Giardia duodenum wata kwayar cuta ce mai saurin kamuwa da cuta wacce ke haifar da giardiasis, kamuwa da cutar hanji musamman ga yara kanana da alamun cutar gudawa.Mun riga mun bayar da rahoton cewa extracellular G. duodenalis yana haifar da kunnawa na oligomerization na intracellular oligomerization-kamar mai karɓa na 3 (NLRP3) mai ɗaure nucleotides kuma yana daidaita martani mai kumburi ta hanyar ɓoyewar ƙwayoyin cuta (EV).Duk da haka, ainihin ma'anar kwayoyin halitta na duodenococcal duodenococcal EV (GEV) da ke da alaƙa a cikin wannan tsari da kuma rawar da NLRP3 mai kumburi a cikin giardiasis ya kasance a bayyane.
Recombinant eukaryotic magana plasmids pcDNA3.1 (+) -alpha-2 da alpha-7.3 giardins a cikin GEV an gina su, an canza su zuwa linzamin kwamfuta na farko na peritoneal macrophages, kuma an gano su ta hanyar auna ma'auni na kumburin kwayoyin caspase-1.An duba matakin magana p20..G. duodenalis alpha-2 da alpha-7.3 giardines an samo asali ne ta hanyar aunawa NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 da caspase-1 p20), ɓoyewar IL.Matakan 1β, furotin da aka gano na apoptotic (ASC) matakan oligomerization, da kuma ƙaddamar da immunofluorescent na NLRP3 da ASC.Matsayin NLRP3 inflammasome a cikin ƙwayar cuta na G. duodenalis an yi la'akari da shi ta hanyar amfani da mice wanda aka katange NLRP3 kunnawa (NLRP3 blocked mice) da kuma canje-canje na pathological a cikin nauyin jiki, nauyin parasitic duodenal, da duodenal nama an kula da su.Bugu da ƙari, mun bincika ko hiaardines alpha-2 da alpha-7.3 suna haifar da ɓoyewar IL-1β a cikin vivo ta hanyar NLRP3 inflammasome kuma ya ƙayyade rawar da waɗannan kwayoyin halitta ke cikin kwayar cutar G. duodenalis a cikin mice.
Alpha-2 da alpha-7.3 giardines suna haifar da kunnawa na NLRP3 inflammasome a cikin vitro.Wannan ya haifar da kunna p20 caspase-1, karuwa a cikin matakan magana na NLRP3, pro-IL-1β, da kuma pro-caspase-1 sunadaran, karuwa mai yawa a cikin ɓoyewar IL-1β, samuwar ASA spots a cikin cytoplasm, da kuma shigar da ASA oligomerization.NLRP3 kumburi hasara na azzakari ya kara tsananta yanayin G. duodenalis a cikin mice.Mice da aka bi da su tare da cysts ta hanyar gavage daga NLRP3-katangar mice sun nuna karuwar adadin trophozoites da mummunar lalacewa ga duodenal villi, wanda ke da alamun necrotic crypts tare da shrunken da reshe.A cikin gwaje-gwajen vivo sun nuna cewa giardines alpha-2 da alpha-7.3 na iya haifar da ɓoyewar IL-1β ta hanyar NLRP3 inflammasome, kuma rigakafi tare da giardines alpha-2 da alpha-7.3 sun rage rashin lafiyar G. duodenalis a cikin mice.
A hade tare, sakamakon wannan binciken ya nuna cewa giardia alpha-2 da alpha-7.3 suna haifar da haɓakar kumburin NLRP3 mai masaukin baki da kuma rage kamuwa da cutar G. duodenalis a cikin mice, wanda ke da alamar da aka yi alkawarin hana giardiasis.
Giardia duodenum wani parasite ne na protozoan na waje wanda ke rayuwa a cikin ƙananan hanji kuma yana haifar da cututtukan giardiasis miliyan 280 a kowace shekara, musamman a tsakanin yara ƙanana a kasashe masu tasowa [1].Mutane suna kamuwa da cutar ta hanyar ruwan sha ko abinci da suka gurɓace da M. duodenum cysts, daga nan sai su shiga ciki kuma a fitar da su a cikin ruwan ciki.Giardia duodenum trophozoites suna haɗe zuwa ga duodenal epithelium, haifar da tashin zuciya, amai, zawo, ciwon ciki, da asarar nauyi.Mutanen da ke da ƙarancin rigakafi da cystic fibrosis suna iya kamuwa da kamuwa da cuta.Kamuwa da cuta kuma na iya faruwa ta hanyar jima'i ta baka da ta dubura [2].Magunguna irin su metronidazole, tinidazole, da nitazoxanide sune zaɓuɓɓukan magani da aka fi so don cututtukan duodenal [3].Koyaya, waɗannan magungunan chemotherapy suna haifar da mummunan sakamako kamar tashin zuciya, carcinogenesis, da genotoxicity [4].Don haka, ana buƙatar samar da dabaru masu inganci don hana kamuwa da cutar G. duodenalis.
Inflammasomes wani nau'i ne na rukunin furotin cytosolic waɗanda ke cikin ɓangaren amsawar rigakafi na asali, suna taimakawa wajen kare kai daga mamayewar ƙwayoyin cuta da daidaita martanin kumburi [5].Daga cikin waɗannan ƙumburi, nucleotide-binding oligomerization (NOD) mai karɓa 3 (NLRP3) nucleotide-binding oligomerization (NLRP3) nucleotide-binding-like inflammasome an yi nazari sosai saboda ana iya gano shi ta hanyoyi daban-daban na kwayoyin cuta / lalacewa-haɗe-haɗe (PAMP/ DAMP), ya gane, yana kunna tsarin rigakafi na asali.kuma yana daidaita homeostasis na hanji a yawancin cututtuka masu kumburi [6,7,8].Ya ƙunshi mai karɓar ƙirar ƙira (PRR) NLRP3, adaftar furotin da aka hange (ASC), da procaspase-1 ko procaspase-11.Rashin kumburin NLRP3 yana aiki azaman mai watsa shiri akan mamayewar pathogen, kamar yadda aka lura a cikin Neospora caninum [9], Paracoccidioides brasiliensis [10], da kuma karatun Leishmania.[11].Dangane da bincikenmu na baya, mun bayar da rahoton cewa G. duodenalis na waje yana haifar da kunna intracellular kunnawa na NLRP3 kumburi kuma yana daidaita martani mai kumburi ta hanyar ɓoye vesicles na waje (EVs) [13].Duk da haka, aikin NLRP3 mai kumburi a cikin G. duodenalis kamuwa da cuta a cikin vivo ya kasance don ƙayyade.
Giardins an kwatanta su ne a matsayin kayan aikin tsarin G. duodenalis cytoskeleton kuma suna taka muhimmiyar rawa a cikin motsi na trophozoite da kuma haɗin sel na epithelial a cikin ƙananan hanji.Don mafi dacewa da yanayin da kuma ƙara yawan ƙwayoyin cuta, G. duodenalis trophozoites sun haɓaka tsarin cytoskeletal na musamman wanda ya ƙunshi 8 flagella, 1 tsakiyar jiki, da 1 ventral disc [14].trophozoites na Giardia duodenum suna amfani da cytoskeleton don shiga cikin ƙananan hanji na sama, musamman duodenum, kuma suna haɗuwa da enterocytes.Suna yin ƙaura akai-akai kuma suna haɗe zuwa sel epithelial ta amfani da ƙwayar sel.Saboda haka, akwai dangantaka ta kud da kud tsakanin cytoskeleton da virulence.Giardines na musamman don Giardia duodenum sune sassan tsarin cytoskeleton [15] kuma an raba su zuwa aji hudu: α-, β-, γ-, da δ-giardines.Akwai mambobi 21 na dangin α-giardin, dukansu suna da ikon dogaro da calcium don ɗaure phospholipids [16].Suna kuma haɗa cytoskeleton zuwa membrane cell.A cikin mutanen da ke fama da gudawa ta hanyar G. duodenalis, α-giardins suna bayyana sosai da kuma rigakafi yayin kamuwa da cuta [17].Alurar rigakafin da aka danganta da Giardia alfa-1 an kare su daga giardiasis a cikin mice kuma sune yuwuwar antigens na ɗan takara don haɓaka rigakafin [18].Alpha-8 giardin, wanda aka sanya shi a cikin membrane na plasma da flagella, amma ba a cikin diski na ventral ba, yana haɓaka motsi da girma na trophozoites a cikin G. duodenalis [19].Alpha-14 giardin yana haɗawa da tsarin microtubule akan flagella kuma yana rinjayar yiwuwar G. duodenalis [20].Alpha-11 giardine yana samuwa a cikin yalwace a duk tsawon rayuwar rayuwa, kuma wuce gona da iri na alpha-11 giardine yana lalata G. duodenalis kanta [21].Duk da haka, ba a sani ba ko alpha-2 giardine da alpha-7.3 giardine suna da kariya daga kamuwa da G. duodenalis da kuma hanyoyin da suke da su.
A cikin wannan binciken, recombinant eukaryotic magana plasmids pcDNA3.1 (+) -alpha-2 giardine da pcDNA3.1 (+) -alpha-7.3 giardine an canza su zuwa linzamin kwamfuta na farko na peritoneal macrophages don kunna mai watsa shiri NLRP3.Daga nan an duba abubuwan da ke da kumburi.Mun kuma kimanta muhimmancin NLRP3 inflammasome a cikin pathogenicity na G. duodenalis, bincika ko alpha-2 da alpha-7,3 giardines haifar da kunnawa na NLRP3 inflammasome a cikin vivo, da kuma ƙaddara cewa wadannan biyu ayyuka na giardines a cikin pathogenicity. G. duodenal.Manufarmu ta gama gari ita ce haɓaka maƙasudai masu ban sha'awa don rigakafin kamuwa da G. duodenalis.
Nau'in daji (WT) C57BL/6 berayen mata masu shekaru 5-8 makonni an siyi su daga Cibiyar Gwajin Dabbobi ta Liaoning Changsheng (Liaoning, China).Beraye suna da damar samun ruwa kyauta, sun sami abinci mai haifuwa kuma an kiyaye su a cikin haske / zagayowar duhu na awa 12/12.Kafin kamuwa da cuta, mice sun karɓi maganin rigakafi ad libitum a cikin ruwan sha wanda aka haɓaka tare da ampicillin (1 mg / mL), vancomycin (1 mg / mL), da neomycin (1.4 mg / mL) (duk wanda aka saya daga Shanghai, China, ƙwayoyin wucin gadi) [22] ].].Berayen da suka rasa ikon ci da sha na tsawon awanni 24 kuma suka rasa ≥ 20% nauyin jiki sun mutu ta hanyar tarwatsewar mahaifa.
WB G. duodenalis trophozoites (Tarin Al'adu na Nau'in Amurka, Manassas, Amurka) an ƙara su da kashi 12.5% ​​na ƙwayar ƙwayar tayi (FBS; Kowane Green, Zhejiang, China) da 0.1% bile na bovine (Sigma-Aldrich, St. Missouri, Amurka) ).Amurka) ƙarƙashin yanayin microaerobic.An tattara trophozoites masu haɗuwa akan kankara kuma an wuce su a cikin rabo na 1: 4 don ƙarin haifuwa.
Giardia duodenum cysts an jawo su kamar yadda aka bayyana a baya [23], trophozoites an girbe a logarithmic lokaci sa'an nan kuma diluted tare da encapsulation inducing matsakaici, pH 7.1 (gyara TYI-S-33) zuwa wani karshe taro na 1 × 106 trophozoites / mL.bile maida hankali 0.05% matsakaici).An yi al'adar Trophozoites a ƙarƙashin yanayin anaerobic a 37 ° C har zuwa lokacin haɓaka logarithmic.Canja matsakaici zuwa cyst inducing matsakaici (pH 7.8; modified TYI-S-33 matsakaici tare da 1% bile maida hankali) da al'adu G. duodenalis a 37 ° C for 48-96 hours, a lokacin da samuwar cysts aka lura a karkashin wani microscope.Bayan yawancin trophozoites an jawo su don samar da cysts, an girbe cakuda al'ada kuma an sake dawo da su a cikin ruwa mai tsabta don lyse sauran trophozoites.An ƙidaya ƙwayoyin cysts kuma an adana su a 4 ° C don bincike na gaba ta hanyar bututun ciki a cikin mice.
Giardia extracellular vesicles (GEVs) an wadata su kamar yadda aka bayyana a baya [13].Trophozoites a cikin lokacin girma na logarithmic an sake dakatar da su a cikin matsakaicin TYI-S-33 da aka gyara tare da ƙarancin ƙarancin FBS (Masana'antun Halittu, Beit-Haemek, Isra'ila) zuwa ƙaddamarwar ƙarshe na 1 × 106 parasites / mL kuma an haɗa shi don 12 hours.An ware su daga al'adar al'ada ta hanyar centrifugation a 2000 g na minti 10, 10,000 g na 45 min, da 100,000 g na 60 min.An narkar da hazo a cikin phosphate buffered saline (PBS), wanda aka ƙididdige ta amfani da kit ɗin gwajin furotin na BCA (Thermo Fisher Scientific, Waltham, MA, Amurka) kuma an adana shi a -80 ° C. ko amfani da shi kai tsaye don ƙarin nazari.
An shirya macrophages na peritoneal na farko kamar yadda aka bayyana a baya [24].A taƙaice, an yi allurar beraye (masu shekaru 6-8 makonni) (intrapereritoneally [ip]) tare da 2.5 ml na 2.98% Difco ruwa thioglycol matsakaici (BD, Franklin Lakes, NJ, Amurka) kuma an ciyar da 3-4 palates.An tattara dakatarwar macrophages daga kogon ciki na mice bayan euthanasia kuma an sanya shi sau 3 a 1000 g na 10 min.An gano ƙwayoyin da aka girbe ta hanyar cytometry mai gudana ta amfani da alamar CD11b har sai da tsabtar tantanin halitta> 98%, sannan an ƙara su zuwa faranti na al'adun tantanin halitta 6-da kyau (sel 4.5 x 106 / rijiyar) kuma an haɗa su tare da 10% FBS (Bioindustry) a 37 ° C.da kuma 5% CO2.
An fitar da RNA daga 1 × 107 trophozoites a cikin 1 ml na TRIzol reagent (Vazyme, Nanjing, China), an fitar da DNA na genomic daga jimlar G. duodenalis RNA ta amfani da MonScript dsDNase (Monad, Wuhan, China) kuma an haɗa DNA na gaba (cDNA) ta amfani da MonScript RTIIII Super Mix (Monad) bisa ga umarnin masana'anta.
An samo bayanan jerin CDS don manufa G. duodenalis gene daga NCBI GenBank.Yi amfani da Primer 5.0 don ƙirƙira ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙwayar cuta don kowane nau'in ƙwayar cuta.Na gaba firamare (5'-3′) ya ƙunshi sassa uku: wani jeri jere tare da linearized vector pcDNA3.1 (+) EcoRV (TGGTGGAATTCTGCAGAT) da kuma fara codons ATG da GNN (idan na farko tushe ba G).Ana yin haka ne don inganta ingantaccen magana.Bugu da ƙari, aƙalla 16 bp haɗin tushe (abin ciki na GC 40-60% / Tm kimanin 55 ° C).Reverse primer (5′-3′) ya ƙunshi sassa biyu, jerin jeri tare da EcoRV-linearized vector pcDNA3.1(+) (GCCGCCACTGTGCTGAT) da haɗin tushe na aƙalla 16 bp.(ban da tasha biyu na ƙarshe).tushe) codon kamar AA ko GA don ba da damar sake haɗawa da plasmids don bayyana sunadaran sunadaran su).An jera jerin abubuwan farko a cikin Tebur 1 kuma Kangmet Biotechnology Co., Ltd. (Changchun, China) ne ya hada su.
An haɓaka maƙasudi ta amfani da Pfu DNA polymerase (Tiangen, Beijing, China) ko Ex-taq (Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, China) ta amfani da G. duodenalis cDNA da aka shirya azaman samfuri.Maganar eukaryotic vector plasmid pcDNA3.1(+) an daidaita shi tare da ƙuntatawa enzyme EcoRV da dephosphorylated ta amfani da Fast AP (Thermo Fisher Scientific).An tsarkake ɓangarorin pcDNA3.1 (+) mai layi da ƙaƙƙarfan ɓangarorin ƙwayoyin cuta ta amfani da kayan aikin tsarkakewa na DNA (Tiangen) kuma an ƙididdige su ta amfani da Nanodrop ND-2000 (Thermo Fisher Scientific).An sake haɗa guntuwar pcDNA3.1 (+) da kowane gutsuttsarin kwayar halittar da aka yi niyya ta amfani da MonClone single Assembly cloning mix (Monad Biotech Co., Ltd., Suzhou, China) kuma an tabbatar da shi ta hanyar jerin DNA ta hanyar amfani da Kamfanin Comate Bioscience Company Limited (Changchun, China) ..
pcDNA3.1 (+) -alpha-2 da pcDNA3.1 (+) -alpha-7.3 an samar da su ta amfani da SanPrep Endotoxin-free Plasmid Mini Kit (Sangon Biotech).An kiyaye maida hankali sama da 500 ng/µl don tabbatar da cewa EDTA a cikin buffer elution bai tsoma baki tare da gwajin canzawa ba.An haɓaka macrophages na farko na linzamin kwamfuta a cikin faranti 6-riji tare da cikakken RPMI 1640 matsakaici (Masana'antun Halittu) na tsawon sa'o'i 12, sa'an nan kuma an wanke sel sau 3 a cikin PBS mai dumi don cire penicillin da streptomycin, sannan a cikin matsakaici tare da cikakken matsakaici.Endotoxin-free plasmids pcDNA3.1 (+) -alpha-2 da pcDNA3.1 (+) -alpha-7.3 (2.5 μg) an diluted a cikin 125 μl na Opti-MEM rage matsakaicin matsakaici (Gibco, Thermo Fisher Scientific) ..Sa'an nan 5 µl na Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific) aka diluted a cikin 125 µl na low serum Opti-MEM matsakaici.Shirya rukunin liposome-DNA ta hanyar haɗa plasmid da ba shi da ƙarancin endotoxin tare da Lipofectamine 2000 da barin cakuda ya tsaya a zafin jiki na mintuna 5.Canja wurin hadaddun daban zuwa sel a kowace rijiya kuma a gauraya a hankali.Bayan sa'o'i 4, an maye gurbin matsakaicin al'adun tantanin halitta tare da 2 ml na cikakken RPMI 1640 matsakaici kuma an ci gaba da al'ada har tsawon sa'o'i 24.An ƙara matsakaicin al'adar sabbin ƙwayoyin sel a cikin sel kuma an sanya su don wuraren lokaci daban-daban dangane da ƙirar ƙima.
An shirya samfurorin furotin daga supernatants da cell lysates kamar yadda aka bayyana a baya [25].Siffofin canja wurin Membrane don pro-IL-1β, pro-caspase-1, caspase-1 p20, NLRP3, β-actin, da alamar sa sune 200 mA/90 min.Don interleukin 1β (IL-1β; R&D Systems, Minneapolis, Minnesota, Amurka), caspase-1 (p20) (Adipogen, Switzerland) da NLRP3 (Adipogen SA, Epalinges, Switzerland) da 1: 5000 da ke niyya ta alamar sa (Amylet Scientific, Wuhan, China) da β-actin (Proteintech, Wuhan, China).
An yi haɗe-haɗe tare da disuccinimide suberate (DSS) kamar yadda aka bayyana a baya [26].An wanke sel sau 3 tare da PBS mai sanyi kuma an lulluɓe su gaba ɗaya tare da allurar ma'auni na 27 a cikin 50 µl ASC reaction buffer (pH 8.0) mai ɗauke da 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES da 125 mM NaHCO3.An yi amfani da cakuda a 5000 g na 3 min kuma an dinka pellet tare da 10 µl DSS (25 mM a cikin DMSO) da 40 µl ASC amsawa na 30 min a 37 ° C.Bayan centrifugation a 5000 g na 10 min, pellet an narkar da a cikin wani bayani na 40 µl na ASC dauki buffer da 10 µl na 6x furotin loading buffer (TransGen, Beijing, China), sa'an nan da bayani da aka quenched a dakin zafin jiki na 15. min., Sannan tafasa minti 10.Sa'an nan kuma an yi amfani da samfurorin sunadaran suna zubar da jini ta Yammacin Turai ta hanyar amfani da magungunan rigakafi na farko na anti-ASC (Wanleibio, Shenyang, China) a cikin rabon dilution na 1:500.
Bayan hanyar da aka bayyana a baya [13], an girbe ma'auni na al'adun tantanin halitta kuma an ƙaddara asirin cytokine IL-1β mai kumburi ta amfani da linzamin kwamfuta IL-1 Beta ELISA kit (Invitrogen, Thermo Fisher Scientific).Mayar da ƙimar OD450nm zuwa adadin furotin ta amfani da ma'auni na IL-1β.
Kwayoyin da aka lullube su a hankali an wanke su sau 3 a cikin PBS mai dumi, an gyara su a cikin gyaran ƙwayar nama (Biosharp, Beijing, China) don 10 min a dakin da zafin jiki (RT), a cikin 0.1% Triton X-Permeabilize a 100 (diluted a PBS; Biosharp). ) na tsawon minti 20 a dakin da zafin jiki kuma toshe a cikin 5% bovine serum albumin (a cikin PBS) na tsawon awanni 2 a dakin da zafin jiki.Sannan an sanya ƙwayoyin sel a cikin dare a 4 ° C tare da ƙwayoyin rigakafi na farko da ASC (1: 100 dilution) ko NLRP3 (1: 100 dilution), bi da bi, da Cy3 mai suna goat anti-zomo IgG (H + L) (1:400; EarthOx). , San Francisco, CA, Amurka) ko FITC-conjugated goat anti-mouse IgG (1:400; Earthox) na dare a 37 ° C a cikin duhu don 1 hour.An lalata ƙwayoyin nuclei tare da Hoechst 33258 (10 μg/ml; UE, Suzhou, China) na mintuna 5 kuma an lura da su a ƙarƙashin na'urar hangen nesa (Olympus Corporation, Tokyo, Japan).
An raba mice zuwa ƙungiyoyi hudu (n = 7 a kowace ƙungiya): (i) PBS-magungunan kulawa mara kyau (PBS kawai; gavage 100 µl / linzamin kwamfuta PBS tare da allurar intraperitoneal na yau da kullum 100 µl / linzamin kwamfuta PBS 3 hours daga baya) ., ci gaba har tsawon kwanaki 7);(ii) ƙungiyar kulawa mara kyau da aka bi da su tare da mai hana MCC950 [27] (100 µl / linzamin kwamfuta ta hanyar PBS gavage, 3 hours daga baya, 10 mg / kg nauyin jiki [BW] MCC950 [a cikin PBS] an gudanar da shi a cikin intraperitoneally kowace rana, tsawon kwanaki 7);(iii) G. duodenalis cyst kamuwa da cuta kungiyar (1.5 x 106 cysts / linzamin kwamfuta ta gavage, 3 hours daga baya, 100 μl / linzamin kwamfuta PBS intraperitoneally gudanar kowace rana don 7 kwanaki);(iv) G. duodenalis cyst hade kamuwa da cuta kungiyar MCC950 inhibitor magani kungiyar (1.5 × 106 cysts / linzamin kwamfuta via gavage, 10mg / kg jiki nauyi MCC950 intraperitoneally kullum ga 7 kwanaki a 3h).Ana kula da nauyin jikin kowane linzamin kwamfuta kullun kuma duk berayen an kashe su a rana ta 7.An yanke duodenum da aka girbe (tsawon 3 cm) a cikin ƙananan ƙananan a cikin 1 ml PBS, an lalata cysts a cikin dare a cikin PBS a 4 ° C, da G. duodenalis trophozoites.Fresh duodenum (tsawon cm 1) an keɓe shi don tabo na hematoxylin da eosin (H&E).
An raba beraye zuwa ƙungiyoyi biyu: (i) ƙungiyar kula da MOCK da (ii) ƙungiyar hanawa MCC950.Akwai jiyya guda biyar a kowace ƙungiya (n = 7 / ƙungiyar kulawa): (i) PBS ƙungiyar kulawa mara kyau (PBS kawai; 100 µl / linzamin kwamfuta PBS, intramuscular (IM) allura (tibialis na gaba) [28, 29];( ii) pcDNA3.1 (+) plasmid korau iko kungiyar (100 µg / linzamin kwamfuta DNA, via intramuscular allura); ƙungiyar da aka yi da plasmid pcDNA3.1 (+) -alpha-2 (100 μg / linzamin kwamfuta DNA, ta hanyar allurar intramuscular), da (v) ƙungiyar da aka yi da pcDNA3.1 (+) -alpha-7.3 (100 μg / linzamin kwamfuta) DNA, bayan sa'o'i 12 na wucewa, mice a cikin ƙungiyar masu hanawa MCC950 sun sami allurar intraperitoneal na yau da kullum na MCC950 (10 mg / kg jiki) na tsawon kwanaki 7, yayin da mice a cikin kungiyar MOCK sun sami daidaitaccen adadin maganin PBS. An samo samfurin jini. An tattara su daga berayen ido kuma an bar su a cikin dare a 4 ° C samfurori na Serum an ware su ta hanyar amfani da enzyme-linked immunosorbent assay (ELISA) don da ma'auni na IL-1β.
An raba beraye talatin da biyar zuwa rukuni biyar (n=7/rukuni).Rukuni na 1 ya kasance ƙungiyar kulawa mara kyau da aka bi da ita tare da PBS: beraye sun karɓi 100 μl na PBS a cikin muscular da kwanaki 3 daga baya ta hanyar gavage.Rukuni na 2 shine ƙungiyar kulawa mai kyau wanda ya kamu da G. duodenalis cysts: an yi wa mice allura tare da 100 μl na PBS, kuma kwanaki 3 bayan 1.5 x 106 cysts / linzamin kwamfuta an yi musu allura a cikin ciki.Ƙungiyar ta uku - rigakafin plasmid tare da pcDNA3.1 (+) a hade tare da ƙungiyar kulawa don duodenal cyst kamuwa da cuta: mice sun karbi 100 μg na plasmid DNA pcDNA3.1 (+) (im) baki, 1.5 × 106 cysts / linzamin kwamfuta 3 don da yawa kwanaki.Ƙungiyoyin 4 da 5 sun kasance masu sake haɗawa pcDNA3.1 (+) -alpha-2 giardine plasmid ko pcDNA3.1 (+) -alpha-7.3 giardine plasmid a hade tare da G. duodenalis cyst kamuwa da cuta.Ƙungiyar gwaji: beraye sun karɓi 100 μg na pcDNA3.1 (+) - giardine plasmid DNA (im), sannan bayan kwanaki 3, an yi allurar 1.5 × 106 cysts / linzamin kwamfuta ta hanyar gavage.An kula da nauyin jikin kowane linzamin kwamfuta bayan gabatarwar G. duodenalis cyst ta cikin bututu.An tattara sabo duodenum don auna ma'aunin nauyin parasitic da nazarin tabo na HE.
An yi nazarin sauye-sauye na tarihi bisa ga tsarin da aka buga a baya [30].Fresh duodenum an gyara shi tare da gyaran sel na nama, an saka shi a cikin paraffin, a yanka a cikin sassan μm 4, an lalata shi da H&E kuma an yi nazari a ƙarƙashin ma'aunin haske.Canje-canje na cututtukan cututtuka na wakilci a cikin sassan nama guda bakwai daga mice masu zaman kansu guda bakwai an kimanta su ta hanyar likitan ilimin likitancin da bai san maganin ba kuma an kama shi a 200x magnification.Tsawon villi da zurfin crypts an auna su daidai da hanyoyin da aka bayyana a baya.
Sakamakon in vitro da in vivo an samu su sau uku.An ƙirƙira zane-zane ta amfani da GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, Amurka).An bincika bambance-bambance tsakanin ƙungiyoyi biyu ta hanyar t-test, yayin da bambance-bambancen tsakanin ≥3 ƙungiyoyi an bincikar su ta hanyar nazarin bambance-bambancen guda ɗaya (ANOVA) ta amfani da software na SPSS (version 22.0; SPSS IBM Corp., Armonk, NY, Amurka).An yi nazarin bayanai don daidaituwar bambancin ta amfani da gwajin Levene wanda Bonferroni ya biyo bayan gwajin hoc (B).An bayyana mahimmanci a matsayin P <0.05, P <0.01, da P <0.001 (ba mahimmanci [ns]) (P> 0.05).
Bincikenmu na baya na GEV proteomics a cikin Kyoto Encyclopedia of Genes and Genomes (KEGG) ya nuna cewa yawancin hari na iya shiga cikin kunna hanyoyin siginar kumburi [13].Mun zaɓi maƙasudai biyu masu ban sha'awa, alpha-2 da alpha-7.3 giardins, haɓaka waɗannan ƙwayoyin kuma amfani da su don gina pcDNA3.1(+) eukaryotic magana vector.Bayan jerin, recombinant pcDNA3.1 (+) -alpha-2 da alpha-7.3 giardine magana plasmids an canza su zuwa farkon linzamin kwamfuta peritoneal macrophages, da kuma caspase-1 p20 sa hannu furotin na kumburi (wani guntu na kunna caspase-1) aka gano. kamar yadda ke bayyana maɓalli masu mahimmanci waɗanda zasu iya haifar da kumburi.Sakamakon ya nuna cewa alpha-2 da alpha-7.3 giardines na iya haifar da p20 caspase-1 magana mai kama da GEV.Ba a sami wani tasiri akan kunnawar caspase-1 a cikin kulawa mara kyau ba (PBS kawai) da pcDNA3.1 (+) mai sarrafa plasmid (Hoto 1).
Ma'auni na p20 caspase-1 kunnawa ta pcDNA3.1(+) -alpha-2 da alpha-7.3 giardins.Recombinant eukaryotic expression plasmids pcDNA3.1(+) -alpha-2 da alpha-7.3 giardines (a sama da kowane layi) an canza su zuwa macrophages na linzamin kwamfuta na farko kuma an girbe al'adun gargajiya sa'o'i 24 bayan haka.An yi amfani da gogewar yamma don auna matakan furci na sa hannu-1 p20 furotin mai kumburi.Ƙungiyar jiyya ta PBS-kawai (layin C) da pcDNA3.1 (+) ƙungiyar monotherapy (pcDNA3.1 layin) an yi amfani da su azaman iko mara kyau, kuma an yi amfani da ƙungiyar jiyya ta GEV azaman kulawa mai kyau.An tabbatar da bayanin furotin da aka sake haɗawa ta hanyar gano alamar histidine a cikin kowane furotin, kuma nau'in furotin da ake sa ran shine alpha-2 giardine (38.2 kDa) da alpha-7.3 giardine (37.2 kDa).GEV, Giardia duodenum extracellular vesicles, pcDNA3.1 (+), EcoRV-linearized vector, SUP, supernatant
Don sanin ko alpha-2 giardine da alpha-7.3 giardine sun haifar da p20 caspase-1 magana kuma suna taka rawa wajen kunna mai watsa shiri NLRP3 mai kumburi, pcDNA3.1 (+) -alpha-2 giardine da pcDNA3.1 (+) -alpha -7.3 giardin an canza shi zuwa cikin macrophages na peritoneal na linzamin kwamfuta na farko tare da DNA plasmid recombinant, da matakan magana, yanki, da oligomerization na mahimman ƙwayoyin ƙwayoyin cuta masu kumburi NLRP3 an ƙaddara.A cikin wannan gwaji, an yi amfani da GEV a matsayin ƙungiyar kulawa mai kyau, kuma babu ƙungiyar kulawa (PBS kawai) ko pcDNA3.1 (+) ƙungiyar maganin canja wuri shine ƙungiyar mara kyau.Sakamakon ya nuna cewa, kamar yadda a cikin ƙungiyar GEV, recombinant plasmid DNA na giardin pcDNA3.1 (+) -alpha-2 da giardin pcDNA3.1 (+) -alpha-7.3 ya haifar da haɓakar NLRP3, pro-IL-1β da procaspase-1 da caspase-1 kunnawa (Fig. 2a).Bugu da ƙari, duka giardines sun haifar da mahimmancin IL-1β (pcDNA3.1: ANOVA, F (4, 10) = 1.625, P = 0.1000; alpha-2 giardine: ANOVA, F (4, 10) = 1.625, P = 0.0007 ).;alpha-7.3 giardine: ANOVA, F (4, 10) = 1.625, P<0.0001;GEV: ANOVA, F(4, 10) = 1.625, P = 0.0047) (Hoto na 2b).Yawancin sunadaran ASC sun kasance monomeric a cikin ƙungiyar marasa magani ko a cikin ƙungiyar kulawa da aka canza tare da pcDNA3.1 (+) plasmid, sabanin pcDNA3.1 (+) -alpha-2 ko pcDNA3.1 (+) -alpha- 7.3 gillar.ASC oligomerization ya faru a cikin recombinant plasmid DNA na GEV tabbatacce iko kungiyar ko rukuni, nuna wani oligomeric form (Figure 2c).Wadannan bayanan farko sun nuna cewa alpha-2 giardine da alpha-7,3 giardine na iya haifar da kunna kumburin NLRP3.Nazarin immunofluorescent na gaba na ƙaddamarwar ASC da NLRP3 ya nuna cewa a cikin rukunin kulawa mara kyau, furotin ASC ya warwatse cikin cytoplasm kuma ya bayyana azaman siginar dige akan haɓakar pcDNA3.1 (+) -alpha-2 tare da giardine ko pcDNA3.1 (+) -alpha-7,3 ƙungiyar giardine ko ƙungiyar kulawa ta GEV (Hoto 2d).A cikin mummunan iko da plasmid-treated pcDNA 3.1 kungiyoyin, ba a gano siginar furotin na NLRP3 ba, yayin da alamar siginar sigina a mayar da martani ga pcDNA3.1 (+) -alpha-2 giardine ko pcDNA3.1 (+) -alpha-7.3 an gano..Ana samun giardine a cikin cytoplasm ko a kan ƙarfafa HEV (Fig. 2e).Wadannan bayanan sun kara nuna cewa G. duodenalis giardin alpha-2 da giardin alpha-7.3 suna kunna NLRP3 inflammasome a cikin linzamin kwamfuta na farko na peritoneal macrophages.
pcDNA3.1 (+) -alpha-2 giardin da pcDNA3.1 (+) -alpha-7.3 giardin kunna NLRP3 inflammasome a cikin linzamin kwamfuta peritoneal macrophages.Canja wurin recombinant eukaryotic magana plasmids pcDNA3.1 (+) -alpha-2 giardin da pcDNA3.1 (+) -alpha-7.3 giardin cikin murine peritoneal macrophages da sel, ko girbi supernatant cikin 24 h don nazarin magana, oligomerization. , asiri.da kuma gano maɓalli na sunadaran masu kumburi.An yi amfani da ƙungiyar PBS-kawai (C) da pcDNA3.1 (+) ƙungiyar jiyya guda ɗaya azaman kulawa mara kyau, kuma an yi amfani da ƙungiyar jiyya ta GEV azaman ƙungiya mai kyau.a Key kumburi sunadaran NLRP3, ciki har da NLRP3, pro-IL-1β, pro-caspase-1, da kuma p20 caspase-1, an gano ta Western blotting.b An ƙaddara matakan ɓoye na IL-1β a cikin masu girma da yawa ta hanyar amfani da enzyme-linked immunosorbent assay (ELISA).An yi nazarin bambance-bambance tsakanin sarrafawa da ƙungiyoyin gwaji ta hanyar nazarin bambance-bambancen hanya ɗaya (ANOVA) ta amfani da sigar software ta SPSS 22.0.Asterisks suna nuna bambance-bambance masu mahimmanci tsakanin ƙungiyoyi **P <0.01 da *** P <0.001.c ASC oligomerization matakan a cikin pellets an ƙaddara ta hanyar binciken haɗin gwiwar haɗin gwiwar DSS, yayin da matakan ASC a cikin lysates tantanin halitta an yi amfani da su azaman sarrafa kaya.d Kallon gani na ISC localization ta amfani da immunofluorescence.e Immunofluorescence an yi amfani da shi don hangen nesa na NLRP3.ASC, nau'in furotin na apoptotic speck;IL, interleukin;NLRP3, nucleotide-daure oligomerization-kamar mai karɓa 3;ns, ba mahimmanci ba (P> 0.05)
Dukansu G. duodenalis da GEVs da yake ɓoye suna kunna kumburin NLRP3 kuma suna daidaita martanin mai kumburi a cikin vitro.Don haka, rawar da NLRP3 mai kumburi a cikin ƙwayoyin cuta na G. duodenalis ya kasance ba a sani ba.Don bincika wannan batu, mun tsara gwaji tsakanin mice da ke kamuwa da G. duodenalis cyst da mice kamuwa da G. duodenalis cyst + MCC950 inhibitor magani da kuma idan aka kwatanta NLRP3 inflammasome magana lokacin da kamuwa da G. duodenalis cyst.An nuna cikakken makircin gwajin a cikin siffa 3a.Canje-canje a cikin nauyin jikin mice a cikin ƙungiyoyin jiyya daban-daban an kula da su don kwanaki 7 bayan kamuwa da cuta tare da cysts, kuma an nuna sakamakon a cikin siffa 3b.Idan aka kwatanta da ƙungiyar da aka yi da PBS mai tsabta, sakamakon ya nuna cewa (i) nauyin jikin mice da ke kamuwa da G. duodenalis cyst ya ragu daga ranar 3 zuwa ranar 7 bayan kamuwa da cuta;(ii) magani tare da mai hana MCC950 ba shi da wani tasiri mai mahimmanci akan nauyin jikin mice..Idan aka kwatanta da ƙungiyar kamuwa da cuta guda ɗaya, BW na ƙungiyar kamuwa da cuta ta duodenal da aka bi da su tare da MCC950 ya ragu zuwa digiri daban-daban (Ranar 1: ANOVA, F (3, 24) = 1.885, P = 0.0148; Ranar 2: ANOVA, F (3, 24) ) = 0.4602, P<0.0001; Day 3: ANOVA, F (3, 24) = 0.8360, P = 0.0010; Day 4: ANOVA, F (3, 24) = 1.683, P = 0.0052; F, 5: A (3, 24) = 0.6497, P=0.0645; Day 6: ANOVA, F(3, 24)=5.457, P=0.0175; Day 7: ANOVA, F(3, 24) = 2.893, P = 0.0202).Wadannan bayanan sun nuna cewa NLRP3 mai kumburi yana kare mice daga asarar nauyi mai yawa a farkon matakan (2-4 days) na kamuwa da cutar duodenal.Sai muka yi nufin gano G. duodenalis trophozoites a cikin duodenal lavage ruwa da kuma sakamakon da aka nuna a Figure 3c.Idan aka kwatanta da kungiyar G. duodenalis cyst kamuwa da cuta, adadin trophozoites a cikin duodenum ya karu sosai bayan da aka hana NLRP3 inflammasome (t (12) = 2.902, P = 0.0133).Duodenal kyallen takarda tare da HE ya nuna, idan aka kwatanta da mummunan iko da aka bi da su tare da PBS da MCC950 kadai: (i) G. duodenalis cyst kamuwa da cuta ya haifar da lalacewa ga duodenal villi (ANOVA, F(3, 24) = 0.4903, P= 0.0488 ) da crypt atrophy (ANOVA, F (3, 24) = 0.4716, P = 0.0089);(ii) duodenum daga berayen da suka kamu da G. duodenalis cysts kuma ana bi da su tare da masu hana MCC950.Duodenal villi sun lalace kuma sun mutu (ANOVA, F (3, 24) = 0.4903, P = 0.0144) tare da atrophy da crypt branching (ANOVA, F (3, 24) = 0.4716, P = 0, 0481) (Fig. 3d- f) .Wadannan sakamakon sun nuna cewa NLRP3 inflammasome yana taka rawa wajen rage ƙwayar cutar G. duodenalis.
Matsayin NLRP3 mai kumburi a cikin Giardia duodenum kamuwa da cuta.An yi amfani da berayen (iv) tare da cysts duodenococcal sannan a bi da su tare da ko ba tare da MCC950 (ip).An yi amfani da ƙungiyoyin jiyya guda ɗaya tare da PBS ko MCC950 azaman sarrafawa.Ƙungiyar gwaji da tsarin kulawa.b An kula da nauyin jikin berayen a cikin kowane rukunin magunguna daban-daban na kwanaki 7.Bambanci tsakanin ƙungiyar G. duodenalis kamuwa da cuta da G. duodenalis + MCC950 ƙungiyar maganin kamuwa da cuta an bincika ta hanyar t-gwajin ta amfani da sigar software ta SPSS 22.0.Asterisks suna nuna bambance-bambance masu mahimmanci a * P <0.05, ** P <0.01, ko *** P <0.001.c An ƙaddara nauyin parasitic ta hanyar kirga adadin trophozoites a cikin ruwan lavage duodenal.Bambanci tsakanin ƙungiyar G. duodenalis kamuwa da cuta da G. duodenalis + MCC950 ƙungiyar maganin kamuwa da cuta an bincika ta hanyar t-gwajin ta amfani da sigar software ta SPSS 22.0.Asterisks suna nuna bambance-bambance masu mahimmanci a * P <0.05.d Hematoxylin da eosin (H&E) sakamakon tabo na duodenal histopathology.Jajayen kiban suna nuna lalacewa ga villi, koren kibiyoyi suna nuna lalacewar crypts.Matsakaicin girman: 100µm.e, f Nazarin ƙididdiga na tsayin duodenal villus da tsayin linzamin kwamfuta.Asterisks suna nuna bambance-bambance masu mahimmanci a * P <0.05 da ** P <0.01.Ana ɗaukar sakamakon daga gwaje-gwajen nazarin halittu masu zaman kansu guda 7.BW, nauyin jiki;ig, hanyar isar da intragastric;ip, hanyar isar da intraperitoneal;ns, ba mahimmanci ba (P> 0.05);PBS, phosphate buffered saline;WT, nau'in daji
Sirrin IL-1β alama ce ta kunna kumburi.Don sanin ko G. duodenalis alpha-2 giardine da alpha-7.3 giardine suna kunna mai watsa shiri na NLRP3 inflammasome a cikin vivo, mun yi amfani da mice WT marasa magani (ƙungiyar sham) da kuma NLRP3 inflammasome-blocked mice (MCC950 hana ƙungiyar kulawa).An nuna cikakken makircin gwajin a cikin siffa 4a.Ƙungiyoyin gwaji sun ƙunshi ƙuƙuka da aka yi da PBS, G. duodenalis cyst magani ta gavage, allurar intramuscular na pcDNA3.1, da allurar intramuscular na pcDNA3.1 (+) -alpha-2 giardine ko pcDNA3.1-alpha-7.3 giardine.A ranar 7th bayan gudanarwar intramuscularly na recombinant plasmid, an tattara ruwan magani kuma an ƙayyade matakin IL-1β a kowace ƙungiya.Kamar yadda aka nuna a cikin Hoto 4b, a cikin ƙungiyar MOCK: (i) idan aka kwatanta da ƙungiyar PBS, jiyya na pcDNA3.1 ba shi da tasiri mai mahimmanci akan ɓoyewar IL-1β (ANOVA, F (4.29) = 4.062, P = 0.9998), duk da haka, Sirri na IL-β ya kasance mai girma a cikin G. duodenalis cyst group (ANOVA, F (4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine da pcDNA3.1- Intramuscular allurar alpha-7.3 giardine ya karu da yawa matakan IL-1β (ANOVA, F (4, 29) = 4.062, P <0.0001);(iii) pcDNA3.1-alpha-7,3 giardine ya haifar da manyan matakan IL -1β a cikin pcDNA3.1-alpha-2 giardine intramuscular injection group (ANOVA, F (4, 29) = 4.062, P = 0.0333) .Idan aka kwatanta da kowane rukuni a cikin ƙungiyar kulawa ta MCC950 da ƙungiyar MOCK: (i) IL-1β matakan ɓoyewa a cikin ƙungiyar kula da PBS da ƙungiyar kulawa ta pcDNA3.1 sun ragu zuwa wani lokaci bayan da aka hana MCC950 mai hanawa, amma bambancin bai kasance ba. mahimmanci (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F (9, 58) = 3.540, P = 0.5949);(ii) bayan toshe MCC950., IL-1β asirin ya ragu sosai a cikin G. duodenalis cyst-cutar rukuni, ƙungiyar pcDNA3.1-alpha-2 giardine, da pcDNA3.1-alpha-7.3 giardine group (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) ) = 3.540, P = 0.0164).Wadannan sakamakon sun nuna cewa alpha-2 giardine da alpha-7.3 giardine suna yin sulhu da kunnawa na NLRP3 inflammasome a cikin vivo.
pcDNA3.1 (+) -giardines kunna NLRP3 rundunar inflammasome a cikin vivo.An yi rigakafin beraye (IM) tare da recombinant eukaryotic magana plasmid pcDNA3.1 (+) -alpha-2 giardine ko pcDNA3.1 (+) -alpha-7.3 giardine sa'an nan kuma bi da su tare da MCC950 (ip; MCC950 kungiyar) ko a'a (dummy group) ).An yi amfani da PBS ko pcDNA3.1 (+) ƙungiyar maganin plasmid a matsayin kulawa mara kyau, an yi amfani da ƙungiyar G. duodenalis cyst a matsayin kulawa mai kyau.Ƙungiyar gwaji da tsarin kulawa.b Matakan jini na IL-1β a cikin mice an auna su a ranar 7 ta hanyar gwajin ELISA.An yi nazarin bambance-bambance tsakanin ƙungiyoyi a cikin ƙungiyar MOCK ta amfani da ANOVA ta hanya ɗaya, kuma an bincika bambance-bambance tsakanin ƙungiyar MOCK da ƙungiyar MCC950 ta amfani da t-gwajin na SPSS software version 22.0.Asterisks suna nuna manyan bambance-bambance tsakanin kungiyoyin jiyya a cikin ƙungiyar MOCK, * P <0.05 da *** P <0.001;Alamun dala ($) suna nuna bambance-bambance masu mahimmanci tsakanin kowace ƙungiya a cikin ƙungiyar MOCK da ƙungiyar MCC950 a P<0.05.Sakamako na gwaje-gwajen nazarin halittu masu zaman kansu guda bakwai.i, alluran intramuscular, ns, ba mahimmanci ba (P> 0.05)
Don bincika sakamakon alpha-2 da alpha-7.3 giardine-mai kunnawa na NLRP3 mai watsa shiri mai kumburi a kan G. duodenalis kamuwa da cuta, mun yi amfani da WT C57BL / 6 mice da allurar alpha-2 giardine da alpha-7.3 giardine.An yi allurar plasmid a cikin jiki, bayan kwanaki 3 ta hanyar bututun ciki na G. duodenalis cyst, bayan haka an lura da mice na kwanaki 7.An nuna cikakken makircin gwajin a cikin siffa 5a.An auna nauyin jikin kowane linzamin kwamfuta a kowace rana, an tattara samfurori na nama na duodenal sabo a ranar 7th bayan gudanarwa ta hanyar bututun ciki, an auna adadin trophozoites, kuma an lura da canje-canje na tarihi.Kamar yadda aka nuna a cikin Hoto 5b, tare da haɓaka lokacin ciyarwa, BW na mice a kowace ƙungiya ya karu a hankali.MT na mice ya fara raguwa a ranar 3rd bayan gudanarwar intragastric na G. duodenalis cysts, sannan a hankali ya karu.Kunna NLRP3 mai kumburi wanda aka haifar da allurar intramuscular na alpha-2 giardine da alpha7.3 giardine yana rage asarar nauyi a cikin mice (Ranar 1: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 1.399, P = 0.9754 Day 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30)=1.399, P=0.9987 Day 2: pcDNA3.1-alpha-2 giardine, ANOVA, F( 4, 30) = 0.3172, P = 0.9979; Day 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.3172, P = 0.8409; Day 3: pcDNA3.1-alpha-2, F 4, 30) = 0.8222, P = 0.0262 Day 3: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.8222, P = 0.0083; Day 4: pcDNA3.1-alpha-2 giardine , F (4, 30) = 0.5620, P = 0.0012, Day 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.5620, P <0.0001, Day 5: pcDNA3.1-alpha - 2 giardine, ANOVA, F (4, 30) = 0.9728, P <0.0001 Day 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Day 6: pcDNA3 . alpha-2 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0012, Day 6: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0006;Ranar 7: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.5369, P<0.0001 Day 7: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5369, P <0.0001).An kimanta nauyin parasitic a cikin duodenum (Fig. 5c).Idan aka kwatanta da ingantaccen kulawar da ba a kula da shi ba da kuma ƙungiyar da aka yi tare da pcDNA3.1 vector mara kyau, adadin G. duodenalis trophozoites ya ragu sosai a cikin ƙungiyoyin da aka allura tare da α-2 giardine da 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).Bugu da ƙari, giardine alfa-7.3 ya fi kariya a cikin mice fiye da giardine alfa-2 (ANOVA, F (3, 24) = 1.209, P = 0.0081).Ana nuna sakamakon tabon HE a cikin fig.5 d-f.Mice da aka yi da alpha-2 giardine da alpha-7.3 giardine suna da ƙananan raunuka na duodenal nama, wanda ya bayyana ta hanyar lalacewar villus, idan aka kwatanta da mice da aka allura tare da G. duodenalis da berayen allura tare da G. duodenalis a hade tare da komai na pcDNA3 vector .1 Zoom.(pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 2.466, P = 0.0035 ko P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F (3, 24) = 2.466, P = 0.0028 ko P = 0.0055) da kuma rage crypt atrophy (pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 1.470, P = 0.0264 ko P = 0.0158; pcDNA3.1-alpha: 7.3 giardine , F (3, 24) = 1.470, P = 0.0371 ko P = 0.0191).Wadannan sakamakon sun nuna cewa alpha-2 giardine da alpha-7,3 giardine sun rage rashin lafiyar G. duodenalis ta hanyar kunna NLRP3 inflammasome a cikin vivo.
Matsayin pcDNA3.1 (+) -giardins a cikin G. duodenal kamuwa da cuta.An yi rigakafin beraye (IM) tare da recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine ko pcDNA3.1 (+) -alpha-7.3 giardine sannan kuma an kalubalanci G. duodenalis cysts (ig).An yi amfani da ƙungiyar PBS da pcDNA3.1 (+) + duodenal cyst treatment group a matsayin ƙungiyoyi masu kulawa mara kyau, kuma an yi amfani da ƙungiyar maganin cyst duodenal a matsayin ƙungiyar kulawa mai kyau.Ƙungiyar gwaji da tsarin kulawa.b An kula da MT na mice a cikin kowane ɗayan ƙungiyoyin jiyya na tsawon kwanaki 7 bayan ƙalubalen.Asterisks suna nuna manyan bambance-bambance tsakanin ƙungiyoyi a cikin rukunin G. duodenalis da ƙungiyar pcDNA3.1 (+) -alpha-2 ƙungiyar giardine, * P <0.05, ** P <0.01, da *** P <0.001;alamar dala ($) tana nuna babban bambanci tsakanin kowane rukuni na G. duodenalis da pcDNA3.1 (+) -alpha-7.3 jardine group, $$P <0.01 da $$$P <0.001.c An ƙaddara nauyin parasitic ta hanyar kirga adadin trophozoites a cikin 1 ml na duodenal lavage daga duodenum (tsawon 3 cm) kuma an bayyana shi azaman adadin parasites a kowane cm na duodenum.Bambance-bambance tsakanin G. duodenalis kamuwa da cuta kungiyar, da pcDNA3.1 (+) -alpha-2 giardine kungiyar, da pcDNA3.1 (+) -alpha-7.3 giardine kungiyar an yi nazari ta hanyar ANOVA hanya daya ta amfani da SPSS software version 22.0.Asterisks suna nuna bambance-bambance masu mahimmanci a ** P <0.01 da *** P <0.001.d Canje-canje na Histopathological a cikin duodenum.Jajayen kiban suna nuna lalacewa ga villi, koren kibiyoyi suna nuna lalacewar crypts.Matsakaicin girman: 100µm.e, f Ƙididdigar ƙididdiga na tsayin duodenal villus linzamin kwamfuta (e) da tsayin crypt (f).An bincika bambance-bambance tsakanin ƙungiyoyi a cikin Hoto 1d ta hanyar ANOVA ta hanya ɗaya ta amfani da sigar software ta SPSS 22.0.Asterisks suna nuna bambance-bambance masu mahimmanci a * P <0.05 da ** P <0.01.Sakamako na gwaje-gwajen nazarin halittu masu zaman kansu guda bakwai.ns, ba mahimmanci ba (P> 0.05)
Giardia duodenum sanannen kwayar cutar hanji ne na mutane da sauran dabbobi masu shayarwa waɗanda ke haifar da giardiasis.A cikin 2004, an haɗa shi cikin shirin WHO da aka yi watsi da Cututtuka saboda yawan yaɗuwarta sama da shekaru 6, musamman a cikin al'ummomin da ke da ƙarancin yanayin zamantakewa [32].Tsarin rigakafi na asali yana taka muhimmiyar rawa a cikin amsawar rigakafi ga G. duodenalis kamuwa da cuta.An ba da rahoton macrophages na linzamin kwamfuta don cinyewa da kashe G. duodenalis ta hanyar sakin tarkuna na waje [33].Nazarin mu na baya sun nuna cewa G. duodenalis, wani nau'i mai banƙyama mai banƙyama, yana kunna p38 MAPK, ERK, NF-κB p65, da kuma NLRP3 hanyoyin siginar ƙumburi a cikin macrophages na linzamin kwamfuta don daidaita martanin mai kumburi mai watsa shiri, kuma an saki GEV na iya haɓaka wannan tsari.13], 24].Duk da haka, ainihin PAMPs da ke cikin NLRP3 ƙumburi mai kumburi a cikin GEV da kuma rawar NLRP3 inflammasome a cikin giardiasis ya kasance a bayyane.Don ƙarin haske a kan waɗannan tambayoyi biyu, mun gudanar da wannan binciken.
Inflammasome NLRP3 yana cikin cytoplasm na sel na rigakafi kuma ana iya kunna su ta wasu barbashi kamar su lu'ulu'u na uric acid, gubobi, ƙwayoyin cuta, ƙwayoyin cuta, da parasites.A cikin nazarin ƙwayoyin cuta, an gano gubobi a matsayin mahimman PAMPs waɗanda ke kunna firikwensin kumburi, wanda ke haifar da kumburi da mutuwar tantanin halitta [34].Wasu gubobi daban-daban, irin su hemolysin daga Staphylococcus aureus [35] da Escherichia coli [36], hemolysin BL (HBL) daga enterotoxin (NHE) [37], haifar da kunna kumburin NLRP3.Nazarin hoto ya nuna cewa sunadaran ƙwayoyin cuta irin su SARS-COV-2 ambulaf (E) furotin [38] da furotin NS5 na cutar Zika [39] sune mahimman PAMPs da mai karɓar NLRP3 ya gane.A cikin nazarin parasites, yawancin ƙwayoyin cuta an ba da rahoton cewa suna da alaƙa da kunnawa mai kumburi, kamar Toxoplasma gondii, Trichomonas vaginalis [40], Trypanosoma cruzi [41], da Leishmania [42].Sunadaran sunadaran granule GRA35, GRA42, da GRA43, waɗanda ke da alaƙa da virulence na Toxoplasma gondii, ana buƙatar shigar da pyroptosis a cikin Lewis rat macrophages [43].Bugu da ƙari, wasu nazarin Leishmania sun mayar da hankali kan kwayoyin halitta guda ɗaya da ke cikin NLRP3 mai kumburi, irin su m membrane lipophosphoglycan [44] ko zinc metalloprotease [45].Daga cikin dangin alpha-giardin na annexin-kamar alfa-giardin, an nuna alpha-1 giardin a matsayin ɗan takarar rigakafin rigakafi wanda ke ba da kariya ga G. duodenalis a cikin ƙirar linzamin kwamfuta [18].A cikin bincikenmu, mun zaɓi G. duodenalis virulence factor alpha-2 da alpha-7,3 giardines, waɗanda ke da mahimmanci ga giardia amma ba da rahoto ba.Wadannan kwayoyin halitta guda biyu da aka yi niyya an haɗa su cikin pcDNA3.1 (+) eukaryotic expression vector don nazarin kunnawa kumburi.
A cikin ƙirar linzamin kwamfutanmu, guntuwar caspase da aka fashe suna zama alamomin kunna kumburi.Bayan ƙarfafawa, NLRP3 yana hulɗa tare da ASC, ƙaddamar da ƙaddamarwa, kuma yana haifar da caspases masu aiki waɗanda ke raba pro-IL-1β da pro-IL-18 zuwa cikin balagagge IL-1β da IL-18, bi da bi -18.Caspases masu ƙumburi (caspases-1, -4, -5 da -11) dangin da aka kiyaye su ne na ƙwayoyin cuta na cysteine ​​waɗanda ke da mahimmanci don kariya ta asali kuma suna shiga cikin kumburi da tsarin mutuwar kwayar halitta [46].Caspase-1 yana kunna ta ta hanyar inflammasomes na canonical [47], yayin da caspases-4, -5, da -11 suna katsewa yayin samuwar inflammasomes mai ƙima [48].A cikin wannan binciken, mun yi amfani da linzamin kwamfuta na peritoneal macrophages a matsayin samfurin kuma bincika p20 caspase-1 cleaved caspase-1 a matsayin alama na mai watsa shiri NLRP3 ƙonewa kunnawa a cikin nazarin G. duodenalis kamuwa da cuta.Sakamakon ya nuna cewa yawancin alpha-giardins suna da alhakin kunnawa na yau da kullun na kumburi, wanda ya yi daidai da gano mahimman ƙwayoyin ƙwayoyin cuta da ke cikin ƙwayoyin cuta da ƙwayoyin cuta.Duk da haka, bincikenmu shine kawai allo na farko kuma akwai wasu kwayoyin da za su iya kunna ƙumburi maras kyau, kamar yadda bincikenmu na baya ya samo duka na gargajiya da na gargajiya a cikin G. duodenalis kamuwa da cuta [13].Don ƙarin ƙayyade ko p20 caspase-1 da aka haifar yana da alaƙa da kumburin NLRP3, mun canza alpha-2 da alpha-7.3 giardins a cikin macrophages peritoneal linzamin kwamfuta don ƙayyade matakan furotin na ƙwayoyin ƙwayoyin cuta da matakan ASC oligomerization, yana mai tabbatar da cewa duka α-giardins suna kunna. mai kumburi NLRP3.Sakamakonmu ya ɗan bambanta da na Manko-Prykhoda et al., Wanda ya ruwaito cewa ƙarfafawar ƙwayoyin Caco-2 tare da G. muris ko E. coli EPEC nau'i ne kawai zai iya ƙara ƙarfin haske na NLRP3, ASC, da caspase-1, ko da yake ba mahimmanci ba, yayin da yadda tsadar G. muris da E. coli ya karu da matakan sunadaran guda uku [49].Wannan bambance-bambancen na iya zama saboda bambance-bambance a cikin zaɓin nau'in Giardia, layin salula, da sel na farko.Mun kuma yi a vivo assay ta amfani da MCC950 a cikin 5-mako-mako mace WT C57BL/6 mice, wanda ya fi saukin kamuwa da G. duodenalis.MCC950 mai ƙarfi ne kuma zaɓi ƙananan ƙwayoyin ƙwayoyin NLRP3 mai hanawa wanda ke toshe kunnawa NLRP3 na canonical da mara-canonical a ƙididdigar nanomolar.MCC950 yana hana NLRP3 kunnawa amma baya shafar kunnawar AIM2, NLRC4, da NLRP1 hanyoyin kumburi ko hanyoyin siginar TLR [27].MCC950 yana toshe NLRP3 kunnawa amma baya hana NLRP3 ƙaddamarwa, K+ efflux, Ca2+ influx, ko hulɗar tsakanin NLRP3 da ASC;maimakon haka, yana hana NLRP3 kunnawa mai kumburi ta hanyar toshe ASC oligomerization [27].Sabili da haka, mun yi amfani da MCC950 a cikin nazarin in vivo don ƙayyade rawar NLRP3 mai kumburi bayan allurar giardine.Kaspase-1 p10 da aka kunna ya raba pro-inflammatory cytokines pro-IL-1β da pro-IL-18 zuwa cikin balagagge IL-1β da IL-18 [50].A cikin wannan binciken, an yi amfani da matakan IL-1β na jini a cikin berayen da aka yi wa maganin giardine tare da ko ba tare da MCC950 ba a matsayin mai nuna alamar ko an kunna kumburin NLRP3.Kamar yadda ake tsammani, jiyya na MCC950 ya rage yawan matakan IL-1β na jini.Wadannan bayanan sun nuna a fili cewa G. duodenalis giardin alfa-2 da giardin alfa-7.3 suna iya kunna linzamin kwamfuta na NLRP3.
Mahimman bayanai da aka tara a cikin shekaru goma da suka gabata sun nuna cewa IL-17A shine babban mai kula da rigakafi da G. muris, yana haifar da siginar IL-17RA, samar da peptides na antimicrobial, da kuma daidaita aikin kunnawa [51].Duk da haka, kamuwa da cutar Giardia yana faruwa akai-akai a cikin samari, kuma an ba da rahoton cewa ciwon Giardia a cikin ƙananan beraye ba ya kunna amsawar IL-17A don yin tasirin kariya [52], yana sa masu bincike su nemi sauran Giardia immunomodulatory.Hanyoyin cututtuka na helminth.Marubutan wani bincike na baya-bayan nan sun ruwaito cewa G. muris na iya kunna NLRP3 inflammasome ta E. coli EPEC, wanda ke inganta samar da peptides na antimicrobial kuma yana rage yawan abin da aka haɗe shi da kuma adadin trophozoites a cikin hanji, ta haka ne rage girman ƙwayar hanji. cututtuka da ke haifar da bacilli [49].NLRP3 inflammasome yana shiga cikin ci gaban cututtuka daban-daban.Nazarin ya nuna cewa Pseudomonas aeruginosa yana haifar da autophagy a cikin macrophages don kauce wa mutuwar tantanin halitta, kuma wannan tsari ya dogara da kunna NLRP3 inflammasome [53].Don N. caninum, nau'in oxygen mai amsawa-matsakaicin kunnawa na NLRP3 mai kumburi yana iyakance kwafinsa a cikin mai watsa shiri, yana mai da shi maƙasudin warkewa [9].Paracoccidioides brasiliensis an samo shi don haifar da kunnawa na NLRP3 mai kumburi a cikin ƙwayoyin dendritic da aka samu na kasusuwa na kasusuwa, wanda ya haifar da sakin cytokine mai kumburi IL-1β, wanda ke taka muhimmiyar rawa wajen kare tsaro [10].Yawancin nau'in Leishmania, ciki har da L. amazonensis, L. major, L. braziliensis, da L. infantum chagasi, kunna NLRP3 da ASC-dogara caspase-1 a cikin macrophages, da kamuwa da cutar Leishmania.An haɓaka kwafin ƙwayoyin cuta a cikin ƙarancin beraye a cikin NLRP3/ASC/caspase-1 gene [11].Zamboni et al.An ba da rahoton kamuwa da cutar Leishmania don haifar da kunna kumburin NLRP3 a cikin macrophages, wanda ke iyakance kwafin ƙwayar cuta ta cikin salula.Don haka, Leishmania na iya hana kunna NLRP3 azaman dabarun gujewa.A cikin nazarin vivo, NLRP3 inflammasome ya ba da gudummawa ga kawar da Leishmania, amma bai shafi kyallen takarda ba [54].Sabanin haka, a cikin nazarin helminthiasis, kunnawa na NLRP3 inflammasome ya hana garkuwar kariya daga helminthiasis na ciki [12].Shigella na daya daga cikin manyan kwayoyin cutar da ke haddasa gudawa a duniya.Wadannan ƙwayoyin cuta na iya haifar da samar da IL-1β ta hanyar P2X7 mai karɓar mai karɓa na K + efflux, nau'in oxygen mai amsawa, lysosomal acidification, da lalacewar mitochondrial.A NLRP3 inflammasome mara kyau yana sarrafa phagocytosis da ayyukan ƙwayoyin cuta na macrophages akan Shigella [55].Nazarin Plasmodium ya nuna cewa AIM2, NLRP3 ko caspase-1 ƙananan berayen da suka kamu da Plasmodium suna samar da manyan matakan nau'in 1 interferon kuma sun fi jure kamuwa da cutar Plasmodium [56].Duk da haka, rawar alpha-2 giardine da alpha-7.3 giardine a cikin haifar da kunnawa na ƙwayoyin cuta na NLRP3 kumburi a cikin mice ba a sani ba.
A cikin wannan binciken, hanawa na NLRP3 inflammasome ta MCC950 ya rage BW kuma ya karu da adadin trophozoites a cikin ruwan lavage na hanji a cikin mice, wanda ya haifar da canje-canje masu tsanani na pathological a cikin duodenal nama.Alpha-2 giardine da alpha-7.3 giardine suna kunna linzamin kwamfuta na NLRP3 mai kumburi, haɓaka nauyin jikin linzamin kwamfuta, rage adadin trophozoites a cikin ruwan lavage na hanji, da kuma rage cututtukan duodenal na pathological.Wadannan sakamakon sun nuna cewa G. duodenalis na iya kunna mai watsa shiri na NLRP3 mai kumburi ta hanyar alpha-2 giardine da alpha-7,3 giardine, rage ƙwayar cutar G. duodenalis a cikin mice.
Gaba ɗaya, sakamakonmu ya nuna cewa alpha-2 da alpha-7.3 giardines suna haifar da kunnawa na NLRP3 mai watsa shiri mai kumburi da kuma rage cututtuka na G. duodenalis a cikin mice.Sabili da haka, waɗannan kwayoyin halitta sune maƙasudin manufa don rigakafin giardiasis.
       Data supporting the results of this study can be obtained from the respective author at gongpt@jlu.edu.cn.
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