Kuyeza mmene methylene blue imakhudzira mitokondiriya: oksijeni, ATP, ndi mphamvu ya magetsi pa nembanemba

Tiyerekeze kuti labotale yawonjezera methylene blue ku mitokondiriya imene kupuma kwake kwasokonekera, ndipo ikuona kugwiritsidwa ntchito kwa oksijeni kukuwonjezeka. Mawu amene angabwere mosavuta polemba ndi akuti utotowo wabwezeretsa ntchito ya mitokondiriya. Mawuwo angakhale olakwika. Liwiro la kugwiritsidwa ntchito kwa oksijeni, kupangidwa kwa ATP, ndi mphamvu ya magetsi pa nembanemba ndi zinthu zitatu zosiyana. Zimagwiritsa ntchito chiwalo chimodzi cha selo, monga momwe kusunga kopi yosungirako, kugwirizanitsa mafayilo, ndi kugawana mafayilo zimagwiritsira ntchito foda imodzi ya pa mtambo: zimagwirizana, koma zimayendetsedwa ndi malamulo osiyana. Mukasokoneza chimodzi ndi chinzake, kutanthauzira kumalephera, ngakhale muyeso uliwonse wachitika molondola mwaukadaulo.

Methylene blue imapangitsa kusokoneza kumeneku kukhala kosavuta chifukwa imatenga nawo mbali m'dongosolo limene ikuyezedwalo. Imadutsa mobwerezabwereza pakati pa oxidation ndi reduction, kulandira maelekitironi kumayambiriro kwa njira ndi kuwapereka kutsogolo kwake. Ndi katayoni wokonda mafuta amene amaunjikana m'mitokondiriya yokhala ndi mphamvu, komanso ndi mankhwala abuluu kwambiri amene amayamwa ndi kutulutsa kuwala m'magulu omwewo a kutalika kwa mafunde monga zipangizo zambiri zoyezera. Muyezo umodzi sungathe kufotokoza zonsezi. Njira yodalirika, imene ikuwoneka m'maphunziro a mitokondiriya a ku Budapest, ndi kuyeza liwiro la kugwiritsidwa ntchito kwa oksijeni pamodzi ndi liwiro la kupangidwa kwa ATP, mphamvu ya magetsi pa nembanemba, ndi kutulutsidwa kwa hydrogen peroxide, pogwiritsa ntchito mayeso otsimikizira okhudza utotowo pa gawo lililonse.

Kuyeza kupuma kumayeza liwiro la kugwiritsidwa ntchito kwa oksijeni, osati mphamvu yopangidwa

Kuyeza kupuma mwatsatanetsatane kwambiri, kaya ndi Oroboros Oxygraph kapena Seahorse extracellular flux analyzer, kumasonyeza chinthu chimodzi: liwiro limene oksijeni ikulandirira maelekitironi. Sikumanena kuti mapurotoni angati anapopedwa pa elekitironi iliyonse, kuti kusiyana kwa mphamvu kunasungidwa kotani, kapena ngati ATP synthase inagwiritsa ntchito kusiyanako.

Mawu amene amagwiritsidwa ntchito ndi ofunika. M'mitokondiriya yopatulidwa, zinthu zodyetsera njira popanda ADP zimapereka kupuma chifukwa cha kutayikira kwa mapurotoni, zinthu zodyetsera njira zokhala ndi ADP yokwanira kwambiri zimapereka mphamvu yopangira ATP mwa oxidative phosphorylation, ndipo kuwonjezera uncoupler pang'onopang'ono kumasonyeza mphamvu yaikulu kwambiri yosamutsa maelekitironi. M'maselo athunthu, miyeso yofanana ya Seahorse ndi kupuma koyambira, kupuma kogwirizana ndi ATP komwe kumakhudzidwa ndi oligomycin, kutayikira kwa mapurotoni, kupuma kwakukulu kwambiri kotanthauzidwa ndi FCCP, ndi kugwiritsidwa ntchito kwa oksijeni kosachokera ku mitokondiriya komwe kumapitirira ngakhale pali rotenone ndi antimycin. Kutchula zotsatira za Seahorse kuti State 3 ndi State 4 kumapereka chithunzi cha kulondola komwe mtundu wa zinthu zoyezedwazo sungachirikize, monga momwe dongosolo la Agilent Mito Stress Test limafotokozera.

Methylene blue imasokoneza lingaliro losavuta lakuti oksijeni wambiri amatanthauza mphamvu zambiri. M'mitokondiriya yopatulidwa ya ubongo wa nkhumba za guinea, utoto wa 100 nM mpaka 1 uM unawonjezera kugwiritsidwa ntchito kwa oksijeni pa nthawi yopuma yosayambitsidwa ndi ADP, pogwiritsa ntchito glutamate pamodzi ndi malate, succinate, ndi alpha-glycerophosphate monga zinthu zodyetsera njira. Kuwonjezekako kunali pafupifupi 64 peresenti pa alpha-glycerophosphate, koma kupuma koyambitsidwa ndi ADP m'mitokondiriya yathanzi sikunasinthe kwenikweni, malinga ndi kafukufuku wa mitokondiriya yopatulidwa ya ubongo. Chifukwa kupuma kotayikira kunawonjezeka pamene liwiro loyambitsidwa ndi ADP silinasinthe, chiŵerengero chowerengeredwa cha kuwongolera kupuma chimatsika. Kutsika kumeneku sikutanthauza kuti utotowo unawononga mitokondiriya. Kumatanthauza kuti utotowo unawonjezera kuzungulira kwa redox komwe kumagwiritsa ntchito oksijeni pamwamba pa kupuma kwabwinobwino. Mayeso a kugwiritsidwa ntchito kwa oksijeni ndi methylene blue amene amangonena za kuwonjezekako ayesa zochitika zenizeni za mankhwala, koma sananene chilichonse chokhudza kusungidwa kwa mphamvu.

Mayeso obwezeretsa ntchito amasonyeza kusiyana komweku pamlingo waukulu. Complex I itatsekedwa, liwiro la kugwiritsidwa ntchito kwa oksijeni linabwerera pafupifupi katatu, pamene kupangidwa kwa ATP kunabwerera pafupifupi 1.5 kokha. Oksijeni wambiri anafika pa elekiturodi. Gawo locheperapo la oksijeni ameneyo linathandiza kupanga ATP. Mawu aliwonse oti oxidative phosphorylation yasintha kukhala yabwino amafunika umboni wa mbali ya ATP, makamaka wofotokozedwa monga chiŵerengero cha ATP pa oksijeni, ndi kufotokoza njira yowerengera oksijeni amene waikidwa pansi pa chiŵerengerocho.

Mayeso a ATP amayeza liwiro la kupangidwa kwake, limene kuchuluka kwake sikungalowe m'malo

Mayeso a ATP amayankha funso lina: kodi ATP imapangidwa mochuluka bwanji pa nthawi inayake? Kuyeza kosonyeza kusintha pakapita nthawi, kaya ndi kutsatira kuwala kochokera ku luciferase ya ziphaniphani kwa mphindi zingapo kapena ndi mayeso ophatikiza hexokinase ndi glucose-6-phosphate dehydrogenase omwe amawerengedwa pa 340 nm, kumapereka liwiro. Muyezo umodzi wa ATP yonse kumapeto umapereka kuchuluka komwe kulipo. Kuchuluka kumeneku kumasonyeza kupangidwa kuchotsapo kugwiritsidwa ntchito, ndipo kokha sikumanena zambiri za oxidative phosphorylation.

Gulu la ku Budapest linagwiritsa ntchito njira ya ma enzyme ophatikizidwa, litachepetsa ntchito ya adenylate kinase, ndipo linaona kuti methylene blue ya 1 mpaka 2 uM inawonjezera kupangidwa kwa ATP pang'ono koma m'njira yofunika pa ziwerengero m'mitokondiriya yomwe inathiridwa rotenone ndi yomwe inathiridwa antimycin. Uku ndi kubwezeretsa kwenikweni, koma ndi kwa mbali yokha. Kafukufuku wa poizoni wa TEGDMA anapeza yankho lofanana m'chitsanzo china: monoma wa mano wa TEGDMA wa 5 mM unatsitsa kupuma kogwirizana ndi Complex I kuchokera pa pafupifupi 286 kufika pa 28 mayunitsi a liwiro, ndipo utoto wa 2 uM unakubwezeretsa kufika pa pafupifupi 86.

Mayeso awiri otsimikizira sangasiyidwe mukamagwiritsa ntchito mankhwala a mtundu amene amatenga nawo mbali mu redox. Choyamba, gwiritsani ntchito oligomycin kuti mudziwe kuchuluka kwa kupangidwa kwa ATP koyezedwako komwe kumadalira ATP synthase. Chachiwiri, chitani mayeso opanda maselo oona ngati ATP yodziwika kuchuluka kwake, yomwe yawonjezedwa, imayesedwa bwino, pogwiritsa ntchito ATP imeneyo pamodzi ndi zinthu zonse za mayeso ndi milingo yeniyeni ya utoto yomwe yagwiritsidwa ntchito, osachepera 0, 100 nM, 300 nM, 1 uM, ndi 2 uM. Utotowo ungayamwe kuwala kotulutsidwa, kulepheretsa enzyme yosonyeza zotsatira, kapena kusintha zochitika za redox za zinthu za mayeso. Ndipo malangizo a mayeso a luciferase amalimbikitsa mayeso ena otsimikizira a biochemical chifukwa cha kusokoneza kwa mtundu umenewu. Musaganize kuti 1 uM ilibe kusokoneza kwa muyezo chifukwa chakuti kuchepetsedwa kwa kuwala nthawi zambiri kumakhala kwakukulu pa milingo yapamwamba.

Zipangizo zoyezera mphamvu ya magetsi pa nembanemba zimayeza kugawikana kwa utoto, osati liwiro la kayendedwe

Safranine O, TMRM ndi TMRE, rhodamine 123, ndi JC-1 onse ndi makat ayoni amene amadutsa nembanemba. Amaunjikana mu matrix yokhala ndi chaji yoipa malinga ndi kufanana kwa mphamvu za magetsi ndi za mankhwala. Choncho fluorescence yonse imasonyeza mphamvu ya magetsiyo mosalunjika kudzera pamalo omwe choyezera chili. Palibe mwa zimenezi chimene chimayeza ATP kapena liwiro la kayendedwe ka maelekitironi.

Choyezera chilichonse chili ndi mtundu wa zinthu zoyezedwa womwe chimagwirizana nawo bwino. Safranine O imagwirizana ndi mitokondiriya yopatulidwa komanso maselo otsegulidwa nembanemba kuti zinthu zidutse. Nthawi zambiri imagwiritsidwa ntchito pafupi ndi 2 uM, ndi kuwala koyambitsa pafupi ndi 495 nm ndi kotuluka pafupi ndi 585 nm, m'njira yochepetsera kuwala imene kulowa kwake kumasintha fluorescence yonse. TMRM ndi TMRE zimagwirizana ndi maselo athunthu zikagwiritsidwa ntchito pa milingo yochepa ya nanomolar m'njira yosachepetsa kuwala. Mmenemo, kutayika kwa kusiyana kwa mphamvu ya magetsi kumatulutsa utoto ndipo fluorescence ya mitokondiriya imatsika; pa milingo yochuluka, kumene chizindikiro chikupita kungasinthike chifukwa utotowo umachepetsa kuwala kwake wokha. JC-1 imapereka chiŵerengero chosavuta cha kuwala kobiriwira poyerekeza ndi kofiira, koma chimadalira kuchuluka kwa utoto wolowetsedwa ndi kuchuluka kwa mitokondiriya. Izi zimapangitsa kukhala yofooka pa ntchito yowerengera molondola kuposa TMRM yoyendetsedwa mosamala. Ubwino ndi zoperewera zimenezi zafotokozedwa mwachidule mu ndemanga yogwiritsidwa ntchito kwambiri ya zipangizo zoyezera mphamvu ya magetsi pa nembanemba ya mitokondiriya.

Methylene blue imasokoneza kuyeza kwa kuwala pa milingo yeniyeni yomwe imagwiritsidwa ntchito. Magulu ake oyamwa kuwala pafupi ndi 600 mpaka 665 nm amalowererana ndi kuwala kofiira kotulutsidwa ndi zoyezera. Choncho, zotsatira za kusefa kuwala mkati, kuchepetsedwa kwa kuwala, ndi kuyamwa kuwala koyambitsa zonse zingachitike. Utotowo ulinso katayoni wachiwiri wokonda mafuta, amene kugawikana kwake kumasintha ndi mphamvu ya magetsi yomwe ikuyezedwa. Njira yodzitetezera ndi kugwiritsa ntchito miyeso yobwerezanso ndi zitsanzo zopanda chinthu choyezedwa: choyezera chokha, utoto wokha, choyezera pamodzi ndi mlingo uliwonse wa utoto, kuchotsa kwathunthu kusiyana kwa mphamvu ya magetsi ndi FCCP monga muyezo wa ziro, komanso, ngati n'kotheka, choyezera chachiwiri chokhala ndi makhalidwe osiyana a kuwala. Kafukufuku wa kudutsa pambali pa Complex III unapereka chitsanzo chimenechi pobwereza kuyeza kubwerera kwa mphamvu ya magetsi pa nembanemba ndi safranine O komanso TMRM pambuyo pa kulepheretsa ndi antimycin ndi myxothiazol, ndi kupeza kubwerera kofanana pa mtundu wa zotsatira. Chosonyeza chimodzi cha kuwala chimapereka lingaliro. Zosonyeza ziwiri zodziimira paokha zimapereka umboni.

Mitundu itatu ya zinthu zoyezedwa, mafunso atatu osiyana

Mitokondiriya yopatulidwa, maselo otsegulidwa nembanemba kuti zinthu zidutse, ndi maselo athunthu sizili milingo yosinthana ya kuzindikira kwa mayeso amodzi. Ndi zinthu zosiyana zokhala ndi mfundo zosiyana za kagwiridwe ka ntchito.

Mitokondiriya yopatulidwa imasunga unyolo wosamutsa maelekitironi, nembanemba yamkati, ndi makina a mu matrix, koma imachotsa nembanemba ya selo, cytosol, ndi kapangidwe ka netiweki. Kuchotsa kumeneko ndiko cholinga chake: zinthu zodyetsera njira, ADP, zolepheretsa, ndi milingo ya utoto zimadziwika molondola, motero njira ingadziwike. Chomwe chimatayika ndi kuperekedwa kwa zinthu zodyetsera njira mwachibadwa, kutumizirana zizindikiro, ndi kufunika kwa mphamvu.

Maselo otsegulidwa nembanemba amasunga mitokondiriya m'kapangidwe kambiri ka selo lake, koma amalola zinthu zodyetsera njira zochokera kunja ndi ADP kudutsa mu nembanemba ya selo yotsegulidwayo. Ndi chida choyenera chodziwira vuto la njira yogwiritsa ntchito zinthu zodyetsera mkati mwa selo. Kutsegula kwambiri kumawononga nembanemba yakunja, ndipo kuwongolera kochokera mu cytosol kumachotsedwa ndi kutsuka.

Maselo athunthu amasunga zonse: chotchinga cha nembanemba ya selo, zinthu zopereka maelekitironi za mu cytosol, kusamutsidwa kwa zinthu zodyetsera zopangidwa mkati, netiweki ya mitokondiriya, ndi kufunika kwa ATP. Ndi amene ali pafupi kwambiri ndi mmene thupi limagwirira ntchito, koma ali kutali kwambiri ndi kuzindikira njira yeniyeni, chifukwa kulowa kwa utoto, kulandira kwake maelekitironi, ndi kusungidwa kwake m'madera ena zonse zimakhudza kuchuluka kwa utoto komwe mitokondiriya imakumana nako.

Kafukufuku wa TEGDMA unagwiritsa ntchito milingo yonse itatu mwadala: mitokondiriya yopatulidwa ya ubongo poyesa kubwezeretsa oxidation ndi mphamvu ya magetsi ndi utoto; maselo a tsinde ochokera mkati mwa mano, otsegulidwa nembanemba, makamaka pofuna kudziwa kuti vuto linali pa oxidation ya zinthu zodyetsera yogwirizana ndi Complex I; ndi maselo athunthu a tsinde poyesa zotsatira zonse pa kagwiridwe ka selo za utoto wa 2 uM pa kugwiritsidwa ntchito kwa oksijeni komwe kunachepetsedwa. Kwa owerenga oyerekeza miyeso ya mitokondiriya pakati pa mitundu ya zinthu zoyezedwa, nkhani yowonjezera yofotokoza mmene methylene blue imasamutsira maelekitironi mu unyolo wa kupuma imapereka maziko a njirayo, ndipo bukhu la kuwerenga maphunziro a methylene blue okhudza kupuma limafotokoza mfundo za milingo ndi zolepheretsa.

Mayeso otsimikizira amene utoto wotenga nawo mbali mu redox umafunikira

Chinthu chimene chimazungulira mobwerezabwereza mu redox chimafuna mndandanda wautali wa mayeso otsimikizira kuposa cholepheretsa wamba, chifukwa zochitika za mankhwala za utoto wokha zingatsanzire zochitika za zamoyo.

Poyeza kupuma, yesani chipinda chokhala ndi buffer yokha kuti muone kusuntha kwa muyezo, chipinda chokhala ndi buffer ndi utoto popanda zinthu za zamoyo, utoto pamodzi ndi chinthu chilichonse chopereka maelekitironi chakunja kwa maselo chomwe chili m'mayesowo, komanso rotenone ndi antimycin kumapeto kuti mudziwe kugwiritsidwa ntchito kwa oksijeni kosachokera ku mitokondiriya m'maselo athunthu. Dziwani kuti utoto womwe walandira maelekitironi umabwereranso ku oxidation popereka maelekitironi kwa oksijeni ndi kupanga hydrogen peroxide. Choncho chitsanzo cha utoto wokha chopanda wopereka maelekitironi chimachepetsa chiŵerengero cha kugwiritsidwa ntchito kwa oksijeni popanda maselo komwe kumaoneka NADH kapena NADPH ikapezeka.

Poyeza hydrogen peroxide, sankhani Amplex UltraRed yoyezedwa ndi miyezo yodziwika pamodzi ndi horseradish peroxidase m'malo mwa utoto wa DCFDA wosasankha chinthu chimodzi. Tsimikizirani ndi catalase, ndipo yesani ngati zotsatira zimadalira kuwala, chifukwa utotowo umayambitsa zochitika za mankhwala ukawunikiridwa. Kafukufuku wa mitokondiriya ya ubongo anapeza kuti kuchuluka kwa peroxide kunakwera kuposa kanayi ndi utoto wa 100 nM wokha, pamene kuchotsedwa kwake kunachedwa. Peroxide inapangidwa mwachindunji ndi utoto womwe walandira maelekitironi, popanda superoxide kukhala gawo lapakati. Tsatanetsatane wa kusankha choyezera ndi mayeso otsimikizira okhudza kuwala uli m'nkhani yowonjezera ya kuyeza mitundu ya oksijeni yochita zochitika mosavuta m'mayesero a utoto, ndipo funso lalikulu la nthawi imene peroxide imagwira ntchito ngati chizindikiro m'malo mokhala cholemetsa lafotokozedwa pamodzi ndi kulinganika kwa redox m'maselo ndi zonena za antioxidants.

Pazonena zokhudza njira, yesani malo onse awiri olepheretsera Complex III: antimycin A pa malo a Qi ndi myxothiazol pa malo a Qo, komanso muyezo wakumapeto wa Complex IV pogwiritsa ntchito cyanide. Kafukufuku wakale wa ubongo wa mbewa unanena kuti utoto unabwezeretsa kupuma kotsekedwa ndi rotenone, koma osati kotsekedwa ndi antimycin. Koma kafukufuku wotsatira wa mitundu itatu ya nyama unapeza kubwezeretsa kwa mbali ndi zolepheretsa zonse ziwiri komanso umboni wakuti cytochrome c inalandira maelekitironi mwachindunji. Tengani kudutsa pambaliko ngati kogwirizana ndi deta yatsopano, osati ngati nkhani yotsimikizidwa kotheratu, chifukwa mlingo wa utoto, malo a cholepheretsa, mmene zinthu zodyetsera zilili, ndi kugawikana kwa utoto zonse zimasintha njira imene imaoneka.

Tebulo losankha mayeso

Gwiritsani ntchito tebulo ili posankha muyezo malinga ndi funso, osati kusankha funso malinga ndi muyezo.

Zonena zochepa zimene zingakhutiritse zimafunika kugwiritsa ntchito milingo yofanana ya utoto, 100 nM, 300 nM, 1 uM, ndi 2 uM, poyeza liwiro la kugwiritsidwa ntchito kwa oksijeni pa mikhalidwe ya kutayikira, ADP, oligomycin, ndi uncoupler; liwiro la kupangidwa kwa ATP; mphamvu ya magetsi pa nembanemba, makamaka ndi zoyezera ziwiri; komanso kutulutsidwa kwa peroxide koyezedwa ndi miyezo yodziwika, pogwiritsa ntchito catalase ndi mayeso otsimikizira opanda maselo. Nenani miyeso yonse yolumikizanayi: liwiro la kugwiritsidwa ntchito kwa oksijeni pamodzi ndi liwiro la ATP, ATP pa oksijeni, mphamvu ya magetsi pa nembanemba, ndi peroxide. Nambala iliyonse mwa zimenezi ikakhala yokha ndi muyezo umene ukufunafuna tanthauzo.