Ukuchazwa kwe-methylene blue, i-Nrf2, nokwakheka kwama-mitochondria amasha

Umusho owodwa uvame ukuvela emibhalweni nge-methylene blue: ivuselela i-Nrf2 futhi yakhe ama-mitochondria amasha. Lo musho uzwakala njengesimangalo esisodwa, kodwa uqukethe imiqondo emibili ehlukene, efundwe ezinhlelweni ezahlukene, ngezinhlobo ezahlukene zobufakazi. Owokuqala uyindlela yokuthumela izimpawu, inzwa evula izakhi zofuzo zokuzivikela. Owesibili uwuhlelo lokwakha, lapho ingqamuzana lenza izingxenye ezengeziwe zemishini yama-mitochondria. Isimpawu singaqalisa ukwakha, kodwa ukukhombisa ukuthi isimpawu sisebenzile akufani nokukhombisa ukuthi isakhiwo sakhiwe, futhi ukukhombisa izinto zokwakha ezengeziwe akufani nokukhombisa ukunikezwa kwamandla okungcono. Lesi sihloko sihlukanisa lezo zinto ezintathu.

Umqondo we-Nrf2: inzwa, hhayi isihlangu

I-Nrf2 (nuclear factor erythroid 2-related factor 2) iyisici sokulawula ukubhalwa kolwazi lwezakhi zofuzo, iphrotheni emsebenzi wayo ukuvula izakhi zofuzo. Ezimweni zokuphumula ibanjwa ku-cytoplasm yiphrotheni eyinzwa ebizwa nge-Keap1, eyidlulisela emishinini yengqamuzana yokulahla izinto ukuze ihlale ibhujiswa. Lapho ama-oxidant noma ama-electrophile eguqula ama-cysteine asebenzayo aku-Keap1, lokhu kulahlwa kuyama. I-Nrf2 esanda kwakhiwa iyanqwabelana, ingene ku-nucleus, ihlangane namaphrotheni amancane e-Maf, bese ibopha ukulandelana kwe-DNA okubizwa ngezindawo zokusabela kuma-antioxidant. Lokho kubopha kuvula iqoqo lezakhi zofuzo zokuzivikela.

Izinkomba ezijwayelekile zale ndlela ziqondile. I-NQO1 (NAD(P)H quinone dehydrogenase 1) ne-TrxR1 (thioredoxin reductase 1) ziphakathi kwezakhi zofuzo ezihlosiwe ze-Nrf2 ezinokwethenjelwa kakhulu. I-HO-1 (heme oxygenase-1) ingavuselelwa kakhulu, kodwa futhi isabela nakwezinye izilawuli. Ama-enzyme afana ne-SOD ne-catalase ayizingxenye zemishini yokulwa nama-oxidant, kodwa amazinga awo alawulwa yizici eziningi zokulawula ukubhalwa kolwazi lwezakhi zofuzo, ngakho ukwenyuka kwe-SOD kukodwa akufakazeli ukuthi i-Nrf2 iyona eyimbangela. Ubufakazi obusondele kakhulu ekuthunyelweni kwesimpawu ukunqwabelana kwe-Nrf2 ku-nucleus, ukusebenza kwenkomba yokuhlola ye-ARE, noma ukuvuselelwa kwezakhi ezihlosiwe ezijwayelekile okuphelayo lapho i-Nrf2 isuswa. Konke okunye, amazinga aphansi ama-oxidant, ukusinda okungcono ngaphansi kwengcindezi, i-glutathione ephakeme, kuyimiphumela elandelayo engakhiqizwa yizindlela eziningi.

Umphumela wokuqala we-methylene blue kule ndaba usondela kakhulu kuleso sigaba sobufakazi. Ama-fibroblast achayeke ku-methylene blue esezingeni lama-nanomolar abonisa ukubambezeleka kokuguga kwamangqamuzana, kanye nokuqukethwe okuphezulu kwe-complex IV, ukusetshenziswa komoya-mpilo okuphezulu, nokuvuselelwa kwama-enzyme esigaba-2 i-TrxR1 ne-NQO1, njengoba kubikiwe ocwaningweni olwaqala ukuxhumanisa i-methylene blue nokuthuthukiswa kwama-mitochondria nokubambezeleka kokuguga kwamangqamuzana. I-TrxR1 ne-NQO1 yizona kanye izakhi ezihlosiwe ezijwayelekile ze-Nrf2 umfundi oqaphelayo afuna ukuzibona. Okungakhonjiswanga ucwaningo ukuthunyelwa kwesimpawu uqobo: kwakungekho ukulinganiswa kwe-Nrf2 ku-nucleus, kungekho nkomba yokuhlola ye-ARE, futhi kungekho ukuhlolwa kokuqhathanisa lapho i-Nrf2 ikhishwe khona ngokuphelele. Ngakho ukufunda lokhu ngokwethembeka kusho ukuthi i-methylene blue ivuselela izakhi zofuzo ezaziwayo ezihloswe yi-Nrf2 kulolu hlelo, okuhambisana nokuvuselelwa kwe-Nrf2 kodwa okungayihlukanisi i-Nrf2 njengembangela. Ukuvuselelwa kwesakhi sofuzo esihlosiwe kuyisinyathelo esisodwa esilandela ukuvulwa kweswishi.

Umqondo wokwakheka kwama-mitochondria amasha: uhlelo olunamaphuzu amathathu okuhlola

Ukwakheka kwama-mitochondria amasha kuwuhlelo ingqamuzana elandisa ngalo amandla alo okusebenza ngama-mitochondria. Ukulandelana kwalo okuchazwa ezincwadini zokufunda kudlula emaphuzwini amathathu okuhlola. Okokuqala, isivuseleli esisebenzisana nezinye, i-PGC-1alpha, sivula uhlelo. Okwesibili, izici eziku-nucleus ezilawula ukubhalwa kolwazi lwezakhi zofuzo (okudidayo ukuthi esinye sibizwa nge-NRF1, nuclear respiratory factor 1, okuyiphrotheni ehlukile ku-Nrf2) ziqhuba ukuvezwa kwezakhi zofuzo zama-mitochondria, okuhlanganisa i-TFAM, egcina futhi ikopishe i-DNA yama-mitochondria. Okwesithathu, ingqamuzana ligcina selinama-genome amaningi ama-mitochondria kanye nemishini eyengeziwe yokuphefumula kwamangqamuzana, okungalinganiswa ngenani lamakhophi e-mtDNA, ukusebenza kwe-citrate synthase, noma ngobuningi nangokusebenza kwama-complex okuphefumula kwamangqamuzana.

Iphuzu ngalinye lokuhlola linikeza isimangalo esibuthakathaka noma esiqinile kunesangaphambili. Amazinga aphezulu e-RNA yesithunywa ye-PGC-1alpha noma ye-TFAM akhombisa ukuthi uhlelo luceliwe, hhayi ukuthi luqediwe. I-mtDNA eningi noma iphrotheni ye-complex IV eningi ikhombisa izinto ezengeziwe, kodwa ama-mitochondria ahlelwe kabusha nawo angabonakala ngendlela efanayo. Ukuphefumula kwamangqamuzana okungcono, ukusetshenziswa komoya-mpilo okuphezulu noma ukusebenza okuphezulu kwama-complex, kukhombisa ukuthi ukusebenza kushintshile, kodwa ukusebenza kungathuthuka ngaphandle kwama-mitochondria amasha nhlobo. Isimangalo esiqinile sokwakheka kwama-mitochondria amasha sihlanganisa konke kokuthathu: isilawuli, okuqukethwe, nokusebenza.

Ucwaningo olulandelayo lwama-fibroblast lwalandela ingxenye enkulu yalolo chungechunge. Emangqamuzaneni abantu e-IMR90 afakwe i-methylene blue engu-100 nanomolar, isilinganiso se-NAD ku-NADH senyuka okwesikhashana phakathi kwemizuzu engu-15 sabuyela ezingeni lokuqala kungakapheli usuku. Leso simpawu samandla sesikhashana salandelwa ukufakwa kweqembu le-phosphate okwesikhashana enzweni yamandla i-AMPK, kwase kulandela ukuvuselelwa kwe-PGC-1alpha ne-SURF1, iphrotheni edingekayo ukuze kuhlanganiswe i-complex IV esebenzayo. Ukusebenza kwe-complex IV kwacishe kwaphindeka kabili, ama-oxidant angaphakathi kwamangqamuzana ehla cishe ngamaphesenti angu-28, futhi ama-telomere ancipha kancane. Ukulandelana okuphelele kuchazwe ephepheni elixhumanisa ukusebenza kwe-redox kwe-methylene blue ne-AMPK nokwakheka kwama-mitochondria amasha.

Imininingwane emibili kulelo phepha idinga ukunakwa ngoba iwuhlobo lwemininingwane evame ukushiywa ngaphandle. Okokuqala, iphethini yayibaluleke ngaphezu kokuba khona kokuvuselelwa: i-methylene blue yavula i-AMPK okwesikhashana, kuyilapho umuthi wokuqhathanisa i-AICAR wayivula ngokuqhubekayo, futhi isivuseleli sesikhashana sabambezela ukuguga kwamangqamuzana ngempumelelo ecishe ibe kasishiyagalombili. Ngakho isimangalo esithi "i-methylene blue ivuselela i-AMPK" asiphelele; isimangalo esibalulekile ukuthi iyivuselela okwesikhashana, futhi ukuqhathanisa kusikisela ukuthi ukuvuselelwa okuqhubekayo akwenzi umsebenzi ofanayo. Okwesibili, i-methylene blue ayizange iguqule umjikelezo wengqamuzana, okususa enye incazelo elula kakhulu: ukuthi amangqamuzana ayevele ehlukana kancane futhi ngenxa yalokho abukeka esemasha.

Igatsha lamagundane: ukuziphatha, ama-genome, nesiqalisi esiphikisanayo ngamabomu

Umsebenzi wokukhulisa amangqamuzana wanwetshwa waya ezilwaneni yiqembu elalifunda amagundane asebudaleni obuphakathi. Ngemva kwezinsuku ezingu-60 enikezwa i-methylene blue ngomlomo, amagundane ayeyitholile alondoloza ukukhuthazela kwawo ngokomzimba nokuziphatha kwawo kokuhlola indawo ngezinga elikhulu, futhi abonisa ukuziphatha okuncane okufana nokukhathazeka kunamagundane asekhulile ayengayinikwanga. Ezicutshini zobuchopho, izakhi zofuzo ezazicindezelwe ukuguga, i-NRF1, i-TFAM, i-SOD2, nengxenye ye-complex IV i-COX1, zabuyiselwa ngas emazingeni obudala obuvuthiwe, futhi inani lamakhophi e-mtDNA lenyuka. Leyo miphumela, ukuziphatha kanye nesilawuli kanye nobuningi bama-genome, ibikiwe ocwaningweni lwamagundane asebudaleni obuphakathi nge-methylene blue nokwakheka kwama-mitochondria amasha ebuchosheni.

Isiqalisi esihlongozwayo yisona esithakazelisayo, ngoba siphikisana nendaba elula yokuthi i-methylene blue iyi-antioxidant. Amagundane ayeyinikiwe abonisa ukukhiqizwa okuphezulu kwezinhlobo zomoya-mpilo ezisebenzayo kuma-mitochondria obuchopho, ikakhulukazi i-hydrogen peroxide. Umbono wababhali: isimpawu esincane se-peroxide yiso esivuselela indlela ye-Nrf2, yona bese iqhuba uhlelo lokwakheka kwama-mitochondria amasha. Kule modeli i-methylene blue ikhuthaza i-oxidation esinyathelweni sokuqalisa futhi ilwa nama-oxidant esinyathelweni somphumela, iphethini ochwepheshe bemithi abayibiza nge-hormesis, lapho ingcindezi encane ivusa ukuzivikela okukhulu kunengcindezi uqobo. Ukuthi lokho kwenyuka kwe-peroxide kuyimbangela ngempela yokuvuselelwa kwe-Nrf2 akuzange kuhlolwe ngokuqondile; kuwukuxhumana okuhlongozwayo, hhayi ukuxhumana okulinganisiwe.

Ucwaningo oluhambisana nalolu oluvela eqenjini elifanayo lwahlola ngokuqinile uhlangothi lokuphepha lwalowo mbono. Kuma-mitochondria obuchopho ahlukanisiwe, i-methylene blue yenyusa ukukhiqizwa kwe-hydrogen peroxide emazingeni okujiya aphansi afinyelela ku-50 nanomolar, kodwa amazinga okujiya asetshenziswa ekwelapheni awazange abangele umonakalo otholakalayo ku-DNA yama-mitochondria. Kuphela ku-100 micromolar, okungaphezu kakhulu kwamazinga okwelapha, lapho izingcezu ezithile ze-mtDNA zabonisa umonakalo, futhi ukuhlanganiswa kodayi ongu-20 micromolar nokukhanya okubomvu kwakhiqiza umoya-mpilo we-singlet owonakalisa i-mtDNA kabanzi, okuwukuziphatha okwaziwayo kukadayi okwenza izinto zizwele ekukhanyeni, kunokuba kube ukusebenza kwawo kwemithi lapho kungekho ukukhanya. Ukusetshenziswa kwawo ngaphambi kokuchayeka kwaze kwavikela ama-mitochondria asengxenyeni engezansi yobuchopho obuphakathi ekulimaleni kwe-mtDNA okubangelwa i-rotenone. Ukusabela ngokwezinga lomthamo nokuhlolwa kokuqhathanisa kokulimala kwezakhi zofuzo kuchazwe ocwaningweni ngokukhiqizwa kwe-peroxide yi-methylene blue ngaphandle kokulimaza izakhi zofuzo.

Okulinganisiwe nokuhlongozwayo: ukufunda umdwebo

Umdwebo wendlela ongezansi wenzelwe lesi sihloko ukuze ugcizelele umehluko umbhalo obulokhu uwenza. Amabhokisi anemingcele yemigqa eqhubekayo ayizinyathelo ezisekelwa ngokuqondile ukuhlolwa emibhalweni yocwaningo nge-methylene blue: ukujikeleza kwe-redox okushintsha i-NAD/NADH, ukufakwa kweqembu le-phosphate ku-AMPK okwesikhashana, ukuvuselelwa kwe-PGC-1alpha ne-SURF1, ukusebenza okuphezulu kwe-complex IV, ukuvuselelwa kwe-NQO1 ne-TrxR1, ukubuyiselwa kokuvezwa kwezakhi zofuzo zokwakheka kwama-mitochondria amasha nenani lamakhophi e-mtDNA ebuchosheni bamagundane, kanye nokwenyuka kwe-peroxide ngaphandle kokulimala kwe-mtDNA ngemithamo yokwelapha. Amabhokisi anemingcele yemigqa edwetshwe izikhala ayizixhumanisi ezihlongozwayo: ukuzwa kwe-Keap1 isimpawu se-peroxide, ukunqwabelana kwe-Nrf2 ku-nucleus okuqhuba izakhi zofuzo zokuzivikela, nomcibisholo wembangela osuka ekuvuselelweni kwe-Nrf2 uye ohlelweni lokwakheka kwama-mitochondria amasha. Lezo zinyathelo ezinemigqa enezikhala ziyakholeka, zihambisana nedatha, futhi ziyi-biology ejwayelekile yezincwadi zokufunda, kodwa kula maphepha azizange zilinganiswe ngezivivinyo zokuqhathanisa (i-Nrf2 ku-nucleus, izinkomba zokuhlola ze-ARE, ukukhishwa ngokuphelele kwe-Nrf2) ebezingazikhuphulela emabhokisini anemingcele eqhubekayo.

Izinto ezintathu ezididayo ziyabonakala lapho umdwebo usuphambi kwakho. Okokuqala, i-NRF1 ne-Nrf2 kungamaphrotheni ahlukene anamagama acishe afane; ucwaningo lwamagundane lwalinganisa i-nuclear respiratory factor 1, isilawuli sokwakheka kwama-mitochondria amasha, hhayi isici sokulawula ukubhalwa kolwazi lwezakhi zofuzo esilwa nama-oxidant, futhi ukuwaxuba kwenza wonke umdwebo ube yiphuzu elilodwa. Okwesibili, ukuvezwa kwezakhi zofuzo kuyisiqalo sendaba, hhayi ukuphela kwayo; ukubuyiselwa kwemiyalezo ye-TFAM noma ye-COX1 kusekela isicelo sokwakheka kwama-mitochondria amasha, kuyilapho inani lamakhophi e-mtDNA nokusebenza kwe-complex IV kuwubufakazi bokuthi isicelo sifeziwe. Okwesithathu, umphumela wokulwa nama-oxidant awufakazeli indlela ye-Nrf2; ukwehla kwama-oxidant ngamaphesenti angu-28 kuma-fibroblast nokuvikelwa ku-rotenone kumagundane kuyimiphumela yangempela elandelayo, kodwa izindlela eziningana zihlangana kuyo.

Yini engaguqula ibhokisi elinomngcele onezikhala libe nelinomngcele oqhubekayo

Ukuhlolwa okushodayo kuwukuhlolwa okujwayelekile. Ukulinganisa iphrotheni ye-Nrf2 ku-nucleus ngemva kokuchayeka ku-methylene blue, ngokulandela isikhathi esihambisana nesimpawu sesikhashana se-AMPK, kungahlola ukuthi la magatsha amabili asebenza ndawonye ngempela yini, njengoba kusho umbono wokuvuselelwa okuhambisanayo. Inkomba yokuhlola eqhutshwa yi-ARE ingaqinisekisa ukusebenza kokubhalwa kolwazi lwezakhi zofuzo, kunokuba lokho kucatshangwe ngokubheka izakhi zofuzo ezihlosiwe. Ukuphinda izilinganiso zokwakheka kwama-mitochondria amasha lapho i-Nrf2 yehlisiwe noma ikhishwe ngokuphelele kungahlola umcibisholo wembangela ngokuqondile: uma ukuvuselelwa kwe-complex IV nokwenyuka kwe-mtDNA kuqhubeka ngaphandle kwe-Nrf2, ukuxhumana akulungile futhi igatsha le-AMPK lithwala umphumela lodwa. Ohlangothini lwezilwane, ucwaningo lokunikezwa kwemithamo isikhathi eside olunamaphuzu amathathu afanayo okuhlola, isilawuli, okuqukethwe, ukusebenza, lungahlola ukuthi uhlelo luhlala luvuliwe yini noma luyazivumelanisa. Kuze kube yileso sikhathi, ukuvuselelwa okuhambisanayo kwezindlela zamandla nezokuzivikela kusalokhu kuyilokho ababhali bakho abakubiza ngakho: isici esiyinhloko esingase sibe khona, hhayi indlela eseyiqinisekisiwe.

Abafundi abavela emkhakheni obanzi wesayensi yokusebenza kwemithi bangase bafune umongo ozungezile: indlela i-methylene blue edlulisa ngayo ama-electron ezingeni lama-mitochondria, okunye ekwenzayo kuma-mitochondria nasemandleni amangqamuzana, ukuthi okutholakele ngokuguga kwamangqamuzana kungena kuphi ocwaningweni lokuguga, indlela izimangalo zokwakheka kwama-mitochondria amasha ezihlobana ngayo nokusebenza kwamakhono okuqonda, nomlando wokuphepha obeka imingcele kuyo yonke ingxoxo ngemithamo. Ngakunye kwalokhu kuwumqondo ohlukile onobufakazi bawo, futhi ngakunye kufanele ukuhlukaniswa okufanayo phakathi kokulinganisiwe nokuhlongozwayo lesi sihloko esikwenze ku-Nrf2.