Umuti ugabanya ubukana bwa anthelmintic N,N-diethyl-m-toluamide (DEET) byavuzwe ko bibuza AChE (acetylcholinesterase) kandi ishobora gutera kanseri bitewe no kwiyongera cyane kw'imitsi. Muri iyi nyandiko, tugaragaza ko DEET ishishikariza by'umwihariko uturemangingo tw'inyuma tw'amaraso dutuma angiogenesis itera imbere, bityo bikongera gukura kwa kanseri. DEET ikora ku buryo bw'umwihariko inzira z'uturemangingo zituma angiogenesis itera imbere, harimo no kwiyongera, kwimuka, no gufatana. Ibi bifitanye isano no kwiyongera kwa NO production no kugaragaza VEGF mu turemangingo tw'inyuma. Gucecekesha M3 cyangwa gukoresha imiti ibuza M3 gukuraho izi ngaruka zose, bigaragaza ko angiogenesis iterwa na DEET ishobora kwangirika kuri M3. Igerageza rijyanye no gutanga ibimenyetso bya calcium mu turemangingo tw'inyuma tw'amaraso n'utw'inyuma tw'amaraso ya M3, ndetse n'inyigo zo gufatana no gufungana, bigaragaza ko DEET ikora nk'umuyoboro wa allosteric wa receptors za M3. Byongeye kandi, DEET ibuza AChE, bityo yongera ubushobozi bwa acetylcholine bwo gufatana na receptors za M3, kandi yongera ingaruka za proangiogenic binyuze mu kugenzura allosteric.
Udukoko tw’ibanze twakuwe mu muyoboro w’amaraso w’imbeba zo mu Busuwisi. Uburyo bwo gukuramo bwahinduwe hakurikijwe protocole ya Kobayashi 26. Udukoko tw’imbere mu mubiri twatewe mu buryo bwa EBM-2 hiyongereyeho 5% bya FBS idakoresha ubushyuhe kugeza ku gice cya kane cy’umubiri.
Ingaruka z'ibipimo bibiri bya DEET ku kwiyongera kwa HUVEC, U87MG, cyangwa BF16F10 zasesenguwe hakoreshejwe CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Muri make, uturemangingo 5.103 kuri buri ruhu twatewe mu isahani y'ibiziba 96, turekerwa gufatana ijoro ryose, hanyuma duterwa na DEET mu gihe cy'amasaha 24. Nyuma yo gukuraho uburyo bwo gukuraho, shyiramo umuti wo gufata irangi kuri buri ruhu rw'inyuma y ...
HUVEC yatewe mu masahani y’ibiziba 96 ku bucucike bwa selile 104 kuri buri kiroba. Selile zavuwe na DEET mu gihe cy’amasaha 24. Hapimwe ubushobozi bw’ingirabuzimafatizo hakoreshejwe ikizamini cya MTT gifite colorimetric (Sigma-Aldrich, M5655). Agaciro k’ubucucike bw’amajwi kabonetse ku gikoresho cyo gusoma microplate cya multimode (Mithras LB940) ku bunini bwa 570 nm.
Ingaruka za DEET zakoreweho ubushakashatsi hakoreshejwe ibizamini bya angiogenesis muri vitro. Ubuvuzi hakoreshejwe 10-8 M cyangwa 10-5 M DEET bwongereye uburebure bwa capillary muri HUVECs (Ishusho ya 1a, b, utubari tw'umweru). Ugereranije n'itsinda ryagenzuwe, ubuvuzi bufite ingano ya DEET kuva kuri 10-14 kugeza kuri 10-5 M bwagaragaje ko uburebure bwa capillary bwageze ku gice cyo hejuru kuri 10-8 M DEET (Ishusho y'inyongera S2). Nta tandukaniro rigaragara ryabonetse mu ngaruka za HUVECs zavuwe na DEET muri vitro mu cyiciro cya 10-8 M na 10-5 M.
Kugira ngo tumenye ingaruka za DEET ku gutera amaraso mu mitsi, twakoze inyigo ku gutera amaraso mu mitsi mu mubiri. Nyuma y'iminsi 14, imbeba zatewe uturemangingo tw’imbere twatewe mbere y’uko tubyara 10-8 M cyangwa 10-5 M DEET yagaragaje ubwiyongere bugaragara bwa hemoglobine (Ishusho ya 1c, utubari tw’umweru).
Byongeye kandi, inyigo yakozwe ku mbeba zifite xenograft ya U87MG zaterwaga DEET buri munsi (ip) ku gipimo kizwiho gutera igipimo cya 10-5 M mu maraso, ibi bikaba ari ibisanzwe ku bantu bagaragaye. muri 23. Ibibyimba bigaragara (ni ukuvuga ibibyimba >100 mm3) byagaragaye iminsi 14 nyuma yo guterwa uturemangingo twa U87MG mu mbeba. Ku munsi wa 28, gukura kwa kanseri kwariyongereye cyane mu mbeba zavuwe na DEET ugereranije n'imbeba zagenzuwe (Ishusho ya 1d, kare). Byongeye kandi, irangi rya CD31 ku bibyimba ryagaragaje ko DEET yongereye cyane ubuso bw'imitsi ariko ntabwo yongereye ubucucike bw'imitsi mito. (Ishusho ya 1e-g).
Kugira ngo hamenyekane uruhare rwa muscarinic receptors mu kwiyongera kw'ibinyabutabire biterwa na DETA, hakoreshejwe 10-8 M cyangwa 10-5 M DETA imbere ya pFHHSiD (10-7 M, umurwanya M3 receptor utoranijwe). Kuvura HUYEC. pFHHSiD yahagaritse burundu imiterere yo kwiyongera kwa DETA ku bwinshi bwose (Imbonerahamwe ya 1).
Muri ibi bihe, twanasuzumye niba DEET yakongera uburebure bwa capillary mu turemangingo twa HUVEC. Mu buryo nk'ubwo, pFHHSiD yabujije cyane uburebure bwa capillary buterwa na DEET (Ishusho ya 1a, b, imvi). Byongeye kandi, igerageza nk'iryo ryakozwe kuri siRNA ya M3. Nubwo siRNA yo kugenzura itaragize ingaruka nziza mu gutuma capillary ishingwa, gucecekesha receptor ya M3 muscarinic byakuyeho ubushobozi bwa DEET bwo kongera uburebure bwa capillary (Ishusho ya 1a, b, ibara ry'umukara).
Byongeye kandi, gutera imitsi ya 10-8 M cyangwa 10-5 M DEET byatewe na vitro na neovascularization mu mubiri byafunzwe burundu na pFHHSiD (Ishusho ya 1c, d, uruziga). Ibi bisubizo bigaragaza ko DEET itera angiogenesis binyuze mu nzira ishobora kwanduza antagonists za M3 cyangwa M3 siRNA.
AChE ni yo DEET ikoresha mu gupima AChE. Imiti nka donepezil, ikora nk'imiti ibuza AChE, ishobora gutera imbaraga EC angiogenesis muri vitro no mu miterere ya ischemia y'imbeba inyuma y'amaguru14. Twagerageje ingaruka z'ibipimo bibiri bya DEET ku bikorwa bya enzyme ya AChE muri HUVEC. Ibipimo biri hasi (10-8 M) n'ibipimo biri hejuru (10-5 M) bya DEET byagabanyije ibikorwa bya AChE mu ngirabuzimafatizo ugereranije n'imiterere yo kugenzura (Ishusho ya 2).
Ibipimo byombi bya DEET (10-8 M na 10-5 M) byagabanyije imikorere ya acetylcholinesterase kuri HUYEC. BW284c51 (10-5 M) yakoreshejwe nk'uburyo bwo kugenzura imiti igabanya acetylcholinesterase. Ibisubizo bigaragazwa nk'ijanisha ry'ibikorwa bya AChE kuri HUVEC byavuwe n'ibipimo bibiri bya DEET ugereranije n'uturemangingo twavuwe n'imodoka. Agaciro kagaragazwa nk'ikigereranyo ± SEM cy'igerageza ritandatu ryigenga. *p < 0.05 ugereranije n'uburyo bwo kugenzura (ikizamini cyo kugereranya Kruskal-Wallis na Dunn).
Okiside ya nitrike (NO) igira uruhare mu mikorere ya angiogenic 33, bityo, umusaruro wa NO muri HUCECs zatewe inkunga na DEET wizweho. Umusaruro wa NO wa endothelial wa NO wavuwe na DEET wongerewe ugereranije n'uturemangingo two kugenzura, ariko wageze ku gaciro ka 10-8 M gusa (Ishusho ya 3c). Kugira ngo hamenyekane impinduka za molekile zigenzura umusaruro wa NO watewe na DEET, imikorere ya eNOS n'imikorere yayo byasesenguwe hakoreshejwe Western blotting. Nubwo ubuvuzi bwa DEET butahinduye imikorere ya eNOS, bwongereye cyane phosphorylation ya eNOS aho ikora (Ser-1177) mu gihe bugabanya aho ikora (Thr-495) ugereranije n'uturemangingo twavuwe muri phosphorylation ya eNOS (Ishusho ya 3d). Byongeye kandi, igipimo cya eNOS ya phosphorylated aho ikora n'aho ikora cyabazwe nyuma yo kuvugurura ingano ya eNOS ya phosphorylated ku gipimo cyose cya enzyme. Iki gipimo cyariyongereye cyane muri HUCECs zavuwe na buri gipimo cya DEET ugereranije n'uturemangingo twavuwe (Ishusho ya 3d).
Amaherezo, igaragara rya VEGF, imwe mu mpamvu zikomeye zituma habaho ihindagurika ry’amaraso, yasesenguwe hakoreshejwe uburyo bwa Western blotting. DEET yongereye cyane igaragara rya VEGF, mu gihe pFHHSiD yahagaritse burundu iyi garagara.
Kubera ko ingaruka za DEET zishobora kwanduza imiterere y’imiti ndetse no kugabanya uburyo M3 receptors zikoreshwa, twagerageje igitekerezo cy’uko DEET ishobora kongera uburyo kalisiyumu ikoreshwa. Igitangaje ni uko DEET yananiwe kongera kalisiyumu ya cytoplasmic muri HUVEC (amakuru ntagaragazwa) na HEK/M3 (Ishusho ya 4a, b) kuri buri ngano yakoreshejwe.
Igihe cyo kohereza: Ukuboza-30-2024