Giardia duodenum ndi tizilombo tating'onoting'ono tomwe timayambitsa giardiasis, matenda am'mimba omwe amapezeka kwambiri mwa ana omwe ali ndi zizindikiro za matenda otsekula m'mimba.Tanena kale kuti extracellular G. duodenalis imayambitsa kuyambitsa kwa intracellular oligomerization-like receptor 3 (NLRP3) yomanga ma nucleotides ndipo imayendetsa mayankho okhudzana ndi kutupa kudzera mu extracellular vesicle (EV) secretion.Komabe, ndondomeko yeniyeni ya maselo a pathogen-associated duodenococcal EV (GEV) yomwe ikukhudzidwa ndi njirayi komanso ntchito ya NLRP3 inflammasome mu giardiasis imakhalabe yodziwika bwino.
Recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 ndi alpha-7.3 giardins mu GEV anamangidwa, kusamutsidwa mu mbewa primary peritoneal macrophages, ndipo anazindikira mwa kuyeza kutupa chandamale molekyulu caspase-1.Mulingo wa mawu a p20 adawonetsedwa..G. duodenalis alpha-2 ndi alpha-7.3 giardines poyamba adadziwika poyesa NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 ndi caspase-1 p20), IL secretion.1β, ma apoptotic spotted protein (ASC) oligomerization levels, ndi immunofluorescent localization ya NLRP3 ndi ASC.Ntchito ya NLRP3 inflammasome mu pathogenicity ya G. duodenalis ndiye inayesedwa pogwiritsa ntchito mbewa zomwe NLRP3 activation inatsekedwa (NLRP3 mbewa zotsekedwa) ndi kusintha kwa pathological kulemera kwa thupi, duodenal parasitic load, ndi duodenal tissue zimayang'aniridwa.Kuonjezera apo, tinafufuza ngati hiardines alpha-2 ndi alpha-7.3 imapangitsa kuti IL-1β ikhale yotsekemera mu vivo kudzera mu NLRP3 inflammasome ndipo tinazindikira udindo wa mamolekyuwa mu pathogenicity ya G. duodenalis mu mbewa.
Alpha-2 ndi alpha-7.3 giardines amachititsa kutsegula kwa NLRP3 inflammasome in vitro.Izi zinayambitsa kuyambitsa kwa p20 caspase-1, kuwonjezeka kwa mawu a NLRP3, pro-IL-1β, ndi pro-caspase-1 mapuloteni, kuwonjezeka kwakukulu kwa IL-1β secretion, kupanga mawanga a ASA mu cytoplasm, ndi kulowetsedwa kwa ASA oligomerization.Kutupa kwa NLRP3 Kutaya kwa mbolo kumawonjezera mphamvu ya G. duodenalis mu mbewa.Makoswe omwe amachiritsidwa ndi cysts ndi gavage kuchokera ku mbewa zotsekedwa za NLRP3 zimasonyeza kuchuluka kwa trophozoites ndi kuwonongeka kwakukulu kwa duodenal villi, yodziwika ndi necrotic crypts yokhala ndi shrunken ndi nthambi.Mayesero a vivo awonetsa kuti giardines alpha-2 ndi alpha-7.3 angapangitse kutsekemera kwa IL-1β kudzera mu NLRP3 inflammasome, ndipo katemera ndi giardines alpha-2 ndi alpha-7.3 anachepetsa matenda a G. duodenalis mu mbewa.
Kutengera pamodzi, zotsatira za phunziroli zimasonyeza kuti giardia alpha-2 ndi alpha-7.3 imayambitsa kutupa kwa NLRP3 ndi kuchepetsa matenda a G. duodenalis mu mbewa, zomwe zimalonjeza zolinga zopewera giardiasis.
Giardia duodenum ndi kachilombo ka protozoan kamene kamakhala m'matumbo aang'ono ndipo kumayambitsa matenda otsegula m'mimba okwana 280 miliyoni pachaka, makamaka pakati pa ana ang'onoang'ono m'mayiko osauka [1].Anthu amatenga kachilomboka ndi madzi akumwa kapena chakudya chokhala ndi zilonda zam'mimba za M. duodenum, zomwe zimalowa m'mimba ndikutuluka mumadzi am'mimba.Giardia duodenum trophozoites amamatira ku duodenal epithelium, kumayambitsa nseru, kusanza, kutsekula m'mimba, kupweteka m'mimba, komanso kuwonda.Anthu omwe ali ndi immunodeficiency ndi cystic fibrosis amatha kutenga matenda.Matendawa amathanso kuchitika kudzera mu kugonana mkamwa ndi kumatako [2].Mankhwala monga metronidazole, tinidazole, ndi nitazoxanide ndi njira zomwe amakonda kwambiri pa matenda a duodenal [3].Komabe, mankhwalawa amayambitsa zotsatira zoyipa monga nseru, carcinogenesis, ndi genotoxicity [4].Choncho, njira zothandiza kwambiri ziyenera kukhazikitsidwa pofuna kupewa matenda a G. duodenalis.
Inflammasomes ndi gulu la mapuloteni a cytosolic omwe ali mbali ya chitetezo cha mthupi, chomwe chimathandiza kuteteza ku tizilombo toyambitsa matenda ndi kuyankhira mayankho otupa [5].Pakati pa inflammasomes, nucleotide-binding oligomerization (NOD) receptor 3 (NLRP3) nucleotide-binding oligomerization (NLRP3) nucleotide-binding-like inflammasome yaphunziridwa mozama chifukwa imatha kuzindikiridwa ndi tizilombo toyambitsa matenda / zowonongeka-zogwirizana ndi mamolekyu / PAMP. DAMP), imazindikira, imayendetsa chitetezo cham'mimba.ndipo imayang'anira matumbo a homeostasis m'matenda ambiri otupa [6,7,8].Amakhala ndi pateni recognition receptor (PRR) NLRP3, adapter apoptotic spotted protein (ASC), ndi effector procaspase-1 kapena procaspase-11.NLRP3 inflammasome imakhala ngati gulu lolimbana ndi kuukira kwa tizilombo toyambitsa matenda, monga tawonera mu Neospora caninum [9], Paracoccidioides brasiliensis [10], ndi maphunziro a Leishmania.[11], koma zanenedwanso kuti kuyambitsa kwa NLRP3 inflammasome kumachepetsa mayankho oteteza chitetezo chamthupi ndikukulitsa kukula kwa matenda, mwachitsanzo, mu nyongolotsi [12].Kutengera zomwe tapeza m'mbuyomu, tidanena kuti ma extracellular G. duodenalis amayambitsa kuyambika kwa intracellular kutupa kwa NLRP3 ndikuwongolera mayankho obwera chifukwa chotupa potulutsa ma extracellular vesicles (EVs) [13].Komabe, ntchito ya NLRP3 inflammasome mu matenda a G. duodenalis mu vivo iyenera kutsimikiziridwa.
Ma Giardins poyambirira adafotokozedwa ngati zigawo za G. duodenalis cytoskeleton ndipo amagwira ntchito yofunika kwambiri pakuyenda kwa trophozoite komanso kulumikizidwa kwa cell m'matumbo aang'ono.Kuti agwirizane bwino ndi chilengedwe ndikuwonjezera matenda awo, G. duodenalis trophozoites anapanga mawonekedwe apadera a cytoskeletal omwe ali ndi 8 flagella, 1 thupi lapakati, ndi 1 ventral disc [14].Ma trophozoites a Giardia duodenum amagwiritsa ntchito cytoskeleton yawo kulowa m'matumbo ang'onoang'ono, makamaka duodenum, ndikuphatikizana ndi ma enterocyte.Amasamuka nthawi zonse ndikumangirira ma cell a epithelial pogwiritsa ntchito cell metabolism.Chifukwa chake, pali ubale wapamtima pakati pa cytoskeleton yawo ndi virulence.Giardines enieni a Giardia duodenum ndi zigawo za cytoskeleton structure [15] ndipo amagawidwa m'magulu anayi: α-, β-, γ-, ndi δ-giardines.Pali mamembala a 21 a banja la α-giardin, onse omwe ali ndi mphamvu yodalira calcium kuti amange phospholipids [16].Amagwirizanitsa cytoskeleton ndi nembanemba ya selo.Kwa anthu omwe ali ndi matenda otsekula m'mimba omwe amayamba chifukwa cha G. duodenalis, α-giardins amawonetsedwa kwambiri komanso amachepetsa chitetezo cha mthupi panthawi ya matenda [17].Katemera wa Heterologous wozikidwa pa Giardia alfa-1 otetezedwa ku giardiasis mu mbewa ndipo ndi ma antigen omwe angathe kupanga katemera [18].Alpha-8 giardin, yomwe imapezeka mu membrane ya plasma ndi flagella, koma osati mu ventral disc, imathandizira motility ndi kukula kwa trophozoites mu G. duodenalis [19].Alpha-14 giardin imamangiriza ku ma microtubule pa flagella ndipo imakhudza mphamvu ya G. duodenalis [20].Alpha-11 giardine ilipo mochuluka m'moyo wonse, ndipo kufotokoza mopambanitsa kwa alpha-11 giardine kumawononga G. duodenalis yokha [21].Komabe, sizikudziwika ngati alpha-2 giardine ndi alpha-7.3 giardine ndi zoteteza ku matenda a G. duodenalis ndi njira zawo zoyambira.
Mu phunziro ili, recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine ndi pcDNA3.1 (+) -alpha-7.3 giardine anasamutsidwa mu mbewa primary peritoneal macrophages kuti yambitsa khamu NLRP3.Zolinga za inflammasome zinafufuzidwa.Tinayesanso ntchito ya NLRP3 inflammasome mu pathogenicity ya G. duodenalis, kufufuza ngati alpha-2 ndi alpha-7,3 giardines amachititsa kuti NLRP3 inflammasome ikhale yogwira ntchito mu vivo, ndipo tinatsimikiza kuti maudindo awiriwa a giardines mu pathogenicity of G. duodenalis.Cholinga chathu chimodzi chinali kupanga zolinga zodalirika zopewera matenda a G. duodenalis.
Mtundu wakutchire (WT) C57BL/6 mbewa zazikazi zazaka zapakati pa 5–8 zinagulidwa ku Liaoning Changsheng Experimental Animal Center (Liaoning, China).Mbewa zinali ndi mwayi wopeza madzi kwaulere, zimalandira chakudya chosawilitsidwa ndipo zimasungidwa mumayendedwe a 12/12 ola / mdima.Asanatenge matenda, mbewa zinalandira maantibayotiki ad libitum m'madzi akumwa owonjezera ampicillin (1 mg/mL), vancomycin (1 mg/mL), ndi neomycin (1.4 mg/mL) (zonse zogulidwa ku Shanghai, China, zamoyo zopanga) [22] ].].Mbewa zomwe zinasiya kudya ndi kumwa kwa > maola a 24 ndikutaya ≥ 20% kulemera kwa thupi kunathandizidwa mwaumunthu ndi kusokonezeka kwa khomo lachiberekero.
WB G. duodenalis trophozoites (American Type Culture Collection, Manassas, USA) adawonjezeredwa ndi 12.5% ​​​​fetal bovine serum (FBS; Every Green, Zhejiang, China) ndi 0.1% bovine bile (Sigma-Aldrich, St. Missouri, USA ).USA) pansi pamikhalidwe ya microaerobic.Ma trophozoite osakanikirana adasonkhanitsidwa pa ayezi ndikudutsa pa chiŵerengero cha 1: 4 kuti aberekenso.
Giardia duodenum cysts adakopeka monga momwe tafotokozera kale [23], trophozoites adakololedwa mu gawo la logarithmic ndiyeno amachepetsedwa ndi encapsulation inducing medium, pH 7.1 (yosinthidwa TYI-S-33) mpaka kumapeto kwa 1 × 106 trophozoites / mL.Kuchuluka kwa bile 0.05% sing'anga).Ma trophozoite adakulitsidwa pansi pamikhalidwe ya anaerobic pa 37 ° C mpaka gawo la kukula kwa logarithmic.Sinthani sing'anga ku cyst inducing sing'anga (pH 7.8; kusinthidwa kwa TYI-S-33 sing'anga ndi 1% bile ndende) ndi chikhalidwe G. duodenalis pa 37 ° C kwa maola 48-96, pamene mapangidwe cysts ankawoneka pansi pa microscope.Ma trophozoite ambiri atakopeka kuti apange cysts, chikhalidwe chosakanikirana chinakololedwa ndikuyimitsidwanso m'madzi osabala omwe amapangidwa kuti asungunuke ma trophozoite otsalawo.Ma cysts adawerengedwa ndikusungidwa pa 4 ° C kuti afufuzidwe motsatira kudzera mu chubu cham'mimba mu mbewa.
Giardia extracellular vesicles (GEVs) adalemeretsedwa monga tafotokozera kale [13].Ma Trophozoites mu gawo la kukula kwa logarithmic adayimitsidwanso mu sing'anga yosinthidwa ya TYI-S-33 yokonzedwa ndi FBS (Biological Industries, Beit-Haemek, Israel) yocheperako mpaka yomaliza ya 1 × 106 parasites/mL ndikuyikidwa kwa maola 12.Anasiyanitsidwa ndi chikhalidwe cha supernatant ndi centrifugation pa 2000 g kwa 10 min, 10,000 g kwa 45 min, ndi 100,000 g kwa 60 min.Madziwo amasungunuka mu mchere wa phosphate buffered saline (PBS), wogwiritsidwa ntchito pogwiritsa ntchito zida zoyesa mapuloteni a BCA (Thermo Fisher Scientific, Waltham, MA, USA) ndi kusungidwa pa -80 ° C. kapena kugwiritsidwa ntchito mwachindunji kuti afufuze.
Ma macrophages oyambira a mbewa adakonzedwa monga tafotokozera kale [24].Mwachidule, mbewa (zaka 6-8 masabata) anabayidwa (intraperitoneally [ip]) ndi 2.5 ml ya 2.98% Difco liquid thioglycol medium (BD, Franklin Lakes, NJ, USA) ndi kudyetsedwa 3-4 palates.Kuyimitsidwa kwa macrophages kunasonkhanitsidwa kuchokera pamimba ya mbewa pambuyo pa euthanasia ndi centrifuged 3 pa 1000 g kwa mphindi 10.Maselo okololedwa adadziwika ndi cytometry yothamanga pogwiritsa ntchito chizindikiro cha CD11b mpaka kuyera kwa selo kunali> 98%, kenaka kuwonjezeredwa ku mbale za 6-well cell culture plates (4.5 x 106 cells/well) ndi kuikidwa ndi 10% FBS (Bioindustry) pa 37 ° C.ndi 5% CO2.
RNA inatulutsidwa kuchokera ku 1 × 107 trophozoites mu 1 ml ya TRIzol reagent (Vazyme, Nanjing, China), genomic DNA inachotsedwa ku G. duodenalis RNA yonse pogwiritsa ntchito MonScript dsDNase (Monad, Wuhan, China) ndi DNA yowonjezera (cDNA) inapangidwa. kugwiritsa ntchito MonScript RTIIII Super Mix (Monad) molingana ndi malangizo a wopanga.
Zambiri za CDS za jini ya G. duodenalis yomwe mukufuna idapezeka kuchokera ku NCBI GenBank.Gwiritsani ntchito Primer 5.0 kuti mupange zoyambira zopanda msoko zamtundu uliwonse womwe mukufuna.Choyambira kutsogolo (5'-3′) chimakhala ndi magawo atatu: kutsatizana kotsatizana kokhala ndi vekitala yoyendera mizere pcDNA3.1(+) EcoRV (TGGTGGAATTCTGCAGAT) ndi kuyambitsa ma codon ATG ndi GNN (ngati maziko oyamba si G).Izi zimachitidwa kuti ziwongolere bwino mawuwo.Kuonjezera apo, osachepera 16 bp ophatikizana maziko (GC okhutira 40-60% / Tm pafupifupi 55 °C).The reverse primer (5′-3′) imakhala ndi magawo awiri, motsatana modutsana ndi EcoRV-linearized vector pcDNA3.1(+) (GCCGCCACTGTGCTGGAT) ndi maziko ophatikiza osachepera 16 bp.(kupatulapo maimidwe awiri omaliza).bases) codon monga AA kapena GA kulola ma plasmids ophatikizana kuti afotokoze mapuloteni awo olembedwa).Magawo oyambawa adalembedwa mu Table 1 ndipo adapangidwa ndi Kangmet Biotechnology Co., Ltd. (Changchun, China).
Zolinga zidakulitsidwa pogwiritsa ntchito Pfu DNA polymerase (Tiangen, Beijing, China) kapena Ex-taq (Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, China) pogwiritsa ntchito G. duodenalis cDNA yokonzedwa ngati template.Eukaryotic expression vector plasmid pcDNA3.1(+) idalumikizidwa ndi enzyme yoletsa EcoRV ndi dephosphorylated pogwiritsa ntchito Fast AP (Thermo Fisher Scientific).Zidutswa za mzere wa pcDNA3.1(+) ndi zidutswa za jini zokulirakulira zidayeretsedwa pogwiritsa ntchito zida zoyeretsera ma gel osakaniza a DNA (Tiangen) ndikuyesedwa pogwiritsa ntchito Nanodrop ND-2000 (Thermo Fisher Scientific).Chidutswa cha pcDNA3.1(+) ndi chidutswa chilichonse cha jini chomwe mukufuna chinaphatikizidwanso pogwiritsa ntchito MonClone single assembly cloning mix (Monad Biotech Co., Ltd., Suzhou, China) ndipo zatsimikiziridwa ndi kutsatizana kwa DNA pogwiritsa ntchito Comate Bioscience Company Limited (Changchun, China) ..
Ma plasmids opanda Endotoxin pcDNA3.1(+)-alpha-2 ndi pcDNA3.1(+)-alpha-7.3 anapangidwa pogwiritsa ntchito SanPrep Endotoxin-free Plasmid Mini Kit (Sangon Biotech).Kukhazikikako kunasungidwa pamwamba pa 500 ng / µl kuti zitsimikizire kuti EDTA mu buffer ya elution sichinasokoneze kuyesa kufalitsa.Primary mouse peritoneal macrophages anakula mu 6-chitsime mbale ndi wathunthu RPMI 1640 sing'anga (Biological Industries) kwa maola 12, ndiye maselo anatsuka 3 mu PBS otentha kuchotsa penicillin ndi streptomycin, ndiyeno sing'anga kuwonjezeredwa ndi sing'anga wathunthu.Ma plasmids opanda Endotoxin pcDNA3.1(+)-alpha-2 ndi pcDNA3.1(+)-alpha-7.3 (2.5 μg) adasungunuka mu 125 μl ya Opti-MEM yochepetsera sing'anga ya seramu (Gibco, Thermo Fisher Scientific)..Kenako 5 µl ya Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific) inachepetsedwa mu 125 µl ya low serum Opti-MEM medium.Konzani ma complex a liposome-DNA posakaniza plasmid yopanda endotoxin yosungunuka ndi Lipofectamine 2000 ndikulola kuti kusakaniza kuyime kutentha kwa mphindi zisanu.Tumizani ma complexes padera ku maselo mu chitsime chilichonse ndikusakaniza pang'onopang'ono.Pambuyo pa maola 4, sing'anga ya chikhalidwe cha cell idasinthidwa ndi 2 ml yathunthu ya RPMI 1640 sing'anga ndipo chikhalidwe chinapitilizidwa kwa maola 24.Ma cell achikhalidwe chatsopano adawonjezedwa m'maselo ndikuwunikiridwa kwa nthawi zosiyanasiyana kutengera kapangidwe kake.
Zitsanzo zamapuloteni zochokera ku supernatants ndi ma cell lysates zidakonzedwa monga tafotokozera kale [25].Miyezo yotumizira ma membrane ya pro-IL-1β, pro-caspase-1, caspase-1 p20, NLRP3, β-actin, ndi His-tag inali 200 mA / 90 min.Kwa interleukin 1β (IL-1β; R&D Systems, Minneapolis, Minnesota, USA), caspase-1 (p20) (Adipogen, Switzerland) ndi NLRP3 (Adipogen SA, Epalinges, Switzerland) ndi 1:5000 yolunjika His tag ( Amylet Scientific, Wuhan, China) ndi β-actin (Proteintech, Wuhan, China).
Kulumikizana ndi disuccinimide subrate (DSS) kunachitika monga tafotokozera kale [26].Maselo amatsukidwa maulendo atatu ndi PBS yozizira ndikuyamwa kwathunthu ndi singano ya 27 mu 50 µl ASC reaction buffer (pH 8.0) yokhala ndi 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES ndi 125 mM NaHCO3.Chosakanizacho chinapangidwa ndi centrifuged pa 5000 g kwa 3 min ndipo pellet idapangidwa ndi 10 µl DSS (25 mM mu DMSO) ndi 40 µl ASC reaction buffer kwa 30 min pa 37 ° C.Pambuyo pa centrifugation pa 5000 g kwa mphindi 10, pellet idasungunuka mu njira ya 40 µl ya ASC reaction buffer ndi 10 µl ya 6x protein loading buffer (TransGen, Beijing, China), ndiyeno yankholo linazimitsidwa kutentha kwa firiji kwa 15. min., Ndiye wiritsani 10 Mphindi.Zitsanzo zamapuloteni zidayikidwa ku Western blotting pogwiritsa ntchito ma anti-ASC antibodies (Wanleibio, Shenyang, China) pa dilution ratio ya 1:500.
Potsatira ndondomeko yomwe yafotokozedwa kale [13], opambana a chikhalidwe cha maselo adakololedwa ndipo kutulutsidwa kwa pro-inflammatory cytokine IL-1β kunatsimikiziridwa pogwiritsa ntchito mbewa IL-1 Beta ELISA kit (Invitrogen, Thermo Fisher Scientific).Sinthani ma OD450nm kukhala ma protein ambiri pogwiritsa ntchito IL-1β yokhazikika.
Maselo ophimbidwa pa zophimba amatsukidwa mofatsa nthawi za 3 mu PBS yotentha, yokhazikika mu minofu yokhazikika (Biosharp, Beijing, China) kwa mphindi 10 kutentha kwa firiji (RT), mu 0.1% Triton X-Permeabilize pa 100 (kuchepetsedwa mu PBS; Biosharp ) kwa mphindi 20 kutentha kwa firiji ndikutchinga mu 5% bovine serum albumin (mu PBS) kwa maola awiri kutentha.Maselo adayikidwa usiku wonse pa 4 ° C ndi ma antibodies oyambirira otsutsana ndi ASC (1: 100 dilution) kapena NLRP3 (1: 100 dilution), motero, ndi mbuzi yotchedwa Cy3 yotchedwa anti-rabbit IgG (H + L) (1: 400; EarthOx) , San Francisco, CA, USA) kapena FITC-conjugated goat anti-mouse IgG (1:400; Earthox) usiku wonse pa 37°C mumdima kwa ola limodzi.Ma nuclei anali odetsedwa ndi Hoechst 33258 (10 μg/ml; UE, Suzhou, China) kwa mphindi 5 ndipo amawonedwa pansi pa microscope ya fluorescence (Olympus Corporation, Tokyo, Japan).
Mbewa zinagawidwa m'magulu anayi (n = 7 mu gulu lirilonse): (i) PBS-treated negative control gulu (PBS yokha; gavage 100 µl / mouse PBS yotsatiridwa ndi jekeseni wa intraperitoneal tsiku lililonse 100 µl / mouse PBS 3 maola pambuyo pake)., mosalekeza kwa masiku 7);(ii) gulu lowongolera loyipa lomwe limathandizidwa ndi MCC950 inhibitor [27] (100 µl / mbewa kudzera pa PBS gavage, maola a 3 pambuyo pake, 10 mg / kg kulemera kwa thupi [BW] MCC950 [mu PBS] inkaperekedwa intraperitoneally tsiku ndi tsiku, nthawi ya 7 masiku);+(iv) G. duodenalis chotupa kuphatikiza matenda gulu MCC950 inhibitor mankhwala gulu (1.5 × 106 cysts/mbewa kudzera gavage, 10mg/kg kulemera kwa thupi MCC950 intraperitoneally tsiku 7 masiku 3h).Kulemera kwa thupi la mbewa iliyonse kumayang'aniridwa tsiku ndi tsiku ndipo mbewa zonse zinatulutsidwa pa tsiku la 7th.Duodenum yokolola (utali wa 3 cm) inadulidwa mu zidutswa zing'onozing'ono mu 1 ml PBS, ma cysts anawonongedwa usiku wonse mu PBS pa 4 ° C, ndi G. duodenalis trophozoites.Duodenum yatsopano (utali wa 1 cm) idapatulidwa kuti iwononge hematoxylin ndi eosin (H&E).
Makoswe adagawidwa m'magulu awiri: (i) gulu lolamulira la MOCK ndi (ii) gulu la inhibitor la MCC950.Panali mankhwala asanu mu gulu lirilonse (n = 7 / gulu la mankhwala): (i) PBS mankhwala osokoneza bongo (PBS okha; 100 µl / mouse PBS, intramuscular (IM) jekeseni (tibialis anterior) [28, 29]; ( ii) pcDNA3.1(+) plasmid negative control group (100 µg/mouse DNA, via intramuscular jekeseni) (iii) G. duodenalis cyst infections positive control group (1.5 x 106 cysts/mbewa, via gavage) (iv) a gulu lomwe limathandizidwa ndi plasmid pcDNA3.1 (+) -alpha-2 (100 μg/mbewa DNA, ndi jakisoni wa muscular), ndi (v) gulu lomwe limathandizidwa ndi plasmid pcDNA3.1 (+) -alpha-7.3 (100 µg/mbewa) DNA, pambuyo pa maola a 12, mbewa mu gulu la MCC950 inhibitor linalandira jekeseni wa intraperitoneal wa MCC950 (10 mg / kg kulemera kwa thupi) kwa masiku 7, pamene mbewa za gulu la MOCK zinalandira chithandizo chofanana cha PBS. Zitsanzo za magazi zinali anasonkhanitsidwa kuchokera ku mbewa za maso ndipo anasiya usiku wonse pa 4 ° C Zitsanzo za seramu zidalekanitsidwa pogwiritsa ntchito enzyme-linked immunosorbent assay (ELISA) ndi miyeso ya IL-1β.
Makoswe makumi atatu ndi asanu adagawidwa m'magulu asanu (n = 7 / gulu).Gulu 1 linali gulu lowongolera loyipa lomwe limathandizidwa ndi PBS: mbewa zidalandira 100 μl ya PBS intramuscularly ndi 3 patatha masiku atatu ndi gavage.Gulu 2 ndi gulu lolamulira labwino lomwe lili ndi G. duodenalis cysts: mbewa zinayikidwa ndi 100 μl ya PBS, ndipo patatha masiku 3 1.5 x 106 cysts / mbewa intragastrically.Gulu lachitatu - katemera wa plasmid ndi pcDNA3.1 (+) pamodzi ndi gulu lolamulira la matenda a duodenal cysts: mbewa zinalandira 100 μg ya plasmid DNA pcDNA3.1 (+) (im) pamlomo, 1.5 × 106 cysts / mbewa 3 kwa angapo. masiku.Magulu 4 ndi 5 anali recombinant pcDNA3.1 (+) -alpha-2 giardine plasmid kapena pcDNA3.1 (+) -alpha-7.3 giardine plasmid pamodzi ndi G. duodenalis chotupa matenda.Gulu loyesera: mbewa zinalandira 100 µg ya pcDNA3.1.Kulemera kwa thupi la mbewa iliyonse kumayang'aniridwa pambuyo poyambitsa G. duodenalis chotupa kudzera mu chubu.Duodenum yatsopano idasonkhanitsidwa poyezera kuchuluka kwa parasitic ndikuwunika madontho a HE.
Kusintha kwa histopathological kudawunikidwa malinga ndi njira yomwe idasindikizidwa kale [30].Duodenum yatsopano idakhazikitsidwa ndi ma cell cell fixative, ophatikizidwa mu parafini, odulidwa mu magawo a 4 μm, odetsedwa ndi H&E ndikuwunikidwa pansi pa microscope.Kusintha koyimilira kwapathological m'magawo asanu ndi awiri a minofu kuchokera ku mbewa zisanu ndi ziwiri zodziyimira pawokha adawunikidwa ndi katswiri wazachipatala osadziwa chithandizocho ndipo adagwidwa pakukula kwa 200x.Kutalika kwa villi ndi kuya kwa crypts kunayesedwa motsatira njira zomwe zafotokozedwa kale.
Zotsatira za mu vitro ndi mu vivo zidapezeka katatu.Zithunzi zidapangidwa pogwiritsa ntchito GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, USA).Kusiyanitsa pakati pa magulu awiri kunayesedwa ndi t-test, pamene kusiyana pakati pa ≥3 magulu adawunikidwa ndi njira imodzi yowunikira kusiyana (ANOVA) pogwiritsa ntchito mapulogalamu a SPSS (mtundu 22.0; SPSS IBM Corp., Armonk, NY, USA).Deta idawunikidwa kuti iwonetse kusiyanasiyana kwamitundu yosiyanasiyana pogwiritsa ntchito mayeso a Levene otsatiridwa ndi mayeso a post hoc a Bonferroni (B).Kufunika kumawonetsedwa ngati P<0.05, P<0.01, ndi P<0.001 (osafunikira [ns]) (P>0.05).
Kusanthula kwathu koyambirira kwa GEV proteomics mu Kyoto Encyclopedia of Genes and Genomes (KEGG) kunawonetsa kuti zolinga zambiri zitha kuphatikizidwa pakuyambitsa njira zowonetsera zotupa [13].Tinasankha zolinga ziwiri zolonjeza, alpha-2 ndi alpha-7.3 giardins, kukulitsa mamolekyuwa ndikuwagwiritsa ntchito popanga pcDNA3.1 (+) eukaryotic expression vector.Pambuyo potsatizana, recombinant pcDNA3.1 (+) -alpha-2 ndi alpha-7.3 giardine expression plasmids adasamutsidwa kukhala macrophages oyambira a mbewa, ndipo caspase-1 p20 siginecha ya protein ya kutupa (chidutswa cha caspase-1) idadziwika. monga kufotokoza mamolekyu ofunika omwe angayambitse kutupa.Zotsatira zinawonetsa kuti alpha-2 ndi alpha-7.3 giardines angapangitse p20 caspase-1 mawu ofanana ndi GEV.Palibe zotsatira za caspase-1 activation yomwe inapezedwa mu ulamuliro wosasamalidwa bwino (PBS okha) ndi plasmid control pcDNA3.1 (+) (Chithunzi 1).
Kuyeza kwa p20 caspase-1 activation ndi pcDNA3.1 (+) -alpha-2 ndi alpha-7.3 giardins.Ma recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 ndi alpha-7.3 giardines (pamwamba pa msewu uliwonse) adasamutsidwa kukhala macrophages oyambira a mbewa a peritoneal macrophages ndi chikhalidwe chapamwamba adakololedwa patatha maola 24.Western blotting idagwiritsidwa ntchito kuyeza milingo ya siginecha ya caspase-1 p20 inflammasome protein.Gulu lothandizira la PBS (lane C) ndi pcDNA3.1 (+) monotherapy gulu (pcDNA3.1 lane) linagwiritsidwa ntchito ngati njira yolakwika, ndipo gulu la mankhwala la GEV linagwiritsidwa ntchito ngati njira yabwino.Kufotokozera kwa mapuloteni ophatikizananso kunatsimikiziridwa pozindikira chizindikiro cha histidine mu puloteni iliyonse, ndipo magulu a mapuloteni omwe amayembekezeredwa anali alpha-2 giardine (38.2 kDa) ndi alpha-7.3 giardine (37.2 kDa).GEV, Giardia duodenum extracellular vesicles, pcDNA3.1(+), EcoRV-linearized vector, SUP, supernatant
Kuti mudziwe ngati alpha-2 giardine ndi alpha-7.3 giardine amalimbikitsa p20 caspase-1 mawu ndikuthandizira kuyambitsa kuyankha kotupa kwa NLRP3, pcDNA3.1 (+) -alpha-2 giardine ndi pcDNA3.1 (+) -alpha -7.3 giardin adasamutsidwa ku macrophages oyambirira a mbewa a peritoneal ndi recombinant plasmid DNA, ndi milingo ya mawu, malo, ndi oligomerization ya mapuloteni ofunika kwambiri otupa a NLRP3 anatsimikiziridwa.Pakuyesa uku, GEV idagwiritsidwa ntchito ngati gulu lolamulira labwino, ndipo gulu lopanda chithandizo (PBS kokha) kapena pcDNA3.1 (+) gulu lochizira matenda opatsirana linali gulu loyipa.Zotsatira zinasonyeza kuti, monga mu gulu la GEV, recombinant plasmid DNA ya giardin pcDNA3.1 (+) -alpha-2 ndi giardin pcDNA3.1 (+) -alpha-7.3 inachititsa kuti NLRP3, pro-IL-1β ndi procaspase-1 ndi caspase-1 kutsegula (mkuyu 2a).Kuphatikiza apo, ma giardines onse adapangitsa kuti IL-1β itulutsidwe (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) (Chithunzi 2b).Mapuloteni ambiri a ASC anali monomeric mu gulu lopanda chithandizo kapena gulu lachipatala lomwe linasinthidwa ndi pcDNA3.1 (+) plasmid, mosiyana ndi pcDNA3.1 (+) -alpha-2 kapena pcDNA3.1 (+) -alpha- 7.3 giardine.ASC oligomerization inachitika mu recombinant plasmid DNA ya GEV positive control gulu kapena gulu, kusonyeza oligomeric mawonekedwe (Chithunzi 2c).Deta yoyambirirayi imasonyeza kuti alpha-2 giardine ndi alpha-7,3 giardine angapangitse NLRP3 kuyambitsa kuyambitsa kutupa.Kafukufuku wotsatira wa immunofluorescent wa kukhazikitsidwa kwa ASC ndi NLRP3 adawonetsa kuti mu gulu lowongolera loyipa, mapuloteni a ASC adamwazika mu cytoplasm ndipo adawoneka ngati chizindikiro cha dontho pakukondoweza kwa pcDNA3.1 (+) -alpha-2 ndi giardine kapena pcDNA3.1 (+) -alpha-7,3 giardine gulu kapena GEV gulu lolamulira labwino (Chithunzi 2d).M'magulu olakwika a pcDNA 3.1 omwe amathandizidwa ndi plasmid, chizindikiro cha mapuloteni a NLRP3 sichinawoneke, pamene dothi la chizindikiro cha fluorescent poyankha pcDNA3.1 (+) -alpha-2 giardine kapena pcDNA3.1 (+) -alpha-7.3 adadziwika..giardine amapezeka mu cytoplasm kapena pa kukondoweza kwa HEV (mkuyu 2e).Deta iyi ikuwonetsanso kuti G. duodenalis giardin alpha-2 ndi giardin alpha-7.3 imayambitsa NLRP3 inflammasome mu mouse primary peritoneal macrophages.
pcDNA3.1(+)-alpha-2 giardin ndi pcDNA3.1(+)-alpha-7.3 giardin yambitsa NLRP3 inflammasome mu mouse peritoneal macrophages.Sinthani recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardin ndi pcDNA3.1(+)-alpha-7.3 giardin mu primary murine peritoneal macrophages ndi maselo, kapena kukolola supernatant mkati 24 h kusanthula mawu, oligomerization , chinsinsi.ndi kukhazikitsidwa kwa mapuloteni ofunikira kwambiri.Gulu la PBS-okha (C) ndi pcDNA3.1 (+) gulu limodzi la mankhwala linagwiritsidwa ntchito ngati kulamulira koipa, ndipo gulu la mankhwala a GEV linagwiritsidwa ntchito ngati gulu labwino.Mapuloteni ofunika kwambiri a NLRP3, kuphatikizapo NLRP3, pro-IL-1β, pro-caspase-1, ndi p20 caspase-1, adadziwika ndi Western blotting.b Miyezo ya secretion ya IL-1β m'ma supernatants adatsimikiziridwa pogwiritsa ntchito enzyme-linked immunosorbent assay (ELISA).Kusiyana pakati pa magulu olamulira ndi oyesera adawunikidwa ndi njira imodzi yowunikira kusiyana (ANOVA) pogwiritsa ntchito pulogalamu ya SPSS 22.0.Nyenyezi zimasonyeza kusiyana kwakukulu pakati pa magulu **P<0.01 ndi ***P<0.001.c Miyezo ya oligomerization ya ASC m'ma pellets idatsimikiziridwa ndi kusanthula kolumikizana kwa DSS, pomwe milingo ya ASC mu ma cell lysates idagwiritsidwa ntchito ngati kuwongolera.d Kuwona kwa ISC komweko pogwiritsa ntchito immunofluorescence.e Immunofluorescence idagwiritsidwa ntchito kuwonetsa kukhazikika kwa NLRP3.ASC, mapuloteni a apoptotic-ngati mawanga;IL, interleukin;NLRP3, nucleotide-binding oligomerization-like receptor 3;ns, osafunikira (P> 0.05)
Onse a G. duodenalis ndi GEVs omwe amawatulutsa amayambitsa NLRP3 inflammasome ndikuwongolera mayankho okhudzana ndi kutupa mu vitro.Choncho, ntchito ya NLRP3 inflammasome mu pathogenicity ya G. duodenalis imakhalabe yosadziwika.Kuti tifufuze nkhaniyi, tinapanga kuyesa pakati pa mbewa zomwe zili ndi G. duodenalis cyst ndi mbewa zomwe zili ndi G. duodenalis cyst + MCC950 inhibitor chithandizo ndikuyerekeza NLRP3 inflammasome expression ikagwidwa ndi G. duodenalis cyst.Chiwembu chatsatanetsatane cha kuyesa chikuwonetsedwa mumkuyu 3a.Kusintha kwa kulemera kwa mbewa m'magulu osiyanasiyana ochizira kunayang'aniridwa kwa masiku 7 pambuyo pa matenda a cysts, ndipo zotsatira zikuwonetsedwa mkuyu 3b.Poyerekeza ndi gulu lomwe linachitidwa ndi PBS yoyera, zotsatira zake zinasonyeza kuti (i) kulemera kwa mbewa zomwe zili ndi G. duodenalis cysts zinachepa kuyambira tsiku la 3 mpaka 7 pambuyo pa matenda;(ii) Kuchiza ndi MCC950 inhibitor sikunakhudze kwambiri kulemera kwa mbewa..Poyerekeza ndi gulu limodzi la matenda, BW wa gulu la matenda a duodenal omwe amathandizidwa ndi MCC950 adatsika mpaka madigiri osiyanasiyana (Tsiku 1: ANOVA, F (3, 24) = 1.885, P = 0.0148; Tsiku 2: ANOVA, F (3, 24) ) = 0.4602, P<0.0001; Tsiku 3: ANOVA, F(3, 24) = 0.8360, P = 0.0010; Tsiku 4: ANOVA, F(3, 24) = 1.683, P = 0.0052; (3, 24)=0.6497, P=0.0645; Tsiku 6: ANOVA, F(3, 24)=5.457, P=0.0175; Tsiku 7: ANOVA, F(3, 24) = 2.893, P = 0.0202).Deta iyi imasonyeza kuti NLRP3 inflammasome imateteza mbewa kuti zisawonongeke kwambiri kumayambiriro (masiku 2-4) a matenda a duodenal.Kenako tinkafuna kudziwa G. duodenalis trophozoites mu duodenal lavage fluid ndipo zotsatira zake zikuwonetsedwa mu Chithunzi 3c.Poyerekeza ndi gulu la G. duodenalis cyst infections, chiwerengero cha trophozoites mu duodenum chinawonjezeka kwambiri pambuyo poletsa NLRP3 inflammasome (t (12) = 2.902, P = 0.0133).Mitsempha ya Duodenal yodetsedwa ndi HE ikuwonetsa, poyerekeza ndi kuwongolera koyipa komwe kumachitidwa ndi PBS ndi MCC950 yokha: (i) G. ) ndi crypt atrophy (ANOVA, F (3, 24) = 0.4716, P = 0.0089);(ii) duodenum kuchokera ku mbewa zomwe zili ndi G. duodenalis cysts ndikuchiritsidwa ndi MCC950 inhibitors.duodenal villi inawonongeka ndi kufa (ANOVA, F (3, 24) = 0.4903, P = 0.0144) ndi atrophy ndi crypt branching (ANOVA, F (3, 24) = 0.4716, P = 0, 0481) (Mkuyu 3d- f).Zotsatirazi zikusonyeza kuti NLRP3 inflammasome imathandizira kuchepetsa G. duodenalis.
Udindo wa NLRP3 inflammasome mu matenda a Giardia duodenum.Mbewa zinaphwanyidwa (iv) ndi ma duodenococcal cysts kenaka kuthandizidwa ndi kapena popanda MCC950 (ip).Magulu ochiritsira amodzi omwe ali ndi PBS kapena MCC950 adagwiritsidwa ntchito ngati zowongolera.Gulu loyesera ndi dongosolo lamankhwala.b Kulemera kwa thupi la mbewa m'magulu osiyanasiyana a mankhwala ankayang'aniridwa kwa masiku 7.Kusiyana pakati pa gulu la matenda a G. duodenalis ndi G. duodenalis + MCC950 gulu lochiza matenda linawunikidwa ndi t-mayeso pogwiritsa ntchito SPSS software version 22.0.Nyenyezi zimasonyeza kusiyana kwakukulu pa *P<0.05, **P<0.01, kapena ***P<0.001.c Parasitic katundu anatsimikiza powerenga chiwerengero cha trophozoite mu duodenal lavage madzimadzi.Kusiyana pakati pa gulu la matenda a G. duodenalis ndi G. duodenalis + MCC950 gulu lochiza matenda linawunikidwa ndi t-mayeso pogwiritsa ntchito SPSS software version 22.0.Nyenyezi zimawonetsa kusiyana kwakukulu pa *P <0.05.d Hematoxylin ndi eosin (H&E) zotsatira zoyipa za duodenal histopathology.Mivi yofiira imasonyeza kuwonongeka kwa villi, mivi yobiriwira imasonyeza kuwonongeka kwa crypts.Scale bar: 100 µm.e, f Kusanthula kwachiwerengero cha duodenal villus kutalika ndi kutalika kwa mbewa ya mbewa.Nyenyezi zimasonyeza kusiyana kwakukulu pa *P<0.05 ndi **P<0.01.Zotsatira zatengedwa kuchokera ku zoyeserera 7 zodziyimira pawokha zachilengedwe.BW, kulemera kwa thupi;ig, njira yoperekera intragastric;ip, njira yoperekera intraperitoneal;ns, osafunikira (P> 0.05);PBS, phosphate buffered saline;WT, mtundu wakutchire
Kutsekemera kwa IL-1β ndi chizindikiro cha kuyambitsa kutupa.Kuti tidziwe ngati G. duodenalis alpha-2 giardine ndi alpha-7.3 giardine yambitsa NLRP3 host inflammasome mu vivo, tinagwiritsa ntchito mbewa za WT zosagwiritsidwa ntchito (sham group) ndi NLRP3 inflammasome-blocked mbewa (MCC950 inhibited treatment group).Chiwembu chatsatanetsatane cha kuyesa chikuwonetsedwa mumkuyu 4a.Magulu oyesera anali ndi mbewa zothandizidwa ndi PBS, G. duodenalis cyst treatment ndi gavage, intramuscular injection ya pcDNA3.1, ndi intramuscular injection ya pcDNA3.1 (+) -alpha-2 giardine kapena pcDNA3.1-alpha-7.3 giardine.Pa tsiku la 7 pambuyo poyendetsa intramuscular of recombinant plasmid, seramu inasonkhanitsidwa ndipo mlingo wa IL-1β mu gulu lirilonse unatsimikiziridwa.Monga momwe tawonetsera mu Chithunzi 4b, mu gulu la MOCK: (i) poyerekeza ndi gulu la PBS, chithandizo cha pcDNA3.1 sichinakhudze kwambiri IL-1β secretion (ANOVA, F (4.29) = 4.062, P = 0.9998), komabe, Kutsekemera kwa IL-β kunakwezedwa kwambiri mu gulu la G. duodenalis cyst (ANOVA, F (4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine ndi pcDNA3.1- jakisoni wa intramuscular wa alpha-7.3 giardine adachulukitsa kwambiri seramu IL-1β (ANOVA, F (4, 29) = 4.062, P <0.0001);(iii) pcDNA3.1-alpha-7,3 giardine inachititsa kuchuluka kwa IL -1β secretion mu pcDNA3.1-alpha-2 giardine intramuscular injection group (ANOVA, F (4, 29) = 4.062, P = 0.0333) .Poyerekeza ndi gulu lirilonse mu gulu la mankhwala la MCC950 ndi gulu la MOCK: (i) IL-1β secretion levels mu gulu lolamulira la PBS ndi gulu lolamulira la pcDNA3.1 linachepa mpaka pang'ono pambuyo poletsa MCC950 inhibitor, koma kusiyana sikunali zofunika (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F (9, 58) = 3.540, P = 0.5949);(ii) atatsekereza MCC950., Kutsekemera kwa IL-1β kunachepetsedwa kwambiri mu gulu la G. duodenalis cyst-infected, pcDNA3.1-alpha-2 giardine gulu, ndi pcDNA3.1-alpha-7.3 giardine gulu (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).Zotsatirazi zikusonyeza kuti alpha-2 giardine ndi alpha-7.3 giardine amagwirizanitsa kuyambitsa kwa NLRP3 inflammasome mu vivo.
pcDNA3.1 (+) -giardines yambitsa NLRP3 host inflammasome mu vivo.Mbewa zinapatsidwa katemera (IM) ndi recombinant eukaryotic expression plasmid pcDNA3.1 (+)-alpha-2 giardine kapena pcDNA3.1 (+) -alpha-7.3 giardine kenako anachiritsidwa ndi MCC950 (ip; MCC950 gulu) kapena ayi (dummy group) ).Gulu la mankhwala a PBS kapena pcDNA3.1 (+) la plasmid linagwiritsidwa ntchito ngati kulamulira koipa, gulu la mankhwala a G. duodenalis cysts linagwiritsidwa ntchito ngati chiwongolero chabwino.Gulu loyesera ndi dongosolo lamankhwala.b Miyezo ya seramu ya IL-1β mu mbewa inayesedwa pa tsiku la 7 ndi kuyesa kwa ELISA.Kusiyana pakati pa magulu a gulu la MOCK kudawunikidwa pogwiritsa ntchito njira imodzi ya ANOVA, ndipo kusiyana pakati pa gulu la MOCK ndi gulu la MCC950 kudawunikidwa pogwiritsa ntchito t-test ya SPSS software version 22.0.Nyenyezi zimasonyeza kusiyana kwakukulu pakati pa magulu a chithandizo mu gulu la MOCK, * P <0.05 ndi *** P <0.001;zizindikiro za dollar ($) zimasonyeza kusiyana kwakukulu pakati pa gulu lirilonse mu gulu la MOCK ndi gulu la MCC950 pa P<0.05.Zotsatira za mayeso asanu ndi awiri odziyimira pawokha achilengedwe.i, jakisoni wa intramuscular, ns, osafunikira (P> 0.05)
Kuti tifufuze zotsatira za alpha-2 ndi alpha-7.3 giardine-mediated activation ya NLRP3 host inflammasome pa G. duodenalis infectivity, tinagwiritsa ntchito WT C57BL / 6 mbewa ndi jekeseni alpha-2 giardine ndi alpha-7.3 giardine.plasmid intramuscularly intramuscularly, pambuyo pa masiku atatu kupyolera mu chubu chapamimba cha G. duodenalis cyst, kenako mbewa zinawonedwa kwa masiku 7.Chiwembu chatsatanetsatane cha kuyesa chikuwonetsedwa mumkuyu 5a.Kulemera kwa thupi la mbewa iliyonse kumayesedwa tsiku ndi tsiku, zitsanzo za minofu yatsopano ya mmimba inasonkhanitsidwa pa tsiku la 7 pambuyo poyang'anira kudzera mu chubu chamimba, chiwerengero cha trophozoite chinayesedwa, ndipo kusintha kwa histopathological kunawonedwa.Monga momwe tawonetsera pa Chithunzi 5b, ndi nthawi yowonjezereka yodyetsa, BW ya mbewa pagulu lililonse inakula pang'onopang'ono.MT ya mbewa inayamba kuchepa pa tsiku la 3 pambuyo pa intragastric administration ya G. duodenalis cysts, ndipo pang'onopang'ono inakula.Kutsegula kwa NLRP3 inflammasome yoyambitsidwa ndi jekeseni wa intramuscular wa alpha-2 giardine ndi alpha7.3 giardine kwambiri kuchepetsa kulemera kwa mbewa (Tsiku 1: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 1.399, P = 0 .9754 Tsiku 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30)=1.399, P=0.9987 Tsiku 2: pcDNA3.1-alpha-2 giardine, ANOVA, F( 4, 30) = 0.3172, P = 0.9979; Tsiku 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.3172, P = 0.8409; Tsiku 3: pcDNA3.1-alpha-2 giardine, ANOVA 4, 30) = 0.8222, P = 0.0262 Tsiku 3: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.8222, P = 0.0083; Tsiku 4: pcDNA3.1-alpha-2 , F(4, 30) = 0.5620, P = 0.0012, Tsiku 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5620, P <0.0001, Tsiku 5: pcDNA3.1-alpha - 2 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Tsiku 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Tsiku 6. -1 pcDNA alpha-2 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0012, Tsiku 6: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0006;Tsiku 7: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.5369, P<0.0001 Tsiku 7: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5369, P <0.0001).The parasitic katundu anayesedwa mu duodenum (mkuyu. 5c).Poyerekeza ndi kayendetsedwe kabwino kopanda chithandizo ndi gulu lojambulidwa ndi pcDNA3.1 vector yopanda kanthu, chiwerengero cha G. duodenalis trophozoites chinachepetsedwa kwambiri m'magulu omwe amabayidwa ndi α-2 giardine ndi 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).Kuphatikiza apo, giardine alfa-7.3 inali yoteteza kwambiri mbewa kuposa giardine alfa-2 (ANOVA, F (3, 24) = 1.209, P = 0.0081).Zotsatira za HE staining zikuwonetsedwa mkuyu.5d–f.Mbewa zojambulidwa ndi alpha-2 giardine ndi alpha-7.3 giardine zinali ndi zotupa zochepa zamtundu wa duodenal, zomwe zimawonetsedwa ndi kuwonongeka kwa villus, poyerekeza ndi mbewa zojambulidwa ndi G. duodenalis ndi mbewa zojambulidwa ndi G. duodenalis kuphatikiza ndi pcDNA3 vector yopanda kanthu .1 Zoom.(pcDNA3.1-alpha-2 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0035 kapena P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0028 kapena P = 0.0055) ndi kuchepetsa crypt atrophy (pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 1.470, P = 0.0264 kapena P = 0.0158; pcDNA3.1-alpha-7.3 ANOVA , F(3, 24) = 1.470, P = 0.0371 kapena P = 0.0191).Zotsatirazi zimasonyeza kuti alpha-2 giardine ndi alpha-7,3 giardine amachepetsa kudwala kwa G. duodenalis mwa kuyambitsa NLRP3 inflammasome mu vivo.
Udindo wa pcDNA3.1 (+) -giardins mu matenda a G. duodenalis.Mbewa zinapatsidwa katemera (IM) ndi recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine kapena pcDNA3.1 (+) -alpha-7.3 giardine ndipo kenako anatsutsidwa ndi G. duodenalis cysts (ig).Gulu la PBS ndi pcDNA3.1 (+) + gulu la chithandizo cha duodenal cysts linagwiritsidwa ntchito ngati magulu olamulira oipa, ndipo gulu la chithandizo cha duodenal cyst linagwiritsidwa ntchito ngati gulu lolamulira labwino.Gulu loyesera ndi dongosolo lamankhwala.b The MT ya mbewa m'magulu osiyanasiyana a mankhwala ankayang'aniridwa kwa masiku a 7 pambuyo pa zovuta.Nyenyezi zimasonyeza kusiyana kwakukulu pakati pa magulu a G. duodenalis gulu ndi pcDNA3.1 (+) -alpha-2 giardine gulu, * P <0.05, ** P <0.01, ndi *** P <0.001;chizindikiro cha dollar ($) chimasonyeza kusiyana kwakukulu pakati pa gulu lililonse la G. duodenalis ndi pcDNA3.1 (+)-alpha-7.3 jardine gulu, $$P<0.01 ndi $$$P<0.001.c Parasitic katundu anatsimikiza ndi kuwerengera chiwerengero cha trophozoites mu 1 ml ya duodenal lavage ku duodenum (3 masentimita yaitali) ndi anasonyeza monga chiwerengero cha tiziromboti pa masentimita a duodenum.Kusiyanitsa pakati pa gulu la G. duodenalis matenda, pcDNA3.1 (+) -alpha-2 giardine gulu, ndi pcDNA3.1 (+) -alpha-7.3 giardine gulu linafufuzidwa ndi njira imodzi ANOVA pogwiritsa ntchito SPSS software version 22.0.Nyenyezi zimasonyeza kusiyana kwakukulu pa **P <0.01 ndi ***P <0.001.d Kusintha kwa histopathological mu duodenum.Mivi yofiira imasonyeza kuwonongeka kwa villi, mivi yobiriwira imasonyeza kuwonongeka kwa crypts.Scale bar: 100 µm.e, f Kusanthula kwa mbewa duodenal villus kutalika (e) ndi kutalika kwa crypt (f).Kusiyana pakati pa magulu mu Chithunzi 1d kudawunikidwa ndi njira imodzi ya ANOVA pogwiritsa ntchito pulogalamu ya SPSS 22.0.Nyenyezi zimasonyeza kusiyana kwakukulu pa *P<0.05 ndi **P<0.01.Zotsatira za mayeso asanu ndi awiri odziyimira pawokha achilengedwe.ns, osafunikira (P> 0.05)
Giardia duodenum ndi tizilombo tating'onoting'ono ta anthu ndi zinyama zina zomwe zimayambitsa giardiasis.Mu 2004, idaphatikizidwa mu bungwe la WHO Neglected Diseases Initiative chifukwa cha kufalikira kwakukulu kwa zaka 6, makamaka m'madera omwe ali ndi chikhalidwe chochepa cha chikhalidwe cha anthu [32].Chitetezo chobadwa nacho chimagwira ntchito yofunika kwambiri pakuyankhira kwa G. duodenalis.Akuti macrophages a mbewa ameza ndi kupha G. duodenalis potulutsa misampha ya extracellular [33].Kafukufuku wathu wam'mbuyomu wasonyeza kuti G. duodenalis, tizilombo toyambitsa matenda tomwe timapanga tizilombo toyambitsa matenda, timayambitsa p38 MAPK, ERK, NF-κB p65, ndi njira zowonetsera zotupa za NLRP3 mu macrophages a mbewa kuti athetsere mayankho okhudzana ndi kutupa, ndipo kutulutsidwa kwa GEV kungapangitse njirayi.[Chithunzi patsamba 13], 24].Komabe, ma PAMPs enieni omwe akukhudzidwa ndi kutupa kwa NLRP3 yokhudzana ndi inflammasome mu GEV ndi udindo wa NLRP3 inflammasome mu giardiasis akuyenera kufotokozedwa.Kuti timvetse bwino mafunso awiriwa, tinachititsa phunziroli.
NLRP3 inflammasome ili mu cytoplasm ya maselo a chitetezo cha mthupi ndipo imatha kuyendetsedwa ndi tinthu tating'ono tosiyanasiyana monga makristasi a uric acid, poizoni, mabakiteriya, mavairasi, ndi tizilombo toyambitsa matenda.M'maphunziro a bakiteriya, poizoni adadziwika ngati PAMPs zazikulu zomwe zimayambitsa zotupa zotupa, zomwe zimapangitsa kutupa ndi kufa kwa cell [34].Ma poizoni ena osiyanasiyana, monga hemolysin kuchokera ku Staphylococcus aureus [35] ndi Escherichia coli [36], hemolysin BL (HBL) kuchokera ku enterotoxin (NHE) [37], imayambitsa kuyambitsa kwa NLRP3 kutupa.Kafukufuku wa ma virus awonetsa kuti ma virulence proteins monga SARS-COV-2 envelope (E) protein [38] ndi Zika virus NS5 protein [39] ndi ma PAMPs ofunikira omwe amadziwika ndi NLRP3 receptor.M'maphunziro a tiziromboti, majeremusi ambiri amanenedwa kuti amalumikizidwa ndi kuyambitsa kwa inflammasome, monga Toxoplasma gondii, Trichomonas vaginalis [40], Trypanosoma cruzi [41], ndi Leishmania [42].Mapuloteni ochuluka a granule GRA35, GRA42, ndi GRA43, okhudzana ndi virulence ya Toxoplasma gondii, amafunikira kuti alowetse pyroptosis mu Lewis rat macrophages [43].Kuphatikiza apo, maphunziro ena a Leishmania amayang'ana kwambiri mamolekyu omwe amakhudzidwa ndi NLRP3 inflammasome, monga parasite membrane lipophosphoglycan [44] kapena zinc metalloprotease [45].Pakati pa annexin-monga alpha-giardin banja la majini, alpha-1 giardin yasonyezedwa kuti ikhoza kukhala katemera wotetezera ku G. duodenalis mu chitsanzo cha mbewa [18].Mu phunziro lathu, tinasankha G. duodenalis virulence factor alpha-2 ndi alpha-7,3 giardines, zomwe zimakhala zosiyana ndi giardia koma ndizochepa.Majini awiriwa adapangidwa mu pcDNA3.1 (+) eukaryotic expression system vector kuti afufuze momwe kutupa kumayambira.
Muchitsanzo chathu cha mbewa, zidutswa za caspase zong'ambika zimakhala ngati zizindikiro zoyambitsa kutupa.Polimbikitsa, NLRP3 imagwirizana ndi ASC, imalemba ma procaspases, ndikupanga ma caspases omwe amadula pro-IL-1β ndi pro-IL-18 kukhala okhwima IL-1β ndi IL-18, motero -18.Ma caspases otupa (caspases-1, -4, -5 ndi -11) ndi banja lotetezedwa la ma cysteine ​​​​proteases omwe ndi ofunikira kwambiri pachitetezo cha chibadwa ndipo amakhudzidwa ndi kutupa komanso kufa kwa cell [46].Caspase-1 imayendetsedwa ndi canonical inflammasomes [47], pamene caspases-4, -5, ndi -11 imadulidwa panthawi yopanga inflammasomes ya atypical [48].Mu phunziro ili, tinagwiritsa ntchito mouse peritoneal macrophages monga chitsanzo ndi kufufuza p20 caspase-1 cleaved caspase-1 monga chizindikiro cha host NLRP3 kutupa activation mu maphunziro a G. duodenalis matenda.Zotsatira zake zidawonetsa kuti ma alpha-giardins ambiri ndi omwe amachititsa kuti kutupa, komwe kumagwirizana ndi kupezeka kwa mamolekyu ofunikira omwe amakhudzidwa ndi mabakiteriya ndi ma virus.Komabe, phunziro lathu ndi chithunzi choyambirira chokha ndipo pali mamolekyu ena omwe amatha kuyambitsa ma inflammasomes omwe sali achikulire, monga momwe kafukufuku wathu wam'mbuyomu adapeza ma inflammasomes amtundu wa G. duodenalis [13].Kuti tidziwenso ngati p20 caspase-1 yopangidwa ikugwirizana ndi NLRP3 inflammasome, tidasamutsira ma alpha-2 ndi alpha-7.3 giardins mu mbewa peritoneal macrophages kuti tidziwe kuchuluka kwa mapuloteni a molekyulu ndi ASC oligomerization, kutsimikizira kuti ma α-giardins onse amagwira ntchito. zotupa NLRP3.Zotsatira zathu ndizosiyana pang'ono ndi za Manko-Prykhoda et al., omwe adanena kuti kusonkhezera kwa maselo a Caco-2 okhala ndi G. muris kapena E. coli EPEC zovuta zokha zimatha kuwonjezera mphamvu ya fluorescence ya NLRP3, ASC, ndi caspase-1, ngakhale osati kwambiri, pamene mtengo wa G. muris ndi E. coli unawonjezera kuchuluka kwa mapuloteni atatu [49].Kusiyana kumeneku kungakhale chifukwa cha kusiyana kwa kusankha kwa mitundu ya Giardia, mizere ya maselo, ndi maselo oyambirira.Tinapanganso mu vivo assays pogwiritsa ntchito MCC950 mu mbewa zazikazi za WT C57BL/6 za masabata 5, zomwe zimakhala zosavuta ku G. duodenalis.MCC950 ndi yamphamvu komanso yosankha kamolekyu yaying'ono ya NLRP3 inhibitor yomwe imatchinga kutsegulidwa kwa canonical ndi non-canonical NLRP3 pamagulu a nanomolar.MCC950 imalepheretsa kuyambitsa kwa NLRP3 koma sizikhudza kuyambitsa kwa AIM2, NLRC4, ndi njira zotupa za NLRP1 kapena njira zowonetsera TLR [27].MCC950 imaletsa kuyambitsa kwa NLRP3 koma sikuletsa kuyambitsa kwa NLRP3, K+ efflux, kuchuluka kwa Ca2+, kapena kuyanjana pakati pa NLRP3 ndi ASC;m'malo mwake, imalepheretsa kuyambitsa kwa NLRP3 inflammasome poletsa oligomerization ya ASC [27].Choncho, tinagwiritsa ntchito MCC950 mu phunziro la vivo kuti tidziwe udindo wa NLRP3 inflammasome pambuyo pa jekeseni wa giardine.Activated caspase-1 p10 imadula ma cytokines oyambitsa-kutupa pro-IL-1β ndi pro-IL-18 kukhala okhwima IL-1β ndi IL-18 [50].Mu phunziro ili, ma seramu a IL-1β mu mbewa zowonongeka ndi giardine kapena opanda MCC950 anagwiritsidwa ntchito ngati chizindikiro chosonyeza ngati NLRP3 inflammasome inatsegulidwa.Monga momwe zimayembekezeredwa, chithandizo cha MCC950 chinachepetsa kwambiri ma seramu a IL-1β.Deta iyi ikuwonetseratu kuti G. duodenalis giardin alfa-2 ndi giardin alfa-7.3 amatha kuyambitsa NLRP3 mouse inflammasome.
Zambiri zomwe zasonkhanitsidwa m'zaka khumi zapitazi zawonetsa kuti IL-17A ndiye woyang'anira chitetezo chamthupi motsutsana ndi G. muris, kuchititsa IL-17RA chizindikiro, kupanga ma peptides antimicrobial, ndikuwongolera kuthandizira kowonjezera [51].Komabe, matenda a Giardia amapezeka kawirikawiri mwa achinyamata, ndipo zanenedwa kuti matenda a Giardia mu mbewa zazing'ono sayambitsa yankho la IL-17A kuti likhale ndi chitetezo chake [52], zomwe zimachititsa ofufuza kuti ayang'ane Giardia ina yoteteza thupi.njira za matenda a helminth.Olemba kafukufuku waposachedwapa adanena kuti G. muris akhoza kuyambitsa NLRP3 inflammasome ndi E. coli EPEC, yomwe imalimbikitsa kupanga ma peptides oletsa tizilombo toyambitsa matenda komanso kuchepetsa mphamvu yake yophatikizira ndi chiwerengero cha trophozoites m'matumbo a m'mimba, motero kuchepetsa kuopsa kwa colon. matenda oyambitsidwa ndi bacilli [49].NLRP3 inflammasome imakhudzidwa ndi chitukuko cha matenda osiyanasiyana.Kafukufuku wasonyeza kuti Pseudomonas aeruginosa imayambitsa autophagy mu macrophages kuti apewe kufa kwa selo, ndipo izi zimadalira kuyambitsa kwa NLRP3 inflammasome [53].Kwa N. caninum, kuyambitsa kwamtundu wa oxygen-mediated wa NLRP3 inflammasome kumachepetsa kubwereza kwake m'chipindacho, zomwe zimapangitsa kuti zikhale chithandizo chamankhwala [9].Paracoccidioides brasiliensis yapezeka kuti imayambitsa kuyambitsa kwa NLRP3 inflammasome m'maselo a dendritic omwe amapangidwa ndi mbewa, zomwe zimapangitsa kuti cytokine IL-1β itulutsidwe, yomwe imagwira ntchito yofunika kwambiri poteteza asilikali [10].Mitundu ingapo ya Leishmania, kuphatikizapo L. amazonensis, L. yaikulu, L. braziliensis, ndi L. infantum chagasi, yambitsani NLRP3 ndi ASC-dependent caspase-1 mu macrophages, komanso matenda a Leishmania.Kubwereza kwa parasite kumakulitsidwa mu mbewa zoperewera mu jini ya NLRP3/ASC/caspase-1 [11].Zamboni et al.Matenda a Leishmania adanenedwa kuti amapangitsa kuti NLRP3 inflammasome ikhale mu macrophages, yomwe imalepheretsa kubwereza kwa tizilombo toyambitsa matenda.Chifukwa chake, Leishmania ikhoza kuletsa kuyambitsa kwa NLRP3 ngati njira yopewera.Mu maphunziro a vivo, NLRP3 inflammasome inathandizira kuthetsa Leishmania, koma sizinakhudze minofu [54].Mosiyana ndi zimenezi, mu maphunziro a helminthiasis, kutsegula kwa NLRP3 inflammasome kunalepheretsa chitetezo cha chitetezo cha m'mimba helminthiasis [12].Shigella ndi amodzi mwa mabakiteriya omwe amayambitsa matenda otsekula m'mimba padziko lonse lapansi.Mabakiteriyawa amatha kuyambitsa kupanga IL-1β kudzera mu P2X7 receptor-mediated K + efflux, mitundu yokhazikika ya okosijeni, lysosomal acidification, ndi kuwonongeka kwa mitochondrial.NLRP3 inflammasome imayendetsa molakwika phagocytosis ndi bactericidal ntchito ya macrophages motsutsana ndi Shigella [55].Kafukufuku wa Plasmodium wasonyeza kuti AIM2, NLRP3 kapena caspase-1 mbewa zoperewera zomwe zili ndi Plasmodium zimapanga mitundu yambiri ya interferon yamtundu wa 1 ndipo zimagonjetsedwa kwambiri ndi matenda a Plasmodium [56].Komabe, ntchito ya alpha-2 giardine ndi alpha-7.3 giardine poyambitsa kuyambitsa kwa NLRP3 kutupa kwa mbewa sikudziwika bwino.
Mu kafukufukuyu, kuletsa kwa NLRP3 inflammasome ndi MCC950 kunachepetsa BW ndikuwonjezera kuchuluka kwa ma trophozoite m'matumbo otsuka madzi mu mbewa, zomwe zimapangitsa kuti pakhale kusintha kwakukulu kwa matenda amtundu wa duodenal.Alpha-2 giardine ndi alpha-7.3 giardine yambitsa mbewa NLRP3 inflammasome, kuwonjezera mbewa kulemera kwa thupi, kuchepetsa chiwerengero cha trophozoites mu intestinal lavage madzimadzi, ndi kuchepetsa pathological duodenal zotupa.Zotsatirazi zimasonyeza kuti G. duodenalis ikhoza kuyambitsa NLRP3 host inflammasome kudzera alpha-2 giardine ndi alpha-7,3 giardine, kuchepetsa matenda a G. duodenalis mu mbewa.
Pamodzi, zotsatira zathu zimasonyeza kuti alpha-2 ndi alpha-7.3 giardines imayambitsa kuyambitsa kwa NLRP3 host inflammasome ndi kuchepetsa matenda a G. duodenalis mu mbewa.Chifukwa chake, mamolekyuwa akulonjeza zolinga zopewera giardiasis.
       Data supporting the results of this study can be obtained from the respective author at gongpt@jlu.edu.cn.
Liang AKS, Liang AAM, Huang AHC, Sergi KM, Kam JKM.Giardiasis: mwachidule.Posachedwapa zidawululidwa kuti Pat Inflamm ndi wosagwirizana ndi mankhwala.2019; 13:134–43.
Escobedo AA, Tsimerman S. Giardiasis: ndemanga ya pharmacotherapy.Malingaliro a Katswiri a pharmacist.2007;8: 1885-902.
Tian Huafeng, Chen Bin, Wen Jianfeng.Giardiasis, kukana mankhwala ndi kupezeka kwa zolinga zatsopano.Imawononga zolinga za mankhwala a Disord.2010; 10:295–302.
Wang Z, Zhang X, Xiao Yi, Zhang W, Wu X, Qin T, etc. NLRP3 inflammasome ndi matenda opatsirana.Oxide Med Cell Longev.2020;2020:4063562.
Chen GY, Núñez G. Udindo wa inflammasome mu kutupa kwamatumbo ndi khansa.Gastroenterology.2011;141:1986–99 .
Pellegrini C, Antonioli L, Lopez-Castejon G, Blandizzi C, Fornai M. Canonical ndi atypical NLRP3 inflammasome activation pamphambano za kulekerera kwa chitetezo cha mthupi ndi kutupa kwamatumbo.chitetezo chamthupi.2017; 8:36.
Li L, Wang XC, Gong PT, Zhang N, Zhang X, Li S, et al.ROS-mediated NLRP3 inflammasome activation ikukhudzidwa ndi matenda a N. caninum.Vector ya parasite.2020; 13:449.