Shuɗin methylene da ferroptosis: tsarin peroxidation na lipid

Ferroptosis mutuwar ƙwayar halitta ce sakamakon lalacewar membran. Kitsen da ke cikin membran wanda yake da mahaɗai biyu-biyu da yawa yana yin hadawan abu da oxygen a cikin jerin halayen sinadarai, membran ya gaza, sannan ƙwayar ta mutu. Ana buƙatar ƙarfe domin waɗannan halayen sinadarai su faru. Wannan jimlar ta ƙunshi dukkan ra'ayin, kuma duk abin da ke cikin wannan labarin gwaji ne na wani ɓangare nasa.

Tambayar nan ta fi wannan takamaimai: mene ne shuɗin methylene yake yi wa wannan jerin a gwaje-gwajen ƙwayoyin halitta, kuma mene ne sakamakon yake nunawa a zahiri? Amsa a taƙaice ita ce, a binciken dakin gwaje-gwaje guda biyu, shuɗin methylene a matakin nanomolar ya rage peroxidation na lipid a mitochondria kuma ya sa ƙwayoyin halitta suka ci gaba da rayuwa bayan amfani da abubuwan da kan jawo ferroptosis. Amsa mai tsawo ta fi muhimmanci, domin an gwada kowace mahaɗa a cikin jerin da wani daban da aka yi amfani da shi, kuma kowanne yana da iyakokinsa.

Ferroptosis a sakin layi ɗaya

Phospholipid na membran wanda yake ɗauke da wutsiyar acid na kitse mai mahaɗai biyu-biyu da yawa yana da wurin rauni: atom ɗin hydrogen da suke da sauƙin ɗauka. Da zarar wani radical ya ɗauki ɗaya, oxygen yana haɗuwa da lipid, radical na lipid peroxyl ya samu, sannan wannan radical ya ɗauka daga lipid na gaba. Halin sinadarin yana ci gaba da yaɗuwa a membran sai dai idan wani abu ya dakatar da shi.

Babban abin da ke dakatar da wannan shi ne wani enzyme mai suna GPX4, wanda yake amfani da glutathione wajen sauya lipid peroxides zuwa lipid alcohols marasa illa ta hanyar reduction. Abubuwan da ake amfani da su a dakin gwaje-gwaje wajen jawo wannan suna kai hari ga wannan kariya a takamaiman wurare. Erastin yana hana shigar cystine ta tsarin xc-minus, wanda hakan yake hana ƙwayar samun cysteine da ake buƙata don haɗa glutathione. Buthionine sulfoximine yana hana haɗa glutathione kai tsaye. RSL3 yana hana GPX4 kansa aiki. Dukkansu uku suna barin peroxides ba tare da an kawar da su ba, amma suna yin hakan a matakai daban-daban, don haka sakamakon wani daga cikinsu ba ya nufin sauran za su ba da irin sakamakon kai tsaye.

Ferroptosis ya bambanta da apoptosis a siffofin da suke bayyana shi. Babu jerin kunna caspase kuma babu gutsattsarin ƙwayar halitta na apoptosis. Abubuwan da suke ɗaure ƙarfe kamar deferoxamine da abubuwan da suke kama radicals kamar ferrostatin-1 suna hana shi. Wannan shi ne gwajin da masu bincike suke amfani da shi don tantance mutuwa a matsayin ferroptosis maimakon necrosis kawai. Faɗaɗɗen bayani kan hanyoyin mutuwar ƙwayoyin halitta da ake sarrafawa yana sanya wannan bambanci cikin mahallinsa, kuma yadda ƙwayoyin halitta suke sarrafa ƙarfen da yake da sauƙin shiga halayen sinadarai yana bayyana dalilin da ya sa ɗaure ƙarfe yake zama muhimmin gwajin tabbatarwa.

Inda mitochondria suke shiga, da dalilin saɓani a kai

Binciken farko ya nuna cewa ƙwayoyin halitta da ba su da DNA na mitochondria har yanzu suna fuskantar ferroptosis, wanda ya nuna cewa mitochondria ba lallai ba ne. Binciken da aka yi daga baya ya gano cewa rage mitochondria, ko hana aikin samar da makamashi ta numfashin ƙwayar halitta, na iya hana ferroptosis da rashin cysteine yake jawowa, amma ba wanda hana GPX4 kai tsaye yake jawowa ba. Duk sakamakon biyu na iya zama gaskiya idan gudummawar mitochondria ta dogara da abin da ya jawo lamarin, nau'in ƙwayar halitta, da wurin da peroxidation mai muhimmanci yake faruwa.

Binciken da wannan labarin ya dogara da shi ya tunkari saɓanin da wani abin gano sauyi da yake aiki a takamaiman sashe na ƙwayar halitta. MitoCLox wani nau'i ne na abin nuna peroxidation na lipid da aka tsara ya nufi mitochondria: hasken fluorescence nasa yana sauyawa idan ya haɗu da radicals na lipid peroxyl a cikin mitochondria. A fibroblasts da aka yi wa amfani da erastin, hadawan abu da oxygen na abin ganowa a mitochondria ya bayyana a ƙwayoyin halitta da suke raye kafin su mutu, kuma ya bambanta tsakanin ƙwayoyin. Wannan lokacin da ya bayyana yana da muhimmanci. Peroxidation da yake faruwa kafin mutuwa yana cikin abubuwan da ake zargin za su iya jawo mutuwar. Peroxidation da aka auna a matattun ƙwayoyin halitta kawai ragowar lalacewa ne. Wani jagora mai alaƙa kan gwaje-gwajen peroxidation na lipid da abin da kowane abin ganowa yake nunawa ya bayyana dalilin da ya sa wurin abin ganowa yake ƙayyade ma'anar wata alama.

Irin wannan tsari ya bayyana a samfurin gwaji na biyu. Fibroblasts daga wani mai cutar Leber hereditary optic neuropathy, waɗanda suke ɗauke da sauyin kwayar halitta na complex I kuma suke da matsin hadawan abu da oxygen mafi girma tun farko, sun mutu ta hanyar ferroptosis lokacin da aka rage glutathione, yayin da fibroblasts na kwatantawa suka jure. Hadawan abu da oxygen na abin ganowa a mitochondria ya sake zuwa da farko. Bayanan suna cikin binciken fibroblasts kan peroxidation na lipid a mitochondria a lokacin ferroptosis.

Abin da shuɗin methylene ya sauya a waɗannan gwaje-gwaje

Shuɗin methylene mai ɗaukar redox ne. Nau'insa da ya yi oxidation yana karɓar electrons daga halayen sinadarai masu alaƙa da NADH a complex I, sannan nau'insa da ya yi reduction yana ba da su ga cytochrome c, wanda yake kai su zuwa complex IV. Wannan yana samar da hanyar kaucewa wani ɓangare na jerin inda electrons kan kuɓuce zuwa oxygen a matsayin superoxide. Masu karatu da suke son cikakken bayanin wannan tsarin miƙa electrons su fara da yadda shuɗin methylene yake motsa electrons ta mitochondria.

A fibroblasts na huhu da aka yi wa amfani da erastin, shuɗin methylene a 250 nanomolar ya kiyaye rayuwar ƙwayoyin halitta a gwajin auna tsira, tare da abubuwan hana oxidation masu nufar mitochondria, SkQ1 da MitoTEMPO. Gwaje-gwajen kwatantawa ne suke ƙayyade abin da za a iya cewa. Ferrostatin-1, alpha-tocopherol, da deferoxamine ma sun ba da kariya, wanda yake tabbatar da cewa mutuwar ferroptosis ce. Nau'in sinadari mai kama da SkQ1 amma ba shi da rukunin hana oxidation bai ba da kariya ba, wanda yake nuna cewa halayen sinadarai na redox ne dalili, ba taruwar sinadarin kawai ba. Kariya ta ragu sama da kusan 200 nanomolar na SkQ1, daidai da sanannen halin abubuwan hana oxidation na quinone na ƙara oxidation idan yawansu ya yi yawa. Shuɗin methylene ma yana da irin wannan halin fuska biyu dangane da yawansa: yana ba da kariya a ƙananan matakan nanomolar a waɗannan farantan gwaji, amma yana iya samar da hydrogen peroxide ta zagayawar redox idan aka ƙara yawansa ko aka haska shi sosai. An tattauna irin wannan mahallin yawa a nau'o'in oxygen masu saurin shiga halayen sinadarai a mitochondria.

Daga nan, gwaje-gwajen mitochondria da aka ware sun bambanta tushen ROS. Idan ƙarfen ferrous yana nan, sinadaran da suke ciyar da complex I tare da rotenone sun ƙara hadawan abu da oxygen na abin ganowa. Rotenone yana toshe hanyar bayan wurin flavin, don haka masu ɗaukar electrons da suke kafin wurin suna ci gaba da kasancewa a yanayin reduction, kuma electrons suna kuɓucewa zuwa oxygen daga flavin da yake fuskantar matrix. Sinadaran da suke ciyar da complex III tare da antimycin A sun samar da hydrogen peroxide mai yawa amma ba su jawo peroxidation na lipid ba. Alamar ROS ta gaba ɗaya iri ɗaya, wurin daban, sakamakon daban. SkQ1 da shuɗin methylene kowannensu ya hana peroxidation da complex I yake jawowa ba tare da hana samar da hydrogen peroxide kansa ba, wanda yake sanya aikinsu bayan samuwar ROS, a matakin peroxidation na lipid.

Ɓangaren yawan ƙarfe da alamar lipofuscin

Bincike na biyu ya duba tambayar daga ɓangaren ƙarfe. Ƙwayoyin tsokar zuciya na H9c2 da aka fallasa wa ferric ammonium citrate a matakin ƙasa da millimolar sun samar da ROS na mitochondria, suka sami peroxidation na lipid a mitochondria, sannan ferroptosis, wanda taruwar wani sinadarin launi mai kama da lipofuscin ta biyo baya cikin sa'o'i 24. Lipofuscin shi ne ragowar lipids da furotin da suka yi oxidation, mai ɗauke da ƙarfe kuma mai fitar da fluorescence da kansa, wanda yake taruwa yayin da shekaru suke ƙaruwa. Bayyanarsa da sauri a nan tana haɗa peroxidation mai tsanani da ƙarfe yake jawowa da sananniyar alamar tsufa. Shuɗin methylene a matakin nanomolar da SkQ1 kowannensu ya hana peroxidation, mutuwar, da taruwar sinadarin launin. Mitochondria na zuciya da aka ware sun tabbatar da wannan tsari: rotenone tare da ƙarfen ferrous sun jawo peroxidation da alama mai kama da ta sinadarin launi, kuma dukkan abubuwan biyu sun hana hakan. An ba da rahoton cikakken jerin a binciken yawan ƙarfe a ƙwayoyin tsokar zuciya na H9c2. Ɓangaren sinadarin launin na wannan bayani yana da alaƙa da lipofuscin da alamomin tsufar ƙwayoyin halitta.

Abin da gwaje-gwajen tsirar ƙwayoyin halitta ba sa nunawa

Sakamakon auna rayuwar ƙwayoyin halitta kamar CellTiterBlue yana nuna yawan ƙwayoyin da suke cikin farantin gwaji waɗanda har yanzu suke gudanar da ayyukan sarrafa sinadarai bayan amfani da abin jawo lamarin tare da wani da ake gwadawa a matsayin mai kariya. Wannan gwaji ne na tsarin aiki a wani tsari da aka ƙayyade. Ba sakamakon magani ba ne. Babu ɗaya daga cikin waɗannan gwaje-gwajen da ya ba mara lafiya sinadarin, ya auna sakamakon cuta, ko ya tabbatar da zangon yawa da zai iya zama mai amfani kuma mai aminci a jiki.

Ya dace a bayyana iyakoki huɗu a sarari. Na farko, nau'o'in ƙwayoyin halittar kaɗan ne: fibroblasts na huhu da aka sauya, fibroblasts na fata da aka samo kai tsaye ciki har da layin ƙwayoyi ɗaya mai cutar mitochondria, da layin ƙwayoyin tsokar zuciya na bera, tare da mitochondria na zuciyar bera da aka ware. Sauƙin shafuwarsu ya bambanta tsakanin layukan, kuma an zaɓi layin LHON ne saboda kasancewarsa mai rauni fiye da yadda aka saba. Na biyu, abubuwan jawo lamarin tsauraran yanayi ne na dakin gwaje-gwaje: cikakken toshe jigilar sinadari ko hana haɗa shi, da yawan ƙarfe da babu wata tsokar jiki ko nama da zai fuskanta cikin ɗan gajeren lokaci. Na uku, ba za a iya sauya tattaruwar sinadari zuwa adadin amfani ta hanyar ninkawa kawai ba. Nanomolar a ruwan renon ƙwayoyin halitta ba adadin da za a ba mutum ba ne. Yadda shuɗin methylene yake rarrabuwa, sauyawarsa ta reduction zuwa nau'insa marar launi a jini da naman jiki, fallasa shi ga haske, da bambancin ƙarfin lantarki a membran duk suna shiga tsakanin farantin gwaji da mutum. Na huɗu, abubuwan ganowa suna nuna abin da yake faruwa a sashensu ne kawai. Hadawan abu da oxygen na MitoCLox yana nuna cewa radicals na lipid sun samu a mitochondria; ba ya nuna cewa an hana fashewar membran na waje na ƙwayar halitta, wanda wataƙila shi ne matakin ƙarshe, kai tsaye maimakon an kauce masa ta hana wani mataki na farko.

Don haka abin da za a iya faɗa da gaskiya shi ne: a waɗannan samfurori, ROS da ya fito daga complex I tare da ƙarfe suna jawo peroxidation na lipid a mitochondria wanda yake faruwa kafin ferroptosis, kuma shuɗin methylene a ƙananan tattaruwa yana katse wannan ɓangaren. Duk wani abu da ya wuce wannan ɓangaren yana buƙatar wasu gwaje-gwaje a dabbobi, sannan a mutane.