I-methylene blue ne-ferroptosis: intsimbi, i-peroxidation yee-lipid, nokufa kweeseli

I-ferroptosis kukufa kweseli ngenxa yokonakala kwenwebu. Amafutha akwiinwebu aneebhondi ezininzi ezingahluthanga afumana i-oxidation kuthotho lweempendulo zekhemikhali, inwebu iyasilela, ize iseli ife. Intsimbi iyafuneka kule nkqubo yekhemikhali. Loo sivakalisi siwushwankathela wonke lo mbono, yaye yonke into ekweli nqaku ivavanya inxalenye yawo.

Umbuzo apha ucace ngakumbi: yenza ntoni i-methylene blue kolo thotho kuvavanyo lweeseli, yaye eso siphumo sibonisa ntoni ngokwenene? Impendulo emfutshane kukuba, kwizifundo ezimbini zaselabhoratri, i-methylene blue ekumanqanaba e-nanomolar yanciphisa i-peroxidation yee-lipid ze-mitochondria yaza yagcina iiseli ziphila emva kokusetyenziswa kwezinto ezivusa i-ferroptosis ngokuqhelekileyo. Impendulo ende ibaluleke ngakumbi, kuba ikhonkco ngalinye kolu thotho lavavanywa ngongenelelo olwahlukileyo, yaye ungenelelo ngalunye lunemida.

I-ferroptosis kumhlathi omnye

I-phospholipid yenwebu enomsila we-asidi enamafutha aneebhondi ezininzi ezingahluthanga inendawo ebuthathaka: iiathom ze-hydrogen ekulula ukuzihlutha. Xa i-radical ihlutha enye, ioksijini idibana ne-lipid, kuvele i-lipid peroxyl radical, ize loo radical ihluthe kwenye i-lipid. Le mpendulo yekhemikhali iqhubeka inwenwa kwinwebu ngaphandle kokuba kukho into eyinqandayo.

Eyona nto iyinqandayo yi-enzyme ebizwa ngokuba yi-GPX4, esebenzisa i-glutathione ukuguqula ii-lipid peroxide zibe zii-lipid alcohol ezingenabungozi. Izinto ezivusa le nkqubo elabhoratri zihlasela olu khuselo kwiindawo ezichaziweyo. I-erastin ivala ukungeniswa kwe-cystine ngenkqubo i-xc-minus, nto leyo eshiya iseli ingenayo i-cysteine eyaneleyo yokwenza i-glutathione. I-buthionine sulfoximine ivala ukwenziwa kwe-glutathione ngokuthe ngqo. I-RSL3 ithintela i-GPX4 ngokwayo. Zontathu zishiya ii-peroxide zingasuswanga, kodwa oko zikwenza kumanyathelo ahlukeneyo, ngoko isiphumo esifunyenwe ngenye kuzo asisebenzi ngokuzenzekelayo kwezinye.

I-ferroptosis yahlukile kwi-apoptosis ngeempawu eziyichazayo. Akukho thotho lokusebenza kwee-caspase, yaye akukho mizimba ye-apoptosis. Izinto ezibophelela intsimbi, njenge-deferoxamine, nezinto ezibamba ii-radical, njenge-ferrostatin-1, ziyayithintela. Olu luvavanyo olusetyenziswa ngabaphandi ukuchaza ukufa njenge-ferroptosis kunokuba kube yi-necrosis nje. Ukujonga ngokubanzi iindlela zokufa kweeseli ezilawulwayo kunceda ukuqonda lo mahluko, yaye indlela iiseli ezilawula ngayo intsimbi ekulula ukuyitshintsha ngokwekhemikhali ichaza ukuba kutheni ukubophelela intsimbi kuluvavanyo olugqibayo lokuthelekisa.

Indawo ye-mitochondria, nesizathu sokuba kwaphikiswana ngayo

Uphando lwangaphambili lwaxela ukuba iiseli ezingenayo i-DNA ye-mitochondria zisafumana i-ferroptosis, nto leyo eyacebisa ukuba i-mitochondria ayisoloko iyimfuneko. Uphando lwamva lwafumanisa ukuba ukunciphisa i-mitochondria, okanye ukuvala ukuphefumla kweeseli, kungathintela i-ferroptosis ebangelwa kukunqongophala kwe-cysteine, kodwa kungayithinteli ebangelwa kukuthintelwa kwe-GPX4 ngokuthe ngqo. Zombini ezi ziphumo zinokuba yinyani ukuba igalelo le-mitochondria lixhomekeke kwinto evusa inkqubo, kuhlobo lweseli, nakwindawo apho kwenzeka khona i-peroxidation egqibayo.

Isifundo esisiseko seli nqaku sajongana nale mpikiswano sisebenzisa isalathisi esisebenza kwicandelo elithile leseli. I-MitoCLox luhlobo lwesalathisi se-peroxidation yee-lipid olujoliswe kwi-mitochondria: sitshintsha ukukhanya kwaso kwe-fluorescence xa sidibana nee-lipid peroxyl radical ngaphakathi kwi-mitochondria. Kwii-fibroblast eziphathwe nge-erastin, i-oxidation yesalathisi se-mitochondria yavela kwiiseli ezazisaphila ngaphambi kokufa, yaye yayingafani kuzo zonke iiseli. Elo xesha ibonakala ngalo libalulekile. I-peroxidation eyenzeka ngaphambi kokufa inokuba ngunobangela wako. I-peroxidation elinganiswa kwiiseli ezifileyo kuphela yintsalela. Isikhokelo esihambelana neli nqaku esimalunga novavanyo lwe-peroxidation yee-lipid noko kuboniswa sisalathisi ngasinye sichaza ukuba kutheni indawo yesalathisi imisela intsingiselo yomqondiso.

Kwavela indlela efanayo kwimodeli yesibini. Ii-fibroblast ezivela kwisigulana esinesifo semithambo-luvo yamehlo esifunyanwa njengelifa i-Leber, ezinotshintsho lwemfuza kwikhompleksi I noxinzelelo lwe-oxidation oluphezulu kwasekuqaleni, zafa nge-ferroptosis xa i-glutathione yancitshiswa, ngoxa ii-fibroblast ezazisetyenziselwa ukuthelekisa zakwazi ukumelana noko. Kwakhona, i-oxidation yesalathisi se-mitochondria yenzeka kuqala. Iinkcukacha zikwisifundo see-fibroblast malunga ne-peroxidation yee-lipid ze-mitochondria kwi-ferroptosis.

Oko kwatshintshwa yi-methylene blue kolu vavanyo

I-methylene blue ngumthwali we-redox. Uhlobo lwayo olufumene i-oxidation lwamkela ii-elektroni kwiinkqubo zekhemikhali ezinxulumene ne-NADH kwikhompleksi I, luze uhlobo lwayo oluncitshisiweyo luzinike i-cytochrome c, ezidlulisela kwikhompleksi IV. Oko kudala indlela yokudlula ngokuyinxenye kwicandelo lothotho apho ii-elektroni zidla ngokuvuza ziye kwioksijini zize zenze i-superoxide. Abafundi abafuna ukuqonda ngokupheleleyo oku kudluliselwa mab aqale ngokufunda indlela i-methylene blue ehambisa ngayo ii-elektroni kwi-mitochondria.

Kwii-fibroblast zemiphunga eziphathwe nge-erastin, i-methylene blue kwi-250 nanomolar yagcina iiseli ziphila ngokomlinganiselo wovavanyo lokuphila kweeseli, ngokunjalo nee-antioxidant ezijoliswe kwi-mitochondria i-SkQ1 ne-MitoTEMPO. Izinto ezisetyenziselwe ukuthelekisa zimisela oko kunokuthiwa ngesiphumo. I-ferrostatin-1, i-alpha-tocopherol, ne-deferoxamine nazo zakhusela iiseli, nto leyo eqinisekisa ukuba ukufa kwakubangelwa yi-ferroptosis. Ikhompawundi efana ne-SkQ1 kodwa engenalo iqela le-antioxidant ayizange ikhusele, nto leyo ekhomba kwinkqubo ye-redox kunokuqokelelana nje okungajoliswanga. Ukhuseleko lwancipha ngaphezu kwe-200 nanomolar ye-SkQ1, ngokuhambelana nokuziphatha okwaziwayo kwee-antioxidant ze-quinone, ezinokukhuthaza i-oxidation xa zininzi. I-methylene blue nayo inokuziphatha ngezi ndlela zimbini ngokuxhomekeke kwisixa: iyakhusela kumanqanaba asezantsi e-nanomolar kwezi zitya zovavanyo, kodwa inokwenza i-hydrogen peroxide ngokujikeleza kwe-redox xa inyuselwa phezulu okanye ikhanyiselwa ngamandla. Lo mba wokuxhomekeka kwisixa uxutyushwa kwinqaku ngeentlobo zeoksijini ezisebenzayo kwi-mitochondria.

Uvavanyo lwe-mitochondria eyahluliweyo lwaza lwahlula imithombo ye-ROS. Xa kukho intsimbi ekwimo ye-ferrous, ii-substrate ezondla ikhompleksi I kunye ne-rotenone zakhuthaza i-oxidation yesalathisi. I-rotenone ivala uthotho emva kwendawo ye-flavin, ngoko abathwali abangaphambi kwayo bahlala bekwimo encitshisiweyo baze bavuzise ii-elektroni kwioksijini zisuka kwi-flavin ejonge kwicala le-matrix. Ii-substrate ezondla ikhompleksi III kunye ne-antimycin A zavelisa i-hydrogen peroxide eninzi, kodwa azizange ziqhube i-peroxidation yee-lipid. Umqondiso uwonke we-ROS uyafana, kodwa indawo yahlukile nesiphumo sahlukile. I-SkQ1 ne-methylene blue nganye yathintela i-peroxidation eqhutywa yikhompleksi I ngaphandle kokuthintela ukwenziwa kwe-hydrogen peroxide ngokwayo, nto leyo ebeka isenzo sazo emva kokwenziwa kwe-ROS, kwinyathelo le-peroxidation yee-lipid.

Icandelo lentsimbi egqithisileyo nomkhondo we-lipofuscin

Isifundo sesibini sajonga lo mbuzo kwicala lentsimbi. Ii-cardiomyocyte ze-H9c2 ezivezwe kwi-ferric ammonium citrate ekumanqanaba angaphantsi kwe-millimolar zavelisa i-ROS ye-mitochondria, i-peroxidation yee-lipid ze-mitochondria, zaza zafumana i-ferroptosis, elandelwa kukuqokelelana kombala ofana ne-lipofuscin kwiiyure ezingama-24. I-lipofuscin yintsalela yee-lipid neeproteni ezifumene i-oxidation, enentsimbi nezikhanyisela ngokwayo nge-fluorescence, eqokelelana njengoko ubudala busanda. Ukuvela kwayo ngokukhawuleza apha kunxulumanisa i-peroxidation ekhawulezileyo eqhutywa yintsimbi nesalathisi esiqhelekileyo sokwaluphala. I-methylene blue ekumanqanaba e-nanomolar ne-SkQ1 nganye zathintela i-peroxidation, ukufa, nokuqokelelana kombala. I-mitochondria yentliziyo eyahluliweyo yaqinisekisa le ndlela: i-rotenone kunye nentsimbi ekwimo ye-ferrous zaqhuba i-peroxidation nomqondiso ofana nowombala, zaza zombini ezi zinto zawuthintela. Lonke olu landelelwano luxeliwe kwisifundo sentsimbi egqithisileyo kwii-cardiomyocyte ze-H9c2. Icala lombala kulo mba lihambelana nenqaku nge-lipofuscin nezalathisi zokwaluphala kweeseli.

Oko kungaboniswa luvavanyo lokuphila kweeseli

Umlinganiselo wokuphila, njenge-CellTiterBlue, ubonisa ukuba zingaphi iiseli ezisesityeni ezisasebenza ngokwemetabolism emva kokufakwa kwento evusa inkqubo kunye nento evavanyelwa ukhuseleko. Olu luvavanyo lwendlela yokusebenza kwinkqubo echaziweyo. Ayisosiphumo sonyango. Akukho nanye kwezi mvavanyo enike isigulana isixa, yalinganisa isiphumo esichaziweyo sesifo, okanye yamisela uluhlu lwezixa ezisebenzayo nezikhuselekileyo kunyango emzimbeni.

Imida emine ifanele ichazwe ngokucacileyo. Okokuqala, iintlobo zeeseli zimbalwa: ii-fibroblast zemiphunga eziguquliweyo, ii-fibroblast zokuqala zolusu eziquka umgca omnye onesifo se-mitochondria, nomgca wee-cardiomyocyte zempuku, kunye ne-mitochondria eyahluliweyo yentliziyo yempuku. Ubuntununtunu babusahluka ngomgca weeseli, yaye umgca we-LHON wawukhethwe ngenxa yokuba wawusengozini engaqhelekanga. Okwesibini, izinto ezivusa inkqubo ziimeko ezigqithisileyo zaselabhoratri: ukuvalwa ngokupheleleyo kokuthuthwa okanye ukwenziwa kwezinto, nezixa zentsimbi ezingasoze zihlangatyezwe sisihlunu ngexesha elifutshane. Okwesithathu, ukuxinana akuguquleki kube sisixa esinikwa umntu ngokuphindaphinda nje. Inqanaba le-nanomolar kulwelo lokukhulisa iiseli alisosixa somntu. Ukusasazeka kwe-methylene blue, ukuncitshiswa kwayo ibe luhlobo olungenambala egazini nakwizihlunu, ukuvezwa ekukhanyeni, nomahluko wombane kwinwebu zizinto zonke ezahlula isitya zovavanyo kumntu. Okwesine, izalathisi zibonisa okwenzeka kwicandelo lazo kuphela. I-oxidation ye-MitoCLox ibonisa ukuba ii-radical zee-lipid ze-mitochondria zenzekile; ayibonisi ukuba ukukrazuka kwenwebu yangaphandle yeseli, okunokuba sisiganeko sokugqibela, kuthintelwe ngokuthe ngqo kunokuba kuthintelwe ngamanyathelo angaphambili.

Ngoko oko kunokuthiwa ngokunyanisekileyo kukuba: kwezi modeli, i-ROS evela kwikhompleksi I kunye nentsimbi ziqhuba i-peroxidation yee-lipid ze-mitochondria eyenzeka ngaphambi kwe-ferroptosis, yaye i-methylene blue ekuxinaneni okuphantsi iphazamisa elo candelo. Nantoni na engaphaya kwelo candelo ifuna uvavanyo olwahlukileyo kwizilwanyana, kuze emva koko kuvavanywe ebantwini.