Ukukala izinhlobo ze-oksijini ezisebenzayo ezivivinyweni ze-methylene blue

Amalabhorethri amabili enza lokho ngayinye ekubiza ngokuthi isivivinyo sokulwa nokuxutshwa ne-oksijini se-methylene blue. Enye ibika ukuthi udayi ucindezela izinhlobo ze-oksijini ezisebenzayo. Enye ibika ukuthi uyazikhiqiza. Zombili zibonisa amajika acacile okusabela kuye ngenani elisetshenzisiwe. Zombili zibika ngobuqotho. Umehluko awubangelwa ukukhwabanisa noma ukungakwazi ukwenza umsebenzi. Ubangelwa ukuthi la maqembu amabili akale izinto ezihlukene ngaphansi kokukhanya okuhlukene, ngezihloli eziphendula imibuzo ehlukene, abe esechaza zombili izimpendulo ngegama elifanayo: ROS.

I-methylene blue iyi-molecule enzima ukuyikala ngokunembile ngoba ibamba iqhaza ekusebenzeni kwamakhemikhali okufanele ikubheke. Ebumnyameni idlulisa ama-electron phakathi kwabanikelayo nabamukelayo, bese ibuyela esimweni sayo sokuqala, ilungele ukuphinda umjikelezo. Ngaphansi kokukhanya iba yi-photosensitiser, ifinyelele esimweni se-triplet esivuselelwe amandla, lapho ingadlulisela khona amandla ku-oksijini ukuze yakhe i-singlet oxygen noma idlulisele ama-electron ukuze yakhe ama-radical ne-hydrogen peroxide. Noma yisiphi isihloli esifakwa kulolo hlelo singaxutshwa ne-oksijini, sithole ama-electron, siphelelwe umbala, noma isignali yaso yokukhanya ifihlwe udayi uqobo. Ngakho umbuzo wokuqala wanoma yikuphi okufundwayo kwe-ROS awukaze ube ukuthi inombolo ithini. Uba ukuthi ukuhlolwa kuthola ini ngempela, nokuthi udayi wenzani ekuhlolweni.

I-Amplex Red: ukuhlolwa kwe-hydrogen peroxide okulinganiswe ngezindinganiso

Inhlanganisela ye-methylene blue ne-Amplex Red iyona enobufakazi obuqine kakhulu kule mibhalo, uma ihlelwe kahle. Ukusebenza kwamakhemikhali kuqondile: i-horseradish peroxidase isebenzisa i-hydrogen peroxide ukuxuba i-Amplex substrate ne-oksijini, iyiguqule ibe ngumkhiqizo okhipha i-fluorescence, i-resorufin, ngesilinganiso samamolekyuli esicishe sibe okukodwa kokukodwa. I-peroxide evela kusampula iba yi-fluorescence emshinini wokuthola isignali. Akukho okunye emndenini ovamile we-ROS okushayela lokho kusabela ngokuqondile.

Inzuzo ebalulekile ukulinganisa ngezindinganiso. Umsebenzi ocophelelayo uhlola izindinganiso ze-peroxide ezaziwayo kusixazululo esifanayo esigcina i-pH, ngamazinga afanayo e-peroxidase ne-substrate, ngesikhathi esifanayo sokufukamela, nangezilungiselelo ezifanayo zokukhanya, bese efunda amasampula ngokuqhathanisa nalelo jika. Ocwaningweni lwama-mitochondria obuchopho ahlukanisiwe oluyisisekelo sale ndlela, ababhali benza lokho kanye, bengeza amanani aziwayo e-peroxide ekugcineni kokuhlolwa ngakunye, futhi izinombolo zibikwa njengamazinga ngama-picomole ngomzuzu ngemiligremu yephrotheni, esikhundleni samayunithi e-fluorescence angalinganiswanga ngezindinganiso. Yilokho okuguqula ukukhanya kube yisilinganiso.

Imininingwane emithathu yalolo cwaningo ifanele ukulingiswa. Okokuqala, ababhali baqinisekisa isignali nge-catalase, ecekela phansi i-peroxide. Ukukhuphuka kwe-fluorescence okubangelwa yi-methylene blue kwanqamuka lapho kufakwa i-catalase, kwaqinisekisa ukuthi isignali yayiyi-peroxide kunokuba ibe yiphutha elidalwe ukukhanya. Okwesibili, bahlola ukukhanyisa ngokuqondile ngokuvala nokuvula ugongolo lokuvuselela, ukuze amasampula athole ingxenye eyodwa kweziyisishiyagalombili yokukhanya okuvamile, futhi isignali yehla ngamaphesenti ayisishiyagalolunye kuphela. Ngaphezu kwamaphesenti angamashumi ayisishiyagalolunye e-peroxide abayibikayo kwakungancikile ekukhanyeni kokukala. Okwesithathu, bakala ukususwa kwe-peroxide ngokwehlukana nge-electrode, bathola ukuthi udayi wawukhuphula ukukhiqizwa kwayo futhi wehlisa ijubane lokususwa kwayo. Isihloli esibika kuphela i-fluorescence esele sasiyohlanganisa leyo miphumela emibili.

Ukuhlolwa kusenezindlela okungahluleka ngazo, futhi i-methylene blue ithinta eziningana zazo. Udayi umunca ukukhanya endaweni ebanzi ebomvu efanayo naleyo i-resorufin ekhipha kuyo ukukhanya, ngakho lapho udayi uminyene kakhulu isampula lingafihla isignali yalo ngenxa yemiphumela yokuhlunga ukukhanya ngaphakathi. Ngakho izindinganiso kufanele zibe nezinga lodayi elifanayo nelamasampula. I-Amplex Red nayo ingaxutshwa ne-oksijini ngenxa yokukhanya ngokwayo, futhi umkhiqizo wayo i-resorufin ungangena emijikelezweni ye-photoredox ekhiqiza i-superoxide ne-peroxide, okuyinto ebaluleke kakhulu lapho isivivinyo siphinde sikhanyise i-photosensitiser. Isixazululo esisebenzayo ukukhanyisa amasampula e-methylene blue kuqala, bese kuhlolwa izingxenye zawo ezilinganisiwe ngezinto zokuhlola ze-Amplex ebumnyameni, kunokukhanyisa udayi nesihloli ndawonye. Ekugcineni, i-superoxide dismutase ayimane icindezele isignali ye-Amplex: ukuguqula i-superoxide ibe yi-peroxide kungayikhuphula. Isilawuli se-SOD esandisa i-fluorescence asihlulekanga. Sithole ukuthi i-superoxide iwuhlobo olungaphambi kwe-peroxide ochungechungeni lokusabela.

I-DCFDA: inkomba yokuxutshwa ne-oksijini engaqondile ohlotsheni olulodwa

Ukuhlanganiswa kwe-methylene blue ne-DCFDA kungumthombo wokudideka okuningi. I-DCFH-DA iyinto eyandulela isihloli, efakwa ngaphakathi kweseli. Ama-esterase angaphakathi kweseli asusa amaqembu ayo e-acetate, kusale i-DCFH engakhiphi i-fluorescence, bese ukuxutshwa kwe-DCFH ne-oksijini kukhiqiza i-DCF ekhipha i-fluorescence. Izinto eziyixuba ne-oksijini ziningi: ama-hydroxyl radical, izinto eziphakathi kokusabela kwe-peroxidase ne-heme, i-nitrogen dioxide, i-hypochlorous acid, nama-radical avela ku-peroxynitrite. I-hydrogen peroxide iyodwa ayiyixubi ne-oksijini ngokuqondile nganoma yiliphi ijubane eliwusizo; i-fluorescence encike ku-peroxide ngokuvamile idinga i-ayoni, amaphrotheni e-heme, noma ukusebenza kwamakhemikhali kwe-peroxidase kulolu chungechunge.

Ukuhlaziywa okugxekayo okufanele kucashunwe kuzo zonke izingxenye ezichaza izindlela kuvela ekuhlaziyweni kwe-DCFH njengesihloli okwenziwa nguKalyanaraman nozakwabo, njengoba kufingqiwe ocwaningweni lwama-mitochondria obuchopho olwenqaba i-DCFDA ngenxa yalesi sizathu. Babonisa ukuthi udayi usabela kancane, ngesilinganiso samamolekyuli esingakachazwa, ngaphandle kokulinganiswa ngezindinganiso okuvumelekile, futhi usabela nakumakhemikhali angewona ama-ROS. Isihloli siphinde sikhulise lokho esikukalayo: ukulahlekelwa kwe-DCFH yi-electron eyodwa kwakha i-DCF radical esabela ne-oksijini ukuze yakhe i-superoxide, yona eguquka nge-dismutation ibe yi-peroxide eyengeziwe. Okufundwayo kukhiqiza ngokwengxenye ukusebenza kwamakhemikhali ekubikayo.

Akukho kulokhu okwenza i-DCFDA ingabi wusizo. Kuyenza ibe ukuhlola kokuqala, hhayi indlela yokuhlonza. Ukukhuphuka kwe-fluorescence ye-DCF kusho ukuthi ukuxutshwa kwesihloli ne-oksijini okuncike entweni exuba ne-oksijini kwandile. Akusho ukuthi i-peroxide yandile, futhi ngokuqinisekile akusho ukuthi ingqikithi ye-ROS yandile, ngoba alikho isihloli esisodwa esikala lelo nani. Umsebenzi wangaphambili nge-methylene blue owancika ekuxutshweni kwe-DCF ne-oksijini emazingeni angaphezu kwe-micromolar eyodwa wabika imiphumela ekhuthaza ukuxutshwa ne-oksijini, kuyilapho ezinye izinhlelo zangaleso sikhathi zabika ukuvikelwa ekufeni kwamaseli okubangelwa yi-peroxide. Kokubili kungaba yiqiniso ngodayi ofanayo ezimweni ezihlukene ze-redox, futhi umphumela we-DCF uwodwa awukwazi ukusho ukuthi iyiphi into exuba ne-oksijini eyadala isignali. Ukubaluleka komsebenzi wangaphambili wama-mitochondria obuchopho ukuthi wathatha indawo yalokhu kuhlola kokuqala ngokukala i-peroxide ngezindinganiso, wathola ukwanda kwayo ngokuqondile: ngisho nodayi osezingeni lama-nanomolar ayikhulu wandisa ukukhishwa kwe-peroxide izikhathi eziningana kuyo yonke i-substrate nakuzo zonke izimo zokuphefumula ezahlolwa.

I-DCFDA inomunye ugibe oqondene ne-methylene blue. Udayi unombala ojulile futhi uyasebenza ku-redox, ngakho ungafiphaza i-fluorescence, ucime ukukhishwa kokukhanya, noma uxube amakhemikhali esihloli ne-oksijini ngaphandle kokuncika ku-ROS yamaseli. Isilawuli esingenamaseli, esinodayi nesihloli kodwa esingenawo amaseli, asiyona nje into yokuhlobisa engakhethwa. Yisivivinyo esikutshela ukuthi udayi uyasabela yini nesihloli.

Ukubamba i-spin nokusetshenziswa kwe-oksijini: ukuhlonza nokugeleza kokusabela

Lapho izihloli ze-fluorescence zibika ukuthi kuvele okuthile okungaxutshwa ne-oksijini, i-electron spin resonance esebenzisa izibambi ze-spin ibika okuthile okusondela kakhudlwana ekutheni bekuyini. Ucwaningo oluyisisekelo lwe-photochemistry ye-methylene blue lwasebenzisa i-ESR ukuthola i-ascorbate radical evela kudayi, ku-ascorbate nasekukhanyeni, lwabe selubonisa ukwakheka kwe-peroxide ezivivinyweni zokusetshenziswa kwe-oksijini, kanye nokwakheka kwama-hydroxyl radical okusheshiswa yi-ayoni ezivivinyweni zokubamba i-spin. Indlela ngayinye yaphendula umbuzo ezinye ezazingakwazi ukuwuphendula: i-ESR yahlonza ama-radical, ukusetshenziswa kwe-oksijini kwabonisa ukugeleza kokusabela ngaphandle kokuncika kunoma yisiphi isihloli se-fluorescence, futhi ukuhlanganiswa kwazo kwabonisa ukuthi izindlela ze-singlet oxygen nezama-radical zazisebenza zombili ohlelweni olufanayo olukhanyisiwe.

Lowo mphumela wango-1984 usafundeka njengesixwayiso. Lapho kukhona ukukhanya nento enikela ngama-electron, udayi wakhiqiza i-peroxide, futhi lapho kukhona i-ayoni uhlelo lwakhiqiza i-hydroxyl radical, uhlobo olusebenza kakhulu kuleli qoqo. Ababhali baxhumanisa lokhu ne-ascorbate esesweni, bangabaza ukusetshenziswa kwayo emehlweni ngaphandle kokuqaphela. Abafundi banamuhla kufanele baqaphele izimo: ukukhanya kanye nento enikela ngama-electron kanye ne-ayoni. Uma kususwa noma yikuphi kwalokhu, indlela yokusabela iba buthaka noma iyanyamalala.

Ama-electrode e-oksijini abaluleka ngendlela ehlukile. I-electrode yohlobo lwe-Clark ikala ukusetshenziswa kwe-oksijini okuphelele ngemva kokubalwa kokukhiqizwa kwayo, okuqinisekisa ukuthi ukusebenza kwamakhemikhali kwe-redox okuqhutshwa udayi kudla i-oksijini yamamolekyuli, futhi kunikeza imininingwane yejubane lokusabela engenakulingiswa yiphutha le-fluorescence. Ayihlonzi umkhiqizo. I-peroxide, i-superoxide, amanzi, nokubola kwe-singlet oxygen konke kudla i-oksijini. Ukugeleza kokusabela ngaphandle kokuhlonza akuphelele njengokuhlonza ngaphandle kokugeleza kokusabela, yingakho amaphepha aqinile ehlanganisa i-electrode nokuhlolwa kwe-peroxide okulinganiswe ngezindinganiso noma nesibambi.

Kungani imiphumela yokulwa nokuxutshwa ne-oksijini iphikisana

Beka lezi zindlela ezintathu zokufunda eceleni kwenye nenye, bese ukuphikisana okubonakala emibhalweni ye-methylene blue kuchazeka ngomehluko wezindlela kanye namaqiniso amathathu ebhayoloji.

Okokuqala, udayi uyisidlulisi sama-electron futhi ungumthombo we-peroxide. Ngokuphambukisa ama-electron ezindaweni zokuphefumula lapho i-oksijini ibingaguquka khona ibe yi-superoxide, unganciphisa uhlobo olulodwa lwe-ROS, kuyilapho isimo sawo esithole ama-electron sidlulisela ama-electron ku-oksijini ngokuqondile siyiguqule ibe yi-peroxide, sandise olunye uhlobo. Ngakho ilabhorethri ebhekene nokusebenza kwamakhemikhali kwe-redox kuma-mitochondria kanye nendima yokudlula enye indlela kufanele ilindele izimpendulo ezincike esihlolini: okufundwayo okuzwela i-superoxide kuyehla, kuyilapho okufundwayo kwe-peroxide kukhuphuka ku-cuvette efanayo.

Okwesibili, i-peroxide emazingeni aphansi iyisignali, hhayi umonakalo nje kuphela. Ucwaningo lwama-mitochondria obuchopho begundane olwalulandela umsebenzi wangaphambili lwathola ukukhiqizwa kwe-peroxide ngodayi ongaba sezingeni eliphansi lama-nanomolar angamashumi amahlanu ngaphandle kokulimala kwe-DNA yama-mitochondria emazingeni okwelapha, kanye nokuvikelwa ekulimaleni okubangelwa yi-rotenone ngemva kokwelashwa kwangaphambili. Udayi walimaza izingcezu ezikhethiwe ze-DNA kuphela ezingeni lama-micromolar ayikhulu, elingaphezulu kakhulu kwebanga lokwelapha, kanti ukukhanya okubomvu kanye nodayi osezingeni lama-micromolar angamashumi amabili kwakhiqiza i-singlet oxygen eyalimaza zonke izingcezu ezahlolwa. Indlela ehlongozwayo iyi-hormesis: ukukhiqizwa okuncane kwe-peroxide kuvusa izivikelo zokulwa nokuxutshwa ne-oksijini ezixhumene ne-Nrf2 kanye nokwakheka kwama-mitochondria amasha, ngakho okufundwayo kwesikhathi esifushane okubonisa ukukhuthazwa kokuxutshwa ne-oksijini kwandulela umphumela wesikhathi eside wokulwa nokuxutshwa ne-oksijini. Ukuhlolwa okufundwa ngehora elilodwa nokufundwa ngosuku olulodwa kungaphikisana ngoba ibhayoloji ishintshile phakathi kwalezo zikhathi.

Okwesithathu, ukukhanya kunquma ngisho nokuthi iyiphi i-molecule ekhona. Ukukhanyisa kususa amaqembu e-methyl kudayi, kuwuguqulele ku-Azure B nakweminye imikhiqizo ehlobene, futhi isibani se-microscope esiphezu kwesitsha sanele ukuwenza usebenze njenge-photosensitiser. Izivivinyo ezenziwa ebumnyameni nezikhanyisiwe ziyizivivinyo ezihlukene, futhi ingxenye yezilawuli zokukhanyisa nokuphathwa kukadayi yanoma iyiphi inqubo kufanele iziphathe kanjalo.

Iqoqo lezilawuli elidingekayo okungenani, nendlela yokufunda noma yisiphi isimangalo

Ukuqhathanisa okungezansi kuwumthombo wokuqala oyireferensi yalesi sihloko. Ngaphambi kokwamukela noma yimuphi umphumela we-ROS we-methylene blue, hlola ukuthi ababhali bagcwalise muphi umugqa nokuthi beqe muphi.

Kulandela umthetho osebenzayo wokufunda. I-Amplex ene-peroxidase, elinganiswe ngezindinganiso, yahlolwa nge-catalase, iwubufakazi nge-peroxide. I-DCFDA iwubufakazi bokuxutshwa kwesihloli ne-oksijini okungaqondile ohlotsheni olulodwa, akukho okungaphezu kwalokho. Ukubamba i-spin ngezilawuli zezinto ezibamba ama-radical kuwubufakazi bokuhlonza ama-radical. Ukusetshenziswa kwe-oksijini kuwubufakazi bokugeleza kokusabela. Umusho osho okungaphezu kwalokho ukuhlolwa kwawo okungakusekela, noma ubhalwe ngokuzethemba kangakanani, ungumusho omayelana nesihloli, hhayi odayi. Izingxoxo ezihlobene zokuncika enanini elisetshenzisiwe nebanga le-hormesis, ukugeleza kwama-electron kuma-mitochondria, ukuphazamiseka kokuhlolwa kumasampula afakwe udayi, namaphutha okukhanyisa nge-microscope zinweba ngalinye lala maphuzu, futhi ngalinye lihlala lisebenza lapho indawo yesikhombisi sereferensi yesikhashana ithathwa yireferensi yangempela.