Giardia duodenum chirwere cheparasitic chinokonzera giardiasis, chirwere chemudumbu chinowanzoitika muvana vaduku vane zviratidzo zvekliniki zvemanyoka.Takambotaura kuti extracellular G. duodenalis inokonzera kushandiswa kwe-intracellular oligomerization-like receptor 3 (NLRP3) inosunga nucleotides uye inogadzirisa mhinduro dzehutachiona kuburikidza ne extracellular vesicle (EV) secretion.Zvisinei, maitiro chaiwo emakemikari epathogen-associated duodenococcal EV (GEV) inobatanidzwa mukuita uku uye basa reNLRP3 inflammasome mu giardiasis rinoramba richijekeswa.
Recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 uye alpha-7.3 giardins muGEV akavakwa, akachinjirwa mugonzo rekutanga peritoneal macrophages, uye akaonekwa nekuyera kuzvimba chinangwa chemorekuru caspase-1.Iyo p20 yekutaura level yakaongororwa..G. duodenalis alpha-2 uye alpha-7.3 giardines yakatanga kuonekwa nekuyera NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 uye caspase-1 p20), IL secretion.1β mazinga, apoptotic spotted protein (ASC) oligomerization mazinga, uye immunofluorescent localization yeNLRP3 uye ASC.Basa reNLRP3 inflammasome mu pathogenicity yeG. duodenalis yakabva yaongororwa kushandisa mbeva umo NLRP3 activation yakavharwa (NLRP3 yakavharwa mice) uye kuchinja kwepathological muhuremu hwemuviri, duodenal parasitic load, uye duodenal tissue yakatariswa.Mukuwedzera, takaongorora kana hiardines alpha-2 uye alpha-7.3 inokonzera IL-1β secretion mu vivo kuburikidza neNLRP3 inflammasome uye takasarudza basa remamorekuru aya mu pathogenicity yeG. duodenalis mumakonzo.
Alpha-2 uye alpha-7.3 giardines inokonzera kushandiswa kweNLRP3 inflammasome in vitro.Izvi zvakakonzera kushandiswa kwep20 caspase-1, kuwedzera kwehuwandu hwemashoko eNLRP3, pro-IL-1β, uye pro-caspase-1 mapuroteni, kuwedzera kukuru kweIL-1β secretion, kuumbwa kweASA mavara mu cytoplasm, uye kuiswa kweASA oligomerization.NLRP3 kuzvimba Penile kurasikirwa kunowedzera pathogenicity yeG. duodenalis mumakonzo.Mice inobatwa ne cysts ne gavage kubva kuNLRP3-yakavharidzirwa mbeva yakaratidza kuwedzera kwenhamba ye trophozoites uye kukuvadza kwakanyanya kune duodenal villi, inoratidzwa necrotic crypts ine shrunken uye branching.In vivo miedzo yakaratidza kuti giardines alpha-2 uye alpha-7.3 inogona kukonzera secretion ye IL-1β kuburikidza neNLRP3 inflammasome, uye kuvhara ne giardines alpha-2 uye alpha-7.3 yakaderedza pathogenicity yeG. duodenalis mumakonzo.
Kutorwa pamwe chete, zvigumisiro zvechidzidzo ichi zvinoratidza kuti giardia alpha-2 uye alpha-7.3 inokonzera kukwidziridzwa kwemukuru weNLRP3 kuzvimba uye kuderedza hutachiona hweG. duodenalis mumakonzo, ayo ari kuvimbisa zvinangwa zvekudzivirira giardiasis.
Giardia duodenum is extracellular protozoan parasite inogara mudumbu diki uye inokonzera 280 miriyoni zviitiko zvegiardiasis nemanyoka gore negore, kunyanya pakati pevana vadiki vari munyika dzichiri kusimukira [1].Vanhu vanotapukirwa nemvura yekunwa kana chikafu chakasvibiswa neM. duodenum cysts, iyo inobva yapinda mudumbu uye inobuda mujusi yemudumbu.Giardia duodenum trophozoites inonamatira kune duodenal epithelium, ichikonzera kusvotwa, kurutsa, manyoka, kurwadziwa mudumbu, uye kurasikirwa nehuremu.Vanhu vane immunodeficiency uye cystic fibrosis vanogona kutapukirwa.Utachiona hunogonawo kuitika kuburikidza nemuromo uye nepabonde [2].Mishonga yakadai semetronidazole, tinidazole, uye nitazoxanide ndiyo inosarudzwa nzira dzekurapa duodenal infections [3].Nekudaro, idzi chemotherapy zvinodhaka zvinokonzeresa mhedzisiro yakaipa senge kusvotwa, carcinogenesis, uye genotoxicity [4].Nokudaro, dzimwe nzira dzinoshanda dzinoda kugadzirwa kudzivirira G. duodenalis utachiona.
Inflammasomes ikirasi ye cytosolic protein complexes iyo iri chikamu chemukati wekuzvidzivirira kwezvirwere, zvichibatsira kudzivirira kubva pathogen invasion uye kupindira mhinduro dzekuputika [5].Pakati peizvi inflammasomes, nucleotide-binding oligomerization (NOD) receptor 3 (NLRP3) nucleotide-binding oligomerization (NLRP3) nucleotide-binding-like inflammasome yakanyatsodzidza nokuti inogona kuonekwa nemhando dzakasiyana-siyana dzepathogen / kukuvara-kwakabatana nema molecular maitiro (PAMP). DAMP), inoziva, inomutsa immune system yemukati.uye inogadzirisa intestinal homeostasis mune dzakawanda zvirwere zvinoputika [6,7,8].Inosanganisira maitiro ekuzivikanwa receptor (PRR) NLRP3, an adapter apoptotic spotted protein (ASC), uye an effector procaspase-1 kana procaspase-11.Iyo NLRP3 inflammasome inoshanda seyakarwisa kurwisa kwepathogen, sekuonekwa muNeospora caninum [9], Paracoccidioides brasiliensis [10], uye Leishmania zvidzidzo.[11], asi zvakare yakashumwa kuti kushandiswa kweNLRP3 inflammasome kunogadzirisa mhinduro dzekudzivirira zvirwere uye kunowedzera kuwedzera kwechirwere, semuenzaniso, muhonye [12].Kubva pane zvatakawana kare, takashuma kuti extracellular G. duodenalis inokonzera intracellular activation yeNLRP3 kuzvimba uye inogadzirisa mhinduro dzekuvhiringidza kuburikidza nekuvhara extracellular vesicles (EVs) [13].Zvisinei, basa reNLRP3 inflammasome muG. duodenalis utachiona mu vivo rinoramba rakatemwa.
Giardins yakatanga kurondedzerwa sezvikamu zvehutano zveG. duodenalis cytoskeleton uye inoita basa rinokosha mu trophozoite motility uye epithelial cell attachment mudumbu duku.Kuti zvigadzirise zviri nani kune zvakatipoteredza uye kuwedzera pathogenicity yavo, G. duodenalis trophozoites yakagadzira chimiro chakasiyana checytoskeletal chinosanganisira 8 flagella, 1 muviri wepakati, uye 1 ventral disc [14].Matrophozoite eGiardia duodenum anoshandisa cytoskeleton yawo kupinda mudumbu repamusoro, kunyanya duodenum, uye anonamatira kuma enterocytes.Vanogara vachitama uye vanonamatira kumasero epithelial vachishandisa cell metabolism.Naizvozvo, pane hukama hwepedyo pakati pecytoskeleton yavo uye virulence.Giardines chaiyo yeGiardia duodenum zvikamu zve cytoskeleton structure [15] uye yakakamurwa kuva makirasi mana: α-, β-, γ-, uye δ-giardines.Kune nhengo dze21 dzemhuri ye-α-giardin, iyo yose ine calcium-inotsamira kukwanisa kusunga phospholipids [16].Ivo zvakare vanobatanidza iyo cytoskeleton kune cell membrane.Muvanhu vane manyoka anokonzerwa neG. duodenalis, α-giardins inoratidzwa zvakanyanya uye inodzivirira panguva yehutachiona [17].Mishonga yeHeterologous yakavakirwa paGiardia alfa-1 yakadzivirirwa kubva kune giardiasis mumakonzo uye inogona kuve maantigen ekuvandudza vaccine [18].Alpha-8 giardin, inowanikwa mu plasma membrane uye flagella, asi kwete mu ventral disc, inowedzera motility uye kukura kwe trophozoites muG duodenalis [19].Alpha-14 giardin inonamatira kune microtubule zvivako pa flagella uye inokanganisa kushanda kweG duodenalis [20].Alpha-11 giardine iripo muhuwandu mukati mehupenyu hwose, uye overexpression yealpha-11 giardine inokuvadza G. duodenalis pachayo [21].Zvisinei, hazvina kujeka kana alpha-2 giardine uye alpha-7.3 giardine inodzivirira kubva kuG. duodenalis utachiona uye maitiro avo ari pasi.
Muchidzidzo ichi, recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine uye pcDNA3.1 (+) -alpha-7.3 giardine yakachinjwa kuva mouse primary peritoneal macrophages kuti ishandise host NLRP3.Zvinangwa zveInflammasome zvakabva zvaongororwa.Isu takaongororawo basa reNLRP3 inflammasome mu pathogenicity yeG. duodenalis, takaongorora kana alpha-2 uye alpha-7,3 giardines inokonzera kushandiswa kweNLRP3 inflammasome in vivo, uye takasarudza kuti aya maviri mabasa egiardines mu pathogenicity ye. G. duodenalis.Chinangwa chedu chakafanana ndechekugadzira zvinangwa zvinovimbisa zvekudzivirira G. duodenalis infection.
Wild type (WT) C57BL/6 mbeva dzechikadzi dzine makore 5–8 mavhiki dzakatengwa kubva kuLiaoning Changsheng Experimental Animal Center (Liaoning, China).Mbeva dzaive nemahara ekuwana mvura, dzakagashira chikafu chine sterilized uye dzakachengetwa mu12/12 awa yechiedza / rima.Pamberi pehutachiona, mbeva dzakagamuchira mishonga inorwisa mabhakitiriya ad libitum mumvura yekunwa yakawedzerwa ne ampicillin (1 mg / mL), vancomycin (1 mg / mL), uye neomycin (1.4 mg / mL) (zvose zvakatengwa kubva kuShanghai, China, zvipenyu zvekugadzira) [22 ].].Mbeva dzakarasikirwa nekukwanisa kudya uye kunwa kwe> 24 maawa uye dzakarasika ≥ 20% uremu hwemuviri dzakagadziriswa nevanhu nekervical dislocation.
WB G. duodenalis trophozoites (American Type Culture Collection, Manassas, USA) yakawedzerwa ne12.5% ​​yefetal bovine serum (FBS; Yese Green, Zhejiang, China) uye 0.1% bovine bile (Sigma-Aldrich, St. Missouri, USA )USA) pasi pemamiriro e microaerobic.Confluent trophozoites yakaunganidzwa pachando uye yakapfuura nechiyero che1: 4 kuti iwedzere kubereka.
Giardia duodenum cysts yakaitwa sezvakatsanangurwa kare [23], trophozoites yakakohwewa mu logarithmic phase ndokuzoderedzwa ne encapsulation inducing medium, pH 7.1 (yakagadziridzwa TYI-S-33) kusvika kumagumo ekupedzisira e1 × 106 trophozoites / mL.bile concentration 0.05% yepakati).Trophozoites yakasimudzwa pasi peanaerobic mamiriro pa37 ° C kusvika iyo logarithmic kukura chikamu.Shandura svikiro kusvika ku cyst inducing medium (pH 7.8; yakagadziridzwa TYI-S-33 yepakati ne 1% bile concentration) uye tsika G. duodenalis pa 37 ° C kwemaawa 48-96, panguva iyo maitiro ekugadzira akaonekwa pasi pe microscope.Mushure mekunge ma trophozoite mazhinji aitwa kuti agadzire ma cysts, musanganiswa wetsika wakakohwewa uye wakamiswa mumvura isina tsvina yakasvibiswa kuti iite lyse yasara trophozoite.Cysts yakaverengwa uye yakachengetwa pa 4 ° C kuitira kuongororwa kwakatevera kuburikidza negastric tube mumakonzo.
Giardia extracellular vesicles (GEVs) yakafumiswa sezvakatsanangurwa kare [13].Trophozoites muchikamu chekukura kwelogarithmic yakamiswazve mune yakagadziridzwa TYI-S-33 svikiro yakagadzirwa ne exosome-yakapera FBS (Biological Industries, Beit-Haemek, Israel) kusvika kuchikamu chekupedzisira che 1 × 106 parasites/mL uye yakanyudzwa kwemaawa gumi nemaviri.vakaparadzaniswa kubva kune tsika supernatant ne centrifugation pa 2000 g ye 10 min, 10,000 g ye 45 min, uye 100,000 g ye 60 min.Precipitates yakanyungudutswa mu phosphate buffered saline (PBS), yakagadziriswa uchishandisa BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) uye yakachengetwa pa -80 ° C. kana kushandiswa zvakananga kuti iwedzere kuongororwa.
Primary mouse peritoneal macrophages yakagadzirirwa sezvakatsanangurwa kare [24].Muchidimbu, mbeva (dzine makore 6-8 mavhiki) dzakaiswa jekiseni (intraperitoneally [ip]) ne 2.5 ml ye2.98% Difco liquid thioglycol medium (BD, Franklin Lakes, NJ, USA) uye yakadyiswa 3-4 palates.Kumiswa kwema macrophages kwakaunganidzwa kubva mudumbu mhango yemakonzo mushure meeuthanasia uye centrifuged 3 nguva pa1000 g ye10 min.Masero akakohwa akaonekwa nekuyerera kwe cytometry achishandisa CD11b marker kusvika kuchena kwesero > 98%, ndokuzowedzerwa kune 6-well cell culture plates (4.5 x 106 masero / zvakanaka) uye incubated ne 10% FBS (Bioindustry) pa 37 ° C.uye 5% CO2.
RNA yakatorwa kubva ku1 × 107 trophozoites mu 1 ml yeTRIzol reagent (Vazyme, Nanjing, China), genomic DNA yakatorwa kubva kuG. duodenalis RNA yakazara uchishandisa MonScript dsDNase (Monad, Wuhan, China) uye inopindirana DNA (cDNA) yakagadzirwa. uchishandisa MonScript RTIIII Super Mix (Monad) zvinoenderana nemirairo yemugadziri.
CDS sequence information yechinangwa cheG. duodenalis gene yakawanikwa kubva kuNCBI GenBank.Shandisa Primer 5.0 kugadzira chaiyo isina musono cloning primers yejini rega rega rinotarirwa.Iyo yekutanga primer (5'-3′) ine zvikamu zvitatu: kutevedzana kwakatevedzana ine linearized vector pcDNA3.1(+) EcoRV (TGGTGGAATTCTGCAGAT) uye kutanga macodons ATG neGNN (kana hwaro hwekutanga isiri G).Izvi zvinoitirwa kuvandudza kushanda kwekutaura.Pamusoro pezvo, kanenge 16 bp yakasanganiswa mabhesi (GC content 40–60%/Tm approx. 55 °C).Reverse primer (5′-3′) ine zvikamu zviviri, kutevedzana kunopindirana neEcoRV-linearized vector pcDNA3.1(+) (GCCGCCACTGTGCTGGAT) uye hwaro hwakabatanidzwa hunosvika gumi nematanhatu bp.(kunze kwekumira kuviri kwekupedzisira).mabhesi) kodoni yakadai seAA kana GA kubvumira maplasmids anodzokororwa kuratidza mapuroteni avo akanyorwa).The primer sequences dzakanyorwa muTable 1 uye dzakagadzirwa neKangmet Biotechnology Co., Ltd. (Changchun, China).
Zvinangwa zvakawedzerwa uchishandisa Pfu DNA polymerase (Tiangen, Beijing, China) kana Ex-taq (Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, China) vachishandisa yakagadzirwa G. duodenalis cDNA se template.Iyo eukaryotic kutaura vector plasmid pcDNA3.1 (+) yakarongedzerwa nerestriction enzyme EcoRV uye dephosphorylated uchishandisa Fast AP (Thermo Fisher Scientific).Linearized pcDNA3.1 (+) zvimedu uye akawedzera chinangwa chemajini zvimedu zvakacheneswa pachishandiswa DNA gel yekunatsa kit (Tiangen) uye yakaverengerwa pachishandiswa Nanodrop ND-2000 (Thermo Fisher Scientific).Iyo pcDNA3.1(+) chidimbu uye chega chega chimedu chejini zvakabatanidzwazve pachishandiswa MonClone single assembly cloning mix (Monad Biotech Co., Ltd., Suzhou, China) uye yakasimbiswa neDNA kutevedzana uchishandisa Comate Bioscience Company Limited (Changchun, China) ..
Endotoxin-isina plasmids pcDNA3.1 (+)-alpha-2 uye pcDNA3.1 (+)-alpha-7.3 yakagadzirwa uchishandisa SanPrep Endotoxin-isina Plasmid Mini Kit (Sangon Biotech).Iko kusungirirwa kwakachengetwa pamusoro pe500 ng / µl kuti ive nechokwadi chokuti EDTA mu-elution buffer haina kupindira nekutsvaga kwekutsvaga.Primary mouse peritoneal macrophages yakagadzirwa mu6-tsime mahwendefa ane yakakwana RPMI 1640 yepakati (Biological Industries) kwemaawa gumi nemaviri, ipapo masero akashambidzwa katatu muPBS inodziya kuti abvise penicillin uye streptomycin, uye ipapo pakati akawedzerwa nepakati yakakwana.Endotoxin-isina plasmids pcDNA3.1 (+)-alpha-2 uye pcDNA3.1 (+)-alpha-7.3 (2.5 μg) yakaderedzwa mu125 μl yeOpti-MEM yakaderedzwa serum medium (Gibco, Thermo Fisher Scientific) ..Zvino 5 µl yeLipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific) yakaderedzwa mu125 µl yeyakaderera serum Opti-MEM yepakati.Gadzirira liposome-DNA complexes nekusanganisa iyo yakanyungudutswa endotoxin-isina plasmid neLipofectamine 2000 uye kubvumira musanganiswa kuti umire pakupisa kwekamuri kwemaminetsi mashanu.Tumira macomplexes akasiyana kumaseru mune imwe neimwe tsime uye sanganisa zvishoma nezvishoma.Mushure memaawa mana, iyo sero tsika yepakati yakatsiviwa ne 2 ml yakakwana yeRPMI 1640 yepakati uye tsika yakaenderera kwemaawa makumi maviri nemana.Fresh cell tsika yepakati yakawedzerwa kumaseru uye incubated kweakasiyana nguva mapoinzi zvichienderana neassay dhizaini.
Mapuroteni samples kubva kune supernatants uye cell lysates akagadzirirwa sezvakatsanangurwa kare [25].Membrane transfer parameters for pro-IL-1β, pro-caspase-1, caspase-1 p20, NLRP3, β-actin, uye His-tag yaiva 200 mA / 90 min.For interleukin 1β (IL-1β; R&D Systems, Minneapolis, Minnesota, USA), caspase-1 (p20) (Adipogen, Switzerland) uye NLRP3 (Adipogen SA, Epalinges, Switzerland) uye 1:5000 inotarisa tag Yake ( Amylet Scientific, Wuhan, China) uye β-actin (Proteintech, Wuhan, China).
Kuyambuka-kubatanidza ne disuccinimide subrate (DSS) yakaitwa sezvakatsanangurwa kare [26].Masero akagezwa 3 nguva nePBS inotonhora uye akanyatsoiswa lysed ne 27 gauge tsono mu 50 µl ASC reaction buffer (pH 8.0) ine 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES uye 125 mM NaHCO3.Musanganiswa wakaiswa centrifuged pa5000 g kwemaminetsi matatu uye pellet yakashongedzwa ne10 µl DSS (25 mM muDMSO) uye 40 µl ASC reaction buffer yemaminetsi makumi matatu pa37°C.Mushure me centrifugation pa 5000 g ye 10 min, pellet yakanyungudutswa mumhinduro ye 40 µl yeASC reaction buffer uye 10 µl ye6x protein loading buffer (TransGen, Beijing, China), uyezve mhinduro yacho yakadzimwa pakamuri tembiricha yegumi nemashanu. min., Zvadaro bika maminitsi gumi.Maprotein samples akabva aiswa kuWestern blotting achishandisa primary anti-ASC antibodies (Wanleibio, Shenyang, China) pachiyero che dilution che1:500.
Kutevera nzira yakambotsanangurwa [13], sero tsika supernatants dzakakohwewa uye secretion ye-pro-inflammatory cytokine IL-1β yakagadziriswa kushandisa mouse IL-1 Beta ELISA kit (Invitrogen, Thermo Fisher Scientific).Shandura OD450nm kukosha kuita mapuroteni akawandisa uchishandisa iyo IL-1β yakajairwa curve.
Masero akavharwa pazvivharo akashambidzwa zvinyoro 3 nguva muPBS inodziya, yakagadziriswa mutissue cell fixative (Biosharp, Beijing, China) kwemaminetsi gumi pamhepo tembiricha (RT), mu 0.1% Triton X-Permeabilize pa100 (diluted muPBS; Biosharp ) kwemaminetsi makumi maviri pakupisa kwekamuri uye block mu 5% bovine serum albumin (muPBS) kwemaawa maviri patembiricha yekamuri.Masero akazoiswa usiku hwose pa4°C aine masoja ekudzivirira chirwere ekutanga ASC (1:100 dilution) kana NLRP3 (1:100 dilution), zvichiteerana, uye Cy3 yakanyorwa mbudzi inorwisa tsuro IgG(H+L) (1:400; EarthOx) , San Francisco, CA, USA) kana FITC-conjugated mbudzi anti-mouse IgG (1:400; Earthox) usiku hwose pa37°C murima kweawa imwe.Iyo nuclei yakasvibiswa neHoechst 33258 (10 μg/ml; UE, Suzhou, China) kwemaminetsi mashanu uye yakacherechedzwa pasi peiyo fluorescence microscope (Olympus Corporation, Tokyo, Japan).
Mbeva dzakakamurwa kuita mapoka mana (n = 7 muboka rimwe nerimwe): (i) PBS-yakabatwa zvisina kunaka kudzora boka (PBS chete; gavage 100 µl/mouse PBS inoteverwa nemazuva ese intraperitoneal jekiseni 100 µl/mouse PBS 3 maawa gare gare)., nguva dzose kwemazuva manomwe);(ii) boka rekutonga risina kunaka rinobatwa neMCC950 inhibitor [27] (100 µl / mouse kuburikidza nePBS gavage, maawa 3 gare gare, 10 mg / kg uremu hwemuviri [BW] MCC950 [muPBS] yaishandiswa intraperitoneally zuva nezuva, nguva ye7 mazuva);(iii) G. duodenalis cyst infection group (1.5 x 106 cysts / mouse ne gavage, maawa matatu gare gare, 100 μl / mouse PBS intraperitoneally inotungamirirwa zuva nezuva kwemazuva 7);(iv) G. duodenalis cyst yakasanganiswa utachiona boka MCC950 inhibitor kurapwa boka (1.5 × 106 cysts / mouse via gavage, 10mg / kg uremu hwemuviri MCC950 intraperitoneally zuva nezuva kwemazuva 7 pa3h).Huremu hwemuviri wembeva yega yega hwaiongororwa zuva nezuva uye mbeva dzese dzakaburitswa pazuva re7th.Yakakohwa duodenum (3 cm kureba) yakatemwa kuita zvidimbu zviduku mu 1 ml PBS, cysts dzakaparadzwa usiku hwose muPBS pa4 ° C, uye G. duodenalis trophozoites.Fresh duodenum (1 cm kureba) yaive yakatsaurwa yehematoxylin uye eosin (H&E) tsvina.
Mice dzakakamurwa kuita mapoka maviri: (i) MOCK control group uye (ii) MCC950 inhibitor boka.Paiva nemishonga mishanu muboka rimwe nerimwe (n = 7 / boka rekurapa): (i) PBS kurapa kwakashata kudzora boka (PBS chete; 100 µl / mouse PBS, intramuscular (IM) jekiseni (tibialis anterior) [28, 29]; ( ii) pcDNA3.1 (+) plasmid negative control boka (100 µg/mouse DNA, via intramuscular injection) (iii) G. duodenalis cyst infection positive control group (1.5 x 106 cysts/ mouse, via gavage) (iv) a) boka rinobatwa neplasmid pcDNA3.1 (+) -alpha-2 (100 μg/mouse DNA, nejekiseni remumuscular), uye (v) boka rinobatwa neplasmid pcDNA3.1 (+) -alpha-7.3 (100 µg/mbeva) DNA, mushure memaawa gumi nemaviri ekufamba, mbeva dziri muboka reMCC950 inhibitor dzakagamuchira jekiseni rezuva nezuva reMCC950 (10 mg/kg uremu hwemuviri) kwemazuva 7, nepo mbeva dziri muboka reMOCK dzakagamuchira huwandu hwakaenzana hwePBS kurapwa. yakaunganidzwa kubva kumakonzo emeso uye yakasara usiku humwe pa4 ° C Serum samples dzakaparadzaniswa dzichishandisa enzyme-yakabatanidzwa immunosorbent assay (ELISA) uye zviyero zveIL-1β mazinga.
Makumi makumi matatu neshanu mbeva dzakakamurwa kuita mapoka mashanu (n = 7 / boka).Boka 1 raive boka rinodzora rakashata rakabatwa nePBS: mbeva dzakagamuchira 100 μl yePBS intramuscularly uye 3 mazuva gare gare negavage.Boka 2 iboka rakanaka rekutonga rinotapukirwa neG. duodenalis cysts: mbeva dzakaiswa jekiseni ne 100 μl yePBS, uye mazuva matatu gare gare 1.5 x 106 cysts / mouse yakajowa intragastrically.Boka rechitatu - kubaya kweplasmid ne pcDNA3.1 (+) pamwe chete neboka rekutonga re duodenal cyst infection: mbeva dzakagamuchira 100 μg ye plasmid DNA pcDNA3.1 (+) (im) nemuromo, 1.5 × 106 cysts / mouse 3 kune akati wandei. mazuva.Mapoka 4 uye 5 aiva recombinant pcDNA3.1 (+) -alpha-2 giardine plasmid kana pcDNA3.1 (+) -alpha-7.3 giardine plasmid pamwe chete neG. duodenalis cyst infection.Boka rekuedza: mbeva dzakagamuchira 100 µg ye pcDNA3.1.Huremu hwemuviri wembeva yega yega hwakatariswa mushure mekuiswa kweG. duodenalis cyst kuburikidza nechubhu.Fresh duodenum yakaunganidzwa kuitira kuyerwa kweparasitic load uye HE staining analysis.
Histopathological shanduko yakaongororwa maererano neyakambodhindwa nzira [30].Fresh duodenum yakagadziriswa ne tissue cell fixative, yakaiswa muparafini, yakachekwa muzvikamu zve 4 μm, yakasvibiswa neH & E uye yakaongororwa pasi pe microscope yakajeka.Representative pathological change muzvikamu zvinomwe zvematishu kubva kumakonzo manomwe akazvimiririra akaongororwa neapathologist asingazivi nezvekurapa uye akabatwa pa200x magnification.Kureba kwe villi uye kudzika kwe crypts kwakayerwa maererano nemaitiro akarondedzerwa kare.
Mhedzisiro mu vitro uye mu vivo yakawanikwa mumatatu.Magirafu akagadzirwa pachishandiswa GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, USA).Misiyano pakati pemapoka maviri yakaongororwa ne t-test, nepo kusiyana pakati pe ≥3 mapoka akaongororwa nenzira imwe chete yekuongorora kusiyana (ANOVA) uchishandisa SPSS software (version 22.0; SPSS IBM Corp., Armonk, NY, USA).Dhata yakaongororwa kune homogeneity yekusiyana uchishandisa bvunzo yaLevene yakateverwa neBonferroni's post hoc bvunzo (B).Kukosha kunoratidzwa seP<0.05, P<0.01, uye P<0.001 (hazvina kukosha [ns]) (P>0.05).
Ongororo yedu yapfuura yeGEV proteomics muKyoto Encyclopedia yeGenes uye Genomes (KEGG) yakaratidza kuti zvinangwa zvakawanda zvingave zvakabatanidzwa mukushandiswa kwekuzvimba kusaina nzira [13].Takasarudza zvinangwa zviviri zvinovimbisa, alpha-2 uye alpha-7.3 giardins, wedzera mamorekuru aya uye toashandisa kugadzira pcDNA3.1 (+) eukaryotic expression vector.Mushure mekutevedzana, recombinant pcDNA3.1 (+) -alpha-2 uye alpha-7.3 giardine kutaura plasmids yakachinjirwa muprimary mouse peritoneal macrophages, uye caspase-1 p20 siginecha protein yekuzvimba (chidimbu che activated caspase-1) chakaonekwa. sekujekesa mamorekuru akakosha anogona kukonzera kuzvimba.Zvigumisiro zvakaratidza kuti alpha-2 uye alpha-7.3 giardines inogona kukonzera p20 caspase-1 kutaura kwakafanana neGEV.Hapana mhedzisiro pane caspase-1 activation yakawanikwa mune isina kubatwa yakashata control (PBS chete) uye plasmid control pcDNA3.1 (+) (Mufananidzo 1).
Kuyera kwep20 caspase-1 activation ne pcDNA3.1 (+) -alpha-2 uye alpha-7.3 giardins.Recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 uye alpha-7.3 giardines (pamusoro penzira imwe neimwe) yakachinjirwa muprimary mouse peritoneal macrophages uye tsika dzepamusoro dzakakohwewa maawa makumi maviri nemana gare gare.Western blotting yakashandiswa kuyera mazinga ekutaura kweiyo siginecha caspase-1 p20 inflammasome protein.Iboka rePBS-chete rekurapa (lane C) uye pcDNA3.1 (+) monotherapy boka (pcDNA3.1 lane) yakashandiswa sekutonga kusina kunaka, uye boka rekurapa reGEV rakashandiswa sekutonga kwakanaka.Kuratidzwa kweprotein yakagadziriswa kwakasimbiswa nekuona tag ye histidine muprotein yega yega, uye mapuroteni aitarisirwa mabhendi aive alpha-2 giardine (38.2 kDa) uye alpha-7.3 giardine (37.2 kDa).GEV, Giardia duodenum extracellular vesicles, pcDNA3.1(+), EcoRV-linearized vector, SUP, supernatant
Kuti uone kana alpha-2 giardine uye alpha-7.3 giardine inokurudzira p20 caspase-1 kutaura uye inobata basa mukugadzirisa muiti weNLRP3 mhinduro yekuzvimba, pcDNA3.1 (+) -alpha-2 giardine uye pcDNA3.1 (+) -alpha -7.3 giardin yakashandurwa kuva primary mouse peritoneal macrophages ine recombinant plasmid DNA, uye mazinga ekutaura, nzvimbo, uye oligomerization yemapuroteni makuru ekuputira NLRP3 akatemwa.Muchiedzo ichi, GEV yakashandiswa seboka rakanaka rekutonga, uye hapana boka rekurapa (PBS chete) kana pcDNA3.1 (+) boka rekurapa kwekutapurirana ndiro boka rakashata.Zvigumisiro zvakaratidza kuti, sezvakaita boka reGEV, recombinant plasmid DNA ye giardin pcDNA3.1 (+) -alpha-2 uye giardin pcDNA3.1 (+) -alpha-7.3 yakakonzera kukwidziridzwa kweNLRP3, pro-IL-1β uye procaspase-1 uye caspase-1 activation (Fig. 2a).Mukuwedzera, zvose giardines zvakakonzera zvakakosha IL-1β secretion (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) (Mufananidzo 2b).Mapuroteni mazhinji eASC aive monomeric muboka risingarapike kana muboka rekurapa rakachinjirwa ne pcDNA3.1 (+) plasmid, kusiyana ne pcDNA3.1 (+) -alpha-2 kana pcDNA3.1 (+) -alpha- 7.3 giardine.ASC oligomerization yakaitika mune recombinant plasmid DNA yeGEV yakanaka control boka kana boka, ichiratidza oligomeric fomu (Mufananidzo 2c).Aya ekutanga data anoratidza kuti alpha-2 giardine uye alpha-7,3 giardine inogona kukonzera NLRP3 kuzvimba activation.Zvidzidzo zvinotevera zve immunofluorescent zvenzvimbo yeASC neNLRP3 zvakaratidza kuti muboka rakashata rekutonga, puroteni yeASC yakapararira mukati me cytoplasm uye yakaonekwa sechiratidzo che dot pakukurudzira pcDNA3.1 (+) -alpha-2 ine giardine kana pcDNA3.1 (+) -alpha-7,3 giardine boka kana GEV positive control boka (Mufananidzo 2d).Mukutonga kwakashata uye plasmid-yakabatwa pcDNA 3.1 mapoka, NLRP3 protein chiratidzo haina kuonekwa, nepo fluorescent chiratidzo dot mukupindura pcDNA3.1 (+) -alpha-2 giardine kana pcDNA3.1 (+) -alpha-7.3 yaonekwa..giardine inowanikwa mu cytoplasm kana pakukurudzira kweHEV (Fig. 2e).Iyi data inowedzera kuratidza kuti G. duodenalis giardin alpha-2 uye giardin alpha-7.3 inoshandisa NLRP3 inflammasome mu mouse primary peritoneal macrophages.
pcDNA3.1 (+) -alpha-2 giardin uye pcDNA3.1 (+)-alpha-7.3 giardin inomutsa NLRP3 inflammasome mu mouse peritoneal macrophages.Shandura recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardin uye pcDNA3.1(+)-alpha-7.3 giardin mu primary murine peritoneal macrophages uye masero, kana kukohwa supernatant mukati me24 h kuti iongororwe yekutaura, oligomerization. , secretion.uye kugarisana kwemapuroteni akakosha ekuputika.Iboka rePBS-chete (C) uye pcDNA3.1 (+) boka rekurapa rimwechete rakashandiswa sekutonga kusina kunaka, uye boka rekurapa reGEV rakashandiswa seboka rakanaka.A Key inflammatory proteins NLRP3, kusanganisira NLRP3, pro-IL-1β, pro-caspase-1, uye p20 caspase-1, yakaonekwa neWestern blotting.b Mazinga ekuvharidzirwa kweIL-1β mumasimba makuru akagadziriswa kushandisa enzyme-linked immunosorbent assay (ELISA).Misiyano pakati pekutonga uye mapoka ekuedza akaongororwa nenzira imwe chete yekuongorora musiyano (ANOVA) uchishandisa SPSS software version 22.0.Nyeredzi dzinoratidza kusiyana kukuru pakati pemapoka ** P <0.01 uye *** P <0.001.c ASC oligomerization mazinga mumapellets akatemerwa neDSS cross-linking ongororo, nepo ASC mazinga mumasero lysates akashandiswa seyekurodha.d Kuonekwa kweISC yenzvimbo uchishandisa immunofluorescence.e Immunofluorescence yakashandiswa kufungidzira nzvimbo yeNLRP3.ASC, apoptotic speck-like protein;IL, interleukin;NLRP3, nucleotide-binding oligomerization-like receptor 3;ns, haina kukosha (P> 0.05)
Ose G. duodenalis uye maGEV aanoburitsa anoita kuti NLRP3 inflammasome ishande uye inogadzirisa mhinduro dzekuzvimba muvitro.Nokudaro, basa reNLRP3 inflammasome mu pathogenicity yeG duodenalis inoramba isingazivikanwi.Kuti tiongorore nyaya iyi, takagadzira chiedzo pakati pembeva dzine G. duodenalis cyst uye mbeva dzine G. duodenalis cyst + MCC950 inhibitor kurapwa uye tikaenzanisa NLRP3 inflammasome kutaura pawakabatwa neG. duodenalis cyst.Chirongwa chakadzama chekuedza chinoratidzwa mumufananidzo 3a.Kuchinja kwehuremu hwemakonzo mumapoka akasiyana-siyana ekurapa akaongororwa kwemazuva 7 mushure mekutapukirwa ne cysts, uye zvigumisiro zvinoratidzwa muFig. 3b.Kuenzaniswa neboka rinobatwa nePBS yakachena, zvigumisiro zvakaratidza kuti (i) uremu hwemakonzo ane utachiona hweG. duodenalis cyst yakaderera kubva pazuva 3 kusvika zuva 7 mushure mehutachiona;(ii) kurapwa neMCC950 inhibitor kwaisava nemhedzisiro yakakosha pahuremu hwemuviri wemakonzo..Kuenzaniswa neboka rimwechete rehutachiona, BW yeboka rechirwere che duodenal rakabatwa neMCC950 yakaderera kusvika kune madhigirii akasiyana (Zuva 1: ANOVA, F (3, 24) = 1.885, P = 0.0148; Zuva 2: ANOVA, F (3, 24) ) = 0.4602, P<0.0001; Zuva 3: ANOVA, F(3, 24) = 0.8360, P = 0.0010; Zuva 4: ANOVA, F(3, 24) = 1.683, P = 0.0052; (3, 24)=0.6497, P=0.0645; Zuva rechitanhatu: ANOVA, F(3, 24)=5.457, P=0.0175; Zuva 7: ANOVA, F(3, 24) = 2.893, P = 0.0202).Iyi data inoratidza kuti NLRP3 inflammasome inodzivirira mice kubva pakurasikirwa kukuru kwekureruka mumatanho ekutanga (2-4 mazuva) ehutachiona hwe duodenal.Takazovavarira kuona G. duodenalis trophozoites muduodenal lavage fluid uye mhedzisiro inoratidzwa muFigure 3c.Kuenzaniswa neboka reG. duodenalis cyst infection, nhamba ye trophozoite mu duodenum yakawedzera zvakanyanya mushure mekuvhara NLRP3 inflammasome (t (12) = 2.902, P = 0.0133).Duodenal tissues yakasvibiswa neHE yakaratidza, ichienzaniswa nekutonga kusina kunaka kwakabatwa nePBS uye MCC950 chete: (i) G. duodenalis cyst utachiona hwakakonzera kukanganisa kune duodenal villi (ANOVA, F (3, 24) = 0.4903, P = 0.0488 ) uye crypt atrophy (ANOVA, F (3, 24) = 0.4716, P = 0.0089);(ii) duodenum kubva kumakonzo ane G. duodenalis cysts uye akabatwa neMCC950 inhibitors.duodenal villi yakakuvadzwa uye yakafa (ANOVA, F (3, 24) = 0.4903, P = 0.0144) ine atrophy uye crypt branching (ANOVA, F (3, 24) = 0.4716, P = 0, 0481) (Fig. 3d- f) .Izvi zvinoratidza kuti NLRP3 inflammasome inoita basa mukuderedza pathogenicity yeG duodenalis.
Basa reNLRP3 inflammasome muGiardia duodenum utachiona.Mbeva dzakaiswa gavaged (iv) ne duodenococcal cysts ndokuzorapwa kana isina MCC950 (ip).Mapoka ekurapa ega ane PBS kana MCC950 akashandiswa sekutonga.Boka rekuedza uye chirongwa chekurapa.b Huremu hwemuviri wemakonzo mune imwe neimwe yemapoka akasiyana ekurapa akatariswa kwemazuva manomwe.Musiyano pakati peG. duodenalis utachiona boka uye G. duodenalis + MCC950 boka rekurapa hutachiona rakaongororwa net-test uchishandisa SPSS software version 22.0.Nyeredzi dzinoratidza kusiyana kukuru pa *P<0.05, **P<0.01, kana ***P<0.001.c Parasitic load yakatemwa nekuverenga nhamba ye trophozoite mu duodenal lavage fluid.Musiyano pakati peG. duodenalis utachiona boka uye G. duodenalis + MCC950 boka rekurapa hutachiona rakaongororwa net-test uchishandisa SPSS software version 22.0.Asterisks inoratidza misiyano yakakura pa *P <0.05.d Hematoxylin uye eosin (H&E) inosvibisa mhedzisiro ye duodenal histopathology.Miseve tsvuku inoratidza kukanganisa kune villi, miseve yakasvibirira inoratidza kukanganisa kune crypts.Scale bar: 100 µm.e, f Statistical analysis ye duodenal villus urefu uye mouse crypt urefu.Nyeredzi dzinoratidza kusiyana kukuru pa * P <0.05 uye ** P <0.01.Mhedzisiro yacho inotorwa kubva ku7 yakazvimiririra yebiological experiments.BW, uremu hwemuviri;ig, intragastric yekuendesa nzira;ip, intraperitoneal yekuendesa nzira;ns, kwete yakakosha (P> 0.05);PBS, phosphate buffered saline;WT, mhando yemusango
Iyo secretion ye IL-1β chiratidzo chekuzvimba activation.Kuti tione kana G. duodenalis alpha-2 giardine uye alpha-7.3 giardine activate NLRP3 host inflammasome in vivo, takashandisa WT mbeva dzisina kugadziriswa (sham group) uye NLRP3 inflammasome-blocked mice (MCC950 inhibited treatment group).Chirongwa chakadzama chekuedza chinoratidzwa mumufananidzo 4a.Mapoka ekuedza aisanganisira mbeva dzakabatwa nePBS, G. duodenalis cyst kurapwa ne gavage, intramuscular injection ye pcDNA3.1, uye intramuscular injection ye pcDNA3.1 (+) -alpha-2 giardine kana pcDNA3.1-alpha-7.3 giardine.Pazuva re7 mushure mekutonga kwe intramuscular of recombinant plasmid, serum yakaunganidzwa uye chiyero che IL-1β muboka rimwe nerimwe chakatemwa.Sezvinoratidzwa muMufananidzo 4b, muboka reMOCK: (i) yakaenzaniswa neboka rePBS, pcDNA3.1 kurapwa kwakanga kusina chinhu chakakosha paIL-1β secretion (ANOVA, F (4.29) = 4.062, P = 0.9998), zvisinei, IL-β secretion yakanyanya kukwidziridzwa muG. duodenalis cyst group (ANOVA, F (4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine uye pcDNA3.1- Intramuscular injection ye alpha-7.3 giardine yakawedzera zvakanyanya serum IL-1β mazinga (ANOVA, F (4, 29) = 4.062, P <0.0001);(iii) pcDNA3.1-alpha-7,3 giardine yakakonzera mazinga akakwirira eIL -1β secretion mu pcDNA3.1-alpha-2 giardine intramuscular injection group (ANOVA, F (4, 29) = 4.062, P = 0.0333) .Kuenzaniswa neboka rimwe nerimwe muboka reMCC950 rekurapa uye boka reMOCK: (i) IL-1β secretion levels muboka rekutonga rePBS uye pcDNA3.1 boka rekutonga rakaderera kusvika kune imwe nhanho mushure mekuvhara MCC950 inhibitor, asi kusiyana kwakanga kusina. zvakakosha (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F (9, 58) = 3.540, P = 0.5949);(ii) mushure mekuvhara MCC950., IL-1β secretion yakaderedzwa zvakanyanya muG. duodenalis cyst-infected group, pcDNA3.1-alpha-2 giardine boka, uye pcDNA3.1-alpha-7.3 giardine boka (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).Izvi zvinoratidza kuti alpha-2 giardine uye alpha-7.3 giardine inopindirana kushandiswa kweNLRP3 inflammasome in vivo.
pcDNA3.1 (+) -giardines activate the NLRP3 host inflammasome in vivo.Mbeva dzakabaiwa (IM) ine recombinant eukaryotic kutaura plasmid pcDNA3.1 (+) -alpha-2 giardine kana pcDNA3.1 (+) -alpha-7.3 giardine uye ndokurapwa neMCC950 (ip; MCC950 boka) kana kwete (dummy boka )PBS kana pcDNA3.1 (+) boka rekurapa replasmid rakashandiswa sekutonga kusina kunaka, G. duodenalis cyst kurapwa boka rakashandiswa sekutonga kwakanaka.Boka rekuedza uye chirongwa chekurapa.b Serum mazinga e IL-1β mumakonzo akayerwa pazuva 7 neELISA assay.Misiyano pakati pemapoka muboka reMOCK yakaongororwa pachishandiswa nzira imwe chete ANOVA, uye misiyano pakati peboka reMOCK neboka reMCC950 yakaongororwa pachishandiswa t-test yeSPSS software version 22.0.Asterisks inoratidza kusiyana kukuru pakati pemapoka ekurapa muboka reMOCK, * P <0.05 uye *** P <0.001;zviratidzo zvemadhora ($) zvinoratidza kusiyana kukuru pakati peboka rega rega muboka reMOCK neboka reMCC950 paP <0.05.Mibairo yezviyedzo zvinomwe zvakazvimirira zvebhayoloji.ini, intramuscular jekiseni, ns, kwete yakakosha (P> 0.05)
Kuti tiongorore kushanda kwealpha-2 uye alpha-7.3 giardine-mediated activation yeNLRP3 host inflammasome paG. duodenalis infectivity, takashandisa WT C57BL/6 mice uye injected alpha-2 giardine uye alpha-7.3 giardine.iyo plasmid yakaiswa intramuscularly, mushure memazuva matatu kuburikidza negastric tube yeG. duodenalis cyst, mushure mokunge mbeva dzakaonekwa kwemazuva manomwe.Chirongwa chakadzama chekuedza chinoratidzwa muFig. 5a.Huremu hwemuviri wembeva yega yega hwakayerwa zuva rega rega, sampuli dzezvinyoro duodenal tishu dzakaunganidzwa pazuva re7th mushure mekutonga kuburikidza negastric tube, nhamba yetrophozoite yakayerwa, uye histopathological shanduko yakaonekwa.Sezvakaratidzwa muMufananidzo 5b, nekuwedzera nguva yekudya, BW yemakonzo muboka rimwe nerimwe yakawedzera zvishoma nezvishoma.Iyo MT yemakonzo yakatanga kuderera pazuva re3 mushure mekutonga kwe intragastric yeG. duodenalis cysts, uye zvishoma nezvishoma yakawedzera.Kushandiswa kweNLRP3 inflammasome inokonzerwa nejekiseni re intramuscular ye alpha-2 giardine uye alpha7.3 giardine zvakanyanya kuderedza uremu mumakonzo (Zuva 1: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 1.399, P = 0 .9754 Zuva 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30)=1.399, P=0.9987 Zuva rechipiri: pcDNA3.1-alpha-2 giardine, ANOVA, F( 4, 30) = 0.3172, P = 0.9979; Zuva 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.3172, P = 0.8409; Zuva 3: pcDNA3.1-alpha-2 giardine, 4, 30) = 0.8222, P = 0.0262 Zuva rechitatu: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.8222, P = 0.0083; Zuva 4: pcDNA3.1-alpha-2 , F(4, 30) = 0.5620, P = 0.0012, Zuva 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5620, P <0.0001, Zuva 5: pcDNA3.1-alpha - 2 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Zuva 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Zuva 6: -1 pcDNA alpha-2 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0012, Zuva rechitanhatu: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0006;Zuva rechinomwe: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.5369, P<0.0001 Zuva rechinomwe: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5369, P <0.0001).Iyo parasitic mutoro wakaongororwa mu duodenum (Fig. 5c).Kuenzaniswa nekusagadziriswa kwakanaka kwekutonga uye boka rakaiswa jekiseni ne pcDNA3.1 vector isina chinhu, nhamba yeG. duodenalis trophozoites yakaderedzwa zvakanyanya mumapoka akaiswa jekiseni ne α-2 giardine uye 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).Mukuwedzera, giardine alfa-7.3 yakanga yakanyanya kudzivirira mumakonzo kupfuura giardine alfa-2 (ANOVA, F (3, 24) = 1.209, P = 0.0081).Mhedzisiro yeHE staining inoratidzwa mufig.5d–f.Mbeva dzakaiswa jekiseni nealpha-2 giardine uye alpha-7.3 giardine dzakanga dzine maronda mashoma eduodenal tissue, anoratidzwa nehutachiona hwehutachiona, zvichienzaniswa nembeva dzakabaiwa neG. duodenalis uye mbeva dzakabaiwa neG. duodenalis pamwe chete nepcDNA3 vector isina chinhu .1 Zoom.(pcDNA3.1-alpha-2 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0035 kana P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0028 kana P = 0.0055) uye yakaderedzwa crypt atrophy (pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 1.470, P = 0.0264 kana P = 0.0158; pcDNA3.1-alpha-7.3 ANOVA , F(3, 24) = 1.470, P = 0.0371 kana P = 0.0191).Migumisiro iyi inoratidza kuti alpha-2 giardine uye alpha-7,3 giardine inoderedza hutachiona hweG duodenalis nekuita kuti NLRP3 inflammasome in vivo.
Basa re pcDNA3.1 (+) -giardins muG. duodenalis utachiona.Mbeva dzakabaiwa (IM) ne recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine kana pcDNA3.1 (+) -alpha-7.3 giardine uye ndokupikiswa neG. duodenalis cysts (ig).Boka rePBS uye pcDNA3.1 (+) + duodenal cyst kurapwa boka rakashandiswa semapoka ekudzora asina kunaka, uye duodenal cyst kurapwa boka rakashandiswa seyakanaka kutonga boka.Boka rekuedza uye chirongwa chekurapa.b The MT yemakonzo mune rimwe nerimwe remapoka akasiyana-siyana ekurapa akatariswa kwemazuva 7 mushure mekupikisa.Asterisks inoratidza kusiyana kukuru pakati pemapoka muboka reG. duodenalis uye pcDNA3.1 (+) -alpha-2 giardine boka, * P <0.05, ** P <0.01, uye *** P <0.001;chiratidzo chedhora ($) chinoratidza kusiyana kukuru pakati peboka rimwe nerimwe reG. duodenalis uye pcDNA3.1 (+)-alpha-7.3 jardine boka, $ $ P <0.01 uye $ $ $ P <0.001.c Parasitic load yakatemwa nekuverenga nhamba ye trophozoite mu 1 ml ye duodenal lavage kubva muduodenum (3 cm kureba) uye inoratidzwa sehuwandu hwehutachiona pasendimita ye duodenum.Kusiyana pakati peboka reG. duodenalis infection, pcDNA3.1 (+) -alpha-2 giardine boka, uye pcDNA3.1 (+) -alpha-7.3 giardine boka rakaongororwa nenzira imwe chete ANOVA uchishandisa SPSS software version 22.0.Nyeredzi dzinoratidza kusiyana kukuru pa ** P <0.01 uye *** P <0.001.d Histopathological shanduko mu duodenum.Miseve tsvuku inoratidza kukanganisa kune villi, miseve yakasvibirira inoratidza kukanganisa kune crypts.Scale bar: 100 µm.e, f Statistical analysis of mouse duodenal villus height (e) uye crypt urefu (f).Misiyano pakati pemapoka muMufananidzo 1d yakaongororwa neimwe nzira ANOVA uchishandisa SPSS software vhezheni 22.0.Nyeredzi dzinoratidza kusiyana kukuru pa * P <0.05 uye ** P <0.01.Mibairo yezviyedzo zvinomwe zvakazvimirira zvebhayoloji.ns, haina kukosha (P> 0.05)
Giardia duodenum inonyanyozivikanwa intestinal parasite yevanhu nedzimwe mhuka dzinoyamwisa dzinokonzera giardiasis.Muna 2004, yakabatanidzwa muWHO Neglected Diseases Initiative nekuda kwekuwanda kwayo kwemakore anopfuura 6, kunyanya munharaunda dzehupfumi hwehupfumi [32].Chirwere chemuviri chemuviri chemuviri chinotora basa rinokosha mukupindura kwezvirwere zveG. duodenalis.Mouse macrophages anonzi akamedza nekuuraya G. duodenalis nekuburitsa misungo inonzi extracellular [33].Zvidzidzo zvedu zvekare zvakaratidza kuti G. duodenalis, isina-invasive extracellular parasite, inomutsa p38 MAPK, ERK, NF-κB p65, uye NLRP3 nzira dzechiratidzo chekuputika mu mouse macrophages kuti dzigadzirise mhinduro dzinopisa, uye GEV yakasunungurwa inogona kusimbisa iyi nzira.13], 24].Zvisinei, iyo chaiyo PAMPs inobatanidzwa muNLRP3 inflammasome-yakagadziriswa kuzvimba muGEV uye basa reNLRP3 inflammasome mu giardiasis rinoramba richijekeswa.Kuti tijekese mibvunzo miviri iyi, takaitisa chidzidzo ichi.
Iyo NLRP3 inflammasome iri mu cytoplasm yemasero ekudzivirira uye inogona kushandiswa nezvikamu zvakasiyana-siyana zvakadai se uric acid crystals, chepfu, mabhakitiriya, mavhairasi, uye zvipembenene.Muzvidzidzo zvebhakitiriya, chepfu yakaonekwa seyiyi PAMPs inobata ma sensors ekupisa, zvichiita kuti kuzvimba uye kufa kwesero [34].Mamwe matukisi akasiyana-siyana, akadai sehemolysin kubva kuStaphylococcus aureus [35] uye Escherichia coli [36], hemolysin BL (HBL) kubva ku enterotoxin (NHE) [37], inoita kuti NLRP3 ishande.Zvidzidzo zvehutachiona zvakaratidza kuti virulence mapuroteni akadai SARS-COV-2 envelope (E) protein [38] uye Zika virus NS5 protein [39] yakakosha PAMPs inozivikanwa neNLRP3 receptor.Mune zvidzidzo zveparasite, zvipembenene zvakawanda zvakanzi zvinosanganiswa nehost inflammasome activation, yakadai seToxoplasma gondii, Trichomonas vaginalis [40], Trypanosoma cruzi [41], uye Leishmania [42].Iyo dense granule mapuroteni GRA35, GRA42, uye GRA43, yakabatana nehutachiona hweToxoplasma gondii, inodiwa pakuiswa kwepyroptosis muLewis rat macrophages [43].Mukuwedzera, zvimwe zvidzidzo zveLeishmania zvakatarisa kune mamwe mamorekuru anobatanidzwa muNLRP3 inflammasome, yakadai separasite membrane lipophosphoglycan [44] kana zinc metalloprotease [45].Pakati pemhuri yeannexin-yakafanana nealpha-giardin yemajini, alpha-1 giardin yakaratidzwa kuva inogona kuva mumiriri wekudzivirira kudzivirira G. duodenalis mumuenzaniso wembeva [18].Muchidzidzo chedu, takasarudza G. duodenalis virulence factor alpha-2 uye alpha-7,3 giardines, iyo yakasiyana negiardia asi isingatauri zvishoma.Aya maviri emajini akatarwa akaiswa mu pcDNA3.1 (+) eukaryotic expression system vector yekuongorora kwekuzvimba activation.
Mune yedu mbeva modhi, akatsemurwa caspase zvimedu zvinoshanda sezviratidzo zvekuzvimba activation.Pakukurudzira, NLRP3 inopindirana neASC, inotora procaspases, uye inogadzira caspases inoshanda inoparadzanisa pro-IL-1β uye pro-IL-18 mukukura IL-1β uye IL-18, maererano -18.Inflammatory caspases (caspases-1, -4, -5 uye -11) imhuri yakachengetedzwa yecysteine ​​​​proteases iyo yakakosha pakudzivirira kwekuzvarwa uye inobatanidzwa mukuputika uye yakarongwa sero kufa [46].Caspase-1 inoshandiswa necanonical inflammasomes [47], nepo caspases-4, -5, uye -11 inotsemurwa panguva yekuumbwa kweatypical inflammasomes [48].Muchidzidzo ichi, takashandisa mouse peritoneal macrophages semuenzaniso uye takaongorora p20 caspase-1 cleaved caspase-1 sechiratidzo chekugamuchira NLRP3 kuzvimba kushandiswa muzvidzidzo zveG. duodenalis infection.Mhedzisiro yacho yakaratidza kuti mazhinji maalpha-giardins ane basa reiyo yakajairika activation yekuzvimba, izvo zvinoenderana nekuwanikwa kweakakosha virulence mamorekuru anosanganisirwa mubhakitiriya nemavhairasi.Zvisinei, chidzidzo chedu chinongova chidzitiro chekutanga uye kune mamwe mamorekuru anogona kushandura non-classical inflammasomes, sezvo chidzidzo chedu chekare chakawana zvose zvekare uye zvisingasviki inflammasomes muG. duodenalis infection [13].Kuenderera mberi kuona kana iyo yakagadzirwa p20 caspase-1 yakabatana neNLRP3 inflammasome, isu takashandura alpha-2 uye alpha-7.3 giardins mugonzo peritoneal macrophages kuona akakosha mamorekuru mapuroteni ekutaura mazinga uye ASC oligomerization mazinga, zvichisimbisa kuti ese ma-α-giardins anoshanda. inflammasome NLRP3.Zvigumisiro zvedu zvakasiyana zvishoma kubva kune zveManko-Prykhoda et al., uyo akashuma kuti kukurudzirwa kweCaco-2 masero ane G. muris kana E. coli EPEC strains chete inogona kuwedzera fluorescence intensity yeNLRP3, ASC, uye caspase-1, kunyange zvazvo zvisinganyanyi kukosha, nepo mari yeG muris uye E. coli yakawedzera mazinga emapuroteni matatu [49].Kusiyana uku kunogona kunge kuri nekuda kwekusiyana kwekusarudzwa kwemarudzi eGiardia, mitsara yemasero, uye masero ekutanga.Isu takaitawo mu vivo assays tichishandisa MCC950 mu5-vhiki-yekuzvarwa yevakadzi WT C57BL/6 mbeva, idzo dzinonyanya kubatwa neG. duodenalis.MCC950 ine simba uye inosarudza diki molecule NLRP3 inhibitor inovhara canonical uye isiri-canonical NLRP3 activation pane nanomolar concentrations.MCC950 inhibits NLRP3 activation asi haina kukanganisa kushandiswa kweAIM2, NLRC4, uye NLRP1 nzira dzinopisa kana TLR zviratidzo nzira [27].MCC950 inovhara NLRP3 activation asi haidziviriri kutanga kweNLRP3, K + efflux, Ca2 + kupinda, kana kupindirana pakati peNLRP3 neASC;pane kudaro, inodzivisa NLRP3 inflammasome activation nekuvhara ASC oligomerization [27].Naizvozvo, takashandisa MCC950 mune in vivo kudzidza kuona basa reNLRP3 inflammasome mushure mejekiseni regiardine.Activated caspase-1 p10 inotsemura pro-inflammatory cytokines pro-IL-1β uye pro-IL-18 mukukura IL-1β uye IL-18 [50].Muchidzidzo ichi, serum IL-1β mazinga mumakonzo akabatwa negiardine ane kana asina MCC950 akashandiswa sechiratidzo chekuti NLRP3 inflammasome yakaitwa.Sezvaitarisirwa, kurapwa kweMCC950 kwakaderedza zvakanyanya serum IL-1β mazinga.Aya data anoratidza zvakajeka kuti G. duodenalis giardin alfa-2 uye giardin alfa-7.3 vanokwanisa kushandura NLRP3 mouse inflammasome.
Yakakosha data yakaunganidzwa mumakore gumi apfuura yakaratidza kuti IL-17A ndiyo inodzora hutachiona hweG. muris, ichiita IL-17RA kusaina, inogadzira antimicrobial peptides, uye inogadzirisa inopindirana activation [51].Nekudaro, hutachiona hweGiardia hunoitika kazhinji muvakuru vechidiki, uye zvakashumwa kuti hutachiona hweGiardia mumakonzo madiki haitese iyo IL-17A mhinduro kuti iite kudzivirira kwayo [52], zvichiita kuti vaongorori vatsvage imwe immunomodulatory Giardia.nzira dzehutachiona hwe helminth.Vanyori vekuongorora kwechangobva kuitika vakashuma kuti G. muris inogona kushandisa NLRP3 inflammasome neE. coli EPEC, iyo inokurudzira kugadzirwa kwepeptide inorwisa mabhakitiriya uye inoderedza simba rayo rekubatanidza uye nhamba ye trophozoite mumatumbo emukati, nokudaro kuderedza kuoma kwekoloni. zvirwere zvinokonzerwa nebacilli [49].Iyo NLRP3 inflammasome inobatanidzwa mukukura kwezvirwere zvakasiyana-siyana.Zvidzidzo zvakaratidza kuti Pseudomonas aeruginosa inokonzera autophagy mu macrophages kudzivirira kufa kwesero, uye izvi zvinoenderana nekushandiswa kweNLRP3 inflammasome [53].Nokuda kweN. caninum, reactive oxygen species-mediated activation yeNLRP3 inflammasome inogadzirisa kudzokorora kwayo mumusangano, zvichiita kuti ive chinangwa chekurapa [9].Paracoccidioides brasiliensis yakawanikwa ichiita kuti kushandiswa kweNLRP3 inflammasome mu mouse bone marrow-derived dendritic cells, zvichiita kuti kusunungurwa kwe cytokine inoputika IL-1β, iyo inobata basa rinokosha mukudzivirira mauto [10].Mhando dzakawanda dzeLeishmania, dzinosanganisira L. amazonensis, L. chikuru, L. braziliensis, uye L. infantum chagasi, shandisa NLRP3 uye ASC-inotsamira caspase-1 mu macrophages, pamwe neLeishmania utachiona.Kudzokorora kweparasite kunowedzerwa mumakonzo asina kukwana muNLRP3/ASC/caspase-1 gene [11].Zamboni et al.Chirwere cheLeishmania chakanzi chinokonzera kushandiswa kweNLRP3 inflammasome mu macrophages, iyo inogadzirisa intracellular parasite replication.Saka, Leishmania inogona kurambidza NLRP3 activation senzira yekudzivisa.Mune zvidzidzo zve vivo, iyo NLRP3 inflammasome yakabatsira mukubviswa kweLeishmania, asi haina kukanganisa matishu [54].Kusiyana neizvi, mune zvidzidzo zvehelminthiasis, kushandiswa kweNLRP3 inflammasome kwakaderedza kuchengetedzwa kwemuiti wekudzivirira kurwisa gastrointestinal helminthiasis [12].Shigella nderimwe reutachiona hunokonzera manyoka pasi rose.Aya mabhakitiriya anogona kukonzera IL-1β kugadzirwa kuburikidza neP2X7 receptor-mediated K + efflux, reactive oxygen marudzi, lysosomal acidification, uye mitochondrial kukuvara.Iyo NLRP3 inflammasome inodzora zvisina kunaka phagocytosis uye bactericidal basa re macrophages kurwisa Shigella [55].Zvidzidzo zvePlasmodium zvakaratidza kuti AIM2, NLRP3 kana caspase-1 mbeva dzisina kukwana dzakabatwa nePlasmodium dzinobudisa mazinga akakwirira emhando 1 interferon uye inonyanya kurwisana nePlasmodium infection [56].Zvisinei, basa re alpha-2 giardine uye alpha-7.3 giardine mukuita pathogenic activation yeNLRP3 kuzvimba mumakonzo hazvina kujeka.
Muchidzidzo ichi, inhibition yeNLRP3 inflammasome neMCC950 yakaderedza BW uye yakawedzera nhamba ye trophozoites mumatumbo ekuchenesa mvura mumakonzo, zvichiita kuti pave nekuchinja kwakanyanya kwepathological mu duodenal tissue.Alpha-2 giardine uye alpha-7.3 giardine activate the host mbeva NLRP3 inflammasome, kuwedzera mbeva uremu hwemuviri, kuderedza nhamba ye trophozoite mu intestinal lavage fluid, uye kuderedza pathological duodenal maronda.Izvi zvinoratidza kuti G. duodenalis inogona kushandura NLRP3 host inflammasome kuburikidza nealpha-2 giardine uye alpha-7,3 giardine, kuderedza pathogenicity yeG. duodenalis mumakonzo.
Pamwe chete, zvigumisiro zvedu zvinoratidza kuti alpha-2 uye alpha-7.3 giardines inokonzera kushandiswa kweNLRP3 host inflammasome uye kuderedza hutachiona hweG. duodenalis mumakonzo.Naizvozvo, mamorekuru aya ari kuvimbisa zvinangwa zvekudzivirira giardiasis.
       Data supporting the results of this study can be obtained from the respective author at gongpt@jlu.edu.cn.
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