Ubururu bwa methylene na ferroptosis: uburyo peroksidasiyo ya lipide ikora

Ferroptosis ni urupfu rw'akaremangingo ruterwa no kwangirika kw'agahu kako. Ibinure bifite imiyoboro myinshi y'inkubwe ebyiri biri mu duhu bikorerwa okisidasiyo mu ruhererekane rw'ibikorwa, agahu kakareka gukora, maze akaremangingo kagapfa. Ubutare burakenewe kugira ngo ibyo bikorwa bya shimi bibeho. Iyo nteruro ikubiyemo igitekerezo cyose, kandi buri kintu muri iyi nyandiko ni igerageza ry'igice kimwe cyacyo.

Ikibazo kiri hano kirihariye kurushaho: ubururu bwa methylene bukora iki kuri urwo ruhererekane mu bushakashatsi ku turemangingo, kandi ibyo bwagezeho byerekana iki koko? Igisubizo kigufi ni uko mu bushakashatsi bubiri bwo muri laboratwari, ubururu bwa methylene ku gipimo cya nanomolar bwagabanyije peroksidasiyo ya lipide muri mitokondiriya kandi bugatuma uturemangingo dukomeza kubaho nyuma yo gushyirwamo ibintu ubusanzwe bitera ferroptosis. Igisubizo kirambuye ni cyo cy'ingenzi kurushaho, kuko buri ntambwe muri urwo ruhererekane yageragejwe hakoreshejwe uburyo butandukanye, kandi buri buryo bufite aho bugarukira.

Ferroptosis mu gika kimwe

Fosifolipide yo mu gahu ifite umurizo w'asidi y'ibinure ifite imiyoboro myinshi y'inkubwe ebyiri igira ahantu h'intege nke: atome za idorojeni zoroshye gukurwaho. Iyo radikali ikuyemo imwe, ogisijeni yiyomeka kuri lipide, hagakorwa radikali ya peroksile ya lipide, maze iyo radikali ikavana atome kuri lipide ikurikiyeho. Icyo gikorwa gikomeza gukwirakwira mu gahu keretse hari ikigihagaritse.

Ikintu cy'ingenzi gihagarika urwo ruhererekane ni anzime yitwa GPX4, ikoresha glutathione kugira ngo ihindure perogiside za lipide mo alukolo za lipide zitangiza. Ibintu bikoreshwa muri laboratwari mu gutera ferroptosis byibasira ubu bwirinzi ku ntambwe zizwi. Erastin ibuza cystine kwinjira mu karemangingo inyura muri sisitemu xc-minus, bigatuma akaremangingo kabura cysteine yo gukora glutathione. Buthionine sulfoximine ibuza ikorwa rya glutathione mu buryo butaziguye. RSL3 ibuza GPX4 ubwayo gukora. Byose uko ari bitatu bituma perogiside zidakurwaho, ariko bikabikora ku ntambwe zitandukanye; bityo ibisubizo byabonetse hakoreshejwe kimwe ntibihita biba ukuri no ku bindi.

Ferroptosis itandukanye na apoptosis mu biyiranga. Nta ruhererekane rw'ikorwa rya caspase rubaho kandi nta dusigazwa tw'uturemangingo turanga apoptosis tuboneka. Ibintu bifata ubutare, nka deferoxamine, n'ibintu bifata radikali, nka ferrostatin-1, birayikumira. Iryo ni ryo gerageza abashakashatsi bifashisha kugira ngo bemeze ko urupfu rw'akaremangingo ari ferroptosis aho kuba necrosis gusa. Kureba muri rusange inzira z'urupfu rw'uturemangingo rugengwa n'imikorere yabyo bifasha kumva iri tandukaniro, kandi uburyo uturemangingo ducunga ubutare bworoshye kwitabira ibikorwa bya shimi busobanura impamvu kubufata ari igerageza ry'ingenzi ryo kugenzura ibyo bisubizo.

Uruhare rwa mitokondiriya n'impamvu rwagibwagaho impaka

Ubushakashatsi bwa mbere bwagaragaje ko uturemangingo tudafite ADN ya mitokondiriya na two dupfa na ferroptosis, ibyo bikaba byaratumye hakekwa ko mitokondiriya zitari ngombwa. Ubushakashatsi bwakurikiyeho bwabonye ko kugabanya mitokondiriya cyangwa guhagarika ibikorwa byazo byo guhumeka bishobora gukumira ferroptosis iterwa no kubura cysteine, ariko ntibikumire iterwa no guhagarika GPX4 mu buryo butaziguye. Ibyo bisubizo byombi bishobora kuba ukuri niba uruhare rwa mitokondiriya rushingiye ku kintu gitera ferroptosis, ku bwoko bw'akaremangingo no ku hantu peroksidasiyo igena iby'ingenzi ibera.

Ubushakashatsi iyi nyandiko ishingiyeho bwasesenguye izo mpaka bukoresheje igikoresho cyo gupima cyibanda ku gice runaka cy'akaremangingo. MitoCLox ni ubwoko bw'igikoresho kigaragaza peroksidasiyo ya lipide, cyagenewe kwerekeza muri mitokondiriya: gihindura floresansi iyo gihuye na radikali za peroksile ya lipide muri mitokondiriya. Mu turemangingo twa fibroblast twashyizwemo erastin, okisidasiyo y'icyo gikoresho muri mitokondiriya yagaragaye uturemangingo tukiri tuzima, mbere y'uko dupfa, kandi ntiyari ku rugero rumwe muri twose. Icyo gihe yabereyeho ni ingenzi. Peroksidasiyo ibanziriza urupfu ishobora kuba ari yo irutera. Peroksidasiyo ipimwe gusa mu turemangingo twapfuye iba ari igisigazwa. Inyandiko ijyana n'iyi yerekeye ibipimo bya peroksidasiyo ya lipide n'ibyo buri gikoresho cyo gupima kigaragaza isobanura impamvu aho igikoresho kiri hagena icyo ikimenyetso gitanga gisobanura.

Uko gukurikirana kwongeye kugaragara mu rugero rwa kabiri rw'ubushakashatsi. Uturemangingo twa fibroblast twavuye ku murwayi ufite indwara y'umutsi w'ijisho ya Leber ihererekanywa mu muryango, dufite ihinduka ry'ingirabuzimafatizo ryibasira urusobe I ndetse n'urwego rusanzwe rw'umuvuduko wa okisidasiyo ruri hejuru, twapfuye na ferroptosis igihe glutathione yagabanywaga, mu gihe uturemangingo twa fibroblast two kugereranya twabyihanganiye. Na none, okisidasiyo y'igikoresho cyo gupima muri mitokondiriya yabanje kubaho. Ibisobanuro birambuye biri mu bushakashatsi ku turemangingo twa fibroblast bwerekeye peroksidasiyo ya lipide muri mitokondiriya mu gihe cya ferroptosis.

Ibyo ubururu bwa methylene bwahinduye muri aya magerageza

Ubururu bwa methylene ni ikintu gitwara elegitoroni mu bikorwa bya okisidasiyo n'igabanya. Imiterere yabwo yakorewe okisidasiyo yakira elegitoroni zivuye mu bikorwa bya shimi bifitanye isano na NADH mu rusobe I, naho imiterere yabwo yagabanyijwe ikaziha cytochrome c, na yo ikazigeza ku rusobe IV. Ibyo bishyiraho inzira y'inyongera inyura ku ruhande rw'igice cy'uruhererekane aho elegitoroni zisanzwe zisohokera zijya kuri ogisijeni zikabyara superoxide. Abasomyi bifuza kumenya neza uko uko guhererekanya kugenda bakwiye guhera ku nyandiko ivuga uko ubururu bwa methylene bwimura elegitoroni muri mitokondiriya.

Mu turemangingo twa fibroblast two mu bihaha twashyizwemo erastin, ubururu bwa methylene kuri 250 nanomolar bwatumye dukomeza kubaho nk'uko byagaragajwe n'igipimo cy'ubuzima bw'uturemangingo, kimwe na SkQ1 na MitoTEMPO, ibintu birwanya okisidasiyo byagenewe kwerekeza muri mitokondiriya. Amagerageza yo kugenzura ni yo agena icyo twakwemeza. Ferrostatin-1, alpha-tocopherol na deferoxamine na byo byararindaga, bikemeza ko urupfu rwari ferroptosis. Ikintu gifite imiterere isa n'iya SkQ1 ariko kidafite itsinda rirwanya okisidasiyo nticyarindaga, ibyo bikerekeza ku bikorwa bya shimi bya okisidasiyo n'igabanya aho kuba ukwiyegeranya kutihariye. Ubwirinzi bwaragabanukaga iyo SkQ1 yarenzaga hafi 200 nanomolar, bihuye n'imyitwarire izwi y'ibintu birwanya okisidasiyo byo mu bwoko bwa quinone, bishobora kuyitera iyo biri ku rugero rwinshi. Ubururu bwa methylene na bwo bugira iyo myitwarire ibiri bitewe n'ingano yabwo: burarinda ku rugero ruto rwa nanomolar muri ibi bikoresho birimo uturemangingo, ariko bushobora kubyara perogiside ya idorojeni binyuze mu ruhererekane rwa okisidasiyo n'igabanya iyo ingano yabwo yongerewe cyangwa bumurikiwe cyane. Uruhare rw'ingano muri ubu buryo rusobanurwa mu nyandiko yerekeye ubwoko bwa ogisijeni bwitabira ibikorwa bya shimi muri mitokondiriya.

Amagerageza kuri mitokondiriya zatandukanyijwe n'uturemangingo yaje gutandukanya inkomoko za ROS. Mu gihe harimo ubutare bwo mu miterere ya ferrous, ibikoresho bitanga elegitoroni ku rusobe I hamwe na rotenone byatumaga habaho okisidasiyo y'igikoresho cyo gupima. Rotenone ihagarika uruhererekane nyuma y'ahantu hari flavin, bityo ibitwara elegitoroni biri mbere yaho bikaguma mu miterere yagabanyijwe kandi elegitoroni zikava kuri flavin ireba mu gice cy'imbere cya mitokondiriya zikajya kuri ogisijeni. Ibikoresho bitanga elegitoroni ku rusobe III hamwe na antimycin A byabyaraga perogiside ya idorojeni nyinshi, nyamara ntibyateraga peroksidasiyo ya lipide. Ikimenyetso rusange cya ROS cyari kimwe, ariko aho zaturukaga n'ingaruka zabyo byari bitandukanye. SkQ1 n'ubururu bwa methylene, buri kimwe ukwacyo, byakumiraga peroksidasiyo iterwa n'urusobe I bitabujije ikorwa rya perogiside ya idorojeni ubwayo. Ibyo byerekana ko ibikorwa byabyo biba nyuma y'ikorwa rya ROS, ku ntambwe ya peroksidasiyo ya lipide.

Igice cy'ubushakashatsi ku butare burenze urugero n'icyo lipofuscin yerekana

Ubushakashatsi bwa kabiri bwabajije icyo kibazo bwibanda ku butare. Uturemangingo tw'imikaya y'umutima twa H9c2 twashyizwe mu ferric ammonium citrate ku rugero ruri munsi ya millimolar twagize ROS muri mitokondiriya, peroksidasiyo ya lipide muri mitokondiriya, hanyuma ferroptosis, bikurikirwa no kwiyegeranya kw'ikinyabutabire cy'amabara gisa na lipofuscin mu masaha 24. Lipofuscin ni igisigazwa cya lipide na poroteyine byakorewe okisidasiyo, kirimo ubutare kandi gitanga floresansi ubwacyo, kigenda cyiyongera uko imyaka ishira. Kugaragara kwayo vuba hano bihuza peroksidasiyo yihuse iterwa n'ubutare n'ikimenyetso kizwi cyo gusaza. Ubururu bwa methylene ku rugero rwa nanomolar na SkQ1, buri kimwe ukwacyo, byabujije peroksidasiyo, urupfu n'ukwiyegeranya kw'icyo kinyabutabire cy'amabara. Mitokondiriya z'umutima zatandukanyijwe n'uturemangingo zemeje uko gukurikirana: rotenone hamwe n'ubutare bwo mu miterere ya ferrous byateye peroksidasiyo n'ikimenyetso gisa n'icy'ikinyabutabire cy'amabara, kandi ibyo bintu byombi byarabikumiriye. Urwo ruhererekane rwose rwatangajwe mu bushakashatsi ku butare burenze urugero mu turemangingo tw'imikaya y'umutima twa H9c2. Igice cyerekeye icyo kinyabutabire cy'amabara kijyana n'inyandiko ivuga lipofuscin n'ibimenyetso byo gusaza kw'uturemangingo.

Ibyo ibipimo byo gukomeza kubaho kw'uturemangingo biterekana

Igipimo cy'ubuzima bw'uturemangingo nka CellTiterBlue kigaragaza umubare w'uturemangingo mu gikoresho dukomeza kugira ibikorwa bya metabolisme nyuma yo gushyirwamo ikintu gitera ferroptosis hamwe n'ikindi gishobora kuturinda. Iryo ni igerageza ry'uburyo imikorere igenda muri sisitemu isobanuwe neza. Si igisubizo cy'ubuvuzi. Nta na rimwe muri aya magerageza ryahaye umurwayi igipimo cy'umuti, ryapimye igipimo cy'ingaruka ku ndwara cyangwa ryagaragaje urugero rw'ikoreshwa ry'umuti rufite inyungu kandi rutekanye mu mubiri.

Hari aho ibi bisubizo bigarukira mu buryo bune bukwiye kuvugwa mu buryo bweruye. Icya mbere, ubwoko bw'uturemangingo bwari buke: uturemangingo twa fibroblast two mu bihaha twahinduwe, uturemangingo twa fibroblast twakuwe ku ruhu tukiri mu miterere yatwo y'ibanze, turimo ubwoko bumwe bufite indwara ya mitokondiriya, n'ubwoko bw'uturemangingo tw'imikaya y'umutima tw'imbeba, hamwe na mitokondiriya z'umutima w'imbeba zatandukanyijwe n'uturemangingo. Uko byagirwagaho ingaruka byatandukanaga bitewe n'ubwoko, kandi ubwoko bwa LHON bwatoranyijwe kubera ko bwibasirwaga bidasanzwe. Icya kabiri, ibintu byateraga ferroptosis byari ibipimo bikabije byo muri laboratwari: guhagarika burundu itwarwa ry'ibintu cyangwa ikorwa ryabyo, n'ingano z'ubutare nta nyama z'umubiri zahura na zo mu gihe gito. Icya gatatu, igipimo cy'ubwinshi ntigihinduka igipimo cyo gukoresha mu muntu binyuze mu gukuba gusa. Nanomolar mu muvange uturemangingo dukuriramo si igipimo cyo guha umuntu. Ukwirakwira kw'ubururu bwa methylene, kugabanywa kwabwo bukajya mu miterere yabwo itagira ibara mu maraso no mu nyama z'umubiri, guhura n'urumuri n'itandukaniro ry'ingufu z'amashanyarazi ku mpande z'agahu, byose bigira uruhare mu itandukaniro hagati y'igikoresho kirimo uturemangingo n'umuntu. Icya kane, ibikoresho byo gupima bigaragaza gusa ibibera mu gice birimo. Okisidasiyo ya MitoCLox yerekana ko habayeho radikali za lipide muri mitokondiriya; ntiyerekana ko gucika kw'agahu k'akaremangingo, gushobora kuba ari cyo gikorwa cya nyuma, kwakumiriwe mu buryo butaziguye aho kwirindwa hakumiriwe ibikorwa byabanje.

Bityo, icyo twakwemeza tudakabya ni iki: muri izi ngero z'ubushakashatsi, ROS ziva mu rusobe I hamwe n'ubutare bitera peroksidasiyo ya lipide muri mitokondiriya ibanziriza ferroptosis, kandi ubururu bwa methylene ku gipimo gito buhagarika icyo gice cy'uruhererekane. Ikindi cyose kirenze icyo gice gikeneye amagerageza atandukanye ku nyamaswa, hanyuma no ku bantu.