Umuthi we-anthelmintic N,N-diethyl-m-toluamide (I-DEET) kuye kwabikwa ukuthi kuvimbela i-AChE (i-acetylcholinesterase) futhi inezici ezingase zibe ne-carcinogenic ngenxa ye-vascularization ngokweqile. Kuleli phepha, sibonisa ukuthi i-DEET ikhuthaza ngokuqondile amangqamuzana e-endothelial akhuthaza i-angiogenesis, ngaleyo ndlela andise ukukhula kwesimila. I-DEET yenza kusebenze izinqubo zamaselula eziholela ku-angiogenesis, okuhlanganisa ukwanda, ukufuduka, nokunamathela. Lokhu kuhlotshaniswa nokukhuphuka kokukhiqizwa kwe-NO kanye nenkulumo ye-VEGF kumaseli e-endothelial. Ukuthuliswa kwe-M3 noma ukusebenzisa ama-M3 inhibitors ekhemisi kuqede yonke le miphumela, okuphakamisa ukuthi i-angiogenesis eyenziwe nge-DEET izwela i-M3. Ukuhlolwa okubandakanya ukusayina kwe-calcium kuma-endothelial kanye namaseli e-HEK adlulisa ngokweqile ama-M3 receptors, kanye nezifundo zokubopha nokudokha, zibonisa ukuthi i-DEET isebenza njengemoduli ye-allosteric ye-M3 receptors. Ngaphezu kwalokho, i-DEET ivimbela i-AChE, ngaleyo ndlela ikhulise i-bioavailability ye-acetylcholine kanye nokubophezela kwayo kuma-receptors e-M3, futhi ithuthukise imiphumela ye-proangiogenic ngokusebenzisa ukulawulwa kwe-allosteric.
Ama-EC ayisisekelo ahlukaniswa ne-aorta yamagundane aseSwitzerland. Indlela yokukhipha ithathelwe ku-Kobayashi protocol 26. Ama-Murine EC akhuliswe endaweni ye-EBM-2 yengezwe nge-FBS engasebenzi ngo-5% kwaze kwaba kusigaba sesine.
Umphumela wokugxilisa okubili kwe-DEET ekwandeni kwe-HUVEC, U87MG, noma i-BF16F10 uhlaziywe kusetshenziswa i-CyQUANT Cell Proliferation Assay Kit (Ama-Molecular Probes, C7026). Kafushane, amaseli angu-5.103 emthonjeni ngamunye afakwe epuletini lemithombo engu-96, avunyelwe ukunamathisela ubusuku bonke, bese ephathwa nge-DEET amahora angu-24. Ngemva kokukhipha indawo yokukhula, engeza isisombululo sokubopha udayi emthonjeni ngamunye we-microplate bese ufukamela amaseli ku-37 °C imizuzu engu-30. Amazinga e-Fluorescence anqunywa kusetshenziswa isifundi se-Mithras LB940 se-multimode microplate (Berthold Technologies, Bad Wildbad, Germany) esihlome ngezihlungi ze-excitation ze-485 nm kanye nezihlungi ze-530 nm emission.
I-HUVEC ifakwe emapuletini anemithombo engama-96 ekumineni kwamaseli ayi-104 emthonjeni ngamunye. Amaseli alashwe nge-DEET amahora angama-24. Ukusebenza kweseli kwahlolwa kusetshenziswa isivivinyo se-MTT esinemibala (Sigma-Aldrich, M5655). Amanani we-Optical density atholwe kumfundi we-multimode microplate (Mithras LB940) nge-wavelength engu-570 nm.
Imiphumela ye-DEET yacwaningwa kusetshenziswa ama-in vitro angiogenesis assays. Ukwelashwa nge-10-8 M noma i-10-5 M DEET kwandise ukwakheka kobude be-capillary kuma-HUVEC (Umfanekiso 1a, b, imigoqo emhlophe). Uma kuqhathaniswa neqembu lokulawula, ukwelashwa ngokugxila kwe-DEET kusukela ku-10-14 kuya ku-10-5 M kubonise ukuthi ubude be-capillary bufinyelele endaweni ephansi ku-10-8 M DEET (Supplementary Fig. S2). Awukho umehluko obalulekile otholakele kumphumela we-in vitro proangiogenic wama-HUVEC aphathwa nge-DEET ebangeni lokuhlushwa lika-10-8 M kanye no-10-5 M.
Ukunquma umphumela we-DEET ku-neovascularization, senze izifundo ze-vivo neovascularization. Ngemuva kwezinsuku ezingu-14, amagundane ajovwe ngamaseli e-endothelial precultured ne-10-8 M noma i-10-5 M DEET abonise ukwanda okukhulu kokuqukethwe kwe-hemoglobin (Fig. 1c, imigoqo emhlophe).
Ngaphezu kwalokho, i-DEET-induced neovascularization yacwaningwa kumagundane athwele i-xenograft e-U87MG ayejovwe nsuku zonke (ip) nge-DEET ngethamo elaziwa ngokungenisa ukugxiliswa kwe-plasma kwe-10-5 M, okuyinto evamile kubantu abaveziwe. ku-23. Izimila ezitholakalayo (okungukuthi izimila> 100 mm3) zabonwa ezinsukwini ezingu-14 ngemva kokujova amaseli e-U87MG kumagundane. Ngosuku lwe-28, ukukhula kwe-tumor kwathuthukiswa kakhulu kumagundane aphethwe yi-DEET uma kuqhathaniswa namagundane okulawula (Fig. 1d, izikwele). Ngaphezu kwalokho, ukungcoliswa kwe-CD31 kwezimila kubonise ukuthi i-DEET yandise kakhulu indawo ye-capillary kodwa hhayi ukuminyana kwe-microvessel. (Umdwebo 1e–g).
Ukuze unqume indima yama-muscarinic receptors ekwandeni kwe-DETA-induced, i-10-8 M noma i-10-5 M DETA phambi kwe-pFHHSiD (10-7 M, i-M3 receptor antagonist ekhethiwe) isetshenzisiwe. Ukwelashwa kwe-HUVEC. I-pFHHSiD ivimbe ngokuphelele izakhiwo ezikhulayo ze-DETA kukho konke ukugxila (Ithebula 1).
Ngaphansi kwalezi zimo, siphinde sahlola ukuthi ingabe i-DEET ingabunyusa yini ubude be-capillary kumaseli e-HUVEC. Ngokufanayo, i-pFHHSiD ivimbele ngokuphawulekayo ubude be-capillary obubangelwa i-DEET (Fig. 1a, b, gray bar). Ngaphezu kwalokho, ukuhlolwa okufanayo kwenziwa nge-M3 siRNA. Nakuba i-siRNA yokulawula ayizange iphumelele ekukhuthazeni ukwakheka kwe-capillary, ukuthuliswa kwe-M3 ye-muscarinic receptor kwaqeda ikhono le-DEET lokwandisa ubude be-capillary (Fig. 1a, b, imigoqo emnyama).
Ngaphezu kwalokho, kokubili i-10-8 M noma i-10-5 M ye-DEET-induced vascularization in vitro kanye neovascularization in vivo ivinjwe ngokuphelele yi-pFHHSiD (Fig. 1c, d, circles). Le miphumela ibonisa ukuthi i-DEET iphromotha i-angiogenesis ngokusebenzisa indlela ezwelayo kubaphikisi abakhethiwe be-M3 receptor noma i-M3 siRNA.
I-AChE iyimpoqo yamangqamuzana e-DEET. Izidakamizwa ezifana ne-donepezil, ezisebenza njenge-AChE inhibitors, zingashukumisa i-EC angiogenesis in vitro kanye namamodeli e-ischemia e-hindlimb yegundane14. Sihlole umphumela wokugxila okubili kwe-DEET kumsebenzi we-enzyme ye-AChE ku-HUVEC. Ukugxila okuphansi (10-8 M) nokuphezulu (10-5 M) kwe-DEET kwehle umsebenzi we-AChE we-endothelial uma kuqhathaniswa nezimo zokulawula (Fig. 2).
Kokubili ukugxila kwe-DEET (10-8 M kanye no-10-5 M) kwehlise umsebenzi we-acetylcholinesterase ku-HUVEC. I-BW284c51 (10-5 M) yasetshenziswa njengokulawula ama-acetylcholinesterase inhibitors. Imiphumela ichazwa njengephesenti lomsebenzi we-AChE ku-HUVEC ephathwa ngokugxilisa okubili kwe-DEET uma kuqhathaniswa namaseli aphathwe imoto. Amanani avezwa njengencazelo ± SEM yokuhlolwa okuyisithupha okuzimele. *p <0.05 uma kuqhathaniswa nokulawula (ukuhlolwa kokuqhathanisa okuningi kwe-Kruskal-Wallis no-Dunn).
I-nitric oxide (NO) ibandakanyeka enqubweni ye-angiogenic 33, ngakho-ke, AKUKHO ukukhiqizwa kuma-HUVEC avuselelwe i-DEET kwacwaningwa. Ukukhiqizwa kwe-Endothelial NO okulashwe nge-DEET kwanyuswa uma kuqhathaniswa namaseli okulawula, kodwa kwafinyelela ukubaluleka kuphela ngedosi ye-10-8 M (Fig. 3c). Ukuze kunqunywe izinguquko zamangqamuzana ezilawula ukukhiqizwa kwe-NO okubangelwa i-DEET, isisho se-eNOS nokwenziwa kusebenze kwahlaziywa ngokucishwa kwe-Western. Nakuba ukwelashwa kwe-DEET akuzange kuguqule inkulumo ye-eNOS, kwandisa kakhulu i-eNOS phosphorylation endaweni yayo yokusebenza (i-Ser-1177) ngenkathi kunciphisa indawo yayo yokuvimbela (Thr-495) uma kuqhathaniswa namaseli angalashwanga ku-eNOS phosphorylation (Fig. 3d). Ngaphezu kwalokho, isilinganiso se-eNOS ye-phosphorylated esizeni sokuvula kanye nesiza sokuvimbela siye sabalwa ngemva kokujwayelekile inani le-phosphorylated eNOS enanini eliphelele le-enzyme. Lesi silinganiso sande kakhulu kuma-HUVEC aphathwa ngokuhlushwa ngakunye kwe-DEET uma kuqhathaniswa namaseli angalashwa (Fig. 3d).
Ekugcineni, inkulumo ye-VEGF, enye yezinto eziyinhloko ze-proangiogenic, yahlaziywa ukuchithwa kwe-Western. I-DEET inyuse kakhulu isisho se-VEGF, kuyilapho i-pFHHSiD ivimbe ngokuphelele lesi sisho .
Njengoba imiphumela ye-DEET izwela kukho kokubili ukuvinjelwa kwemithi kanye nokwehliswa kokulawulwa kwama-M3 receptors, sihlole umbono wokuthi i-DEET ingase ithuthukise ukusayinda kwe-calcium. Ngokumangalisayo, i-DEET yehlulekile ukukhulisa i-cytoplasmic calcium ku-HUVEC (idatha engabonisiwe) kanye ne-HEK/M3 (Fig. 4a, b) kukho kokubili ukugxilisa ingqondo okusetshenzisiwe.
Isikhathi sokuthumela: Dec-30-2024