Inqaku
I-methylene blue ne-autophagy: i-AMPK, i-mTOR, novavanyo lwee-neuron
Khawucinge usenza uvavanyo lweeseli, wongeza i-methylene blue, uze ubone ibhanti yeproteni ebizwa ngokuba yi-LC3-II isiba mnyama ngakumbi kwi-blot yakho. Kulula ukuhendeka ubhengeze ukuba le khompawundi “yonyusa i-autophagy” uze udlulele phambili. Eso sigqibo sithathwa kwangethuba. Ibhanti ye-LC3-II emnyama ngakumbi inokuthetha ukuba iseli yenza iingxowa ezincinci zokucoca ezingakumbi, okanye inokuthetha ukuba ezi ngxowa ziyaqokelelana kuba inyathelo lokulahla livaliwe. Ezi ngcaciso zimbini zikhomba kumacala aphikisanayo, yaye uncwadi lophando nge-methylene blue kwiiseli zemithambo-luvo lunemizekelo yobungqina bohlobo ngalunye. Eli nqaku lichaza oko kwalinganiswa ngokwenene, uvavanyo ngalunye ngokwahlukeneyo.
I-macroautophagy, uhlobo olufundwayo apha, yinkqubo yeseli yokurisayikilisha izinto ngobuninzi. Ingxowa eyenziwe yinwebu, ebizwa ngokuba yi-phagophore, ikhula ijikeleze iiproteni nezakhiwo zangaphakathi zeseli ezigugileyo, ivaleke ibe yi-autophagosome, idibane ne-lysosome, zize izinto ezikuyo ziqhekezwe zikhutshwe ukuze zisetyenziswe kwakhona. Ii-neuron zixhomekeke kule nkqubo kuba azahluli, ngoko azikwazi ukunciphisa umlinganiselo weendawo ezonakeleyo ngokwahlukana. Xa ukurisayikilisha kuxinga, inkunkuma iyaqokelelana. Xa kuqhubeka, iseli iphinda ifumane izinto zokwakha xa iphantsi koxinzelelo.
Oko kwaboniswa luvavanyo olunye lwe-hippocampus
Uphononongo oluphambili lokuqaliswa kwe-autophagy lwasebenzisa iiseli ze-HT22 ze-hippocampus yempuku ezazifakwe phantsi koxinzelelo ngokuvinjwa i-serum kangangeeyure ezingama-24. Le meko isusa izinto ezikhuthaza ukukhula nemiqondiso exhasa ukondleka nokuphila kweeseli, ize ivuse zombini i-apoptosis ne-autophagy. Abaphandi baxela ukuba i-methylene blue yanciphisa ukufa kweeseli ngokuxhomekeke kwidosi, yaye olu khuselo lwaluhamba neempawu ze-macroautophagy.
Iziphawuli ezalandelwayo zazicacile. Ukudaya kwe-LC3B okwakusasazekile kwaphinda kwasasazwa kwaba ngamachaphaza ajikeleze umbindi weseli, kuqala malunga ne-20 nM ye-methylene blue, yaye eyona mpendulo inamandla ye-LC3-I ne-LC3-II yayikufutshane ne-1 μM. Zombini i-LC3B-I ne-LC3B-II zonyuka ngokuxhomekeke kwidosi nakwixesha. Okubalulekileyo kukuba iqela alizange liphelele kwi-blot ye-LC3 ebonisa imeko yomzuzu omnye. Longeze i-10 μM ye-chloroquine, ethintela ukususwa kwezinto ngee-lysosome, laza lafumanisa i-LC3-II engaphezulu kunye ne-p62 egcinwe ingaqhekezwa kwiiseli ezifakwe i-methylene blue. Le ndlela yeziphumo ixhasa inkqubo yokuqhekezwa nokususwa kwezinto exhomekeke kwi-lysosome, kunokuqokelelana nje. Bakwabonisa ukuba i-chloroquine yanciphisa ngokuyinxenye isiphumo sokuthintela i-apoptosis, nto leyo enxulumanisa ukhuseleko nendlela ye-autophagy nee-lysosome kule modeli.
Isiphumo semiqondiso sesona sidla ngokucatshulwa ngokungachanekanga. Imiqondiso ye-AMPK, elinganiswa nge-AMPK efakwe iqela le-phosphate kunye nento ekujoliswe kuyo emva kwayo, i-ACC efakwe iqela le-phosphate, yonyuka kwiiyure ezili-12 nezingama-24 ngokuxhomekeke kwidosi. Isithinteli se-AMPK esibizwa ngokuba yi-Compound C, esasifakwe kwi-20 μM kwiiyure ezi-4 zokugqibela, sanciphisa kokubini ukufakwa kweqela le-phosphate kwi-ACC nokuvuselelwa kwe-LC3. Ngokwahlukileyo, i-mTOR efakwe iqela le-phosphate ayizange ibonise kucinezelwa kunokubonwa. Ngoko ke, kwiiseli ze-hippocampus eziphantsi koxinzelelo, kwafunyaniswa ukuqaliswa okunxulunyaniswa ne-AMPK ngendlela ebonakala ingaxhomekekanga kwi-mTOR. Kwaolu hlobo lweziphumo lwabonakala kwiimpuku eziphilayo, apho ukufakwa kwe-methylene blue kwavuselela iziphawuli ze-macroautophagy kwi-cortex nakwi-hippocampus.
Ukuqalisa akuthethi ukuhamba kwenkqubo de igqitywe
Apha umahluko phakathi kwezi ngcamango ubaluleke kakhulu. Ukuqalisa kuthetha ukuba izixhobo ezisebenza ekuqaleni kwenkqubo zenza ii-autophagosome ezingakumbi. Ukuhamba kwenkqubo, okanye i-flux, kuthetha ukuba izinto ezithwelweyo zihamba yonke indlela: ukuvalelwa, ukuvuthwa, ukudibana ne-lysosome, ukuqhekezwa, nokurisayikilishwa. Nakuphi na ukunyuka kwe-LC3-II kubonisa ukuqaliswa kuphela de uvavanyo lwe-flux luqinisekise inxalenye eseleyo yohambo.
Uvavanyo oluqhelekileyo lwe-flux lulinganisa i-LC3-II xa kukho naxa kungekho kuvalwa okwethutyana kwee-lysosome, njengokusebenzisa i-bafilomycin A1 okanye i-chloroquine. Ukuba into efakiweyo iyakukhawulezisa ngokwenene ukwenziwa, ukuthintela ukuqhekezwa kufanele kuyinyuse nangakumbi i-LC3-II. Isiphawuli esidityanisiweyo se-mCherry-GFP-LC3 songeza enye indlela yokujonga: izakhiwo zikhazimla zityheli ngaphambi kokudibana ne-lysosome, zize zikhazimle zibomvu kuphela emva kokuba i-asidi icime umqondiso we-GFP. Ngoko amachaphaza amaninzi abomvu kuphela axhasa ukuqhubela phambili ukuya kwii-autolysosome ezine-asidi. Ukwehla kweproteni ye-p62 kunokuxhasa ukuba izinto ezithwelweyo ziyaqhekezwa zize zisuswe, kodwa kufuneka kuqatshelwe ukuba nokukhutshelwa kolwazi lwemfuza lwe-p62 kuyasabela kuxinzelelo, yaye kufuneka kuhlolwe kwinqanaba le-mRNA.
Olu luhlu lokuhlola lungezantsi lunxulumanisa uvavanyo ngalunye olusetyenzisiweyo kolu ncwadi noko lukulinganisayo kunye noko lungenako ukukungqina lodwa. Abafundi banokuphinda balusebenzise nakweliphi na ibango le-macroautophagy elizayo, kuquka iingxoxo ngokuzila ukutya nokuphila ixesha elide ezisebenzisa ezi ziphawuli.
| Uvavanyo | Oko lukulinganisayo | Oko lungenako ukukungqina lodwa |
|---|---|---|
| Inqanaba le-LC3B-I | Ubungakanani bovimba wezandulela okwi-cytosol ofumanekayo ukuze kongezwe ii-lipid | Umsebenzi we-autophagy; ukwenziwa nokulungiswa nako kuyalitshintsha |
| Inqanaba le-LC3B-II | Ubungakanani be-LC3 ebotshelelwe kwinwebu yezakhiwo ze-autophagy | Ukuhamba kwenkqubo de igqitywe; ukunyuka kunokuthetha ukwenziwa okukhawulezayo okanye ukususwa okuvaliweyo |
| Amachaphaza e-LC3 nokuqokelelana okujikeleze umbindi weseli | Ukusasazwa ngokutsha kwe-LC3 kwizakhiwo zeengxowa ezincinci | Icala eliya kulo utshintsho; imifanekiso yomzuzu omnye ayikwazi ukwahlula ukukhawuleza kokuqhubeka ekuxingeni |
| I-LC3-II xa kukho naxa kungekho chloroquine okanye bafilomycin A1 | Ukuba ii-autophagosome ezintsha ziyaqhubeka na ukwakheka xa ukususwa kuvaliwe | Ukuqhekezwa ngokupheleleyo kwezinto ezithwelweyo; kufuneka ukufakwa kwesithinteli okwethutyana okulungiselelwe uvavanyo |
| Inqanaba leproteni ye-p62 | Ukuqhekezwa nokutshintshwa kweproteni eqhagamshela izinto ezithwelweyo, edla ngokuqhekezwa kwii-autolysosome | I-flux yodwa; iindlela zoxinzelelo nazo zilawula ukukhutshelwa kolwazi lwemfuza lwe-p62 |
| Isiphawuli esidityanisiweyo se-mCherry-GFP-LC3 | Ukuqhubela phambili ukuya kwii-autolysosome ezine-asidi (ukutshintsha ukusuka kumthubi ukuya kubomvu kuphela) | Ukuqhekezwa ngokupheleleyo kohlobo ngalunye lwezinto ezithwelweyo |
| I-Phospho-AMPK ne-phospho-ACC | Ukusebenza kwesiva soxinzelelo lwamandla kunye nomqondiso ophuma kuso | Ukuba i-autophagy ibangele isiphumo sokusinda; i-AMPK ilawula amasebe amaninzi emetabolism |
| I-Phospho-mTOR, p70S6K, 4EBP1 | Umsebenzi wendlela yemiqondiso yokukhula ethintela i-autophagy | I-flux; le yimiqondiso evumela inkqubo ekuqaleni kwayo, hayi imilinganiselo yokulahla |
Qaphela umgibe omnye wamagama okhuselwa yile theyibhile. I-ACC efakwe iqela le-phosphate luhlobo olunomsebenzi we-enzayim othintelweyo. Ukunyuka kwayo kubonisa umqondiso ophuma kwi-AMPK, hayi ukuvuselelwa kwe-ACC.
Ibinzana elithi “ukuthintelwa kwe-macroautophagy kwaluphelisa ukhuseleko lweeseli zemithambo-luvo” lifuna ukuchaneka okufanayo. Kwimodeli ye-HT22 lithetha ukuba ukuphazamisa indlela ye-autophagy nee-lysosome kwasusa inzuzo yokusinda eyayinikelwa yi-methylene blue xa iiseli zazivinjwe i-serum. Lixhasa ukuba i-autophagy yayiyimfuneko okanye yayinegalelo kwezo seli zazikwisitya sovavanyo. Alimiseli ukuba i-autophagy kuphela kwendlela yokhuselo yale khompawundi, alingqini ukuba i-chloroquine isebenza nge-autophagy kuphela, yaye ukuphazanyiswa ngokwemfuza kwe-ATG5, ATG7, okanye ULK1 bekuya kufuneka ukuze kuqiniswe ibango lokubangela isiphumo.
Iindlela ezimbini eziya kwizixhobo ezifanayo
Imephu eyenziwe lula iyanceda. Uxinzelelo lwamandla lonyusa i-AMPK, i-AMPK ivuselela indibanisela ye-ULK1, ize i-autophagy iqale. Izinto ezikhuthaza ukukhula nezondlo zonyusa i-mTORC1, i-mTORC1 ithintela i-ULK1, ize i-autophagy ihlale ingasebenzi. Emva kwe-ULK1, indibanisela ye-Beclin-1 ne-VPS34 iqalisa ukwakheka kwe-phagophore, kongezwe ii-lipid kwi-LC3-I ukuze ibe yi-LC3-II, i-autophagosome ivaleke, idibane ne-lysosome, zize izinto ezithwelweyo ziqhekezwe kunye ne-p62.
Lo mzobo ubeka isiphumo ngasinye se-methylene blue kule mephu: isiphumo se-hippocampus ecaleni kwesebe le-AMPK, nesiphumo seeseli ezingabanduleli ecaleni kwesebe le-mTOR.
Le ndlela yokujonga icacisa ukungavisisani okubonakala kukho nophononongo lwesibini luka-Xie. Kwiiseli zempuku ezingabanduleli beeseli zemithambo-luvo ezazisanda, uvavanyo olwahlukileyo lwafumanisa ukuba i-5 μM ye-methylene blue yathintela ukwanda yaza yakhuthaza imeko yokungasebenzi, iiseli ezinophawu lwe-Ki67 zincipha ngosuku loku-1 nolwesi-3, ngoxa ukwahluka kwazo zibe zii-neuron emva kokuzibophelela kuloo ndlela kwahlala kungaphazamisekanga. Apho indlela yokusebenza yayiquka ukucinezelwa kwemiqondiso ye-mTOR: ukuncipha kwe-mTOR iyonke ne-mTOR efakwe iqela le-phosphate, kunye nokwehla kokubonakaliswa kwe-p70S6K ne-4EBP1, kwanokuncipha kokubonakaliswa kwee-cyclin E1, B1, D1, ne-D2. Olona phawu lwalubalasele yayikukungasebenzi kweeseli ezingabanduleli, nto leyo ababhali abayinxulumanisa nokulibaziseka kokuguga kweeseli, hayi ukuhlangulwa kweeseli ze-hippocampus eziphantsi koxinzelelo.
Akukho kuphikisana phakathi kwala maphepha mabini. I-AMPK ne-mTOR zidibanisa imiqondiso ngokuxhomekeke kwimeko, azizozitshixo ezinokusebenza okusisigxina. Iseli efana ne-neuron ephantsi koxinzelelo inokusebenzisa i-AMPK ngaphandle kokuthintelwa kwe-mTOR okunokulinganiswa, ngoxa iseli engumanduleli eqhutywa ngumqondiso okhuthaza ukwahlukana inokusabela kwakwikhompawundi enye ngokucutha izixhobo zokukhula ezixhomekeke kwi-mTOR. Uhlobo lweseli, imeko yoxinzelelo, uxinaniso, nexesha lokulinganisa zigqiba ukuba leliphi isebe elilawulayo. Yiyo loo nto iziphumo ze-methylene blue kumandla ee-mitochondria nakwi-redox kufuneka ziqwalaselwe kumfanekiso omnye: njengoko uphononongo lweendlela zokukhusela iiseli zemithambo-luvo luchaza, le khompawundi isebenza njengomnye umthwali wee-electron, ivuselela imiqondiso yokuthintela i-oxidation ne-apoptosis, ithintela i-MAO ne-NOS, ize inxulumane nokuhlaziywa kwee-mitochondria ngokwenziwa kwezintsha ne-autophagy. I-autophagy lelinye isebe lekhompawundi eneziphumo ezininzi, ayilobali liphela.
Oko kungaboniswa luvavanyo
Ukuvinjwa i-serum kulinganisa uxinzelelo lokunqongophala kwenkxaso yokondleka nokuphila kunye noxinzelelo lwemetabolism kwisitya sovavanyo. Akulinganisi ukuzila ukutya kwesidalwa sonke: i-glucose, ii-amino acid, neetyuwa zihlala zikhona kumxube osisiseko wokukhulisa iiseli, yaye akukho kuziphatha kokutya. Uxinaniso lokukhulisa iiseli olusuka kwi-20 nM ukuya kwi-5 μM alunakuguqulelwa kwiidosi zabantu ngezibalo nje, kuba ukufunxwa, ukubotshelelwa kwiiproteni, imeko ye-redox, imetabolism, ukuqokelelana kwizicwili, nexesha lokuchanabeka zonke ziyahluka.
Akukho lingo ebantwini eliyilelwe ukubonisa ukuhamba kwenkqubo ye-autophagy okuvuselelwe yi-methylene blue kwizigulana. Ubungqina bendlela yokusebenza busahleli kuphando lweeseli, lwezicwili ezingaphandle komzimba, nolwezilwanyana, yaye uphononongo lusachaza ubungqina bokudlulisela ezi ziphumo ekusetyenzisweni ebantwini njengobokuqala. Ngoko ezi zifundo azibonisi ukuba i-methylene blue iphinda ivelise imeko yokuzila ukutya, yandisa ubude bobomi babantu nge-autophagy, okanye igqibezela ukuhamba kwenkqubo ye-autophagy kwingqondo yomntu. Amabango okulinganisa ukuzila ukutya nawokuphila ixesha elide afuna ubungqina bawo bezonyango, yaye amalingo e-methylene blue ebantwini ngezinye iinjongo awabi ngamalingo e-autophagy ngenxa yokunxulunyaniswa nje. Uphando ngeemodeli zoxinzelelo lokunqongophala kwezondlo nezokuphila ixesha elide kufuneka lufundwe kugcinwe loo mda.
Kwiphepha elilandelayo olifundayo, sebenzisa olu luhlu lokuhlola ngokulandelelana. Qala ubuze ukuba leliphi isebe elitshintshileyo: i-AMPK, i-mTOR, okanye akukho nanye kuzo. Emva koko buza ukuba isiphumo se-LC3 sikhatshwe luvavanyo lwe-flux okanye sizimele sodwa. Emva koko jonga ukuba ukuthintela indlela kuyisusile na inzuzo, nokuba kusetyenziswe yiphi indlela. Uphononongo oluphendula yomithathu le mibuzo lunesiseko sebango elomeleleyo lendlela yokusebenza. Oluphendula owokuqala kuphela lubonise umqondiso ovumela inkqubo, yaye inxalenye eseleyo yohambo ayikacaciswa.