Methylene blue da mitochondria: sufurin electrons da shaidu

Ka yi tunanin samfuran gwaji biyu a gefe da gefe. Dukansu suna ɗauke da mitochondria na ƙwaƙwalwa da aka gurbata da rotenone, wani sinadari da ke toshe Complex I na sarkar numfashin salula. A ɗaya, mai gwaji yana amfani da pipette ya ƙara adadin methylene blue a ma'aunin micromolar. Amfani da oxygen yana dawowa a wani ɓangare. Bambancin ƙarfin lantarki a membrane yana dawowa a wani ɓangare. Ana sake fara samar da ɗan ATP. A cuvette da ke kusa, na'urar auna peroxide tana bayyana sauran ɓangaren labarin: fitar hydrogen peroxide ta ninku sau da dama, a kowane adadin da aka gwada, da kowane substrate da aka yi amfani da shi.

Duk ma'aunan biyu daidai ne. Methylene blue mai jigilar electrons ne wanda zai iya ci gaba da tafiyar da su ta hanyar kewaye ɓangaren sarkar numfashin salula da ya lalace, kuma wannan jigilar tana da farashin ƙarin peroxide yayin da take aiki. Idan an riƙe waɗannan hujjoji biyu tare, rubuce-rubucen bincike kan methylene blue da aikin mitochondria za su zama masu sauƙin fahimta. Idan aka riƙe ɗaya kawai, za su yi kama da mu'ujiza ko barazana, gwargwadon takardar binciken da aka fara karantawa.

Jigilar, a bayyane

Sarkar numfashin salula jerin tashoshi ne masu miƙa electrons ga juna. Electrons suna shiga ta Complex I (daga NADH) ko ta Complex II da enzymes na flavin masu alaƙa (daga succinate da makamantan masu ba da electrons), su wuce ta Complex III zuwa cytochrome c mai ɗaukar su daga wuri zuwa wuri, sannan ta Complex IV zuwa oxygen. Kowace miƙawa tana tura protons su ƙetare membrane na ciki, kuma bambancin da hakan ke haifarwa yana tafiyar da samar da ATP. Idan aka toshe kowace tasha, aikin ɓangaren da ke gabanta yana tsayawa.

Methylene blue yana shiga a matsayin wani mai jigila a layi na daban. Siffarsa da ta rasa electrons tana karɓar electrons daga masu ɗaukar su da ke gaba a sarkar, ta koma sifarsa marar launi da ta karɓi electrons, wato leucomethylene blue. Wannan sai ya miƙa electrons ɗin zuwa ɓangaren da ke gaba a hanyar tafiya, ya kuma koma shuɗi. An bayyana siffofin biyu a bayanin nau'ikan biyu na redox, yayin da aka tattauna faɗaɗɗen tsarin miƙa electrons a bayanin yadda yake aiki gaba ɗaya. Tambayar da har yanzu take buɗe ita ce inda mai jigilar yake komawa cikin sarkar: a aljihun ɗaure quinol na Complex III da ake kira wurin Qo, ko kuma kai tsaye a cytochrome c. Hanyar fitar da electrons da ta fi rinjaye tana dogara da yanayin gwajin, kamar yadda nazarin kewaye Complex III ya bayyana dalla-dalla.

Abin da bincike uku kan dabbobin rodent suka auna a zahiri

Dukkan binciken uku sun yi amfani da mitochondria na ƙwaƙwalwa da aka ware, wato ƙananan sassan salula da aka cire daga salular aka sanya cikin ruwan buffer mai sanannen tsari. Ba a yi amfani da salula cikakku ba, ko dabbobin da aka ba sinadarin, ko mutane a matsayin mahalarta gwaji. Matsakaicin sinadarin da aka ambata yana nufin wanda yake cikin ruwan da ke kewaye da waɗannan ƙananan sassan salula, ba adadin da mutum zai sha ta baki ba. Idan aka kiyaye wannan mahallin, lambobin suna magana a sarari.

Binciken farko, kan mitochondria da aka ware daga ƙwaƙwalwar guinea pig a 100 nanomolar zuwa 2 micromolar, ya bayyana fa'ida da farashi a samfurin gwaji guda. A mitochondria masu lafiya, rinin ya ƙara amfani da oxygen a lokacin hutu amma bai canza numfashin da ADP ke ƙarfafawa ba, don haka samar da ATP da ke haɗe da numfashin bai inganta ba. Yayin toshe Complex I, 2 micromolar ya dawo da samar da ATP daga 46 zuwa 93 nanomoles a minti ɗaya a kowace milligram, idan aka kwatanta da ƙimar al'ada ta kusan 1109. Yayin toshe Complex III, adadin nan ɗaya ya dawo da ATP daga kusan 46 zuwa 63, idan aka kwatanta da ƙimar al'ada ta kusan 910. Bambancin ƙarfin lantarki da shigar calcium sun dawo a wani ɓangare. Ba a iya kewaye toshewar Complex IV da cyanide ya haifar ba, wanda ke nuna cewa hanyar fita mai amfani ta electrons tana kafin Complex IV mai aiki. Kowane dawowar aiki ya kasance na gaske bisa ƙididdiga, amma ƙarami: kaso masu lamba ɗaya cikin ɗari na samar da ATP na al'ada.

Bincike na biyu, kan mitochondria na ɓawon ƙwaƙwalwar mouse a kusan 1 micromolar, ya tabbatar da kewaye Complex I kuma ya nuna iyaka a Complex III. Rotenone ya rage numfashi daga kusan 121 zuwa ƙasa da 2 nanomoles na oxygen a minti ɗaya a kowace milligram, rinin kuma ya ɗaga shi zuwa kusan 21, wato kusan kashi ɗaya cikin shida na al'ada, yayin da bambancin ƙarfin lantarki a membrane ya dawo kusan gaba ɗaya. Amma bayan toshe Complex III, numfashi da bambancin ƙarfin lantarki ba su dawo ba a duk matakan 1 zuwa 5 micromolar, kuma toshewar ta danne siginar peroxide da rinin ke haifarwa. Marubutan sun ba da ra'ayin cewa rinin da ya karɓi electrons yana miƙa su a wurin Qo na Complex III: idan wannan wurin ya kasance a toshe, mai jigilar ba shi da inda zai kai su.

Bincike na uku, kan mitochondria na ƙwaƙwalwar mouse, rat da guinea pig, galibi a 2 micromolar, ya gwada wannan sabanin kai tsaye kuma ya gano cewa ana iya kewaye toshewar Complex III. Lokacin da myxothiazol, mai hana aikin wurin Qo, ya rage numfashin asali da kashi 88 cikin ɗari, rinin ya dawo da amfani da oxygen zuwa kusan kashi 62 cikin ɗari na ƙimar da ake samu ba tare da toshewa ba. A gwaji kai tsaye, rabin micromolar ya ba cytochrome c da aka ƙara daga waje electrons ko da Complex III yana toshe, yana goyon bayan hanyar daga NADH ta rinin zuwa cytochrome c sannan zuwa Complex IV. Amma numfashin da aka dawo da shi ya yi aiki ba kamar yadda aka saba ba: ƙara ADP ya rage amfani da oxygen maimakon ya ƙara shi, kuma hana samar da ATP ya ƙarfafa numfashi. Bambancin ƙarfin lantarki ya dawo ne kawai a wani ɓangare. Yawan amfani da oxygen tare da alaƙar numfashi da samar da ATP da ta juya akasin yadda aka saba ba samar da makamashi na al'ada ba ne.

Dalilin da ya sa dakunan gwaje-gwaje suka saɓa, da abin da ke warware sabanin

Bayani na cewa bambancin jinsin dabbobi ne ya jawo hakan shi ne na farko da ba ya dacewa da shaidar. Ƙungiya ta uku ta maimaita aikin a nau'ikan rodent uku kuma ta samu irin wannan kewaye a dukansu, har da nau'in mouse da aka ruwaito cewa ba a samu kewaye a cikinsa ba. Abubuwan da suka bambanta a maimakon haka su ne matsakaicin sinadarin (1 idan aka kwatanta da 2 micromolar), adadin sinadaran toshewa, ruwan buffer, hanyoyin ware mitochondria, kuma sama da komai, yanayin makamashin da aka kwatanta: kewaye ya fi bayyana kafin ƙara ADP, daidai lokacin da martanin ADP mai saɓa da tsammani yake bayyana. Saboda haka, zane mai dacewa da shaidu yana nuna hanyoyin fita biyu masu dogaro da yanayi, ɗaya a wurin Qo, ɗaya kuma a cytochrome c. Mai jigilar yana kai electrons inda yanayin sinadaran wurin ya ba shi damar kaiwa.

Farashin peroxide

Shaidar farashin da ake biya tana da daidaito kamar shaidar dawowar aiki. A aikin guinea pig, rinin ya ƙara fitar peroxide a kowane yanayi da aka gwada: hutu, samar da ATP da nakasar da sinadarai suka haifar, tare da duk substrates ukun. Lokacin samar da ATP, fitar peroxide ta tashi daga kusan 148 zuwa sama da 1500 picomoles a minti ɗaya a kowace milligram a 1 micromolar, kuma ko 100 nanomolar ya ƙara siginar fiye da sau huɗu. Rinin kuma ya rage saurin kawar da peroxide da kusan sau 2.6, don haka siginar tana nuna samarwa da ya fi sauri tare da kawarwa da ya fi jinkiri. Tsarin sinadaran da aka ba da shawara shi ne rinin da ya karɓi electrons yana ba oxygen electrons kai tsaye ba tare da superoxide da za a iya ganowa ba, duk da cewa wannan har yanzu fassara ce maimakon mataki da aka tabbatar.

Aikin mouse yana ƙara sarƙaƙiya ga hoton ta hanya mai amfani. Peroxide ya ƙaru sosai yayin ƙara adadin sinadarin, daga ƙimar asali ta kusan 76 zuwa kusan 2000 picomoles a minti ɗaya a kowace milligram a 2 micromolar, amma toshe Complex III ya danne ɓangaren da rinin ke haifarwa. Idan peroxide ya fito ne kawai daga rinin da ya karɓi electrons yana yin martani da oxygen, toshe sarkar bai kamata ya yi tasiri sosai ba. Amma yana yi, don haka sarkar tana shiga cikin aikin. Nazarin aikin hana oxidation da ƙarfafa oxidation ya sanya wannan yanayi mai fuska biyu cikin mahalli mafi faɗi, kuma nazarin oxygen idan aka kwatanta da ATP ya bayyana dalilin da ya sa lambobin oxygen kaɗai ba za su taɓa tabbatar da ikirarin makamashi ba.

Dawo da aiki ba gyara ba ne

Maganar da ya kamata a daina amfani da ita ita ce "shin methylene blue yana gyara mitochondria." Gyara zai nufi daidaitawa mai ɗorewa: sake gina rukunonin sarkar, alaƙar numfashi da samar da ATP da ke ci gaba bayan maganin ya fita, dawowar aiki a salula cikakku, sannan ingantaccen aiki a rayayyar halitta. Babu ɗaya daga cikin takardun binciken uku da ya nuna haka. Suna nuna sauya hanyar electrons na gajeren lokaci a ƙananan sassan salula da aka ware kwanan nan yayin toshewa ta wucin gadi da sinadarai suka haifar, tare da samar da ATP a ƙananan kaso na al'ada da kuma alaƙar numfashi da samar da ATP wadda wani lokaci take da ban mamaki sosai. Rotenone da antimycin kayan aikin dakin gwaje-gwaje ne, ba tsarin kwaikwayon tsufa ko cututtukan mitochondria na mutane ba, kuma babu abin da ke nan da yake nufin ƙarin makamashi ga mutum mai lafiya. Wannan kuwa tambaya ce ta shaidu dabam wadda take da nata rubuce-rubucen bincike kan mutane.

Iyakoki uku suna sa duk wani hukunci game da biohacking ya kasance mai bin shaidu. Na farko, matsakaicin sinadarin cikin ruwan gwaji ba adadin da za a sha ba ne: 0.1 zuwa 2 micromolar a kewaye da ƙananan sassan salula da aka ware ba ya nuna adadin da za a sha ta baki wanda zai samar da wannan matsakaicin sinadari mai 'yanci a mitochondrion na ƙwaƙwalwar mutum, musamman domin rinin yana taruwa ko fita gwargwadon bambancin ƙarfin lantarki na membrane kansa. Na biyu, oxygen ba makamashi ba ne: rinin zai iya ƙara amfani da oxygen ta zagayawar electrons da zubewarsu, da kuma ta phosphorylation mai samar da ATP. Don haka auna numfashi ba tare da ma'aunan ATP da bambancin ƙarfin lantarki ba ba zai ba da hujjar ikirarin makamashi ba. Na uku, fa'ida da nauyi suna cikin magana ɗaya: duk bayanin dawowar bambancin ƙarfin lantarki ko ATP da bai ambaci ƙaruwar peroxide da ke faruwa tare da shi ba yana ba da rahoton gwajin ba daidai ba. Ƙarin abubuwan lura masu alaƙa sun haɗa da daidaiton redox na NAD, siffar alaƙar adadin sinadari da martani da jagorar gwaje-gwajen auna aiki.

Ɗaya daga cikin bayanan ƙarshe masu amfani: ikon jigilar electrons halayyar wannan takamaiman ƙwayar sinadari ne. Sinadarai masu alaƙa da shi da suka rasa rukunin methyl, kamar Azure B, su ma suna aiki a redox, amma halayensu sun bambanta. Saboda haka, ruwan sinadari da ya lalace a wani ɓangare yana samar da gwaji dabam da rinin da bai lalace ba. Wannan dalili ɗaya ne na dagewa kan cikakkun ƙa'idojin kundin magunguna da takardar bayanan rukunin samarwa da aka wallafa, maimakon gwajin ƙarafa kaɗai. Ayyukan sinadaran da haske ke tafiyarwa suna kawo wani ƙarin abin lura ga amfani da ake yi a ƙarƙashin haske, wanda aka tattauna a haɗawa da jan haske.

Takaitaccen ikirarin yana nan daram duk da wannan, kuma ya cancanci a riƙe shi: methylene blue zai iya zama mai jigilar electrons mai dogaro da yanayi a mitochondria da aka ware kuma suka lalace, yana kiyaye wani ɓangare na tafiyar electrons amma da farashin ƙarin peroxide. Duk abin da ya wuce wannan, ko makamashi, tsawon rai, ko gyara, yana jiran shaidar da waɗannan binciken ba a tsara su don bayarwa ba.