Methylene blue ndi ferroptosis: mmene peroxides amapangikira m'malipidi

Ferroptosis ndi kufa kwa selo chifukwa cha kuwonongeka kwa nembanemba. Mafuta a m'manembanemba okhala ndi ma bondi awiri angapo amasintha mwa oxidation yofalikira ngati unyolo, nembanemba imalephera kugwira ntchito, ndipo selo limafa. Chitsulo chimafunika kuti kusintha kwamankhwala kumeneku kuchitike. Chiganizo chimenechi chikufotokoza mfundo yonseyo, ndipo zonse m'nkhaniyi zikuyesa mbali imodzi ya mfundo imeneyi.

Funso la pano ndi lachindunji kwambiri: kodi methylene blue imachita chiyani pa unyolo umenewu m'mayesero a maselo, ndipo zotsatira zake zimasonyeza chiyani kwenikweni? Yankho lachidule ndi lakuti m'maphunziro awiri a mu labotale, methylene blue pa mlingo wa nanomolar inachepetsa kupangika kwa peroxides m'malipidi a mitochondria ndi kusunga maselo amoyo atakumana ndi zoyambitsa zomwe popanda iyo zimachititsa ferroptosis. Yankho lalitali ndilofunika kwambiri, chifukwa gawo lililonse la unyolowo linayesedwa pogwiritsa ntchito njira yosiyana, ndipo njira iliyonse ili ndi malire ake.

Ferroptosis m'ndime imodzi

Phospholipid ya nembanemba yokhala ndi mchira wa fatty acid wokhala ndi ma bondi awiri angapo ili ndi malo ofooka: ma atomu a haidrojeni osavuta kulandidwa. Radical ikalanda atomu imodzi, okosijeni imalumikizana ndi lipidi, lipid peroxyl radical imapangika, ndipo radical imeneyo imalanda atomu ku lipidi yotsatira. Kusinthaku kumafalikira m'nembanemba pokhapokha chinachake chikakuletsa.

Chinthu chachikulu choletsa kusinthaku ndi enzyme yotchedwa GPX4, yomwe imagwiritsa ntchito glutathione kuchepetsa lipid peroxides kukhala lipid alcohols osavulaza. Zoyambitsa za mu labotale zimasokoneza chitetezo chimenechi pa magawo odziwika. Erastin imaletsa kulowetsedwa kwa cystine kudzera mu system xc-minus, motero selo limasowa cysteine yopangira glutathione. Buthionine sulfoximine imaletsa kupangidwa kwa glutathione mwachindunji. RSL3 imaletsa GPX4 yeniyeniyo. Zonse zitatu zimasiya peroxides osachotsedwa, koma zimatero pa magawo osiyanasiyana, choncho zotsatira za choyambitsa chimodzi sizingangotengedwa kuti zikugwiranso ntchito pa zina.

Ferroptosis imasiyana ndi apoptosis pa makhalidwe amene amaizindikiritsa. Palibe unyolo woyambitsana wa caspase ndipo palibe tizidutswa ta selo totchedwa apoptotic bodies. Zinthu zomanga chitsulo, monga deferoxamine, ndi zinthu zogwira ma radical, monga ferrostatin-1, zimachepetsa ferroptosis. Amenewa ndi mayeso amene ofufuza amagwiritsa ntchito kunena kuti kufa kwa selo ndi kwa ferroptosis osati necrosis yokha. Kumvetsa njira zosiyanasiyana zoyendetsedwa za kufa kwa maselo kumathandiza kufotokoza kusiyana kumeneku, ndipo mmene maselo amasamalirira chitsulo chosavuta kutenga nawo mbali pa kusintha kwamankhwala kumafotokoza chifukwa chake kumanga chitsulo ndi njira yofunika kwambiri yotsimikizira zotsatira.

Kumene mitochondria imalowerera, ndi chifukwa chake panali mkangano

Kafukufuku woyambirira ananena kuti maselo opanda DNA ya mitochondria amakumanabe ndi ferroptosis, zomwe zinasonyeza kuti mitochondria mwina si yofunika kuti izi zichitike. Kafukufuku wotsatira anapeza kuti kuchepetsa mitochondria, kapena kuletsa kupuma kwa selo, kungachepetse ferroptosis yochokera ku kusowa kwa cysteine koma osati yochokera ku kuletsedwa kwa GPX4 mwachindunji. Zotsatira zonsezi zingakhale zoona ngati ntchito ya mitochondria imadalira choyambitsa, mtundu wa selo, ndi malo amene kupangika kwa peroxides kofunika kwambiri kumachitikira.

Kafukufuku amene nkhaniyi yazikidwapo anafufuza mkanganowu pogwiritsa ntchito chinthu choyezera chogwira ntchito m'gawo linalake la selo. MitoCLox ndi mtundu wa chizindikiro choyezera kupangika kwa peroxides m'malipidi womwe umalunjika ku mitochondria: umasintha kuwala kwake kwa fluorescence ukakumana ndi lipid peroxyl radicals mkati mwa mitochondria. M'maselo a fibroblast omwe anapatsidwa erastin, oxidation ya choyezera cha mitochondria inaonekera m'maselo omwe anali adakali amoyo asanafe, ndipo sinali yofanana m'maselo onse. Nthawi imeneyo ndi yofunika. Kupangika kwa peroxides komwe kumachitika selo lisanafe kungakhale chimodzi mwa zoyambitsa kufako. Kupangika kwa peroxides koyezedwa m'maselo akufa okha ndi zotsalira za kuwonongeka. Buku lotsagana nalo lofotokoza mayeso a kupangika kwa peroxides m'malipidi ndi zimene choyezera chilichonse chimasonyeza limafotokoza chifukwa chake malo a choyezera amatsimikizira tanthauzo la chizindikirocho.

Mchitidwe womwewo unaonekera m'chitsanzo chachiwiri. Maselo a fibroblast ochokera kwa wodwala matenda obadwa nawo a mitsempha ya maso a Leber, omwe ali ndi kusintha kwa majini kwa complex I komanso vuto la oxidation lokwera kuyambira pachiyambi, anafa mwa ferroptosis pamene glutathione inachepetsedwa, pamene maselo a fibroblast oyerekezera anapirira. Oxidation ya choyezera cha mitochondria inayambanso kuonekera kufa kusanachitike. Tsatanetsatane ali mu kafukufuku wa maselo a fibroblast wokhudza kupangika kwa peroxides m'malipidi a mitochondria pa ferroptosis.

Zimene methylene blue inasintha m'mayesero amenewa

Methylene blue ndi chonyamula cha redox. Mtundu wake womwe wasintha mwa oxidation umalandira ma elekitironi kuchokera ku kusintha kwamankhwala kogwirizana ndi NADH pa complex I, ndipo mtundu wake wochepetsedwa mwa reduction umawapereka ku cytochrome c, yomwe imawapititsa ku complex IV. Izi zimapanga njira yodutsira pambali pa gawo la unyolo kumene ma elekitironi nthawi zambiri amatayikira ku okosijeni n'kupanga superoxide. Owerenga amene akufuna chithunzi chonse cha kupatsirana kumeneku ayenera kuyamba ndi mmene methylene blue imayendetsera ma elekitironi kudzera mu mitochondria.

M'maselo a fibroblast a m'mapapo omwe anapatsidwa erastin, methylene blue pa 250 nanomolar inasunga maselo amoyo malinga ndi muyeso wa kupulumuka kwa maselo, mofanana ndi zinthu zoletsa oxidation zolunjika ku mitochondria, SkQ1 ndi MitoTEMPO. Zinthu zoyerekezera zimafotokoza malire a zimene tinganene. Ferrostatin-1, alpha-tocopherol, ndi deferoxamine zinatetezanso maselo, zomwe zimatsimikizira kuti kufako kunali kwa ferroptosis. Mtundu wofanana ndi SkQ1 koma wopanda gulu loletsa oxidation sunateteze maselo, zomwe zimaloza ku kusintha kwamankhwala kwa redox osati kungowunjikana kopanda ntchito yapadera. Chitetezo chinachepa pamene SkQ1 inaposa pafupifupi 200 nanomolar, mogwirizana ndi khalidwe lodziwika la zinthu zoletsa oxidation za quinone loyambitsa oxidation zikachuluka. Methylene blue ilinso ndi khalidwe losiyana malinga ndi mlingo: inateteza pa milingo yochepa ya nanomolar m'mbale za mayeso amenewa, koma ingapange hydrogen peroxide mwa kusinthasintha kwa redox mlingo ukakwezedwa kapena ikawala ndi kuwala kwamphamvu. Kufunika kwa mlingo kotereku kumafotokozedwa mu mitundu ya okosijeni yogwira ntchito kwambiri mu mitochondria.

Mayesero a mitochondria yolekanitsidwa ndi maselo kenako anasiyanitsa magwero a ROS. Pakakhala chitsulo cha ferrous, zinthu zopatsa complex I ma elekitironi pamodzi ndi rotenone zinalimbikitsa oxidation ya choyezera. Rotenone imatsekereza njira pambuyo pa malo a flavin, choncho zonyamula za magawo oyambirira zimakhalabe mumtundu wochepetsedwa ndipo zimatayitsa ma elekitironi kupita ku okosijeni kuchokera ku flavin yoyang'ana ku matrix. Zinthu zopatsa complex III ma elekitironi pamodzi ndi antimycin A zinapanga hydrogen peroxide yambiri koma sizinayambitse kupangika kwa peroxides m'malipidi. Chizindikiro cha ROS yonse chinali chofanana, koma malo anali osiyana ndipo zotsatira zinali zosiyana. SkQ1 ndi methylene blue chilichonse chinachepetsa kupangika kwa peroxides koyendetsedwa ndi complex I popanda kuchepetsa kupangidwa kwa hydrogen peroxide komweko. Izi zikusonyeza kuti zimagwira ntchito ROS itapangidwa kale, pa gawo la kupangika kwa peroxides m'malipidi.

Gawo la chitsulo chochuluka ndi chizindikiro cha lipofuscin

Kafukufuku wachiwiri anafunsa funsoli kuchokera ku mbali ya chitsulo. Maselo a minofu ya mtima a H9c2 omwe anapatsidwa ferric ammonium citrate pa mlingo wa submillimolar anapanga ROS ya mitochondria, kenako peroxides m'malipidi a mitochondria, kenako anakumana ndi ferroptosis, ndipo pambuyo pake mtundu wofanana ndi lipofuscin unawunjikana mkati mwa 24 hours. Lipofuscin ndi zotsalira za malipidi ndi mapuloteni omwe asintha mwa oxidation, zokhala ndi chitsulo komanso zowala zokha mwa fluorescence, zomwe zimawunjikana munthu akamakalamba. Kuonekera kwake mwamsanga pano kumagwirizanitsa kupangika kwadzidzidzi kwa peroxides koyendetsedwa ndi chitsulo ndi chizindikiro chodziwika cha kukalamba. Methylene blue pa mlingo wa nanomolar ndi SkQ1 chilichonse chinaletsa kupangika kwa peroxides, kufa, ndi kuwunjikana kwa mtunduwo. Mitochondria yolekanitsidwa ndi maselo a mtima inatsimikizira mchitidwewo: rotenone pamodzi ndi chitsulo cha ferrous zinayambitsa kupangika kwa peroxides ndi chizindikiro chofanana ndi mtunduwo, ndipo zinthu zonse ziwiri zinaziletsa. Ndondomeko yonseyi yafotokozedwa mu kafukufuku wa chitsulo chochuluka m'maselo a minofu ya mtima a H9c2. Mbali ya nkhaniyi yokhudza mtunduwo ikugwirizana ndi lipofuscin ndi zizindikiro za kukalamba kwa maselo.

Zimene mayeso a kupulumuka kwa maselo sasonyeza

Muyezo wa kukhala moyo monga CellTiterBlue umasonyeza kuchuluka kwa maselo m'bale omwe akupitiriza kuchita ntchito za kagayidwe atapatsidwa choyambitsa pamodzi ndi chinthu choyesedwa ngati choteteza. Amenewa ndi mayeso a njira yogwirira ntchito m'mikhalidwe yodziwika. Si zotsatira za chithandizo. Palibe mayeso amenewa amene anapereka mlingo kwa wodwala, kuyeza zotsatira za matenda, kapena kukhazikitsa malire a mlingo wa chithandizo wogwira ntchito komanso wotetezeka m'thupi.

Malire anayi ayenera kunenedwa momveka. Choyamba, mitundu ya maselo inali yochepa: maselo a fibroblast a m'mapapo osinthidwa, maselo oyambirira a fibroblast a pakhungu kuphatikizapo mzere umodzi wa maselo okhala ndi matenda a mitochondria, ndi mzere wa maselo a minofu ya mtima wa khoswe, kuphatikizapo mitochondria yolekanitsidwa ndi maselo a mtima wa khoswe. Kukhudzidwa kunasiyana malinga ndi mzere wa maselo, ndipo mzere wa LHON unasankhidwa chifukwa unali wosavuta kuvulazidwa kuposa nthawi zonse. Chachiwiri, zoyambitsazo ndi mikhalidwe yoopsa ya mu labotale: kutsekereza kwathunthu kwa kayendedwe ka zinthu kapena kupangidwa kwa zinthu, komanso kuchuluka kwa chitsulo komwe palibe minofu ingakumane nako mwadzidzidzi. Chachitatu, kuchuluka kwa chinthu m'njira yoyesera sikungasandulidwe kukhala mlingo wa munthu mwa kungochulukitsa. Nanomolar m'zamadzimadzi zokulitsira maselo si mlingo woperekedwa kwa munthu. Kufalikira kwa methylene blue, kusanduka kwake mwa reduction kukhala mtundu wopanda mtundu m'magazi ndi m'minofu, kukumana ndi kuwala, ndi kusiyana kwa mphamvu yamagetsi pa nembanemba zonse zimapangitsa kusiyana pakati pa mbale ya mayeso ndi munthu. Chachinayi, zoyezera zimasonyeza zimene zikuchitika m'gawo lawo lokha. Oxidation ya MitoCLox imasonyeza kuti ma radical a malipidi a mitochondria apangika; sisonyeza kuti kusweka kwa nembanemba yakunja ya selo, komwe mwina ndi chochitika chomaliza, kunaletsedwa mwachindunji m'malo mongopewedwa chifukwa chosokoneza magawo oyambirira.

Choncho zimene tinganene moona mtima ndi izi: m'machitsanzo amenewa, ROS yochokera ku complex I pamodzi ndi chitsulo zimayendetsa kupangika kwa peroxides m'malipidi a mitochondria komwe kumachitika ferroptosis isanayambe, ndipo methylene blue pa kuchuluka kochepa imadula gawo limenelo. Chilichonse chopitirira gawo limenelo chimafuna mayesero ena pa nyama kenako pa anthu.