Isihloko
I-methylene blue ne-autophagy: i-AMPK, i-mTOR, nezivivinyo zamaseli ezinzwa
Cabanga wenza isivivinyo ngamaseli, ufaka i-methylene blue, bese ubona ibhande lephrotheni ebizwa nge-LC3-II liya liba mnyama esivivinyweni sakho se-blot. Kuyalinga ukumemezela ukuthi le nhlanganisela yamakhemikhali "ikhulisa i-autophagy" bese uqhubeka. Leso siphetho siphuthuma kakhulu. Ibhande le-LC3-II elimnyama kakhulu lingasho ukuthi iseli lakha izikhwanyana zolwelwesi eziningi zokuhlanza, noma lingasho ukuthi lezi zikhwanyana ziyanqwabelana ngoba isinyathelo sokulahla imfucuza sivimbekile. Lezi zincazelo ezimbili zikhomba ezinhlangothini eziphikisanayo, futhi imibhalo yocwaningo nge-methylene blue kumaseli ezinzwa inezibonelo zohlobo ngalunye lobufakazi. Lesi sihloko sihlola lokho okwalinganiswa ngempela, isivivinyo ngesivivinyo.
I-macroautophagy, uhlobo olucwaningwe lapha, iyindlela iseli elisebenzisa ngayo kabusha izinto ngobuningi. Isikhwama solwelwesi esibizwa nge-phagophore sikhula sizungeze amaphrotheni agugile nezakhi ezingaphakathi kweseli, sivale sibe yi-autophagosome, sihlangane ne-lysosome, bese okukuso kubhidlizwa kukhishwe ukuze kusetshenziswe kabusha. Amaseli ezinzwa ancike kule nqubo ngoba awahlukani futhi awakwazi ukunciphisa ingxenye yezakhi ezilimele ngokuhlukana. Lapho ukusetshenziswa kabusha kuma, imfucuza iyanqwabelana. Lapho kuqhubeka, iseli libuyisa izinto zokwakha lapho libhekene nengcindezi.
Lokho okwaboniswa yisivivinyo esisodwa se-hippocampus
Isifundo esiyinhloko sokuqalisa sasebenzisa amaseli e-HT22 avela ku-hippocampus yegundane, afakwa ngaphansi kwengcindezi ngokuncishwa i-serum amahora angu-24. Lesi simo sisusa izici ezikhuthaza ukukhula nezimpawu ezisekela ukuphila kwamaseli, futhi sivusa kokubili i-apoptosis ne-autophagy. Abacwaningi babika ukuthi i-methylene blue yanciphisa ukufa kwamaseli ngokwezinga lomthamo, nokuthi lokhu kuvikelwa kwakuhambisana nezinkomba ze-macroautophagy.
Ukulandelana kwezinkomba kwakucacile. Ukufakwa kombala kwe-LC3B okwakusabalele kwaphinde kwasabalala kwaba amachashazi azungeze i-nucleus, kuqala cishe ku-20 nM we-methylene blue, kanti ukusabela okuqine kakhulu kwe-LC3-I ne-LC3-II kwaba seduze no-1 μM. Kokubili i-LC3B-I ne-LC3B-II kwakhuphuka ngokwezinga lomthamo nesikhathi. Okubalulekile ukuthi iqembu ladlulela ngalé kwe-blot ye-LC3 ekhombisa isimo somzuzu owodwa. Lafaka u-10 μM we-chloroquine, ovimba ukususwa kwezinto ngama-lysosome, lathola i-LC3-II ephakeme kanye ne-p62 ezinzile kumaseli afakwe i-methylene blue. Leli phethini lisekela ukubhidlizwa nokuvuselelwa kwezinto okuncike kuma-lysosome, kunokuba kube ukunqwabelana nje. Labonisa nokuthi i-chloroquine yawunciphisa ngokwengxenye umphumela wokuvimbela i-apoptosis, okuyinto exhumanisa ukuvikelwa nendlela ye-autophagy nama-lysosome kule modeli.
Umphumela wokudluliselwa kwezimpawu yiwona ovame ukucashunwa ngendlela engafanele. Ukudluliselwa kwezimpawu kwe-AMPK, okwalinganiswa nge-AMPK efakwe iqembu le-phosphate nange-ACC efakwe iqembu le-phosphate okuyithagethi yayo elandelayo, kwakhuphuka emahoreni angu-12 nangu-24 ngokwezinga lomthamo. I-Compound C, eyisivimbeli se-AMPK, yafakwa ku-20 μM phakathi namahora angu-4 okugcina, yanciphisa kokubili ukufakwa kwamaqembu e-phosphate ku-ACC nokuvuselelwa kwe-LC3. Ngokuphambene, i-mTOR efakwe iqembu le-phosphate ayizange ibonise ukucindezelwa okwakungatholakala ngesilinganiso. Ngakho, kumaseli e-hippocampus ayengaphansi kwengcindezi, okwatholakala kwakuwukuqalisa okuhlobene ne-AMPK ngendlela eyayibonakala ingancikile ku-mTOR. Iphethini efanayo yabonakala kumagundane aphilayo, lapho ukufakwa kwe-methylene blue kwaqalisa izinkomba ze-macroautophagy ku-cortex naku-hippocampus.
Ukuqalisa akukhona ukugeleza okuphelele
Lapha umehluko phakathi kwemiqondo ubaluleke kakhulu. Ukuqalisa kusho ukuthi izingxenye ezisekuqaleni kwenqubo zakha ama-autophagosome amaningi. Ukugeleza kusho ukuthi izinto ezithwelwe yilezi zikhwama zihamba yonke indlela: ukubiyelwa, ukuvuthwa, ukuhlangana ne-lysosome, ukubhidlizwa, nokusetshenziswa kabusha. Konke ukwanda kwe-LC3-II kuwubufakazi bokuqalisa kuphela kuze kube yilapho isivivinyo sokugeleza siqinisekisa ingxenye esele yohambo.
Isivivinyo esijwayelekile sokugeleza silinganisa i-LC3-II lapho kunokuvinjelwa kwama-lysosome kwesikhashana, njengokusebenzisa i-bafilomycin A1 noma i-chloroquine, nalapho kungekho lokho kuvinjelwa. Uma okufakiwe kusheshisa ngempela ukukhiqizwa, ukuvimba ukubhidlizwa kufanele kukhuphule i-LC3-II nakakhulu. Isikhombisi esihlanganisa i-mCherry-GFP-LC3 sengeza enye indlela yokubheka: izakhi zikhanya ngokuphuzi ngaphambi kokuhlangana ne-lysosome, bese zikhanya ngokubomvu kuphela ngemva kokuba i-asidi icime isignali ye-GFP. Ngakho amachashazi amaningi abomvu kuphela asekela ukuqhubekela kuma-autolysosome ane-asidi. Ukwehla kwephrotheni ye-p62 kungaqinisekisa ukubhidlizwa nokuvuselelwa kwezinto ezithwelwe, kodwa kufanele kukhunjulwe ukuthi ukubhalwa kolwazi lofuzo lwe-p62 nakho kusabela engcindezini futhi kufanele kuhlolwe ezingeni le-mRNA.
Uhlu lokuhlola olungezansi luhlanganisa isivivinyo ngasinye esisetshenziswe kule mibhalo yocwaningo nalokho esikulinganisayo nalokho esingeke sikufakazele sodwa. Abafundi bangalusebenzisa futhi kunoma yisiphi isimangalo esizayo nge-macroautophagy, kuhlanganise nezimpikiswano ngokuzila ukudla nokuphila isikhathi eside ezisebenzisa lezi zinkomba.
| Isivivinyo | Lokho esikulinganisayo | Lokho esingeke sikufakazele sodwa |
|---|---|---|
| Izinga le-LC3B-I | Ubukhulu beqoqo lezanduleli eziku-cytosol ezitholakalayo ukuze kuhlanganiswe ama-lipid kuzo | Ukusebenza kwe-autophagy; ukukhiqizwa nokucutshungulwa nakho kuyakulishintsha |
| Izinga le-LC3B-II | Inani le-LC3 eboshelwe kulwelwesi ezakhiweni ze-autophagy | Ukugeleza okuphelele; ukwanda kungasho ukukhiqizwa okusheshayo noma ukususwa okuvinjiwe |
| Amachashazi e-LC3 nokunqwabelana okuzungeze i-nucleus | Ukusabalala kabusha kwe-LC3 iye ezakhiweni zezikhwanyana zolwelwesi | Isiqondiso soshintsho; izithombe zesimo somzuzu owodwa azikwazi ukuhlukanisa ukusheshisa nokuminyana kwenqubo |
| I-LC3-II lapho kukhona i-chloroquine noma i-bafilomycin A1 uma kuqhathaniswa nalapho kungekho | Ukuthi ama-autophagosome amasha ayaqhubeka yini ukwakheka lapho ukususwa kuvinjiwe | Ukubhidlizwa okuphelele kwezinto ezithwelwe; kudingeka ukuvezwa esivimbelini kwesikhashana okulungiselelwe kahle |
| Izinga lephrotheni ye-p62 | Ukubhidlizwa nokuvuselelwa kwephrotheni exhumanisa izinto ezithwelwe, evame ukubhidlizwa kuma-autolysosome | Ukugeleza kuphela; izindlela zengcindezi nazo zilawula ukubhalwa kolwazi lofuzo lwe-p62 |
| Isikhombisi esihlanganisa i-mCherry-GFP-LC3 | Ukuqhubekela kuma-autolysosome ane-asidi (ukushintsha kokuphuzi kube okubomvu kuphela) | Ukubhidlizwa okuphelele kwazo zonke izinhlobo zezinto ezithwelwe |
| I-AMPK ne-ACC ezifakwe amaqembu e-phosphate | Ukusebenza kwenzwa yengcindezi yamandla kanye nomphumela wayo | Ukuthi i-autophagy yiyo ebangele umphumela wokusinda; i-AMPK ilawula amagatsha amaningi okushintshaniswa kwezinto emzimbeni |
| I-mTOR efakwe iqembu le-phosphate, i-p70S6K, i-4EBP1 | Ukusebenza kwendlela yezimpawu zokukhula ebamba i-autophagy | Ukugeleza; lezi yizimpawu zokuvumela ezisekuqaleni kwenqubo, hhayi izilinganiso zokulahlwa kwezinto |
Qaphela ugibe olulodwa lwamagama leli thebula elisiza ukulugwema. I-ACC efakwe iqembu le-phosphate iyifomu umsebenzi walo we-enzyme ovinjelwe. Ukwanda kwayo kubonisa umphumela we-AMPK, hhayi ukuvuselelwa kwe-ACC.
Inkulumo ethi "ukuvinjelwa kwe-macroautophagy kwaqeda ukuvikelwa kwamaseli ezinzwa" idinga ukunemba okufanayo. Kumodeli ye-HT22 isho ukuthi ukuphazamisa indlela ye-autophagy nama-lysosome kwasusa inzuzo yokusinda eyayilethwa yi-methylene blue lapho amaseli encishwe i-serum. Isekela umqondo wokuthi i-autophagy iyadingeka noma inesandla kuleso sitsha sokucwaninga. Ayibonisi ukuthi i-autophagy iwukuphela kwendlela yokuvikela yale nhlanganisela, ayifakazeli ukuthi i-chloroquine isebenza nge-autophagy kuphela, futhi ukuphazanyiswa kofuzo lwe-ATG5, i-ATG7, noma i-ULK1 kungadingeka ukuze kuqiniswe isimangalo sokuthi iyimbangela.
Izindlela ezimbili ezingena ohlelweni olufanayo
Imephu eyenziwe lula iyasiza. Ingcindezi yamandla ikhuphula i-AMPK, i-AMPK ivuselela iqoqo le-ULK1, bese i-autophagy iqala. Izici zokukhula nezakhamzimba zikhuphula i-mTORC1, i-mTORC1 ivimbe i-ULK1, bese i-autophagy ihlala ingasebenzi. Ezigabeni ezilandela i-ULK1, iqoqo le-Beclin-1 ne-VPS34 liqalisa ukwakheka kwe-phagophore, i-LC3-I ihlanganiswe nama-lipid ibe yi-LC3-II, i-autophagosome ivale, ihlangane ne-lysosome, bese izinto ezithwelwe zibhidlizwa kanye nokubhidlizwa nokuvuselelwa kwe-p62.
Umdwebo ubeka okutholakele ngakunye nge-methylene blue kule mephu: umphumela we-hippocampus eceleni kwegatsha le-AMPK, nomphumela wamaseli angaphambi kokuvuthwa eceleni kwegatsha le-mTOR.
Le ndlela yokuchaza isusa ukungahambisani okubonakalayo nesifundo sesibili sika-Xie. Kumaseli angaphambi kokuvuthwa ezinzwa zegundane ayanda, isivivinyo esihlukile sathola ukuthi u-5 μM we-methylene blue wavimbela ukwanda futhi wakhuthaza isimo sokuphumula, lapho amaseli abonisa i-Ki67 ancipha ngosuku 1 nangosuku 3, kuyilapho ukuhlukaniseka kwamaseli asezinikele ukuba amaseli ezinzwa kwahlala kungaphazamisekile. Lapho, indlela yokusebenza yayihilela ukucindezelwa kokudluliselwa kwezimpawu kwe-mTOR: ukuncipha kwe-mTOR ephelele ne-mTOR efakwe iqembu le-phosphate, kanye nokwehla kokukhiqizwa kwe-p70S6K ne-4EBP1, nokwehliswa kwamazinga ama-cyclin E1, B1, D1, no-D2. Isici esivelele kwakuwukuphumula kwamaseli angaphambi kokuvuthwa, abalobi abakuxhumanisa nokubambezeleka kokuguga kwamaseli, hhayi ukusindiswa kwamaseli e-hippocampus ayengaphansi kwengcindezi.
Akukho ukuphikisana phakathi kwalawa maphepha amabili. I-AMPK ne-mTOR zihlanganisa izimpawu ngokwesimo, aziwona amaswishi angaguquki. Iseli elifana neseli lezinzwa elingaphansi kwengcindezi lingasebenzisa i-AMPK ngaphandle kokuvinjelwa kwe-mTOR okulinganisekayo, kuyilapho iseli elingaphambi kokuvuthwa eliqhutshwa yizimpawu ezikhuthaza ukuhlukana lingasabela kuyo le nhlanganisela ngokunciphisa ukusebenza kohlelo lokukhula oluncike ku-mTOR. Uhlobo lweseli, isimo sengcindezi, ukugxila kwenhlanganisela, nesikhathi sokulinganisa kunquma ukuthi yiliphi igatsha elibusayo. Yingakho nemiphumela ye-methylene blue emandleni ama-mitochondria nasezinhlelweni ze-redox kufanele ifakwe kulokhu kubuka okufanayo: njengoba ukubuyekezwa kwezindlela zokuvikela amaseli ezinzwa kuchaza, le nhlanganisela isebenza njengomthwali ohlukile wama-electron, ivuse ukudluliselwa kwezimpawu ezilwa nokonakala okubangelwa i-oxidation nezivimbela i-apoptosis, ivimbe i-MAO ne-NOS, futhi ixhumane nokuvuselelwa kwama-mitochondria ngokwakheka kwawo okusha nange-autophagy. I-autophagy iyigatsha elilodwa leqoqo lemiphumela ehlukahlukene, hhayi yonke indaba.
Lokho izivivinyo ezingakubonisi
Ukuncishwa i-serum kumodela ingcindezi yokwesekwa kokuphila kweseli nokushintshaniswa kwezinto esitsheni sokucwaninga. Akumodeli ukuzila ukudla komzimba wonke: i-glucose, ama-amino acid, nosawoti kusala oketshezini oluyisisekelo lokukhulisa amaseli, futhi akukho ukuziphatha kokudla. Ukugxila kwezinto okusetshenziselwa ukukhulisa amaseli kusukela ku-20 nM kuya ku-5 μM akunakuguqulwa kube yimithamo yabantu ngezibalo, ngoba ukumuncwa, ukubophezela emaphrothenini, isimo se-redox, ukushintshaniswa kwezinto emzimbeni, ukunqwabelana ezicutshini, nesikhathi sokuchayeka konke kuyahluka.
Akukho sivivinyo kubantu esiklanyelwe ukubonisa ukugeleza kwe-autophagy okuqaliswe yi-methylene blue ezigulini. Ubufakazi bendlela yokusebenza busalokhu buvela ocwaningweni lwamaseli, olwenzeka ezicutshini ezikhishwe emzimbeni, nolwezilwane, futhi ukubuyekezwa kusachaza ubufakazi bokudlulisela lokhu ekusetshenzisweni kubantu njengobokuqala. Ngakho lezi zifundo azibonisi ukuthi i-methylene blue ikhiqiza kabusha imiphumela yokuzila ukudla, yelula ubude bokuphila kwabantu nge-autophagy, noma iqedela ukugeleza kwe-autophagy ebuchosheni bomuntu. Izimangalo zokulingisa ukuzila ukudla nokuphila isikhathi eside zidinga ubufakazi bazo bomtholampilo, futhi izivivinyo ze-methylene blue kubantu ezimayelana nokunye ukusetshenziswa aziphenduki izivivinyo ze-autophagy ngenxa nje yokuhlotshaniswa nayo. Ucwaningo ngamamodeli engcindezi yezakhamzimba nokuphila isikhathi eside kufanele lufundwe kugcinwe lowo mngcele.
Ephepheni elilandelayo olifundayo, sebenzisa uhlu lokuhlola ngokulandelana. Qala ubuze ukuthi yiliphi igatsha elashintsha: i-AMPK, i-mTOR, noma alikho kulawa. Bese ubuza ukuthi umphumela we-LC3 wawuhambisana yini nesivivinyo sokugeleza noma wawumi wodwa. Bese uhlola ukuthi ukuvimba indlela yokusebenza kwayisusa yini inzuzo, nokuthi kwenziwa ngayiphi indlela. Isifundo esiphendula yonke le mibuzo emithathu sinobufakazi obusekela isimangalo esiqinile sendlela yokusebenza. Esiphendula owokuqala kuphela sibonise isignali yokuvumela, kanti ingxenye esele yohambo isalokhu ingakadwetshwa emephini.