Imethylene blue, i-Nrf2, nokwenziwa kweemitochondria: oko kwalinganiswa noko kusacetywayo

Isivakalisi esinye sivela rhoqo kwimibhalo ephathelele imethylene blue: ivuselela i-Nrf2 ize yakhe iimitochondria ezintsha. Esi sivakalisi sifundeka njengebango elinye, kodwa sineengcamango ezimbini ezahlukeneyo, eziphononongwe kwiinkqubo ezahlukeneyo, kusetyenziswa iintlobo ezahlukeneyo zobungqina. Eyokuqala yindlela yokudlulisa imiqondiso, isivamvo esivula imizila yemfuza yokhuselo. Eyesibini yinkqubo yokwakha, apho iseli yenza izixhobo ezininzi ngakumbi zeemitochondria. Umqondiso unokuqalisa ulwakhiwo, kodwa ukubonisa ukuba umqondiso uthunyelwe akufani nokubonisa ukuba isakhiwo sakhiwe, yaye ukubonisa izinto zokwakha ezininzi ngakumbi akufani nokubonisa unikezelo lwamandla olungcono. Eli nqaku ligcina ezo zinto zintathu zahlukene.

Ingcamango ye-Nrf2: sisivamvo, hayi ikhaka

I-Nrf2 (nuclear factor erythroid 2-related factor 2) yiprotheyini elawula ukukhutshelwa kolwazi lwemfuza, umsebenzi wayo ikukuvula imizila yemfuza. Phantsi kweemeko eziqhelekileyo ibanjwa kwicytoplasm yiprotheyini esisivamvo ebizwa ngokuba yi-Keap1, eyinikezela kwizixhobo zeseli zokulahla izinto ukuze isoloko itshatyalaliswa. Xa izinto ezibangela ioksideshini okanye ii-electrophile ziguqula ii-cysteine ezisabela lula ezikwi-Keap1, oko kutshatyalaliswa kuyayeka. I-Nrf2 esandul’ ukwenziwa iyaqokelelana, ingene kwinucleus, idibane neeprotheyini ezincinci ze-Maf, ize ibopheleleke kulandelelwano lwe-DNA olubizwa ngokuba ziziqalelo zempendulo yokhuselo kwioksideshini. Oko kubopheleleka kuvula uthotho lwemizila yemfuza yokhuselo.

Izalathisi eziqhelekileyo ezilinganiswa kule ndlela zizinto ezithile. I-NQO1 (NAD(P)H quinone dehydrogenase 1) kunye ne-TrxR1 (thioredoxin reductase 1) ziphakathi kweyona mizila yemfuza ithembekileyo ekujoliswe kuyo yi-Nrf2. I-HO-1 (heme oxygenase-1) ivuseleleka kakhulu, kodwa ilawulwa nangabanye abalawuli. Ii-enzyme ezifana ne-SOD kunye ne-catalase zizixhobo zokhuselo kwioksideshini, kodwa amanqanaba azo alawulwa ziiprotheyini ezininzi ezilawula ukukhutshelwa kolwazi lwemfuza, ngoko ukunyuka kwe-SOD kukodwa akungqini ukuba i-Nrf2 ibingunobangela. Obona bungqina busondeleyo kwisiganeko sokudlulisa umqondiso kukuqokelelana kwe-Nrf2 kwinucleus, ukuvuselelwa kwesalathisi se-ARE, okanye ukuvuselelwa kwemizila yemfuza eqhelekileyo ekujoliswe kuyo okunyamalalayo xa i-Nrf2 isusiwe. Yonke enye into, amanqanaba asezantsi ezinto ezibangela ioksideshini, ukusinda okungcono phantsi koxinzelelo, i-glutathione ephezulu, ziziphumo ezilandelayo ezinokuveliswa ziindlela ezininzi.

Isiphumo sokuqala semethylene blue kule ngxelo siwela kufutshane nelo cala lobungqina. Ii-fibroblast ezichanabeke kwimethylene blue ekumanqanaba e-nanomolar zabonisa ukulibaziseka kokuguga kweeseli kunye nobuninzi obuphezulu be-complex IV, ukusetyenziswa okuphezulu kweoksijini, nokuvuselelwa kwee-enzyme zesigaba-2, i-TrxR1 kunye ne-NQO1, njengoko kwaxelwa kuphononongo lokuqala olwanxulumanisa imethylene blue nokuphuculwa kweemitochondria nokulibaziseka kokuguga kweeseli. I-TrxR1 kunye ne-NQO1 kanye yimizila yemfuza eqhelekileyo ekujoliswe kuyo yi-Nrf2 umfundi oqwalaselayo afuna ukuyibona. Into olungayibonisanga uphononongo sisiganeko sokudlulisa umqondiso ngokwaso: kwakungekho mlinganiselo we-Nrf2 kwinucleus, kungekho salathisi se-ARE, kungekho vavanyo lothelekiso apho i-Nrf2 isuswe ngokupheleleyo. Ngoko ukufundwa okunyanisekileyo kukuba imethylene blue ivuselela imizila yemfuza eyaziwayo ekujoliswe kuyo yi-Nrf2 kule nkqubo, nto leyo ehambelana nokuvuselelwa kwe-Nrf2 kodwa engayibeki yodwa i-Nrf2 njengonobangela. Ukuvuselelwa komzila wemfuza ekujoliswe kuwo kulinyathelo elinye emva kwesivuli.

Ingcamango yokwenziwa kweemitochondria: inkqubo enamanqanaba amathathu okuhlola

Ukwenziwa kweemitochondria yinkqubo iseli eyandisa ngayo amandla ayo e-mitochondria. Ulandelelwano lwayo oluchazwa kwiincwadi zezifundo ludlula kumanqanaba amathathu okuhlola. Okokuqala, umncedisi wokuvuselela i-PGC-1alpha uvula inkqubo. Okwesibini, iiprotheyini ezikwinucleus ezilawula ukukhutshelwa kolwazi lwemfuza (okudidayo kukuba enye ibizwa ngokuba yi-NRF1, nuclear respiratory factor 1, iprotheyini eyahlukileyo kwi-Nrf2) ziqhuba ukubonakaliswa kwemizila yemfuza ye-mitochondria, kuquka i-TFAM, egcina ize iphindaphinde i-DNA ye-mitochondria. Okwesithathu, iseli iphela ineejenom ze-mitochondria ezininzi ngakumbi kunye nezixhobo ezininzi ngakumbi zokuphefumla kweseli, ezinokulinganiswa ngenani leekopi ze-mtDNA, ngomsebenzi we-citrate synthase, okanye ngobuninzi nomsebenzi we-complex zokuphefumla kweseli.

Inqanaba ngalinye lokuhlola lixhasa ibango elibuthathaka okanye elomeleleyo kunelangaphambili. Amanqanaba aphezulu e-RNA yesithunywa ye-PGC-1alpha okanye ye-TFAM abonisa ukuba inkqubo iceliwe, hayi ukuba igqityiwe. I-mtDNA eninzi ngakumbi okanye iprotheyini ye-complex IV eninzi ngakumbi ibonisa izinto ezininzi ngakumbi, kodwa iimitochondria ezihlengahlengisiweyo zinokubonakala ngendlela efanayo. Ukuphefumla kweseli okungcono, ukusetyenziswa okuphezulu kweoksijini okanye umsebenzi ophezulu we-complex, kubonisa ukuba umsebenzi utshintshile, kodwa umsebenzi unokuphucuka ngaphandle kokuba kubekho iimitochondria ezintsha kwaphela. Ibango elomeleleyo lokwenziwa kweemitochondria lidibanisa zonke zontathu: umlawuli, ubuninzi bezinto, nomsebenzi.

Uphononongo olulandelayo lwee-fibroblast lwadlula phantse kulo lonke olo landelelwano. Kwiiseli zabantu ze-IMR90 ezanyangwa nge-100 nanomolar yemethylene blue, umlinganiselo we-NAD ukuya kwi-NADH wenyuka okwethutyana kwimizuzu eli-15 waza wabuyela kwinqanaba lokuqala kwisithuba sosuku. Loo mqondiso wamandla wethutyana walandelwa kukufakwa kwephosphate okwethutyana kwisivamvo samandla i-AMPK, kwaza kwalandela ukuvuselelwa kwe-PGC-1alpha kunye ne-SURF1, iprotheyini efunekayo ukuze kudityaniswe i-complex IV esebenzayo. Umsebenzi we-complex IV waphantse waphindeka kabini, izinto ezibangela ioksideshini kwiiseli zehla malunga nama-28 ekhulwini, yaye ii-telomere zancipha ngokucothayo ngakumbi. Ulandelelwano olupheleleyo luchazwe kwiphepha elinxulumanisa umsebenzi wemethylene blue wokunciphisa noweoksideshini ne-AMPK nokwenziwa kweemitochondria.

Iinkcukacha ezimbini kwelo phepha zifuna ukuqwalaselwa kuba ziluhlobo lweenkcukacha ezidla ngokushiywa ngaphandle. Okokuqala, indlela okwenzeka ngayo yayibaluleke ngaphezu kobukho bayo: imethylene blue yavula i-AMPK okwethutyana, ngoxa iyeza lothelekiso i-AICAR layivula ngokuqhubekayo, yaye into eyayivuselela okwethutyana yalibazisa ukuguga kweeseli malunga namaxesha asibhozo ngempumelelo engaphezulu. Ibango elithi “imethylene blue ivuselela i-AMPK” ke ngoko aliphelelanga; ibango elichanekileyo lelokuba ivuselela i-AMPK okwethutyana, yaye uthelekiso lubonisa ukuba ukuvuselela okuqhubekayo akwenzi umsebenzi ofanayo. Okwesibini, imethylene blue ayizange iguqule umjikelo weseli, nto leyo esusa eyona ngcaciso ilula engenye: ukuba iiseli zazisahluleka nje ngokucothayo ngakumbi zaza ngenxa yoko zabonakala ziselula.

Icandelo leempuku: indlela yokuziphatha, iijenom, nento evuselelayo ephikisana ngabom noko kulindelekileyo

Umsebenzi wokukhulisa iiseli elabhoratri wandiselwa kwizilwanyana liqela elaliphonononga iimpuku ezikwiminyaka ephakathi. Emva kweentsuku ezingama-60 zokunikwa imethylene blue ngomlomo, iimpuku ezanyangwayo zagcina unyamezelo lwazo lomzimba nokuziphatha kokuhlola ngakumbi, zaza zabonisa ukuziphatha okufana nexhala okuncinane kuneempuku ezindala ezinganyangwanga. Kwizicubu zobuchopho, imizila yemfuza eyayicinezelwe kukuguga, i-NRF1, i-TFAM, i-SOD2, kunye necandelo le-complex IV eliyi-COX1, yabuyiselwa kufutshane namanqanaba eempuku ezikhule ngokupheleleyo, yaye inani leekopi ze-mtDNA lenyuka. Ezo ziphumo, indlela yokuziphatha kunye nomlawuli kunye nobuninzi beejenom, zixelwe kuphononongo lweempuku ezikwiminyaka ephakathi ngemethylene blue nokwenziwa kweemitochondria zobuchopho.

Into ekucetywa ukuba iqalise le nkqubo yeyona inika umdla, kuba iphikisana nengcaciso elula yokuba imethylene blue yinto ekhusela kwioksideshini. Iimpuku ezanyangwayo zabonisa ukuveliswa okuphezulu kweentlobo zeoksijini ezisabela lula kwiimitochondria zobuchopho, ngokukodwa i-hydrogen peroxide. Umbono wababhali wawusithi: ngumqondiso omncinane weperoxide ovuselela indlela ye-Nrf2, ethi emva koko iqhube inkqubo yokwenziwa kweemitochondria. Kule modeli imethylene blue ikhuthaza ioksideshini kwinyathelo lokuqalisa ize ikhusele kwioksideshini kwinyathelo lesiphumo, indlela iingcali zamayeza eziyibiza ngokuba yi-hormesis, apho uxinzelelo oluncinane luvusa ukhuseleko olukhulu kunoxinzelelo ngokwalo. Ukuba oko kunyuka kweperoxide kubangela ngokwenene ukuvuselelwa kwe-Nrf2 akuzange kuvavanywe ngokuthe ngqo; lunxibelelwano olucetywayo, hayi olulinganisiweyo.

Uphononongo oluhambelanayo oluvela kwakwelo qela lwavavanya ngokungqongqo icala lokhuseleko lalo mbono. Kwiimitochondria zobuchopho ezahlulweyo, imethylene blue yandisa ukuveliswa kwe-hydrogen peroxide kumanqanaba asezantsi njenge-50 nanomolar, kodwa amanqanaba asetyenziswa kunyango awabangela monakalo ubonakalayo kwi-DNA ye-mitochondria. Kuphela kwi-100 micromolar, ekude kakhulu ngaphezu kwamanqanaba onyango, apho amaqhekeza athile e-mtDNA abonisa umonakalo, yaye ukudityaniswa kwedayi eyi-20 micromolar nokukhanya okubomvu kwavelisa i-singlet oxygen eyonakalisa i-mtDNA ngokubanzi, nto leyo eyindlela eyaziwayo idayi esebenza ngayo njengento evuselelwa kukukhanya kunokuba ibe yindlela yayo yokusebenza ngaphandle kokukhanya. Ukunyangwa kwangaphambili kwade kwakhusela iimitochondria ezikwicala elisezantsi lobuchopho obuphakathi kumonakalo we-mtDNA obangelwa yi-rotenone. Indlela impendulo etshintsha ngayo ngokwedosi kunye neemvavanyo zothelekiso zomonakalo kwimfuza zichazwe ngokweenkcukacha kuphononongo lokuveliswa kweperoxide yimethylene blue ngaphandle kokonakalisa imfuza.

Okulinganisiweyo xa kuthelekiswa nokucetywayo: ukufunda umzobo

Umzobo wendlela engezantsi wazotyelwa eli nqaku ukuze ucacise umahluko obusenziwa sisicatshulwa. Iibhokisi ezinemida eqinileyo ngamanyathelo anenkxaso yovavanyo oluthe ngqo kwimibhalo yophando ngemethylene blue: umjikelo wokunciphisa noweoksideshini oguqula i-NAD/NADH, ukufakwa kwephosphate okwethutyana kwi-AMPK, ukuvuselelwa kwe-PGC-1alpha kunye ne-SURF1, umsebenzi ophezulu we-complex IV, ukuvuselelwa kwe-NQO1 kunye ne-TrxR1, ukubuyiselwa kokubonakaliswa kwemizila yemfuza yokwenziwa kweemitochondria nenani leekopi ze-mtDNA kubuchopho beempuku, nokunyuka kweperoxide ngaphandle komonakalo we-mtDNA kwiidosi zonyango. Iibhokisi ezinemida eyenziwe yimigca eqhawukileyo lunxibelelwano olucetywayo: i-Keap1 ebona umqondiso weperoxide, ukuqokelelana kwe-Nrf2 kwinucleus okuqhuba imizila yemfuza yokhuselo, kunye notolo olubonisa unobangela osuka ekuvuselelweni kwe-Nrf2 uye kwinkqubo yokwenziwa kweemitochondria. Loo manyathelo anemigca eqhawukileyo anokukholeleka, ayahambelana nedatha, yaye ayibhayoloji eqhelekileyo kwiincwadi zezifundo, kodwa kula maphepha awazange alinganiswe kusetyenziswa iimvavanyo zothelekiso (i-Nrf2 kwinucleus, izalathisi ze-ARE, ukususwa ngokupheleleyo kwe-Nrf2) ebezinokuwenza abe ziibhokisi ezinemida eqinileyo.

Ukudideka okuthathu kuyacaca xa umzobo uphambi kwakho. Okokuqala, i-NRF1 kunye ne-Nrf2 ziiprotheyini ezahlukeneyo ezinamagama aphantse afane; uphononongo lweempuku lwalinganisa i-nuclear respiratory factor 1, umlawuli wokwenziwa kweemitochondria, hayi iprotheyini elawula ukukhutshelwa kolwazi lwemfuza yokhuselo kwioksideshini, yaye ukuzixuba kudibanisa wonke umzobo ube yindawo enye. Okwesibini, ukubonakaliswa kwemizila yemfuza sisiqalo sebali, hayi isiphelo; imiyalezo ye-TFAM okanye ye-COX1 ebuyiselweyo ixhasa isicelo sokwenziwa kweemitochondria, ngoxa inani leekopi ze-mtDNA nomsebenzi we-complex IV zibubungqina bokuba isicelo sizalisekisiwe. Okwesithathu, isiphumo sokhuselo kwioksideshini asiyingqini indlela ye-Nrf2; ukwehla kwezinto ezibangela ioksideshini ngama-28 ekhulwini kwii-fibroblast kunye nokhuselo kwi-rotenone kwiimpuku ziziphumo ezilandelayo zokwenene, kodwa iindlela ezininzi ziyahlangana kuzo.

Yintoni enokuguqula ibhokisi enemigca eqhawukileyo ibe neyomda oqinileyo

Iimvavanyo ezingekhoyo zeziqhelekileyo. Ukulinganisa iprotheyini ye-Nrf2 kwinucleus emva kokuchanabeka kwimethylene blue, kulandelwa amaxesha ahambelana nomqondiso wethutyana we-AMPK, bekuya kuvavanya ukuba omabini amasebe ayavuseleleka ngokwenene kunye na, njengoko ingcinga yokuvuselelwa ngaxeshanye isitsho. Isalathisi esiqhutywa yi-ARE besiya kuqinisekisa umsebenzi wokukhutshelwa kolwazi lwemfuza kunokuba uqikelelwe ngokujonga imizila yemfuza ekujoliswe kuyo. Ukuphinda kulinganiswe izalathisi zokwenziwa kweemitochondria xa i-Nrf2 incitshisiwe okanye isusiwe ngokupheleleyo bekuya kuvavanya utolo lonobangela ngokuthe ngqo: ukuba ukuvuselelwa kwe-complex IV nokwanda kwe-mtDNA kuqhubeka ngaphandle kwe-Nrf2, olo nxibelelwano aluchanekanga yaye lisebe le-AMPK lodwa elibangela isiphumo. Kwicala lezilwanyana, uphononongo lokunikwa kweedosi ixesha elide olunamanqanaba amathathu afanayo okuhlola, umlawuli, ubuninzi bezinto, nomsebenzi, beluya kuvavanya ukuba inkqubo ihlala ivuliwe okanye iyaziqhelanisa. Kude kube ngoko, ukuvuselelwa ngaxeshanye kweendlela zamandla nezokhuselo kuhlala kuyile nto ababhali bako babeyibiza ngayo: into ephambili enokuba negalelo, hayi indlela yokusebenza eqinisekisiweyo.

Abafundi abavela kuphando olubanzi lwendlela amayeza asebenza ngayo basenokufuna umxholo ojikelezileyo: indlela imethylene blue ehambisa ngayo ii-electron kwinqanaba le-mitochondria, ezinye izinto ezenzayo kwiimitochondria nakumandla eeseli, indawo yeziphumo zokuguga kweeseli kuphando lokuguga, indlela amabango okwenziwa kweemitochondria anxulumana ngayo nokusebenza kwengqondo, kunye nengxelo yokhuseleko emisela imida yayo yonke ingxoxo yeedosi. Nganye kwezo yingcamango eyahlukileyo enobungqina bayo, yaye nganye ifanele ukwahlulelwa ngendlela efanayo phakathi kokulinganisiweyo nokucetywayo eli nqaku liyisebenzisileyo kwi-Nrf2.