Ichiza le-anthelmintic N,N-diethyl-m-toluamide (DEET) kuye kwabikwa ukuba inqanda i-AChE (i-acetylcholinesterase) kwaye ineempawu ezinokuthi zenzeke ngenxa ye-vascularization. Kweli phepha, sibonisa ukuba i-DEET ivuselela ngokukodwa iiseli ze-endothelial ezikhuthaza i-angiogenesis, ngokwandisa ukukhula kwethumba. I-DEET ivula iinkqubo zeselula ezikhokelela kwi-angiogenesis, kubandakanywa ukwanda, ukufuduka, kunye nokunamathela. Oku kuhambelana nokunyuka kwe-NO imveliso kunye nokubonakaliswa kwe-VEGF kwiiseli ze-endothelial. Ukuthuliswa kwe-M3 okanye ukusebenzisa i-pharmacological M3 inhibitors kupheliswe zonke ezi ziphumo, kubonisa ukuba i-DEET-induced angiogenesis yi-M3-sensitive. Iimvavanyo ezibandakanya ukubonakaliswa kwe-calcium kwi-endothelial kunye neeseli ze-HEK ezigqithise i-M3 receptors, kunye nezifundo zokubopha kunye ne-docking, zibonisa ukuba i-DEET isebenza njenge-modulator ye-allosteric ye-M3 receptors. Ngaphezu koko, i-DEET inqanda i-AChE, ngaloo ndlela inyusa i-bioavailability ye-acetylcholine kunye nokubophelela kwayo kwi-M3 receptors, kunye nokuphucula imiphumo ye-proangiogenic ngokusebenzisa ukulawulwa kwe-allosteric.
Ii-EC eziphambili zazibekwe zodwa kwi-aorta yeempuku zaseSwitzerland. Indlela yokukhupha yatshintshwa kwi-protocol ye-Kobayashi 26. I-Murine ECs zaye zakhuliswa kwi-EBM-2 medium yongezwa kunye ne-5% ye-FBS engasetyenziswanga ukushisa kude kube yivesi yesine.
Umphumo wezinto ezimbini zokugxilwa kwe-DEET kwi-proliferation ye-HUVEC, i-U87MG, okanye i-BF16F10 yahlalutywa kusetyenziswa i-CyQUANT Cell Proliferation Assay Kit (i-Molecular Probes, C7026). Ngokufutshane, iiseli ze-5.103 ngomthombo ngamnye zifakwe kwi-plate ye-96-well plate, zivunyelwe ukuba zifake ubusuku bonke, kwaye ziphathwe nge-DEET kwi-24 h. Emva kokususa i-medium yokukhula, yongeza isisombululo sokubopha idayi kwiqula ngalinye le-microplate kwaye udibanise iiseli kwi-37 ° C kwi-30 min. Amanqanaba e-Fluorescence agqitywe ngokusebenzisa i-Mithras LB940 i-multimode microplate reader (i-Berthold Technologies, i-Bad Wildbad, eJamani) ixhotyiswe ngeefilitha ze-485 nm ze-excitation kunye ne-530 nm emission filters.
I-HUVEC yatyalwa kwiipleyiti ezinamaqula angama-96 kuxinaniso lweeseli ezili-104 kwiqula ngalinye. Iiseli zaphathwa nge-DEET i-24 h. Ukusebenza kweseli kwavavanywa kusetyenziswa i-MTT ye-colorimetric assay (Sigma-Aldrich, M5655). Amanani oxinaniso lwe-Optical density afunyenwe kwi-multimode microplate reader (Mithras LB940) kwi-wavelength ye-570 nm.
Iziphumo ze-DEET zafundwa kusetyenziswa i-in vitro angiogenesis assays. Unyango nge-10-8 M okanye i-10-5 M DEET yandise ukubunjwa kobude be-capillary kwi-HUVECs (Umfanekiso 1a, b, imivalo emhlophe). Xa kuthelekiswa neqela lolawulo, unyango kunye ne-DEET yogxininiso ukusuka kwi-10-14 ukuya kwi-10-5 M ibonise ukuba ubude be-capillary bufikelele kwi-plateau kwi-10-8 M DEET (i-Supplementary Fig. S2). Akukho mahluko abalulekileyo afunyenwe kwi-in vitro proangiogenic effect ye-HUVECs ephathwa nge-DEET kwi-concentration range ye-10-8 M kunye ne-10-5 M.
Ukumisela umphumo we-DEET kwi-neovascularization, senze izifundo ze-vivo neovascularization. Emva kweentsuku ze-14, iigundane ezifakwe kwiiseli ze-endothelial precultured kunye ne-10-8 M okanye i-10-5 M DEET ibonise ukwanda okukhulu kwi-hemoglobin content (Umfanekiso 1c, imivalo emhlophe).
Ngaphezu koko, i-DEET-induced neovascularization yafundwa kwi-U87MG i-xenograft-bearing mice eyayijojowe imihla ngemihla (ip) kunye ne-DEET kwidosi eyaziwayo ngokubangela i-plasma concentrations ye-10-5 M, eqhelekileyo kubantu abavezwayo. ngama-23. Amathumba abonakalayo (oko kukuthi amathumba> 100 mm3) abonwa kwiintsuku ezili-14 emva kokutofwa kweeseli ze-U87MG kwiimpuku. Ngomhla we-28, ukukhula kwe-tumor kwaphuculwa kakhulu kwiigundane eziphathwe nge-DEET xa kuthelekiswa neegundane zokulawula (umzobo 1d, izikwere). Ngaphaya koko, i-CD31 staining of tumors ibonise ukuba i-DEET inyuse kakhulu indawo ye-capillary kodwa hayi ukuxinana kwe-microvessel. (Umfanekiso 1e–g).
Ukumisela indima ye-muscarinic receptors kwi-DETA-induced proliferation, i-10-8 M okanye i-10-5 M i-DETA phambi kwe-pFHHSiD (10-7 M, i-M3 receptor antagonist) isetyenzisiweyo. Unyango lwe-HUVEC. I-pFHHSiD izithintele ngokupheleleyo iipropathi zokwandisa zeDETA kuzo zonke iinkalo (iThebhile 1).
Phantsi kwezi meko, siphinde savavanya ukuba i-DEET ingabunyusa na ubude be-capillary kwiiseli ze-HUVEC. Ngokufanayo, i-pFHHSiD ithintele kakhulu ubude be-capillary eyenziwe nge-DEET (Umfanekiso 1a, b, imivalo engwevu). Ngapha koko, iimvavanyo ezifanayo zenziwa nge-M3 siRNA. Nangona i-siRNA yolawulo yayingasebenziyo ekukhuthazeni ukubunjwa kwe-capillary, ukuthuliswa kwe-M3 ye-muscarinic receptor kwacima amandla e-DEET ukwandisa ubude be-capillary (Umfanekiso 1a, b, imivalo emnyama).
Ngaphezu koko, zombini i-10-8 M okanye i-10-5 M i-DEET-induced vascularization in vitro kunye neovascularization in vivo ivalwe ngokupheleleyo yi-pFHHSiD (Umfanekiso 1c, d, izangqa). Ezi ziphumo zibonisa ukuba i-DEET ikhuthaza i-angiogenesis ngokusebenzisa indlela echaphazelekayo kwi-M3 receptor antagonists okanye i-M3 siRNA.
I-AChE yithagethi yemolekyuli yeDEET. Iziyobisi ezifana ne-donepezil, ezisebenza njenge-AChE inhibitors, zinokuvuselela i-EC angiogenesis in vitro kunye ne-mouse hindlimb ischemia models14. Sivavanye umphumo wokugxininiswa kwezinto ezimbini ze-DEET kumsebenzi we-enzyme ye-AChE kwi-HUVEC. I-Low (10-8 M) kunye ne-high (10-5 M) i-DEET iyancipha umsebenzi we-AChE we-endothelial xa kuthelekiswa neemeko zokulawula (umzobo 2).
Zombini izigxina ze-DEET (10-8 M kunye ne-10-5 M) zinciphisa umsebenzi we-acetylcholinesterase kwi-HUVEC. I-BW284c51 (10-5 M) yayisetyenziswe njengolawulo lwe-acetylcholinesterase inhibitors. Iziphumo zichazwa njengepesenti yomsebenzi we-AChE kwi-HUVEC enyangwa ngogxininiso olubini lwe-DEET xa kuthelekiswa neeseli eziphathwa yimoto. Amaxabiso achazwa njengentsingiselo ± SEM yemifuniselo emithandathu ezimeleyo. * p <0.05 xa kuthelekiswa nolawulo (Kruskal-Wallis kunye noDunn uvavanyo lokuthelekisa ezininzi).
I-nitric oxide (NO) ibandakanyeka kwinkqubo ye-angiogenic 33, ngoko ke, AKUKHO mveliso kwi-DEET-stimulated HUVECs yafundwa. I-DEET-treated endothelial NO imveliso yanyuswa xa kuthelekiswa neeseli zokulawula, kodwa yafikelela ukubaluleka kuphela kwi-dose ye-10-8 M (Umfanekiso 3c). Ukumisela utshintsho lweemolekyuli ezilawula i-DEET-induced NO production, i-eNOS ibonakaliso kunye nokusebenza kuhlalutyelwe yi-Western blotting. Nangona unyango lwe-DEET aluzange luguqule ukubonakaliswa kwe-eNOS, kwandisa kakhulu i-eNOS phosphorylation kwindawo yayo yokusebenza (i-Ser-1177) ngelixa iyancipha indawo yayo yokuvimbela (i-Thr-495) xa kuthelekiswa neeseli ezingaphendulwanga kwi-eNOS phosphorylation (Fig. 3d). Ngaphezu koko, umlinganiselo we-eNOS ye-phosphorylated kwindawo yokuvuselela kunye ne-inhibitory site ibalwa emva kokulinganisa inani le-phosphorylated eNOS kwisixa esipheleleyo se-enzyme. Lo mlinganiso wanda kakhulu kwi-HUVECs ephathwayo kunye noxinzelelo ngalunye lwe-DEET xa kuthelekiswa neeseli ezingaphathwanga (Umfanekiso 3d).
Ekugqibeleni, ukubonakaliswa kwe-VEGF, enye yezona zinto ziphambili ze-proangiogenic, zahlaziywa yi-Western blotting. I-DEET inyuse kakhulu intetho yeVEGF, ngelixa i-pFHHSiD iye yavala ngokupheleleyo le ntetho .
Ekubeni iimpembelelo ze-DEET zinovakalelo kuzo zombini i-pharmacological blockade kunye nokunciphisa i-M3 receptors, siye savavanya i-hypothesis yokuba i-DEET inokunyusa ukubonakaliswa kwe-calcium. Okumangalisa kukuba, i-DEET ayiphumelelanga ukwandisa i-cytoplasmic calcium kwi-HUVEC (idatha engaboniswa) kunye ne-HEK / M3 (Umfanekiso 4a, b) kuzo zombini izigxina ezisetyenzisiweyo.
Ixesha lokuposa: Dec-30-2024