Kuyeza mitundu ya okosijeni yogwira kwambiri mu mayesero a methylene blue

Ma labotale awiri akuchita zomwe aliyense amatcha mayesero a methylene blue oletsa oksideshoni. Limodzi likunena kuti utotowo umachepetsa mitundu ya okosijeni yogwira kwambiri. Lina likunena kuti umapanga mitundu imeneyi. Onse akuwonetsa mizere yosalala yosonyeza mmene zotsatira zimasinthira ndi kuchuluka kwa utoto. Onse akunena zoona malinga ndi zomwe apeza. Kusiyanaku sikuchokera ku chinyengo kapena kusadziwa ntchito. Kumachokera poti magulu awiriwa anayeza zinthu zosiyanasiyana pansi pa kuwala kosiyanasiyana, pogwiritsa ntchito zowunikira zomwe zimayankha mafunso osiyanasiyana, kenako anafotokoza mayankho onse ndi liwu limodzi: ROS.

Methylene blue ndi molekyulu yovuta kuyeza molondola chifukwa imachita nawo machitidwe a kemikali omwe iyenera kungowunikira. Mu mdima, imanyamula maelekitironi pakati pa zopereka ndi zolandira maelekitironi, kenako imabwerera ku mawonekedwe ake oyambirira, yokonzeka kubwereza njirayo. Pansi pa kuwala, imakhala chinthu choyambitsa machitidwe a kemikali ndi kuwala, n'kufika pa mkhalidwe wa mphamvu yowonjezeka wa triplet. Kuchokera pamenepo, ingapereke mphamvu kwa okosijeni kuti ipange singlet oxygen, kapena kusamutsa maelekitironi kuti ipange ma radical ndi hydrogen peroxide. Chowunikira chilichonse choikidwa mu dongosololi chingachitidwe oksideshoni, ridakeshoni, kutayitsidwa mtundu, kapena kubisidwa pa muyeso wa kuwala ndi utoto womwewo. Choncho funso loyamba pa zotsatira zilizonse za ROS silikhala lakuti nambalayo ndi iti. Limakhala lakuti mayesowo amazindikira chiyani kwenikweni, ndipo utotowo umachita chiyani pa mayesowo.

Amplex Red: mayeso a hydrogen peroxide osanjidwa ndi miyeso yodziwika

Kuphatikiza kwa methylene blue ndi Amplex Red ndi njira yodalirika kwambiri m'mabuku a kafukufuku amenewa, ngati yakonzedwa bwino. Machitidwe ake a kemikali ndi olunjika pa chinthu chimodzi: horseradish peroxidase imagwiritsa ntchito hydrogen peroxide kuchititsa oksideshoni ya chinthu cha Amplex kuti chikhale resorufin, yomwe imatulutsa kuwala kwa fuloresensi, pa chiŵerengero cha mamolekyulu cha pafupifupi chimodzi ndi chimodzi. Peroxide ya m'chitsanzo imasanduka kuwala kwa fuloresensi pa chipangizo chozindikira. Palibe chinthu china cha m'gulu lodziwika la ROS chomwe chimayendetsa mwachindunji machitidwe amenewa.

Ubwino waukulu ndi kusanja miyeso pogwiritsa ntchito miyezo yodziwika. Wogwira ntchito mosamala amayesa peroxide ya kuchuluka kodziwika mu madzi omwewo osunga pH, pogwiritsa ntchito kuchuluka kofanana kwa peroxidase ndi chinthu chomwe imachita nacho, nthawi yofanana yosungira kuti machitidwe achitike, ndi makonzedwe ofanana a zida za kuwala. Kenako amayerekezera zitsanzo ndi mzere wa miyeso imeneyo. Mu kafukufuku wa mitochondria yopatukana ndi ubongo womwe ndi maziko a njirayi, olemba anachita zimenezi ndendende: anawonjezera peroxide ya kuchuluka kodziwika kumapeto kwa mayeso aliwonse. Ziŵerengero zawo zinalembedwa ngati liwiro la kupanga mu picomoles pa mphindi pa milligram ya mapuloteni, osati mayunitsi a fuloresensi opanda muyezo wodziwika. Zimenezi n'zimene zimasandutsa kuwala kukhala muyeso.

Mfundo zitatu za kafukufuku ameneyo ndi zoyenera kutsanzira. Choyamba, olemba anatsimikizira chizindikirocho ndi catalase, yomwe imawononga peroxide. Kuwonjezeka kwa fuloresensi koyambitsidwa ndi methylene blue kunasiya pamene catalase inawonjezedwa, kutsimikizira kuti chizindikirocho chinali cha peroxide osati cholakwika cha muyeso wa kuwala. Chachiwiri, anayesa kuunikidwa mwachindunji potseka ndi kutsegula mtengo wa kuwala koyambitsa fuloresensi, kotero kuti zitsanzo zinalandira gawo limodzi mwa magawo asanu ndi atatu la kuwala komwe zimalandira nthawi zonse. Chizindikirocho chinatsika ndi zisanu ndi zinayi peresenti zokha. Peroxide yopitirira makumi asanu ndi anayi peresenti ya zomwe ananena sinali chifukwa cha kuwala koyezera. Chachitatu, anayeza kuchotsedwa kwa peroxide payokha ndi elekiturodi, ndipo anapeza kuti utotowo umawonjezera kupanga kwake komanso umachedwetsa kuchotsedwa kwake. Chowunikira chomwe chimangonena fuloresensi yotsalira chikanaphatikiza zotsatira ziwirizi ngati chinthu chimodzi.

Mayesowa akadali ndi njira zomwe angalakwikire, ndipo methylene blue imayambitsa zingapo mwa izo. Utotowo umayamwa kuwala m'gawo lalikulu lofiira lomwelo momwe resorufin imatulutsa kuwala. Choncho utoto ukakhala wochuluka kwambiri, chitsanzo chingabise chizindikiro chake chomwe kudzera m'zotsatira za kusefera kwa kuwala mkati. Chifukwa chake miyezo yoyerekezerayi iyenera kukhala ndi kuchuluka kwa utoto kofanana ndi kwa m'zitsanzo. Amplex Red imathanso kuchitidwa oksideshoni ndi kuwala payokha, ndipo resorufin yomwe imapanga ingalowe m'machitidwe obwerezabwereza a redox oyendetsedwa ndi kuwala, omwe amapanga superoxide ndi peroxide. Izi zimakhala zofunika kwambiri ngati mayeserowo akuunikiranso chinthu choyambitsa machitidwe a kemikali ndi kuwala. Njira yothandiza ndi kuunikira zitsanzo za methylene blue choyamba, kenako kuyeza magawo ang'onoang'ono a zitsanzozo ndi zinthu za Amplex mu mdima, m'malo mounikira utoto ndi chowunikira nthawi imodzi. Pomaliza, superoxide dismutase sikuti imangochepetsa chizindikiro cha Amplex: kusandutsa superoxide kukhala peroxide kungachiwonjezere. Mayeso owongolera a SOD omwe amawonjezera fuloresensi sali olephera. Azindikira kuti superoxide ndi chinthu chomwe chili koyambirira kwa njirayo.

DCFDA: chizindikiro cha oksideshoni chosaloza chinthu chimodzi chokha

Kuphatikiza kwa methylene blue ndi DCFDA ndi komwe kumayambitsa chisokonezo chochuluka. DCFH-DA ndi chinthu choyambirira chomwe chimalowetsedwa m'maselo. Ma enzyme a esterase a m'selo amachotsa magulu ake a acetate n'kusiya DCFH yosatulutsa fuloresensi, ndipo oksideshoni ya DCFH imapanga DCF yotulutsa fuloresensi. Zinthu zomwe zimachititsa oksideshoni yake ndi zambiri: ma hydroxyl radical, zinthu zapakatikati za peroxidase ndi heme, nitrogen dioxide, hypochlorous acid, ndi ma radical ochokera ku peroxynitrite. Hydrogen peroxide yokha siichititsa oksideshoni yake mwachindunji pa liwiro lothandiza; fuloresensi yodalira peroxide nthawi zambiri imafunika ayironi, mapuloteni a heme, kapena machitidwe a peroxidase kuti igwire ntchito.

Kuwunika kosonyeza zofooka kumene gawo lililonse lofotokoza njira liyenera kutchula kumachokera ku kusanthula kwa DCFH ngati chowunikira kochitidwa ndi Kalyanaraman ndi anzake. Monga momwe kwafotokozedwera mwachidule mu kafukufuku wa mitochondria ya ubongo womwe unakana DCFDA pachifukwa chimenechi, iwo anasonyeza kuti chowunikiracho chimachita machitidwe pang'onopang'ono, chili ndi chiŵerengero cha mamolekyulu chosafotokozedwa bwino, chilibe kusanja kovomerezeka kwa miyeso, ndipo chimachitanso machitidwe ndi mankhwala omwe si ROS. Chowunikiracho chimawonjezeranso zomwe chikuyesa: oksideshoni ya DCFH yotaya elekitironi imodzi imapanga DCF radical, yomwe imachita machitidwe ndi okosijeni kupanga superoxide. Superoxide imeneyo imasanduka peroxide yowonjezera kudzera mu dismutation. Choncho gawo lina la machitidwe a kemikali omwe muyesowo ukunena limapangidwa ndi muyesowo wokha.

Zimenezi sizitanthauza kuti DCFDA ndi yopanda ntchito. Zimatanthauza kuti ndi njira yoyang'anira koyambirira, osati yozindikira chinthu chenicheni. Kuwonjezeka kwa fuloresensi ya DCF kumatanthauza kuti oksideshoni ya chowunikira yodalira zinthu zochititsa oksideshoni yawonjezeka. Sikutanthauza kuti peroxide yawonjezeka, ndipo ndithudi sikutanthauza kuti ROS yonse yawonjezeka, chifukwa palibe chowunikira chimodzi chomwe chimayeza kuchuluka kumeneko. Kafukufuku wakale wa methylene blue wodalira oksideshoni ya DCF pa kuchuluka kopitirira micromolar imodzi unanena zotsatira zolimbikitsa oksideshoni, pamene machitidwe ena a nthawi yomweyo anasonyeza kutetezedwa ku imfa ya maselo yoyambitsidwa ndi peroxide. Zomwe anaona zonsezi zingakhale zoona za utoto womwewo m'mikhalidwe yosiyanasiyana ya redox, ndipo zotsatira za DCF zokha sizinganene kuti ndi chinthu chiti chochititsa oksideshoni chomwe chinapereka chizindikirocho. Chofunika pa kafukufuku wakale wa mitochondria ya ubongo n'chakuti unalowa m'malo mwa njira yoyang'anira imeneyi ndi muyeso wa peroxide wosanjidwa bwino, ndipo unapeza kuwonjezekako mwachindunji: ngakhale utoto wa nanomolar zana limodzi unawonjezera kutulutsidwa kwa peroxide kangapo pa chinthu chilichonse chopereka mphamvu ndi pa mkhalidwe uliwonse wa kupuma kwa mitochondria womwe unayesedwa.

DCFDA ili ndi msampha winanso wokhudza methylene blue mwapadera. Utotowo uli ndi mtundu wamphamvu ndipo umachita nawo redox, choncho ungafooketse fuloresensi, kuzimitsa kutulutsidwa kwa kuwala, kapena kuchititsa oksideshoni ya chowunikira mosadalira ROS ya m'maselo. Mayeso owongolera opanda maselo, okhala ndi utoto ndi chowunikira chokha, si chowonjezera chosafunika. Ndi mayeso omwe amakuwuzani ngati utotowo ukuchita machitidwe ndi chowunikiracho.

Kutchera ma radical ndi kuyeza kugwiritsidwa ntchito kwa okosijeni: mtundu ndi liwiro la machitidwe

Pamene zowunikira za fuloresensi zimanena kuti china choti chichitidwe oksideshoni chawonekera, electron spin resonance yokhala ndi zotchera ma radical imapereka yankho loyandikira kwambiri pa zomwe chinthucho chinali. Kafukufuku wakale wodziwika wa machitidwe a methylene blue oyendetsedwa ndi kuwala unagwiritsa ntchito ESR kuzindikira ascorbate radical yochokera ku utoto, ascorbate ndi kuwala. Kenako unasonyeza kupanga kwa peroxide m'mayesero a kugwiritsidwa ntchito kwa okosijeni, komanso kupanga kwa hydroxyl radical koyambitsidwa ndi ayironi m'mayesero a kutchera ma radical. Njira iliyonse inayankha funso lomwe zina sizikanatha kuyankha: ESR inazindikira mtundu wa radical, kuyeza kugwiritsidwa ntchito kwa okosijeni kunasonyeza liwiro la machitidwe mosadalira chowunikira chilichonse cha fuloresensi, ndipo kuphatikiza kwake kunasonyeza kuti njira za singlet oxygen ndi za ma radical zonse zinkagwira ntchito m'dongosolo limodzi lounikiridwa.

Zotsatira za 1984 zimenezo zikuwerengedwabe ngati chenjezo. Pakakhala kuunikira ndi chinthu chochititsa ridakeshoni, utotowo unapanga peroxide. Pakakhalanso ayironi, dongosololo linapanga hydroxyl radical, yomwe ndi yogwira kwambiri kuposa zinthu zina za m'gululi. Olemba analumikiza zimenezi ndi ascorbate ya m'diso ndipo anakayikira kugwiritsa ntchito utotowo m'maso mosasamala. Owerenga a masiku ano ayenera kuzindikira mikhalidweyo: kuwala kuphatikiza chinthu chochititsa ridakeshoni kuphatikiza ayironi. Chotsani chilichonse mwa zitatuzi ndipo njirayo imafooka kapena kutha.

Maelekiturodi a okosijeni amakhala othandiza mwanjira ina. Elekiturodi ya mtundu wa Clark imayeza kugwiritsidwa ntchito konse kwa okosijeni pambuyo pochotsa womwe wapangidwa. Izi zimatsimikizira kuti machitidwe a redox oyendetsedwa ndi utoto akugwiritsa ntchito okosijeni wa mamolekyulu, ndipo zimapereka liwiro la machitidwe lomwe cholakwika cha fuloresensi sichingapangitse kukhala labodza. Sizizindikira chinthu chomwe chapangidwa. Kupanga peroxide, superoxide, madzi, komanso kuwonongeka kwa singlet oxygen, zonse zimagwiritsa ntchito okosijeni. Liwiro losadziwa mtundu wa chinthu ndi losakwanira, monganso kudziwa mtundu popanda liwiro. N'chifukwa chake mapepala amphamvu a kafukufuku amaphatikiza elekiturodi ndi mayeso a peroxide osanjidwa bwino kapena ndi chotchera ma radical.

Chifukwa chake zotsatira za kuletsa oksideshoni zimatsutsana

Ikani zotsatira za njira zitatuzi pamodzi, ndipo zotsutsana zooneka m'mabuku a kafukufuku wa methylene blue zimamveka ngati kusiyana kwa njira zoyezera kuphatikiza mfundo zitatu za zamoyo.

Choyamba, utotowo ndi wonyamula maelekitironi komanso gwero la peroxide. Ukapatutsa maelekitironi ku malo a kupuma kwa mitochondria kumene okosijeni akanakhala superoxide, ungachepetse ROS imodzi. Koma mawonekedwe ake omwe achitidwa ridakeshoni angachititse ridakeshoni ya okosijeni mwachindunji kukhala peroxide, n'kuwonjezera ina. Choncho labotale yomwe ikufufuza machitidwe a redox mu mitochondria ndi ntchito ya methylene blue yopatutsa njira ya maelekitironi iyenera kuyembekezera mayankho odalira chowunikira: muyeso wozindikira superoxide umatsika pamene muyeso wa peroxide ukukwera m'chiwiya chimodzi choyezera kuwala.

Chachiwiri, peroxide ya kuchuluka kochepa ndi chizindikiro, osati kungowononga kokha. Kafukufuku wa mitochondria ya ubongo wa mbewa womwe unatsatira ntchito yakaleyo unapeza kupangidwa kwa peroxide ndi utoto wochepa kufika pa nanomolar makumi asanu, popanda kuwonongeka kwa DNA ya mitochondria pa milingo yogwiritsidwa ntchito pochiza, komanso kutetezedwa ku kuwonongeka koyambitsidwa ndi rotenone pambuyo pogwiritsa ntchito utotowo pasadakhale. Utotowo unawononga zigawo zina zosankhidwa za DNA pokhapokha pa micromolar zana limodzi, kuchuluka koposa kwambiri milingo yogwiritsidwa ntchito pochiza. Ndipo kuwala kofiira kuphatikiza utoto wa micromolar makumi awiri kunapanga singlet oxygen yomwe inawononga zigawo zonse zomwe zinayesedwa. Njira yomwe akuganiza kuti imachitika ndi ya hormesis: kupanga peroxide pang'ono kumayambitsa chitetezo choletsa oksideshoni cholumikizidwa ndi Nrf2 komanso kupanga mitochondria yatsopano. Choncho muyeso wa nthawi yochepa wosonyeza kulimbikitsidwa kwa oksideshoni umatsogolera zotsatira za nthawi yaitali zosonyeza kuletsedwa kwa oksideshoni. Mayeso owerengedwa patatha ola limodzi ndi ena owerengedwa patatha tsiku limodzi angasiyane chifukwa machitidwe a zamoyo asintha pakati pa nthawi ziwirizo.

Chachitatu, kuwala kumasankha ngakhale molekyulu yomwe ilipo. Kuunikira kumachotsa magulu a methyl pa utotowo, kuwusintha kupita ku Azure B ndi zinthu zina zofanana, ndipo nyali ya maikulosikopu yomwe ili pamwamba pa mbale ya zitsanzo ndi yokwanira kuyambitsa machitidwe ake oyendetsedwa ndi kuwala. Mayesero a mu mdima ndi mayesero ounikiridwa ndi mayesero osiyana, ndipo gawo la zowongolera kuunikidwa ndi kusamalira utoto la ndondomeko iliyonse liyenera kuwachitira motero.

Mayeso owongolera ofunika osachepera, ndi mmene mungawerengere zonena zilizonse

Kuyerekezera komwe kuli pansipa ndi chida choyambirira cha nkhaniyi choti mugwiritse ntchito ngati cholozera. Musanavomereze zotsatira zilizonse za ROS mu methylene blue, onani mzere womwe olemba anachita ndi womwe anadumpha.

Mfundo yothandiza powerenga ndi iyi. Amplex yokhala ndi peroxidase, yosanjidwa ndi miyeso yodziwika komanso yotsimikiziridwa ndi catalase, imapereka umboni wokhudza peroxide. DCFDA imapereka umboni wa oksideshoni ya chowunikira yosaloza chinthu chimodzi chokha, osati china chilichonse kuposa pamenepo. Kutchera ma radical pamodzi ndi mayeso owongolera ogwiritsa ntchito zinthu zogwira ma radical kumapereka umboni wa mtundu wa radical. Kuyeza kugwiritsidwa ntchito kwa okosijeni kumapereka umboni wa liwiro la machitidwe. Chiganizo chomwe chimanena zambiri kuposa zimene mayeso ake angatsimikizire, ngakhale chilembedwe molimba mtima bwanji, ndi chiganizo chokhudza chowunikira, osati utoto. Zokambirana zina zokhudza kudalira kwa zotsatira pa kuchuluka ndi milingo ya hormesis, kuyenda kwa maelekitironi mu mitochondria, kusokonezedwa kwa mayeso m'zitsanzo zokhala ndi utoto, ndi zolakwika zoyambitsidwa ndi kuunikira kwa maikulosikopu zimakulitsa mfundo zonsezi. Ndipo mfundo iliyonse imakhalabe yolondola cholozera chake chosungira malo chikadzalowedwa m'malo ndi cholozera chenicheni.