Methylene blue ndi kutumiza zizindikiro za kutupa: ma inflammasome, IL-6, ndi STAT3

Tiyerekeze kuti malo atatu ofufuza anena kuti chinthu chimodzi chabuluu chimachepetsa kutupa. Malo amodzi akusonyeza kuti IL-1beta yochokera ku ma macrophage yachepa. Ena akusonyeza kuti enzayimu ya iNOS yachepa mu maselo a mtundu womwewo. Enanso akusonyeza kuti IL-6 yachepa m'magazi ndipo ntchito ya STAT3 yachepa mu ubongo. Zimakhala zokopa kuphatikiza zonse zitatu kukhala mfundo imodzi. Kuphatikiza kumeneko kumabisa zimene zapezeka.

Zotsatira zilizonse zimakhudzana ndi njira yosiyana yotumizira zizindikiro, yomwe ili ndi choyambitsa chake, sitepe yake yosonkhanitsa zigawo, ndi choyezera chake. Methylene blue imakhudza njira iliyonse pa malo osiyana. Kaya zimenezi zingathandize pa zotsatira za thanzi ndi funso lina, lomwe likufotokozedwa mu chidule chathu cha methylene blue ndi kutupa. Tsamba lino limangofotokoza njira za m'kati mwa maselo.

Njira 1: kusonkhanitsidwa kwa inflammasome ndi caspase-1

Inflammasome ndi chimango cha mapuloteni chomwe chimapangidwa pakafunika mkati mwa selo la myeloid. Zigawo zake ndi chozindikira, cholumikizira chotchedwa ASC, ndi enzayimu yogwira ntchito yotchedwa caspase-1. Zikasonkhanitsidwa, caspase-1 imadula pro-IL-1beta ndi pro-IL-18 kukhala mitundu yawo yotulutsidwa kunja kwa selo ndipo ingayambitse pyroptosis, mtundu wa kufa kwa selo womwe umayambitsa kutupa. Chozindikira chimatsimikizira mtundu wake: NLRP3 imayankha makhiristo ndi poizoni wopanga mabowo monga nigericin ndi ATP, NLRC4 imayankha flagellin ya mabakiteriya, AIM2 imayankha DNA ya zingwe ziwiri yomwe ili mu cytosol, ndipo njira yosatsatira dongosolo lodziwika imayankha lipopolysaccharide yomwe ili mkati mwa selo.

Kuyambitsa ntchito kumafuna zizindikiro ziwiri. Chizindikiro choyamba ndi kukonzekeretsa: chinthu chomangirira pa cholandirira cha mtundu wa Toll chimayendetsa kukopera uthenga wa majini a NLRP3 ndi pro-IL-1beta kodalira NF-kB kuti zigawozi zikhalepo. Chizindikiro chachiwiri ndi kusonkhanitsa: choyambitsa china chimachititsa kuti mamolekyu a ASC alumikizane kukhala tinthu tating'ono, caspase-1 idulidwe kukhala chidutswa chake cha p20, ndipo IL-1beta p17 yokhwima itulutsidwe. Mitundu ya okosijeni yogwira ntchito kwambiri yochokera mu mitochondria ndi kulowetsedwa kwa makhiristo mu selo zimadziwika kuti zimathandizira chizindikiro chachiwiri cha NLRP3.

Kafukufuku wa mu 2017 wa kulepheretsa mitundu yosiyanasiyana ya inflammasome mu ma macrophage anayesa methylene blue pa zizindikiro zonse ziwiri. Mu ma macrophage a m'mafupa a mbewa omwe anakonzekeretsedwa ndi lipopolysaccharide, nigericin, ATP, ndi makhiristo a monosodium urate chilichonse chinayambitsa kutulutsidwa kwa IL-1beta ndi caspase-1 komanso kupangidwa kwa tinthu ta ASC. Methylene blue pa 20 mpaka 200 micromolar inachepetsa zonse zitatu molingana ndi kuchuluka kwake popanda kupha maselowo. Flagellin ndi Salmonella pa NLRC4, DNA ya zingwe ziwiri ndi Listeria pa AIM2, komanso lipopolysaccharide yolowetsedwa mu maselo kapena E. coli yamoyo pa njira yosatsatira dongosolo lodziwika zinasonyeza mchitidwe womwewo. Chinthucho chokha sichinayambitse kutulutsidwa, ndipo mbale za mayesero oyerekezera sizinasonyeze kuti chimapha kapena kulepheretsa mabakiteriya. Choncho zotsatira zake zikukhudzana ndi kuyankha kwa chamoyo chomwe chili ndi mabakiteriyawo.

Pa masitepe oyambirira, zinthu zitatu zomwe zinasinthidwa zinayesedwa. Superoxide ya mu mitochondria yoyambitsidwa ndi rotenone inachepa ndi methylene blue, ndipo kutulutsidwa kwa IL-1beta koyambitsidwa ndi rotenone kunachepanso. Kulowetsedwa kwa timikanda towala mu maselo kunachepa, mogwirizana ndi kuchepa kwa phagocytosis, yomwe ndi kumeza zinthu ndi selo, makhiristo asanazindikiridwe. Choyezera choyendetsedwa ndi gawo loyambitsa kukopera uthenga wa jini la NLRP3, lokhala ndi malo awiri omangirira NF-kB, chinasonyeza ntchito yochepa ndi methylene blue. Koma gawo loyambitsalo litafupikitsidwa ndi kuchotsa malo amenewo silinayankhe. Mu chubu chopanda maselo, methylene blue inachepetsanso mwachindunji ntchito ya caspase-1 ya munthu yopangidwa ndi njira zosintha majini. Kuika chinthucho pa kukonzekeretsa kokha kapena pa kuyambitsa ntchito kokha kunachepetsabe kutulutsidwa kwa IL-1beta, IL-18, ndi caspase-1, choncho masitepe onse awiri amakhudzidwa.

Mayesero a mbewa anagwirizana ndi kusankha kwakeku. Kuchuluka kwa lipopolysaccharide kopha kunapha mbewa za gulu loyerekezera m'maola ochepa, pamene methylene blue inawonjezera kupulumuka; IL-1beta ya m'kati mwa mimba inachepa koma IL-6 ya m'madzi omwewo sinachepe. Mu chitsanzo choyesera kutupa kwa nembanemba ya m'kati mwa mimba chifukwa cha Listeria, IL-1beta ya m'kati mwa mimba inachepa pamene chiwerengero chonse cha maselo sichinasinthe. Ma monocyte a munthu a THP-1 anachita ngati ma macrophage a mbewa pa mitundu yonse inayi ya inflammasome. Zotsatira za inflammasome zimafotokoza kukhwima kwa IL-1beta ndi IL-18, osati cytokine iliyonse. N'chifukwa chakenso maonekedwe a maselo a chitetezo cha m'thupi mu ubongo mu mayesero a microglia ndi astrocyte amayankha funso losiyana ndi la kusonkhanitsidwa kwa chimango.

Njira 2: kukopera uthenga wa jini la iNOS kudzera mu kumangirira kwa NF-kB ndi STAT1

Inducible nitric oxide synthase, kapena iNOS, imakhalapo pang'ono kwambiri selo likakhala losayambitsidwa, ndipo uthenga wa jini lake umakopedwa pakafunika. Lipopolysaccharide imayiyambitsa makamaka kudzera mu kuwonongeka kwa IkB-alpha, komwe kumatsatiridwa ndi kuwonjezeredwa kwa magulu a phosphate pa NF-kB ndi kulowa kwake mu nyukiliyasi. Interferon-gamma imayiyambitsa makamaka kudzera mu kuwonjezeredwa kwa magulu a phosphate pa STAT1 ndi Janus kinase, komwe kumatsatiridwa ndi kulowa kwa STAT1 mu nyukiliyasi. Kenako puloteni iliyonse imamangirira pa malo ake pa gawo loyambitsa kukopera uthenga wa jini la iNOS. Ntchito ya enzayimu ndi kuyambitsidwa kwa jini ndi malo osiyana olamulira, ndipo mankhwala angakhudze amodzi kapena onse awiri.

Kafukufuku wa mu 2015 wa kuyambitsidwa kwa iNOS mu ma macrophage ndi mbewa zokhala ndi endotoxin m'magazi anaika zotsatira zake pa uthenga wa jini. Mu maselo a RAW 264.7 ndi ma macrophage otengedwa mwachindunji ku mbewa, 1 micromolar ya methylene blue yoikidwa mphindi 30 lipopolysaccharide, interferon-gamma, kapena zonse ziwiri zisanaikidwe inachepetsa puloteni ya iNOS ndi mRNA m'maola 8 mpaka 24. Nitrite inachepa m'madzi olereramo maselo ndipo maselowo sanataye mphamvu yokhala ndi moyo. Mu mbewa zopatsidwa lipopolysaccharide ya 25 mg pa kg, methylene blue ya 5 mg pa kg yoperekedwa kamodzi ola limodzi m'mbuyomo inachepetsa puloteni ya iNOS ndi mRNA m'mapapo, m'chiwindi, ndi mumtima.

Mfundo yapadera ndi zomwe sizinasinthe. Kuwonongeka kwa IkB-alpha, kuwonjezeredwa kwa magulu a phosphate pa NF-kB ndi STAT1, komanso kuchulukana kwa puloteni iliyonse mu nyukiliyasi sizinakhudzidwe. Kusokonekera kunachitika pa sitepe yotsatira: zinthu zotengedwa mu nyukiliyasi zinasonyeza kuchepa kwa kumangirira kwa NF-kB pa malo ake a DNA pambuyo pa lipopolysaccharide ndi kuchepa kwa kumangirira kwa STAT1 pambuyo pa interferon-gamma. Njira ya chromatin immunoprecipitation inatsimikizira kuti mapuloteni onsewa anamangirira mochepa pa gawo loyambitsa kukopera uthenga wa jini la iNOS mkati mwa maselo. Tumor necrosis factor alpha, yomwe gawo lake loyambitsa kukopera uthenga wa jini limagwiritsanso ntchito NF-kB, inachepanso. Mapuloteniwa anali atayambitsidwa ndipo anafika, koma sanamangirire bwino pa DNA yomwe amayenera kugwirapo ntchito.

Onani kusiyana kwake ndi njira 1. Izi zikukhudza kumangirira kwa puloteni pa DNA, zimagwiritsa ntchito STAT1 osati STAT3, ndipo zimayeza mRNA, puloteni, ndi nitrite m'malo mwa tinthu ta ASC ndi zidutswa za caspase-1. Mapu athu a zinthu zomwe methylene blue imakhudza m'mayesero osiyanasiyana a maselo amasiyanitsa gawo limeneli la kukopera uthenga wa majini ndi kuyeza mwachindunji ma enzayimu ndi mitochondria.

Njira 3: IL-6 ya mu seramu ndi kuyambitsidwa kwa STAT3 mu minofu

IL-6 imatumiza zizindikiro kudzera mu banja la zolandirira za gp130 kupita mu mndandanda wa Janus kinase kupita ku STAT3. STAT3 yowonjezeredwa magulu a phosphate imalumikizana awiriawiri, imalowa mu nyukiliyasi, ndipo imayendetsa mapulogalamu oyambitsa kutupa. IL-6 yokhayo ndi chimodzi mwa zinthu zoyendetsedwa ndi NF-kB, choncho ziwirizi zimapanga kuzungulira kwa zizindikiro zobwererana komwe kaŵirikaŵiri kumatchedwa njira ya IL-6 kupita ku STAT3. Miyezo yodziwika ndi IL-6 ya mu seramu pa masitepe oyambirira, chiŵerengero cha STAT3 yowonjezeredwa magulu a phosphate poyerekezera ndi STAT3 yonse, ndi ma enzayimu ogwira ntchito monga iNOS ndi COX2 pa masitepe otsatira.

Kafukufuku wa mbewa wa mu 2023 wa IL-6 ndi STAT3 pambuyo pa lipopolysaccharide anayeza njirayi m'madera osiyanasiyana a thupi. Mbewa zazimuna za C57BL/6 zinapatsidwa lipopolysaccharide ya 1 mg pa kg tsiku lililonse kwa masiku atatu, pamodzi ndi methylene blue ya 5 kapena 10 mg pa kg mphindi 30 pambuyo pa mlingo uliwonse. Pa tsiku lachitatu, IL-6 ya mu seramu inali yochepa m'magulu onse awiri a methylene blue kuposa mu gulu lopatsidwa lipopolysaccharide yokha, pamene ma cytokine ena ambiri omwe anayezedwa sanasinthe ndi zomwe zinaperekedwa. Chiŵerengero cha STAT3 yowonjezeredwa magulu a phosphate poyerekezera ndi STAT3 yonse chinali chochepa mu cortex, hippocampus, ndi khungu la khutu. Panalinso kuchepa kwa kupaka utoto wa Iba-1 pa microglia, kuchepa kwa iNOS ndi COX2, komanso kuchepetsedwa pang'ono kwa kutayika kwa kulemera.

Pali mfundo ziwiri zofunika kusamala nazo pano. Kuchepa kwa IL-6 ya mu seramu pamodzi ndi kuchepa kwa kuwonjezeredwa kwa magulu a phosphate pa STAT3 mu minofu kumagwirizana ndi kusinthidwa kwa njirayi, koma sikusonyeza malo oyambirira omwe akhudzidwa. Zili choncho chifukwa kuchepa kwa cytokine pa masitepe oyambirira, kuchepa kwa zizindikiro zochokera pa cholandirira, kapena kusintha kwa ntchito ya phosphatase chilichonse chingapereke chiŵerengero chomwecho. Komanso kulemera pa masiku atatu a kuyesa ndi choyambitsa champhamvu ndi muyeso wa nyama yonse, osati muyeso wa zizindikiro za matenda kapena wa kuchepa kwa matenda. Kumawonjezera mfundo zina kuposa chithunzi cha mapuloteni choyezedwa popanda kusintha zotsatira za kutumiza zizindikiro kukhala mfundo yonena kuti ndi chithandizo. Njira zosiyana za makemikolo zokhudza kusamalira redox mu mitochondria zimachitika limodzi ndi zimenezi zokhudza ma cytokine ndipo siziyenera kuphatikizidwa nazo.

Kuyerekezera njira: malo okhudzidwa ndi miyezo ya zotsatira

Tebuloli limaletsa kugwiritsa ntchito zotsatira za njira imodzi kutsimikizira ina m'njira zitatu zosayenera. Zotsatira za tinthu ta ASC sizitsimikizira zotsatira za STAT3. Zotsatira za STAT1 pa gawo loyambitsa kukopera uthenga wa jini la iNOS sizitsimikizira zotsatira za STAT3, ngakhale mapuloteni onse awiri ali m'banja la STAT. Zotsatira za IL-6 ya mu seramu sizitsimikizira kuchepa kwa kusonkhanitsidwa kwa inflammasome: mu kuyesa kwa m'kati mwa mimba, IL-1beta inachepa pamene IL-6 sinachepe. Mzere uliwonse umafuna choyambitsa chake ndi muyezo wake wa zotsatira. N'chifukwa chake powerenga kafukufuku wa methylene blue mu maselo, nyama, ndi anthu m'pofunika kugwirizanitsa mfundo iliyonse ndi gawo lomwe linayezedwa.

Palibe mwa zimenezi chomwe chimatsimikizira mlingo woyenera, chitetezo, kapena phindu mwa anthu. Mayesero a lipopolysaccharide ndi zoyambitsa zolamulidwa poyesa mmene njira imagwirira ntchito, osati zitsanzo zofanana ndi matenda okhalitsa. Kupereka mlingo pamodzi ndi choyambitsacho kumayesa kupewa kusintha koyambitsidwa, osati kubwezeretsa mkhalidwe wa matenda omwe alipo kale. Siyanitsani kufotokozera njira kumeneku ndi zotsatira mwa odwala, ndipo weruzani mfundo iliyonse yokhudza thanzi potengera kafukufuku wa odwala womwe ukuichirikiza.