ʻO ka Giardia duodenum kahi mea ʻino e hoʻoulu ai i ka giardiasis, kahi maʻi ʻōpū i maʻamau i nā keiki liʻiliʻi me nā hōʻailona lapaʻau o ka maʻi maʻi.Ua hōʻike mua mākou i ka extracellular G. duodenalis e hoʻoulu i ka hoʻoulu ʻana o ka intracellular oligomerization-like receptor 3 (NLRP3) hoʻopaʻa i nā nucleotides a hoʻoponopono i nā pane inflammatory host ma o ka huna ʻana o extracellular vesicle (EV).Eia nō naʻe, ʻo nā ʻano molekala pololei o ka pathogen-associated duodenococcal EV (GEV) i komo i kēia kaʻina hana a me ke kuleana o ka inflammasome NLRP3 i ka giardiasis e hoʻomau ʻia e elucidated.
Ua kūkulu ʻia nā plasmids hōʻike eukaryotic recombinant pcDNA3.1(+)-alpha-2 a me alpha-7.3 giardins i GEV, ua hoʻololi ʻia i loko o nā macrophages peritoneal mua o ka ʻiole, a ʻike ʻia ma ke ana ʻana i ka caspase-1.Ua nānā ʻia ka pae hōʻike p20..ʻO G. duodenalis alpha-2 a me alpha-7.3 giardines i ʻike mua ʻia ma ke ana ʻana i ka inflammasome NLRP3 (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 a me caspase-1 p20), huna huna IL.1β mau pae, apoptotic spotted protein (ASC) oligomerization pae, a immunofluorescent localization o NLRP3 a me ASC.ʻO ke kuleana o ka inflammasome NLRP3 i ka pathogenicity o G. duodenalis ua loiloi ʻia me ka hoʻohana ʻana i nā ʻiole kahi i kāohi ʻia ai ka hoʻoulu ʻana o NLRP3 (NLRP3 blocked mice) a me nā loli pathological i ke kaumaha o ke kino, duodenal parasitic load, a me ka duodenal tissue i nānā ʻia.Eia hou, ua noiʻi mākou inā hoʻokomo ka hiardines alpha-2 a me alpha-7.3 i ka huna ʻana o IL-1β i loko o ka vivo ma o ka inflammasome NLRP3 a hoʻoholo i ke kuleana o kēia mau mole i ka pathogenicity o G. duodenalis i nā ʻiole.
ʻO Alpha-2 a me alpha-7.3 giardines e hoʻoulu i ka hoʻouluʻana o ka NLRP3 inflammasome in vitro.Ua alakaʻi kēia i ka hoʻoulu ʻana o ka p20 caspase-1, ka piʻi ʻana o nā pae hōʻike o NLRP3, pro-IL-1β, a me nā protein pro-caspase-1, kahi piʻi nui o ka huna ʻana o IL-1β, ka hoʻokumu ʻana o nā kiko ASA i ka cytoplasm, a me ka induction o ASA oligomerization.NLRP3 ho'āhu Penile lilo ho'onui i ka pathogenicity o G. duodenalis i loko o nāʻiole.ʻO nā ʻiole i mālama ʻia me nā cysts e nā ʻiole i hoʻopaʻa ʻia e NLRP3 ua hōʻike i ka nui o nā trophozoites a me nā pōʻino nui i ka duodenal villi, i hōʻike ʻia e nā necrotic crypts me ka shrunken a me ka lālā.Ua hōʻike ʻia nā hoʻokolohua vivo e hiki i nā giardine alpha-2 a me alpha-7.3 ke hoʻoulu i ka huna ʻana o IL-1β ma o ka inflammasome NLRP3, a ʻo ka pale ʻana me giardines alpha-2 a me alpha-7.3 i hoʻemi i ka pathogenicity o G. duodenalis i nā ʻiole.
Hoʻohui pū ʻia, hōʻike nā hopena o kēia noiʻi ʻana i ka giardia alpha-2 a me alpha-7.3 ke kumu i ka hoʻoponopono ʻana i ka ʻeha o ka host NLRP3 a hoʻemi i ka infectivity o G. duodenalis i nā ʻiole, ʻo ia nā mea e hoʻohiki ai no ka pale ʻana i ka giardiasis.
ʻO Giardia duodenum kahi maʻi protozoan extracellular e noho ana i loko o ka ʻōpū liʻiliʻi a ke kumu i 280 miliona mau hihia o ka giardiasis me ka maʻi maʻi i kēlā me kēia makahiki, ʻoi aku hoʻi i waena o nā keiki liʻiliʻi ma nā ʻāina ulu.Loaʻa ka poʻe i ka maʻi ma ka inu ʻana i ka wai a i ʻole ka meaʻai i haumia ʻia me M. duodenum cysts, a laila komo i loko o ka ʻōpū a hoʻokuʻu ʻia i loko o ka wai ʻōpū.Hoʻopili ʻo Giardia duodenum trophozoites i ka epithelium duodenal, e hoʻoulu ai i ka nausea, ka luaʻi, ka ʻōpū, ka ʻeha ʻōpū, a me ka pohō kaumaha.Hiki i nā kānaka me ka immunodeficiency a me ka cystic fibrosis i ka maʻi.Hiki ke loaʻa ka maʻi ma o ka waha a me ka anal sex [2].ʻO nā lāʻau lapaʻau e like me metronidazole, tinidazole, a me nitazoxanide nā koho lapaʻau maikaʻi loa no nā maʻi duodenal [3].Eia nō naʻe, ʻo kēia mau lāʻau lapaʻau chemotherapy ke kumu i nā hopena ʻino e like me ka nausea, carcinogenesis, a me genotoxicity [4].No laila, pono e hoʻomohala ʻia nā hoʻolālā ʻoi aku ka maikaʻi e pale ai i ka maʻi G. duodenalis.
ʻO nā Inflammasomes kahi papa o nā cytosolic protein complexes he ʻāpana o ka pane kūlohelohe kūlohelohe, e kōkua ana i ka pale ʻana i ka hoʻouka ʻana o ka pathogen a me ka hoʻoponopono ʻana i nā pane inflammatory [5].Ma waena o kēia mau inflammasomes, ua aʻo nui ʻia ka nucleotide-binding oligomerization (NOD) receptor 3 (NLRP3) nucleotide-binding oligomerization (NLRP3) nucleotide-binding-like inflammasome no ka mea hiki ke ʻike ʻia e nā ʻano mole / pōʻino pili i ka pathogen (PAMP). ʻO DAMP), ʻike, hoʻāla i ka ʻōnaehana immune innate.a hoʻoponopono i ka homeostasis intestinal i nā maʻi inflammatory he nui [6,7,8].Aia i loko o ke kumu hoʻokipa hoʻomaopopo (PRR) NLRP3, kahi mea hoʻopili apoptotic spotted protein (ASC), a me kahi procaspase-1 a i ʻole procaspase-11.Hana ʻia ka inflammasome NLRP3 ma ke ʻano he pūʻali e kūʻē i ka hoʻouka kaua pathogen, e like me ka ʻike ʻia ma Neospora caninum [9], Paracoccidioides brasiliensis [10], a me Leishmania study.[11], akā ua hōʻike pū ʻia ʻo ka hoʻāla ʻana o ka inflammasome NLRP3 e kaupalena i nā pane pale pale a hoʻonui i ka piʻi ʻana o ka maʻi, no ka laʻana, i nā ilo [12].Ma muli o kā mākou ʻike mua, ua hōʻike mākou i ka extracellular G. duodenalis e hoʻoulu i ka intracellular activation o NLRP3 mumū a hoʻololi i nā pane inflammatory host ma ka huna ʻana i nā vesicle extracellular (EVs) [13].Eia naʻe, e hoʻoholo ʻia ke kuleana o ka inflammasome NLRP3 i ka maʻi G. duodenalis in vivo.
Ua wehewehe mua ʻia ʻo Giardins ma ke ʻano he ʻāpana o ka G. duodenalis cytoskeleton a he mea nui i ka motility trophozoite a me ka hoʻopili epithelial cell i loko o ka ʻōpū liʻiliʻi.No ka hoʻololi maikaʻi ʻana i ke kaiapuni a hoʻonui i ko lākou pathogenicity, ua hoʻomohala ʻo G. duodenalis trophozoites i kahi ʻano cytoskeletal kūʻokoʻa me 8 flagella, 1 kino waena, a me 1 ventral disc [14].Hoʻohana nā trophozoites o Giardia duodenum i kā lākou cytoskeleton e komo i loko o ka ʻōpū liʻiliʻi o luna, ʻoi aku ka duodenum, a hoʻopili i nā enterocytes.Ke neʻe mau nei lākou a hoʻopili i nā cell epithelial me ka hoʻohana ʻana i ka metabolism cell.No laila, aia kahi pilina pili ma waena o kā lākou cytoskeleton a me ka virulence.ʻO nā Giardines kikoʻī no Giardia duodenum he mau ʻāpana o ke ʻano cytoskeleton [15] a ua māhele ʻia i ʻehā mau papa: α-, β-, γ-, a me δ-giardines.Aia he 21 mau lālā o ka ʻohana α-giardin, aia nā mea āpau i ka hiki ke pili i ka calcium e hoʻopaʻa i nā phospholipids [16].Hoʻopili pū lākou i ka cytoskeleton i ka membrane cell.I nā poʻe me ka maʻi paʻi i hana ʻia e G. duodenalis, ua hōʻike nui ʻia nā α-giardins a me ka immunoreactive i ka wā maʻi [17].ʻO nā lāʻau lapaʻau Heterologous e pili ana iā Giardia alfa-1 i pale ʻia mai ka giardiasis i nā ʻiole a he mau antigens moho no ka hoʻomohala ʻana i ka maʻi [18].ʻO Alpha-8 giardin, i loko o ka membrane plasma a me ka flagella, akāʻaʻole i loko o ka disc ventral, hoʻonui i ka motility a me ka uluʻana o nā trophozoites ma G. duodenalis [19].Hoʻopili ʻo Alpha-14 giardin i nā hale microtubule ma ka flagella a pili i ka ola o G. duodenalis [20].Loaʻa ka Alpha-11 giardine i ka nui o ke ola holoʻokoʻa, a ʻo ka overexpression o ka alpha-11 giardine e pōʻino iā G. duodenalis ponoʻī [21].Eia naʻe, ʻaʻole maopopo inā he pale ka alpha-2 giardine a me alpha-7.3 giardine i ka maʻi G. duodenalis a me kā lākou mau ʻano kumu.
I loko o kēia haʻawina, ua hoʻololi ʻia nā plasmids hōʻike eukaryotic recombinant pcDNA3.1(+)-alpha-2 giardine a me pcDNA3.1(+)-alpha-7.3 giardine i loko o nā macrophages peritoneal mua o ka ʻiole e hoʻāla i ka host NLRP3.A laila ua nānā ʻia nā pahuhopu inflammasome.Ua loiloi pū mākou i ke kuleana o ka inflammasome NLRP3 i ka pathogenicity o G. duodenalis, ua noiʻi inā he alpha-2 a me alpha-7,3 giardines i hoʻoulu i ka hoʻoulu ʻana o ka inflammasome NLRP3 i loko o vivo, a ua hoʻoholo i kēia mau kuleana ʻelua o giardines i ka pathogenicity o G. duodenalis.ʻO kā mākou pahuhopu maʻamau ka hoʻomohala ʻana i nā pahuhopu hoʻohiki no ka pale ʻana i ka maʻi G. duodenalis.
Ua kūʻai ʻia nā ʻiole wahine ʻano ʻāhiu (WT) C57BL/6 mai ka Liaoning Changsheng Experimental Animal Center (Liaoning, Kina).Loaʻa ka ʻiole i ka wai, loaʻa ka meaʻai sterilized a mālama ʻia i loko o kahi pōʻaiapuni māmā/ʻeleʻele 12/12.Ma mua o ka maʻi, ua loaʻa i nā ʻiole nā ​​lāʻau antibiotic ad libitum i ka wai inu i hoʻohui ʻia me ka ampicillin (1 mg / mL), vancomycin (1 mg / mL), a me neomycin (1.4 mg / mL) (kūʻai ʻia mai Shanghai, Kina, nā mea kūlohelohe) [22 ].].ʻO nā ʻiole i nalowale i ka hiki ke ʻai a inu no > 24 mau hola a nalowale ≥ 20% ke kaumaha o ke kino i hoʻopau ʻia e ke kanaka euthanized e ka cervical dislocation.
Ua hoʻonui ʻia ʻo WB G. duodenalis trophozoites (American Type Culture Collection, Manassas, USA) me 12.5% ​​fetal bovine serum (FBS; ʻO kēlā me kēia ʻōmaʻomaʻo, Zhejiang, Kina) a me 0.1% bovine bile (Sigma-Aldrich, St. Missouri, USA. ).USA) ma lalo o nā kūlana microaerobic.Ua hōʻiliʻili ʻia nā trophozoites confluent ma luna o ka hau a kau ʻia ma ka ratio o 1:4 no ka hana hou ʻana.
Giardia duodenum cysts ua hoʻouluʻia e like me ka mea i ho'ākāka muaʻia [23], ua hōʻiliʻiliʻia nā trophozoites i ka māhele logarithmic a laila hoʻoheheʻeʻia me ka encapsulation inducing medium, pH 7.1 (hoʻololiʻia TYI-S-33) i ka hopena hope loa o 1 × 106 trophozoites / mL.ka nui o ka bile 0.05% waena).Hoʻoulu ʻia nā Trophozoites ma lalo o nā kūlana anaerobic ma 37 ° C a hiki i ka wā ulu logarithmic.E hoʻololi i ka mea hoʻololi i ka mea hoʻoulu kime (pH 7.8; hoʻololi ʻia ʻo TYI-S-33 medium me ka 1% bile concentration) a me ka moʻomeheu G. duodenalis ma 37 ° C no 48-96 mau hola, i ka wā i ʻike ʻia ai nā cysts hoʻokumu ma lalo o kahi microscope.Ma hope o ka hoʻoulu ʻia ʻana o ka hapa nui o nā trophozoites e hana i nā cysts, ua ʻohi ʻia ka hui moʻomeheu a hoʻomaha ʻia i loko o ka wai deionized sterile e lyse i nā trophozoites i koe.Ua helu ʻia nā cysts a mālama ʻia ma 4 ° C no ka nānā ʻana ma hope o kahi paipu ʻōpū i nā ʻiole.
Ua hoʻonui ʻia ʻo Giardia extracellular vesicles (GEVs) e like me ka mea i hōʻike mua ʻia [13].Ua hoʻokuʻu hou ʻia nā Trophozoites i loko o ka logarithmic growth phase i hoʻololi ʻia me TYI-S-33 medium i hoʻomākaukau ʻia me ka exosome-depleted FBS (Biological Industries, Beit-Haemek, Israel) i ka hopena hope o 1 × 106 parasites / mL a hoʻomoʻa ʻia no 12 mau hola.ua hoʻokaʻawale ʻia mai ka supernatant moʻomeheu e ka centrifugation ma 2000 g no 10 min, 10,000 g no 45 min, a me 100,000 g no 60 min.Ua hoʻoheheʻe ʻia nā precipitates i loko o ka phosphate buffered saline (PBS), i helu ʻia me ka BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) a mālama ʻia ma -80° C. a i hoʻohana pololei ʻia no nā loiloi hou aʻe.
Ua hoʻomākaukau ʻia nā macrophages peritoneal kiʻi mua e like me ka mea i wehewehe mua ʻia [24].ʻO ka pōkole, ua hoʻokomo ʻia nā ʻiole (nā makahiki 6-8 mau wiki) (intraperitoneally [ip]) me 2.5 ml o 2.98% Difco liquid thioglycol medium (BD, Franklin Lakes, NJ, USA) a hānai ʻia i nā palate 3-4.Ua hōʻiliʻili ʻia kahi hoʻokuʻu o nā macrophages mai ka ʻōpū o ka ʻiole ma hope o ka euthanasia a centrifuged 3 mau manawa ma 1000 g no 10 min.Ua ʻike ʻia nā cell i hōʻiliʻili ʻia e ka cytometry kahe e hoʻohana ana i ka marker CD11b a hiki i ka maʻemaʻe o ka cell ma>98%, a laila hoʻohui ʻia i nā papa moʻomeheu cell 6-well (4.5 x 106 cell/well) a hoʻomoʻi ʻia me 10% FBS (Bioidustry) ma 37°C.a me 5% CO2.
Ua unuhiʻia ka RNA mai ka 1 × 107 trophozoites i ka 1 ml o TRIzol reagent (Vazyme, Nanjing, Kina), ua unuhiʻia ka DNA genomic mai ka G. duodenalis RNA a pau e hoʻohana ana i ka MonScript dsDNase (Monad, Wuhan, Kina) a ua hoʻohuiʻia ka DNA (cDNA) e hoʻohana ana i ka MonScript RTIII Super Mix (Monad) e like me nā ʻōlelo a ka mea hana.
Ua loaʻa mai ka ʻike kaʻina CDS no ka gene G. duodenalis target mai ka NCBI GenBank.E hoʻohana i ka Primer 5.0 no ka hoʻolālā ʻana i nā kumu hoʻomaka cloning seamless no kēlā me kēia ʻano gene.ʻO ka mua mua (5′-3′) he ʻekolu ʻāpana: kahi kaʻina overlapping me kahi vector linearized pcDNA3.1(+) EcoRV (TGGTGGAATTTCTGCAGAT) a hoʻomaka i nā codons ATG a me GNN (inā ʻaʻole G ka kumu mua).Hana ʻia kēia i mea e hoʻomaikaʻi ai i ka pono o ka ʻōlelo.Eia hou, ma ka liʻiliʻi loa he 16 bp kumu i hui pū ʻia (GC maʻiʻo 40–60%/Tm ma kahi o 55 °C).ʻO ka mua hope (5′-3′) he ʻelua ʻāpana, kahi kaʻina overlapping me ka EcoRV-linearized vector pcDNA3.1(+) (GCCGCCACTGTGCTGGAT) a me kahi kumu hui o ka liʻiliʻi 16 bp.(koe i na pani hope elua).bases) he codon e like me AA a i ʻole GA e ʻae i nā plasmids recombinant e hōʻike i kā lākou mau protein i kapa ʻia).Ua helu ʻia nā kaʻina hana mua ma ka Papa 1 a ua hana ʻia e Kangmet Biotechnology Co., Ltd. (Changchun, Kina).
Hoʻonui ʻia nā pahuhopu me ka hoʻohana ʻana i ka Pfu DNA polymerase (Tiangen, Beijing, Kina) a i ʻole Ex-taq (Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, Kina) me ka hoʻohana ʻana i ka G. duodenalis cDNA i hoʻomākaukau ʻia ma ke ʻano he hoʻohālike.ʻO ka eukaryotic expression vector plasmid pcDNA3.1(+) i linearized me ka restriction enzyme EcoRV a dephosphorylated me ka hoʻohana ʻana i ka Fast AP (Thermo Fisher Scientific).Hoʻomaʻemaʻe ʻia nā ʻāpana pcDNA3.1(+) Linearized a me nā ʻāpana gene target i hoʻonui ʻia me ka hoʻohana ʻana i kahi pahu hoʻomaʻemaʻe DNA gel (Tiangen) a helu ʻia me ka Nanodrop ND-2000 (Thermo Fisher Scientific).Ua hui hou ʻia ka ʻāpana pcDNA3.1(+) a me kēlā me kēia ʻāpana gene target me ka hoʻohana ʻana iā MonClone single assembly cloning mix (Monad Biotech Co., Ltd., Suzhou, Kina) a hōʻoia ʻia e ka DNA sequencing me ka hoʻohana ʻana i ka Comate Bioscience Company Limited (Changchun, Kina)..
Hoʻokumu ʻia nā plasmids-free Endotoxin pcDNA3.1(+)-alpha-2 a me pcDNA3.1(+)-alpha-7.3 me ka hoʻohana ʻana i ka SanPrep Endotoxin-free Plasmid Mini Kit (Sangon Biotech).Ua mālamaʻia ka manaʻo ma luna o 500 ng / µl e hōʻoia i ka EDTA i loko o ka elution bufferʻaʻole i hoʻopilikia i ka ho'āʻo transfection.Ua kanu ʻia nā macrophages peritoneal ʻiole mua i loko o nā pā 6-well me ka RPMI 1640 piha (Biological Industries) no 12 mau hola, a laila holoi ʻia nā cell i 3 mau manawa i ka PBS mahana e wehe i ka penicillin a me streptomycin, a laila ma ke ʻano i hoʻohui ʻia me ke ʻano piha.Endotoxin-free plasmids pcDNA3.1(+)-alpha-2 a me pcDNA3.1(+)-alpha-7.3 (2.5 μg) i hoʻoheheʻe ʻia ma 125 μl o Opti-MEM i hoʻemi ʻia serum medium (Gibco, Thermo Fisher Scientific)..A laila ua hoʻoheheʻe ʻia ka 5 µl o Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific) i loko o 125 µl o ka mea haʻahaʻa serum Opti-MEM.E hoʻomākaukau i ka liposome-DNA complexes ma ka hui ʻana i ka plasmid endotoxin-free diluted me Lipofectamine 2000 a ʻae i ka hui ʻana e kū i ka lumi wela no 5 mau minuke.E hoʻokaʻawale i nā mea paʻakikī i nā cell i kēlā me kēia pūnāwai a hui mālie.Ma hope o 4 mau hola, ua hoʻololi ʻia ka moʻomeheu cell me 2 ml o ka medium RPMI 1640 piha a ua hoʻomau ʻia ka moʻomeheu no 24 mau hola.Ua hoʻohui ʻia ke ʻano moʻomeheu cell hou i nā cell a hoʻomoʻi ʻia no nā manawa like ʻole e pili ana i ka hoʻolālā assay.
Ua hoʻomākaukau ʻia nā ʻāpana protein mai nā supernatants a me nā cell lysates e like me ka mea i wehewehe mua ʻia [25].ʻO nā palena hoʻololi membrane no ka pro-IL-1β, pro-caspase-1, caspase-1 p20, NLRP3, β-actin, a me kāna-tag he 200 mA / 90 min.No ka interleukin 1β (IL-1β; R&D Systems, Minneapolis, Minnesota, USA), caspase-1 (p20) (Adipogen, Switzerland) a me NLRP3 (Adipogen SA, Epalinges, Switzerland) a me 1: 5000 e kuhikuhi ana i kāna tag (Amylet Scientific, Wuhan, Kina) a me β-actin (Proteintech, Wuhan, Kina).
ʻO ka hoʻopili ʻana me ka disuccinimide suberate (DSS) i hana ʻia e like me ka mea i hōʻike mua ʻia [26].Ua holoi ʻia nā kelepona i 3 mau manawa me ka PBS anu a hoʻopaʻa ʻia me kahi nila 27 ana ma 50 µl ASC reaction buffer (pH 8.0) i loaʻa ka 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES a me 125 mM NaHCO3.Hoʻopili ʻia ka hui ʻana ma 5000 g no 3 min a ua hoʻopaʻa ʻia ka pellet me 10 µl DSS (25 mM i DMSO) a me 40 µl ASC reaction buffer no 30 min ma 37 ° C.Ma hope o ka centrifugation ma 5000 g no 10 min, ua hoʻoheheʻe ʻia ka pellet i kahi hopena o 40 µl o ASC reaction buffer a me 10 µl o 6x protein loading buffer (TransGen, Beijing, Kina), a laila ua kinai ʻia ka hopena ma ka lumi wela no 15. min., A laila e hoʻolapalapa i 10 mau minuke.Hoʻopili ʻia nā laʻana protein i ka blotting Western me ka hoʻohana ʻana i nā antibodies anti-ASC mua (Wanleibio, Shenyang, Kina) ma kahi ratio dilution o 1:500.
Ma hope o kahi kaʻina hana i wehewehe mua ʻia [13], ua ʻohi ʻia nā supernatants moʻomeheu cell a ua hoʻoholo ʻia ka huna ʻana o ka cytokine pro-inflammatory IL-1β me ka hoʻohana ʻana i ka kit IL-1 Beta ELISA kit (Invitrogen, Thermo Fisher Scientific).E hoʻohuli i nā waiwai OD450nm i nā ʻano protein me ka hoʻohana ʻana i ka pihi maʻamau IL-1β.
Ua holoi mālie ʻia nā kelepona i uhi ʻia ma nā uhi uhi i 3 mau manawa i ka PBS mahana, i hoʻopaʻa ʻia i loko o ka cell cell fixative (Biosharp, Beijing, Kina) no 10 min ma ka lumi wela (RT), ma 0.1% Triton X-Permeabilize ma 100 (hoʻoheheʻe ʻia i PBS; Biosharp ) no 20 mau minuke ma ka lumi wela a poloka i ka 5% bovine serum albumin (ma PBS) no 2 mau hola ma ka lumi wela.Hoʻokomo ʻia nā cell i ka pō ma 4 ° C me nā antibodies mua e kūʻē iā ASC (1:100 dilution) a i ʻole NLRP3 (1:100 dilution), a me Cy3 i kapa ʻia ʻo kao anti-rabbit IgG(H+L) (1:400; EarthOx. , San Francisco, CA, USA) a i ʻole FITC-conjugated goat anti-mouse IgG (1:400; Earthox) i ka pō ma 37°C i ka pōʻeleʻele no 1 hola.Ua hoʻopaʻa ʻia ka nuclei me Hoechst 33258 (10 μg/ml; UE, Suzhou, Kina) no 5 mau minuke a nānā ʻia ma lalo o kahi microscope fluorescence (Olympus Corporation, Tokyo, Iapana).
Ua māhele ʻia nā ʻiole i ʻehā mau pūʻulu (n = 7 i kēlā me kēia hui): (i) PBS-treated negative control group (PBS only; gavage 100 µl/mouse PBS ma hope o kēlā me kēia lā intraperitoneal injection 100 µl/mouse PBS 3 mau hola ma hope)., mau no 7 lā);(ii) ka hui hoʻomalu maikaʻi ʻole i mālama ʻia me MCC950 inhibitor [27] (100 µl/mouse ma o PBS gavage, 3 mau hola ma hope, 10 mg/kg kino kaumaha [BW] MCC950 [i PBS] i lawelawe ʻia intraperitoneally i kēlā me kēia lā, lōʻihi 7 lā);(iii) G. duodenalis cyst infection group (1.5 x 106 cysts/mouse by gavage, 3 hours later, 100 μl/mouse PBS intraperitoneally lawelawe i kēlā me kēia lā no 7 lā);(iv) G. duodenalis cyst hui maʻi pūʻulu MCC950 inhibitor lāʻau pūʻulu (1.5×106 cysts/iole ma o gavage, 10mg/kg kino kaumaha MCC950 intraperitoneally i kela la i keia la no 7 la ma 3h).Ua nānā ʻia ke kaumaha o ke kino o kēlā me kēia ʻiole i kēlā me kēia lā a ua hoʻopau ʻia nā ʻiole āpau i ka lā 7th.Ua ʻoki ʻia ka duodenum i ʻohi ʻia (3 cm lōʻihi) i loko o nā ʻāpana liʻiliʻi i 1 ml PBS, ua luku ʻia nā cysts i ka pō ma PBS ma 4 ° C, a me G. duodenalis trophozoites.Ua hoʻokaʻawale ʻia ka duodenum hou (1 knm ka lōʻihi) no ke kala ʻana hematoxylin a me eosin (H&E).
Ua māhele ʻia nā ʻiole i ʻelua pūʻulu: (i) pūʻulu hoʻomalu MOCK a me (ii) pūʻulu inhibitor MCC950.ʻElima mau lāʻau lapaʻau i kēlā me kēia hui (n = 7 / hui lapaʻau): (i) PBS mālama kino ʻole pūʻulu (PBS wale nō; 100 µl / mouse PBS, intramuscular (IM) injection (tibialis anterior) [28, 29] ;( ii) pcDNA3.1(+) pūʻulu mana ʻino ʻole plasmid (100 µg/ʻiole DNA, ma o ka hoʻokele intramuscular injection) (iii) G. duodenalis cyst infection pūʻulu hoʻomalu maikaʻi (1.5 x 106 cysts/mouse, via gavage) (iv) a hui i mālama ʻia me ka plasmid pcDNA3.1(+)-alpha-2 (100 μg/mouse DNA, ma ka intramuscular injection), a me (v) kahi hui i mālama ʻia me plasmid pcDNA3.1(+)-alpha-7.3 (100 µg/mouse ʻO DNA, ma hope o nā hola 12 o ka hele ʻana, loaʻa nā ʻiole i ka hui inhibitor MCC950 i kēlā me kēia lā intraperitoneal injection o MCC950 (10 mg / kg kino kaumaha) no nā lā 7, ʻoiai ʻo nā ʻiole i ka hui MOCK i loaʻa i ka nui like o ka mālama ʻana i ka PBS. ʻohi ʻia mai nā ʻiole maka a waiho ʻia i ka pō ma 4 °C ua hoʻokaʻawale ʻia nā laʻana Serum me ka hoʻohana ʻana i ka enzyme-linked immunosorbent assay (ELISA) a me nā ana o nā pae IL-1β.
Ua māhele ʻia he kanakolukumamālima mau ʻiole i ʻelima hui (n=7/hui).ʻO ka hui 1 he pūʻulu mana maikaʻi ʻole i mālama ʻia me PBS: loaʻa i nā ʻiole 100 μl o PBS intramuscularly a me 3 mau lā ma hope o ka gavage.ʻO ka hui 2 he pūʻulu hoʻomalu maikaʻi i loaʻa i ka G. duodenalis cysts: ua hoʻokomoʻia nāʻiole me ka 100 μl o PBS, a me nā lā 3 ma hope o 1.5 x 106 cysts / mouse i hoʻokomoʻia i loko o ka'ōpū.ʻO ke kolu o ka hui - ka pale ʻana i ka plasmid me ka pcDNA3.1(+) i hui pū ʻia me kahi pūʻulu hoʻomalu no ka maʻi duodenal cyst: loaʻa nā ʻiole 100 μg o ka plasmid DNA pcDNA3.1(+)(im) ma ka waha, 1.5×106 cysts/mouse 3 no kekahi. lā.ʻO nā hui 4 a me 5 he pcDNA3.1 (+)-alpha-2 giardine plasmid a i ʻole pcDNA3.1 (+)-alpha-7.3 giardine plasmid i hui pū ʻia me G. duodenalis cyst infection.Pūʻulu hoʻokolohua: loaʻa i nā ʻiole 100 µg o pcDNA3.1(+)-giardine plasmid DNA (im), a laila 3 mau lā ma hope, 1.5 × 106 cysts/mouse i hoʻopaʻa ʻia ma o gavage.Ua nānā ʻia ke kaumaha o ke kino o kēlā me kēia ʻiole ma hope o ka hoʻokomo ʻia ʻana o ka G. duodenalis cyst ma o ka paipu.Ua hōʻiliʻili ʻia ka duodenum hou no nā ana haʻahaʻa parasitic a me ka nānā ʻana i ka hoʻopaʻa ʻana HE.
Ua nānā ʻia nā loli histopathological e like me kahi kaʻina hana i paʻi ʻia ma mua [30].Ua hoʻopaʻa ʻia ka duodenum hou me ka hoʻopaʻa ʻana o ka cell tissue, hoʻokomo ʻia i loko o ka paraffin, ʻoki ʻia i loko o nā ʻāpana 4 μm, hoʻopaʻa ʻia me ka H&E a nānā ʻia ma lalo o kahi microscope māmā.Ua loiloi ʻia nā loli pathological o nā ʻāpana ʻehiku mai ʻehiku mau ʻiole kūʻokoʻa e kahi pathologist ʻike ʻole i ka mālama ʻana a ua hopu ʻia ma 200x magnification.Ua anaʻia ka lōʻihi o ka villi a me ka hohonu o nā crypts e like me nāʻano i ho'ākāka muaʻia.
Loaʻa ʻia nā hopena in vitro a me in vivo ma ka ʻekolu.Ua hana ʻia nā kiʻi me ka GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, USA).Ua kālailai ʻia nā ʻokoʻa ma waena o nā pūʻulu ʻelua e ka t-test, aʻo nā ʻokoʻa ma waena o nā pūʻulu ≥3 i kālailai ʻia e ka hoʻohana ʻana i ka ʻano like ʻole (ANOVA) me ka hoʻohana ʻana i ka polokalamu SPSS (version 22.0; SPSS IBM Corp., Armonk, NY, USA).ʻIke ʻia ka ʻikepili no ka homogeneity o ka ʻokoʻa me ka hoʻohana ʻana i ka hoʻāʻo a Levene a ukali ʻia e ka hoʻāʻo post hoc a Bonferroni (B).Hōʻike ʻia ka manaʻo nui e like me P<0.05, P<0.01, a me P<0.001 (ʻaʻole nui [ns]) (P>0.05).
ʻO kā mākou loiloi mua o GEV proteomics ma ka Kyoto Encyclopedia of Genes and Genomes (KEGG) i hōʻike ʻia he nui nā pahuhopu i komo i ka hoʻāla ʻana o nā ala hōʻailona inflammatory [13].Ua koho mākou i ʻelua mau pahuhopu hoʻohiki, alpha-2 a me alpha-7.3 giardins, e hoʻonui i kēia mau molekala a hoʻohana iā lākou e kūkulu i ka pcDNA3.1(+) eukaryotic expression vector.Ma hope o ke kaʻina ʻana, ua hoʻololi ʻia ka pcDNA3.1 (+) -alpha-2 a me alpha-7.3 giardine expression plasmids i loko o nā macrophages peritoneal kiʻi mua, a ua ʻike ʻia ka caspase-1 p20 signature protein o ka mumū (kahi ʻāpana o ka caspase-1) i ʻike ʻia. e like me ka wehewehe ʻana i nā molekole kī e hiki ke hoʻoulu i ka mumū.Ua hōʻike nā hopena i hiki i nā giardine alpha-2 a me alpha-7.3 ke hoʻoulu i ka p20 caspase-1 e like me GEV.ʻAʻole i loaʻa ka hopena ma ka caspase-1 activation i ka mana maikaʻi ʻole i mālama ʻia (PBS wale nō) a me ka mana plasmid pcDNA3.1 (+) (Figure 1).
Ana o p20 caspase-1 ho'ā 'ia e pcDNA3.1(+)-alpha-2 a alpha-7.3 giardins.Recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 a me alpha-7.3 giardines (ma luna o kēlā me kēia ala) ua hoʻololi ʻia i loko o nā macrophages peritoneal kiʻekiʻe mua a ua ʻohi ʻia nā supernatants moʻomeheu 24 mau hola ma hope.Ua hoʻohana ʻia ka Western blotting e ana i nā pae hōʻike o ka pūlima caspase-1 p20 inflammasome.Ua hoʻohana ʻia ka hui lapaʻau PBS wale nō (lane C) a me ka hui monotherapy pcDNA3.1(+) (pcDNA3.1 lane) ma ke ʻano he mana maikaʻi ʻole, a ua hoʻohana ʻia ka hui mālama GEV ma ke ʻano he mana maikaʻi.Ua hōʻoia ʻia ka hōʻike ʻana o ka protein recombinant ma ka ʻike ʻana i kahi hōʻailona histidine i kēlā me kēia protein, a ʻo nā kaula protein i manaʻo ʻia he alpha-2 giardine (38.2 kDa) a me alpha-7.3 giardine (37.2 kDa).GEV, Giardia duodenum vesicles extracellular, pcDNA3.1(+), EcoRV-linearized vector, SUP, supernatant
No ka hoʻoholo ʻana inā hoʻokomo ka alpha-2 giardine a me alpha-7.3 giardine i ka hōʻike p20 caspase-1 a pāʻani i ka hana i ka hoʻoulu ʻana i ka pane inflammatory NLRP3 host, pcDNA3.1(+)-alpha-2 giardine a me pcDNA3.1(+)-alpha -7.3 giardin ua lawe ʻia i loko o nā macrophages peritoneal kiʻi mua me ka DNA plasmid recombinant, a ua hoʻoholo ʻia nā pae o ka ʻōlelo, localization, a me ka oligomerization o nā protein inflammatory kī NLRP3.Ma kēia hoʻokolohua, ua hoʻohana ʻia ʻo GEV ma ke ʻano he pūʻulu hoʻomalu maikaʻi, a ʻo ka pūʻulu mālama ʻole (PBS wale nō) a i ʻole ka hui lapaʻau transfection pcDNA3.1(+) ʻo ia ka hui ʻino.Ua hōʻike ʻia nā hopena, e like me ka hui GEV, recombinant plasmid DNA o giardin pcDNA3.1(+)-alpha-2 a me giardin pcDNA3.1(+)-alpha-7.3 i hopena i ka upregulation o NLRP3, pro-IL-1β a me procaspase-1 a me caspase-1 ho'āla (Fig. 2a).Eia kekahi, ua hoʻokomo nā giardine ʻelua i ka huna nui 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) (Kiʻi 2b).ʻO ka hapa nui o nā protein ASC he monomeric i loko o ka hui no-treatment a i ʻole i ka hui lapaʻau i hoʻololi ʻia me ka pcDNA3.1(+) plasmid, i hoʻohālikelike ʻia me pcDNA3.1(+)-alpha-2 a i ʻole pcDNA3.1(+)-alpha- 7.3 giardine.Ua loaʻa ka oligomerization ASC i ka DNA plasmid recombinant o ka hui mana maikaʻi GEV a i ʻole hui, e hōʻike ana i kahi ʻano oligomeric (Figure 2c).Hōʻike kēia mau ʻikepili mua e hiki i ka alpha-2 giardine a me alpha-7,3 giardine ke hoʻoulu i ka hoʻoulu ʻana o ka mumū NLRP3.Ua hōʻike ʻia nā haʻawina immunofluorescent ma hope o ka localization o ASC a me NLRP3 i loko o ka pūʻulu mana maikaʻi ʻole, ua hoʻopuehu ʻia ka protein ASC i loko o ka cytoplasm a ua ʻike ʻia he hōʻailona kiko ma ka hoʻoulu ʻana o pcDNA3.1 (+) -alpha-2 me giardine a i ʻole pcDNA3.1(+)-alpha-7,3 hui giardine a i ʻole GEV pūʻulu mana maikaʻi (Figure 2d).I ka mana maikaʻi ʻole a me nā pūʻulu pcDNA 3.1 i mālama ʻia i ka plasmid, ʻaʻole i ʻike ʻia ka hōʻailona protein NLRP3, ʻoiai he kiko hōʻailona fluorescent i pane i ka pcDNA3.1 (+) -alpha-2 giardine a i ʻole pcDNA3.1 (+) -alpha-7.3 ua ʻike ʻia..Loaʻa ka giardine i ka cytoplasm a i ʻole ma ka hoʻoulu ʻana o ka HEV (Fig. 2e).Hōʻike hou kēia mau ʻikepili i ka G. duodenalis giardin alpha-2 a me giardin alpha-7.3 e hoʻāla i ka NLRP3 inflammasome i loko o nā macrophages peritoneal mua.
pcDNA3.1(+)-alpha-2 giardin a me pcDNA3.1(+)-alpha-7.3 giardin e ho'ā i ka NLRP3 inflammasome i loko o nā macrophages peritoneal mouse.Hoʻololi i ka recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardin a me pcDNA3.1(+)-alpha-7.3 giardin i loko o nā macrophages peritoneal murine mua a me nā pūnaewele, a i ʻole e hōʻiliʻili i ka supernatant i loko o 24 h no ka nānā ʻana i ka ʻōlelo, oligomerization , huna.a me ka localization o nā kī inflammatory proteins.ʻO ka hui PBS-wale (C) a me ka hui pcDNA3.1 (+) hoʻokahi lāʻau lapaʻau i hoʻohana ʻia e like me ka mana maikaʻi ʻole, a ua hoʻohana ʻia ka hui mālama GEV ma ke ʻano he hui maikaʻi.a Key inflammatory proteins NLRP3, me NLRP3, pro-IL-1β, pro-caspase-1, a me p20 caspase-1, ua ʻike ʻia e ka Western blotting.b Ua hoʻoholo ʻia nā pae o ka huna ʻana o IL-1β i nā supernatants me ka hoʻohana ʻana i ka enzyme-linked immunosorbent assay (ELISA).Ua kālailai ʻia nā ʻokoʻa ma waena o ka mana a me nā pūʻulu hoʻokolohua e ka hoʻohana ʻana i ka polokalamu SPSS version 22.0.Hōʻike nā Asterisk i nā ʻokoʻa koʻikoʻi ma waena o nā hui ** P<0.01 a me ***P<0.001.Ua hoʻoholo ʻia nā pae oligomerization ASC i nā pellets e ka DSS cross-linking analysis, aʻo nā pae ASC i nā cell lysates i hoʻohana ʻia ma ke ʻano he mana hoʻouka.d Ka ʻike ʻana o ka localization ISC me ka hoʻohana ʻana i ka immunofluorescence.Ua hoʻohana ʻia ka Immunofluorescence e nānā i ka localization o NLRP3.ASC, apoptotic speck-like protein;IL, interleukin;NLRP3, nucleotide-binding oligomerization-like receptor 3;ns, ʻaʻole koʻikoʻi (P > 0.05)
ʻO G. duodenalis a me nā GEV i hūnā ʻia e hoʻāla i ka inflammasome NLRP3 a hoʻoponopono i nā pane inflammatory host in vitro.No laila, ʻaʻole maopopo ka hana o ka inflammasome NLRP3 i ka pathogenicity o G. duodenalis.No ka noiʻi ʻana i kēia pilikia, ua hoʻolālā mākou i kahi hoʻokolohua ma waena o nā ʻiole i loaʻa i ka G. duodenalis cyst a me nā ʻiole i loaʻa i ka G. duodenalis cyst + MCC950 inhibitor treatment a hoʻohālikelike i ka ʻōlelo inflammasome NLRP3 i ka wā i loaʻa i ka G. duodenalis cyst.Hōʻike ʻia kahi hoʻolālā kikoʻī o ka hoʻokolohua ma Fig. 3a.Ua nānā ʻia nā hoʻololi i ke kaumaha o ke kino o nā ʻiole i nā hui lapaʻau like ʻole no 7 mau lā ma hope o ka maʻi ʻana me nā cysts, a ua hōʻike ʻia nā hopena ma Fig. 3b.Ke hoʻohālikelike ʻia me ka hui i mālama ʻia me ka PBS maʻemaʻe, ua hōʻike nā hopena i (i) ua emi ke kaumaha o ke kino o nā ʻiole i loaʻa i ka G. duodenalis cyst mai ka lā 3 a i ka lā 7 ma hope o ka maʻi;(ii) ʻaʻohe hopena koʻikoʻi o ka mālama ʻana me ka MCC950 inhibitor i ke kaumaha o ke kino o nā ʻiole..Hoʻohālikelike ʻia i ka hui maʻi hoʻokahi, ua emi ka BW o ka hui maʻi duodenal i mālama ʻia me MCC950 i nā degere like ʻole (La 1: ANOVA, F(3, 24) = 1.885, P = 0.0148; Lā 2: ANOVA, F(3, 24). ) = 0.4602, P<0.0001 La 3: ANOVA, F(3, 24) = 0.8360, P = 0.0010 La 4: ANOVA, F(3, 24) = 1.683, P = 0.0052; (3, 24)=0.6497, P=0.0645; La 6: ANOVA, F(3, 24)=5.457, P=0.0175;Hōʻike kēia mau ʻikepili i ka pale ʻana o ka inflammasome NLRP3 i nā ʻiole mai ka pohō kaumaha nui i ka wā mua (2-4 mau lā) o ka maʻi duodenal.A laila, manaʻo mākou e ʻike i nā trophozoites G. duodenalis i loko o ka wai holoi duodenal a hōʻike ʻia nā hopena ma ke Kiʻi 3c.Ke hoʻohālikelikeʻia i ka hui G. duodenalis cyst infection, ua hoʻonui nuiʻia ka nui o nā trophozoites i loko o ka duodenum ma hope o ka paleʻana i ka NLRP3 inflammasome (t (12) = 2.902, P = 0.0133).Ua hōʻike ʻia nā ʻiʻo Duodenal i hoʻopaʻa ʻia me HE, i hoʻohālikelike ʻia me ka mana maikaʻi ʻole i mālama ʻia me PBS a me MCC950 wale nō: ​​(i) G. duodenalis cyst maʻi i hopena i ka pōʻino i ka duodenal villi (ANOVA, F(3, 24)=0.4903, P= 0.0488 ) a me ka crypt atrophy (ANOVA, F(3, 24) = 0.4716, P = 0.0089);(ii) duodenum mai nāʻiole i loaʻa i ka G. duodenalis cysts a mālama ʻia me ka MCC950 inhibitors.duodenal villi ua poino a make (ANOVA, F (3, 24) = 0.4903, P = 0.0144) me ka atrophy a me ka crypt branching (ANOVA, F (3, 24) = 0.4716, P = 0, 0481) (Fig. 3d- f).Hōʻike kēia mau hopena i ka hana ʻana o ka inflammasome NLRP3 i ka hōʻemi ʻana i ka pathogenicity o G. duodenalis.
Ke kuleana o ka inflammasome NLRP3 i ka maʻi Giardia duodenum.Ua hoʻopaʻa ʻia nā ʻiole (iv) me ka duodenococcal cysts a laila mālama ʻia me ka MCC950 (ip) a i ʻole.Ua hoʻohana ʻia nā hui mālama hoʻokahi me PBS a i ʻole MCC950 e like me nā mana.ʻO ka hui hoʻokolohua a me ke ʻano o ka mālama ʻana.b Ua nānā ʻia ke kaumaha o ke kino o nā ʻiole i kēlā me kēia hui lapaʻau no 7 mau lā.Ua nānā ʻia ka ʻokoʻa ma waena o ka hui maʻi G. duodenalis a me ka hui mālama maʻi G. duodenalis + MCC950 e ka ho'āʻo-t me ka hoʻohana ʻana i ka polokalamu SPSS version 22.0.Hōʻike nā asterisk i nā ʻokoʻa nui ma *P<0.05, **P<0.01, a i ʻole ***P<0.001.c Hoʻoholo ʻia ka haʻahaʻa Parasitic ma ka helu ʻana i ka nui o nā trophozoites i loko o ka wai holoi duodenal.Ua nānā ʻia ka ʻokoʻa ma waena o ka hui maʻi G. duodenalis a me ka hui mālama maʻi G. duodenalis + MCC950 e ka ho'āʻo-t me ka hoʻohana ʻana i ka polokalamu SPSS version 22.0.Hōʻike nā asterisk i nā ʻokoʻa nui ma *P <0.05.d Hematoxylin a me eosin (H&E) hopena o ka duodenal histopathology.Hōʻike nā pua ʻulaʻula i ka pōʻino i ka villi, hōʻike nā pua ʻōmaʻomaʻo i ka pōʻino i nā crypts.ʻO ka pā unahi: 100 µm.e, f Ka helu helu o ka duodenal villus kiʻekiʻe a me ka kiʻekiʻe crypt kiʻekiʻe.Hōʻike nā Asterisk i nā ʻokoʻa nui ma *P<0.05 a me **P<0.01.Lawe ʻia nā hopena mai 7 mau hoʻokolohua kūlohelohe kūʻokoʻa.BW, kaumaha kino;ig, ke ala lawe intragastric;ip, ke ala lawe intraperitoneal;ns, ʻaʻole nui (P > 0.05);PBS, phosphate buffered saline;WT, ʻano ʻāhiu
ʻO ka huna ʻana o IL-1β kahi hōʻailona o ka hoʻoulu ʻana o ka mumū.No ka hoʻoholo ʻana inā hoʻāla ʻo G. duodenalis alpha-2 giardine a me alpha-7.3 giardine i ka NLRP3 host inflammasome in vivo, ua hoʻohana mākou i nā mice WT untreated (sham group) a me NLRP3 inflammasome-blocked mice (MCC950 inhibited treatment group).Hōʻike ʻia kahi hoʻolālā kikoʻī o ka hoʻokolohua ma Fig. 4a.ʻO nā hui hoʻokolohua he mau ʻiole i mālama ʻia me ka PBS, G. duodenalis cyst treatment by gavage, intramuscular injection of pcDNA3.1, and intramuscular injection of pcDNA3.1(+)-alpha-2 giardine or pcDNA3.1-alpha-7.3 giardine.Ma ka lā 7 ma hope o ka hoʻokele intramuscular o ka plasmid recombinant, ua hōʻiliʻili ʻia ka serum a ua hoʻoholo ʻia ka pae o IL-1β i kēlā me kēia hui.E like me ka mea i hōʻike ʻia ma ka Figure 4b, i ka hui MOCK: (i) i hoʻohālikelike ʻia me ka hui PBS, ʻaʻohe hopena koʻikoʻi o ka mālama ʻana pcDNA3.1 i ka huna ʻana o IL-1β (ANOVA, F (4.29) = 4.062, P = 0.9998), akā naʻe, Ua hoʻokiʻekiʻe nuiʻia ka huna IL-β i ka hui G. duodenalis cyst (ANOVA, F (4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine a me pcDNA3.1- Intramuscular injection of alpha-7.3 giardine i hoʻonui nui i ka serum IL-1β (ANOVA, F (4, 29) = 4.062, P<0.0001);(iii) pcDNA3.1-alpha-7,3 giardine i hoʻokomo i nā kiʻekiʻe kiʻekiʻe o ka IL -1β huna i loko o ka pcDNA3.1-alpha-2 giardine intramuscular injection group (ANOVA, F (4, 29) = 4.062, P = 0.0333) .Ke hoʻohālikelikeʻia me kēlā me kēia hui i ka hui mālama MCC950 a me ka pūʻulu MOCK: (i) IL-1β nā pae huna i loko o ka pūʻulu mana PBS a me ka pūʻulu mana pcDNA3.1 i emi iho i kekahiʻano ma hope o ka paleʻana i ka mea hoʻopaneʻe MCC950, akā,ʻaʻole kaʻokoʻa. nui (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F (9, 58) = 3.540, P = 0.5949);(ii) ma hope o ka ālai ʻana iā MCC950., Ua ho'ēmi nuiʻia ka hunaʻana o IL-1β i ka hui G. duodenalis cyst-infected, ka hui pcDNA3.1-alpha-2 giardine, a me ka hui 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; ) = 3.540, P = 0.0164).Hōʻike kēia mau hopena i ka alpha-2 giardine a me alpha-7.3 giardine i ka hoʻoulu ʻana o ka NLRP3 inflammasome in vivo.
pcDNA3.1 (+) -giardines ho'āla i ka NLRP3 host inflammasome in vivo.Ua pale ʻia nā ʻiole (IM) me ka recombinant eukaryotic expression plasmid pcDNA3.1(+)-alpha-2 giardine a i ʻole pcDNA3.1(+)-alpha-7.3 giardine a laila mālama ʻia me MCC950 (ip; MCC950 group) a i ʻole (hui dummy. ).Ua hoʻohanaʻia ka PBS a iʻole ka pcDNA3.1 (+) plasmid pūʻulu lapaʻau ma keʻano he mana maikaʻiʻole, ua hoʻohanaʻia ka G. duodenalis cyst treatment group ma keʻano he mana maikaʻi.ʻO ka hui hoʻokolohua a me ke ʻano o ka mālama ʻana.b Ua ana ʻia nā pae serum o IL-1β i nā ʻiole i ka lā 7 e ELISA assay.Ua kālailai ʻia nā ʻokoʻa ma waena o nā pūʻulu o ka pūʻulu MOCK me ka hoʻohana ʻana i ka ANOVA ala hoʻokahi, a ua ʻike ʻia nā ʻokoʻa ma waena o ka pūʻulu MOCK a me ka hui MCC950 me ka hoʻohana ʻana i ka t-test o ka polokalamu polokalamu SPSS version 22.0.Hōʻike nā Asterisk i nā ʻokoʻa koʻikoʻi ma waena o nā hui lapaʻau ma ka hui MOCK, * P <0.05 a me *** P <0.001;hōʻailona kālā ($) hōʻike i nā ʻokoʻa koʻikoʻi ma waena o kēlā me kēia hui ma ka hui MOCK a me ka hui MCC950 ma P<0.05.Nā hualoaʻa o nā hoʻokolohua olaola kūʻokoʻa ʻehiku.i, hoʻokomo intramuscular, ns, ʻaʻole nui (P > 0.05)
No ka noiʻi ʻana i ka hopena o ka alpha-2 a me alpha-7.3 giardine-mediated activation o ka NLRP3 host inflammasome ma G. duodenalis infectivity, ua hoʻohana mākou i ka WT C57BL/6 mice a hoʻokomo i ka alpha-2 giardine a me alpha-7.3 giardine.ua hoʻokomo ʻia ka plasmid intramuscularly, ma hope o 3 mau lā ma o ka ʻōpū o ka ʻōpū o ka G. duodenalis cyst, ma hope o ka nānā ʻana i nā ʻiole no 7 mau lā.Hōʻike ʻia kahi hoʻolālā kikoʻī o ka hoʻokolohua ma Fig. 5a.Ua ana ʻia ke kaumaha o ke kino o kēlā me kēia ʻiole i kēlā me kēia lā, hōʻiliʻili ʻia nā laʻana o ka ʻiʻo duodenal hou i ka lā 7 ma hope o ka hoʻohana ʻana ma o ka ʻōpū ʻōpū, ua ana ʻia ka nui o nā trophozoites, a ua ʻike ʻia nā loli histopathological.E like me ka mea i hōʻike ʻia ma ka Figure 5b, me ka hoʻonui ʻana i ka manawa hānai, ua piʻi mālie ka BW o nā ʻiole i kēlā me kēia hui.Ua hoʻomaka ka MT o nāʻiole i ka emiʻana i ka lā 3rd ma hope o ka hoʻoponopono intragastric o G. duodenalis cysts, a laila mahuahua mālie.ʻO ka hoʻouluʻana o ka NLRP3 inflammasome i hoʻokomoʻia e ka intramuscular injection o alpha-2 giardine a me alpha7.3 giardine i hoʻemi nui i ka poho kaumaha i nāʻiole (Day 1: pcDNA3.1-alpha-2 giardine, ANOVA, F (4, 30) = 1.399, P = 0 .9754 Lā 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30)=1.399, P=0.9987 Lā 2: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.3172, P = 0.9979; Lā 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.3172, P = 0.8409 lā 3 : pcDNA3.1-alpha-2 giardine 4, 30) = 0.8222, P = 0.0262 Lā 3: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4 , 30) = 0.8222, P = 0.0083 lā 4: pcDNA3.1-alpha-2 giardine , F(4, 30) = 0.5620, P = 0.0012, Lā 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5620, P <0.0001, Lā 5: pcDNA3.1-alpha - 2 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Lā 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F(4, 30) = 0.9728, P <0.0001 Lā 6: pcDNA3 . alpha-2 giardine, ANOVA, F(4, 30) = 0.7154, P = 0.0012, Lā 6: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.7154, P = 0.0006;Lā 7: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.5369, P<0.0001 Lā 7: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5369, P <0.0001).Ua heluʻia ka ukana parasitic i loko o ka duodenum (Fig. 5c).Ke hoʻohālikelikeʻia i ka mana maikaʻiʻole a me ka hui i hoʻokomoʻia me ka pcDNA3.1 vector nele, ua hoʻemi nuiʻia ka helu o G. duodenalis trophozoites i nā hui i hoʻokomoʻia me ka α-2 giardine a me ka 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).Eia kekahi, ʻoi aku ka palekana o giardine alfa-7.3 i nā ʻiole ma mua o giardine alfa-2 (ANOVA, F (3, 24) = 1.209, P = 0.0081).Hōʻike ʻia nā hopena o ka hoʻopaʻa ʻana iā HE ma ka fig.5d–f.ʻO nāʻiole i hoʻokomoʻia me ka alpha-2 giardine a me ka alpha-7.3 giardine he liʻiliʻi ka liʻiliʻi o nāʻiʻo duodenal, i hōʻikeʻia e ka pōʻino villus, ke hoʻohālikelikeʻia me nāʻiole i hoʻokomoʻia me G. duodenalis a me nāʻiole i hoʻokomoʻia me G. duodenalis i hui pū me kahi vector pcDNA3 .1 Zoom.(pcDNA3.1-alpha-2 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0035 a i ʻole P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0028 a i ʻole P = 0.0055) a hoʻemi ʻia ka crypt atrophy (pcDNA3.1-alpha-2 giardine: ANOVA, F (3, 24) = 1.470, P = 0.0264 a i ʻole P = 0.0158; pcDNA3.1-alpha-7.3 giardine , F(3, 24) = 1.470, P = 0.0371 a i ʻole P = 0.0191).Hōʻike kēia mau hopena i ka alpha-2 giardine a me alpha-7,3 giardine e hoʻemi i ka infectivity o G. duodenalis ma ka hoʻoulu ʻana i ka inflammasome NLRP3 i loko o vivo.
Ke kuleana o pcDNA3.1 (+) -giardins i ka maʻi G. duodenalis.Hoʻopaʻa ʻia nā ʻiole (IM) me ka recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardine a i ʻole pcDNA3.1(+)-alpha-7.3 giardine a laila hoʻokūkū me G. duodenalis cysts (ig).Ua hoʻohana ʻia ka pūʻulu PBS a me ka hui pcDNA3.1(+) + duodenal cyst ma ke ʻano he mau pūʻulu hoʻomalu maikaʻi ʻole, a ua hoʻohana ʻia ka hui mālama ʻana i ka duodenal cyst ma ke ʻano he pūʻulu mana maikaʻi.ʻO ka hui hoʻokolohua a me ke ʻano o ka mālama ʻana.b Ua nānā ʻia ka MT o nā ʻiole i kēlā me kēia hui lapaʻau no 7 mau lā ma hope o ka hoʻokūkū.Hōʻike nā Asterisk i nā ʻokoʻa nui ma waena o nā hui i ka hui G. duodenalis a me ka hui pcDNA3.1 (+) -alpha-2 giardine, * P <0.05, ** P <0.01, a me *** P <0.001;ʻo ka hōʻailona kālā ($) hōʻike i kahi ʻokoʻa nui ma waena o kēlā me kēia hui o G. duodenalis a me ka pcDNA3.1(+)-alpha-7.3 pūʻulu jardine, $$P<0.01 a me $$$P<0.001.c Hoʻoholo ʻia ka haʻahaʻa Parasitic ma ka helu ʻana i ka helu o nā trophozoites i 1 ml o ka duodenal lavage mai ka duodenum (3 knm ka lōʻihi) a hōʻike ʻia e like me ka helu o nā parasites ma ke knm o duodenum.ʻO nā ʻokoʻa ma waena o ka hui maʻi G. duodenalis, ka pcDNA3.1(+)-alpha-2 giardine pūʻulu, a me ka pcDNA3.1(+)-alpha-7.3 hui giardine i kālailai ʻia e ka ANOVA ala hoʻokahi me ka hoʻohana ʻana i ka polokalamu SPSS version 22.0.Hōʻike nā Asterisk i nā ʻokoʻa koʻikoʻi ma **P<0.01 a me ***P<0.001.d nā loli histopathological i ka duodenum.Hōʻike nā pua ʻulaʻula i ka pōʻino i ka villi, hōʻike nā pua ʻōmaʻomaʻo i ka pōʻino i nā crypts.ʻO ka pā unahi: 100 µm.e, f Ka helu helu o ke kiʻekiʻe o ka villus duodenal iole (e) a me ke kiʻekiʻe crypt (f).Ua ʻike ʻia nā ʻokoʻa ma waena o nā hui ma ke Kiʻi 1d e ka ANOVA ala hoʻokahi me ka hoʻohana ʻana i ka polokalamu polokalamu SPSS 22.0.Hōʻike nā Asterisk i nā ʻokoʻa nui ma *P<0.05 a me **P<0.01.Nā hualoaʻa o nā hoʻokolohua olaola kūʻokoʻa ʻehiku.ns, ʻaʻole koʻikoʻi (P > 0.05)
ʻO Giardia duodenum kahi maʻi maʻi ʻōpū i ʻike ʻia o ke kanaka a me nā mea momona ʻē aʻe e kumu ai ka giardiasis.Ma 2004, ua hoʻokomo ʻia i loko o ka WHO Neglected Diseases Initiative ma muli o kona kiʻekiʻe kiʻekiʻe ma mua o 6 mau makahiki, ʻoi aku hoʻi i nā kaiāulu o ke kūlana socioeconomic haʻahaʻa [32].He kuleana koʻikoʻi ka ʻōnaehana pale kino i ka pane ʻana i ka maʻi G. duodenalis.Ua hōʻike ʻia nā macrophages ʻiole e hoʻopau a pepehi iā G. duodenalis ma ka hoʻokuʻu ʻana i nā pahele extracellular [33].Ua hōʻike kā mākou mau haʻawina mua i ka G. duodenalis, kahi parasite extracellular non-invasive, hoʻāla i ka p38 MAPK, ERK, NF-κB p65, a me NLRP3 nā ala hōʻailona inflammatory i loko o nā macrophages mouse e hoʻoponopono i nā pane inflammatory host, a hoʻokuʻu ʻia ʻo GEV hiki ke hoʻonui i kēia kaʻina.13], 24].Eia nō naʻe, ʻo nā PAMP pololei i pili i ka NLRP3 inflammasome-regulated inflammation ma GEV a me ke kuleana o NLRP3 inflammasome i giardiasis e hoʻomau ʻia e elucidated.No ka wehewehe ʻana i kēia mau nīnau ʻelua, ua hana mākou i kēia noiʻi.
Aia ka inflammasome NLRP3 i loko o ka cytoplasm o nā cell immune a hiki ke hoʻāla ʻia e nā ʻāpana like ʻole e like me ka uric acid crystals, toxins, bacteria, viruses, and parasites.I loko o nā haʻawina bacteria, ua ʻike ʻia nā toxins i nā PAMP koʻikoʻi e hoʻolaʻa i nā sensor inflammatory, e alakaʻi ana i ka mumū a me ka make cell [34].ʻO kekahi mau meaʻawaʻawa likeʻole, e like me ka hemolysin mai Staphylococcus aureus [35] a me Escherichia coli [36], hemolysin BL (HBL) mai enterotoxin (NHE) [37], hoʻoulu i ka hoʻouluʻana o ka mumū NLRP3.Ua hōʻike ʻia nā haʻawina viral i nā protein virulence e like me SARS-COV-2 envelope (E) protein [38] a me Zika virus NS5 protein [39] he mau PAMP koʻikoʻi i ʻike ʻia e ka NLRP3 receptor.I loko o nā haʻawina parasite, ua hōʻike ʻia ka nui o nā parasites e pili ana i ka hoʻoulu ʻana o ka host inflammasome, e like me Toxoplasma gondii, Trichomonas vaginalis [40], Trypanosoma cruzi [41], a me Leishmania [42].Pono nā protein granule GRA35, GRA42, a me GRA43, e pili ana i ka virulence o Toxoplasma gondii, no ka hoʻokomo ʻana i ka pyroptosis i Lewis rat macrophages [43].Eia kekahi, ua kālele kekahi mau haʻawina Leishmania i nā molekala pākahi i komo i ka inflammasome NLRP3, e like me ka lipophosphoglycan membrane parasite [44] a i ʻole zinc metalloprotease [45].Ma waena o ka ʻohana alpha-giardin e like me annexin o nā genes, ua hōʻike ʻia ka alpha-1 giardin i mea hiki ke hoʻopaʻa ʻia i ka pale ʻana iā G. duodenalis ma ke ʻano ʻiole [18].I loko o kā mākou noiʻi, koho mākou i nā kumu virulence G. duodenalis alpha-2 a me alpha-7,3 giardines, he mea kū hoʻokahi i ka giardia akā ʻaʻole i hōʻike ʻia.Ua hoʻopaʻa ʻia kēia mau genes ʻelua i loko o ka pcDNA3.1(+) eukaryotic expression system vector no ka nānā ʻana i ka hoʻōla ʻana.
I kā mākou kumu hoʻohālike ʻiole, ʻo nā ʻāpana caspase i hoʻokaʻawale ʻia e lilo i hōʻailona o ka hoʻōla ʻana.Ma ka hoʻoulu ʻana, hui pū ʻo NLRP3 me ASC, hoʻoulu i nā procaspases, a hoʻoulu i nā caspases ikaika e hoʻokaʻawale i ka pro-IL-1β a me ka pro-IL-18 i IL-1β a me IL-18 makua, kēlā me kēia -18.ʻO nā caspases inflammatory (caspases-1, -4, -5 a me -11) he ʻohana mālama ʻia o ka cysteine ​​​​proteases he mea koʻikoʻi no ka pale ʻana a pili i ka mumū a me ka make cell programmed [46].Hoʻohana ʻia ʻo Caspase-1 e nā canonical inflammasomes [47], ʻoiai ʻo caspases-4, -5, a me -11 i hoʻokaʻawale ʻia i ka wā o ka hoʻokumu ʻana o nā inflammasomes atypical [48].I loko o kēia noiʻi, ua hoʻohana mākou i nā macrophages peritoneal mouse ma ke ʻano he kumu hoʻohālike a ua noiʻi i ka p20 caspase-1 i hoʻokaʻawale i ka caspase-1 ma ke ʻano he hōʻailona o ka host NLRP3 hoʻoulu ʻana i ka mumū i nā haʻawina o ka maʻi G. duodenalis.Ua hōʻike ʻia nā hopena he nui nā alpha-giardins ke kuleana no ka hoʻōla maʻamau o ka mumū, e kūlike me ka loaʻa ʻana o nā molekole virulence nui i pili i ka bacteria a me nā maʻi.Eia naʻe, ʻo kā mākou aʻo ʻana he pale mua wale nō a aia kekahi mau moleke e hiki ke hoʻāla i nā inflammasomes non-classical, e like me kā mākou noiʻi mua i ʻike ai i nā inflammasomes classical a me nā non-classical inflammasomes i ka maʻi G. duodenalis [13].No ka hoʻoholo hou ʻana inā pili ka p20 caspase-1 i ka inflammasome NLRP3, ua hoʻohuli mākou i nā giardins alpha-2 a me alpha-7.3 i loko o nā macrophages peritoneal mouse e hoʻoholo ai i nā pae kiʻekiʻe o ka protein mole mole a me nā pae oligomerization ASC, e hōʻoia ana e hoʻoikaika nā α-giardins ʻelua. inflammasome NLRP3.Heʻokoʻa iki kā mākou mau hopena mai nā Manko-Prykhoda et al., nāna i hōʻike i ka hoʻouluʻana o nā pūnaewele Caco-2 me G. muris a iʻole E. coli EPEC strains hiki ke hoʻonui i ka ikaika o ka fluorescence o NLRP3, ASC, a me caspase-1, ʻoiai ʻaʻole nui, ʻoiai pehea ka costimulation o G. muris a me E. coli i hoʻonui i nā pae o ʻekolu mau protein [49].ʻO kēia ʻokoʻa paha ma muli o nā ʻokoʻa i ke koho ʻana i nā ʻano ʻano Giardia, nā laina kelepona, a me nā cell mua.Ua hana pū mākou i nā ho'āʻo vivo me ka hoʻohana ʻana iā MCC950 i nā ʻiole wahine WT C57BL/6 he 5 mau pule, ʻoi aku ka maʻalahi o G. duodenalis.ʻO ka MCC950 kahi mea hoʻopaneʻe NLRP3 mole liʻiliʻi liʻiliʻi a koho i ka hoʻonā ʻana i ka canonical a me ka non-canonical NLRP3 i nā nanomolar concentrations.Hoʻopili ʻo MCC950 i ka hoʻoulu ʻana o NLRP3 akā ʻaʻole pili i ka hoʻoulu ʻana o AIM2, NLRC4, a me NLRP1 ala inflammatory a i ʻole nā ​​ala hōʻailona TLR [27].Hoʻopaʻa ʻo MCC950 i ka hoʻoulu ʻana o NLRP3 akā ʻaʻole ia e kāohi i ka hoʻomaka ʻana o NLRP3, K+ efflux, Ca2+ influx, a i ʻole ka pilina ma waena o NLRP3 a me ASC;akā, ke kāohi nei ia i ka NLRP3 inflammasome activation ma ke kāohi ʻana i ka oligomerization ASC [27].No laila, ua hoʻohana mākou i ka MCC950 i loko o kahi noiʻi in vivo e hoʻoholo i ke kuleana o ka inflammasome NLRP3 ma hope o ka hoʻokele giardine.Hoʻopau ka caspase-1 p10 i nā cytokine pro-inflammatory pro-IL-1β a me pro-IL-18 i loko o IL-1β a me IL-18 [50].Ma kēia haʻawina, ua hoʻohana ʻia nā pae serum IL-1β i nā ʻiole i mālama ʻia e giardine me ka ʻole o MCC950 ma ke ʻano he hōʻailona no ka hoʻāla ʻia ʻana o ka inflammasome NLRP3.E like me ka mea i manaʻo ʻia, ua hoʻemi nui ka mālama ʻana o MCC950 i nā pae serum IL-1β.Hōʻike maopopo kēia mau ʻikepili hiki iā G. duodenalis giardin alfa-2 a me giardin alfa-7.3 ke hoʻāla i ka NLRP3 mouse inflammasome.
Ua hōʻike ʻia nā ʻikepili koʻikoʻi i hōʻiliʻili ʻia i nā makahiki he ʻumi i hala iho nei ʻo IL-17A ka haku hoʻoponopono o ka pale ʻana iā G. muris, e hoʻoulu ana i ka hōʻailona IL-17RA, e hana ana i nā peptides antimicrobial, a me ka hoʻoponopono ʻana i ka hoʻoulu ʻana [51].Eia naʻe, loaʻa pinepine ka maʻi Giardia i ka poʻe ʻōpio, a ua hōʻike ʻia ʻaʻole hoʻāla ka maʻi Giardia i nā ʻiole ʻōpio i ka pane IL-17A e hoʻokō i kona hopena pale [52], e koi ana i nā mea noiʻi e ʻimi i nā Giardia immunomodulatory ʻē aʻe.nā hana o ka maʻi helminth.Ua hōʻike nā mea kākau o kahi noiʻi hou e hiki iā G. muris ke hoʻoulu i ka inflammasome NLRP3 e E. coli EPEC, e hoʻoikaika ana i ka hana ʻana o nā peptides antimicrobial a hoʻemi i kona hiki ke hoʻopili a me ka helu o nā trophozoites i loko o ka ʻāpana ʻōpū, a laila e hōʻemi ana i ka paʻakikī o ka colon. nā maʻi i hana ʻia e ka bacilli [49].Hoʻopili ka NLRP3 inflammasome i ka ulu ʻana o nā maʻi like ʻole.Ua hōʻike nā haʻawina i ka Pseudomonas aeruginosa e hoʻoulu i ka autophagy i nā macrophages e pale aku i ka make cell, a pili kēia kaʻina i ka hoʻoulu ʻana o ka NLRP3 inflammasome [53].No ka N. caninum, ka ho'āla hou 'ana o ka oxygen species-mediated activation o ka NLRP3 inflammasome e kaupalena 'ia kona replication i loko o ka mea hoʻokipa, e lilo ia i mea therapeutic target [9].Ua ʻike ʻia ʻo Paracoccidioides brasiliensis e hoʻoulu i ka hoʻoulu ʻana o ka inflammasome NLRP3 i loko o nā cell dendritic i loaʻa i ka iwi ʻiole, ka hopena o ka hoʻokuʻu ʻana o ka cytokine inflammatory IL-1β, kahi mea koʻikoʻi i ka pale o ka host [10].ʻO kekahi mauʻano Leishmania, e like me L. amazonensis, L. major, L. braziliensis, a me L. infantum chagasi, ho'āla i ka NLRP3 a me ka ASC-dependent caspase-1 i nā macrophages, a me ka maʻi Leishmania.Hoʻonui ʻia ka hoʻopiʻi ʻana i nā ʻiole i ka NLRP3/ASC/caspase-1 gene [11].Zamboni et al.Ua hōʻike ʻia ka maʻi Leishmania e hoʻoulu i ka hoʻoulu ʻana o ka inflammasome NLRP3 i loko o nā macrophages, e kaupalena ana i ka hoʻopiʻi ʻana o ka parasite intracellular.No laila, hiki i Leishmania ke kāohi i ka hoʻoulu ʻana o NLRP3 ma ke ʻano he hoʻolālā pale.I loko o nā haʻawina vivo, ua kōkua ka inflammasome NLRP3 i ka hoʻopau ʻana o Leishmania, akā ʻaʻole i pili i nā ʻiʻo [54].ʻO ka mea like ʻole, ma nā haʻawina helminthiasis, hoʻāla ʻia ka NLRP3 inflammasome i kāohi i ka pale pale o ka mea hoʻokipa i ka helminthiasis gastrointestinal [12].ʻO Shigella kekahi o nā maʻi bacteria nui e hoʻoulu ai i ka maʻi maʻi ma ka honua holoʻokoʻa.Hiki i kēia mau bacteria ke hoʻoulu i ka hana IL-1β ma o ka P2X7 receptor-mediated K+ efflux, reactive oxygen species, lysosomal acidification, a me ka pōʻino mitochondrial.Hoʻoponopono maikaʻi ka inflammasome NLRP3 i ka phagocytosis a me ka hana bactericidal o macrophages e kū'ē iā Shigella [55].Ua hōʻike ʻia nā haʻawina Plasmodium ʻo AIM2, NLRP3 a i ʻole caspase-1 deficient mice i loaʻa i ka Plasmodium e hana i nā kiʻekiʻe kiʻekiʻe o ka interferon type 1 a ʻoi aku ka kūʻē i ka maʻi Plasmodium [56].Eia naʻe, ʻaʻole maopopo ka hana o ka alpha-2 giardine a me alpha-7.3 giardine i ka hoʻoulu ʻana i ka pathogenic o ka mumū NLRP3 i nā ʻiole.
Ma kēia noiʻi ʻana, ʻo ka inhibition o ka inflammasome NLRP3 e MCC950 i hoʻemi i ka BW a hoʻonui i ka nui o nā trophozoites i loko o ka wai hoʻomaʻemaʻe intestinal i nā ʻiole, e hopena i nā loli pathological koʻikoʻi i ka ʻiʻo duodenal.Alpha-2 giardine a me alpha-7.3 giardine ho'ā i ka host mouse NLRP3 inflammasome, hoʻonui i kaʻiole kino kaumaha, hoemi i ka helu o trophozoites i intestinal lavage wai, a alleviate pathological duodenal lesions.Hōʻike kēia mau hopena e hiki iā G. duodenalis ke hoʻoulu i ka host inflammasome NLRP3 ma o ka alpha-2 giardine a me ka alpha-7,3 giardine, e hōʻemi ana i ka pathogenicity o G. duodenalis i nāʻiole.
ʻO ka hui pū ʻana, hōʻike kā mākou hopena i ka alpha-2 a me alpha-7.3 giardines e hoʻoulu i ka hoʻoulu ʻana o ka host NLRP3 host inflammasome a hoʻemi i ka infectivity o G. duodenalis i nā ʻiole.No laila, ua hoʻohiki kēia mau moleke i nā pahuhopu no ka pale ʻana i ka 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: he ʻike.Ua hōʻike ʻia i kēia manawa ua maʻi ʻo Pat Inflamm i nā lāʻau lapaʻau.2019;13:134–43.
Escobedo AA, Tsimerman S. Giardiasis: he loiloi o pharmacotherapy.Manaʻo loea o ka lāʻau lapaʻau.2007;8: 1885–902.
Tian Huafeng, Chen Bin, Wen Jianfeng.ʻO Giardiasis, kū'ē i ka lāʻau a me ka loaʻaʻana o nā pahuhopu hou.Hoʻopili i nā pahuhopu lāʻau Disord.2010;10:295–302.
Wang Z, Zhang X, Xiao Yi, Zhang W, Wu X, Qin T, etc. NLRP3 inflammasome a me nā maʻi inflammatory.Oxide Med Cell Longev.2020;2020:4063562.
Chen GY, Núñez G. ʻO ke kuleana o ka inflammasome i loko o ka ʻōpū a me ka maʻi kanesa.ʻO ka maʻi gastroenterology.2011;141:1986–99.
Pellegrini C, Antonioli L, Lopez-Castejon G, Blandizzi C, Fornai M. Canonical a me ka atypical NLRP3 inflammasome activation ma ke ala o ka hoʻomanawanui a me ka mumū o ka ʻōpū.pre-immune.2017;8:36.
Li L, Wang XC, Gong PT, Zhang N, Zhang X, Li S, a me al.Hoʻokomo ʻia ka ROS-mediated NLRP3 inflammasome activation i ka pane ʻana i ka maʻi N. caninum.He mea hoʻopāpā.2020;13:449.