I-methylene blue ne-nitric oxide: i-NOS, i-cGMP, izimpawu emithanjeni

Umbuzo uzwakala ulula: ingabe i-methylene blue ivimbela i-nitric oxide? Elabhorethri, impendulo ihlukana ibe imibuzo emine ehlukene, futhi ngamunye unesivivinyo esihlukile sokuhlola. Ukuyixuba yisona sizathu esiyinhloko esenza okubhalwa nge-methylene blue ne-nitric oxide kuzwakale kuqiniseka kodwa kungacacile.

Cabanga ngesignali ye-nitric oxide njengeketanga elinezixhumanisi ezine. Isakhi sofuzo silotshwa ukuze kwakhiwe i-nitric oxide synthase. I-enzyme iguqula i-arginine ibe yi-nitric oxide. Le gesi isakazekela lapho iqondiswe khona ngaphambi kokuba isabele kokunye. I-guanylate cyclase encibilikayo iyayizwa bese yenza i-GMP eyisiyingi. Kukhona imiphumela eshicilelwe ye-methylene blue kusixhumanisi ngasinye, kodwa ubufakazi buvela kumasampula alungiselelwe ngezindlela ezihlukene, emazingeni okugxila ahlukene, nasezinhlotsheni ezihlukene ze-enzyme. Umphumela kusixhumanisi esisodwa awufakazeli ukusebenza kwesinye.

Uchungechunge olujwayelekile, luchazwe ngokusobala

Odongeni lomthambo wegazi, ukulandelana okujwayelekile kuhamba kanje. Isikhuthazi sisebenza engqamuzaneni ye-endothelium. I-nitric oxide synthase ye-endothelium, ebizwa nge-eNOS noma i-NOS3, yenza ukukhishwa okuncane kwesikhashana kwe-nitric oxide. Le gesi isakazekela engqamuzaneni eseduze yomsipha obushelelezi, ibophe ku-heme enensimbi esesimweni esincishisiwe ye-guanylate cyclase encibilikayo, bese isheshisa ukuguqulwa kwe-GTP ibe yi-GMP eyisiyingi. I-GMP eyisiyingi ivuselela i-protein kinase G, indlela yokulawulwa kwe-calcium iyashintsha, bese umsipha uyakhululeka.

Kunochungechunge lwesibili okufanele lugcinwe luhlukile. Ngesikhathi sokutheleleka noma sokuchayeka kuma-cytokine, amangqamuzana alotsha i-enzyme ehlukile, i-nitric oxide synthase evuselelwayo noma i-iNOS, ebizwa nangokuthi i-NOS2. Ikhiqiza ukugeleza kwe-nitric oxide okukhulu nokuhlala isikhathi eside kunokwe-eNOS. Uhlobo lwesithathu, i-NOS yama-neuron noma i-nNOS, lusebenza komunye umongo futhi. Uma iphepha locwaningo lithi i-nitric oxide synthase ngaphandle kokucacisa uhlobo, yima uhlole ukuthi yiluphi olulinganisiwe. Ukubuka konke kwendlela i-methylene blue ehambisa ngayo ama-electron kugcizelela okufanayo ngezinto eziqondiswe ukusabela kwe-redox ngokujwayelekile: ukudlulisa ama-electron akwenzi lutho ngokwakho, futhi umlingani osekugcineni ngakunye nguyena onquma umphumela.

I-GMP eyisiyingi elinganisiwe nayo iwukufundwa komphumela ohlanganisiwe, hhayi ukuhlolwa okuqondile kwe-enzyme. Izinga layo libonisa ukukhiqizwa yi-cyclase kususwe ukubhidlizwa ngama-phosphodiesterase, ikakhulukazi i-PDE5 emsipheni obushelelezi wemithambo yegazi. Ngakho ukwehla kwe-GMP eyisiyingi kusho ukuthi isignali encane ifinyelele kumamukeli. Ngokwakho, lokhu akusho ukuthi yisiphi isixhumanisi esihlulekile.

Isixhumanisi 1: kukhiqizwa i-enzyme ye-iNOS encane

Kuma-macrophage amagundane amancane avuselelwe nge-lipopolysaccharide yamabhaktheriya, i-interferon gamma, noma kokubili, i-methylene blue yavimbela ukukhuphuka okwakulindelwe kwephrotheni ye-iNOS futhi yacindezela kakhulu i-RNA yesithunywa ye-iNOS ezivivinyweni zokulotshwa kofuzo kuma-macrophage. Ingxenye esifundisayo yilokho okungazange kushintshe. Ukubhidlizwa kwesivimbeli i-I kappa B alpha, ukufakwa kwamaqembu e-phosphate ku-NF kappa B naku-STAT1, kanye nokungena kwalezo zici ku-nucleus kwaqhubeka kakhulu kungaphazamisekile. Okwehlayo kwakuwukubopha kwazo ku-DNA, kwaqinisekiswa ngokuhlolwa kwe-chromatin immunoprecipitation okwabonisa ukuncipha kokubopha endaweni ye-promoter ye-iNOS.

Lowo umphumela wokulawula ufuzo oza ngaphambi kokushintsha kwenani le-enzyme, hhayi ukuvinjwa okusheshayo kwe-enzyme esivele ikhona. Uyingxenye yokudluliswa kwezimpawu zokuvuvukala, lapho izici zokulotshwa kofuzo kufanele zibophe ku-promoter ngaphambi kokuba iphrotheni ivele. Awusho lutho ngokuqondile nge-eNOS ekhiqizwa njalo emthanjeni ongavuselelwanga, futhi kufanele uchazwe ngaleyo ndlela.

Isixhumanisi 2: i-enzyme ye-NOS esivele ikhona isebenza kancane

Ucwaningo lwamagundane amakhulu lwahlola izinga elihlukile. Amagundane amakhulu esilisa ayephapheme anikezwa i-lipopolysaccharide ngomthambo wegazi, kwathi ngemva kwamahora amathathu anikezwa isixazululo sikasawoti noma i-methylene blue engu-3 mg nge-kg ngomthambo wegazi. Ukusebenza kwe-nitric oxide synthase engxenyeni ephakathi nesezansi ye-hypothalamus kwalinganiswa ngokuguqulwa kwe-arginine ephawuliwe ibe yi-citrulline ocwaningweni lokusebenza kwe-NOS ku-hypothalamus. Ingcindezi kanye nesixazululo sikasawoti kuphela kwavuselela ukusebenza okuthile okungancikile ku-calcium, kanti i-lipopolysaccharide yakukhuphula kwafinyelela cishe izikhathi eziyishumi ezingeni leqembu lokulawula. I-methylene blue yaqeda zombili izingxenye zokusebenza ezazivuselelwe. Ukusebenza okuvamile okuncike ku-calcium akuzange kushintshe kulezo zicubu ngalelo nani elinikeziwe nangaleso sikhathi sokulinganisa.

Funda lowo mphumela ngendlela obhalwe ngayo: ukusebenza okulinganisiwe kohlobo lwe-iNOS kwacindezeleka ngaphansi kwalezo zimo. Akusona isilinganiso sokuvezwa kofuzo, futhi akubona ubufakazi bokuthi njalo kukhethwa i-iNOS kune-eNOS noma i-nNOS. Umsebenzi owenziwe nge-enzyme ehlanziwe ugcizelela lesi sixwayiso. Ekuhlolweni okuqondile kwe-nitric oxide synthase ehlanziwe, i-methylene blue yavimba ngokuphelele ukwakheka kwe-citrulline ku-30 micromolar. Izinga lokugxila elibangela ukuvinjelwa okuyisigamu laliseduze no-5.3 micromolar ngaphandle kwe-superoxide dismutase, no-9.2 micromolar uma ikhona, njengoba kubikwe ekuqhathanisweni kwe-NOS ehlanziwe ne-guanylate cyclase. Ucwaningo oluhlukile lwe-nNOS ehlanziwe lwabonisa ukuthi i-oxidation ye-heme ebophe ku-enzyme yayihilelekile. Isifundo esisebenzisekayo ukuthi ukuvinjelwa okuqondile kwe-NOS kuyenzeka ngempela ezinhlelweni ezihlukanisiwe, kodwa umphumela we-hypothalamus awunakunwetshwa ube yisimangalo esivamile sokuthi i-methylene blue ayithinti zonke izinhlobo ze-NOS ezikhiqizwa njalo kuyo yonke indawo.

Isixhumanisi 3: i-nitric oxide iyalahleka ngaphambi kokufika

Ubufakazi obucace kakhulu bokuthi okunye ngaphandle kokuvinjelwa kwe-enzyme kungachaza ukwehla kwe-GMP eyisiyingi buvela kumangqamuzana omsipha obushelelezi womthambo wamaphaphu kanogwaja akhuliswe elabhorethri. Indlela yokuhlola yeqa ngokuphelele i-NOS yangaphakathi ngokwengeza ngokuqondile izinto ezikhulula i-nitric oxide, yabe isihlola ukuthi i-methylene blue isayicindezela yini impendulo ye-GMP eyisiyingi ocwaningweni lwe-superoxide emsipheni obushelelezi wemithambo yegazi.

Kwahlolwa izinto ezintathu ezikhulula i-nitric oxide, kusukela ku-1 kuya ku-100 micromolar: i-S nitroso L cysteine, i-sodium nitroprusside, kanye ne-analogue ezinzile ekhishwe iqembu le-amino ebizwa nge-MPANO. Zonke zakhuphula i-GMP eyisiyingi cishe izikhathi eziphakathi kuka-1.5 no-12. I-methylene blue ku-10 micromolar yavimbela ezokuqala ezimbili ngamaphesenti angu-51 kuya ku-100, yashiya impendulo ye-MPANO ingashintshile. Ukube i-methylene blue yayimane ivimbe indawo ye-cyclase eyenza ukusabela, bekuyoba nzima ukuchaza lokho kukhetha phakathi kwezinto ezikhulula i-nitric oxide, ngoba zontathu zigcina zisebenza ku-enzyme efanayo esekugcineni kwalolu chungechunge.

Izivivinyo zokulawula ezintathu zabe sezibophela le ndlela yokusebenza ku-superoxide. Okokuqala, ukukhiqiza i-superoxide ngaphandle kwamangqamuzana nge-xanthine kanye ne-xanthine oxidase kwaphinda iphethini efanayo: i-CYSNO ne-nitroprusside kwavinjelwa, i-MPANO yangathinteka. Okwesibili, i-methylene blue yabangela ukukhiqizwa kwe-superoxide kumangqamuzana omsipha obushelelezi ngendlela encike enanini elinikeziwe nasesikhathini, okwalinganiswa ngokuncishiswa kwe-cytochrome c. Okwesithathu, i-superoxide dismutase yavimbela ngokuphelele lokhu kuvinjelwa kuyilapho i-catalase ingakwenzanga, okukhomba i-superoxide kunokuba kube yi-hydrogen peroxide njengento eyayibangela umphumela ngokuqondile kulawo masampula.

Uhlobo lwesivivinyo lokukhulisa amangqamuzana ndawonye kufanele luchazwe ngoba lubonisa indawo okwenzeka kuyo umphumela. Amangqamuzana e-endothelium ayesezingcezwini zengilazi zokumboza akhuliswa isikhashana ndawonye namangqamuzana omsipha obushelelezi, ngakho into ekhishwa yi-endothelium ezimweni ezijwayelekile yakhuphula i-GMP eyisiyingi yomsipha obushelelezi izikhathi ezintathu kuya kweziyisishiyagalombili. Ukwelapha noma iyiphi yalezi zingxenye kusengaphambili nge-methylene blue kwanciphisa leyo signali, futhi i-superoxide dismutase yabuyisela ngokwengxenye umphumela wokwelashwa kusengaphambili kwe-endothelium. Ngamanye amazwi, i-methylene blue ayidingi ukusebenza ngaphakathi kwengqamuzana yomsipha obushelelezi eyamukela isignali. Inganciphisa isignali ngokususa isithunywa sisahamba. Umhlahlandlela wokufunda ucwaningo lwe-methylene blue ubheka lokhu njengomthetho ojwayelekile: yisho ingxenye lapho ukungenelela kwenzeke khona ngaphambi kokusho into eqondisiwe ezingeni lamangqamuzana ezinto.

Ukubiza lokhu ngokuthi ukuvinjelwa kwe-NOS bekuyoba iphutha. Asikho isinyathelo se-synthase esasidingeka ukuze lezi zinto ezikhulula i-nitric oxide zisebenze, kodwa isignali yehla noma kunjalo.

Isixhumanisi 4: umamukeli ngokwawo usabela kancane

Imiphumela eqondile ku-guanylate cyclase encibilikayo ikhona, kodwa amazinga okugxila abalulekile. Ekuqhathanisweni kwama-enzyme ahlanziwe okucashunwe ngenhla, i-cyclase evuselelwe yi-nitric oxide yayidinga cishe u-60 micromolar we-methylene blue ukuze ivinjelwe ngesigamu, uma kuqhathaniswa nokuvinjelwa okuyisigamu kwe-NOS eduze kuka-5 kuya ku-9 micromolar. Ngisho no-1 millimolar wabanga ukuvinjelwa okukhulu okwakungaphelele. Lowo umehluko ocishe ube izikhathi eziyisithupha kuya kweziyishumi nanye. Ngakho isivivinyo samangqamuzana esisebenzisa izinga eliphansi le-micromolar esinciphisa i-GMP eyisiyingi asikwazi ukubizwa ngokuthi ukuvinjelwa okukhethayo kwe-cyclase ngesisekelo senani le-GMP eyisiyingi kuphela.

Ucwaningo lwakamuva lwe-redox lwengeza isixwayiso sesibili: i-methylene blue yenza i-cyclase evuselelwe yi-nitric oxide ingasebenzi ngendlela ehlukile ku-oxidation elula yensimbi ye-heme eyenziwa umoya. Ngakho umamukeli angaphazamiseka, kodwa indlela yamakhemikhali ayikacaciswa ngokuphelele, futhi umehluko wamazinga okugxila usho ukuthi imiphumela ye-superoxide ne-NOS kufanele iqale ikhishwe njengezincazelo lapho kuchayekwa emazingeni aphansi e-micromolar. Yilapha futhi lapho kufanele khona ukuhlolwa kokulawula i-phosphodiesterase. Ngaphandle kokuthi ukubhidlizwa kukalwe noma kuvinjelwe, ukuncipha kokuqongelela kwe-GMP eyisiyingi kuyahambisana nokukhiqizwa okuncane, ukubhidlizwa okusheshayo, noma kokubili.

Kungani lokhu kungenakufingqwa ngesimangalo esisodwa ngomfutho wegazi

Isixhumanisi ngasinye saqinisekiswa ohlelweni oluhlukile. Ukulotshwa kofuzo kuma-macrophage akufani nokusebenza kwe-enzyme ku-hypothalamus. Ukusebenza ku-hypothalamus ngemva kwe-lipopolysaccharide akufani nezinga lokufinyela kwe-endothelium engavuselelwanga. Into ekhishwa yi-endothelium ezimweni ezijwayelekile lapho amangqamuzana ekhuliswe ndawonye ayifani nokunwebeka kwemithambo okuvuselelwe into evusa umamukeli. Amangqamuzana omthambo wamaphaphu akhuliswe elabhorethri ku-10 micromolar we-methylene blue awafani nokujikeleza kwegazi komuntu. Ukukuhlanganisa kube yisitatimende esinjengokuthi i-methylene blue ikhuphula umfutho wegazi ngokuvimba i-nitric oxide kulahla wona kanye umehluko izivivinyo zokulawula ezazenzelwe ukuwugcina.

Izinto ezintathu okufanele zihlolwe zigcina incazelo ithembekile. Okokuqala, yisho uhlobo lwe-enzyme: i-eNOS ngezinga lokufinyela kwemithambo engavuselelwanga, i-iNOS ngokugeleza okubangelwa ukuvuvukala, i-nNOS ezimweni zama-neuron nakwezinye ezihilela ama-enzyme ahlanziwe. Okwesibili, yisho indlela isignali ye-nitric oxide elethwa ngayo: ukukhiqizwa ngaphakathi, ukukhishwa okujwayelekile yi-endothelium, noma into ethile ekhulula i-nitric oxide njenge-CYSNO, i-nitroprusside, noma i-MPANO, ngoba le ezinzile yamelana ne-methylene blue kuyilapho lezo ezingazinzile zingazange. Okwesithathu, yisho izinga lokugxila nendlela yokubuyisela umphumela: ukubuyiselwa nge-superoxide dismutase kukhomba ukubandakanyeka kwe-superoxide, ukuhluleka kwe-catalase kuphikisana nokuthi i-hydrogen peroxide iyona ebangela umphumela kulawo masampula, ukulingisa nge-xanthine oxidase kubonisa ukuthi le ndlela iyanele ukubanga umphumela, futhi ukuhlolwa kwama-enzyme ahlanziwe kuhlukanisa imiphumela eqondile ekusheshiseni ukusabela nokulahleka kwesignali ngesikhathi ihamba.

Isifinyezo esihlanganisiwe yilesi. Kungcono ukubheka i-methylene blue njengento ephazamisa ezindaweni eziningi ukudluliswa kwezimpawu kusukela ku-nitric oxide kuya ku-GMP eyisiyingi kunokuyibheka njengesivimbeli esikhetha i-cyclase kuphela. Kuye ngamasampula alungiselelwe kanye nezinga lokugxila, inganciphisa ukubopha kwezici zokulotshwa kofuzo kwe-iNOS, ivimbele ngokuqondile ama-enzyme e-NOS, inciphise ukutholakala kwe-nitric oxide ngokukhiqizwa kwe-superoxide, futhi iphazamise i-cyclase evuselelwe yi-nitric oxide. Indima yokudluliswa kwezimpawu nge-GMP eyisiyingi ocwaningweni lwemithambo yegazi kanye nokuqhathaniswa kwezinto ezikhulula i-nitric oxide ezisetshenziswa ekuhlolweni kwamangqamuzana ngakunye kuchaza elinye lalawo magatsha ngokuningiliziwe, kanti izindlela ezisetshenziswa ukuhlukanisa imiphumela ye-redox nemiphumela kuma-enzyme zichaza ukuthi kungani i-superoxide dismutase kanye ne-catalase kanye nokuhlolwa kokulawula ngento ezinzile ekhulula i-nitric oxide kuvela kaningi kangaka kule mibhalo yocwaningo.

Inani eliphansi le-GMP eyisiyingi ngemva kwe-methylene blue libonisa iketanga elinqamukile. Izivivinyo zokulawula kuphela ezisho ukuthi yisiphi isixhumanisi esinqamukile.