I-methylene blue ne-ferroptosis: indlela ye-peroxidation yama-lipid

I-ferroptosis iwukufa kweseli ngenxa yokulimala kolwelwesi. Amafutha ane-polyunsaturation olwelesini enziwa i-oxidation ngokusabela kwamakhemikhali okulandelanayo, ulwelwesi luyahluleka, bese iseli lifa. Le nqubo yamakhemikhali idinga i-ayoni. Lowo musho uqukethe wonke umqondo, futhi konke okukulesi sihloko kuhlola ingxenye ethile yawo.

Umbuzo lapha ugxile kokuncane: i-methylene blue yenzani kulolo chungechunge ekuhlolweni kwamaseli, futhi lowo mphumela ubonisani ngempela? Impendulo emfushane ithi ezifundweni ezimbili zaselabhorethri, i-methylene blue emazingeni e-nanomolar yanciphisa i-peroxidation yama-lipid kuma-mitochondria futhi yagcina amaseli ephila ngemva kokusetshenziswa kwezinto ezivusa i-ferroptosis ngokuvamile. Impendulo ende ibaluleke kakhulu, ngoba isixhumanisi ngasinye kulolu chungechunge sahlolwa ngokungenelela okuhlukile, futhi ukungenelela ngakunye kunemikhawulo.

I-ferroptosis ngesigaba esisodwa

I-phospholipid yolwelwesi enomsila we-fatty acid ene-polyunsaturation inendawo ebuthakathaka: ama-athomu e-hydrogen okulula ukuwasusa. Uma i-radical isisuse elilodwa, i-oxygen inamathela ku-lipid, kwakheke i-lipid peroxyl radical, bese leyo radical isusa i-hydrogen ku-lipid elandelayo. Lokhu kusabela kusakazeka olwelesini ngaphandle uma kukhona okukumisayo.

Isivikelo esiyinhloko esikumisayo yi-enzyme ebizwa ngokuthi i-GPX4, esebenzisa i-glutathione ukuguqula ama-lipid peroxide abe ama-lipid alcohol angenangozi ngenqubo ye-reduction. Izinto ezivusa le nqubo elabhorethri zihlasela lesi sivikelo ezindaweni ezithile. I-erastin ivimba ukungena kwe-cystine ngohlelo i-xc-minus, okwenza iseli liswele i-cysteine edingekayo ekwakheni i-glutathione. I-buthionine sulfoximine ivimba ukwakheka kwe-glutathione ngokuqondile. I-RSL3 ivimbela i-GPX4 uqobo. Zontathu zishiya ama-peroxide engasuswanga, kodwa zenza lokhu ezigabeni ezihlukene, ngakho umphumela otholakala ngento eyodwa evusayo awusebenzi ngokuzenzakalelayo nakwezinye.

I-ferroptosis ihlukile ku-apoptosis ngezici eziyichazayo. Alukho uchungechunge lokusebenza kwama-caspase futhi azikho izicucu zamaseli ezibizwa ngokuthi ama-apoptotic body. Izinto ezibopha i-ayoni, njenge-deferoxamine, nezinto ezibamba ama-radical, njenge-ferrostatin-1, ziyayicindezela. Lokhu kuwukuhlola abacwaningi abakusebenzisayo ukubiza ukufa ngokuthi kubangelwa i-ferroptosis kunokuba kube i-necrosis nje. Umongo obanzi wezindlela zokufa kwamaseli ezilawulwayo usiza ukuqonda lo mehluko, kanti indlela amaseli aphatha ngayo i-ayoni etholakala kalula ukuze isabele ichaza ukuthi kungani ukuyibopha kuwukulawula kokuhlola okunqumayo.

Indima yama-mitochondria, nokuthi kungani kwakuphikiswana ngayo

Ucwaningo lwangaphambili lwabika ukuthi amaseli angenayo i-DNA yama-mitochondria asakwazi ukufa nge-ferroptosis, okwaphakamisa ukuthi ama-mitochondria awadingeki njalo. Ucwaningo lwakamuva lwathola ukuthi ukunciphisa ama-mitochondria, noma ukuvimbela ukuphefumula kwamaseli, kungacindezela i-ferroptosis ebangelwa ukuswela i-cysteine kodwa hhayi ebangelwa ukuvinjelwa kwe-GPX4 ngokuqondile. Yomibili le miphumela ingaba yiqiniso uma indima yama-mitochondria incike entweni evusayo, ohlotsheni lweseli, nasendaweni lapho kwenzeka khona i-peroxidation enqumayo.

Ucwaningo okuyisisekelo salesi sihloko lwabhekana nalokhu kuphikisana ngesikhombisi sokuhlola esiqondene nengxenye ethile yeseli. I-MitoCLox iwuhlobo lwesikhombisi se-peroxidation yama-lipid oluqondiswe kuma-mitochondria: ishintsha i-fluorescence lapho ihlangana nama-lipid peroxyl radical ngaphakathi kwama-mitochondria. Kuma-fibroblast afakwe i-erastin, i-oxidation yesikhombisi esikuma-mitochondria yavela kumaseli ayesaphila ngaphambi kokufa, futhi yayingalingani phakathi kwawo. Lokho kulandelana kwesikhathi kubalulekile. I-peroxidation eyenzeka ngaphambi kokufa ingaba yimbangela yako. I-peroxidation elinganiswa kuphela kumaseli asefile iyinsalela yokulimala. Umhlahlandlela ohambisana nalesi sihloko omayelana nokuhlolwa kwe-peroxidation yama-lipid nalokho okubikwa isikhombisi ngasinye uchaza ukuthi kungani indawo yesikhombisi inquma incazelo yesignali.

Iphethini efanayo yavela kumodeli yesibili. Ama-fibroblast athathwe esigulini esine-Leber hereditary optic neuropathy, anokuguquka kwezakhi zofuzo ze-complex I namazinga aphezulu okucindezeleka okubangelwa i-oxidation ngisho engakavuswa, afa nge-ferroptosis lapho i-glutathione incishiswa, kuyilapho ama-fibroblast asetshenziswa njengokulawula emelana nalokho. I-oxidation yesikhombisi esikuma-mitochondria yaphinde yavela kuqala. Imininingwane itholakala ocwaningweni lwama-fibroblast nge-peroxidation yama-lipid kuma-mitochondria ngesikhathi se-ferroptosis.

Lokho i-methylene blue eyakushintsha kulokhu kuhlolwa

I-methylene blue ingumthwali we-redox. Isimo sayo esenziwe i-oxidation samukela ama-electron ezinqubweni zamakhemikhali ezixhumene ne-NADH ku-complex I, kanti isimo sayo esenziwe i-reduction siwanikeza i-cytochrome c, yona ewathumela ku-complex IV. Lokho kwakha indlela yokudlula ingxenye yochungechunge lapho ama-electron evame ukuphunyukela khona ku-oxygen akhe i-superoxide. Abafundi abafuna ukuqonda lonke lolu hlelo lokudluliselwa kufanele baqale ngendlela i-methylene blue ehambisa ngayo ama-electron kuma-mitochondria.

Kuma-fibroblast amaphaphu afakwe i-erastin, i-methylene blue engu-250 nanomolar yagcina amaseli ephila ngokwesilinganiso sokuphila kwamaseli, njengoba kwenza ama-antioxidant aqondiswe kuma-mitochondria, i-SkQ1 ne-MitoTEMPO. Izinto zokulawula ekuhlolweni zinquma ukuthi yini engashiwo ngomphumela. I-ferrostatin-1, i-alpha-tocopherol, ne-deferoxamine nazo zavikela amaseli, okuqinisekisa ukuthi ukufa kwakubangelwa i-ferroptosis. Into efana ne-SkQ1 kodwa engenalo iqembu elisebenza njenge-antioxidant ayizange ivikele, okukhomba izinqubo zamakhemikhali ze-redox kunokuba kube ukuqoqana nje okungaqondile. Ukuvikelwa kwancipha lapho i-SkQ1 idlula cishe ku-200 nanomolar, okuhambisana nokuziphatha okwaziwayo kwama-antioxidant e-quinone okukhuthaza i-oxidation lapho amanani esephezulu. I-methylene blue nayo inokuziphatha okumbaxambili kuye ngenani: iyavikela emazingeni aphansi e-nanomolar kulezi zitsha zokukhulisa amaseli, kodwa ingakhiqiza i-hydrogen peroxide ngokujikeleza kwe-redox lapho inani layo likhushulwa noma ikhanyiselwa kakhulu. Umongo walolu hlobo ophathelene nenani uchazwa esihlokweni sezinhlobo ze-oxygen ezisabela kalula kuma-mitochondria.

Ukuhlolwa kwama-mitochondria ahlukanisiwe kwabe sekuhlukanisa imithombo ye-ROS. Lapho kukhona i-ayoni esesimweni se-ferrous, ama-substrate anikeza i-complex I ama-electron kanye ne-rotenone akhuthaza i-oxidation yesikhombisi. I-rotenone ivimba ukugeleza ngemva kwendawo ye-flavin, ngakho abathwali abangaphambi kwaleyo ndawo bahlala besesimweni se-reduction futhi badedela ama-electron aye ku-oxygen esuka ku-flavin ebheke ku-matrix. Ama-substrate anikeza i-complex III ama-electron kanye ne-antimycin A akhiqiza i-hydrogen peroxide eningi kodwa awazange aqhube i-peroxidation yama-lipid. Isignali ye-ROS ephelele yayifana, kodwa indawo nomphumela kwakuhlukile. I-SkQ1 ne-methylene blue ngayinye zacindezela i-peroxidation eqhutshwa yi-complex I ngaphandle kokucindezela ukukhiqizwa kwe-hydrogen peroxide uqobo, okubonisa ukuthi zisebenza ngemva kokwakheka kwe-ROS, esigabeni se-peroxidation yama-lipid.

Ingxenye yokugcwala ngokweqile kwe-ayoni nomkhondo we-lipofuscin

Ucwaningo lwesibili lwabheka lo mbuzo ohlangothini lwe-ayoni. Ama-cardiomyocyte e-H9c2 afakwe i-ferric ammonium citrate ezingeni elingaphansi kwe-millimolar aba ne-ROS kuma-mitochondria, kwalandela i-peroxidation yama-lipid kuma-mitochondria, kwase kuba yi-ferroptosis, elandelwa ukuqoqana kwento enombala efana ne-lipofuscin kungakapheli 24 hours. I-lipofuscin iyinsalela yama-lipid namaprotheni enziwe i-oxidation, equkethe i-ayoni futhi ekhipha i-fluorescence ngokwayo, eqoqana ngokukhula kweminyaka. Ukuvela kwayo ngokushesha lapha kuhlobanisa i-peroxidation esheshayo eqhutshwa yi-ayoni nophawu olwaziwayo lokuguga. I-methylene blue ne-SkQ1 emazingeni e-nanomolar ngayinye zavimbela i-peroxidation, ukufa, nokuqoqana kwaleyo nto enombala. Ama-mitochondria enhliziyo ahlukanisiwe aqinisekisa le phethini: i-rotenone kanye ne-ayoni esesimweni se-ferrous kwaqhuba i-peroxidation nesignali efana neyento enombala, futhi zombili lezi zinto zakuvimba. Lonke lolu chungechunge lubikwe ocwaningweni lokugcwala ngokweqile kwe-ayoni kuma-cardiomyocyte e-H9c2. Ingxenye ephathelene nale nto enombala ihambisana nesihloko se-lipofuscin nezimpawu zokuguga kwamaseli.

Lokho ukuhlolwa kokuphila kwamaseli okungakubonisi

Isilinganiso sokuphila kwamaseli esifana ne-CellTiterBlue sibika ukuthi mangaki amaseli esitsheni asasebenza ngokwe-metabolism ngemva kokufakwa kwento evusayo kanye nento ehlolwayo ukuthi ingavikela yini. Lokhu kuwukuhlolwa kwendlela yokusebenza ohlelweni oluchazwe ngokucacile. Akuwona umphumela wokwelashwa. Akukho kulokhu kuhlolwa okwanikeza isiguli umthamo, kwalinganisa umphumela wesifo, noma kwanquma ibanga lamanani angasebenza ekwelapheni emzimbeni.

Kunemikhawulo emine okufanele ishiwo ngokucacile. Okokuqala, izinhlobo zamaseli zincane ngokwehlukahlukana: ama-fibroblast amaphaphu aguquliwe, ama-fibroblast esikhumba athathwe ngokuqondile emzimbeni okuhlanganisa umugqa owodwa wamaseli anesifo sama-mitochondria, nomugqa wama-cardiomyocyte egundane, kanye nama-mitochondria ahlukanisiwe enhliziyo yegundane. Ukuzwela kwakuhlukahluka ngomugqa wamaseli, futhi umugqa we-LHON wawusengozini enkulu ngendlela engavamile ngenxa yokukhethwa kwawo kulolu cwaningo. Okwesibili, izinto ezivusayo ziyizimo ezeqisayo zaselabhorethri: ukuvinjelwa okuphelele kokuthuthwa noma kokwakheka kwento, kanye namanani e-ayoni okungekho sicubu esingahlangabezana nawo ngesikhathi esifushane. Okwesithathu, izinga lokugxila aliguqulelwa kumthamo womuntu ngokuphindaphinda nje. Izinga le-nanomolar oketshezini lokukhulisa amaseli aliwona umthamo womuntu. Ukusakazeka kwe-methylene blue emzimbeni, ukuguqulwa kwayo nge-reduction ibe yisimo esingenambala egazini nasezicutshini, ukuchayeka ekukhanyeni, nomehluko we-voltage olwelesini konke kuhlukanisa okwenzeka esitsheni nokwenzeka kumuntu. Okwesine, izikhombisi zibika okwenzeka engxenyeni ezikuyo kuphela. I-oxidation ye-MitoCLox ibonisa ukuthi kwakheke ama-radical ama-lipid kuma-mitochondria; ayibonisi ukuthi ukudabuka kolwelwesi lweseli, okungenzeka kube yisigameko sokugcina, kwavinjelwa ngokuqondile kunokuba kugwenywe ngokuphazamisa izigaba zangaphambili.

Ngakho lokho okungashiwo ngokwethembeka yilokhu: kula mamodeli, i-ROS evela ku-complex I kanye ne-ayoni kuqhuba i-peroxidation yama-lipid kuma-mitochondria eyenzeka ngaphambi kwe-ferroptosis, futhi i-methylene blue emazingeni aphansi okunqwabelana inqamula leyo ngxenye yochungechunge. Noma yini engaphezu kwaleyo ngxenye idinga ukuhlolwa okuhlukile ezilwaneni, bese kuba kubantu.