Methylene blue ndi autophagy: AMPK, mTOR, ndi zoyesera za maselo a mitsempha

Tiyerekeze kuti mukuchita choyesera cha maselo, mwawonjezera methylene blue, ndipo mukuona kuti mzere wa puloteni wotchedwa LC3-II ukuyamba kuda kwambiri pa pepala lanu la blot. N'zosavuta kufuna kulengeza kuti mankhwalawa “amalimbikitsa autophagy” n'kupitiriza ndi zina. Kutanthauzira kumeneko n'kofulumira kwambiri. Mzere wa LC3-II wakuda kwambiri ungatanthauze kuti selo likupanga matumba ambiri ochotsera zinyalala, kapena ungatanthauze kuti matumbawo akuunjikika chifukwa gawo lotaya zinyalala latsekeka. Matanthauzo awiriwa akuloza mbali zosiyana, ndipo zolemba za kafukufuku wa methylene blue m'maselo a mitsempha zili ndi zitsanzo za umboni wa mtundu uliwonse. Nkhaniyi ikufotokoza zimene zinayezedwa kwenikweni, mayeso ndi mayeso.

Macroautophagy, mtundu umene waphunziridwa pano, ndi njira imene selo limaphwanya ndi kugwiritsanso ntchito zinthu zambiri nthawi imodzi. Thumba lopangidwa ndi nembanemba, lotchedwa phagophore, limakula mozungulira mapuloteni ndi ziwalo za selo zomwe zatha ntchito, limatseka n'kukhala autophagosome, limalumikizana ndi lysosome, ndipo zomwe lanyamula zimaphwanyidwa n'kutulutsidwa kuti zigwiritsidwenso ntchito. Maselo a mitsempha amadalira njira imeneyi chifukwa sagawikana ndipo sangathe kuchepetsa kuchuluka kwa mbali zowonongeka mwa kugawikana. Njira yogwiritsanso ntchito zinthu ikayima, zinyalala zimachuluka. Ikamayenda bwino, selo limapezanso zinthu zomangira pamene likukumana ndi mavuto.

Zimene choyesera chimodzi cha hippocampus chinasonyeza

Kafukufuku waukulu wokhudza kuyambitsa njirayi anagwiritsa ntchito maselo a HT22 ochokera ku hippocampus ya mbewa, omwe anaikidwa m'mavuto mwa kuwachotsera seramu kwa maola 24. Mkhalidwe umenewu umachotsa zinthu zolimbikitsa kukula ndi zizindikiro zothandiza kupulumuka kwa maselo, ndipo umayambitsa apoptosis ndi autophagy. Ofufuzawo ananena kuti methylene blue inachepetsa kufa kwa maselo mogwirizana ndi kuchuluka kwake, ndipo chitetezo chimenechi chinatsagana ndi zizindikiro za macroautophagy.

Zizindikiro zomwe anaona zinali zodziwika bwino. Mtundu wa LC3B womwe unali wofalikira unasamukira m'madontho ozungulira phata la selo kuyambira pafupifupi 20 nM ya methylene blue, ndipo kusintha kwakukulu kwambiri kwa LC3-I ndi LC3-II kunali pafupi ndi 1 μM. LC3B-I ndi LC3B-II zonse zinawonjezeka mogwirizana ndi kuchuluka kwa mankhwalawo komanso nthawi. Chofunika kwambiri n'chakuti gululo linapitirira kungoyeza LC3 ndi blot pa nthawi imodzi. Anawonjezera 10 μM ya chloroquine, yomwe imalepheretsa kuchotsa zinthu kudzera mu lysosome, ndipo anapeza LC3-II yambiri komanso p62 yomwe inakhalabe osaphwanyidwa m'maselo omwe anapatsidwa methylene blue. Zotsatira zimenezi zimathandiza kutsimikizira kuti zinthuzo zinkaphwanyidwa ndi kusinthidwa kudzera mu lysosome, osati kungounjikika. Anaonetsanso kuti chloroquine inachepetsa pang'ono mphamvu yoletsa apoptosis, zomwe zimagwirizanitsa chitetezocho ndi njira ya autophagy ndi lysosome mu chitsanzo chimenechi.

Zotsatira zokhudza kutumiza zizindikiro ndizo zomwe nthawi zambiri zimafotokozedwa molakwika. Kutumiza zizindikiro kwa AMPK, komwe kunayesedwa pogwiritsa ntchito AMPK yowonjezeredwa phosphate ndi ACC yowonjezeredwa phosphate yomwe AMPK imagwira ntchito pa iyo, kunawonjezeka pa maola 12 ndi 24 mogwirizana ndi kuchuluka kwa mankhwalawo. Compound C, choletsa AMPK chomwe chinagwiritsidwa ntchito pa 20 μM m'maola 4 omalizira, chinachepetsa kuwonjezeredwa kwa phosphate pa ACC komanso kuyambitsidwa kwa LC3. Mosiyana ndi zimenezi, mTOR yowonjezeredwa phosphate sinasonyeze kuponderezedwa komwe kukanatha kuyezedwa. Choncho, m'maselo a hippocampus omwe anali m'mavuto, zomwe zinapezeka zinali kuyambitsidwa kogwirizana ndi AMPK kudzera m'njira yomwe ikuoneka kuti sinadalire mTOR. Zotsatira zofananazi zinaonekeranso mu mbewa zamoyo, momwe kupatsidwa methylene blue kunayambitsa zizindikiro za macroautophagy mu cortex ndi hippocampus.

Kuyambitsa sikutanthauza kuti njira yonse yatha

Apa kusiyana kwa mfundozi n'kofunika kwambiri. Kuyambitsa kumatanthauza kuti zinthu zogwira ntchito kumayambiriro kwa njirayi zikupanga ma autophagosome ambiri. Kuyenda kwa njira yonse, kotchedwa flux, kumatanthauza kuti katundu akudutsa magawo onse: kutsekeredwa, kukhwima, kulumikizana ndi lysosome, kuphwanyidwa, ndi kugwiritsidwanso ntchito. Kuwonjezeka kulikonse kwa LC3-II kumangothandiza kunena kuti njirayi yayambitsidwa mpaka mayeso a flux atatsimikizira magawo otsalawo.

Mayeso a flux omwe amagwiritsidwa ntchito nthawi zambiri amayezera LC3-II pamene kuletsa kwakanthawi kochepa kwa lysosome, monga ndi bafilomycin A1 kapena chloroquine, kulipo komanso pamene kulibe. Ngati mankhwala amene akuyesedwa akufulumizadi kupanga, kuletsa kuphwanya kuyenera kuwonjezera LC3-II kwambiri kuposa poyamba. Chizindikiro chophatikiza mCherry-GFP-LC3 chimapereka njira yachiwiri yowonera: zinthu zimawala zachikasu zisanagwirizane ndi lysosome, ndipo zimawala zofiira zokha asidi atazimitsa chizindikiro cha GFP. Choncho, madontho ambiri ofiira okha amathandiza kutsimikizira kupita patsogolo kulowa m'ma autolysosome a asidi. Kuchepa kwa puloteni ya p62 kungathandizenso kutsimikizira kuphwanyidwa ndi kusinthidwa kwa katundu, koma pali chenjezo lakuti kupanga mRNA ya p62 kumasinthanso chifukwa cha mavuto omwe selo likukumana nawo ndipo kuyenera kuyesedwa pa mlingo wa mRNA.

Mndandanda womwe uli pansipa ukufananitsa mayeso aliwonse omwe agwiritsidwa ntchito m'mabuku a kafukufukuyu ndi zimene amayezera komanso zimene sangathe kutsimikizira pawokha. Owerenga angaugwiritsenso ntchito pa zonena zilizonse zam'tsogolo zokhudza macroautophagy, kuphatikizapo mfundo zokhudza kusala kudya ndi kukhala ndi moyo wautali zomwe zimadalira zizindikirozi.

Onani cholakwika chimodzi cha mawu chomwe tebulo limathandiza kupewa. ACC yowonjezeredwa phosphate ndi mtundu womwe ntchito yake ya enzyme yalepheretsedwa. Kuwonjezeka kwake kumasonyeza zotsatira za ntchito ya AMPK, osati kuyambitsidwa kwa ACC.

Mawu akuti “kuletsa macroautophagy kunathetsa chitetezo cha maselo a mitsempha” amafunikanso kulondola kotereku. Mu chitsanzo cha HT22, amatanthauza kuti kusokoneza njira ya autophagy ndi lysosome kunachotsa mwayi wa kupulumuka womwe methylene blue inapereka pamene seramu inachotsedwa. Izi zimathandiza kutsimikizira kuti autophagy inali yofunika kapena inathandizira m'choyeseracho. Sizitsimikizira kuti autophagy ndiyo njira yokhayo imene mankhwalawa amaperekera chitetezo, sizitsimikizira kuti chloroquine imagwira ntchito kudzera mu autophagy yokha, ndipo kusintha majini a ATG5, ATG7, kapena ULK1 kungafunike kuti umboni wa zomwe zinayambitsa zotsatirazo ukhale wolimba.

Njira ziwiri zolowera m'dongosolo lomwelo

Mapu osavuta amathandiza. Mavuto a mphamvu amawonjezera ntchito ya AMPK, AMPK imayambitsa gulu la ULK1, ndipo autophagy imayamba. Zinthu zolimbikitsa kukula ndi zakudya zimawonjezera ntchito ya mTORC1, mTORC1 imalepheretsa ULK1, ndipo autophagy imakhalabe yosayambitsidwa. Pambuyo pa ntchito ya ULK1, gulu la Beclin-1 ndi VPS34 limayambitsa kupanga phagophore, LC3-I imawonjezeredwa lipid n'kukhala LC3-II, autophagosome imatseka, imalumikizana ndi lysosome, ndipo katundu amaphwanyidwa pamene p62 nayonso imaphwanyidwa ndi kusinthidwa.

Chithunzichi chikuika zotsatira zonse za methylene blue pa mapuwa: zotsatira za hippocampus pafupi ndi nthambi ya AMPK, ndipo zotsatira za maselo oyambira pafupi ndi nthambi ya mTOR.

Kufotokoza motere kumathetsa kusiyana komwe kumaoneka ndi kafukufuku wachiwiri wa Xie. M'maselo oyambira a mitsempha ya makoswe omwe anali kuchulukana, choyesera china chinapeza kuti 5 μM ya methylene blue inalepheretsa kuchulukana ndipo inalimbikitsa mkhalidwe wopuma popanda kugawikana. Maselo osonyeza Ki67 anachepa pa masiku 1 ndi 3, pamene kusanduka kwa maselo omwe anali atayamba kale njira yokhala maselo a mitsempha kunakhalabe kosasokonezedwa. Pamenepo njira yogwirira ntchito inali kupondereza kutumiza zizindikiro kwa mTOR: mTOR yonse ndi mTOR yowonjezeredwa phosphate zinachepa, limodzi ndi kuchepa kwa kupanga p70S6K ndi 4EBP1, komanso kuchepetsa kupanga ma cyclin E1, B1, D1, ndi D2. Khalidwe lalikulu lomwe linaoneka linali kupuma kwa maselo oyambira popanda kugawikana. Olembawo analigwirizanitsa ndi kuchedwa kwa kukalamba kwa maselo, osati kupulumutsa maselo a hippocampus omwe anali m'mavuto.

Palibe kutsutsana pakati pa zolemba ziwirizi. AMPK ndi mTOR zimaphatikiza zizindikiro mogwirizana ndi mmene zinthu zilili, osati ngati masiwichi osasintha. Selo lofanana ndi selo la mitsempha lomwe lili m'mavuto lingayambitse AMPK popanda kuletsa mTOR komwe kungayesedwe, pamene selo loyambira lomwe likulimbikitsidwa kugawikana lingayankhe ku mankhwala omwewo mwa kuchepetsa ntchito za kukula zodalira mTOR. Mtundu wa selo, mtundu wa mavuto ake, kuchuluka kwa mankhwala, ndi nthawi yoyezera zimatsimikizira nthambi yomwe imakhala yamphamvu kwambiri. N'chifukwa chakenso zotsatira za methylene blue pa mphamvu ndi zochitika za redox m'mitochondria ziyenera kukhala mbali ya chithunzi chomwechi: monga kuwunika kwa njira zotetezera maselo a mitsempha kukufotokozera, mankhwalawa amagwira ntchito ngati chonyamulira china cha ma electron, amayambitsa kutumiza zizindikiro zolimbana ndi kuwonongeka chifukwa cha okosijeni komanso apoptosis, amaletsa MAO ndi NOS, ndipo amagwirizana ndi kukonzanso mitochondria kudzera mu kupanga zatsopano ndi autophagy. Autophagy ndi nthambi imodzi ya ntchito zosiyanasiyana za mankhwalawa, osati nkhani yonse.

Zimene zoyeserazi sizisonyeza

Kuchotsa seramu kumapereka chitsanzo cha mavuto okhudzana ndi thandizo la kukula ndi kagayidwe ka zinthu m'mbale yoyesera. Sikupereka chitsanzo cha kusala kudya kwa chamoyo chonse: glucose, ma amino acid, ndi mchere zimakhalabe m'madzi oyambira okulitsira maselo, ndipo palibe khalidwe lililonse la kudya. Kuchuluka kwa mankhwala m'maselo okulitsidwa kuchokera pa 20 nM kufika pa 5 μM sikungasinthidwe kukhala milingo ya anthu mwa kungowerengera, chifukwa kuyamwidwa, kumangirirana ndi mapuloteni, mkhalidwe wa redox, kagayidwe ka zinthu, kuunjikana m'minofu, ndi nthawi yomwe mankhwala amakhalapo zonse zimasiyana.

Palibe mayeso a anthu omwe anakonzedwa kuti asonyeze flux ya autophagy yoyambitsidwa ndi methylene blue mwa odwala. Umboni wa njira yogwirira ntchito udakali wa maselo, minofu yochotsedwa m'thupi, ndi nyama, ndipo kuwunika kwa kafukufuku kukufotokozabe umboni wogwiritsa ntchito zotsatirazi mwa anthu kuti ndi woyambirira. Choncho, kafukufukuyu sasonyeza kuti methylene blue imatsanzira kusala kudya, imatalikitsa moyo wa anthu kudzera mu autophagy, kapena imamaliza njira yonse ya autophagy mu ubongo wa munthu. Zonena zakuti imatsanzira kusala kudya kapena imathandiza kukhala ndi moyo wautali zimafunikira umboni wawo wa mayeso azachipatala. Mayeso a methylene blue mwa anthu pa ntchito zina sasanduka mayeso a autophagy chifukwa chongogwirizanitsidwa nawo. Kafukufuku wa zitsanzo za mavuto obwera chifukwa cha kusowa kwa zakudya ndi moyo wautali uyenera kuwerengedwa podziwa malire amenewa.

Mukamawerenga kafukufuku wotsatira, gwiritsani ntchito mndandandawu motsatira dongosolo lake. Choyamba funsani kuti ndi nthambi iti yomwe inasintha: AMPK, mTOR, kapena palibe mwa ziwirizi. Kenako funsani ngati zotsatira za LC3 zinaphatikizidwa ndi mayeso a flux kapena zili zokha. Kenako onani ngati kuletsa njirayi kunachotsa phindu, komanso njira yomwe inagwiritsidwa ntchito. Kafukufuku amene wayankha mafunso onse atatu ali ndi maziko olimba ofotokozera njira yogwirira ntchito. Amene wayankha loyamba lokha wasonyeza chizindikiro cholola njirayi kuyamba, ndipo magawo otsalawo sanadziwikebe.