Maƙala
Shuɗin methylene da autophagy: AMPK, mTOR, da gwaje-gwajen ƙwayoyin jijiyoyi
Ka yi tunanin kana gudanar da gwajin ƙwayoyin halitta, ka ƙara shuɗin methylene, sannan ka ga wani layin furotin da ake kira LC3-II ya ƙara duhu a takardar gwajin blot ɗinka. Abu ne mai jan hankali ka sanar cewa sinadarin yana “ƙarfafa autophagy” ka wuce. Amma yin wannan fassarar ya yi wuri. Layin LC3-II mai ƙarin duhu na iya nufin ƙwayar halitta tana gina ƙarin ƙananan jakunkunan tsaftacewa, ko kuma jakunkunan suna taruwa ne saboda matakin kawar da abubuwan da ke cikinsu ya toshe. Waɗannan fassarori biyu suna nuna abubuwa masu saɓani, kuma wallafe-wallafen shuɗin methylene a ƙwayoyin jijiyoyi sun ƙunshi misalan kowanne irin shaida. Wannan labarin yana bayani kan abin da aka auna a zahiri, gwaji bayan gwaji.
Macroautophagy, nau'in da aka nazarta a nan, ita ce hanyar ƙwayar halitta ta sake sarrafa abubuwa da yawa lokaci guda. Wata jaka mai murfi da ake kira phagophore tana girma ta kewaye furotin da ƙananan sassan cikin ƙwayar halitta da suka lalace, ta rufe ta zama autophagosome, ta haɗu da lysosome, sannan a rushe abubuwan da ke cikinta a sake su don a sake amfani da su. Ƙwayoyin jijiyoyi suna dogara da wannan tsari domin ba sa rarrabuwa, don haka ba za su iya rage yawan sassan da suka lalace ta hanyar rarrabuwa ba. Idan sake sarrafawar ta tsaya, sharar tana taruwa. Idan tana gudana, ƙwayar halitta tana dawo da sinadaran gini a lokacin matsin lamba.
Abin da wani gwajin hippocampus ya nuna
Babban nazarin ƙaddamarwar ya yi amfani da ƙwayoyin hippocampus na bera HT22 da aka sanya cikin matsin lamba ta hanyar hana su serum na tsawon 24 hours, wani yanayi da ke cire abubuwan ƙarfafa girma da saƙonnin tallafa rayuwar ƙwayoyin halitta, kuma yana haifar da apoptosis da autophagy. Masu binciken sun ruwaito cewa shuɗin methylene ya rage mutuwar ƙwayoyin halitta gwargwadon adadin da aka yi amfani da shi, kuma wannan kariyar ta zo tare da alamomin macroautophagy.
Jerin alamomin ya kasance takamaimai. Rinin LC3B da ya bazu ya koma ɗigogi a kewayen cibiyar ƙwayar halitta tun daga kusan 20 nM na shuɗin methylene, tare da mafi ƙarfi a martanin LC3-I da LC3-II kusa da 1 μM. LC3B-I da LC3B-II duk sun ƙaru gwargwadon adadi da lokaci. Abu mafi muhimmanci shi ne, ƙungiyar ta wuce gwajin blot na LC3 na lokaci guda. Sun ƙara 10 μM na chloroquine, wanda ke toshe kawar da abubuwa ta lysosome, suka kuma sami ƙarin LC3-II da p62 da ya tsaya ba tare da rushewa ba a ƙwayoyin da aka yi wa shuɗin methylene. Wannan tsarin yana goyon bayan rushewa da maye gurbin abubuwa da ke dogara da lysosome, maimakon taruwa kawai. Sun kuma nuna cewa chloroquine ya rage tasirin hana apoptosis a wani ɓangare, abin da ya danganta kariyar da hanyar autophagy da lysosome a wannan samfurin gwaji.
Sakamakon isar da saƙonni shi ne ɓangaren da aka fi nakalto ba daidai ba. Isar da saƙonnin AMPK, wadda aka auna ta AMPK mai haɗe da phosphate da ACC mai haɗe da phosphate da yake sarrafawa a mataki na gaba, ta ƙaru a 12 da 24 hours gwargwadon adadi. Mai hana AMPK, Compound C, da aka yi amfani da shi a 20 μM a cikin 4 hours na ƙarshe, ya rage haɗa phosphate da ACC da kunna LC3. Amma mTOR mai haɗe da phosphate bai nuna wani danniya da za a iya ganowa ba. Don haka, a ƙwayoyin hippocampus da ke cikin matsin lamba, abin da aka samu shi ne ƙaddamarwa mai alaƙa da AMPK ta wata hanya da a bayyane take ba ta dogara da mTOR ba. An ga irin wannan tsarin a beraye masu rai, inda amfani da shuɗin methylene ya ƙaddamar da alamomin macroautophagy a cortex da hippocampus.
Ƙaddamarwa ba kammalallen gudana ba ce
A nan ne bambancin ma'anoni ya fi muhimmanci. Ƙaddamarwa na nufin tsarin da ke farkon hanya yana samar da ƙarin autophagosomes. Gudana, wato flux, na nufin abubuwan da ake sarrafawa sun bi dukkan hanyar: killacewa, balaga, haɗuwa da lysosome, rushewa, da sake sarrafawa. Kowace ƙaruwa ta LC3-II tana goyon bayan da'awar ƙaddamarwa ne kawai har sai gwajin gudana ya tabbatar da sauran tafiyar.
Gwajin gudana na yau da kullum yana auna LC3-II tare da kuma ba tare da toshe lysosome na ɗan lokaci da abu kamar bafilomycin A1 ko chloroquine ba. Idan abin da aka yi wa ƙwayoyin da gaske yana hanzarta samarwa, toshe rushewa ya kamata ya ƙara ɗaga LC3-II. Mai nuna alama mai haɗin mCherry-GFP-LC3 yana ƙara wata hanyar dubawa: sassan suna haskaka rawaya kafin haɗuwa da lysosome, sannan ja kaɗai bayan acid ya kashe saƙon GFP; don haka ƙarin ɗigogi masu ja kaɗai suna goyon bayan ci gaba zuwa autolysosomes masu acid. Raguwar furotin p62 na iya tabbatar da rushewa da maye gurbin abubuwan da ake sarrafawa, amma a lura cewa rubuta umarnin p62 daga DNA ma yana amsa matsin lamba, don haka ya kamata a duba shi a matakin mRNA.
Jerin dubawa da ke ƙasa yana danganta kowane gwajin aunawa da aka yi amfani da shi a waɗannan wallafe-wallafen da abin da yake aunawa da abin da ba zai iya tabbatarwa shi kaɗai ba. Masu karatu za su iya sake amfani da shi wajen tantance kowace da'awar macroautophagy a nan gaba, ciki har da hujjojin azumi da tsawon rai da suke amfani da waɗannan alamomin.
| Gwajin aunawa | Abin da yake aunawa | Abin da ba zai iya tabbatarwa shi kaɗai ba |
|---|---|---|
| Matakin LC3B-I | Girman tarin sinadarin farko a ruwan cikin ƙwayar halitta da ke samuwa don haɗa shi da lipid | Ayyukan autophagy; samarwa da sarrafawa su ma suna canza shi |
| Matakin LC3B-II | Adadin LC3 da ke manne da murfin sassan autophagy | Kammalallen gudana; ƙaruwa na iya nufin hanzarin samarwa ko toshe kawarwa |
| Ɗigogin LC3 da taruwarsu a kewayen cibiyar ƙwayar halitta | Sake rarraba LC3 zuwa sassa masu kama da ƙananan jakunkuna | Alkiblar canji; hotunan lokaci guda ba za su iya bambanta hanzari da cunkoson da ya toshe hanya ba |
| LC3-II tare da chloroquine ko bafilomycin A1 idan aka kwatanta da ba tare da su ba | Ko sababbin autophagosomes suna ci gaba da samuwa lokacin da aka toshe kawarwa | Cikakken rushewar abubuwan da ake sarrafawa; ana buƙatar amfani da mai hanawa na ɗan lokaci da aka daidaita yadda ya dace |
| Matakin furotin p62 | Rushewa da maye gurbin furotin mai haɗa abubuwan da ake sarrafawa, wanda aka saba rushewa a autolysosomes | Gudana kaɗai; hanyoyin martani ga matsin lamba su ma suna sarrafa rubuta umarnin p62 daga DNA |
| Mai nuna alama mai haɗin mCherry-GFP-LC3 | Ci gaba zuwa autolysosomes masu acid (sauyi daga rawaya zuwa ja kaɗai) | Cikakken rushewar kowane nau'in abin da ake sarrafawa |
| Phospho-AMPK da phospho-ACC | Shigar na'urar gane matsin ƙarancin makamashi cikin aiki da sakamakon aikinta | Cewa autophagy ce ta haifar da tasirin ci gaba da rayuwa; AMPK tana sarrafa rassan metabolism da yawa |
| Phospho-mTOR, p70S6K, 4EBP1 | Ayyukan hanyar saƙonnin girma da ke takaita autophagy | Gudana; waɗannan saƙonnin ba da izini ne a farkon hanya, ba ma'aunan kawar da abubuwa ba |
A lura da wata tarkon lafazi da teburin ke karewa. ACC mai haɗe da phosphate ita ce sigar da aka hana aikinta na enzyme. Ƙaruwarta tana nuna sakamakon aikin AMPK, ba kunna ACC ba.
Maganar “hana macroautophagy ya kawar da kariyar ƙwayoyin jijiyoyi” tana buƙatar irin wannan takamaiman bayani. A samfurin HT22, tana nufin cewa katsalandan ga hanyar autophagy da lysosome ya kawar da fifikon ci gaba da rayuwa da shuɗin methylene ya bayar lokacin hana serum. Tana goyon bayan cewa autophagy ta kasance wajibi ko mai ba da gudummawa a wannan kwanon gwaji. Ba ta tabbatar da cewa autophagy ita ce kawai hanyar kariya ta sinadarin ba, ba ta tabbatar da cewa chloroquine yana aiki ta autophagy kaɗai ba, kuma ana buƙatar canjin gwaji a ƙwayoyin halittar gado na ATG5, ATG7, ko ULK1 don ƙarfafa da'awar sanadi.
Hanyoyi biyu zuwa tsarin aiki ɗaya
Taswira mai sauƙi tana taimakawa. Matsin ƙarancin makamashi yana ɗaga AMPK, AMPK tana kunna rukunin ULK1, kuma autophagy tana farawa. Abubuwan ƙarfafa girma da sinadaran gina jiki suna ɗaga mTORC1, mTORC1 tana hana ULK1, kuma autophagy tana kasancewa a kashe. A matakan da ke bayan ULK1, rukunin Beclin-1 da VPS34 yana fara samuwar phagophore, ana haɗa LC3-I da lipid ya koma LC3-II, autophagosome yana rufewa, ya haɗu da lysosome, sannan a rushe abubuwan da ke cikinsa tare da rushewa da maye gurbin p62.
Zanen yana sanya kowane sakamakon shuɗin methylene a wannan taswirar: sakamakon hippocampus kusa da reshen AMPK, sakamakon ƙwayoyin asali kuma kusa da reshen mTOR.
Wannan bayani yana warware saɓanin da ake gani da nazarin Xie na biyu. A ƙwayoyin asalin jijiyoyin bera da suke ƙaruwa ta rarrabuwa, wani gwaji dabam ya gano cewa 5 μM na shuɗin methylene ya hana ƙaruwa ta rarrabuwa kuma ya ƙarfafa shiga yanayin hutun rashin rarrabuwa, inda ƙwayoyin da suka nuna Ki67 suka ragu a ranakun 1 da 3, yayin da bambancewa zuwa ƙwayoyin jijiyoyi da suka riga suka ɗauki wannan hanya ta ci gaba ba tare da tangarda ba. A nan tsarin ya bi ta danne saƙonnin mTOR: jimillar mTOR da mTOR mai haɗe da phosphate sun ragu tare da raguwar samar da p70S6K da 4EBP1, da kuma rage samar da cyclins E1, B1, D1, da D2. Babban yanayin da aka gani shi ne hutun rashin rarrabuwa na ƙwayoyin asali, wanda marubutan suka danganta da jinkirta tsufar ƙwayoyin halitta, ba ceto ƙwayoyin hippocampus da ke cikin matsin lamba ba.
Babu saɓani tsakanin takardun biyu. AMPK da mTOR masu haɗa saƙonni ne da ke sauyawa gwargwadon yanayi, ba madannai masu aiki iri ɗaya kullum ba. Ƙwayar halitta mai kama da ƙwayar jijiyar da ke cikin matsin lamba na iya amfani da AMPK ba tare da hanawar mTOR da za a iya aunawa ba, yayin da ƙwayar asali da sinadarin ƙarfafa rarrabuwa ke tura ta za ta iya amsa wannan sinadarin ta hanyar takaita tsarin girma da ke dogara da mTOR. Nau'in ƙwayar halitta, yanayin matsin lamba, yawan sinadarin, da lokacin aunawa suna ƙayyade reshen da zai fi tasiri. Wannan kuma shi ne dalilin da ya sa tasirin shuɗin methylene kan makamashin mitochondria da redox suke cikin wannan bayani ɗaya: kamar yadda wani bita kan hanyoyin kariyar ƙwayoyin jijiyoyi ya bayyana, sinadarin yana aiki a matsayin madadin mai ɗaukar electron, yana shigar da saƙonnin yaƙi da oxidation da hana apoptosis cikin aiki, yana hana MAO da NOS, kuma yana haɗuwa da sabunta mitochondria ta hanyar samar da sababbi da autophagy. Autophagy reshe ɗaya ce na tasirinsa masu yawa, ba ita ce dukkan bayanin ba.
Abin da gwaje-gwajen ba su nuna ba
Hana serum yana kwaikwayon matsin rashin tallafin girma da na metabolism a cikin kwanon gwaji. Ba ya kwaikwayon azumin dukkan jiki: glucose, amino acids, da gishirai suna nan a ruwan gina jiki na asali, kuma babu halayen cin abinci. Ba za a iya sauya yawan sinadarin a ruwan renon ƙwayoyin halitta daga 20 nM zuwa 5 μM zuwa adadin amfani ga mutane ta lissafi kawai ba, domin sha cikin jiki, ɗaurewa da furotin, yanayin redox, metabolism, taruwa a kyallen jiki, da lokacin kasancewar sinadarin duk sun bambanta.
Ba a tsara wani gwajin mutane don nuna gudanar autophagy da shuɗin methylene ya ƙaddamar a marasa lafiya ba. Shaidar hanyoyin aikin tana nan a matakin ƙwayoyin halitta, kyallen da aka cire daga jiki, da nazarin dabbobi, kuma har yanzu bitoci suna bayyana shaidar amfani da sakamakon ga mutane a matsayin ta farko. Don haka waɗannan nazarin ba su nuna cewa shuɗin methylene yana kwaikwayon azumi, yana tsawaita rayuwar ɗan adam ta autophagy, ko yana kammala gudanar autophagy a ƙwaƙwalwar ɗan adam ba. Da'awar kwaikwayon azumi da tsawon rai suna buƙatar nasu shaidar gwajin asibiti, kuma gwaje-gwajen shuɗin methylene a mutane don wasu dalilai ba sa zama gwaje-gwajen autophagy saboda alaƙa kawai. Ya kamata a karanta bincike kan samfuran matsin rashin sinadaran gina jiki da tsawon rai tare da kiyaye wannan iyaka.
A takarda ta gaba da za ka karanta, yi amfani da jerin dubawar a jere. Da farko ka tambayi wane reshe ne ya canza: AMPK, mTOR, ko babu ɗayansu. Sannan ka tambayi ko an haɗa sakamakon LC3 da gwajin gudana, ko kuwa shi kaɗai ne. Bayan haka ka duba ko toshe hanyar ya kawar da amfanin, kuma ta wace hanya aka yi hakan. Nazarin da ya amsa duk ukun ya sami hujjar da'awa mai ƙarfi kan tsarin aiki. Wanda ya amsa ta farko kaɗai ya nuna saƙon ba da izini ne, kuma sauran tafiyar ba a riga an tsara taswirarta ba.