Umuthi we-anthelmintic i-N,N-diethyl-m-toluamide (I-DEET) kubikwe ukuthi kuvimbela i-AChE (i-acetylcholinesterase) futhi kunezimpawu ezingaba umdlavuza ngenxa yokwanda kwemithambo yegazi. Kuleli phepha, sibonisa ukuthi i-DEET ivuselela ngqo amangqamuzana e-endothelial akhuthaza i-angiogenesis, ngaleyo ndlela yandise ukukhula kwesimila. I-DEET isebenzisa izinqubo zamaseli eziholela ku-angiogenesis, okuhlanganisa ukwanda, ukufuduka, kanye nokunamathela. Lokhu kuhlotshaniswa nokwanda kokukhiqizwa kwe-NO kanye nokubonakaliswa kwe-VEGF kumaseli e-endothelial. Ukuthulisa i-M3 noma ukusebenzisa izithibi ze-M3 zemithi kwaqeda yonke le miphumela, okuphakamisa ukuthi i-angiogenesis ebangelwa yi-DEET ibucayi ku-M3. Ukuhlolwa okubandakanya ukusayinwa kwe-calcium kumaseli e-endothelial kanye ne-HEK akhipha ama-receptor e-M3 ngokweqile, kanye nezifundo zokubopha kanye nokufaka, kubonisa ukuthi i-DEET isebenza njenge-modulator ye-allosteric yama-receptor e-M3. Ngaphezu kwalokho, i-DEET ivimbela i-AChE, ngaleyo ndlela yandisa ukutholakala kwe-acetylcholine kanye nokubopha kwayo kuma-receptor e-M3, futhi ithuthukise imiphumela ye-proangiogenic ngokulawulwa kwe-allosteric.
Ama-EC ayinhloko ahlukaniswe ku-aorta yamagundane aseSwitzerland. Indlela yokukhipha yaguqulwa kusukela kuphrothokholi yeKobayashi 26. Ama-EC e-Murine akhuliswa endaweni ephakathi ye-EBM-2 engezwe nge-5% ye-FBS engashisi ukushisa kuze kube yilapho kudlula okwesine.
Umphumela wokuhlushwa okubili kwe-DEET ekwandeni kwe-HUVEC, U87MG, noma i-BF16F10 uhlaziywe kusetshenziswa i-CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Kafushane, amaseli angu-5.103 ngomthombo ngamunye ahlwanyelwe epuletini elinamanzi angu-96, avunyelwe ukunamatheliswa ubusuku bonke, bese ephathwa nge-DEET amahora angu-24. Ngemva kokususa indawo yokukhula, engeza isisombululo sokubopha udayi emthonjeni ngamunye wepuleti elincane bese ufaka amaseli ku-37 °C imizuzu engama-30. Amazinga e-Fluorescence anqunywa kusetshenziswa i-Mithras LB940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany) ehlonyiswe ngezihlungi zokuvuselela ezingama-485 nm kanye nezihlungi zokukhipha ezingama-530 nm.
Ama-HUVEC ahlwanyelwe emapuletini angu-96-well density angamaseli angu-104 ngomthombo ngamunye. Amaseli aphathwe nge-DEET amahora angu-24. Ukuphila kahle kweseli kwahlolwa kusetshenziswa i-colorimetric MTT assay (Sigma-Aldrich, M5655). Amanani okuqina kwe-optical atholakale ku-multimode microplate reader (Mithras LB940) ngobude be-wavelength obungu-570 nm.
Imiphumela ye-DEET yafundwa kusetshenziswa izivivinyo ze-in vitro angiogenesis. Ukwelashwa nge-10-8 M noma i-10-5 M DEET kwandise ukwakheka kobude be-capillary kuma-HUVEC (Isithombe 1a, b, imigoqo emhlophe). Uma kuqhathaniswa neqembu lokulawula, ukwelashwa okunamazinga e-DEET asukela ku-10-14 kuya ku-10-5 M kubonise ukuthi ubude be-capillary bufinyelele ethafeni ku-10-8 M DEET (Isithombe Esingeziwe S2). Akukho mehluko obalulekile otholakale kumphumela we-in vitro proangiogenic wama-HUVEC aphathwe nge-DEET ebangeni lokuhlushwa elingu-10-8 M kanye no-10-5 M.
Ukuze sithole umphumela we-DEET ku-neovascularization, senze izifundo ze-in vivo neovascularization. Ngemva kwezinsuku ezingu-14, amagundane ajovwe ngamaseli e-endothelial atshalwe kusengaphambili nge-10-8 M noma i-10-5 M DEET abonise ukwanda okukhulu kokuqukethwe kwe-hemoglobin (Isithombe 1c, imigoqo emhlophe).
Ngaphezu kwalokho, i-neovascularization ebangelwa yi-DEET yafundwa emagundwini aphethe i-xenograft e-U87MG ajovwa nsuku zonke (ip) ne-DEET ngesilinganiso esaziwa ukuthi sibangela ukugcwala kwe-plasma okungu-10-5 M, okuvamile kubantu abavezwe. ku-23. Amathumba atholakalayo (okungukuthi amathumba >100 mm3) abonwa ezinsukwini eziyi-14 ngemuva kokujova amaseli e-U87MG emagundwini. Ngosuku lwama-28, ukukhula kwamathumba kwanda kakhulu emagundwini aphathwe yi-DEET uma kuqhathaniswa namagundane alawulayo (Isithombe 1d, izikwele). Ngaphezu kwalokho, ukudaya kwamathumba nge-CD31 kubonise ukuthi i-DEET yandise kakhulu indawo yama-capillary kodwa hhayi ubuningi bemithambo emincane. (Isithombe 1e–g).
Ukuze kutholakale indima yama-receptor e-muscarinic ekwandeni okubangelwa yi-DETA, kwasetshenziswa i-10-8 M noma i-10-5 M DETA lapho kukhona i-pFHHSiD (10-7 M, i-M3 receptor antagonist ekhethiwe). Ukwelashwa kwe-HUVEC. I-pFHHSiD ivimbe ngokuphelele izakhiwo zokwanda kwe-DETA kuzo zonke izingxube (Ithebula 1).
Ngaphansi kwalezi zimo, sihlole nokuthi i-DEET izokwandisa ubude be-capillary kumaseli e-HUVEC. Ngokufanayo, i-pFHHSiD ivimbele kakhulu ubude be-capillary obubangelwa yi-DEET (Isithombe 1a, b, imigoqo empunga). Ngaphezu kwalokho, kwenziwa izivivinyo ezifanayo nge-M3 siRNA. Nakuba i-control siRNA ingasebenzi kahle ekukhuthazeni ukwakheka kwe-capillary, ukuthulisa i-M3 muscarinic receptor kwaqeda ikhono le-DEET lokukhulisa ubude be-capillary (Isithombe 1a, b, imigoqo emnyama).
Ngaphezu kwalokho, kokubili i-10-8 M noma i-10-5 M DEET-induced vascularization in vitro kanye ne-neovascularization in vivo kwavinjelwa ngokuphelele yi-pFHHSiD (Isithombe 1c, d, imibuthano). Le miphumela ikhombisa ukuthi i-DEET ikhuthaza i-angiogenesis ngendlela ezwela kuma-anti-receptor antagonists akhethiwe e-M3 noma i-M3 siRNA.
I-AChE iyisisulu sama-molecule se-DEET. Izidakamizwa ezifana ne-donepezil, ezisebenza njengezithibi ze-AChE, zingakhuthaza i-EC angiogenesis in vitro kanye namamodeli e-ischemia yemilenze yangemuva yegundane14. Sivivinye umphumela wamazinga amabili e-DEET emsebenzini we-enzyme ye-AChE ku-HUVEC. Amazinga aphansi (10-8 M) kanye namazinga aphezulu (10-5 M) e-DEET anciphise umsebenzi we-AChE we-endothelial uma kuqhathaniswa nezimo zokulawula (Isithombe 2).
Zombili izingqimba ze-DEET (10-8 M kanye no-10-5 M) zinciphise umsebenzi we-acetylcholinesterase ku-HUVEC. I-BW284c51 (10-5 M) isetshenziswe njengokulawula ama-acetylcholinesterase inhibitors. Imiphumela ivezwa njengephesenti lomsebenzi we-AChE ku-HUVEC ephathwe ngama-concentration amabili e-DEET uma kuqhathaniswa namaseli aphathwe ngemoto. Amanani avezwa njengesilinganiso ± SEM sezivivinyo eziyisithupha ezizimele. *p < 0.05 uma kuqhathaniswa nokulawula (ukuhlolwa kokuqhathanisa okuningi kwe-Kruskal-Wallis kanye ne-Dunn).
I-nitric oxide (NO) ihilelekile enqubweni ye-angiogenic 33, ngakho-ke, ukukhiqizwa kwe-NO kuma-HUVEC avuselelwe yi-DEET kwafundwa. Ukukhiqizwa kwe-endothelial NO okuphathwe yi-DEET kwanda uma kuqhathaniswa namaseli okulawula, kodwa kwafinyelela ukubaluleka kuphela ngesilinganiso esingu-10-8 M (Isithombe 3c). Ukuze kutholakale izinguquko zama-molecule ezilawula ukukhiqizwa kwe-NO okubangelwa yi-DEET, ukuvezwa kwe-eNOS kanye nokusebenza kwayo kwahlaziywa yi-Western blotting. Nakuba ukwelashwa kwe-DEET kungazange kushintshe ukubonakaliswa kwe-eNOS, kwandise kakhulu i-phosphorylation ye-eNOS endaweni yayo esebenzayo (Ser-1177) ngenkathi kunciphisa indawo yayo yokuvimbela (Thr-495) uma kuqhathaniswa namaseli angelashwanga ku-phosphorylation ye-eNOS (Isithombe 3d). Ngaphezu kwalokho, isilinganiso se-eNOS ephethwe yi-phosphorylated endaweni yokusebenza kanye nendawo yokuvimbela sabalwa ngemuva kokulungisa inani le-eNOS ephethwe yi-phosphorylated enanini eliphelele le-enzyme. Lesi silinganiso sanda kakhulu kuma-HUVEC aphathwe ngokuhlushwa ngakunye kwe-DEET uma kuqhathaniswa namaseli angelashwanga (Isithombe 3d).
Ekugcineni, ukuvezwa kwe-VEGF, enye yezinto eziyinhloko ezibangela i-angiogenic, kwahlaziywa yi-Western blotting. I-DEET yandise kakhulu ukuvezwa kwe-VEGF, kanti i-pFHHSiD yavimba ngokuphelele lokhu kuvezwa.
Njengoba imiphumela ye-DEET ibucayi kokubili ekuvinjelweni kwemithi kanye nokwehla kwama-receptor e-M3, sihlole umbono wokuthi i-DEET ingase ithuthukise ukusayinwa kwe-calcium. Ngokumangazayo, i-DEET yehlulekile ukwandisa i-calcium ye-cytoplasmic ku-HUVEC (idatha ayiboniswanga) kanye ne-HEK/M3 (Isithombe 4a, b) kokubili ukugxila okusetshenzisiwe.
Isikhathi sokuthunyelwe: Disemba 30-2024