Mankhwala oletsa ululu a N,N-diethyl-m-toluamide (DEET) zanenedwa kuti zimaletsa AChE (acetylcholinesterase) ndipo zimatha kuyambitsa khansa chifukwa cha kuchuluka kwa mitsempha yamagazi. Mu pepalali, tikuwonetsa kuti DEET imalimbikitsa makamaka maselo a endothelial omwe amalimbikitsa angiogenesis, motero imawonjezera kukula kwa chotupa. DEET imayendetsa njira zama cell zomwe zimapangitsa angiogenesis, kuphatikizapo kuchulukana, kusamuka, ndi kumamatira. Izi zikugwirizana ndi kuwonjezeka kwa kupanga kwa NO ndi kufalikira kwa VEGF m'maselo a endothelial. Kuletsa M3 kapena kugwiritsa ntchito zoletsa za M3 zamankhwala kunathetsa zonsezi, zomwe zikusonyeza kuti angiogenesis yomwe imayambitsa DEET imakhudzidwa ndi M3. Kuyesera kokhudzana ndi calcium signaling mu maselo a endothelial ndi HEK omwe amachulukitsa ma M3 receptors, komanso maphunziro omangirira ndi kuyika, akuwonetsa kuti DEET imagwira ntchito ngati allosteric modulator ya ma M3 receptors. Kuphatikiza apo, DEET imaletsa AChE, motero imawonjezera kupezeka kwa acetylcholine ndi kumamatira kwake ku ma M3 receptors, ndikuwonjezera zotsatira za proangiogenic kudzera mu allosteric regulation.
Ma EC oyambilira adachotsedwa ku aorta ya mbewa zaku Swiss. Njira yochotsera idasinthidwa kuchokera ku protocol ya Kobayashi 26. Ma EC a murine adakulitsidwa mu EBM-2 medium yowonjezera ndi 5% ya FBS yosatenthedwa ndi kutentha mpaka njira yachinayi.
Zotsatira za kuchuluka kwa DEET kawiri pa kuchuluka kwa HUVEC, U87MG, kapena BF16F10 zidawunikidwa pogwiritsa ntchito CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Mwachidule, maselo 5.103 pachitsime chilichonse adabzalidwa mu mbale ya zitsime 96, ndikuloledwa kulumikizidwa usiku wonse, kenako nkuchiritsidwa ndi DEET kwa maola 24. Mukachotsa malo okulirapo, onjezerani yankho lomangira utoto pachitsime chilichonse cha microplate ndikuyika maselowo pa 37 °C kwa mphindi 30. Magawo a fluorescence adadziwika pogwiritsa ntchito chowerengera cha Mithras LB940 multimode microplate (Berthold Technologies, Bad Wildbad, Germany) chokhala ndi zosefera zoyambitsa 485 nm ndi zosefera zotulutsa 530 nm.
HUVEC idabzalidwa m'mabolo 96 a zitsime pamlingo wa maselo 104 pachitsime chilichonse. Maselo adachiritsidwa ndi DEET kwa maola 24. Kugwira ntchito bwino kwa maselo kunayesedwa pogwiritsa ntchito colorimetric MTT assay (Sigma-Aldrich, M5655). Ma Optical density values adapezeka pa multimode microplate reader (Mithras LB940) pamlingo wa 570 nm.
Zotsatira za DEET zinaphunziridwa pogwiritsa ntchito mayeso a angiogenesis mu vitro. Chithandizo cha 10-8 M kapena 10-5 M DEET chinawonjezera kutalika kwa capillary mu HUVECs (Chithunzi 1a, b, mipiringidzo yoyera). Poyerekeza ndi gulu lolamulira, chithandizo chokhala ndi kuchuluka kwa DEET kuyambira 10-14 mpaka 10-5 M chinawonetsa kuti kutalika kwa capillary kunafika pamtunda pa 10-8 M DEET (Chithunzi Chowonjezera S2). Palibe kusiyana kwakukulu komwe kunapezeka mu zotsatira za proangiogenic za HUVECs zothandizidwa ndi DEET mu kuchuluka kwa 10-8 M ndi 10-5 M.
Kuti tidziwe momwe DEET imakhudzira neovascularization, tinachita kafukufuku wa neovascularization m'thupi. Patatha masiku 14, mbewa zomwe zinabayidwa ndi maselo a endothelial omwe anali ndi 10-8 M kapena 10-5 M DEET zinasonyeza kuwonjezeka kwakukulu kwa hemoglobin (Chithunzi 1c, mipiringidzo yoyera).
Kuphatikiza apo, kufalikira kwa mitsempha yamagazi komwe kunachitika chifukwa cha DEET kunaphunziridwa mu mbewa zokhala ndi xenograft za U87MG zomwe zimabayidwa tsiku lililonse (ip) ndi DEET pamlingo wodziwika kuti umayambitsa kuchuluka kwa plasma kwa 10-5 M, zomwe ndi zachilendo kwa anthu omwe ali pachiwopsezo. mu 23. Zotupa zomwe zimawonedwa (monga zotupa >100 mm3) zidawonedwa patatha masiku 14 kuchokera pamene maselo a U87MG adabayidwa m'makoswe. Pa tsiku la 28, kukula kwa chotupa kudakula kwambiri mu mbewa zomwe zidathandizidwa ndi DEET poyerekeza ndi mbewa zowongolera (Chithunzi 1d, masikweya). Kuphatikiza apo, utoto wa CD31 wa zotupa udawonetsa kuti DEET idakulitsa kwambiri dera la capillary koma osati kuchuluka kwa microvessel. (Chithunzi 1e-g).
Kuti adziwe udindo wa ma muscarinic receptors pakukula kwa DETA, 10-8 M kapena 10-5 M DETA pamaso pa pFHHSiD (10-7 M, wotsutsa wa M3 receptor wosankha) adagwiritsidwa ntchito. Chithandizo cha HUYEC. pFHHSiD idaletsa kwathunthu kuchuluka kwa DETA pa kuchuluka konse (Table 1).
Pansi pa mikhalidwe iyi, tinafufuzanso ngati DEET ingawonjezere kutalika kwa ma capillary m'maselo a HUVEC. Mofananamo, pFHHSiD inaletsa kwambiri kutalika kwa ma capillary omwe amayambitsidwa ndi DEET (Chithunzi 1a, b, mipiringidzo yaimvi). Kuphatikiza apo, kuyesa kofananako kunachitika ndi M3 siRNA. Ngakhale kuti siRNA yowongolera sinali yothandiza pakulimbikitsa mapangidwe a ma capillary, kuletsa M3 muscarinic receptor kunathetsa kuthekera kwa DEET kuwonjezera kutalika kwa ma capillary (Chithunzi 1a, b, mipiringidzo yakuda).
Kuphatikiza apo, mitsempha yonse ya 10-8 M kapena 10-5 M yomwe imayambitsa DEET mu vitro ndi neovascularization mu vivo idatsekedwa kwathunthu ndi pFHHSiD (Chithunzi 1c, d, mabwalo). Zotsatirazi zikusonyeza kuti DEET imalimbikitsa angiogenesis kudzera munjira yomwe imakhudzidwa ndi ma M3 receptor antagonists osankhidwa kapena M3 siRNA.
AChE ndiye cholinga cha mamolekyulu a DEET. Mankhwala monga donepezil, omwe amagwira ntchito ngati zoletsa za AChE, amatha kuyambitsa EC angiogenesis mu vitro ndi mu mbewa za hindlimb ischemia models14. Tinayesa zotsatira za kuchuluka kwa DEET kawiri pa ntchito ya enzyme ya AChE mu HUVEC. Kuchuluka kwa DEET kotsika (10-8 M) ndi kwakukulu (10-5 M) kunachepetsa ntchito ya AChE ya endothelial poyerekeza ndi mikhalidwe yolamulira (Chithunzi 2).
Kuchulukana konse kwa DEET (10-8 M ndi 10-5 M) kunachepetsa ntchito ya acetylcholinesterase pa HUVEC. BW284c51 (10-5 M) idagwiritsidwa ntchito ngati chowongolera cha zoletsa za acetylcholinesterase. Zotsatira zake zimawonetsedwa ngati kuchuluka kwa ntchito ya AChE pa HUVEC yothandizidwa ndi kuchulukana kawiri kwa DEET poyerekeza ndi maselo ochiritsidwa ndi magalimoto. Ma Values amawonetsedwa ngati apakati ± SEM ya zoyeserera zisanu ndi chimodzi zodziyimira pawokha. *p < 0.05 poyerekeza ndi chowongolera (Kruskal-Wallis ndi Dunn multiple comparison test).
Nitric oxide (NO) imagwira ntchito mu njira ya angiogenic 33, motero, kupanga kwa NO mu ma HUVEC oyambitsidwa ndi DEET kunaphunziridwa. Kupanga kwa endothelial NO komwe kunathandizidwa ndi DEET kunawonjezeka poyerekeza ndi maselo olamulira, koma kunafika pamlingo wa 10-8 M (Chithunzi 3c). Kuti mudziwe kusintha kwa mamolekyu komwe kumalamulira kupanga kwa NO komwe kunayambitsidwa ndi DEET, kufotokozedwa kwa eNOS ndi kuyambitsa kunasanthulidwa ndi Western blotting. Ngakhale kuti chithandizo cha DEET sichinasinthe kufotokozedwa kwa eNOS, chinawonjezera kwambiri phosphorylation ya eNOS pamalo ake oyambitsa (Ser-1177) pomwe chinachepetsa malo ake oletsa (Thr-495) poyerekeza ndi maselo osachiritsidwa mu phosphorylation ya eNOS (Chithunzi 3d). Kuphatikiza apo, chiŵerengero cha eNOS yomwe ili ndi phosphorylated pamalo oyambitsa ndi malo oletsa chinawerengedwa pambuyo pokonza kuchuluka kwa eNOS yomwe ili ndi phosphorylated ku kuchuluka konse kwa enzyme. Chiŵerengerochi chinawonjezeka kwambiri mu ma HUVEC omwe amathandizidwa ndi kuchuluka kulikonse kwa DEET poyerekeza ndi maselo osachiritsidwa (Chithunzi 3d).
Pomaliza, kufotokozera kwa VEGF, chimodzi mwa zinthu zazikulu zomwe zimayambitsa matenda, kunafufuzidwa ndi Western blotting. DEET inawonjezera kwambiri kufotokozera kwa VEGF, pomwe pFHHSiD inaletsa kwathunthu kufotokozera kumeneku.
Popeza zotsatira za DEET zimakhala zovuta kwambiri chifukwa cha kutsekeka kwa mankhwala komanso kuchepa kwa ma M3 receptors, tinayesa lingaliro lakuti DEET ikhoza kuwonjezera chizindikiro cha calcium. Chodabwitsa n'chakuti, DEET inalephera kuwonjezera calcium ya cytoplasmic mu HUVEC (deta sinawonetsedwe) ndi HEK/M3 (Chithunzi 4a, b) pa kuchuluka konse komwe kwagwiritsidwa ntchito.
Nthawi yotumizira: Disembala-30-2024