Mankhwala anthelmintic N,N-diethyl-m-toluamide (DEET) adanenedwa kuti amaletsa AChE (acetylcholinesterase) ndipo ali ndi mphamvu zowononga khansa chifukwa cha mitsempha yambiri. Mu pepalali, tikuwonetsa kuti DEET imathandizira makamaka ma cell a endothelial omwe amalimbikitsa angiogenesis, motero amakulitsa kukula kwa chotupa. DEET imayendetsa njira zama cell zomwe zimatsogolera ku angiogenesis, kuphatikiza kuchulukana, kusamuka, ndi kumamatira. Izi zimagwirizanitsidwa ndi kuwonjezeka kwa NO kupanga ndi VEGF kufotokoza m'maselo a endothelial. Kuletsa M3 kapena kugwiritsa ntchito mankhwala a M3 inhibitors kunathetsa zotsatira zonsezi, kutanthauza kuti DEET-induced angiogenesis ndi M3-sensitive. Kuyesera kophatikiza ma sign a calcium mu endothelial ndi HEK cell overexpressing M3 receptors, komanso maphunziro omangirira ndi docking, akuwonetsa kuti DEET imagwira ntchito ngati allosteric modulator ya M3 receptors. Kuphatikiza apo, DEET imalepheretsa AChE, motero imakulitsa bioavailability ya acetylcholine ndi kumangiriza kwa M3 zolandilira, komanso kukulitsa zotsatira za proangiogenic kudzera mu malamulo a allosteric.
Ma EC oyambirira anali olekanitsidwa ndi msempha wa mbewa za ku Swiss. Njira yochotsera idasinthidwa kuchokera ku protocol 26 ya Kobayashi. Murine ECs adakulitsidwa mu EBM-2 sing'anga yowonjezeredwa ndi 5% FBS yopanda kutentha mpaka ndime yachinayi.
Zotsatira za magawo awiri a DEET pakukula kwa HUVEC, U87MG, kapena BF16F10 adawunikidwa pogwiritsa ntchito CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Mwachidule, ma cell a 5.103 pachitsime chilichonse adabzalidwa m'mbale ya 96-chitsime, amaloledwa kulumikiza usiku wonse, kenako amathandizidwa ndi DEET kwa 24 h. Mukachotsa sing'anga yokulirapo, onjezerani njira yomangira utoto pachitsime chilichonse cha microplate ndikuyika ma cell pa 37 ° C kwa mphindi 30. Miyezo ya Fluorescence idatsimikiziridwa pogwiritsa ntchito Mithras LB940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany) yokhala ndi 485 nm excitation filters ndi 530 nm emission filters.
HUVEC idabzalidwa m'mbale zachitsime 96 pa kachulukidwe ka ma cell 104 pachitsime chilichonse. Maselo amathandizidwa ndi DEET kwa 24 h. Kuthekera kwa ma cell kunayesedwa pogwiritsa ntchito colorimetric MTT assay (Sigma-Aldrich, M5655). Mawonekedwe kachulukidwe owoneka adapezedwa pa owerenga ma multimode microplate (Mithras LB940) pamtunda wa 570 nm.
Zotsatira za DEET zidawerengedwa pogwiritsa ntchito in vitro angiogenesis assays. Chithandizo ndi 10-8 M kapena 10-5 M DEET chinawonjezera mapangidwe a capillary kutalika mu HUVECs (mkuyu 1a, b, mipiringidzo yoyera). Poyerekeza ndi gulu lolamulira, chithandizo chokhazikika cha DEET kuyambira 10-14 mpaka 10-5 M chinasonyeza kuti kutalika kwa capillary kunafika pamtunda pa 10-8 M DEET (Supplementary Fig. S2). Palibe kusiyana kwakukulu komwe kunapezeka mu in vitro proangiogenic zotsatira za ma HUVEC omwe amathandizidwa ndi DEET mumtundu wa 10-8 M ndi 10-5 M.
Kuti tidziwe momwe DEET imakhudzira neovascularization, tidachita maphunziro a vivo neovascularization. Pambuyo masiku 14, mbewa jekeseni ndi maselo endothelial precultured ndi 10-8 M kapena 10-5 M DEET anasonyeza kwambiri kuwonjezeka hemoglobin okhutira (mkuyu. 1c, mipiringidzo woyera).
Kuphatikiza apo, DEET-induced neovascularization idaphunziridwa mu mbewa zokhala ndi xenograft za U87MG zomwe zidabayidwa tsiku lililonse (ip) ndi DEET pamlingo womwe umadziwika kuti umapangitsa kuchuluka kwa plasma kwa 10-5 M, zomwe ndizabwinobwino kwa anthu omwe ali pachiwopsezo. mu 23. Zotupa zodziwika (ie zotupa> 100 mm3) zinkawonedwa patatha masiku 14 jekeseni wa maselo a U87MG mu mbewa. Patsiku la 28, kukula kwa chotupa kunalimbikitsidwa kwambiri mu mbewa zowonongeka ndi DEET poyerekeza ndi mbewa zowongolera (mkuyu 1d, mabwalo). Kuphatikiza apo, madontho a CD31 a zotupa adawonetsa kuti DEET idachulukitsa kwambiri dera la capillary koma osati kuchuluka kwa ma microvessel. (mkuyu 1e–g).
Kuti mudziwe udindo wa muscarinic receptors mu kuwonjezeka kwa DETA-induced, 10-8 M kapena 10-5 M DETA pamaso pa pFHHSiD (10-7 M, wosankha M3 receptor antagonist) anagwiritsidwa ntchito. Chithandizo cha HUVEC. pFHHSiD idatsekereza kuchulukitsa kwa DETA pamitundu yonse (Table 1).
Pazifukwa izi, tidawunikanso ngati DEET ingachulukitse kutalika kwa capillary m'maselo a HUVEC. Mofananamo, pFHHSiD inalepheretsa kwambiri DEET-induced capillary kutalika (Mkuyu 1a, b, imvi mipiringidzo). Kuphatikiza apo, kuyesa kofananako kunachitika ndi M3 siRNA. Ngakhale kuti siRNA yolamulira sinali yothandiza pakulimbikitsa mapangidwe a capillary, kutonthola kwa M3 muscarinic receptor kunathetsa kuthekera kwa DEET kuonjezera kutalika kwa capillary (Mkuyu 1a, b, mipiringidzo yakuda).
Komanso, onse 10-8 M kapena 10-5 M DEET-induced vascularization in vitro ndi neovascularization mu vivo anali otsekedwa kwathunthu ndi pFHHSiD (Mkuyu 1c, d, mabwalo). Zotsatira izi zikuwonetsa kuti DEET imalimbikitsa angiogenesis kudzera m'njira yomwe imakhudzidwa ndi kusankha M3 receptor antagonists kapena M3 siRNA.
AChE ndiye chandamale cha molekyulu ya DEET. Mankhwala monga donepezil, omwe amakhala ngati AChE inhibitors, amatha kulimbikitsa EC angiogenesis in vitro ndi mu mouse hindlimb ischemia models14. Tinayesa zotsatira za magawo awiri a DEET pa ntchito ya AChE enzyme mu HUVEC. Otsika (10-8 M) ndi apamwamba (10-5 M) a DEET amachepetsa endothelial AChE ntchito poyerekeza ndi machitidwe olamulira (mkuyu 2).
Kuyika konse kwa DEET (10-8 M ndi 10-5 M) kumachepetsa ntchito ya acetylcholinesterase pa HUVEC. BW284c51 (10-5 M) idagwiritsidwa ntchito ngati chiwongolero cha acetylcholinesterase inhibitors. Zotsatira zimawonetsedwa ngati kuchuluka kwa zochitika za AChE pa HUVEC yothandizidwa ndi magawo awiri a DEET poyerekeza ndi ma cell othandizidwa ndi magalimoto. Makhalidwe amafotokozedwa ngati akutanthauza ± SEM pazoyeserera zisanu ndi chimodzi. *p <0.05 poyerekeza ndi kulamulira (Kruskal-Wallis ndi Dunn angapo poyerekeza mayeso).
Nitric oxide (NO) imakhudzidwa ndi angiogenic process 33, motero, PALIBE kupanga mu DEET-stimulated HUVECs kunaphunziridwa. DEET-treated endothelial NO kupanga kunawonjezeka poyerekeza ndi maselo olamulira, koma anafikira kufunikira kokha pa mlingo wa 10-8 M (mkuyu 3c). Kuti mudziwe kusintha kwa mamolekyulu omwe amawongolera kupanga kwa NO kwa DEET, mafotokozedwe a eNOS ndi kuyambitsa adawunikidwa ndi kutseka kwa Western. Ngakhale kuti chithandizo cha DEET sichinasinthe mafotokozedwe a eNOS, chinawonjezera kwambiri eNOS phosphorylation pa malo ake oyambitsa (Ser-1177) pamene kuchepetsa malo ake oletsa (Thr-495) poyerekeza ndi maselo osagwiritsidwa ntchito mu eNOS phosphorylation (Mkuyu 3d). Komanso, chiŵerengero cha phosphorylated eNOS pa kutsegula malo ndi malo oletsa anawerengedwa pambuyo normalizing kuchuluka kwa phosphorylated eNOS kwa chiwerengero cha enzyme. Chiŵerengerochi chinawonjezeka kwambiri mu HUVECs chochitidwa ndi ndende iliyonse ya DEET poyerekeza ndi maselo osatulutsidwa (mkuyu 3d).
Pomaliza, mawu a VEGF, chimodzi mwazinthu zazikulu za proangiogenic, adawunikidwa ndi kutsekedwa kwa Kumadzulo. DEET idakulitsa kwambiri mawu a VEGF, pomwe pFHHSiD idatsekereza mawuwa.
Popeza kuti zotsatira za DEET zimakhudzidwa ndi kutsekedwa kwa mankhwala komanso kutsika kwa ma M3 receptors, tinayesa lingaliro lakuti DEET ikhoza kupititsa patsogolo kashiamu. Chodabwitsa n'chakuti, DEET inalephera kuonjezera calcium ya cytoplasmic mu HUVEC (deta yosasonyezedwa) ndi HEK / M3 (Fig. 4a, b) pazomwe zimagwiritsidwa ntchito.
Nthawi yotumiza: Dec-30-2024