Chinyorwa
Methylene blue neautophagy: AMPK, mTOR, nekuedza pamasero etsinga
Fungidzira uchiita kuedza pamasero, wowedzera methylene blue, woona kuti mutsetse weprotein unonzi LC3-II wava kusviba zvakanyanya pabloti yako. Zvinogona kukwezva kuti utaure kuti chinhu ichi “chinowedzera autophagy” wozopfuudzira mberi. Mhedziso iyoyo inenge yakurumidzirwa. Mutsetse weLC3-II wakasviba zvakanyanya unogona kureva kuti sero riri kugadzira zvihomwe zvakawanda zvekuchenesa, kana kuti zvihomwe izvi zviri kuungana nokuti danho rekurasa zvakatakurwa rakavharika. Dudziro mbiri idzi dzinonongedzera kumativi akapesana, uye zvinyorwa zvetsvakurudzo yemethylene blue pamasero etsinga zvine mienzaniso yeuchapupu hwemhando imwe neimwe. Chinyorwa chino chinotsanangura zvakayerwa chaizvo, bvunzo neimwe neimwe.
Macroautophagy, rudzi rwakadzidzwa pano, inzira inoshandiswa nesero kudzokorodza zvinhu zvakawanda pamwe chete. Chihomwe chemembrane chinonzi phagophore chinokura chichikomberedza maprotein nemaorganelle asakara, chovhara chava autophagosome, chobatana nelysosome, uye zvachakatakura zvinoputswa zvobudiswa kuti zvishandiswezve. Masero etsinga anovimba nemaitiro aya nokuti haapatsanuki uye haagoni kuderedza huwandu hwezvikamu zvakakuvadzwa nekuzvipatsanura. Kana kudzokorodza kwamira, marara anoungana. Kana kuchienderera, sero rinowanazve zvinhu zvekuvaka naro parinenge riri pasi pekushushikana.
Zvakaratidzwa nekumwe kuedza pamasero ehippocampus
Chidzidzo chikuru chekutanga macroautophagy chakashandisa masero eHT22 ehippocampus yembeva, akaiswa pasi pekushushikana nekushayiswa serum kwenguva ye24 hours. Mamiriro aya anobvisa zvinhu zvinokurudzira kukura nemasaini anotsigira masero, achikonzera zvose apoptosis neautophagy. Vatsvakurudzi vakataura kuti methylene blue yakaderedza kufa kwemasero zvichienderana nehuwandu hwayo hwakashandiswa, uye kuti kudzivirirwa uku kwakauya pamwe nezviratidzo zvemacroautophagy.
Zviratidzo zvacho zvaive zvakanyatsojeka. Ruvara rweLC3B rwakanga rwakapararira rwakatangazve kuungana kuita tumapundu twakapoteredza nucleus paanenge 20 nM emethylene blue, uye kuchinja kukuru kweLC3-I neLC3-II kwakawanikwa pedyo ne1 μM. Zvose LC3B-I neLC3B-II zvakawedzera zvichienderana nehuwandu hwakashandiswa nenguva. Chinokosha ndechekuti boka iri harina kungogumira pabloti yeLC3 inoratidza mamiriro epanguva imwe chete. Vakawedzera 10 μM yechloroquine, iyo inovhara kubviswa kwezvinhu nelysosome, vakawana LC3-II yakawanda pamwe nep62 yakaramba iripo mumasero akashandiswa methylene blue. Maitiro aya anotsigira kuputswa nekutsiviwa kwezvinhu kunoenderana nelysosome, kwete kungoungana kwezvinhu chete. Vakaratidzawo kuti chloroquine yakadzorera shure chikamu chemhedzisiro yekudzivirira apoptosis, zvichibatanidza kudzivirirwa uku nenzira yeautophagy nelysosome mumuenzaniso uyu.
Zvakawanikwa pamusoro pekutumira masaini ndizvo zvinowanzotaurwa zvisirizvo. Kutumira masaini kweAMPK, kwakayerwa neAMPK yakaiswa phosphate uye ACC yakaiswa phosphate iyo inobatwa neAMPK pamatanho anotevera, kwakawedzera pa12 ne24 hours zvichienderana nehuwandu hwakashandiswa. Compound C, chinhu chinodzivisa AMPK, chakaiswa pa20 μM munguva yekupedzisira ye4 hours, chakaderedza zvose kuiswa kwephosphate paACC nekumutswa kweLC3. Asi mTOR yakaiswa phosphate haina kuratidza kudzvinyirirwa kwaigona kuonekwa. Saka mumasero ehippocampus aive pasi pekushushikana, zvakawanikwa zvaive kutanga kwemaitiro kwakabatana neAMPK kuburikidza nenzira yairatidzika kusavimba nemTOR. Maitiro mamwe chete akaonekwa mumbeva mhenyu, umo kushandiswa kwemethylene blue kwakamutsa zviratidzo zvemacroautophagy mucortex nehippocampus.
Kutanga maitiro hakusi kupedzwa kweflux
Pano ndipo panonyanya kukosha kusiyanisa pfungwa idzi. Kutanga maitiro kunoreva kuti hurongwa hwematanho ekutanga hunogadzira maautophagosome akawanda. Flux inoreva kuti zvinhu zvinotakurwa zvinopfuura nenzira yose: kuvharirwa, kukura, kubatana nelysosome, kuputswa, nekudzokorodzwa. Kuwedzera kwese kweLC3-II kunongova chirevo chekutanga maitiro kusvikira bvunzo yeflux yasimbisa mamwe matanho erwendo rwacho.
Bvunzo yeflux inowanzoshandiswa inoyera LC3-II paine uye pasina kuvharwa kwenguva pfupi kwelysosome nechinhu chakaita sebafilomycin A1 kana chloroquine. Kana chinhu chashandiswa chichinyatsokurumidzisa kugadzirwa, kuvhara kuparadzwa kunofanira kuita kuti LC3-II iwedzere zvakare. Mucherechedzi unosanganisa mCherry-GFP-LC3 unopa imwe nzira yekuongorora: zvimiro zvinopenya zviri yellow zvisati zvabatana nelysosome uye zvinopenya zviri red chete mushure mekunge acid yadzima chiratidzo cheGFP. Saka kuwanda kwetumapundu tune red chete kunotsigira kufambira mberi kupinda mumaautolysosome ane acid. Kuderera kweprotein yep62 kunogona kutsigira kuti zvinhu zvinotakurwa zviri kuputswa nekutsiviwa, asi panofanira kurangarirwa kuti kunyorwa kwemirayiridzo yekugadzira p62 pachako kunochinja pakushushikana uye kunofanira kuongororwa padanho remRNA.
Rondedzero yekuongorora iri pazasi inobatanidza bvunzo imwe neimwe yakashandiswa muzvinyorwa izvi nezvainoyera uye nezvaisingagoni kuratidza iri yoga. Vaverengi vanogona kuishandisa zvakare pachirevo chipi zvacho chemacroautophagy chichabuda mune ramangwana, kusanganisira nharo dzekutsanya nekurarama kwenguva refu dzinoshandisa zviratidzo izvi.
| Bvunzo | Zvainoyera | Zvaisingagoni kuratidza iri yoga |
|---|---|---|
| Huwandu hweLC3B-I | Ukuru hwehuwandu hwezvinhu zvekutanga zviri mucytosol zvinowanikwa kuti zvibatanidzwe nemalipid | Kushanda kweautophagy; kugadzirwa nekugadziridzwa kwezvinhu kunochinjawo huwandu uhwu |
| Huwandu hweLC3B-II | Huwandu hweLC3 yakasungirirwa pamembrane pazvimiro zveautophagy | Flux yakapedzwa; kuwedzera kunogona kureva kugadzirwa kunokurumidza kana kubviswa kwakavharwa |
| Tumapundu tweLC3 nekuungana kwakapoteredza nucleus | Kugoverwazve kweLC3 muzvimiro zvezvihomwe | Kwakananga kuchinja; mifananidzo yepanguva imwe chete haigoni kusiyanisa kukurumidza kwemaitiro nekuvharika kwekufamba |
| LC3-II paine ichienzaniswa nepasisina chloroquine kana bafilomycin A1 | Kana maautophagosome matsva achiramba achiumbwa apo kubviswa kwezvinhu kwakavharwa | Kuputswa kwakakwana kwezvinhu zvinotakurwa; zvinoda kushandiswa kwechinhu chinodzivisa kwenguva pfupi yakanyatsogadzirirwa bvunzo |
| Huwandu hweprotein yep62 | Kuputswa nekutsiviwa kwechinobatanidza zvinhu zvinotakurwa, chinowanzoparadzwa mumaautolysosome | Flux chete; nzira dzinoshanda pakushushikana dzinodzorawo kunyorwa kwemirayiridzo yekugadzira p62 |
| Mucherechedzi unosanganisa mCherry-GFP-LC3 | Kufambira mberi kupinda mumaautolysosome ane acid (kuchinja kubva payellow kuenda kured chete) | Kuputswa kwakakwana kwerudzi rwose rwezvinhu zvinotakurwa |
| Phospho-AMPK nephospho-ACC | Kushanda kwechinonzwa kushushikana kwesimba uye zvachinobudisa | Kuti autophagy ndiyo yakakonzera kuramba kwemaseru ari mapenyu; AMPK inodzora mapazi akawanda emetabolism |
| Phospho-mTOR, p70S6K, 4EBP1 | Kushanda kwenzira yemasaini ekukura inodzivisa autophagy | Flux; izvi zviratidzo zvinobvumira matanho ekutanga, kwete zviyero zvekurasa zvinhu |
Cherechedza musungo mumwe wemashoko unodzivirirwa netafura iyi. ACC yakaiswa phosphate ndiyo iri muchimiro chine kushanda kweenzyme kwakadziviswa. Kuwedzera kwayo kunoratidza zvinobudiswa nekushanda kweAMPK, kwete kumutswa kweACC.
Mashoko ekuti “kudzivisa macroautophagy kwakabvisa kudzivirirwa kwemasero etsinga” anofanirawo kunyatsorehwa. Mumuenzaniso weHT22 anoreva kuti kuvhiringidza nzira yeautophagy nelysosome kwakabvisa mukana wakapiwa nemethylene blue wekuti masero arambe ari mapenyu pakushayiswa serum. Izvi zvinotsigira kuti autophagy yaidiwa kana kuti yaibatsira mundiro iyoyo yekuedza. Hazviratidzi kuti autophagy ndiyo chete nzira yekudzivirira yechinhu ichi, hazviratidzi kuti chloroquine inoshanda kuburikidza neautophagy chete, uye kuchinja ATG5, ATG7, kana ULK1 nenzira dzemajini kwaizodiwa kusimbisa chirevo chechikonzero nemhedzisiro.
Nzira mbiri dzinopinda muhurongwa humwe chete
Mepu yakapfava inobatsira. Kushushikana kwesimba kunowedzera kushanda kweAMPK, AMPK inomutsa mubatanidzwa weULK1, uye autophagy inotanga. Zvinhu zvinokurudzira kukura nezvinovaka muviri zvinowedzera kushanda kwemTORC1, mTORC1 inodzivisa ULK1, uye autophagy inoramba isina kutanga. Pamatanho anotevera ULK1, mubatanidzwa weBeclin-1 neVPS34 unotanga kuumbwa kwephagophore, LC3-I inosungirirwa kumalipid yova LC3-II, autophagosome inovhara, yobatana nelysosome, uye zvinhu zvayakatakura zvinoputswa pamwe nekuputswa nekutsiviwa kwep62.
Mufananidzo unoisa chinhu chimwe nechimwe chakawanikwa nezvemethylene blue pamepu iyi: zvakawanikwa pahippocampus pedyo nebazi reAMPK, uye zvakawanikwa pamasero anotangira mamwe pedyo nebazi remTOR.
Maonero aya anogadzirisa zvinoita sokusawirirana nechidzidzo chechipiri chaXie. Mumasero anotangira masero etsinga emakonzo aiwanda, kumwe kuedza kwakawana kuti 5 μM yemethylene blue yakadzivisa kuwanda kwemasero uye yakakurudzira kupinda mumamiriro ekuzorora pasina kupatsanuka. Masero ane Ki67 akaderera pamazuva 1 ne3, asi kushanduka kuita masero etsinga kwakanga kwatotanga kwakaramba kusina kukanganiswa. Ipapo nzira yacho yakashanda kuburikidza nekudzvinyirirwa kwekutumira masaini kwemTOR: huwandu hwese hwemTOR nemTOR yakaiswa phosphate hwakaderera pamwe nekuderera kwekugadzirwa kwep70S6K ne4EBP1, uye kuderedzwa kwekugadzirwa kwemacyclin E1, B1, D1, neD2. Shanduko yainyanya kuoneka yaive kupinda kwemasero anotangira mamwe mumamiriro ekuzorora pasina kupatsanuka, uko vanyori vakabatanidza nekunonoka kwekukwegura kwemasero, kwete kununurwa kwemasero ehippocampus aive pasi pekushushikana.
Hapana kupesana pakati pezvinyorwa zviviri izvi. AMPK nemTOR zvinobatanidza masaini zvichienderana nemamiriro aripo; hazvisi maswichi asingachinji. Sero rakaita seresinga riri pasi pekushushikana rinogona kushandisa AMPK pasina kudziviswa kwemTOR kunogona kuyerwa, ukuwo sero rinotangira mamwe richikurudzirwa nezvinhu zvinokonzera kupatsanuka richigona kupindura chinhu chimwe chete nekuderedza kushanda kwehurongwa hwekukura hunovimba nemTOR. Rudzi rwesero, mamiriro ekushushikana, concentration, nenguva yekuyera ndizvo zvinotonga kuti ibazi ripi rinonyanya kushanda. Ndokusakawo mhedzisiro yemethylene blue pasimba remitochondria neparedox ichifanira kuonekwa mumufananidzo mumwe chete: sezvinotsanangurwa neongororo yenzira dzekudzivirira masero etsinga, chinhu ichi chinoshanda semumwe mutakuri wemaelectron, chinoshandisa masaini ekudzivirira oxidation neapoptosis, chinodzivisa MAO neNOS, uye chinobatana nekuvandudzwa kwemitochondria kuburikidza nekugadzirwa kwemitsva neautophagy. Autophagy ibazi rimwe remhedzisiro dzakawanda dzechinhu ichi, kwete nyaya yacho yose.
Zvisingaratidzwi nekuedza uku
Kushayiswa serum kunomiririra kushushikana kunobva pakushaya rutsigiro rwekukura nekuchinja kwemetabolism mundiro yekuedza. Hakumiririri kutsanya kwemuviri wose: glucose, maamino acid, nemunyu zvinoramba zviri mumushonga wekutanga unorimwa masero, uye hapana maitiro ekudya aripo. Maconcentration anoshandiswa pakurima masero kubva pa20 nM kusvika pa5 μM haagoni kushandurwa kuita huwandu hwekushandisa kuvanhu nekuverenga chete, nokuti kupinda mumuviri, kusungirirwa kumaprotein, mamiriro eredox, metabolism, kuungana mumatishu, nenguva yekusangana nechinhu ichi zvose zvinosiyana.
Hapana kuedza pavanhu kwakagadzirirwa kuratidza autophagic flux yakakonzerwa nemethylene blue muvarwere. Uchapupu hwenzira dzekushanda hunoramba huri hwezvidzidzo zvemasero, zvezvikamu zvakabviswa mumuviri, nezvemhuka, uye ongororo dzichiri kutsanangura uchapupu hwekushandisa zvakawanikwa izvi kuvanhu sehwematanho ekutanga. Saka zvidzidzo izvi hazviratidzi kuti methylene blue inotevedzera kutsanya, inowedzera urefu hweupenyu hwevanhu kuburikidza neautophagy, kana kuti inopedzisa autophagic flux muuropi hwevanhu. Zvinotaurwa nezvekutevedzera kutsanya nekurarama kwenguva refu zvinoda uchapupu hwazvo hwekuedza kuvanhu, uye kuedza kwemethylene blue kuvanhu kune zvimwe zvinangwa hakungovi kuedza autophagy nokungobatanidzwa nayo. Tsvakurudzo yemienzaniso yekushushikana kunokonzerwa nezvinovaka muviri neyokurarama kwenguva refu inofanira kuverengwa uchichengeta muganhu iwoyo.
Pachinyorwa chinotevera chetsvakurudzo chaunoverenga, shandisa rondedzero yekuongorora uchitevera marongerwo ayo. Tanga wabvunza kuti ibazi ripi rakachinja: AMPK, mTOR, kana kuti hapana. Wobvunza kana zvakawanikwa paLC3 zvakabatanidzwa nebvunzo yeflux kana kuti zvakamira zvoga. Wozotarisa kana kuvhara nzira yacho kwakabvisa kubatsira kwacho, uye kuti zvakaitwa nenzira ipi. Chidzidzo chinopindura zvose zvitatu chinopa hwaro hwakasimba hwechirevo chenzira yekushanda. Chinopindura chekutanga chete chinenge changoratidza chiratidzo chinobvumira maitiro, uye mamwe matanho erwendo anenge achiri asina kutsanangurwa.