Giardia duodenum waa noole dulin ah oo keena giardiasis, caabuqa mindhicirka gaar ahaan ku badan carruurta yaryar ee leh calaamadaha caafimaad ee shubanka.Waxaan horey u soo sheegnay in ka baxsan unugyada G. duodenalis ay kiciso firfircoonida oligomerization intracellular oligomerization-like reseptor 3 (NLRP3) xiritaanka nucleotides waxayna nidaamisaa jawaabaha bararka martida iyada oo loo marayo dheecaanka ka baxsan unugyada (EV).Si kastaba ha ahaatee, qaababka saxda ah ee molecular ee duodenococcal EV (GEV) ee ku lug leh habkan iyo doorka NLRP3 caabuqa ee giardiasis ayaa weli ah in la caddeeyo.
Recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 iyo alpha-7.3 giardins ee GEV ayaa la dhisay, oo loo beddelay mouse peritoneal macrophages, oo lagu ogaado iyada oo la cabbirayo bararka bartilmaameedka molecule caspase-1.Heerka muujinta p20 waa la eegay..G. duodenalis alpha-2 iyo alpha-7.3 giardines ayaa markii hore lagu aqoonsaday iyadoo la cabbirayo NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 iyo caspase-1 p20), dheecaanka IL.Heerarka 1β, borotiinka spotted protein (ASC) oligomerization heerarka, iyo meelaynta immunofluorescent ee NLRP3 iyo ASC.Doorka NLRP3 inflammasome ee xanuunka G. duodenalis ayaa markaa la qiimeeyay iyada oo la adeegsanayo jiirarka kaas oo NLRP3 firfircoonida la xannibay (jiirarka NLRP3 ee xannibay) iyo isbeddellada pathological ee miisaanka jidhka, culeyska dulinka duodenal, iyo unugyada duodenal ayaa la kormeeray.Intaa waxaa dheer, waxaan baarnay bal in hiaardines alpha-2 iyo alpha-7.3 ay keenaan dheecaanka IL-1β ee vivo iyada oo loo marayo NLRP3 inflammasome oo go'aamiyay doorka molecules-ka ee ku jira xanuunka G. duodenalis ee jiirarka.
Alpha-2 iyo alfa-7.3 giardines waxay kiciyaan firfircoonida NLRP3 inflammasome in vitro.Tani waxay keentay firfircoonida p20 caspase-1, korodhka heerarka muujinta NLRP3, pro-IL-1β, iyo borotiinka pro-caspase-1, koror weyn oo ku yimaada dheecaanka IL-1β, sameynta dhibcaha ASA ee cytoplasm, iyo kicinta ASA oligomerization.NLRP3 caabuqa luminta guska ayaa ka sii dareysa cudur-sidaha G. duodenalis ee jiirarka.Jiirarka lagu daweeyay fiix iyadoo laga soo qaaday jiirarka NLRP3-xiran ayaa muujiyay tiro korodhay oo trophozoites ah iyo dhaawac ba'an oo soo gaadhay villi duodenal, oo lagu garto crypts necrotic oo la gooyay iyo laamo.Tijaabooyinka vivo waxay muujiyeen in giardines alpha-2 iyo alpha-7.3 ay keeni karaan dheecaanka IL-1β iyada oo loo sii marayo NLRP3 inflammasome, iyo tallaalka giardines alpha-2 iyo alpha-7.3 waxay hoos u dhigtay xanuunka G. duodenalis ee jiirarka.
Isku soo wada duuboo, natiijooyinka daraasaddan waxay soo jeedinayaan in giardia alpha-2 iyo alpha-7.3 ay sababaan kor u qaadista caabuqa NLRP3 ee martida loo yahay waxayna yareeyaan infekshinka G. duodenalis ee jiirarka, kuwaas oo ah bartilmaameedyo rajo leh oo looga hortagayo giardiasis.
Giardia duodenum waa dulin ka baxsan unugyada protozoan ah oo ku nool xiidmaha yar oo sababa 280 milyan oo xaaladood oo giardiasis qaba shuban sannadkii, gaar ahaan carruurta yaryar ee dalalka soo koraya.Dadku waxay ku dhacaan biyaha la cabbo ama cuntada ay ku wasakhoobeen cysts M. duodenum, ka dibna caloosha gala oo laga soo saaro dheecaanka gastric.Giardia duodenum trophozoites waxay ku dhegtaa epithelium duodenal, taasoo keenta lallabbo, matag, shuban, calool xanuun, iyo miisaan lumis.Shakhsiyaadka leh difaac la'aanta iyo cystic fibrosis waxay u nugul yihiin caabuqa.Caabuqa sidoo kale wuxuu ku dhici karaa galmada afka iyo dabada [2].Daawooyinka sida metronidazole, tinidazole, iyo nitazoxanide ayaa ah doorashooyinka daawaynta ee la door bidayo ee caabuqyada duodenal-ka.Si kastaba ha ahaatee, daawooyinkan kiimoterabiga ah waxay keenaan waxyeelo xun sida lallabbo, kansar, iyo genotoxicity [4].Sidaa darteed, xeelado waxtar leh ayaa loo baahan yahay in la sameeyo si looga hortago caabuqa G. duodenal.
Inflammasomes waa nooc ka mid ah isku-dhafka borotiinka cytosolic kuwaas oo qayb ka ah jawaab-celinta difaaca jirka, ka caawinta inay iska difaacaan duulaanka pathogene iyo dhexdhexaadinta jawaabaha bararka [5].Waxaa ka mid ah caabuqyadaas, nucleotide-binding oligomerization (NOD) reseptor 3 (NLRP3) nucleotide-binding oligomerization (NLRP3) nucleotide-binding-like inflammasome ayaa si ballaaran loo bartay sababtoo ah waxaa lagu ogaan karaa noocyo kala duwan oo cudur-sidaha / waxyeelada leh (PAMP/ DAMP) , aqoonsada , waxa ay hawlgelisaa habka difaaca jidhka.wuxuuna nidaamiyaa homeostasis xiidmaha ee cudurro badan oo barar ah [6,7,8].Waxay ka kooban tahay qaabeeyaha aqoonsiga naqshadeeyaha (PRR) NLRP3, adabtarada apoptotic spotted protein (ASC), iyo procaspase-1 ama procaspase-11.Bararka NLRP3 wuxuu u dhaqmaa sidii martigeliye ka soo horjeeda duulaanka pathogene, sida lagu arkay Neospora caninum [9], Paracoccidioides brasiliensis [10], iyo daraasadaha Leishmania.[11], laakiin sidoo kale waxaa la soo sheegay in kicinta NLRP3 inflammasome ay xaddidayso jawaabaha difaaca ee difaaca waxayna sii xumaynaysaa horumarka cudurka, tusaale ahaan, gooryaanka [12].Iyada oo ku saleysan natiijooyinkayagii hore, waxaan soo sheegnay in G. duodenalis ee ka baxsan unugyadu ay kiciso firfircoonida intracellular ee caabuqa NLRP3 waxayna wax ka beddeshaa jawaabaha bararka martigelinta iyada oo la qarinayo xuubka unugyada ka baxsan (EVs) [13].Si kastaba ha ahaatee, doorka NLRP3 inflammasome ee caabuqa G. duodenalis ee vivo ayaa weli ah in la go'aamiyo.
Giardins ayaa markii hore lagu tilmaamay inay yihiin qaybo qaab dhismeedka G. duodenalis cytoskeleton waxayna door muhiim ah ka ciyaaraan dhaqdhaqaaqa trophozoite iyo xiritaanka unugyada epithelial ee xiidmaha yar.Si ay si fiican ula qabsadaan deegaanka oo ay u kordhiyaan cudurkooda, G. duodenalis trophozoites waxay soo saareen qaab-dhismeedka cytoskeletal gaar ah oo ka kooban 8 flagella, 1 jirka dhexe, iyo 1 saxan ventral [14].Trophozoites of Giardia duodenum waxay isticmaalaan cytoskeleton si ay u galaan mindhicirka yar ee sare, gaar ahaan duodenum, oo ay ku dhejiyaan enterocytes.Waxay si joogto ah u haajiraan oo ay ku xidhaan unugyada epithelial iyaga oo isticmaalaya dheef-shiid kiimikaadka unugyada.Sidaa darteed, waxaa jira xiriir dhow oo ka dhexeeya cytoskeleton iyo fayraska.Giardines u gaar ah Giardia duodenum waa qaybo ka mid ah qaab-dhismeedka cytoskeleton [15] waxaana loo qaybiyaa afar qaybood: α-, β-, γ-, iyo δ-giardines.Waxaa jira 21 xubnood oo ka tirsan qoyska α-giardin, kuwaas oo dhammaantood leh awood calcium-ku-tiirsan oo lagu xiro fosfolipids [16].Waxay sidoo kale ku xidhaan cytoskeleton iyo xuubka unugyada.Shakhsiyaadka shuban qaba ee uu keeno G. duodenalis, α-giardins ayaa si aad ah loo muujiyaa oo difaaca jirka inta lagu jiro caabuqa [17].Tallaalada heterologous ee ku salaysan Giardia alfa-1 ayaa laga ilaaliyay giardiasis ee jiirarka waxayna yihiin musharaxiinta antigens ee horumarinta tallaalka [18].Alpha-8 giardin, oo ku yaal xuubka balaasmaha iyo calanka, laakiin aan ku jirin saxanka ventral, waxay kor u qaadaysaa dhaqdhaqaaqa iyo heerka koritaanka trophozoites ee G. duodenalis [19].Alpha-14 giardin waxay ku dhejisaa qaababka microtubule ee calanka waxayna saameeyaan suurtagalnimada G. duodenalis [20].Alfa-11 giardine waxa ay ku badan tahay inta lagu jiro wareegga nolosha, iyo muujinta xad dhaafka ah ee alfa-11 giardine waxay dhaawacdaa G. duodenalis lafteeda [21].Si kastaba ha ahaatee, ma cadda in alfa-2 giardine iyo alfa-7.3 giardine ay ka ilaalinayaan caabuqa G. duodenalis iyo hababka hoose.
Daraasaddan, recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine iyo pcDNA3.1 (+) -alpha-7.3 giardine ayaa loo beddelay macrophages hoose ee jiirka si ay u dhaqaajiyaan martida loo yahay NLRP3.Bartilmaameedyada bararsan ayaa markaa la baaray.Waxaan sidoo kale qiimeynay doorka NLRP3 inflammasome ee xanuunka G. duodenalis, baaritaan lagu sameeyay in alpha-2 iyo alpha-7,3 giardines ay kiciyaan firfircoonida NLRP3 inflammasome ee vivo, waxaana go'aamiyay in labadan door ee giardines ee ku-meel-gaarka ah G. duodenalis.Hadafkayagu guud wuxuu ahaa inaan horumarino yoolal rajo leh oo looga hortagayo caabuqa G. duodenalis.
Nooca duurjoogta ah (WT) C57BL/6 jiirarka dheddigga ah ee da'doodu u dhaxayso 5-8 toddobaad ayaa laga soo iibsaday Xarunta Tijaabada ee Liaoning Changsheng (Liaoning, Shiinaha).Jiirarku waxay heleen biyo bilaash ah, waxay heleen cunto nadiif ah waxaana lagu hayay 12/12 saacadood iftiin/wareeg madow.Infekshanka ka hor, jiirarka ayaa helay antibiyootiga ad libitum ee biyaha la cabbo oo lagu daray ampicillin (1 mg / mL), vancomycin (1 mg / mL), iyo neomycin (1.4 mg / mL) (dhammaantood laga soo iibsaday Shanghai, Shiinaha, noolaha macmal) [22] ].].Jiirarka lumiyay awoodda wax-cunidda iyo cabbitaanka > 24 saacadood oo lumisay ≥ 20% miisaanka jidhka ayaa si bini'aadmi ah u nabxiyay kala-baxa afka ilmagaleenka.
WB G. duodenalis trophozoites (Ururinta Dhaqanka Nooca Maraykanka, Manassas, USA) ayaa lagu kabay 12.5% ​​serum bovine bovine (FBS; Cagaar kasta, Zhejiang, Shiinaha) iyo 0.1% bile bovine (Sigma-Aldrich, St. Missouri, USA) ).USA) xaaladaha microaerobic.Trophozoites-ka isku-darka ah ayaa lagu soo ururiyay baraf waxaana la marsiiyay saamiga 1:4 si ay u sii tarmo.
Fayraska duodenum ee Giardia ayaa la soo jiiday sidii hore loogu sharraxay [23], trophozoites ayaa lagu goostay marxaladda logarithmic ka dibna lagu qasi jiray dhexdhexaadinta dhexdhexaadinta, pH 7.1 (la beddelay TYI-S-33) ilaa heerka ugu dambeeya ee 1 × 106 trophozoites / mL.fiirsashada bile 0.05% dhexdhexaad ah).Trophozoites waxaa lagu dhaqay xaaladaha anaerobic ee 37°C ilaa heerka koritaanka logarithmic.U beddel dhexdhexaadinta dhexdhexaadka ah ee kicinta dhexdhexaadka ah (pH 7.8; TYI-S-33 dhexdhexaad ah oo la beddelay oo leh 1% fiirsashada bile) iyo dhaqanka G. duodenalis ee 37 ° C ee saacadaha 48-96, inta lagu jiro taas oo fiilooyinka samaynta lagu arkay mikroskoob.Ka dib markii inta badan trophozoites la kiciyay si ay u sameeyaan cysts, isku dhafka dhaqanka waa la goostay oo dib loogu celiyay biyo nadiif ah oo nadiif ah si loo lyse trophozoites haray.Faallooyinku waa la tiriyey oo lagu kaydiyey 4°C si loo baaro tubo gastric ee jiirarka.
Giardia extracellular vesicles (GEVs) ayaa la hodmay sidii hore loogu sharraxay [13].Trophozoites ee marxaladda kobaca logarithmic ayaa dib loogu celiyay TYI-S-33 dhexdhexaad ah oo wax laga beddelay oo lagu diyaariyey FBS exosome-dhamaystay (Warshadaha noolaha, Beit-Haemek, Israel) ilaa fiirsashada kama dambaysta ah ee 1 × 106 dulin / mL oo lagu beeray saacadaha 12.waxaa laga soocay dhaqamada sare ee centrifugation at 2000 g 10 min, 10,000 g 45 min, iyo 100,000 g 60 daqiiqo.Roobabku waxay ku milmeen phosphate buffered saline (PBS), oo lagu qiyaasey iyadoo la isticmaalayo xirmada baaritaanka borotiinka BCA (Thermo Fisher Scientific, Waltham, MA, USA) oo lagu kaydiyay -80° C. ama si toos ah ayaa loo isticmaalay falanqayn dheeraad ah.
Makrophages-ka jiirka hoose ee peritoneal ayaa loo diyaariyey sidii hore loogu sharraxay [24].Si kooban, jiirarka (da'doodu u dhaxayso 6-8 toddobaad) ayaa lagu duray (intrapereritoneally [ip]) oo leh 2.5 ml oo ah 2.98% Difco dareeraha thioglycol dhexdhexaad ah (BD, Franklin Lakes, NJ, USA) waxaana la quudiyay 3-4 palates.Joojinta macrophages-ka ayaa laga soo ururiyay godka caloosha ee jiirarka ka dib euthanasia waxaana la saaray 3 jeer 1000 g 10 min.Unugyada la goostay waxaa lagu ogaadey cytometry socodka iyadoo la adeegsanayo calaamadda CD11b ilaa ay nadiifnimada unuggu ahayd> 98%, ka dibna lagu daray 6-wanaagga taarikada dhaqanka unugga (4.5 x 106 unug / ceel) waxaana lagu dhex daray 10% FBS (Bioindustry) at 37 ° C.iyo 5% CO2.
RNA waxaa laga soo saaray 1 × 107 trophozoites in 1 ml oo ah TRIzol reagent (Vazyme, Nanjing, China), DNA genomic waxaa laga soo saaray wadarta G. duodenalis RNA iyadoo la adeegsanayo MonScript dsDNase (Monad, Wuhan, Shiinaha) iyo DNA dhammaystiran (cDNA) ayaa la sameeyay. iyadoo la isticmaalayo MonScript RTIIII Super Mix (Monad) sida waafaqsan tilmaamaha soo saaraha.
Macluumaadka isku xigxiga CDS ee hidoowga G. duodenalis bartilmaameedka ayaa laga helay NCBI GenBank.Isticmaal Primer 5.0 si aad u naqshadayso curiyayaasha cloning-ka ee gaarka ah ee hido kasta oo la beegsado.Qoryaha hore (5′-3′) wuxuu ka kooban yahay saddex qaybood: isku xigxig is dulsaaran oo leh pcDNA3.1 (+) EcoRV (TGGTGGAATTCTGCAGAT) toosan oo bilow codons ATG iyo GNN (haddii saldhiga kowaad aanu ahayn G).Tan waxa loo sameeyaa si loo hagaajiyo hufnaanta odhaahda.Intaa waxaa dheer, ugu yaraan 16 bp saldhigyada la isku daray (GC content 40-60%/Tm qiyaastii. 55 °C).Qoryaha gadaale (5′-3′) wuxuu ka kooban yahay laba qaybood, isku xigxiga oo leh EcoRV-linearized vector pcDNA3.1(+) (GCCGCCACTGTGCTGAT) iyo saldhig isku dhafan oo ah ugu yaraan 16 bp.(marka laga reebo labada joogsi ee u dambeeya).saldhigyada) codon sida AA ama GA si loogu ogolaado plasmids recombinant si ay u muujiyaan borotiinada calaamadeysan).Taxanayaasha asaasiga ahi waxay ku taxan yihiin shaxda 1 waxaana soo saaray Kangmet Biotechnology Co., Ltd. (Changchun, Shiinaha).
Bartilmaameedyada ayaa la xoojiyay iyadoo la adeegsanayo Pfu DNA polymerase (Tiangen, Beijing, China) ama Ex-taq (Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, Shiinaha) iyadoo la adeegsanayo G. duodenalis cDNA oo la diyaariyey qaab template ah.Odhaahda eukaryotic vector plasmid pcDNA3.1(+) ayaa lagu toosiyay xadaynta enzyme EcoRV iyo dephosphorylated iyadoo la isticmaalayo Fast AP (Thermo Fisher Scientific).Qaybaha pcDNA3.1(+) toosan iyo jajabyada hiddo-wadaha la xoojiyay ayaa lagu sifeeyay iyada oo la isticmaalayo qalabka sifaynta jel DNA (Tiangen) waxaana lagu qiyaasaa Nandrop ND-2000 (Thermo Fisher Scientific).Jajabka pcDNA3.1 (+) iyo jajab kasta oo hido-side kasta oo bartilmaameed ah ayaa la isku daray iyadoo la adeegsanayo MonClone hal isku-dhafan cloning mix (Monad Biotech Co., Ltd., Suzhou, Shiinaha) waxaana lagu xaqiijiyay taxanaha DNA iyadoo la adeegsanayo Comate Bioscience Company Limited (Changchun, China) ..
Plasmids-free endotoxin pcDNA3.1(+) -alpha-2 iyo pcDNA3.1(+) -alpha-7.3 ayaa la soo saaray iyadoo la isticmaalayo SanPrep Endotoxin-free Plasmid Mini Kit (Sangon Biotech).Isku-duubnida waxaa lagu ilaalinayay in ka badan 500 ng/µl si loo hubiyo in EDTA ee kaydiyaha elution-ka aysan faragelinin baaritaanka gudbinta.Macrophages jiirka aasaasiga ah ee peritoneal macrophages ayaa lagu beeray saxannada 6-well oo dhammaystiran RPMI 1640 dhexdhexaad ah (warshadaha noolaha) 12 saacadood, ka dibna unugyada ayaa lagu maydhay 3 jeer PBS diiran si looga saaro penicillin iyo streptomycin, ka dibna dhexdhexaad ah oo lagu kabo dhexdhexaad dhammaystiran.plasmids-free Endotoxin-free pcDNA3.1 (+) -alpha-2 iyo pcDNA3.1 (+) -alpha-7.3 (2.5 μg) ayaa lagu qasi jiray 125 μl of Opti-MEM dhexdhexaad ah serum (Gibco, Thermo Fisher Scientific) ..Dabadeed 5 µl ee Lipofectamine 2000 reagent wareejinta (Invitrogen, Thermo Fisher Scientific) ayaa lagu qasi jiray 125 µl oo ah serum Opti-MEM dhexdhexaad ah.U diyaari dhismeyaasha liposome-DNA adiga oo ku qasaya plasmid-ka endotoxin-la'aanta ah ee la milmay iyo Lipofectamine 2000 una oggolow isku darka inay ku istaagto heerkulka qolka 5 daqiiqo.Si gooni gooni ah ugu wareeji dhismooyinka unug kasta oo ceel kasta oo si tartiib ah isku qas.4 saacadood ka dib, dhexdhexaadinta dhaqanka unugyada waxaa lagu bedelay 2 ml oo dhamaystiran RPMI 1640 dhexdhexaad ah iyo dhaqanka ayaa la sii waday 24 saacadood.Dhexdhexaad dhaqameed unug cusub ayaa lagu daray unugyada waxaana lagu beeray waqtiyo kala duwan iyadoo ku xiran naqshadda qiimeynta.
Tijaabooyin borotiinno ah oo laga soo qaaday kuwa sare-sare iyo lysates unugyada ayaa loo diyaariyey sidii hore loogu sharraxay [25].Qiyaasaha wareejinta membrane ee pro-IL-1β, pro-caspase-1, caspase-1 p20, NLRP3, β-actin, iyo His-tag waxay ahaayeen 200 mA/90 min.Interleukin 1β (IL-1β; R & D Systems, Minneapolis, Minnesota, USA), Caspase-1 (p20) (Adipogen, Switzerland) iyo NLRP3 (Adipogen SA, Epalinges, Switzerland) iyo 1: 5000 oo lagu beegsanayo sumaddiisa (Amylet Scientific, Wuhan, Shiinaha) iyo β-actin (Proteintech, Wuhan, Shiinaha).
Isku-xidhka isku-tallaabta ee disuccinimide suberate (DSS) ayaa la sameeyay sidii hore loogu sharraxay [26].Unugyada waxaa lagu dhaqay 3 jeer qabow PBS waxaana si buuxda loogu safiyay irbad 27 ah oo ku jirta 50 µl ASC falcelinta falcelinta (pH 8.0) oo ka kooban 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES iyo 125 mM NaHCO3.Isku darka waxaa lagu xareeyay 5000 g 3 daqiiqo iyo pellet waxaa lagu tolay 10 µl DSS (25 mM gudaha DMSO) iyo 40 µl ASC falcelinta 30 min at 37°C.Ka dib markii centrifugation at 5000 g 10 min, pellet waxaa lagu kala direy xal of 40 µl ee falcelinta ASC iyo 10 µl of 6x borotiinka loading bakhaar (TransGen, Beijing, Shiinaha), ka dibna xalku waxaa lagu demiyey heerkulka qolka 15 min., Ka dibna karkari 10 daqiiqo.Shaybaarada borotiinka ayaa markaas lagu sammeeyay xinjirowga reer galbeedka iyadoo la adeegsanayo unugyada difaaca jirka aasaasiga ah ee ASC (Wanleibio, Shenyang, Shiinaha) marka loo eego saamiga milanka ee 1:500.
Iyadoo la raacayo habraac hore loo sharraxay [13], kor-u-qaadayaasha dhaqanka unugga ayaa la goostay oo dheecaanka cytokine pro-inflammatory IL-1β waxaa lagu go'aamiyay iyadoo la adeegsanayo qalabka IL-1 Beta ELISA (Invitrogen, Thermo Fisher Scientific).U beddelo qiyamka OD450nm isu-ururinta borotiinka iyadoo la adeegsanayo qalooca caadiga ah ee IL-1β.
Unugyada ku dahaaran daboolka ayaa si tartiib ah loogu dhaqay 3 jeer PBS diiran, oo lagu hagaajiyay unugyada unugyada unugyada (Biosharp, Beijing, China) 10 min heerkulka qolka (RT), gudaha 0.1% Triton X-Permeabilize at 100 ( lagu qasi jiray PBS; Biosharp ) 20 daqiiqo heerkulka qolka oo ku xidh 5% serum albumin bovine (ku jira PBS) 2 saacadood heerkulka qolka.Unugyada ayaa markaa lagu daray habeenkii 4 ° C oo leh unugyada difaaca aasaasiga ah ee ka soo horjeeda ASC (1: 100 dilution) ama NLRP3 (1: 100 dilution), siday u kala horreeyaan, iyo Cy3 oo ku calaamadsan riyaha ka hortagga bakaylaha IgG (H + L) (1:400; EarthOx , San Francisco, CA, USA) ama FITC-conjugated riyaha ka hortagga jiirka IgG (1:400; Earthox) habeenkii at 37 ° C gudcurka 1 saac.Nuclei-yada waxaa lagu dheehday Hoechst 33258 (10 μg/ml; UE, Suzhou, Shiinaha) daqiiqado 5 waxaana lagu arkay mikroskoob-ka fluorescence (Olympus Corporation, Tokyo, Japan).
Jiirarka ayaa loo qaybiyay afar kooxood (n = 7 koox kasta): (i) PBS-daaweeyay kooxda xakamaynta taban (PBS kaliya; gavage 100 µl / jiirka PBS oo ay ku xigto cirbadeynta maalinlaha ah ee intraperitoneal 100 µl / mouse PBS 3 saacadood ka dib) ., si joogto ah 7 maalmood);(ii) kooxda xakamaynta taban ee lagu daaweeyay MCC950 inhibitor [27] (100 µl / mouse iyada oo loo marayo PBS gavage, 3 saacadood ka dib, 10 mg / kg miisaanka jirka [BW] MCC950 [ee PBS] ayaa la maamulay intraperitoneal maalin kasta, muddada 7 maalmood);(iii) G. duodenalis cyst kooxda caabuqa (1.5 x 106 fiix / mouse by gavage, 3 saacadood ka dib, 100 μl / mouse PBS intraperitoneally maamula maalin kasta 7 maalmood);(iv) G. duodenalis cyst isku dhafan kooxda caabuqa MCC950 inhibitor kooxda daaweynta (1.5 × 106 cysts / mouse via gavage, 10mg / kg jirka MCC950 intraperitoneal maalin kasta 7 maalmood at 3h).Miisaanka jirka ee jiir kasta ayaa la kormeeray maalin kasta, dhammaan jiirarkana waa la nadiifiyey maalintii 7aad.Duodenum-ka la goostay (dhererkiisu yahay 3 cm) ayaa la googooyay qaybo yaryar oo 1 ml PBS ah, fiilooyinka ayaa la burburiyay habeenkii PBS ee 4 ° C, iyo G. duodenalis trophozoites.Duodenum cusub (dherer 1 cm) ayaa loo go'doomiyey hematoxylin iyo eosin (H&E) midabaynta.
Jiirarka ayaa loo kala qaybiyay laba kooxood: (i) kooxda xakamaynta MOCK iyo (ii) kooxda inhibitor MCC950.Waxaa jiray shan daaweyn oo koox kasta ah (n = 7 / kooxda daaweynta): (i) kooxda daaweynta xun ee PBS (PBS kaliya; 100 µl / jiirka PBS, muruqa (IM) cirbadeynta (tibialis anterior) [28, 29];( ii) pcDNA3.1 (+) kooxda xakamaynta xun ee plasmid (100 µg/jiirka DNA, iyada oo la adeegsanayo duritaanka muruqa); kooxda lagu daweeyay plasmid pcDNA3.1(+) -alpha-2 (100 μg/mouse DNA, oo lagu duray muruqa gudaha ah), iyo (v) koox lagu daweeyay plasmid pcDNA3.1(+) -alpha-7.3 (100 μg/mouse) DNA, ka dib saacadaha 12 ee marinka, jiirarka ee kooxda MCC950 inhibitor waxay heleen maalin kasta cirbadeynta intraperitoneal ee MCC950 (10 mg / kg miisaanka jidhka) ee maalmaha 7, halka jiirarka kooxda MOCK ay heleen mugga siman ee daaweynta PBS. Shaybaarada dhiigga ayaa la sameeyay. laga soo ururiyay jiirarka indhaha oo laga tagay habeenki 4 °C shaybaarada Serum ayaa la go'doomiyay iyadoo la isticmaalayo baaritaanka difaaca jirka ee enzyme-linked (ELISA) iyo cabbirada IL-1β.
Shan iyo soddon jiir ayaa loo qaybiyay shan kooxood (n=7/koox).Kooxda 1 waxay ahayd kooxda xakamaynta taban ee lagu daweeyay PBS: jiirarku waxay heleen 100 μl oo PBS ah gudaha murqaha iyo 3 maalmood kadib gavage.Kooxda 2 waa koox xakameyn togan oo qaba G. duodenalis cysts: jiirarka ayaa lagu duray 100 μl oo PBS ah, iyo 3 maalmood ka dib 1.5 x 106 cysts / mouse ayaa lagu duray gudaha caloosha.Kooxda saddexaad - tallaalka plasmid ee pcDNA3.1 (+) oo ay weheliso kooxda kantaroolka ee caabuqa cyst duodenal: jiirarka ayaa helay 100 μg oo ah DNA plasmid pcDNA3.1 (+) (im) afka, 1.5 × 106 cysts / mouse 3 dhowr maalmo.Kooxaha 4 iyo 5 waxay ahaayeen recombinant pcDNA3.1 (+) -alpha-2 giardine plasmid ama pcDNA3.1 (+) -alpha-7.3 giardine plasmid oo ay weheliso G. duodenalis cyst infectionKooxda tijaabada ah: jiirarka ayaa helay 100 μg oo pcDNA3 ah.1 (+) - giardine plasmid DNA (im), ka dibna 3 maalmood ka dib, 1.5 × 106 cysts / mouse ayaa lagu duray iyada oo loo marayo gavage.Miisaanka jirka ee jiir kasta ayaa la kormeeray ka dib markii la soo galiyay cyst G. duodenalis iyada oo loo marayo tuubada.Duodenum cusub ayaa loo ururiyay cabbirrada culeyska dulinka iyo falanqaynta wasakhaynta HE.
Isbeddellada Histopathological waxaa lagu falanqeeyay iyadoo loo eegayo habraac hore loo daabacay [30].Duodenum cusub ayaa lagu hagaajiyay unug unug hagaajinaya, oo lagu dhex daray paraffin, la gooyay qaybaha 4 μm, waxaa lagu wajahay H&E waxaana lagu lafa guray mikroskoob hoostiis.Isbeddellada cudur-sidaha ee matalayaasha ee toddoba qaybood oo unug ah oo ka yimid toddoba jiir oo madaxbannaan ayaa lagu qiimeeyay cilmi-nafsi yaqaanka oo aan ka warqabin daaweynta waxaana lagu qabtay 200x weyneynta.Dhererka villi iyo qoto dheer ee crypts ayaa lagu qiyaasay si waafaqsan hababka hore loo sharaxay.
Natiijooyinka in vitro iyo in vivo waxaa lagu helay saddex jeer.Garaafyada waxaa la sameeyay iyadoo la adeegsanayo GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, USA).Farqiga u dhexeeya labada kooxood ayaa lagu falanqeeyay t-test, halka farqiga u dhexeeya kooxaha ≥3 lagu falanqeeyay falanqayn hal dhinac ah oo kala duwanaansho ah (ANOVA) iyadoo la adeegsanayo software SPSS (nooca 22.0; SPSS IBM Corp., Armonk, NY, USA).Xogta waxaa lagu falanqeeyay isku-mid ahaanshiyaha kala duwanaanshiyaha iyadoo la adeegsanayo tijaabada Levene oo ay ku xigto Bonferroni's post hoc test (B).Muhiimada waxaa lagu muujiyey sida P<0.05, P<0.01, iyo P<0.001 (ma ahan mid muhiim ah [ns]) (P>0.05).
Falanqayntayadii hore ee GEV proteomics ee Kyoto Encyclopedia of Genes and Genomes (KEGG) waxay muujisay in bartilmaameedyo badan laga yaabo inay ku lug yeeshaan firfircoonida waddooyinka calaamadaha caabuqa [13].Waxaan dooranay laba bartilmaameed oo rajo leh, alfa-2 iyo alfa-7.3 giardins, kor u qaad molecule-yada oo u isticmaal si aan u dhisno pcDNA3.1(+) vector muujinta eukaryotic.Isku xigxiga ka dib, recombinant pcDNA3.1 (+) -alpha-2 iyo alpha-7.3 giardine expression plasmids ayaa loo beddelay macrophages mouse peritoneal macrophages, iyo kaspase-1 p20 borotiinka saxeexa caabuqa sida caddaynaya molecules muhiimka ah ee kicin kara bararka.Natiijooyinka waxay muujiyeen in alfa-2 iyo alfa-7.3 giardines ay keeni karaan p20 caspase-1 odhaah la mid ah GEV.Wax saameyn ah kuma yeelan firfircoonida kaspase-1 ayaa laga helay kantaroolka taban ee aan la daweyn (PBS kaliya) iyo kantaroolka plasmid pcDNA3.1 (+) (Jaantuska 1).
Cabbirka p20 caspase-1 firfircoonida by pcDNA3.1(+) -alpha-2 iyo alfa-7.3 giardins.Recombinant eukaryotic expression plasmids pcDNA3.1(+) -alpha-2 iyo alpha-7.3 giardines (ka korreeya haad kasta) ayaa loo bedelay makrofajiyada jiirka hoose ee xuubka xuubka iyo dhaqamada sare ee 24 saac kadib.Burburinta reer galbeedka ayaa loo isticmaalay in lagu cabbiro heerarka muujinta ee borotiinka bararsan ee caspase-1 p20.Kooxda daawaynta ee PBS-kaliya (lane C) iyo pcDNA3.1 (+) kooxda monotherapy (pcDNA3.1 haadka) ayaa loo adeegsaday xakamaynta taban, kooxda daawaynta GEV ayaa loo isticmaalay sidii xakamayn togan.Muujinta borotiinka dib-u-habaynta ayaa lagu xaqiijiyay in la ogaado sumadda histidine ee borotiin kasta, iyo xirmooyinka borotiinka ee la filayo waxay ahaayeen alfa-2 giardine (38.2 kDa) iyo alfa-7.3 giardine (37.2 kDa).GEV, Giardia duodenum vesicles ka baxsan, pcDNA3.1(+), EcoRV-linearized vector, SUP, supernatant
Si loo go'aamiyo in alfa-2 giardine iyo alfa-7.3 giardine ay keenaan p20 caspase-1 muujinta oo ay door ka ciyaaraan firfircoonida martigeliyaha NLRP3 jawaabta caabuqa, pcDNA3.1 (+) -alpha-2 giardine iyo pcDNA3.1 (+) -alpha -7.3 giardin ayaa loo beddelay macrophages jiirka hoose ee peritoneal macrophages oo leh DNA-da plasmid recombinant, iyo heerarka muujinta, meelaynta, iyo oligomerization ee borotiinnada bararka muhiimka ah ee NLRP3 ayaa la go'aamiyay.Tijaabadan, GEV waxaa loo adeegsaday kooxda kantaroolka togan, iyo kooxda daawaynta (PBS kaliya) ama pcDNA3.1 (+) kooxda daawaynta gudbinta waxay ahayd kooxda taban.Natiijooyinku waxay muujiyeen in, sida kooxda GEV, DNA plasmid recombinant of giardin pcDNA3.1 (+) -alpha-2 iyo giardin pcDNA3.1 (+) -alpha-7.3 waxay keentay kor u kaca NLRP3, pro-IL-1β iyo procaspase-1 iyo kaspase-1 firfircoonida (Jaantus. 2a).Intaa waxaa dheer, labada giardine waxay keeneen dheecaanka IL-1β ee muhiimka ah (pcDNA3.1: ANOVA, F (4, 10) = 1.625, P = 0.1000; alfa-2 giardine: ANOVA, F (4, 10) = 1.625, P = 0.0007 ).;alfa-7.3 giardine: ANOVA, F(4, 10) = 1.625, P<0.0001;GEV: ANOVA, F(4, 10) = 1.625, P = 0.0047) (Jaantuska 2b).Inta badan borotiinada ASC waxay ahaayeen monomeric kooxda daawaynta la'aanta ama kooxda daawaynta ee lagu beddelay pcDNA3.1 (+) plasmid, marka loo eego pcDNA3.1 (+) -alpha-2 ama pcDNA3.1 (+) -alpha- 7.3 Giardine.ASC oligomerization waxay ku dhacday DNA-da plasmid-ka ee dib-u-dhiska ah ee kooxda xakamaynta togan ee GEV, oo muujinaysa qaabka oligomeric (Jaantus 2c).Xogtan hordhaca ah waxay soo jeedinaysaa in alfa-2 giardine iyo alpha-7,3 giardine ay keeni karaan firfircoonida caabuqa NLRP3.Daraasadaha soo socda ee immunofluorescent ee meelaynta ASC iyo NLRP3 waxay muujiyeen in kooxda xakamaynta taban, borotiinka ASC uu ku kala firidhsan yahay cytoplasm wuxuuna u muuqday calaamad muujinaysa kicinta pcDNA3.1 (+) -alpha-2 oo leh giardine ama pcDNA3.1 (+) -alpha-7,3 kooxda giardine ama kooxda xakamaynta togan ee GEV (Jaantuska 2d).Xakamaynta taban iyo kooxaha pcDNA 3.1 ee plasmid-lagu daweeyay, calaamada borotiinka NLRP3 lama ogaanin, halka dhibicda calaamada fluorescent ee jawaabta pcDNA3.1 (+) -alpha-2 giardine ama pcDNA3.1 (+) -alpha-7.3 ayaa la ogaaday..giardine waxaa laga helaa cytoplasm ama marka la kicinayo HEV (Jaantus. 2e).Xogtaasi waxay sii muujinaysaa in G. duodenalis giardin alpha-2 iyo giardin alpha-7.3 ay kiciyaan caabuqa NLRP3 ee macrophages hoose ee peritoneal mouse.
pcDNA3.1 (+) -alpha-2 giardin iyo pcDNA3.1 (+) -alpha-7.3 giardin waxay kicisaa caabuqa NLRP3 ee mouse peritoneal macrophages.U beddelo muujinta eukaryotic recombinant plasmids pcDNA3.1 (+) -alpha-2 giardin iyo pcDNA3.1 (+) -alpha-7.3 giardin galay macrophages xuubka murine aasaasiga ah iyo unugyo, ama goosto supernatant gudaha 24 h si loo falanqeeyo muujinta, oligomerization , dheecaaniyo meelaynta borotiinnada caabuqa ee muhiimka ah.Kooxda PBS-keliya (C) iyo pcDNA3.1 (+) kooxda daawaynta kaliya ayaa loo isticmaalay sidii xakamaynta taban, kooxda daawaynta GEV ayaa loo isticmaalay sidii kooxda togan.NLRP3 borotiinada muhiimka ah ee bararka, oo ay ku jiraan NLRP3, pro-IL-1β, pro-caspase-1, iyo p20 caspase-1, ayaa lagu ogaaday xannibaadda Galbeedka.b Heerarka dheecaanka IL-1β ee ka sarreeya kuwa sare waxaa lagu go'aamiyay iyadoo la adeegsanayo baaritaanka difaaca jirka ee ku xiran enzyme (ELISA).Farqiga u dhexeeya kantaroolka iyo kooxaha tijaabada ah ayaa lagu falanqeeyay falanqayn hal dhinac ah oo kala duwanaanshiyaha ah (ANOVA) iyadoo la adeegsanayo nooca software SPSS 22.0.Asterisks ayaa tilmaamaya farqi weyn oo u dhexeeya kooxaha **P<0.01 iyo ***P<0.001.c Heerarka oligomerization ASC ee pellets waxaa lagu go'aamiyay falanqaynta isku-xidhka DSS, halka heerarka ASC ee lysates-ka unugyada loo isticmaalay sidii xakamaynta loading.d Aragtida meelaynta ISC iyadoo la adeegsanayo immunofluorescence.e Immunofluorescence ayaa loo isticmaalay si loo arko meelaynta NLRP3.ASC, borotiinka speck-sida apoptotic;IL, interleukin;NLRP3, nucleotide-binding oligomerization-like reseptor 3;ns, aan muhiim ahayn (P> 0.05)
Labada G. duodenalis iyo GEV-yada ay siraysaa waxay dhaqaajiyaan bararka NLRP3 waxayna nidaamiyaan jawaabaha bararka martida loo yahay ee vitro.Sidaa darteed, doorka NLRP3 inflammasome ee cudur-sidaha G. duodenalis ayaa weli ah mid aan caddayn.Si loo baaro arrintan, waxaan naqshadeynay tijaabo u dhexeeya jiirarka qaba G. duodenalis cyst iyo jiirarka qaba G. duodenalis cyst + MCC950 daaweynta inhibitor iyo marka la barbardhigo NLRP3 muujinta caabuqa marka uu ku dhaco G. duodenalis cyst.Nidaam faahfaahsan oo tijaabo ah ayaa lagu muujiyay sawirka 3a.Isbeddellada miisaanka jidhka ee jiirarka ee kooxaha daawaynta ee kala duwan ayaa la kormeeray 7 maalmood ka dib caabuqa fiix, iyo natiijooyinka waxaa lagu muujiyay sawirka 3b.Marka la barbardhigo kooxda lagu daaweeyay PBS saafi ah, natiijadu waxay muujisay in (i) miisaanka jidhka ee jiirarka ee G. duodenalis cyst uu hoos u dhacay maalinta 3 ilaa maalinta 7 ka dib caabuqa;(ii) daaweynta MCC950 inhibitor wax saameyn ah kuma yeelan miisaanka jirka ee jiirarka..Marka la barbardhigo kooxda caabuqa kaliya, BW ee kooxda infekshanka duodenal ee lagu daaweeyay MCC950 waxay hoos ugu dhacday heerar kala duwan (Maalinta 1: ANOVA, F (3, 24) = 1.885, P = 0.0148; Maalinta 2: ANOVA, F (3, 24 ) = 0.4602, P <0.0001; Maalinta 3: ANOVA, F (3, 24) = 0.8360, P = 0.0010; Maalinta 4: ANOVA, F (3, 24) = 1.683, P = 0.0052; (3, 24) = 0.6497, P=0.0645; Maalinta 6: ANOVA, F(3, 24)=5.457, P=0.0175; Maalinta 7: ANOVA, F(3, 24) = 2.893, P = 0.0202).Xogtaasi waxay muujineysaa in bararka NLRP3 uu ka ilaaliyo jiirarka miisaan lumis weyn marxaladaha hore (2-4 maalmood) ee caabuqa duodenal.Waxaan markaa ujeednay inaan ogaano G. duodenalis trophozoites ee dareeraha duodenal lavage natiijadiina waxaa lagu muujiyay sawirka 3c.Marka la barbardhigo kooxda G. duodenalis cyst infekshinka, tirada trophozoites ee duodenum ayaa si weyn u kordhay ka dib markii la xannibay caabuqa NLRP3 (t (12) = 2.902, P = 0.0133).Unugyada duodenal ee lagu shubay HE waxay muujiyeen, marka la barbardhigo xakamaynta taban ee lagu daaweeyay PBS iyo MCC950 oo keliya: (i) G. duodenalis cyst infekshanku wuxuu keenay dhaawac soo gaadhay villi duodenal (ANOVA, F (3, 24) = 0.4903, P= 0.0488 ) iyo crypt atrophy (ANOVA, F (3, 24) = 0.4716, P = 0.0089);(ii) duodenum ka soo baxa jiirarka oo qaba fiix G. duodenalis waxaana lagu daweeyay inhibitors MCC950.Duodenal villi ayaa waxyeelo soo gaartay oo dhintay (ANOVA, F (3, 24) = 0.4903, P = 0.0144) oo leh atrophy iyo crypt branching (ANOVA, F (3, 24) = 0.4716, P = 0, 0481) (Sawir 3d- f)Natiijooyinkani waxay soo jeedinayaan in NLRP3 inflammasome uu door ka ciyaaro dhimista cudur-sidaha G. duodenalis.
Doorka NLRP3 caabuqa caabuqa duodenum ee Giardia.Jiirarka ayaa lagu gafay (iv) oo leh fiix duodenococcal ka dibna lagu daaweeyay ama la'aanteed MCC950 (ip).Kooxaha daawaynta kaliya ee leh PBS ama MCC950 ayaa loo isticmaalay sidii kontaroolo.Kooxda tijaabada ah iyo habka daaweynta.b Miisaanka jidhka ee jiirarka mid kasta oo ka mid ah kooxaha daaweynta kala duwan ayaa la kormeeray 7 maalmood.Farqiga u dhexeeya kooxda caabuqa ee G. duodenalis iyo G. duodenalis + MCC950 kooxda daawaynta caabuqa ayaa lagu falanqeeyay t-tijaab iyadoo la adeegsanayo nooca software SPSS 22.0.Xiddigogu waxay muujinayaan kala duwanaansho weyn xagga * P <0.05, **P <0.01, ama *** P <0.001.c Culayska dulinka waxaa lagu go'aamiyay tirinta tirada trophozoites ee dareeraha lavage duodenal.Farqiga u dhexeeya kooxda caabuqa ee G. duodenalis iyo G. duodenalis + MCC950 kooxda daawaynta caabuqa ayaa lagu falanqeeyay t-tijaab iyadoo la adeegsanayo nooca software SPSS 22.0.Xiddigogu waxay muujinayaan kala duwanaansho weyn xagga * P <0.05.d Hematoxylin iyo eosin (H&E) natiijooyinka midabaynta ee histopathology duodenal.Fallaadhaha guduudan waxay muujinayaan dhaawac soo gaaray villi, fallaadhaha cagaaran waxay muujinayaan dhaawaca crypts.Cabirka: 100 µm.e, f Falanqaynta tirakoobka ee dhererka villus duodenal iyo dhererka mouse crypt.Xiddigogu waxay muujinayaan kala duwanaansho weyn xagga * P<0.05 iyo **P <0.01.Natiijooyinka waxaa laga soo qaatay 7 tijaabo oo bayooloji oo madax banaan.BW, miisaanka jidhka;ig, marinka dhalmada intragastric;ip, marinka gudbinta intraperitoneal;ns, aan muhiim ahayn (P> 0.05);PBS, cusbo fosfooraska buffered;WT, nooca duurjoogta ah
Siinta IL-1β waa calaamad muujinaysa firfircoonida caabuqa.Si loo go'aamiyo in G. duodenalis alpha-2 giardine iyo alpha-7.3 giardine ay kiciyaan NLRP3 martida loo yahay inflammasome in vivo, waxaan isticmaalnay jiirarka WT ee aan la daweyn (kooxdii shaambada) iyo NLRP3 jiirarka caabuqa-xiran (MCC950 kooxda daawaynta ee la joojiyay).Nidaam faahfaahsan oo tijaabo ah ayaa lagu muujiyay sawirka 4a.Kooxaha tijaabada ah waxay ka kooban yihiin jiirar lagu daaweeyay PBS, G. duodenalis cyst daawaynta by gavage, duritaanka intramuscular ee pcDNA3.1, iyo duritaanka intramuscular pcDNA3.1 (+) -alpha-2 giardine ama pcDNA3.1-alpha-7.3 giardine.Maalintii 7-aad ka dib maamulka muruqyada ee plasmid recombinant, serum ayaa la ururiyey waxaana la go'aamiyay heerka IL-1β ee koox kasta.Sida ku cad Jaantuska 4b, kooxda MOCK: (i) marka la barbar dhigo kooxda PBS, daawaynta pcDNA3.1 wax saameyn ah kuma yeelan dheecaanka IL-1β (ANOVA, F (4.29) = 4.062, P = 0.9998), si kastaba ha ahaatee, Qarsoodi IL-β ayaa si weyn kor loogu qaaday kooxda G. duodenalis cyst (ANOVA, F (4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine iyo pcDNA3.1- Cirridka muruqa ee alfa-7.3 giardine ayaa si weyn u kordhay heerarka serum IL-1β (ANOVA, F (4, 29) = 4.062, P <0.0001);(iii) pcDNA3.1-alpha-7,3 giardine waxay keentay heerarka sare ee IL -1β ee dheecaanka pcDNA3.1-alpha-2 giardine intramuscular injection group (ANOVA, F (4, 29) = 4.062, P = 0.0333) .Marka la barbardhigo koox kasta oo ka tirsan kooxda daaweynta MCC950 iyo kooxda MOCK: (i) IL-1β heerarka qarsoodiga ah ee kooxda xakamaynta PBS iyo kooxda kontoroolka pcDNA3.1 ayaa hoos u dhacday ilaa xad ka dib markii ay xannibeen inhibitor MCC950, laakiin farqigu ma ahayn muhiim ah (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F (9, 58) = 3.540, P = 0.5949);(ii) Ka dib markii la xannibay MCC950., dheecaanka IL-1β ayaa si weyn hoos ugu dhacay kooxda G. duodenalis cyst-infection, kooxda pcDNA3.1-alpha-2 giardine, iyo pcDNA3.1-alpha-7.3 kooxda giardine (G. duodenalis: ANOVA, F (9) , 58) = 3.540, P = 0.0120; pcDNA3.1-alpha-2 giardine: ANOVA, F (9, 58) = 3.540, P = 0.0447; pcDNA3.1-alpha-7.3 giardine: ANOVA, F ) = 3.540, P = 0.0164).Natiijooyinkani waxay soo jeedinayaan in alfa-2 giardine iyo alfa-7.3 giardine ay dhexdhexaadiyaan firfircoonida caabuqa NLRP3 ee vivo.
pcDNA3.1(+) -giardines waxay kicisaa NLRP3 martida loo yahay hurgunka gudaha vivo.Jiirarka ayaa lagu tallaalay (IM) oo leh recombinant eukaryotic expression plasmid pcDNA3.1 (+) -alpha-2 giardine ama pcDNA3.1 (+) -alpha-7.3 giardine ka dibna lagu daaweeyay MCC950 (ip; MCC950 group) ama maya (koox dummy). ).PBS ama pcDNA3.1 (+) kooxda daawaynta plasmid ayaa loo isticmaalay xakamaynta xun, kooxda daawaynta cyst ee G. duodenalis ayaa loo isticmaalay sidii xakamayn togan.Kooxda tijaabada ah iyo habka daaweynta.b Heerarka serum ee IL-1β ee jiirarka ayaa lagu cabbiray maalinta 7 by ELISA assay.Farqiga u dhexeeya kooxaha kooxda MOCK ayaa lagu falanqeeyay iyada oo la adeegsanayo hal dhinac oo ANOVA ah, farqiga u dhexeeya kooxda MOCK iyo kooxda MCC950 ayaa la falanqeeyay iyada oo la adeegsanayo t-tijaabka nooca software SPSS 22.0.Asterisks waxay tilmaamaysaa farqi weyn oo u dhexeeya kooxaha daawaynta ee kooxda MOCK, * P <0.05 iyo *** P <0.001;calaamadaha dollarka ($) waxay muujinayaan faraqa weyn oo u dhexeeya koox kasta oo ka tirsan kooxda MOCK iyo kooxda MCC950 ee P<0.05.Natiijooyinka todoba tijaabo oo bayooloji oo madax banaan.i, duritaanka muruqa, ns, aan muhiim ahayn (P> 0.05)
Si loo baaro saameynta alfa-2 iyo alfa-7.3 giardine-dhexdhexaadinta firfircoonida NLRP3 martida loo yahay caabuqa G. duodenalis, waxaan isticmaalnay jiirarka WT C57BL / 6 waxaana lagu duray alfa-2 giardine iyo alfa-7.3 giardine.plasmid-ka ayaa lagu duray muruqa, ka dib 3 maalmood iyada oo loo marayo tuubada gastric ee cyst G. duodenalis, ka dib jiirarka ayaa la arkay 7 maalmood.Nidaam faahfaahsan oo tijaabo ah ayaa lagu muujiyay sawirka 5a.Miisaanka jirka ee jiir kasta ayaa la qiyaasay maalin kasta, muunado cusub oo duodenal ah ayaa la ururiyey maalintii 7aad ka dib markii la maamulay iyada oo loo marayo tuubo gastric, tirada trophozoites ayaa la qiyaasay, iyo isbeddello histopathological ah ayaa la arkay.Sida ku cad Jaantuska 5b, wakhtiga quudinta oo kordhaysa, BW ee jiirarka ee koox kasta ayaa si tartiib tartiib ah u kordhay.MT ee jiirarka ayaa bilaabay inay hoos u dhigaan maalinta 3aad ka dib maamulka intragastric ee cysts G. duodenalis, ka dibna si tartiib tartiib ah u kordhay.Dhaqdhaqaaqa NLRP3 inflammasome oo ay keentay cirbadeynta muruqa ee alfa-2 giardine iyo alpha7.3 giardine ayaa si weyn u yareeyey miisaanka luminta jiirarka (Maalinta 1: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 1.399, P = 0.9754 Maalinta 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30)=1.399, P=0.9987 Maalinta 2: pcDNA3.1-alpha-2 giardine, ANOVA, F( 4, 30) = 0.3172, P = 0.9979; Maalinta 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.3172, P = 0.8409; Maalinta 3: pcDNA3.1-alpha-2, F 4, 30) = 0.8222, P = 0.0262 Maalinta 3: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.8222, P = 0.0083; Maalinta 4: pcDNA3.1-alpha-2 giardine , F (4, 30) = 0.5620, P = 0.0012, Maalinta 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5620, P <0.0001, Maalinta 5: pcDNA3.1-alpha - 2 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Maalinta 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Maalinta 6: pcDNA3 alfa-2 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0012, Maalinta 6: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0006;Maalinta 7: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.5369, P<0.0001 Maalinta 7: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5369, P <0.0001).Culayska dulin ayaa lagu qiimeeyay duodenum (Jaantus. 5c).Marka la barbardhigo xakamaynta togan ee aan la daweynin iyo kooxda lagu duro pcDNA3.1 vector madhan, tirada G. duodenalis trophozoites ayaa si weyn hoos ugu dhacday kooxaha lagu duray α-2 giardine iyo 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).Intaa waxaa dheer, giardine alfa-7.3 wuxuu ahaa mid ka ilaalin badan jiirka marka loo eego giardine alfa-2 (ANOVA, F (3, 24) = 1.209, P = 0.0081).Natiijooyinka midabaynta HE waxaa lagu muujiyey berdaha.5d–f.Jiirarka lagu duray alfa-2 giardine iyo alfa-7.3 giardine waxay lahaayeen nabarro unugyo duodenal ah oo yar, oo ay ka muuqato waxyeellada villus, marka la barbar dhigo jiirarka lagu duray G. duodenalis iyo jiirarka lagu duray G. duodenalis oo ay weheliso faaruq pcDNA3 vector .1 Zoom.(pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 2.466, P = 0.0035 ama P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F (3, 24) = 2.466, P = 0.0028 ama P = 0.0055) iyo hoos u dhaca atrophy crypt (pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 1.470, P = 0.0264 ama P = 0.0158; pcDNA3.1-alpha-24.3 giardine , F (3, 24) = 1.470, P = 0.0371 ama P = 0.0191).Natiijooyinkani waxay soo jeedinayaan in alfa-2 giardine iyo alfa-7,3 giardine ay yareeyaan infekshanka G. duodenalis iyaga oo kicinaya NLRP3 inflammasome in vivo.
Doorka pcDNA3.1(+) -giardins ee caabuqa G. duodenalisJiirarka ayaa lagu tallaalay (IM) oo leh recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine ama pcDNA3.1 (+) -alpha-7.3 giardine ka dibna lagu xujeeyay G. duodenalis cysts (ig).Kooxda PBS iyo pcDNA3.1(+) + kooxda daawaynta cyst duodenal ayaa loo isticmaalay sidii kooxaha xakamaynta xun, kooxda daaweynta cyst duodenal ayaa loo isticmaalay sidii kooxda xakamaynta togan.Kooxda tijaabada ah iyo habka daaweynta.b MT ee jiirarka mid kasta oo ka mid ah kooxaha daawaynta ee kala duwan ayaa la kormeeray 7 maalmood kahor tartanka.Asterisks waxay tilmaamaysaa farqi weyn oo u dhexeeya kooxaha G. duodenalis group iyo pcDNA3.1(+) -alpha-2 kooxda giardine, *P <0.05, **P <0.01, iyo ***P <0.001;calaamada dollarka ($) waxay tusinaysaa farqi wayn oo u dhexeeya koox kasta oo ah G. duodenalis iyo pcDNA3.1(+)-alpha-7.3 jardine group, $$P <0.01 iyo $$$P<0.001.c culeyska dulinka waxaa lagu go'aamiyay tirinta tirada trophozoites ee 1 ml oo ah duodenal lavage duodenum (dheer 3 cm) oo lagu muujiyay sida tirada dulin per cm ee duodenum.Farqiga u dhexeeya kooxda caabuqa ee G. duodenalis, pcDNA3.1 (+) -alpha-2 giardine group, iyo pcDNA3.1 (+) -alpha-7.3 kooxda giardine ayaa lagu falanqeeyay hal dhinac ANOVA iyadoo la adeegsanayo nooca software SPSS 22.0.Asterisks waxay muujinayaan kala duwanaansho muhiim ah **P<0.01 iyo ***P<0.001.d Isbeddellada Histopathological ee Duodenum.Fallaadhaha guduudan waxay muujinayaan dhaawac soo gaaray villi, fallaadhaha cagaaran waxay muujinayaan dhaawaca crypts.Cabirka: 100 µm.fFarqiga u dhexeeya kooxaha Jaantuska 1d waxaa falanqeeyay hal dhinac oo ANOVA ah iyadoo la adeegsanayo nooca software SPSS 22.0.Xiddigogu waxay muujinayaan kala duwanaansho weyn xagga * P<0.05 iyo **P <0.01.Natiijooyinka todoba tijaabo oo bayooloji oo madax banaan.ns, aan muhiim ahayn (P> 0.05)
Giardia duodenum waa dulin xiidmaha si fiican loo yaqaan ee aadanaha iyo naasleyda kale ee sababa Giardiasis.2004tii, waxaa lagu daray Hay'adda WHO ee Dayacan Cudurrada iyadoo ay ugu wacan tahay faafitaankeeda sare ee sanadaha 6, gaar ahaan bulshooyinka heerka dhaqaale ee hooseeya [32].Nidaamka difaaca jirka ayaa door muhiim ah ka ciyaara jawaabta difaaca ee caabuqa G. duodenalis.Makrophages-ka mouse-ka ayaa lagu soo warramey in ay qariyeen oo ay dilaan G. duodenalis iyaga oo siidaaya dabinada unugyada ka baxsan [33].Daraasadaheenii hore waxay muujiyeen in G. duodenalis, dulin-ka-baxsan oo aan fiicneyn, oo kicinaya p38 MAPK, ERK, NF-κB p65, iyo NLRP3 waddooyinka calaamadaha caabuqa ee macrophages mouse si loo xakameeyo jawaabaha caabuqa ee martida loo yahay, oo la sii daayo GEV ayaa kor u qaadi karta habkan.13], 24].Si kastaba ha ahaatee, PAMP-yada saxda ah ee ku lug leh NLRP3 caabuqa habaysan ee caabuqa ee GEV iyo doorka NLRP3 inflammasome ee giardiasis ayaa weli ah in la caddeeyo.Labadan su’aalood si aan u iftiimino, waxaanu samaynay daraasaddan.
Inflammasome NLRP3 wuxuu ku yaalaa cytoplasm ee unugyada difaaca waxaana lagu hawlgelin karaa qaybo kala duwan sida kiristaalo uric acid, sunta, bakteeriyada, fayrasyada, iyo dulinnada.Daraasadaha bakteeriyada, sunta ayaa loo aqoonsaday inay yihiin PAMP-yada muhiimka ah ee dhaqaajiya dareemayaasha caabuqa, taasoo keenta bararka iyo dhimashada unugyada [34].Qaar ka mid ah sunta qaabdhismeedka kala duwan, sida hemolysin oo ka yimid Staphylococcus aureus [35] iyo Escherichia coli [36], hemolysin BL (HBL) oo ka soo jeeda enterotoxin (NHE) [37], waxay keenaan firfircoonida caabuqa NLRP3.Daraasadaha fayrasku waxay muujiyeen in borotiinnada fayraska sida SARS-COV-2 baqshadda (E) borotiinka [38] iyo borotiinka NS5 fayraska [39] ay yihiin PAMP-yada muhiimka ah ee uu aqoonsaday soo-dhoweeyaha NLRP3.Daraasadaha dulinka, dulin badan ayaa lagu soo warramey inay la xiriiraan firfircoonida bararka martida loo yahay, sida Toxoplasma gondii, Trichomonas vaginalis [40], Trypanosoma cruzi [41], iyo Leishmania [42].Borotiinka granule cufan GRA35, GRA42, iyo GRA43, oo la xidhiidha fayraska Toxoplasma gondii, ayaa looga baahan yahay soo-saarka pyroptosis ee macrophages Lewis rat [43].Intaa waxaa dheer, qaar ka mid ah daraasadaha Leishmania waxay diiradda saareen molecules shakhsi ahaaneed ee ku lug leh caabuqa NLRP3, sida lipophosphoglycan xuubka dulin [44] ama zinc metalloprotease [45].Ka mid ah qoyska annexin-sida alfa-giardin ee hiddo-wadaha, alfa-1 giardin ayaa lagu muujiyay inuu yahay musharax tallaal oo ka hortagaya G. duodenalis ee qaabka jiirka [18].Daraasadayada, waxaanu ku dooranay G. duodenalis virulence factors alpha-2 iyo alpha-7,3 giardines, kuwaas oo u gaar ah giardia laakiin wax yar ayaa la soo sheegay.Labadan hiddo-wade ee bartilmaameedka ah ayaa lagu xidhay pcDNA3.1 (+) habka muujinta eukaryotic vector ee falanqaynta firfircoonida caabuqa.
Qaabka mouse-kayada, jajabyada caspase ee la jeexjeexay waxay u adeegaan calaamado kicinta bararka.Marka la kicinayo, NLRP3 waxay la falgashaa ASC, waxay qortaa procaspases, waxayna abuurtaa kasbasyo firfircoon kuwaas oo u kala jiidaya pro-IL-1β iyo pro-IL-18 qaan IL-1β iyo IL-18, siday u kala horreeyaan -18.Caspases-ka bararka (caspases-1, -4, -5 iyo -11) waa qoys la ilaaliyo oo ka mid ah borotiinka cysteine ​​​​ee muhiim u ah difaaca gudaha waxayna ku lug leeyihiin bararka iyo dhimashada unugyada barnaamijka [46].Caspase-1 waxaa kiciyay inflammasomes canonical [47], halka kaspases-4, -5, iyo -11 la jeexjeexay inta lagu jiro samaynta caabuqa aan caadiga ahayn [48].Daraasaddan, waxaan u isticmaalnay macrophages mouse peritoneal macrophages sida moodel waxaanan baaray p20 caspase-1 oo la jarjaray kasbase-1 oo calaamad u ah firfircoonida caabuqa NLRP3 ee daraasadaha caabuqa G. duodenalis.Natiijooyinku waxay muujiyeen in badan oo alfa-giardins ah ayaa mas'uul ka ah firfircoonida caadiga ah ee caabuqa, taas oo la socota helitaanka unugyada fayrasyada muhiimka ah ee ku lug leh bakteeriyada iyo fayraska.Si kastaba ha ahaatee, daraasaddeenu waa muraayad horudhac ah oo keliya waxaana jira unugyo kale oo kicin kara caabuqyada aan caadiga ahayn, sida daraasaddeennii hore lagu helay inflammasomes classical iyo non-classical infekshanka G. duodenalis [13].Si loo sii go'aamiyo in p20 caspase-1 ee la soo saaray ay la xiriirto caabuqa NLRP3, waxaan u gudbinay alpha-2 iyo alpha-7.3 giardins galay macrophages peritoneal mouse si loo go'aamiyo heerarka muujinta borotiinka molecule muhiimka ah iyo heerarka oligomerization ASC, taasoo xaqiijinaysa in labadaba α-giardins ay dhaqaajiyaan. NLRP3 oo barara.Natiijooyinkayagu wax yar bay ka duwan yihiin kuwa Manko-Prykhoda et al., kuwaas oo sheegay in kicinta unugyada Caco-2 ee G. muris ama E. coli EPEC oo keliya ay kordhin karaan xoojinta fluorescence ee NLRP3, ASC, iyo caspase-1, in kasta oo aan si weyn loo arag, halka sida kharashka G. Muris iyo E. coli ay u kordhiyeen heerarka saddexda borotiin [49].Kala duwanaanshuhu wuxuu noqon karaa kala duwanaanshaha xulashada noocyada Giardia, khadadka unugyada, iyo unugyada asaasiga ah.Waxaan sidoo kale ku samaynay shaybaarka vivo anagoo adeegsanayna MCC950 dheddig 5-todobaad jirta WT C57BL/6 jiirarka, kuwaas oo aad ugu nugul G. duodenalis.MCC950 waa molecule yar yar oo awood leh oo la xushay inhibitor NLRP3 kaas oo xannibaya firfircoonida NLRP3 kanonical iyo non-canonical ee uruurinta nanomolar.MCC950 waxay joojisaa firfircoonida NLRP3 laakiin ma saameynayso firfircoonida AIM2, NLRC4, iyo NLRP1 dariiqyada caabuqa ama dariiqyada calaamadaha TLR [27].MCC950 waxay xannibaysaa firfircoonida NLRP3 laakiin ma joojinayso NLRP3 bilawga, K+ efflux, Ca2+ qulqulka, ama isdhexgalka ka dhexeeya NLRP3 iyo ASC;Taa beddelkeeda, waxay joojisaa firfircoonida NLRP3 ee bararka iyadoo xannibaysa ASC oligomerization [27].Sidaa darteed, waxaan u isticmaalnay MCC950 daraasad ku jirta vivo si loo go'aamiyo doorka caabuqa NLRP3 ka dib duritaanka giardine.Caspase-1 p10 firfircooni waxay u kala dillaacdaa cytokines pro-inflammatory pro-IL-1β iyo pro-IL-18 galay IL-1β iyo IL-18 [50].Daraasaddan, heerarka serum IL-1β ee jiirarka giardine-la daaweeyay ee leh ama aan lahayn MCC950 ayaa loo isticmaalay tilmaame ah in caabuqa NLRP3 la hawlgeliyay.Sida la filayo, daaweynta MCC950 waxay si weyn hoos ugu dhigtay heerarka serum IL-1β.Xogtaasi waxay si cad u muujineysaa in G. duodenalis giardin alfa-2 iyo giardin alfa-7.3 ay awoodaan inay kiciyaan jiirka NLRP3 ee caabuqa.
Xogta muhiimka ah ee la ururiyay tobankii sano ee la soo dhaafay ayaa muujisay in IL-17A ay tahay maamulaha sare ee xasaanadda ka dhanka ah G. Muris, taasoo keentay calaamadda IL-17RA, soo saarista peptides antimicrobial, iyo nidaaminta firfircoonida dhamaystirka [51].Si kastaba ha noqotee, infekshanka Giardia wuxuu ku badan yahay dhalinyarada qaangaarka ah, waxaana la sheegay in caabuqa Giardia ee jiirarka da'da yar uusan kicin jawaabta IL-17A si uu u sameeyo saameyntiisa ilaalinta [52], taasoo keentay cilmi-baarayaasha inay raadiyaan Giardia kale oo difaaca jirka ah.hababka caabuqa helminth.Qorayaasha daraasad dhowaan la sameeyay ayaa sheegay in G. muris uu kicin karo caabuqa NLRP3 ee E. coli EPEC, kaas oo kor u qaadaya soo saarista peptides antimicrobial waxayna yaraynaysaa awoodda ku-xirnaanta iyo tirada trophozoites ee xiidmaha, taas oo yaraynaysa darnaanta xiidanka. cudurada ay keento bacilli [49].Inflammasome NLRP3 wuxuu ku lug leeyahay horumarinta cudurro kala duwan.Daraasaduhu waxay muujiyeen in Pseudomonas aeruginosa ay kiciso autophagy ee macrophages si looga fogaado dhimashada unugyada, habkani wuxuu ku xiran yahay firfircoonida NLRP3 inflammasome [53].Wixii N. caninum, noocyada ogsijiinta falcelinta-dhexdhexaadinta firfircoonida NLRP3 inflammasome waxay xaddidaysaa ku-noqoshadeeda martida loo yahay, taasoo ka dhigaysa bartilmaameed daweyn oo suurtagal ah [9].Paracoccidioides brasiliensis ayaa la ogaaday in ay kiciso firfircoonida NLRP3 inflammasome ee unugyada dendritic ee dhuuxa lafta, taasoo keentay sii deynta cytokine bararka IL-1β, kaas oo door muhiim ah ka ciyaara difaaca martida [10].Dhowr nooc oo Leishmania ah, oo ay ku jiraan L. amazonensis, L. major, L. braziliensis, iyo L. infantum chagasi, waxay dhaqaajiyaan NLRP3 iyo Caspase-ku-tiirsan ASC-1 ee macrophages, iyo sidoo kale caabuqa Leishmania.Ku-noqoshada dulinku waxa lagu xoojiyey jiirarka ku liidata hidda-wadaha NLRP3/ASC/caspase-1 [11].Zamboni iyo al.Infekshanka Leishmania ayaa la soo sheegay inuu kiciyo kicinta caabuqa NLRP3 ee macrophages, kaas oo xaddidaya ku-noqoshada dulinka unugyada.Markaa, Leishmania waxa laga yaabaa inay hor istaagto hawlgelinta NLRP3 sidii istiraatijiyad ka fogaansho ah.Daraasadaha vivo, NLRP3 inflammasome waxay gacan ka gaysatay ciribtirka Leishmania, laakiin ma saameynin unugyada [54].Taa bedelkeeda, daraasadaha helminthiasis, kicinta NLRP3 inflammasome waxay xakamaysay difaaca ka hortagga helminthiasis ee caloosha [12].Shigella waa mid ka mid ah bakteeriyada ugu badan ee keenta shubanka adduunka oo dhan.Bakteeriyadani waxay keeni kartaa wax soo saarka IL-1β iyada oo loo marayo P2X7 reseptor-mediated K+ efflux, noocyada oksijiinta falcelinta, acidification lysosomal, iyo dhaawaca mitochondrial.Cudurka NLRP3 wuxuu si xun u maamulaa phagocytosis iyo dhaqdhaqaaqa bakteeriyada ee macrophages ka dhanka ah Shigella [55].Daraasadaha Plasmodium waxay muujiyeen in AIM2, NLRP3 ama kaspase-1 jiirka yar ee qaba Plasmodium ay soo saaraan heerar sare oo ah nooca 1 interferon waxayna aad uga adkeysanayaan caabuqa Plasmodium [56].Si kastaba ha noqotee, doorka alfa-2 giardine iyo alfa-7.3 giardine ee kicinta firfircoonida caabuqa ee NLRP3 ee jiirarka ma cadda.
Daraasaddan, joojinta NLRP3 caabuqa ee MCC950 waxay hoos u dhigtay BW waxayna kordhisay tirada trophozoites ee dareeraha mindhicirka mindhicirka ee jiirarka, taasoo keentay isbeddello ba'an oo bahal ah oo ku dhaca unugyada duodenal.Alpha-2 giardine iyo alfa-7.3 giardine waxay kiciyaan jiirka martida loo yahay ee NLRP3 hurgun, kordhinta miisaanka jidhka jiirka, waxay yareeyaan tirada trophozoites ee dheecaanka xiidmaha mindhicirka, waxayna yareeyaan dhaawacyada duodenal pathological.Natiijooyinkani waxay soo jeedinayaan in G. duodenalis uu kicin karo NLRP3 martida loo yahay inflammasome iyada oo loo marayo alpha-2 giardine iyo alpha-7,3 giardine, hoos u dhigista cudurada G. duodenalis ee jiirarka.
Si wada jir ah, natiijooyinkayagu waxay muujinayaan in alfa-2 iyo alfa-7.3 giardines ay kiciyaan firfircoonida NLRP3 martida loo yahay inflammasome oo ay yareeyaan caabuqa G. duodenalis ee jiirarka.Sidaa darteed, molecules-yadani waa bartilmaameedyo rajo leh oo ka hortagga 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: dulmar.Waxaa dhawaan la shaaciyay in Pat Inflamm uu xasaasiyad ku leeyahay daroogada.2019;13:134–43.
Escobedo AA, Tsimerman S. Giardiasis: dib u eegis ku saabsan daawaynta.Fikradda khabiirka farmashiistaha.2007;8: 1885-902.
Tian Huafeng, Chen Bin, Wen Jianfeng.Giardiasis, caabbinta daroogada iyo helitaanka bartilmaameedyo cusub.Waxyeeleeya bartilmaameedyada daroogada ee Disord.2010;10:295–302.
Wang Z, Zhang X, Xiao Yi, Zhang W, Wu X, Qin T, iwm. NLRP3 cudurada bararka iyo bararka.Oxide Med Cell Longev.2020;2020:4063562.
Chen GY, Núñez G. Doorka bararka ee caabuqa mindhicirka iyo kansarka.Gastroenterology.2011;141:1986-99.
Pellegrini C, Antonioli L, Lopez-Castejon G, Blandizzi C, Fornai M. Canonical iyo NLRP3 firfircoon firfircoon ee bararsan ee isgoysyada dulqaadka difaaca iyo caabuqa mindhicirka.kahor difaac.2017;8:36.
Li L, Wang XC, Gong PT, Zhang N, Zhang X, Li S, iyo al.ROS-dhexdhexaadin NLRP3 kicinta barara waxay ku lug leedahay ka jawaab celinta caabuqa N. caninum.Faa'iidada dulin.2020;13:449.