Ukulinganisa iintlobo zeoksijini ezisebenzayo kuvavanyo lwemethylene blue

Iilebhu ezimbini ziqhuba oko nganye ikubiza ngokuba luvavanyo lwemethylene blue lokuthintela ioksideshini. Enye inika ingxelo yokuba idayi inciphisa iintlobo zeoksijini ezisebenzayo. Enye inika ingxelo yokuba iyazivelisa. Zombini zibonisa iigrafu ezicacileyo zobudlelwane phakathi kwedosi nempendulo. Zombini zinyanisekile. Umahluko awubangelwa bubuqhophololo okanye kukungakwazi ukwenza umsebenzi. Ubangelwa kukuba la maqela mabini alinganise izinto ezahlukileyo phantsi kokukhanya okwahlukileyo, esebenzisa izikhombisi eziphendula imibuzo eyahlukileyo, aza achaza zombini iimpendulo ngegama elinye: ROS.

Imethylene blue yimolekyuli ekunzima ukuyilinganisa ngokuchanekileyo kuba ithatha inxaxheba kwikhemistri ekufanele ukuba iyijongile. Ebumnyameni ihambisa iielektroni phakathi kwabanikeli nabamkeli ize ibuyele kwimo yayo yokuqala, ilungele ukuqalisa omnye umjikelo. Phantsi kokukhanya iba sisivuseleli sokusabela ekukhanyeni, ifikelele kwimeko yamandla aphezulu ye-triplet apho inokudlulisela amandla kwioksijini ukuze yenze ioksijini ye-singlet okanye idlulisele iielektroni ukuze yenze iiradikhali nehydrogen peroxide. Nasiphi na isikhombisi esifakwa kuloo nkqubo sinokwenziwa ioksideshini, iridakshini, sitshintshelwe umbala, okanye umqondiso waso wokukhanya ugqunywe yidayi ngokwayo. Ngoko umbuzo wokuqala walo naluphi na ufundo lwe-ROS awusoze ube ngowokuba lithini inani. Ngowokuba uvavanyo lufumanisa ntoni kanye kanye, nokuba idayi ilwenza ntoni uvavanyo.

I-Amplex Red: uvavanyo lwehydrogen peroxide olulinganiswe ngemigangatho

Indibaniselwano yemethylene blue ne-Amplex Red yeyona inokuthethelelwa kakuhle kolu ncwadi lophando, xa ilungiselelwe ngokuchanekileyo. Ikhemistri yayo ijolise kwinto ethile: i-horseradish peroxidase isebenzisa ihydrogen peroxide ukwenza ioksideshini yesubstrate ye-Amplex ibe yimveliso ekhupha ukukhanya kwefluoresensi, iresorufin, malunga nomlinganiselo wesitokhiyometri omnye komnye. Iperoxide ekwisampulu iguquka ibe yifluoresensi kwisixhobo sokufumanisa. Ayikho enye into kwiqela eliqhelekileyo le-ROS eqhuba loo mpendulo ngokuthe ngqo.

Inzuzo ebalulekileyo kukulinganisa ngemigangatho. Umphandi osebenza ngononophelo uvavanya imigangatho yeperoxide eyaziwayo kwisithinteli sokutshintsha kwe-pH esinye, enoxinzelelo olufanayo lweperoxidase nesubstrate, ixesha elifanayo lokufukamela, noseto olufanayo lwezixhobo zokukhanya, aze atolike iisampulu ngokuthelekisa naloo grafu. Kuphononongo lweemitochondria ezahlulwe ebuchotsheni olusisiseko sale ndlela, ababhali benza kanye loo nto, befaka izixa ezaziwayo zeperoxide ekupheleni kovavanyo ngalunye, yaye amanani axelwa njengamazinga ngeepicomoles ngomzuzu ngemilligram yeprotheni kunokuba axelwe ngeeyunithi zefluoresensi ezingamiselwanga mgangatho. Yiloo nto eguqula ukukhazimla kube ngumlinganiselo.

Iinkcukacha ezintathu zolo phononongo zifanele ukuxeliswa. Okokuqala, ababhali baqinisekisa umqondiso ngecatalase, etshabalalisa iperoxide. Ukwanda kwefluoresensi okubangelwe yimethylene blue kwanqanda xa kufakwa icatalase, nto leyo eqinisekisa ukuba umqondiso wawuyiperoxide kungekhona impembelelo yobuxoki yokukhanya. Okwesibini, bavavanya ukukhanyisa ngokuthe ngqo ngokuvala umtha wokuvuselela ukuze iisampulu zifumane isinye kwisibhozo sedosi yokukhanya eqhelekileyo, yaye umqondiso wehla ngepesenti ezilithoba kuphela. Ngaphezu kwepesenti ezingamashumi alithoba zeperoxide abayixelayo zazingaxhomekekanga kukukhanya kokulinganisa. Okwesithathu, balinganisa ukususwa kweperoxide ngokwahlukeneyo nge-electrode baza bafumanisa ukuba idayi yayandisa ukuveliswa kwayo yaye icothisa ukususwa kwayo. Isikhombisi esixela kuphela ifluoresensi eseleyo besiya kuzidibanisa ezo mpembelelo zimbini.

Olu vavanyo lusenazo iindlela olunokusilela ngazo, yaye imethylene blue ibangela ezininzi zazo. Idayi ifunxa ukukhanya ngokubanzi kummandla omnye obomvu apho iresorufin ikhupha khona ukukhanya, ngoko xa uxinzelelo lwedayi luphezulu isampulu inokugquma umqondiso wayo ngenxa yeempembelelo zokuhluza ukukhanya ngaphakathi. Ngoko imigangatho kufuneka ibe noxinzelelo lwedayi olufanayo nolweesampulu. I-Amplex Red nayo inokwenza ioksideshini ebangelwa kukukhanya ngokwayo, yaye imveliso yayo, iresorufin, inokungena kumjikelo we-redox oqhutywa kukukhanya ovelisa isuperoxide neperoxide, nto leyo ebaluleke kakhulu xa uvavanyo lukwakhanyisa isivuseleli sokusabela ekukhanyeni. Isisombululo esisebenzayo kukukhanyisa iisampulu zemethylene blue kuqala uze uvavanye iinxalenye zazo ezilinganisiweyo ngeereyijenti ze-Amplex ebumnyameni, kunokukhanyisa idayi nesikhombisi kunye. Okokugqibela, i-superoxide dismutase ayinciphisi nje umqondiso we-Amplex: ukuguqula isuperoxide ibe yiperoxide kunokuwandisa. Isilawuli se-SOD esandisa ifluoresensi asisilelanga. Sichonge isuperoxide njengohlobo olwandulela iperoxide kwinkqubo.

I-DCFDA: isikhombisi seoksideshini esingajolisanga kuhlobo oluthile

Indibaniselwano yemethylene blue ne-DCFDA ingumthombo woninzi lokubhideka. I-DCFH-DA sisandulela esifakwa ngaphakathi kwiseli. Ii-esterase ezingaphakathi kwiseli zisusa amaqela ayo e-acetate zishiye i-DCFH engakhuphi fluoresensi, yaye ioksideshini ye-DCFH ivelisa i-DCF ekhupha fluoresensi. Izinto eziyenza ioksideshini zininzi: iiradikhali zehydroxyl, iimveliso eziphakathi zeperoxidase neheme, initrogen dioxide, ihypochlorous acid, neeradikhali ezivela kwiperoxynitrite. Ihydrogen peroxide iyodwa ayiyenzi ioksideshini ngokuthe ngqo nangaliphi na izinga elisebenzisekayo; ifluoresensi exhomekeke kwiperoxide idla ngokufuna intsimbi, iiprotheni zeheme, okanye ikhemistri yeperoxidase kwinkqubo.

Uhlalutyo olugxekayo ekufanele licatshulwe licandelo ngalinye leendlela zovavanyo luvela ekuhlalutyweni kwe-DCFH njengesikhombisi nguKalyanaraman noogxa bakhe, njengoko lushwankathelwe kuphononongo lweemitochondria zobuchopho olwayikhaba i-DCFDA ngenxa yesi sizathu. Babonisa ukuba isikhombisi sisabela kancinane, ngesitokhiyometri esingachazwanga, ngaphandle kokulinganisa ngemigangatho okusebenzayo, yaye sisabela neekhompawundi ezingeyiyo i-ROS. Isikhombisi sikwandisa oko sikulinganisayo: ioksideshini ye-DCFH ngokususwa kwe-elektroni enye ivelisa iradikhali ye-DCF esabela kwioksijini yenze isuperoxide, ethi iguquke nge-dismutation ibe yiperoxide eyongezelelekileyo. Ufundo ngokwalo luvelisa inxalenye yekhemistri oluyixelayo.

Akukho nanye kwezi zinto eyenza i-DCFDA ingabi namsebenzi. Ziyenza ibe luvavanyo lokuqala lokukhangela, kungekhona uvavanyo lokuchonga. Ukwanda kwefluoresensi ye-DCF kuthetha ukuba ioksideshini yesikhombisi exhomekeke kwizinto ezenza ioksideshini yandile. Akuthethi ukuba iperoxide yandile, yaye ngokuqinisekileyo akuthethi ukuba iyonke i-ROS yandile, umlinganiselo ekungekho sikhombisi sinye siwulinganisayo. Uphando lwangaphambili lwemethylene blue olwaluxhomekeke kwioksideshini ye-DCF ngaphezu kwemicromolar enye lwaxela iimpembelelo ezikhuthaza ioksideshini, ngoxa ezinye iinkqubo kwangelo xesha zaxela ukhuseleko ekufeni kweeseli okubangelwa yiperoxide. Zombini ezi zinto zinokubonwa kwidayi enye kwiimeko ezahlukeneyo ze-redox, yaye isiphumo se-DCF sisodwa asinakuxela ukuba yeyiphi into eyenza ioksideshini eyayithwele umqondiso. Okubalulekileyo ngophando lwangaphambili lweemitochondria zobuchopho kukuba lwathatha indawo yolu vavanyo lokukhangela lwasebenzisa umlinganiselo weperoxide olinganiswe ngemigangatho, lwaza lwafumanisa umthwalo ngokuthe ngqo: nedayi enoxinzelelo lweenanomolar ezilikhulu yandisa ukukhutshwa kweperoxide izihlandlo ezininzi kuzo zonke iisubstrate neemeko zokuphefumla ezavavanywayo.

I-DCFDA inomnye umgibe okhethekileyo kwimethylene blue. Idayi inombala onamandla kwaye iyasebenza kwi-redox, ngoko inokuphelisa ifluoresensi, icime ukukhutshwa kokukhanya, okanye yenze ioksideshini kwikhemistri yesikhombisi ngokuzimeleyo kwi-ROS yeeseli. Isilawuli esingenaziseli, esinedayi nesikhombisi kuphela, asiyonto yokuhombisa onokuyikhetha. Luvavanyo olukuxelela ukuba idayi iyasebenzisana na nesikhombisi.

Ukubanjwa kweeradikhali nokusetyenziswa kweoksijini: ukuchonga nezinga lokuhamba kwempendulo

Apho izikhombisi zefluoresensi zixela ukuba kuvele into enokwenziwa ioksideshini, i-electron spin resonance esebenzisa izibambi zeeradikhali inika ingxelo esondele ngakumbi ekuchongeni ukuba yayiyintoni. Uphononongo oludumileyo lwekhemistri yemethylene blue eqhutywa kukukhanya lwasebenzisa i-ESR ukufumanisa iradikhali ye-ascorbate evela kwidayi, kwi-ascorbate nasekukhanyeni, lwaza lwabonisa ukuveliswa kweperoxide kuvavanyo lokusetyenziswa kweoksijini nokuveliswa kweeradikhali zehydroxyl okukhuthazwa yintsimbi kuvavanyo lokubanjwa kweeradikhali. Indlela nganye yaphendula umbuzo ezinye ezazingenakuwuphendula: i-ESR yachonga iradikhali, ukusetyenziswa kweoksijini kwabonisa izinga lokuhamba kwempendulo elingaxhomekekanga nakwesiphi na isikhombisi sefluoresensi, yaye ukudityaniswa kwazo kwabonisa ukuba zombini indlela yeoksijini ye-singlet neendlela zeeradikhali zazisebenza kwinkqubo enye ekhanyisiweyo.

Eso siphumo sango-1984 sisafundeka njengeleyibhile yesilumkiso. Xa kukho ukukhanya nento eyenza iridakshini, idayi yavelisa iperoxide, yaye xa kukho intsimbi inkqubo yavelisa iradikhali yehydroxyl, eyona ntlobo isebenzayo kweli qela. Ababhali bakunxulumanisa oku ne-ascorbate esweni baza bakuthandabuza ukusetyenziswa kwayo emehlweni ngaphandle kwenkathalo. Abafundi banamhlanje bafanele baqaphele iimeko: ukukhanya kunye nento eyenza iridakshini kunye nentsimbi. Susa nayiphi na kwezi, ibe indlela iyancipha okanye iphele.

Ii-electrode zeoksijini zibaluleka ngendlela eyahlukileyo. I-electrode yohlobo lweClark ilinganisa ukusetyenziswa kweoksijini okushiyekileyo, nto leyo eqinisekisa ukuba ikhemistri ye-redox eqhutywa yidayi isebenzisa ioksijini yeemolekyuli, yaye inika iinkcukacha zesantya sempendulo ezingenakwenziwa bubuxoki befluoresensi. Ayichongi imveliso. Iperoxide, isuperoxide, amanzi nokuphela kweoksijini ye-singlet zonke zisebenzisa ioksijini. Izinga lokuhamba kwempendulo ngaphandle kokuchonga aligqibelelanga njengokuchonga ngaphandle kwezinga lokuhamba kwempendulo, yiyo loo nto amaphepha ophando omeleleyo edibanisa i-electrode novavanyo lweperoxide olulinganiswe ngemigangatho okanye isibambi seeradikhali.

Kutheni iziphumo zokuthintela ioksideshini ziphikisana

Beka ezi ndlela zintathu zokufunda ecaleni kwenye, yaye ukuphikisana okubonakala kuncwadi lophando lwemethylene blue kuchazeka ngomahluko kwiindlela kunye neenyaniso ezintathu zebhayoloji.

Okokuqala, idayi yindlela yokuhambisa iielektroni ikwangumthombo weperoxide. Ngokuphambukisa iielektroni kwiindawo zokuphefumla apho ioksijini ibiya kuguquka ibe yisuperoxide, inokunciphisa uhlobo olunye lwe-ROS ngoxa imo yayo eyenziwe iridakshini yenza iridakshini yeoksijini ngokuthe ngqo ibe yiperoxide, yandise olunye uhlobo. Ngoko ilebhu ejongene nekhemistri ye-redox yeemitochondria nendima yayo yokudlula kwezinye iindlela kufuneka ilindele iimpendulo ezixhomekeke kwisikhombisi: ufundo oluvakalelwa yisuperoxide luyehla ngoxa ufundo lweperoxide lunyuka kwicuvette enye.

Okwesibini, iperoxide kwiidosi ezisezantsi ngumqondiso, ayingomonakalo nje. Uphononongo lweemitochondria zobuchopho beempuku olwalandela uphando lwangaphambili lwafumanisa ukuveliswa kweperoxide ngedayi enoxinzelelo olusezantsi njengeenanomolar ezingamashumi amahlanu ngaphandle komonakalo kwi-DNA yeemitochondria kumanqanaba onyango, kunye nokukhuselwa kumonakalo obangelwa yirotenone emva kokunyangwa kwangaphambili. Kuphela kwiimicromolar ezilikhulu, ezingaphezulu kakhulu koluhlu lonyango, apho idayi yonakalisa iziqwenga ezithile ze-DNA, yaye ukukhanya okubomvu kunye nedayi enoxinzelelo lweemicromolar ezingamashumi amabini kwavelisa ioksijini ye-singlet eyonakalisa zonke iziqwenga ezavavanywayo. Indlela ecetywayo yeyehormesis: ukuveliswa kweperoxide okuncinci kuvuselela ukhuseleko lokuthintela ioksideshini olunxulumene ne-Nrf2 nokuveliswa kweemitochondria, ngoko ufundo lwexesha elifutshane olubonisa ukukhuthazwa kweoksideshini lwandulela isiphumo sexesha elide sokuthintela ioksideshini. Uvavanyo olufundwa emva kweyure enye nolufundwa emva kosuku olunye lunokungavumelani kuba ibhayoloji itshintshile phakathi kwaloo maxesha.

Okwesithathu, ukukhanya kugqiba nokuba yeyiphi imolekyuli ekhoyo. Ukukhanyisa kususa amaqela e-methyl kwidayi kuyiguqulele kwi-Azure B nakwiimveliso ezinxulumeneyo, yaye isibane semayikhroskophu esiphezu kwesitya sanele ukuyivuselela ukuba isabele ekukhanyeni. Uvavanyo olwenziwa ebumnyameni nolwenziwa ekukhanyeni ziimvavanyo ezahlukileyo, yaye icandelo lezilawuli zokukhanyisa nokuphatha idayi lalo naliphi na iprothokholi kufuneka liziphathe ngaloo ndlela.

Iseti esisiseko yezilawuli, nendlela yokutolika naliphi na ibango

Uthelekiso olungezantsi sisixhobo sokuqala sokubhekisa seli nqaku. Ngaphambi kokwamkela nasiphi na isiphumo se-ROS yemethylene blue, jonga ukuba ngowuphi umqolo ababhali abawuzalisileyo nabawutsibileyo.

Kulandela umgaqo osebenzayo wokutolika. I-Amplex ene-peroxidase, elinganiswe ngemigangatho, yaza yaqinisekiswa ngecatalase, ibubungqina malunga neperoxide. I-DCFDA ibubungqina malunga neoksideshini yesikhombisi engajolisanga kuhlobo oluthile, akukho ngaphezu koko. Ukubanjwa kweeradikhali ngezilawuli zezinto ezizibambayo kububungqina malunga nokuchongwa kweeradikhali. Ukusetyenziswa kweoksijini kububungqina malunga nezinga lokuhamba kwempendulo. Isivakalisi esibanga ngaphezu koko uvavanyo lwaso lunokukuxhasa, nokuba sibhalwe ngokuzithemba kangakanani, sisivakalisi esimalunga nesikhombisi, kungekhona idayi. Iingxoxo ezinxulumeneyo zokuxhomekeka kwidosi noluhlu lwehormesis, ukuhamba kwee-elektroni kwiimitochondria, ukuphazamiseka kovavanyo kwiisampulu ezifakwe idayi, neempembelelo zobuxoki zokukhanyisa kwimayikhroskophu zandisa nganye yezi ngongoma, yaye nganye ihlala isebenza xa isalathiso sayo esibekelwe indawo sitshintshwa ngesisigxina.