Iyeza le-anthelmintic i-N,N-diethyl-m-toluamide (I-DEET) kuye kwaxelwa ukuba kuthintelwa i-AChE (i-acetylcholinesterase) kwaye ineempawu ezinokubangela umhlaza ngenxa yokusasazeka kwemithambo yegazi kakhulu. Kule phepha, sibonisa ukuba i-DEET ikhuthaza ngokukodwa iiseli ze-endothelial ezikhuthaza i-angiogenesis, ngaloo ndlela inyusa ukukhula kwethumba. I-DEET ivuselela iinkqubo zeseli ezikhokelela kwi-angiogenesis, kubandakanya ukwanda, ukufuduka, kunye nokunamathela. Oku kunxulunyaniswa nokwanda kwemveliso ye-NO kunye nokubonakaliswa kwe-VEGF kwiiseli ze-endothelial. Ukuthulisa i-M3 okanye ukusebenzisa ii-pharmacological M3 inhibitors kuphelise zonke ezi ziphumo, nto leyo ebonisa ukuba i-angiogenesis ebangelwa yi-DEET i-M3-sensitive. Uvavanyo olubandakanya i-calcium signaling kwiiseli ze-endothelial kunye ne-HEK ezikhupha ii-receptors ze-M3 ngokugqithisileyo, kunye nezifundo zokubopha kunye nokufaka i-docking, zibonisa ukuba i-DEET isebenza njenge-allosteric modulator yee-receptors ze-M3. Ngaphezu koko, i-DEET ithintela i-AChE, ngaloo ndlela inyusa ukufumaneka kwe-acetylcholine kunye nokubopha kwayo kwi-receptors ze-M3, kwaye iphucula iziphumo ze-proangiogenic ngokulawulwa kwe-allosteric.
Ii-EC eziphambili zahlulwe kwi-aorta yeempuku zaseSwitzerland. Indlela yokukhupha yahlengahlengiswa kwiprotocol yeKobayashi 26. Ii-EC zeMurine zakhuliswa kwi-EBM-2 medium eyongeziweyo nge-5% ye-FBS engenakusebenza ngobushushu de kwaba lixesha lesine.
Isiphumo soxinzelelo olubini lwe-DEET ekwandeni kwe-HUVEC, U87MG, okanye i-BF16F10 sihlalutywe kusetyenziswa i-CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Ngamafutshane, iiseli ezi-5.103 ngomthombo ngamnye zityalwe kwipleyiti yemithombo engama-96, zavunyelwa ukuba zincamathiselwe ubusuku bonke, zaze zanyangwa nge-DEET iiyure ezingama-24. Emva kokususa i-growth medium, yongeza isisombululo sokubopha idayi kumthombo ngamnye we-microplate kwaye ufukame iiseli kwi-37 °C imizuzu engama-30. Amanqanaba e-Fluorescence amiselwe kusetyenziswa i-Mithras LB940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany) exhotyiswe ngee-485 nm excitation filters kunye nee-530 nm emission filters.
I-HUVEC ityalwe kwiipleyiti ezingama-96-wells kuxinano lweeseli ezili-104 ngomthombo ngamnye. Iiseli ziphathwe nge-DEET iiyure ezingama-24. Ukuphila kweseli kuhlolwe kusetyenziswa uvavanyo lwe-colorimetric MTT (Sigma-Aldrich, M5655). Amaxabiso oxinano lwe-optical afunyenwe kwi-multimode microplate reader (Mithras LB940) kubude bobude be-570 nm.
Iziphumo ze-DEET zifundwe kusetyenziswa uvavanyo lwe-in vitro angiogenesis. Unyango olusebenzisa i-10-8 M okanye i-10-5 M DEET lonyuse ukwakheka kobude be-capillary kwi-HUVECs (Umzobo 1a, b, imivalo emhlophe). Xa kuthelekiswa neqela lolawulo, unyango olune-DEET concentrations ukusuka kwi-10-14 ukuya kwi-10-5 M lubonise ukuba ubude be-capillary bufikelele kwi-plateau kwi-10-8 M DEET (Umzobo ongezelelweyo S2). Akukho mahluko ubalulekileyo ufunyenweyo kwi-in vitro proangiogenic effect ye-HUVECs enyangwe nge-DEET kuluhlu lwe-concentration lwe-10-8 M kunye ne-10-5 M.
Ukuze sifumanise impembelelo ye-DEET kwi-neovascularization, senze izifundo ze-vivo neovascularization. Emva kweentsuku ezili-14, iimpuku ezifakwe iiseli ze-endothelial ezikhuliswe kwangaphambili nge-10-8 M okanye i-10-5 M DEET zibonise ukwanda okukhulu komxholo we-hemoglobin (Umzobo 1c, imivalo emhlophe).
Ngaphezu koko, i-neovascularization ebangelwe yi-DEET ifundwe kwiimpuku ezine-xenograft ze-U87MG ezazitofwa imihla ngemihla (ip) nge-DEET ngedosi eyaziwayo ukuba ibangela uxinano lwe-plasma lwe-10-5 M, into eqhelekileyo kubantu abachatshazelweyo. kwi-23. Ii-tumor ezibonwayo (oko kukuthi ii-tumor >100 mm3) zabonwa kwiintsuku ezili-14 emva kokufakwa kweeseli ze-U87MG kwiimpuku. Ngomhla wama-28, ukukhula kwe-tumor kwanda kakhulu kwiimpuku eziphathwe yi-DEET xa kuthelekiswa neempuku zolawulo (Umzobo 1d, izikwere). Ngaphezu koko, ukudaywa kwe-CD31 kwee-tumor kubonise ukuba i-DEET yonyusa kakhulu indawo ye-capillary kodwa hayi uxinano lwe-microvessel. (Umzobo 1e–g).
Ukufumanisa indima yee-receptors ze-muscarinic ekukhuleni okubangelwa yi-DETA, kwasetyenziswa i-10-8 M okanye i-10-5 M DETA xa kukho i-pFHHSiD (10-7 M, i-M3 receptor antagonist ekhethiweyo). Unyango lwe-HUVEC. I-pFHHSiD ivale ngokupheleleyo iipropati zokwanda kwe-DETA kuzo zonke iindawo ezixineneyo (Itheyibhile 1).
Phantsi kwezi meko, sikwahlolisise nokuba i-DEET iya kwandisa ubude be-capillary kwiiseli ze-HUVEC. Ngokufanayo, i-pFHHSiD ithintele kakhulu ubude be-capillary obubangelwa yi-DEET (Umzobo 1a, b, ii-grey bars). Ngaphezu koko, iimvavanyo ezifanayo zenziwe nge-M3 siRNA. Nangona i-control siRNA ingasebenzi kakuhle ekukhuthazeni ukwakheka kwe-capillary, ukuthulisa i-M3 muscarinic receptor kwaphelisa amandla e-DEET okwandisa ubude be-capillary (Umzobo 1a, b, ii-black bars).
Ngaphezu koko, zombini i-10-8 M okanye i-10-5 M DEET-induced vascularization in vitro kunye ne-neovascularization in vivo zavalwa ngokupheleleyo yi-pFHHSiD (Umzobo 1c, d, izangqa). Ezi ziphumo zibonisa ukuba i-DEET ikhuthaza i-angiogenesis ngendlela ebuthathaka kwi-selective M3 receptor antagonists okanye i-M3 siRNA.
I-AChE yeyona nto ijoliswe kuyo kwi-DEET. Amayeza afana ne-donepezil, asebenza njenge-AChE inhibitors, anokukhuthaza i-EC angiogenesis kwi-vitro nakwimodeli ze-ischemia ye-mouse hindlimb ischemia14. Sivavanye isiphumo se-DEET ezimbini zomsebenzi we-enzyme ye-AChE kwi-HUVEC. Amanqanaba aphantsi (10-8 M) kunye naphezulu (10-5 M) e-DEET anciphise umsebenzi we-AChE we-endothelial xa kuthelekiswa neemeko zolawulo (Umzobo 2).
Zombini ii-concentrations ze-DEET (10-8 M kunye ne-10-5 M) zinciphise umsebenzi we-acetylcholinesterase kwi-HUVEC. I-BW284c51 (10-5 M) yasetyenziswa njengolawulo lwee-acetylcholinesterase inhibitors. Iziphumo zibonakaliswa njengepesenti yomsebenzi we-AChE kwi-HUVEC ephathwe ngee-concentrations ezimbini ze-DEET xa kuthelekiswa neeseli eziphathwe ngezithuthi. Amaxabiso abonakaliswa njenge-average ± SEM yezilingo ezintandathu ezizimeleyo. *p < 0.05 xa kuthelekiswa nolawulo (uvavanyo lokuthelekisa oluninzi lweKruskal-Wallis kunye neDunn).
I-nitric oxide (NO) ibandakanyeka kwinkqubo ye-angiogenic 33, ngoko ke, imveliso ye-NO kwi-HUVECs ezivuselelwe yi-DEET ifundwe. Imveliso ye-endothelial NO ephathwe yi-DEET inyuswe xa ithelekiswa neeseli zolawulo, kodwa ifikelele kukubaluleka kuphela kwidosi ye-10-8 M (Umzobo 3c). Ukufumanisa utshintsho lweemolekyuli ezilawula imveliso ye-NO ebangelwe yi-DEET, ukubonakaliswa kunye nokusebenza kwe-eNOS kuhlalutywe yi-Western blotting. Nangona unyango lwe-DEET lungazange lutshintshe ukubonakaliswa kwe-eNOS, lunyuse kakhulu i-phosphorylation ye-eNOS kwindawo yayo esebenzayo (Ser-1177) ngelixa lunciphisa indawo yayo yokuthintela (Thr-495) xa kuthelekiswa neeseli ezinganyangwanga kwi-phosphorylation ye-eNOS (Umzobo 3d). Ngaphezu koko, umlinganiselo we-phosphorylated eNOS kwindawo yokuvuselela kunye nendawo yokuthintela ubalwe emva kokulungelelanisa ubungakanani be-phosphorylated eNOS kwinani elipheleleyo le-enzyme. Olu mlinganiselo lunyuswe kakhulu kwi-HUVECs ezinyangwa ngoxinzelelo ngalunye lwe-DEET xa kuthelekiswa neeseli ezinganyangwanga (Umzobo 3d).
Ekugqibeleni, ukubonakaliswa kwe-VEGF, enye yezona zinto ziphambili ezibangela i-angiogenic, kwahlalutywa yi-Western blotting. I-DEET yonyuse kakhulu ukubonakaliswa kwe-VEGF, ngelixa i-pFHHSiD yayivale ngokupheleleyo le ntetho.
Ekubeni iziphumo ze-DEET zinovelwano kokubini kwi-pharmacological blockade kunye nokunciphisa i-M3 receptors, sivavanye ingcamango yokuba i-DEET inokuphucula i-calcium signaling. Okumangalisayo kukuba, i-DEET ayiphumelelanga ukunyusa i-cytoplasmic calcium kwi-HUVEC (idatha ayiboniswanga) kunye ne-HEK/M3 (Umzobo 4a, b) kuzo zombini iindawo ezisetyenzisiweyo.
Ixesha leposi: Disemba-30-2024