β itanga umusaruro binyuze muri P2X7 yakira reseptor-yunganirwa na K + efflux, ubwoko bwa ogisijeni ikora, aside aside ya lysosomal, no kwangiza mitochondial.NLRP3 inflammasome igenga nabi fagocytose nigikorwa cya bagiteri yica macrophage irwanya Shigella [55].Ubushakashatsi bwa Plasmodium bwerekanye ko imbeba zabuze AIM2, NLRP3 cyangwa caspase-1 zanduye Plasmodium zitanga intera yo mu bwoko bwa interferon yo mu bwoko bwa 1 kandi ikaba ishobora kurwanya indwara ya Plasmodium [56].Uruhare rwa alpha-2 giardine na alpha-7.3 giardine mu gutera indwara ziterwa na NLRP3 mu mbeba ntirusobanutse.
Muri ubu bushakashatsi, kubuza NLRP3 inflammasome na MCC950 byagabanije BW kandi byongera umubare wa trophozoite mumazi yo mu mara ya lavage mu mbeba, bikaviramo impinduka zikomeye z’indwara mu ngingo zifata imyanya ndangagitsina.Alpha-2 giardine na alpha-7.3 giardine ikora imbeba yakira NLRP3 inflammasome, kongera uburemere bwimibiri yumubiri, kugabanya umubare wa trophozoite mumazi yo mu mara, no kugabanya ibikomere byindwara ya duodenal.Ibisubizo byerekana ko G. duodenalis ishobora gukora NLRP3 yakira inflammasome ikoresheje alpha-2 giardine na alpha-7,3 giardine, bikagabanya indwara ya G. duodenalis mu mbeba.
Hamwe na hamwe, ibisubizo byacu byerekana ko alpha-2 na alpha-7.3 giardine itera gukora kwa NLRP3 yakira inflammasome kandi bikagabanya kwandura G. duodenalis mu mbeba.Kubwibyo, izo molekile ziratanga intego zo gukumira giardiasis.
       Data supporting the results of this study can be obtained from the respective author at gongpt@jlu.edu.cn.
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