I-methylene blue nokudluliswa kwamasignali okuvuvukala: ama-inflammasome, i-IL-6, ne-STAT3

Ake sithi amalabhorethri amathathu abika ukuthi inhlanganisela efanayo eluhlaza okwesibhakabhaka inciphisa ukuvuvukala. Eyodwa ibonisa i-IL-1beta encane evela kuma-macrophage. Enye ibonisa i-enzyme ye-iNOS encane ohlotsheni olufanayo lwamaseli. Enye ibonisa i-IL-6 encane egazini nomsebenzi omncane we-STAT3 ebuchosheni. Kulula ukufuna ukuhlanganisa le miphumela emithathu ibe yisimangalo esisodwa. Lokho kuhlanganisa kufihla okutholakele.

Umphumela ngamunye ungowomqondo ohlukile wokudluliswa kwamasignali, onesishukumisi sawo, isinyathelo sawo sokuhlanganisa, nesilinganiso sawo somphumela. I-methylene blue ihlangana nomqondo ngamunye endaweni ehlukile. Ukuthi noma yikuphi kwalokhu kuthuthukisa umphumela wezempilo kungumbuzo ohlukile, okukhulunywa ngawo ekubukezweni kwethu kwe-methylene blue nokuvuvukala. Leli khasi ligxila ezindleleni zokudlulisa amasignali.

Indlela 1: ukuhlanganiswa kwe-inflammasome ne-caspase-1

I-inflammasome iwuhlaka lwamaprotheni olwakhiwa lapho ludingeka ngaphakathi kweseli lozalo lwe-myeloid. Izingxenye zalo ziyinzwa, iphrotheni exhumanisayo ebizwa ngokuthi i-ASC, ne-enzyme eyenza umsebenzi ebizwa ngokuthi i-caspase-1. Uma sezihlangene, i-caspase-1 isika i-pro-IL-1beta ne-pro-IL-18 ibe izinhlobo zazo ezikhishelwa ngaphandle kweseli futhi ingavusa i-pyroptosis, okuwuhlobo lokufa kweseli oluhambisana nokuvuvukala. Inzwa ichaza uhlobo: i-NLRP3 isabela kumakristalu nasezintweni ezinobuthi ezakha izimbobo ezifana ne-nigericin ne-ATP, i-NLRC4 isabela ku-flagellin yamagciwane, i-AIM2 isabela ku-DNA enemicu emibili eku-cytosol, kanti indlela engalandeli uhlelo oluvamile isabela ku-lipopolysaccharide engaphakathi kweseli.

Ukuvuselelwa kudinga amasignali amabili. Isignali yokuqala iwukulungiselela: i-ligand yesamukeli sohlobo lwe-Toll ikhuthaza ukubhalwa kolwazi lwe-NLRP3 ne-pro-IL-1beta ku-RNA okuncike ku-NF-kB, ukuze izingxenye zibe khona. Isignali yesibili iwukuhlanganisa: isishukumisi sesibili sibangela ukuthi ama-molecule e-ASC ahlangane abe ngamachashaza, i-caspase-1 isikwe ibe yingxenye yayo ye-p20, bese kukhishwa i-IL-1beta p17 esivuthiwe. Izinhlobo zomoya-mpilo ezisabela kalula ezivela kuma-mitochondria nokungeniswa kwamakristalu eselini kwaziwa njengezinto ezifaka isandla kusignali yesibili ye-NLRP3.

Ucwaningo lwango-2017 lwama-macrophage ngokuvinjelwa kwezinhlobo eziningi zama-inflammasome lwahlola i-methylene blue kuwo womabili amasignali. Kuma-macrophage omnkantsha wamathambo amagundane ayelungiselelwe nge-lipopolysaccharide, i-nigericin, i-ATP, namakristalu e-monosodium urate ngakunye kwabangela ukukhishwa kwe-IL-1beta ne-caspase-1 kanye nokwakheka kwamachashaza e-ASC. I-methylene blue engu-20 kuya ku-200 micromolar yanciphisa yomithathu le miphumela ngendlela eyayincike esilinganisweni esisetshenzisiwe, ngaphandle kokubulala amaseli. I-flagellin ne-Salmonella ye-NLRC4, i-DNA enemicu emibili ne-Listeria ye-AIM2, kanye ne-lipopolysaccharide efakwe ngaphakathi kwamaseli noma i-E. coli ephilayo yendlela engalandeli uhlelo oluvamile, kwabonisa iphethini efanayo. Le nhlanganisela iyodwa ayizange ivuse ukukhishwa, futhi amapuleti okuqhathanisa awabonisanga umphumela wokulwa namagciwane, ngakho umphumela uhlobene nokusabela komzimba owamukele lawo magciwane.

Ezigabeni zangaphambili zale ndlela, kwalinganiswa imithelela emithathu yokungenelela. I-superoxide yama-mitochondria eyayivuswe yi-rotenone yehla lapho kusetshenziswa i-methylene blue, futhi ukukhishwa kwe-IL-1beta okwakuqhutshwa yi-rotenone kwehla nakho. Ukungeniswa kwezinhlayiyana ezikhazimulayo eselini kwehla, okuhambisana nokuncipha kwe-phagocytosis ngaphambi kokuzwa amakristalu. Isistimu yokubika eyayiqhutshwa yindawo ekhuthaza ukubhalwa kofuzo lwe-NLRP3, enezindawo ezimbili zokubopha i-NF-kB, yabonisa umsebenzi ophansi lapho kusetshenziswa i-methylene blue, kanti indawo ekhuthazayo eyayifinyeziwe, ingenazo lezo zindawo, ayizange isabele. Eshubhuni elingenamaseli, i-methylene blue yaphinde yehlisa ngokuqondile umsebenzi we-caspase-1 yomuntu eyakhiwe ngobuchwepheshe bokuhlanganisa ufuzo. Ukwengeza le nhlanganisela ngesikhathi sokulungiselela kuphela noma ngesikhathi sokuvuselela kuphela nakho kwanciphisa ukukhishwa kwe-IL-1beta, i-IL-18, ne-caspase-1, ngakho zombili izinyathelo ziyathinteka.

Ukuhlolwa kwamagundane kwahambisana nalokhu kuthinteka okukhethiwe. Isilinganiso se-lipopolysaccharide esibulalayo sabulala amagundane eqembu lokuqhathanisa emahoreni ambalwa, kuyilapho i-methylene blue yathuthukisa ukusinda; i-IL-1beta oketshezini lomgodi wesisu yehla, kodwa i-IL-6 oketshezini olufanayo ayizange yehle. Kumodeli yokuvuvukala kolwelwesi lomgodi wesisu okubangelwa yi-Listeria, i-IL-1beta oketshezini lomgodi wesisu yehla kuyilapho inani eliphelele lamaseli lingashintshanga. Ama-monocyte omuntu e-THP-1 aziphatha njengama-macrophage amagundane kuzo zonke izinhlobo ezine zama-inflammasome. Umphumela ophathelene ne-inflammasome uchaza ukuvuthwa kwe-IL-1beta ne-IL-18, hhayi wonke ama-cytokine. Yingakho futhi ukuma kwamaseli okuzivikela ebuchosheni ezivivinyweni zama-microglia nama-astrocyte kuphendula umbuzo ohlukile kowokuhlanganiswa kohlaka.

Indlela 2: ukubhalwa kolwazi lwe-iNOS ku-RNA ngokubopha kwe-NF-kB ne-STAT1

I-nitric oxide synthase evuswayo, noma i-iNOS, iba khona kancane kakhulu lapho iseli lingavuselelwanga, futhi ulwazi lwayo lubhalwa ku-RNA lapho kudingeka. I-lipopolysaccharide ikhuthaza lokhu ikakhulu ngokudilizwa kwe-IkB-alpha, okulandelwa ukwengezwa kweqembu le-phosphate ku-NF-kB nokungena kwayo kuyinucleus. I-interferon-gamma ikukhuthaza ikakhulu ngokwengezwa kweqembu le-phosphate ku-STAT1 yi-Janus kinase, okulandelwa ukungena kwe-STAT1 kuyinucleus. Iphrotheni ngayinye ibe isibopha endaweni yayo engxenyeni ekhuthaza ukubhalwa kofuzo lwe-iNOS. Umsebenzi we-enzyme nokuvuswa kofuzo kuyizindawo ezimbili ezihlukene zokulawula, futhi umuthi ungasebenza kunoma iyiphi yazo noma kuzo zombili.

Ucwaningo lwango-2015 lokuvuswa kwe-iNOS kuma-macrophage nasemagundaneni ane-endotoxin egazini lwathola ukuthi umphumela wawusezingeni lomlayezo we-RNA. Kumaseli e-RAW 264.7 nakuma-macrophage athathwe ngokuqondile emagundaneni, i-methylene blue engu-1 micromolar eyanikezwa imizuzu engu-30 ngaphambi kwe-lipopolysaccharide, i-interferon-gamma, noma kokubili, yanciphisa iphrotheni ye-iNOS ne-mRNA phakathi kwamahora angu-8 kuya ku-24, kwaba ne-nitrite ephansi oketshezini lokukhulisa amaseli futhi amandla amaseli okuqhubeka ephila awanciphanga. Emagundaneni anikezwa i-lipopolysaccharide engu-25 mg per kg, isilinganiso esisodwa se-methylene blue esingu-5 mg per kg ihora elilodwa ngaphambili sanciphisa iphrotheni ye-iNOS ne-mRNA emaphashini, esibindini, nasenhliziyweni.

Imininingwane ephawulekayo yilokho okungazange kushintshe. Ukudilizwa kwe-IkB-alpha, ukwengezwa kweqembu le-phosphate ku-NF-kB, ukwengezwa kweqembu le-phosphate ku-STAT1, nokunqwabelana kwalezi zinhlobo zombili zamaprotheni kuyinucleus kwaqhubeka kungaphazamisekile. Ukuphazamiseka kwenzeka esinyathelweni esisodwa kamuva: izinto ezikhishwe kuyinucleus zabonisa ukubopha okuncane kwe-NF-kB engxenyeni yayo ye-DNA ngemva kwe-lipopolysaccharide nokubopha okuncane kwe-STAT1 ngemva kwe-interferon-gamma. Ukuhlolwa kwe-chromatin immunoprecipitation kwaqinisekisa ukuthi womabili la maprotheni ayetholakala kancane endaweni ekhuthaza ukubhalwa kofuzo lwe-iNOS ngaphakathi kwamaseli. I-tumor necrosis factor alpha, indawo yayo ekhuthaza ukubhalwa kofuzo nayo esebenzisa i-NF-kB, yehla nayo. La maprotheni ayesevuselelwe futhi efikile, kodwa ayebopha kabi ku-DNA ayeqondiswe kuyo.

Qaphela umehluko phakathi kwalokhu nendlela 1. Lokhu kuphathelene nokubopha kwamaprotheni ku-DNA, kusebenzisa i-STAT1 esikhundleni se-STAT3, futhi kulinganisa i-mRNA, iphrotheni, ne-nitrite esikhundleni samachashaza e-ASC nezingxenye ze-caspase-1. Imephu yethu yezinto eziqondiswa yi-methylene blue kumamodeli amaseli igcina leli zinga lokubhalwa kolwazi lofuzo ku-RNA lihlukile ezilinganisweni eziqondile zama-enzyme nama-mitochondria.

Indlela 3: i-IL-6 kuserum nokuvuselelwa kwe-STAT3 ezicutshini

I-IL-6 idlulisa amasignali ngomndeni wezamukeli ze-gp130 iye ochungechungeni lwe-Janus kinase kuya ku-STAT3. I-STAT3 eneqembu le-phosphate ihlangana ngamapheya, ingene kuyinucleus, bese ikhuthaza izinhlelo zokuvuvukala. I-IL-6 ngokwayo iyinto eqondiswa yi-NF-kB, ngakho lezi ezimbili zakha umjikelezo wokubuyelana ovame ukubizwa ngokuthi i-eksisi ye-IL-6 kuya ku-STAT3. Izilinganiso ezijwayelekile zemiphumela ziyi-IL-6 kuserum ezigabeni zangaphambili, isilinganiso se-STAT3 eneqembu le-phosphate uma iqhathaniswa nayo yonke i-STAT3, kanye nama-enzyme enza umsebenzi afana ne-iNOS ne-COX2 ezigabeni ezilandelayo.

Ucwaningo lwamagundane lwango-2023 lwe-IL-6 ne-STAT3 ngemva kwe-lipopolysaccharide lwalinganisa le eksisi ezingxenyeni ezahlukene zomzimba. Amagundane angamaduna e-C57BL/6 athola i-lipopolysaccharide engu-1 mg per kg nsuku zonke izinsuku ezintathu, kanye ne-methylene blue engu-5 noma 10 mg per kg imizuzu engu-30 ngemva kwesilinganiso ngasinye. Ngosuku lwesithathu, i-IL-6 kuserum yayiphansi kuwo womabili amaqembu e-methylene blue uma iqhathaniswa neqembu elathola i-lipopolysaccharide kuphela, kuyilapho iningi lamanye ama-cytokine ahlolwa lingashintshanga ngokwelashwa. Izilinganiso ze-STAT3 eneqembu le-phosphate uma iqhathaniswa nayo yonke i-STAT3 zaziphansi ku-cortex, ku-hippocampus, nasesikhunjeni sendlebe, kanye nokubonakala okuncane kwama-microglia ekufakweni kombala nge-Iba-1, i-iNOS ne-COX2 ephansi, nokuncipha kokwehla kwesisindo ngokwengxenye.

Kudingeka kuqashelwe izinto ezimbili lapha. I-IL-6 ephansi kuserum kanye nokwengezwa okuncane kweqembu le-phosphate ku-STAT3 ezicutshini kuhambisana nokuguqulwa kwale eksisi, kodwa akukhombi indawo eyinhloko ethintekayo. Lokhu kungenxa yokuthi i-cytokine encane ezigabeni zangaphambili, ukudluliswa okuncane kwamasignali yisamukeli, noma umsebenzi oshintshile we-phosphatase ngakunye kungakhiqiza isilinganiso esifanayo. Futhi isisindo phakathi kwezinsuku ezintathu zokuchayeka okusheshayo esishukumisweni siyisilinganiso somphumela wesilwane sonke, hhayi isilinganiso sezimpawu noma sokudamba kwesifo. Sengeza umongo ngaphezu komphumela wokuhlolwa kwe-blot ngaphandle kokuguqula umphumela wamasignali ube yisimangalo sokwelapha. Ikhemistri ehlukile yokuphathwa kwe-redox kuma-mitochondria iqhubeka ngokuhambisana nalokhu kuchazwa kwama-cytokine futhi akufanele ihlanganiswe nakho.

Ukuqhathaniswa kwezindlela: izindawo zokungenelela nezilinganiso zemiphumela

Ithebula livimbela ukudluliselwa kweziphetho okuthathu okungavumelekile. Umphumela wamachashaza e-ASC awufakazeli umphumela we-STAT3. Umphumela wokubopha kwe-STAT1 endaweni ekhuthaza ukubhalwa kofuzo lwe-iNOS awufakazeli umphumela we-STAT3, nakuba womabili la maprotheni engamalungu omndeni we-STAT. Umphumela we-IL-6 kuserum awufakazeli ukuncipha kokuhlanganiswa kwe-inflammasome: esivivinyweni somgodi wesisu i-IL-1beta yehla kuyilapho i-IL-6 ingazange yehle. Umugqa ngamunye udinga isishukumisi sawo nesilinganiso sawo somphumela. Yingakho ukufunda ucwaningo lwe-methylene blue lwamaseli, lwezilwane, nolwabantu emtholampilo kudinga ukuthi isimangalo ngasinye sihambisane nezinga elalinganiswa.

Akukho kulokhu okuqinisekisa izilinganiso zokusebenzisa, ukuphepha, noma inzuzo kubantu. Amamodeli e-lipopolysaccharide ayizishukumisi ezilawulwayo zokuhlola inqubo, hhayi imifanekiso ephelele yokugula okungamahlalakhona. Futhi ukunikeza isilinganiso ngesikhathi esihambisana nesishukumisi kuhlola ukuvinjelwa koshintsho oluvusiwe, hhayi ukubuyisela emuva isifo esesivele sikhona. Gcina le ncazelo yezinqubo ihlukile emiphumeleni yeziguli, futhi wahlulele noma yisiphi isimangalo sezempilo ngocwaningo lweziguli olusisekelayo.