Umuti wa anthelmintic N, N-diethyl-m-toluamide (DEET) byavuzwe ko bibuza AChE (acetylcholinesterase) kandi ifite kanseri ishobora gutera kanseri bitewe n'amaraso menshi. Muri iyi nyandiko, twerekana ko DEET itera cyane ingirabuzimafatizo ya endoteliyale itera angiogenez, bityo ikiyongera ryikibyimba. DEET ikora inzira ya selile iganisha kuri angiogenez, harimo gukwirakwizwa, kwimuka, hamwe no gufatira hamwe. Ibi bifitanye isano no kwiyongera NTA musaruro hamwe na VEGF imvugo muri selile endothelia. Gucecekesha M3 cyangwa gukoresha imiti ya M3 inhibitori yakuyeho izo ngaruka zose, byerekana ko angiogenezi iterwa na DEET iterwa na M3. Ubushakashatsi bujyanye na calcium yerekana ibimenyetso muri selile ya endoteliyale na HEK bikabije bikabije byakira M3, hamwe nubushakashatsi bwo guhuza no gufata ibyuma, byerekana ko DEET ikora nka moderi ya allosteric modulator ya M3 yakira. Byongeye kandi, DEET ibuza AChE, bityo ikongera bioavailable ya acetylcholine no guhuza kwakirwa na M3, no kongera ingaruka za proangiogenic binyuze mumabwiriza ya allosteric.
EC yibanze yatandukanijwe na aorta yimbeba zo mubusuwisi. Uburyo bwo kuvoma bwakuwe muri protocole ya Kobayashi 26. Murine ECs yarezwe muri EBM-2 iciriritse hiyongeraho 5% FBS idashyuha kugeza igice cya kane.
Ingaruka yibice bibiri bya DEET ku ikwirakwizwa rya HUVEC, U87MG, cyangwa BF16F10 byasesenguwe hakoreshejwe ibikoresho bya CyQUANT Cell Proliferation Assay Kit (Ikibazo cya Molecular, C7026). Muri make, selile 5.103 kuri buri riba zatewe mu isahani 96-y-iriba, yemererwa gufunga ijoro ryose, hanyuma ivurwa na DEET kumasaha 24. Nyuma yo gukuraho uburyo bwo gukura, ongeramo igisubizo gihuza irangi kuri buri riba rya microplate hanyuma ushiremo selile kuri 37 ° C muminota 30. Urwego rwa Fluorescence rwagenwe hifashishijwe umusomyi wa microplate ya Mithras LB940 (Berthold Technologies, Bad Wildbad, Ubudage) ifite ibikoresho 485 nm byo gushungura hamwe na 530 nm byungurura imyuka.
HUVEC yabibwe mu masahani 96-yuzuye ku bunini bwa selile 104 kuri buri riba. Ingirabuzimafatizo zavuwe na DEET kuri 24 h. Ubushobozi bwakagari bwasuzumwe hifashishijwe ibara rya MTT (Sigma-Aldrich, M5655). Indangagaciro ya optique yabonetse kubasomyi ba microplate ya multimode (Mithras LB940) kumuraba wa 570 nm.
Ingaruka za DEET zakozwe hakoreshejwe vitro angiogenez. Umuti hamwe na 10-8 M cyangwa 10-5 M DEET byongereyeho uburebure bwa capillary muri HUVECs (Igishusho 1a, b, utubari twera). Ugereranije nitsinda rishinzwe kugenzura, kuvura hamwe na DEET yibanze kuva kuri 10-14 kugeza 10-5 M byerekanaga ko uburebure bwa capillary bwageze mubibaya kuri 10-8 M DEET (Ishusho yinyongera. S2). Nta tandukaniro rikomeye ryabonetse muri vitro proangiogenic ingaruka za HUVECs zavuwe na DEET murwego rwo hagati ya 10-8 M na 10-5 M.
Kugirango tumenye ingaruka za DEET kuri neovascularization, twakoze mubushakashatsi bwa vivo neovascularization. Nyuma yiminsi 14, imbeba zatewe na selile endoteliyale zabanjirijwe na 10-8 M cyangwa 10-5 M DEET zerekanye ubwiyongere bukabije bwibintu bya hemoglobine (Ishusho 1c, utubari twera).
Byongeye kandi, DEET iterwa na neovascularization yakozwe mu mbeba zifite U87MG xenograft zatewe inshinge buri munsi (ip) hamwe na DEET ku kigero kizwiho gutera plasma ya 10-5 M, bikaba bisanzwe mubantu bagaragaye. muri 23. Ibibyimba byamenyekanye (ni ukuvuga ibibyimba> 100 mm3) byagaragaye nyuma yiminsi 14 nyuma yo guterwa ingirabuzimafatizo za U87MG mu mbeba. Ku munsi wa 28, imikurire yikibyimba yazamutse cyane mu mbeba zivuwe na DEET ugereranije nimbeba zo kugenzura (Ishusho 1d, kare). Byongeye kandi, CD31 yerekana ibibyimba yerekanaga ko DEET yongereye cyane agace ka capillary ariko ntabwo ari microvessel. (Ishusho 1e - g).
Kugirango umenye uruhare rwimitsi ya muscarinike mukwirakwiza DETA, 10-8 M cyangwa 10-5 M DETA imbere ya pFHHSiD (10-7 M, antagonist ya M3 yatoranijwe). Kuvura HUVEC. pFHHSiD yahagaritse burundu imitungo ikwirakwizwa ya DETA mubitekerezo byose (Imbonerahamwe 1).
Muri ibi bihe, twasuzumye kandi niba DEET yakongera uburebure bwa capillary muri selile HUVEC. Mu buryo nk'ubwo, pFHHSiD yabujije cyane DEET iterwa na capillary uburebure (Igishusho 1a, b, ibara ry'imvi). Byongeye kandi, ubushakashatsi busa bwakozwe na M3 siRNA. Nubwo kugenzura siRNA itagize akamaro mu guteza imbere imiterere ya capillary, gucecekesha reseptor ya M3 muscarinic yakuyeho ubushobozi bwa DEET bwo kongera uburebure bwa capillary (Ishusho 1a, b, utubari twirabura).
Byongeye kandi, byombi 10-8 M cyangwa 10-5 M DEET iterwa na vascularisation iterwa na vitro na neovascularization muri vivo byahagaritswe burundu na pFHHSiD (Ishusho 1c, d, inziga). Ibisubizo byerekana ko DEET iteza angiogenezi binyuze munzira yunvikana kuri M3 yakira antagonist cyangwa M3 siRNA.
AChE ni intego ya molekulari ya DEET. Ibiyobyabwenge nka donepezil, ikora nka inhibitori ya AChE, irashobora gutera EC angiogenez muri vitro no muri imbeba hindlimb ischemia moderi14. Twagerageje ingaruka yibice bibiri bya DEET kubikorwa bya enzyme ya AChE muri HUVEC. Ubushyuhe buke (10-8 M) hamwe nuburebure (10-5 M) bwa DEET bwagabanije ibikorwa bya ACHE ya endoteliyale ugereranije nuburyo bwo kugenzura (Ishusho 2).
Ibitekerezo byombi bya DEET (10-8 M na 10-5 M) byagabanije ibikorwa bya acetylcholinesterase kuri HUVEC. BW284c51 (10-5 M) yakoreshejwe nkigenzura rya acetylcholinesterase inhibitor. Ibisubizo bigaragazwa nkijanisha ryibikorwa bya AChE kuri HUVEC bivurwa hamwe na bibiri bya DEET ugereranije na selile zivura imodoka. Indangagaciro zigaragazwa nkibisobanuro ± SEM yubushakashatsi butandatu bwigenga. * p <0.05 ugereranije no kugenzura (Kruskal-Wallis na Dunn ikizamini cyo kugereranya inshuro nyinshi).
Nitide ya Nitric (OYA) igira uruhare mubikorwa bya angiogenic 33, kubwibyo, NTA musaruro muri DEET-iterwa na HUVECs yakozwe. DEET ivurwa na endoteliyale NTA musaruro wariyongereye ugereranije ningirabuzimafatizo, ariko wageze ku kamaro gusa ku kigero cya 10-8 M (Ishusho 3c). Kugirango umenye impinduka za molekuline zigenzura DEET-iterwa NTA musaruro, imvugo ya eNOS no gukora byasesenguwe na Western blotting. Nubwo kuvura DEET bitahinduye imvugo ya eNOS, byongereye cyane fosifora ya eNOS kurubuga rwayo ikora (Ser-1177) mugihe yagabanije ikibanza cyayo (Thr-495) ugereranije ningirabuzimafatizo zitavuwe muri fosifora ya eNOS (Ishusho 3d). Byongeye kandi, igipimo cya fosifora ya eNOS kurubuga rwa enterineti hamwe na site ya inhibitor yabazwe nyuma yo guhuza ingano ya fosifora ya eNOS nubunini bwa enzyme. Iri gereranya ryiyongereye cyane muri HUVECs zavuwe hamwe na buri kintu cya DEET ugereranije na selile zitavuwe (Ishusho 3d).
Hanyuma, imvugo ya VEGF, kimwe mubintu nyamukuru bya proangiogenic, yasesenguwe na Western blotting. DEET yongereye cyane imvugo ya VEGF, mugihe pFHHSiD yahagaritse rwose iyi mvugo.
Kubera ko ingaruka za DEET zumva neza gukumira no gufata imiti ya M3, twagerageje hypothesis ko DEET ishobora kongera ibimenyetso bya calcium. Igitangaje, DEET yananiwe kongera calcium ya cytoplasmeque muri HUVEC (amakuru aterekanwa) na HEK / M3 (Igishusho 4a, b) kubitekerezo byombi byakoreshejwe.
Igihe cyo kohereza: Ukuboza-30-2024