Giardia duodenum ke kokoana-hloko e bakang giardiasis, tšoaetso ea mala e atileng haholo ho bana ba nang le matšoao a letšollo.Re kile ra tlaleha hore extracellular G. duodenalis e etsa hore ho be le ts'ebetso ea intracellular oligomerization-like receptor 3 (NLRP3) e tlamang li-nucleotide le ho laola likarabo tsa ho ruruha tse amohelang batho ka secretion ea extracellular vesicle (EV).Leha ho le joalo, mekhoa e nepahetseng ea limolek'hule tsa pathogen-associated duodenococcal EV (GEV) e amehang ts'ebetsong ena le karolo ea NLRP3 inflammasome ka giardiasis e ntse e tla hlakisoa.
Recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 le alpha-7.3 giardins ho GEV li ile tsa hahoa, tsa fetisetsoa ho mouse primary peritoneal macrophages, 'me tsa fumanoa ka ho lekanya molek'hule ea sepheo sa ho ruruha caspase-1.Boemo ba polelo ea p20 bo ile ba hlahlojoa..G. duodenalis alpha-2 le alpha-7.3 giardines qalong li ne li khetholloa ka ho lekanya NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 le caspase-1 p20), IL secretion.Maemo a 1β, apoptotic spotted protein (ASC) maemo a oligomerization, le sebaka sa immunofluorescent sa NLRP3 le ASC.Karolo ea NLRP3 inflammasome ho pathogenicity ea G. duodenalis e ile ea hlahlojoa ho sebelisoa litoeba moo ts'ebetso ea NLRP3 e neng e thibetsoe (NLRP3 e thibetsoe litoeba) le liphetoho tsa pathological boima ba 'mele, duodenal parasitic load, le lisele tsa duodenal li ne li hlahlojoa.Ho phaella moo, re ile ra batlisisa hore na hiardines alpha-2 le alpha-7.3 e kenya IL-1β secretion ka vivo ka NLRP3 inflammasome 'me e fumane karolo ea limolek'hule tsena ho pathogenicity ea G. duodenalis ho litoeba.
Alpha-2 le alpha-7.3 giardines li etsa hore ho sebelisoe NLRP3 inflammasome in vitro.Sena se ile sa lebisa ts'ebetsong ea p20 caspase-1, keketseho ea litekanyetso tsa polelo ea NLRP3, pro-IL-1β, le pro-caspase-1 liprotheine, keketseho e kholo ea IL-1β secretion, ho thehoa ha mabala a ASA cytoplasm, le ho kenngoa ha oligomerization ea ASA.Ho ruruha ha NLRP3 Ho lahleheloa ke penile ho mpefatsa pathogenicity ea G. duodenalis ho litoeba.Litoeba tse tšoaroang ka li-cysts ka li-gavage tse tsoang ho litoeba tse koetsoeng tsa NLRP3 li bontšitse palo e ntseng e eketseha ea li-trophozoite le tšenyo e khōlō ho duodenal villi, e khetholloang ke li-crypts tsa necrotic tse nang le shrunken le branching.Liteko tsa vivo li bontšitse hore giardines alpha-2 le alpha-7.3 e ka etsa hore ho be le secretion ea IL-1β ka NLRP3 inflammasome, 'me ente ka giardines alpha-2 le alpha-7.3 e fokolitse pathogenicity ea G. duodenalis ho litoeba.
Ha li kopane, liphello tsa phuputso ena li fana ka maikutlo a hore giardia alpha-2 le alpha-7.3 e baka ho phahama ha ho ruruha ha moeti NLRP3 le ho fokotsa ts'oaetso ea G. duodenalis ho litoeba, e leng lipakane tse tšepisang bakeng sa ho thibela giardiasis.
Giardia duodenum ke parasite ea extracellular protozoan e lulang ka maleng a manyane 'me e baka linyeoe tse limilione tse 280 tsa giardiasis ka letšollo selemo le selemo, haholo-holo ho bana ba banyenyane linaheng tse tsoelang pele.Batho ba tšoaetsoa ke metsi a nooang kapa lijo tse silafalitsoeng ke M. duodenum cysts, ebe li kena ka mpeng ebe li ntšoa ka matute a ka mpeng.Giardia duodenum trophozoites e ikamahanya le epithelium ea duodenal, e bakang ho nyekeloa ke pelo, ho hlatsa, letšollo, bohloko ba ka mpeng le ho theola boima ba 'mele.Batho ba nang le immunodeficiency le cystic fibrosis ba kotsing ea ho tšoaetsoa.Tšoaetso e ka boela ea hlaha ka ho kopanela liphate ka molomo le ka morao [2].Lithethefatsi tse kang metronidazole, tinidazole le nitazoxanide ke mekhoa e khethiloeng ea phekolo bakeng sa tšoaetso ea duodenal [3].Leha ho le joalo, lithethefatsi tsena tsa chemotherapy li baka litlamorao tse mpe joalo ka ho nyekeloa ke pelo, carcinogenesis le genotoxicity [4].Ka hona, mekhoa e sebetsang haholoanyane e lokela ho etsoa ho thibela tšoaetso ea G. duodenalis.
Li-inflammasomes ke sehlopha sa liprotheine tsa cytosolic tseo e leng karolo ea karabelo ea tlhaho ea 'mele ea ho itšireletsa mafung, e thusang ho itšireletsa khahlanong le tlhaselo ea pathogen le likarabo tsa ho ruruha [5].Har'a li-inflammasomes tsena, nucleotide-binding oligomerization (NOD) receptor 3 (NLRP3) nucleotide-binding oligomerization (NLRP3) nucleotide-binding-like inflammasome e 'nile ea ithutoa haholo hobane e ka fumanoa ka mefuta e sa tšoaneng ea likokoana-hloko / tšenyo e amanang le limolek'hule (PAMP). DAMP), ea hlokomela, e kenya tšebetsong sesole sa tlhaho sa tlhaho.le ho laola mala a homeostasis mafung a mangata a ho ruruha [6,7,8].E na le mokhoa oa ho amohela mohlala (PRR) NLRP3, li-adapter apoptotic spotted protein (ASC), le procaspase-1 kapa procaspase-11.NLRP3 inflammasome e sebetsa e le moamoheli khahlano le tlhaselo ea pathogen, joalo ka ha e bonoa ho Neospora caninum [9], Paracoccidioides brasiliensis [10], le lithuto tsa Leishmania.[11], empa ho boetse ho tlalehoa hore ts'ebetso ea NLRP3 inflammasome e fokotsa likarabo tsa ho itšireletsa mafung le ho mpefatsa tsoelo-pele ea mafu, mohlala, liboko [12].Ho latela liphuputso tsa rona tse fetileng, re tlalehile hore extracellular G. duodenalis e etsa hore ho be le ts'ebetso ea intracellular ea ho ruruha ha NLRP3 le ho fetola likarabo tsa ho ruruha ka ho hlahisa li-extracellular vesicles (EVs) [13].Leha ho le joalo, karolo ea NLRP3 inflammasome ho tšoaetso ea G. duodenalis ka vivo e ntse e lokela ho khethoa.
Qalong li-giardins li ne li hlalosoa e le likarolo tsa sebopeho sa G. duodenalis cytoskeleton 'me li phetha karolo ea bohlokoa ho trophozoite motility le epithelial cell attachment ka maleng a manyenyane.E le ho ikamahanya hantle le tikoloho le ho eketsa pathogenicity ea bona, G. duodenalis trophozoites e ile ea hlahisa mohaho o ikhethang oa cytoskeletal o nang le 8 flagella, 1 bohareng ba 'mele, le 1 ventral disc [14].Li-trophozoite tsa Giardia duodenum li sebelisa cytoskeleton ea tsona ho kenella ka har'a mala a manyane a ka holimo, haholo-holo duodenum, 'me li ikamahanya le li-enterocyte.Li lula li falla 'me li ikamahanya le lisele tsa epithelial li sebelisa metabolism ea lisele.Ka hona, ho na le kamano e haufi pakeng tsa cytoskeleton ea bona le virulence.Li-giardine tse khethehileng bakeng sa Giardia duodenum ke likarolo tsa sebopeho sa cytoskeleton [15] 'me li arotsoe ka lihlopha tse' nè: α-, β-, γ-, le δ-giardines.Ho na le litho tse 21 tsa lelapa la α-giardin, tseo kaofela li nang le bokhoni bo itšetlehileng ka khalsiamo ho tlama phospholipids [16].Li boetse li hokahanya cytoskeleton le lera la sele.Ho batho ba nang le letšollo le bakoang ke G. duodenalis, α-giardins e hlahisoa haholo 'me ha e na matla a ho itšireletsa mafung nakong ea tšoaetso [17].Liente tsa Heterologous tse thehiloeng ho Giardia alfa-1 tse sirelelitsoeng khahlano le giardiasis ho litoeba mme ke li-antigen tse ka bang teng bakeng sa nts'etsopele ea ente [18].Alpha-8 giardin, e fumanehang ka har'a membrane ea plasma le flagella, empa eseng ka har'a ventral disc, e ntlafatsa motility le sekhahla sa kholo ea trophozoite ho G. duodenalis [19].Alpha-14 giardin e khomarela mehaho ea microtubule holim'a flagella 'me e ama ho sebetsa ha G. duodenalis [20].Alpha-11 giardine e teng ka bongata ho pholletsa le potoloho ea bophelo, 'me ho feteletsa ha alpha-11 giardine ho senya G. duodenalis ka boeona [21].Leha ho le joalo, ha ho hlake hore na alpha-2 giardine le alpha-7.3 giardine li sireletsa khahlanong le tšoaetso ea G. duodenalis le mekhoa ea bona e ka tlaase.
Thutong ena, recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine le pcDNA3.1 (+) -alpha-7.3 giardine li ile tsa fetisetsoa ho mouse primary peritoneal macrophages ho kenya ts'ebetsong NLRP3.Ka mor'a moo, ho ile ha hlahlojoa lipakane tsa inflammasome.Re ile ra boela ra hlahloba karolo ea NLRP3 inflammasome ho pathogenicity ea G. duodenalis, ra batlisisa hore na alpha-2 le alpha-7,3 giardines li etsa hore ho sebelisoe NLRP3 inflammasome ka vivo, 'me ra etsa qeto ea hore likarolo tsena tse peli tsa giardines ho pathogenicity ea. G. duodenalis.Sepheo sa rona se tšoanang e ne e le ho hlahisa lipakane tse tšepisang bakeng sa ho thibela tšoaetso ea G. duodenalis.
Mofuta o hlaha (WT) C57BL/6 litoeba tse tšehali tsa libeke tse 5–8 li rekiloe ho Liaoning Changsheng Experimental Animal Center (Liaoning, China).Litoeba li ne li na le phihlello ea metsi mahala, li ne li fumana lijo tse hloekisitsoeng 'me li bolokoa ka nako ea 12/12 hora ea leseli / lefifi.Pele ho ts'oaetso, litoeba li ile tsa fumana lithibela-mafu ad libitum metsing a nooang a tlatsitsoeng ka ampicillin (1 mg/mL), vancomycin (1 mg/mL), le neomycin (1.4 mg/mL) (tsohle tse rekiloeng Shanghai, Chaena, likokoana-hloko tsa maiketsetso) [22] ].].Litoeba tse ileng tsa lahleheloa ke matla a ho ja le ho noa bakeng sa > lihora tsa 24 le ho lahleheloa ke ≥ 20% boima ba 'mele li ile tsa kholisoa ka botho ke ho senyeha ha molomo oa popelo.
WB G. duodenalis trophozoites (Pokello ea Setso sa Mofuta oa Amerika, Manassas, USA) e ile ea tlatsetsoa ka 12.5% ​​ea fetal bovine serum (FBS; E 'ngoe le e 'ngoe e tala, Zhejiang, Chaena) le 0.1% ea bile ea likhomo (Sigma-Aldrich, St. Missouri, USA) ).USA) tlas'a maemo a microaerobic.Li-trophozoite tse kopaneng li ile tsa bokelloa holim'a leqhoa 'me tsa fetisoa ka karolelano ea 1: 4 bakeng sa ho ikatisa ho eketsehileng.
Giardia duodenum cysts e ile ea kenngoa joalokaha ho hlalositsoe pejana [23], trophozoites e ile ea kotuloa ka mohato oa logarithmic 'me ea hlakoloa ka mokhoa oa ho kenya li-encapsulation, pH 7.1 (e fetotsoeng TYI-S-33) ho ea ho khatello ea ho qetela ea 1 × 106 trophozoites / mL.matsoai a bile 0,05% mahareng).Li-trophozoite li ne li lengoa tlas'a maemo a anaerobic ho 37 ° C ho fihlela mohato oa kholo ea logarithmic.Fetola sebaka se bohareng ho isa ho cyst inducing medium (pH 7.8; e fetotsoeng TYI-S-33 medium le 1% bile concentration) le setso sa G. duodenalis ho 37 ° C bakeng sa lihora tsa 48-96, nakong eo li-cysts tsa sebopeho li neng li hlokomeloa tlas'a microscope.Ka mor'a hore boholo ba li-trophozoite li susumetsoe hore li thehe li-cysts, motsoako oa setso o ile oa kotuloa 'me oa khutlisetsoa metsing a sa hloekang a hloekileng ho lyse li-trophozoite tse setseng.Li-cysts li ne li baloa 'me tsa bolokoa ho 4 ° C bakeng sa litlhahlobo tse latelang ka tube ea gastric ho litoeba.
Giardia extracellular vesicles (GEVs) e ile ea ntlafatsoa joalo ka ha ho hlalositsoe pejana [13].Li-trophozoite tse karolong ea kholo ea logarithmic li ile tsa tsosolosoa ka mokhoa o fetotsoeng oa TYI-S-33 o lokiselitsoeng ka FBS (Biological Industries, Beit-Haemek, Israel) e nang le exosome-depleted ho qetela ea 1 × 106 parasites / mL le ho kenngoa lihora tse 12.Li ne li arotsoe ho setso sa supernatant ka centrifugation ho 2000 g bakeng sa 10 min, 10,000 g bakeng sa 45 min, le 100,000 g bakeng sa 60 min.Li-precipitates li ile tsa qhibiliha ka phosphate buffered saline (PBS), e lekantsoeng ho sebelisoa BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) 'me ea bolokoa ho -80 ° C. kapa ea sebelisoa ka ho toba bakeng sa litlhahlobo tse ling.
Li-macrophages tsa mantlha tsa peritoneal li ile tsa lokisoa joalo ka ha ho hlalositsoe pejana [24].Ka bokhutšoanyane, litoeba (tse lilemo li 6-8 libeke) li ile tsa kenngoa (intraperitoneally [ip]) ka 2.5 ml ea 2.98% Difco liquid thioglycol medium (BD, Franklin Lakes, NJ, USA) 'me ea fepa 3-4 palates.Ho emisoa ha macrophages ho ile ha bokelloa ho tloha ka mpeng ea litoeba ka mor'a ho bolaoa ke lefu la sethoathoa le centrifuged makhetlo a 3 ho 1000 g bakeng sa 10 min.Lisele tse kotutsoeng li ile tsa fumanoa ka cytometry ea phallo ho sebelisa letšoao la CD11b ho fihlela bohloeki ba sele bo le> 98%, ebe bo kenngoa lipoleiti tsa setso sa lisele tsa 6 (4.5 x 106 lisele / hantle) 'me li kenngoa ka 10% FBS (Bioindustry) ho 37 ° C.le 5% CO2.
RNA e ntšitsoe ho 1 × 107 trophozoites ka 1 ml ea reagent ea TRIzol (Vazyme, Nanjing, Chaena), DNA ea genomic e ntšitsoe ho kakaretso ea G. duodenalis RNA ho sebelisoa MonScript dsDNase (Monad, Wuhan, China) le DNA e tlatsetsang (cDNA) e entsoeng. sebelisa MonScript RTIIII Super Mix (Monad) ho latela litaelo tsa moetsi.
Boitsebiso ba tatellano ea CDS bakeng sa gene ea G. duodenalis e lebisitsoeng e fumanoe ho NCBI GenBank.Sebelisa Primer 5.0 ho rala li-primer tsa cloning tse se nang moeli bakeng sa mofuta o mong le o mong o shebiloeng.Primer (5′-3′) e na le likarolo tse tharo: tatellano e fetang e nang le vector e nang le linearized pcDNA3.1(+) EcoRV (TGGTGGAATTCTGCAGAT) le ho qala li-codons ATG le GNN (haeba setsi sa pele e se G).Sena se etsoa ho ntlafatsa katleho ea polelo.Ho phaella moo, bonyane 16 bp e kopantsoeng metheo (GC content 40-60% / Tm approx. 55 °C).Reverse primer (5′-3′) e na le likarolo tse peli, tatellano e fetang e nang le EcoRV-linearized vector pcDNA3.1(+) (GCCGCCACTGTGCTGGAT) le motheo o kopaneng oa bonyane 16 bp.(ntle le libaka tse peli tsa ho qetela).bases) codon e kang AA kapa GA ho lumella li-plasmids tse kopantsoeng ho hlahisa liprotheine tsa tsona tse ngotsoeng).Tatelano ea mantlha e thathamisitsoe ho Lethathamo la 1 mme e entsoe ke Kangmet Biotechnology Co., Ltd. (Changchun, China).
Lipheo li ile tsa matlafatsoa ho sebelisoa Pfu DNA polymerase (Tiangen, Beijing, China) kapa Ex-taq (Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, China) ho sebelisoa G. duodenalis cDNA e lokiselitsoeng e le template.Eukaryotic expression vector plasmid pcDNA3.1(+) e ne e tsamaisana le enzyme e thibelang EcoRV le dephosphorylated ho sebelisoa Fast AP (Thermo Fisher Scientific).Likhechana tsa linearized pcDNA3.1 (+) le likaroloana tsa liphatsa tsa lefutso tse matlafalitsoeng li ile tsa hloekisoa ho sebelisoa DNA gel purification kit (Tiangen) 'me li lekantsoe ho sebelisoa Nanodrop ND-2000 (Thermo Fisher Scientific).Sekhechana sa pcDNA3.1(+) le sekhechana se seng le se seng sa liphatsa tsa lefutso li ile tsa kopanngoa hape ho sebelisoa MonClone single assembly cloning mix (Monad Biotech Co., Ltd., Suzhou, China) 'me tsa tiisoa ke tatellano ea DNA ho sebelisoa Comate Bioscience Company Limited (Changchun, China) ..
Li-plasmids tse se nang Endotoxin pcDNA3.1(+)-alpha-2 le pcDNA3.1(+)-alpha-7.3 li ile tsa hlahisoa ho sebelisoa Plasmid Mini Kit ea SanPrep Endotoxin-free (Sangon Biotech).Khatiso e ne e bolokoa ka holimo ho 500 ng/µl ho netefatsa hore EDTA ka har'a buffer ea elution ha e kena-kenane le tlhahlobo ea phetisetso.Primary mouse peritoneal macrophages e ne e entsoe ka lipoleiti tse 6 tse nang le RPMI 1640 e feletseng (Biological Industries) bakeng sa lihora tsa 12, ebe lisele li ne li hlatsuoa ka makhetlo a 3 ka PBS e futhumetseng ho tlosa penicillin le streptomycin, ebe ka nako e bohareng e tlatsitsoe ka mokhoa o feletseng.Li-plasmids tse se nang Endotoxin pcDNA3.1(+)-alpha-2 le pcDNA3.1(+)-alpha-7.3 (2.5 μg) li ile tsa hlapolloa ka 125 μl ea Opti-MEM e fokotsehileng serum medium (Gibco, Thermo Fisher Scientific) ..Joale 5 µl ea Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific) e ile ea hlapolloa ka 125 µl ea serum e tlase ea Opti-MEM medium.Lokisetsa liposome-DNA complexes ka ho kopanya plasmid e hlapolotsoeng e se nang endotoxin le Lipofectamine 2000 le ho lumella motsoako ho ema mocheso oa kamore metsotso e 5.Fetisetsa li-complexes ka thoko ho lisele tsa seliba se seng le se seng ebe u kopanya butle.Ka mor'a lihora tsa 4, mokhoa oa setso oa sele o ile oa nkeloa sebaka ke 2 ml ea RPMI 1640 e feletseng 'me setso se ile sa tsoela pele ka lihora tse 24.Setsi se secha sa setso sa lisele se ile sa eketsoa liseleng mme sa kenngoa ka nako e fapaneng ho latela moralo oa tlhahlobo.
Lisampole tsa protheine tse tsoang ho li-supernatants le li-cell lysates li ile tsa lokisoa joalo ka ha ho hlalositsoe pejana [25].Litekanyetso tsa ho fetisa li-membrane bakeng sa pro-IL-1β, pro-caspase-1, caspase-1 p20, NLRP3, β-actin, le His-tag e ne e le 200 mA / 90 min.Bakeng sa interleukin 1β (IL-1β; R&D Systems, Minneapolis, Minnesota, USA), caspase-1 (p20) (Adipogen, Switzerland) le NLRP3 (Adipogen SA, Epalinges, Switzerland) le 1: 5000 e lebisang tag ea Hae ( Amylet Scientific, Wuhan, China) le β-actin (Proteintech, Wuhan, China).
Ho hokahanya le disuccinimide subrate (DSS) ho entsoe joalo ka ha ho hlalositsoe pejana [26].Lisele li ne li hlatsuoa ka makhetlo a 3 ka PBS e batang 'me tsa lysed ka ho feletseng ka nale ea 27 gauge ka 50 µl ASC reaction buffer (pH 8.0) e nang le 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES le 125 mM NaHCO3.Motsoako o ne o le centrifuged ho 5000 g bakeng sa 3 mins mme pellet e ne e sutured ka 10 µl DSS (25 mM ho DMSO) le 40 µl ASC reaction buffer bakeng sa 30 min ho 37°C.Kamora centrifugation ho 5000 g bakeng sa 10 min, pellet e ne e qhibilihisoa ka tharollo ea 40 µl ea ASC reaction buffer le 10 µl ea 6x protheine loading buffer (TransGen, Beijing, China), ebe tharollo e tingoa ka mocheso oa kamore bakeng sa 15. min., Ebe pheha 10 metsotso.Ka nako eo, lisampole tsa protheine li ne li le tlas'a ho thibela Bophirimela ho sebelisa li-anti-ASC antibodies (Wanleibio, Shenyang, Chaena) ka tekanyo ea dilution ea 1: 500.
Ka mor'a mokhoa o hlalositsoeng pele [13], li-supernatants tsa cell culture li ile tsa kotuloa 'me secretion ea pro-inflammatory cytokine IL-1β e ne e ikemiselitse ho sebelisa mouse IL-1 Beta ELISA kit (Invitrogen, Thermo Fisher Scientific).Fetolela boleng ba OD450nm ho tsepamisa maikutlo ho protheine u sebelisa IL-1β standard curve.
Lisele tse koahetsoeng ka likoahelo li ne li hlatsuoa ka bonolo ka makhetlo a 3 ka PBS e futhumetseng, e tsitsitseng ka har'a li-tissue cell fixative (Biosharp, Beijing, Chaena) bakeng sa metsotso ea 10 mocheso oa kamore (RT), ka 0.1% Triton X-Permeabilize ho 100 (diluted ka PBS; Biosharp ) bakeng sa metsotso ea 20 mocheso oa kamore le thibela ho 5% ea serum ea bovine albumin (ka PBS) bakeng sa lihora tse 2 mocheso oa kamore.Lisele li ile tsa kenngoa bosiu bo le bong ho 4 ° C le li-antibodies tsa mantlha khahlanong le ASC (1: 100 dilution) kapa NLRP3 (1: 100 dilution), ka ho latellana, le Cy3 e ngotsoeng ke pōli e khahlanong le rabbit IgG (H + L) (1: 400; EarthOx) , San Francisco, CA, USA) kapa FITC-conjugated goat anti-mouse IgG (1:400; Earthox) ka bosiu bo le bong ka 37 ° C lefifing bakeng sa hora e le 'ngoe.Li-nuclei li ne li silafalitsoe ke Hoechst 33258 (10 μg/ml; UE, Suzhou, China) ka metsotso e 5 'me li shebiloe ka tlas'a microscope ea fluorescence (Olympus Corporation, Tokyo, Japan).
Litoeba li ne li arotsoe ka lihlopha tse 'nè (n = 7 sehlopheng se seng le se seng): (i) Sehlopha sa taolo e mpe e tšoaroang ke PBS (PBS feela; gavage 100 µl / mouse PBS e lateloa ke ente ea letsatsi le letsatsi ea intraperitoneal 100 µl / mouse PBS lihora tsa 3 hamorao)., ka ho tsoelang pele ka matsatsi a 7);(ii) sehlopha sa taolo e mpe e tšoaroang ka MCC950 inhibitor [27] (100 µl / mouse ka PBS gavage, lihora tsa 3 hamorao, 10 mg / kg boima ba 'mele [BW] MCC950 [ka PBS] e ne e tsamaisoa ka intraperitoneally letsatsi le leng le le leng, nako ea matsatsi a 7);(iii) Sehlopha sa ts'oaetso ea G. duodenalis cysts (1.5 x 106 cysts / mouse ka gavage, lihora tsa 3 hamorao, 100 μl / mouse PBS intraperitoneally e tsamaisoa letsatsi le leng le le leng bakeng sa matsatsi a 7);(iv) G. duodenalis cyst e kopantsoeng le sehlopha sa ts'oaetso ea MCC950 inhibitor sehlopha sa phekolo (1.5 × 106 cysts / mouse ka gavage, 10mg / kg boima ba 'mele MCC950 intraperitoneally letsatsi le letsatsi bakeng sa matsatsi a 7 ho 3h).Boima ba 'mele ba toeba e' ngoe le e 'ngoe bo ne bo beoa leihlo letsatsi le leng le le leng mme litoeba tsohle li ile tsa hlakoloa ka letsatsi la 7th.Duodenum e kotutsoeng (bolelele ba 3 cm) e ile ea khaoloa likotoana tse nyenyane ka 1 ml PBS, li-cysts li ile tsa senngoa bosiu bo le bong PBS ka 4 ° C, le G. duodenalis trophozoites.Duodenum e ncha (bolelele ba 1 cm) e ne e arotsoe bakeng sa litšila tsa hematoxylin le eosin (H&E).
Litoeba li ne li arotsoe ka lihlopha tse peli: (i) Sehlopha sa taolo ea MOCK le (ii) MCC950 inhibitor sehlopha.Ho ne ho e-na le mekhoa e mehlano ea phekolo sehlopheng ka seng (n = 7 / sehlopha sa phekolo): (i) PBS sehlopha sa taolo e mpe ea phekolo (PBS feela; 100 µl / mouse PBS, intramuscular (IM) ente (tibialis anterior) [28, 29]; ( ii) pcDNA3.1(+) sehlopha sa taolo e mpe ea plasmid (100 µg/mouse DNA, ka ente ea intramuscular) (iii) sehlopha sa taolo ea tšoaetso ea G. duodenalis cysts (1.5 x 106 cysts / mouse, ka gavage) (iv) a sehlopha se tšoaroang ka plasmid pcDNA3.1 (+) -alpha-2 (100 μg / mouse DNA, ka ente ea intramuscular), le (v) sehlopha se tšoaroang ka plasmid pcDNA3.1 (+) -alpha-7.3 (100 µg / mouse DNA, ka mor'a lihora tsa 12 tsa ho feta, litoeba tsa sehlopha sa MCC950 inhibitor li ile tsa fumana ente ea letsatsi le letsatsi ea MCC950 (10 mg / kg boima ba 'mele) bakeng sa matsatsi a 7, ha litoeba tsa sehlopha sa MOCK li fumana tekanyo e lekanang ea phekolo ea PBS. Mehlala ea mali e ne e le e bokelloa ho tloha litoeba tsa mahlo 'me e siiloe bosiu bo le bong ho 4 ° C Li-samples tsa Serum li ne li khethiloe ka ho sebelisa enzyme-linked immunosorbent assay (ELISA) bakeng sa le litekanyo tsa IL-1β.
Litoeba tse mashome a mararo a metso e mehlano li ne li arotsoe ka lihlopha tse hlano (n=7/group).Sehlopha sa 1 e ne e le sehlopha sa taolo e mpe e tšoaroang ka PBS: litoeba li amohetse 100 μl ea PBS intramuscularly le matsatsi a 3 hamorao ka gavage.Sehlopha sa 2 ke sehlopha se setle sa taolo se tšoaelitsoeng ke G. duodenalis cysts: litoeba li ne li kenngoa ka 100 μl ea PBS, 'me matsatsi a 3 hamorao 1.5 x 106 cysts / mouse e ne e kenngoa ka intragastrically.Sehlopha sa boraro - ente ea plasmid e nang le pcDNA3.1 (+) hammoho le sehlopha sa taolo bakeng sa tšoaetso ea duodenal cyst: litoeba li fumane 100 μg ea plasmid DNA pcDNA3.1 (+) (im) ka molomo, 1.5 × 106 cysts / mouse 3 bakeng sa 'maloa. matsatsi.Lihlopha tsa 4 le 5 e ne e le recombinant pcDNA3.1 (+) -alpha-2 giardine plasmid kapa pcDNA3.1 (+) -alpha-7.3 giardine plasmid hammoho le tšoaetso ea G. duodenalis cyst.Sehlopha sa liteko: litoeba li fumane 100 µg ea pcDNA3.1 (+) -giardine plasmid DNA (im), ebe matsatsi a 3 hamorao, 1.5 × 106 cysts / mouse e ne e kenngoa ka gavage.Boima ba 'mele oa mouse e' ngoe le e 'ngoe bo ile ba behoa leihlo ka mor'a ho kenngoa ha G. duodenalis cyst ka tube.Duodenum e ncha e ile ea bokelloa bakeng sa litekanyo tsa boima ba likokoana-hloko le tlhahlobo ea mabala a HE.
Liphetoho tsa histopathological li ile tsa hlahlojoa ho latela mokhoa o phatlalalitsoeng pele [30].Duodenum e ncha e ne e tsitsitsoe ka "thishu cell fixative", e kentsoeng ka parafine, e khaotsoe ka likarolo tsa 4 μm, e silafalitsoeng ke H & E 'me e hlahlojoe ka tlas'a microscope e khanyang.Liphetoho tse emelang likokoana-hloko likarolong tse supileng tsa lisele tse tsoang ho litoeba tse supileng tse ikemetseng li ile tsa hlahlojoa ke setsebi sa mafu a sa tsebeng phekolo 'me se ile sa tšoaroa ka 200x magnification.Bolelele ba villi le botebo ba li-crypts li ne li lekantsoe ho latela mekhoa e hlalositsoeng pele.
Liphetho tsa in vitro le in vivo li fumanoe ka makhetlo a mararo.Li-graph li ile tsa hlahisoa ho sebelisoa GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, USA).Phapang pakeng tsa lihlopha tse peli e ile ea hlahlojoa ke t-test, ha liphapang pakeng tsa lihlopha tsa ≥3 li ile tsa hlahlojoa ka mokhoa o le mong oa ho hlahloba phapang (ANOVA) ho sebelisa software ea SPSS (version 22.0; SPSS IBM Corp., Armonk, NY, USA).Lintlha li ile tsa hlahlojoa bakeng sa homogeneity ea phapang ho sebelisoa tlhahlobo ea Levene e lateloang ke tlhahlobo ea post hoc ea Bonferroni (B).Bohlokoa bo hlalosoa e le P<0.05, P<0.01, le P<0.001 (eseng bohlokoa [ns]) (P>0.05).
Tlhahlobo ea rona ea nakong e fetileng ea GEV proteomics ho Kyoto Encyclopedia of Genes and Genomes (KEGG) e bontšitse hore lipakane tse ngata li ka 'na tsa ameha ts'ebetsong ea litsela tsa matšoao a ho ruruha [13].Re khethile lipheo tse peli tse tšepisang, alpha-2 le alpha-7.3 giardins, re holisa limolek'hule tsena le ho li sebelisa ho aha pcDNA3.1 (+) vector ea polelo ea eukaryotic.Kamora tatellano, recombinant pcDNA3.1 (+) -alpha-2 le alpha-7.3 giardine expression plasmids li ile tsa fetisetsoa ho macrophages ea mantlha ea peritoneal, 'me caspase-1 p20 signature protheine ea ho ruruha (sekhechana sa caspase-1) se ile sa tsejoa. e le ho hlakisa limolek'hule tsa bohlokoa tse ka bakang ho ruruha.Liphetho li bonts'itse hore alpha-2 le alpha-7.3 giardines li ka etsa hore polelo ea p20 caspase-1 e ts'oana le GEV.Ha ho phello ea ts'ebetso ea caspase-1 e fumanoeng taolong e mpe e sa sebetsoeng (PBS feela) le taolo ea plasmid pcDNA3.1 (+) (Setšoantšo sa 1).
Tekanyo ea ts'ebetso ea p20 caspase-1 ka pcDNA3.1 (+) -alpha-2 le alpha-7.3 giardins.Recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 le alpha-7.3 giardines (ka holimo ho tsela e' ngoe le e 'ngoe) li ile tsa fetisetsoa ho macrophages ea mantlha ea mouse ea peritoneal le li-supernatant tsa setso li ile tsa kotuloa lihora tse 24 hamorao.Western blotting e ne e sebelisetsoa ho lekanya maemo a polelo a saena caspase-1 p20 protheine ea inflammasome.Sehlopha sa kalafo sa PBS-feela (lane C) le pcDNA3.1 (+) sehlopha sa monotherapy (pcDNA3.1 lane) se ile sa sebelisoa e le taolo e mpe, 'me sehlopha sa phekolo ea GEV se ile sa sebelisoa e le taolo e ntle.Pontšo ea protheine e ncha e tiisitsoe ka ho lemoha letšoao la histidine protheine e 'ngoe le e' ngoe, 'me lihlopha tsa protheine tse lebelletsoeng e ne e le alpha-2 giardine (38.2 kDa) le alpha-7.3 giardine (37.2 kDa).GEV, Giardia duodenum extracellular vesicles, pcDNA3.1(+), EcoRV-linearized vector, SUP, supernatant
Ho fumana hore na alpha-2 giardine le alpha-7.3 giardine li kenya p20 caspase-1 polelo le ho bapala karolo ea ho kenya ts'ebetsong karabo ea ho ruruha ea NLRP3, pcDNA3.1 (+) -alpha-2 giardine le pcDNA3.1 (+) -alpha -7.3 giardin e ile ea fetisetsoa ho macrophages ea mantlha ea mouse ea peritoneal e nang le recombinant plasmid DNA, le maemo a polelo, sebaka sa sebaka seo, le oligomerization ea liprotheine tsa bohlokoa tsa ho ruruha NLRP3 li ile tsa khethoa.Tekong ena, GEV e ne e sebelisoa e le sehlopha sa taolo e ntle, 'me sehlopha se se nang phekolo (PBS feela) kapa pcDNA3.1 (+) sehlopha sa phekolo ea phetisetso e ne e le sehlopha se fosahetseng.Liphello li bontšitse hore, joalo ka sehlopha sa GEV, DNA ea plasmid recombinant ea giardin pcDNA3.1 (+) -alpha-2 le giardin pcDNA3.1 (+) -alpha-7.3 e ile ea fella ka ho tsosolosoa ha NLRP3, pro-IL-1β le procaspase-1 le ts'ebetso ea caspase-1 (setšoantšo sa 2a).Ho phaella moo, li-giardine ka bobeli li ile tsa etsa hore ho be le secretion ea bohlokoa ea 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) (Setšoantšo sa 2b).Boholo ba liprotheine tsa ASC e ne e le monomeric sehlopheng se sa phekoloeng kapa sehlopheng sa phekolo se fetisitsoeng ka pcDNA3.1 (+) plasmid, ho fapana le pcDNA3.1 (+) -alpha-2 kapa pcDNA3.1 (+) -alpha- 7.3 giardine.ASC oligomerization e etsahetse ho DNA ea plasmid recombinant ea sehlopha sa taolo e ntle ea GEV kapa sehlopha, se bonts'ang foromo ea oligomeric (Setšoantšo sa 2c).Lintlha tsena tsa pele li fana ka maikutlo a hore alpha-2 giardine le alpha-7,3 giardine e ka etsa hore ho be le ts'ebetso ea ho ruruha ha NLRP3.Liphuputso tse latelang tsa immunofluorescent tsa sebaka sa ASC le NLRP3 li bontšitse hore sehlopheng sa taolo e mpe, protheine ea ASC e ne e hasane ho pholletsa le cytoplasm 'me e hlaha e le letšoao la letheba holim'a ts'usumetso ea pcDNA3.1 (+) -alpha-2 e nang le giardine kapa pcDNA3.1 (+) -alpha-7,3 sehlopha sa giardine kapa sehlopha sa taolo e ntle ea GEV (Setšoantšo sa 2d).Litaolong tse mpe le lihlopha tsa pcDNA 3.1 tse tšoaroang ka plasmid, lets'oao la protheine la NLRP3 ha lea ka la fumanoa, ha letheba la letšoao la fluorescent le arabela pcDNA3.1 (+) -alpha-2 giardine kapa pcDNA3.1 (+) -alpha-7.3 e bonwe..giardine li fumanoa ka cytoplasm kapa holim'a stimulation ea HEV (sa feiga. 2e).Lintlha tsena li bontša hape hore G. duodenalis giardin alpha-2 le giardin alpha-7.3 li kenya ts'ebetso ea NLRP3 inflammasome ho mouse primary peritoneal macrophages.
pcDNA3.1 (+) -alpha-2 giardin le pcDNA3.1 (+)-alpha-7.3 giardin e kenya tšebetsong NLRP3 inflammasome ka har'a mouse peritoneal macrophages.Fetisetsa recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardin le pcDNA3.1(+)-alpha-7.3 giardin ho li-primary murine peritoneal macrophages le lisele, kapa kotula supernatant ka hare ho 24 h bakeng sa tlhahlobo ea polelo, oligomerization , sephiri.le sebaka sa libaka tsa liprotheine tse ka sehloohong tsa ho ruruha.Sehlopha sa PBS-feela (C) le pcDNA3.1 (+) sehlopha sa phekolo se le seng se sebelisitsoe e le taolo e mpe, 'me sehlopha sa phekolo ea GEV se ne se sebelisoa e le sehlopha se setle.Liprotheine tse ka sehloohong tsa ho ruruha NLRP3, ho akarelletsa le NLRP3, pro-IL-1β, pro-caspase-1, le p20 caspase-1, li ile tsa fumanoa ke Western blotting.b Litekanyetso tsa secretion ea IL-1β ho li-supernatants li ile tsa khethoa ho sebelisa enzyme-linked immunosorbent assay (ELISA).Phapang pakeng tsa lihlopha tsa taolo le liteko li ile tsa hlahlojoa ka mokhoa o le mong oa ho hlahloba phapang (ANOVA) ho sebelisa SPSS software version 22.0.Linaleli li bontša phapang e khōlō pakeng tsa lihlopha **P<0.01 le ***P<0.001.c Maemo a oligomerization a ASC ka har'a li-pellets a ile a khethoa ke tlhahlobo ea li-cross-link tsa DSS, athe maemo a ASC ho li-lysate tsa lisele a ne a sebelisoa e le taolo ea ho jarolla.d Ho bona pono ea sebaka sa ISC ho sebelisa immunofluorescence.e Immunofluorescence e ne e sebelisetsoa ho bona pono ea sebaka sa NLRP3.ASC, protheine e kang ea apoptotic speck;IL, interleukin;NLRP3, nucleotide-binding oligomerization-like receptor 3;ns, ha e bohlokoa (P > 0.05)
Ka bobeli G. duodenalis le li-GEV tseo e li hlahisang li kenya tšebetsong NLRP3 inflammasome le ho laola likarabo tsa ho ruruha tse amohelang batho ka har'a vitro.Ka hona, karolo ea NLRP3 inflammasome ho pathogenicity ea G. duodenalis e ntse e sa hlaka.Ho batlisisa taba ena, re entse teko lipakeng tsa litoeba tse tšoaelitsoeng G. duodenalis cyst le litoeba tse tšoaelitsoeng G. duodenalis cyst + MCC950 inhibitor treatment and bapisa NLRP3 inflammasome expression ha e tšoaelitsoe G. duodenalis cyst.Leano le qaqileng la teko le bontšitsoe setšoantšong sa 3a.Liphetoho tsa boima ba 'mele oa litoeba lihlopheng tse fapaneng tsa phekolo li ile tsa hlahlojoa ka matsatsi a 7 ka mor'a tšoaetso ea li-cysts,' me liphello li bontšoa ho Setšoantšo sa 3b.Ha ho bapisoa le sehlopha se tšoaroang ka PBS e hloekileng, liphello li bontšitse hore (i) boima ba 'mele oa litoeba tse tšoaelitsoeng ke G. duodenalis cyst bo fokotsehile ho tloha letsatsing la 3 ho ea ho la 7 ka mor'a tšoaetso;(ii) kalafo ka MCC950 inhibitor e ne e se na phello e kholo boima ba 'mele oa litoeba..Ha ho bapisoa le sehlopha se le seng sa tšoaetso, BW ea sehlopha sa ts'oaetso ea duodenal e tšoaroang le MCC950 e fokotsehile ho ea ho likhato tse fapaneng (Letsatsi la 1: ANOVA, F (3, 24) = 1.885, P = 0.0148; Letsatsi la 2: ANOVA, F (3, 24) ) = 0.4602, P<0.0001; Letsatsi la 3: ANOVA, F (3, 24) = 0.8360, P = 0.0010; Letsatsi la 4: ANOVA, F (3, 24) = 1.683, P = 0.0052; (3, 24)=0.6497, P=0.0645; Letsatsi la 6: ANOVA, F(3, 24)=5.457, P=0.0175; Letsatsi la 7: ANOVA, F(3, 24) = 2.893, P = 0.0202).Lintlha tsena li bontša hore NLRP3 inflammasome e sireletsa litoeba ho lahleheloa ke boima ba 'mele matsatsing a pele (matsatsi a 2-4) a tšoaetso ea duodenal.Ka mor'a moo re ne re ikemiselitse ho lemoha G. duodenalis trophozoites ka duodenal lavage fluid 'me liphello li bontšoa ho Setšoantšo sa 3c.Ha ho bapisoa le sehlopha sa G. duodenalis cyst infection, palo ea trophozoite ka duodenum e ile ea eketseha haholo ka mor'a ho thibela NLRP3 inflammasome (t (12) = 2.902, P = 0.0133).Li-tissue tsa Duodenal tse silafalitsoeng ke HE li bonts'itse, ha li bapisoa le taolo e mpe e tšoaroang ka PBS le MCC950 feela: (i) G. duodenalis cyst infection e bakile tšenyo ea duodenal villi (ANOVA, F (3, 24) = 0.4903, P = 0.0488 ) le crypt atrophy (ANOVA, F (3, 24) = 0.4716, P = 0.0089);(ii) duodenum ho tloha litoeba tse tšoaelitsoeng ke G. duodenalis cysts le ho phekoloa ka MCC950 inhibitors.duodenal villi e ne e senyehile mme e shoele (ANOVA, F (3, 24) = 0.4903, P = 0.0144) e nang le atrophy le crypt branching (ANOVA, F (3, 24) = 0.4716, P = 0, 0481) (Setšoantšo sa 3d- f) .Liphetho tsena li fana ka maikutlo a hore NLRP3 inflammasome e phetha karolo ea ho fokotsa pathogenicity ea G. duodenalis.
Karolo ea NLRP3 inflammasome ho tšoaetso ea Giardia duodenum.Litoeba li ile tsa hlajoa (iv) ka li-cysts tsa duodenococcal ebe li phekoloa ka MCC950 (ip) kapa ntle le eona.Lihlopha tsa kalafo tse le 'ngoe tse nang le PBS kapa MCC950 li ne li sebelisoa e le taolo.Sehlopha sa liteko le mokhoa oa phekolo.b Boima ba 'mele oa litoeba ho se seng le se seng sa lihlopha tse fapaneng tsa kalafo bo ne bo behiloe leihlo ka matsatsi a 7.Phapang pakeng tsa sehlopha sa ts'oaetso ea G. duodenalis le sehlopha sa phekolo ea ts'oaetso ea G. duodenalis + MCC950 e ile ea hlahlojoa ke t-test ho sebelisa SPSS software version 22.0.Linaleli li bontša phapang e khōlō ho *P<0.05, **P<0.01, kapa ***P<0.001.c Moroalo oa likokoana-hloko o ile oa khethoa ka ho bala palo ea li-trophozoite ka har'a mokelikeli oa duodenal lavage.Phapang pakeng tsa sehlopha sa ts'oaetso ea G. duodenalis le sehlopha sa phekolo ea ts'oaetso ea G. duodenalis + MCC950 e ile ea hlahlojoa ke t-test ho sebelisa SPSS software version 22.0.Linaleli li bontša phapang e khōlō ho *P <0.05.d Liphetho tsa litšila tsa Hematoxylin le eosin (H&E) tsa duodenal histopathology.Metsu e khubelu e bontša tšenyo ho villi, metsu e tala e bontša tšenyo ea li-crypts.Scale bar: 100 µm.e, f Tlhahlobo ea lipalo-palo ea bophahamo ba duodenal villus le bophahamo ba crypt ea mouse.Linaleli li bontša phapang e khōlō ho *P<0.05 le **P<0.01.Liphetho li nkiloe litekong tse 7 tse ikemetseng tsa baeloji.BW, boima ba 'mele;ig, tsela ea ho fana ka intragastric;ip, tsela ea ho fana ka intraperitoneal;ns, eseng bohlokoa (P > 0.05);PBS, phosphate buffered saline;WT, mofuta o hlaha
Sephiri sa IL-1β ke letšoao la ts'ebetso ea ho ruruha.Ho fumana hore na G. duodenalis alpha-2 giardine le alpha-7.3 giardine li kenya tšebetsong NLRP3 host inflammasome in vivo, re sebelisitse litoeba tse sa phekoleheng tsa WT (sehlopha sa sham) le NLRP3 litoeba tse koetsoeng ka inflammasome (MCC950 e thibetsoe sehlopha sa kalafo).Leano le qaqileng la teko le bontšitsoe setšoantšong sa 4a.Lihlopha tsa liteko li ne li e-na le litoeba tse tšoaroang ka PBS, G. duodenalis cyst treatment by gavage, intramuscular injection ea pcDNA3.1, le intramuscular injection ea pcDNA3.1 (+) -alpha-2 giardine kapa pcDNA3.1-alpha-7.3 giardine.Ka letsatsi la 7th ka mor'a tsamaiso ea intramuscular ea plasmid e kopantsoeng, serum e ile ea bokelloa 'me boemo ba IL-1β sehlopheng ka seng bo ile ba khethoa.Joalokaha ho bontšitsoe setšoantšong sa 4b, sehlopheng sa MOCK: (i) ha se bapisoa le sehlopha sa PBS, phekolo ea pcDNA3.1 e ne e se na phello e kholo ho IL-1β secretion (ANOVA, F (4.29) = 4.062, P = 0.9998), leha ho le joalo, IL-β secretion e ne e phahame haholo sehlopheng sa G. duodenalis cyst (ANOVA, F (4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine le pcDNA3.1- Intramuscular injection ea alpha-7.3 giardine e ile ea eketsa haholo serum IL-1β maemo (ANOVA, F (4, 29) = 4.062, P <0.0001);(iii) pcDNA3.1-alpha-7,3 giardine e hlahisitse maemo a phahameng a IL -1β secretion ho pcDNA3.1-alpha-2 giardine intramuscular injection group (ANOVA, F (4, 29) = 4.062, P = 0.0333) .Ha ho bapisoa le sehlopha se seng le se seng sa sehlopha sa phekolo sa MCC950 le sehlopha sa MOCK: (i) IL-1β maemo a secretion sehlopheng sa taolo ea PBS le sehlopha sa taolo sa pcDNA3.1 se fokotsehile ho isa tekanyong e itseng ka mor'a ho thibela MCC950 inhibitor, empa phapang e ne e se. bohlokoa (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F (9, 58) = 3.540, P = 0.5949);(ii) ka mor'a ho thibela MCC950., IL-1β secretion e ile ea fokotseha haholo sehlopheng sa G. duodenalis cyst-infected, sehlopha sa pcDNA3.1-alpha-2 giardine, le sehlopha sa pcDNA3.1-alpha-7.3 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).Liphetho tsena li fana ka maikutlo a hore alpha-2 giardine le alpha-7.3 giardine li sebelisana le ts'ebetso ea NLRP3 inflammasome ka vivo.
pcDNA3.1 (+) -giardines e kenya tšebetsong NLRP3 host inflammasome in vivo.Litoeba li ile tsa entoa (IM) ka recombinant eukaryotic expression plasmid pcDNA3.1(+)-alpha-2 giardine kapa pcDNA3.1(+)-alpha-7.3 giardine ebe e phekoloa ka MCC950 (ip; MCC950 sehlopha) kapa che (sehlopha sa dummy ).Sehlopha sa PBS kapa pcDNA3.1 (+) sa phekolo ea plasmid se ne se sebelisoa e le taolo e mpe, sehlopha sa phekolo ea G. duodenalis cyst se ne se sebelisoa e le taolo e ntle.Sehlopha sa liteko le mokhoa oa phekolo.b Maemo a serum a IL-1β ka litoeba a ne a lekantsoe ka letsatsi la 7 ka tlhahlobo ea ELISA.Liphapang pakeng tsa lihlopha tsa sehlopha sa MOCK li ile tsa hlahlobjoa ka mokhoa o le mong oa ANOVA, 'me liphapang pakeng tsa sehlopha sa MOCK le sehlopha sa MCC950 li ile tsa hlahlojoa ho sebelisoa teko ea t ea SPSS software version 22.0.Linaleli li bontša phapang e khōlō pakeng tsa lihlopha tsa phekolo sehlopheng sa MOCK, * P <0.05 le *** P <0.001;matšoao a lidolara ($) a bontša phapang e khōlō pakeng tsa sehlopha ka seng sehlopheng sa MOCK le sehlopha sa MCC950 ho P<0.05.Liphetho tsa liteko tse supileng tse ikemetseng tsa baeloji.i, ente ea intramuscular, ns, ha e bohlokoa (P> 0.05)
Ho batlisisa phello ea alpha-2 le alpha-7.3 giardine-mediated activation ea NLRP3 host inflammasome ka G. duodenalis infectivity, re sebelisitse litoeba tsa WT C57BL / 6 le ho kenya alpha-2 giardine le alpha-7.3 giardine.plasmid e ne e kenngoa ka intramuscularly, ka mor'a matsatsi a 3 ka tube ea gastric ea G. duodenalis cyst, ka mor'a moo litoeba li ile tsa hlokomeloa ka matsatsi a 7.Leano le qaqileng la teko le bontšitsoe setšoantšong sa 5a.Boima ba 'mele ba toeba e' ngoe le e 'ngoe bo ne bo lekanngoa letsatsi le leng le le leng, lisampole tsa lisele tse ncha tsa duodenal li ile tsa bokelloa ka letsatsi la 7 ka mor'a ho tsamaisoa ka gastric tube, palo ea trophozoite e lekantsoe, 'me liphetoho tsa histopathological li ile tsa bonoa.Joalokaha ho bontšitsoe setšoantšong sa 5b, ka nako e ntseng e eketseha ea ho fepa, BW ea litoeba sehlopheng ka seng e ile ea eketseha butle-butle.MT ea litoeba e ile ea qala ho fokotseha ka letsatsi la 3rd ka mor'a tsamaiso ea intragastric ea G. duodenalis cysts, 'me butle-butle ea eketseha.Ts'ebetso ea NLRP3 inflammasome e bakoang ke ente ea intramuscular ea alpha-2 giardine le alpha7.3 giardine e fokolitse boima ba 'mele ho litoeba (Letsatsi la 1: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 1.399, P = 0 .9754 Letsatsi la 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30)=1.399, P=0.9987 Letsatsi la 2: pcDNA3.1-alpha-2 giardine, ANOVA, F( 4, 30) = 0.3172, P = 0.9979; Letsatsi la 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.3172, P = 0.8409; Letsatsi la 3: pcDNA3.1-alpha-2 giardine, ANOVA 4, 30) = 0.8222, P = 0.0262 Letsatsi la 3: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.8222, P = 0.0083; Letsatsi la 4: pcDNA3.1-alpha-2 , F (4, 30) = 0.5620, P = 0.0012, Letsatsi la 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.5620, P <0.0001, Letsatsi la 5: pcDNA3.1-alpha - 2 giardine, ANOVA, F (4, 30) = 0.9728, P <0.0001 Letsatsi la 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F (4, 30) = 0.9728, P <0.0001 Letsatsi la 6. -1 pcDNA alpha-2 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0012, Letsatsi la 6: pcDNA3.1-alpha-7.3 giardine, ANOVA, F (4, 30) = 0.7154, P = 0.0006;Letsatsi la 7: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 0.5369, P<0.0001 Letsatsi la 7: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4 , 30) = 0.5369, P <0.0001).Moroalo oa likokoana-hloko o ile oa hlahlojoa ka duodenum (setšoantšo sa 5c).Ha ho bapisoa le taolo e ntle e sa sebetsoeng le sehlopha se kentsoeng ka pcDNA3.1 vector e se nang letho, palo ea G. duodenalis trophozoites e fokotsehile haholo lihlopheng tse entsoeng ka α-2 giardine le 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).Ho phaella moo, giardine alfa-7.3 e ne e sireletsa ho feta litoeba ho feta giardine alfa-2 (ANOVA, F (3, 24) = 1.209, P = 0.0081).Liphetho tsa HE staining li bontšoa ho feiga.5d–f.Litoeba tse kentsoeng ka alpha-2 giardine le alpha-7.3 giardine li ne li e-na le maqeba a fokolang a lisele tsa duodenal, tse bonahatsoang ke tšenyo ea villus, ha li bapisoa le litoeba tse kenngoeng ka G. duodenalis le litoeba tse kenngoa G. duodenalis hammoho le pcDNA3 vector e se nang letho .1 Zoom.(pcDNA3.1-alpha-2 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0035 kapa P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0028 kapa P = 0.0055) le ho fokotsa crypt atrophy (pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 1.470, P = 0.0264 kapa P = 0.0158; pcDNA3.1-alpha-7.3 ANOVAardine , F(3, 24) = 1.470, P = 0.0371 kapa P = 0.0191).Liphetho tsena li fana ka maikutlo a hore alpha-2 giardine le alpha-7,3 giardine li fokotsa ts'oaetso ea G. duodenalis ka ho kenya NLRP3 inflammasome ka vivo.
Karolo ea pcDNA3.1 (+) -giardins ho tšoaetso ea G. duodenalis.Litoeba li ne li entoa (IM) ka recombinant eukaryotic expression plasmids pcDNA3.1 (+) -alpha-2 giardine kapa pcDNA3.1 (+) -alpha-7.3 giardine ebe e qholotsoa ka G. duodenalis cysts (ig).Sehlopha sa PBS le pcDNA3.1 (+) + sehlopha sa phekolo ea li-duodenal cyst li ne li sebelisoa e le lihlopha tse mpe tsa taolo, 'me sehlopha sa phekolo ea li-duodenal cyst se ne se sebelisoa e le sehlopha sa taolo e ntle.Sehlopha sa liteko le mokhoa oa phekolo.b The MT ea litoeba ho e 'ngoe le e' ngoe ea lihlopha tse fapaneng tsa phekolo e ne e behiloe leihlo bakeng sa matsatsi a 7 ka mor'a phephetso.Linaleli li bontša phapang e khōlō pakeng tsa lihlopha tsa sehlopha sa G. duodenalis le pcDNA3.1 (+) -alpha-2 giardine sehlopha, * P <0.05, ** P <0.01, le *** P <0.001;letšoao la dollar ($) le bontša phapang e khōlō pakeng tsa sehlopha ka seng sa G. duodenalis le pcDNA3.1 (+) -alpha-7.3 jardine sehlopha, $ $ P <0.01 le $ $ $ P <0.001.c Moroalo oa likokoana-hloko o ile oa khethoa ka ho bala palo ea li-trophozoite ka 1 ml ea duodenal lavage ho tloha duodenum (bolelele ba 3 cm) mme e hlalosoa e le palo ea likokoana-hloko ka cm cm ea duodenum.Phapang pakeng tsa sehlopha sa ts'oaetso ea G. duodenalis, pcDNA3.1 (+) -alpha-2 giardine sehlopha, le pcDNA3.1 (+) -alpha-7.3 sehlopha sa giardine se ile sa hlahlojoa ka tsela e le 'ngoe ANOVA e sebelisang SPSS software version 22.0.Linaleli li bontša phapang e khōlō ho **P<0.01 le ***P<0.001.d Liphetoho tsa histopathological ka duodenum.Metsu e khubelu e bontša tšenyo ho villi, metsu e tala e bontša tšenyo ea li-crypts.Scale bar: 100 µm.e, f Tlhahlobo ea lipalo-palo ea bophahamo ba mouse duodenal villus (e) le bophahamo ba crypt (f).Phapang pakeng tsa lihlopha ho Setšoantšo sa 1d e ile ea hlahlojoa ke ANOVA ea tsela e le 'ngoe e sebelisang SPSS software version 22.0.Linaleli li bontša phapang e khōlō ho *P<0.05 le **P<0.01.Liphetho tsa liteko tse supileng tse ikemetseng tsa baeloji.ns, ha e bohlokoa (P > 0.05)
Giardia duodenum ke kokoana-hloko e tsebahalang ea mala a batho le liphoofolo tse ling tse anyesang tse bakang giardiasis.Ka 2004, e kenyelelitsoe ho WHO Neglected Diseases Initiative ka lebaka la ho ata ha eona ho feta lilemo tse 6, haholo-holo metseng ea maemo a tlaase a moruo [32].Tsamaiso ea 'mele ea ho itšireletsa mafung e phetha karolo ea bohlokoa karabelong ea 'mele ea ho itšireletsa mafung ho tšoaetso ea G. duodenalis.Li-macrophages tsa litoeba li tlalehiloe hore li koahetse le ho bolaea G. duodenalis ka ho lokolla maraba a extracellular [33].Liphuputso tsa rona tse fetileng li bontšitse hore G. duodenalis, likokoana-hloko tse sa hlaselehang tsa extracellular, li kenya ts'ebetsong p38 MAPK, ERK, NF-κB p65, le NLRP3 litsela tsa matšoao a ho ruruha ho macrophages ea mouse ho laola likarabo tsa ho ruruha, 'me ho lokolloa GEV ho ka ntlafatsa ts'ebetso ena.[Setšoantšo se leqepheng la 13], 24].Leha ho le joalo, li-PAMP tse tobileng tse amehang ho ruruha ho laoloang ke inflammasome ea NLRP3 ho GEV le karolo ea NLRP3 inflammasome ho giardiasis e ntse e tla hlakisoa.E le ho hlakisa lipotso tsena tse peli, re ile ra khanna thuto ena.
NLRP3 inflammasome e fumaneha ka har'a cytoplasm ea lisele tsa 'mele tsa ho itšireletsa mafung' me e ka sebelisoa ke likaroloana tse sa tšoaneng tse kang likristale tsa uric acid, chefo, libaktheria, likokoana-hloko le likokoana-hloko.Lithutong tsa baktheria, chefo e fumanoe e le li-PAMP tsa bohlokoa tse etsang hore li-sensor tsa ho ruruha li sebetse, tse lebisang ho ruruha le lefu la sele [34].Lichefo tse ling tse nang le mefuta e sa tšoaneng, tse kang hemolysin ho tloha Staphylococcus aureus [35] le Escherichia coli [36], hemolysin BL (HBL) ho tloha enterotoxin (NHE) [37], e etsa hore ho sebelisoe NLRP3 ho ruruha.Liphuputso tsa kokoana-hloko li bontšitse hore liprotheine tsa kokoana-hloko tse kang SARS-COV-2 protheine ea enfelopo (E) [38] le Zika virus NS5 protein [39] ke PAMPs ea bohlokoa e amoheloang ke NLRP3 receptor.Lithutong tsa likokoana-hloko, likokoana-hloko tse ngata li 'nile tsa tlalehoa hore li amahanngoa le ts'ebetso ea inflammasome, e kang Toxoplasma gondii, Trichomonas vaginalis [40], Trypanosoma cruzi [41] le Leishmania [42].Liprotheine tse teteaneng tsa granule GRA35, GRA42, le GRA43, tse amanang le virulence ea Toxoplasma gondii, lia hlokahala bakeng sa ho kenngoa ha pyroptosis ho Lewis rat macrophages [43].Ho phaella moo, lithuto tse ling tsa Leishmania li tsepamisitse maikutlo ho limolek'hule tse amehang ho NLRP3 inflammasome, tse kang parasite membrane lipophosphoglycan [44] kapa zinc metalloprotease [45].Har'a liphatsa tsa lefutso tse kang annexin-like alpha-giardin, alpha-1 giardin e bontšitsoe hore e ka ba moemeli oa ente e fanang ka tšireletso khahlanong le G. duodenalis ka mohlala oa mouse [18].Thutong ea rona, re khethile G. duodenalis virulence factor alpha-2 le alpha-7,3 giardines, e ikhethang ho giardia empa e batla e le tlase ho tlalehoa.Liphatsa tsena tse peli tsa liphatsa tsa lefutso li ile tsa kenngoa ho pcDNA3.1 (+) eukaryotic expression system vector bakeng sa tlhahlobo ea ts'ebetso ea ho ruruha.
Moetsong oa rona oa toeba, likhechana tsa caspase tse petsotsoeng li sebetsa e le matšoao a ts'ebetso ea ho ruruha.Ha e tsosoa, NLRP3 e sebelisana le ASC, e hira li-procaspase, 'me e hlahisa li-caspases tse sebetsang tse arolang pro-IL-1β le pro-IL-18 hore e be IL-1β e hōlileng le IL-18, ka ho latellana -18.Inflammatory caspases (caspases-1, -4, -5 le -11) ke lelapa le sirelelitsoeng la li-cysteine ​​​​protease tse bohlokoa bakeng sa ts'ireletso ea tlhaho 'me li ameha ho ruruha le lefu le hlophisitsoeng la sele [46].Caspase-1 e sebelisoa ke canonical inflammasomes [47], ha caspases-4, -5, le -11 li phunyeletsoa nakong ea ho thehoa ha li-inflammasomes tse sa tloaelehang [48].Thutong ena, re sebelisitse mouse peritoneal macrophages e le mohlala 'me ra batlisisa p20 caspase-1 cleaved caspase-1 e le letšoao la moeti NLRP3 ts'ebetso ea ho ruruha lithutong tsa ts'oaetso ea G. duodenalis.Liphello li bontšitse hore li-alpha-giardins tse ngata li ikarabella bakeng sa ts'ebetso e tloaelehileng ea ho ruruha, e lumellanang le ho sibolloa ha limolek'hule tsa bohlokoa tsa likokoana-hloko tse amehang ho libaktheria le likokoana-hloko.Leha ho le joalo, thuto ea rona ke skrine ea pele feela 'me ho na le limolek'hule tse ling tse ka kenyang li-inflammasome tse sa tloaelehang, joalokaha thuto ea rona e fetileng e fumane li-inflammasomes tsa khale le tse seng tsa khale ho G. duodenalis tšoaetso [13].Ho tsoela pele ho fumana hore na p20 caspase-1 e hlahisitsoeng e amahanngoa le NLRP3 inflammasome, re fetisitse alpha-2 le alpha-7.3 giardins ka har'a mouse peritoneal macrophages ho fumana maemo a bohlokoa a liprotheine tsa molek'hule le maemo a ASC oligomerization, ho tiisa hore bobeli ba α-giardins ba sebetsa. ho ruruha ha NLRP3.Liphetho tsa rona li fapane hanyenyane le tsa Manko-Prykhoda et al., ea ileng a tlaleha hore ho tsosoa ha lisele tsa Caco-2 tse nang le mefuta ea G. muris kapa E. coli EPEC feela ho ka eketsa matla a fluorescence a NLRP3, ASC, le caspase-1, le hoja e se haholo, ha mokhoa oa costimulation oa G. muris le E. coli o ekelitse maemo a liprotheine tse tharo [49].Phapang ena e ka 'na ea bakoa ke ho se tšoane ha khetho ea mefuta ea Giardia, lisele tsa lisele, le lisele tsa mantlha.Re boetse re etsa litlhahlobo tsa vivo re sebelisa MCC950 ho litoeba tsa WT C57BL/6 tsa basali ba libeke tse 5, tse hlaselehang habonolo G. duodenalis.MCC950 ke molek'hule e nyane e matla le e khethang ea NLRP3 inhibitor e thibelang ts'ebetso ea canonical le e seng ea canonical NLRP3 maemong a nanomolar.MCC950 e thibela ts'ebetso ea NLRP3 empa ha e ame ts'ebetso ea AIM2, NLRC4, le NLRP1 litsela tsa ho ruruha kapa litsela tsa lipontšo tsa TLR [27].MCC950 e thibela ts'ebetso ea NLRP3 empa ha e thibele ho qalisoa ha NLRP3, K+ efflux, Ca2 + e phallang, kapa tšebelisano pakeng tsa NLRP3 le ASC;ho e-na le hoo, e thibela ts'ebetso ea NLRP3 ea inflammasome ka ho thibela oligomerization ea ASC [27].Ka hona, re sebelisitse MCC950 thutong ea in vivo ho fumana karolo ea NLRP3 inflammasome ka mor'a ente ea giardine.Activated caspase-1 p10 e qhekella li-cytokines tse pro-inflammatory pro-IL-1β le pro-IL-18 hore e be IL-1β e hōlileng le IL-18 [50].Phuputsong ena, litekanyetso tsa serum IL-1β ka litoeba tse tšoaroang ke giardine tse nang le MCC950 kapa ntle le tsona li ne li sebelisoa e le pontšo ea hore na NLRP3 inflammasome e ne e sebelisoa.Joalokaha ho lebeletsoe, phekolo ea MCC950 e fokolitse haholo maemo a serum IL-1β.Lintlha tsena li bontša ka ho hlaka hore G. duodenalis giardin alfa-2 le giardin alfa-7.3 li khona ho kenya ts'ebetso ea NLRP3 mouse inflammasome.
Lintlha tsa bohlokoa tse bokelletsoeng lilemong tse leshome tse fetileng li bontšitse hore IL-17A ke molaoli ea ka sehloohong oa ho itšireletsa khahlanong le G. muris, ho etsa hore ho be le pontšo ea IL-17RA, ho hlahisa li-peptide tse thibelang likokoana-hloko, le ho laola ts'ebetso ea tlatsetso [51].Leha ho le joalo, tšoaetso ea Giardia e etsahala hangata ho batho ba baholo ba bacha, 'me ho tlalehiloe hore tšoaetso ea Giardia ho litoeba tse nyenyane ha e sebetse karabelo ea IL-17A ho fana ka tšireletso ea eona [52], e leng se etsang hore bafuputsi ba batle Giardia e' ngoe ea immunomodulatory.mekhoa ea tšoaetso ea helminth.Bangoli ba phuputso ea morao tjena ba tlalehile hore G. muris e ka etsa hore NLRP3 inflammasome e sebelisoe ke E. coli EPEC, e khothalletsang tlhahiso ea li-peptide tse thibelang likokoana-hloko le ho fokotsa matla a eona a ho khomarela le palo ea trophozoite ka har'a pampiri ea mala, kahoo e fokotsa boima ba kolone. mafu a bakoang ke bacilli [49].NLRP3 inflammasome e ameha ho nts'etsopele ea mafu a sa tšoaneng.Liphuputso li bontšitse hore Pseudomonas aeruginosa e baka autophagy ka macrophages ho qoba lefu la lisele, 'me mokhoa ona o itšetlehile ka ts'ebetso ea NLRP3 inflammasome [53].Bakeng sa N. caninum, ts'ebetso ea mefuta ea oksijene e sebetsang ea NLRP3 inflammasome e fokotsa ho pheta-pheta ha eona ho moeti, e leng se etsang hore e be sepheo sa phekolo [9].Paracoccidioides brasiliensis e fumanoe e etsa hore ho be le ts'ebetso ea NLRP3 inflammasome ka har'a lisele tsa dendritic tse entsoeng ka lesapo la mouse, e leng se etsang hore ho lokolloe cytokine IL-1β, e nang le karolo ea bohlokoa ho sireletsa moeti [10].Mefuta e mengata ea Leishmania, ho kenyeletsoa L. amazonensis, L. e kholo, L. braziliensis, le L. infantum chagasi, e kenya tšebetsong NLRP3 le caspase-1 e itšetlehileng ka ASC ho macrophages, hammoho le tšoaetso ea Leishmania.Phetoho ea likokoana-hloko e ntlafatsoa ho litoeba tse haelloang ke mofuta oa NLRP3/ASC/caspase-1 [11].Zamboni et al.Ho tlalehoa hore tšoaetso ea Leishmania e etsa hore ho sebelisoe NLRP3 inflammasome ka macrophages, e fokotsang ho ikatisa ha likokoana-hloko tsa intracellular.Kahoo, Leishmania e ka thibela ts'ebetso ea NLRP3 e le leano la ho qoba.Lithutong tsa vivo, NLRP3 inflammasome e kentse letsoho ho felisoeng ha Leishmania, empa ha ea ka ea ama lisele [54].Ka lehlakoreng le leng, lithutong tsa helminthiasis, ts'ebetso ea NLRP3 inflammasome e ile ea hatella tšireletso ea moeti khahlanong le helminthiasis ea mala [12].Shigella ke e 'ngoe ea libaktheria tse ka sehloohong tse bakang letšollo lefatšeng ka bophara.Libaktheria tsena li ka kenya tlhahiso ea IL-1β ka P2X7 receptor-mediated K + efflux, mefuta e sebetsang ea oksijene, lysosomal acidification, le tšenyo ea mitochondrial.NLRP3 inflammasome e laola hampe phagocytosis le ts'ebetso ea bactericidal ea macrophages khahlanong le Shigella [55].Liphuputso tsa Plasmodium li bontšitse hore AIM2, NLRP3 kapa litoeba tse haelloang ke caspase-1 tse tšoaelitsoeng ke Plasmodium li hlahisa mefuta e phahameng ea mofuta oa 1 interferon 'me li hanyetsana le tšoaetso ea Plasmodium [56].Leha ho le joalo, karolo ea alpha-2 giardine le alpha-7.3 giardine ho kenya ts'ebetsong ea pathogenic ea ho ruruha ha NLRP3 ho litoeba ha e hlake.
Phuputsong ena, ho thibela NLRP3 inflammasome ka MCC950 ho fokolitse BW le ho eketsa palo ea trophozoite ka mala a ho hlatsoetsa mala ka har'a litoeba, e leng se ileng sa fella ka liphetoho tse matla haholo tsa pathological meleng ea duodenal.Alpha-2 giardine le alpha-7.3 giardine activate the host toeba NLRP3 inflammasome, eketsa boima ba 'mele oa mouse, fokotsa palo ea trophozoites ka mala a ho hlatsoa mala, le ho kokobetsa liso tsa pathological duodenal.Liphetho tsena li fana ka maikutlo a hore G. duodenalis e ka kenya tšebetsong NLRP3 host inflammasome ka alpha-2 giardine le alpha-7,3 giardine, ho fokotsa pathogenicity ea G. duodenalis ho litoeba.
Ka kakaretso, liphetho tsa rona li bontša hore alpha-2 le alpha-7.3 giardines li etsa hore ho sebelisoe NLRP3 host inflammasome le ho fokotsa ts'oaetso ea G. duodenalis ho litoeba.Ka hona, limolek'hule tsena ke lipakane tse tšepisang bakeng sa thibelo ea giardiasis.
       Data supporting the results of this study can be obtained from the respective author at gongpt@jlu.edu.cn.
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