Cov tshuaj tua kab mob N,N-diethyl-m-toluamide (DEET) tau tshaj tawm tias inhibit AChE (acetylcholinesterase) thiab muaj peev xwm ua rau mob qog noj ntshav vim muaj ntau dhau ntawm cov hlab ntsha. Hauv tsab ntawv no, peb qhia tias DEET tshwj xeeb ua rau cov hlwb endothelial uas txhawb nqa angiogenesis, yog li ua rau mob qog loj hlob ntxiv. DEET ua rau cov txheej txheem cellular ua rau angiogenesis, suav nrog kev loj hlob, kev tsiv teb tsaws chaw, thiab kev sib txuas. Qhov no cuam tshuam nrog kev tsim NO ntau ntxiv thiab VEGF qhia hauv cov hlwb endothelial. Kev ntsiag to ntawm M3 lossis siv cov tshuaj pharmacological M3 inhibitors tshem tawm tag nrho cov teebmeem no, qhia tias DEET-induced angiogenesis yog M3-sensitive. Cov kev sim uas muaj calcium signaling hauv endothelial thiab HEK hlwb overexpressing M3 receptors, nrog rau kev khi thiab docking kev tshawb fawb, qhia tias DEET ua haujlwm ua tus allosteric modulator ntawm M3 receptors. Ntxiv mus, DEET inhibits AChE, yog li ua rau kom muaj bioavailability ntawm acetylcholine thiab nws txoj kev khi rau M3 receptors, thiab txhim kho cov teebmeem proangiogenic los ntawm kev tswj hwm allosteric.
Cov ECs thawj zaug tau raug rho tawm ntawm lub aorta ntawm cov nas Swiss. Txoj kev rho tawm tau hloov kho los ntawm Kobayashi protocol 26. Murine ECs tau cog qoob loo hauv EBM-2 nruab nrab ntxiv nrog 5% cua sov-inactivated FBS kom txog thaum hla plaub.
Qhov cuam tshuam ntawm ob qhov concentration ntawm DEET rau kev loj hlob ntawm HUVEC, U87MG, lossis BF16F10 tau raug tshuaj xyuas siv CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Hauv luv luv, 5.103 lub hlwb ib lub qhov tau cog rau hauv lub phaj 96-qhov, cia kom txuas ib hmos, thiab tom qab ntawd kho nrog DEET rau 24 teev. Tom qab tshem tawm cov khoom noj khoom haus loj hlob, ntxiv cov tshuaj zas xim rau txhua lub qhov ntawm microplate thiab incubate cov hlwb ntawm 37 ° C rau 30 feeb. Cov theem fluorescence tau txiav txim siab siv Mithras LB940 multimode microplate nyeem ntawv (Berthold Technologies, Bad Wildbad, Lub Tebchaws Yelemees) nruab nrog 485 nm excitation lim dej thiab 530 nm emission lim dej.
HUVEC tau cog rau hauv 96-qhov phaj ntawm qhov ceev ntawm 104 lub hlwb ib qhov. Cov hlwb tau kho nrog DEET rau 24 teev. Kev muaj sia nyob ntawm lub hlwb tau soj ntsuam siv cov xim MTT assay (Sigma-Aldrich, M5655). Cov nqi ceev ntawm qhov muag tau txais ntawm tus nyeem ntawv multimode microplate (Mithras LB940) ntawm qhov ntev ntawm 570 nm.
Cov teebmeem ntawm DEET tau kawm los ntawm kev siv cov kev sim angiogenesis hauv vitro. Kev kho mob nrog 10-8 M lossis 10-5 M DEET ua rau muaj qhov ntev ntawm cov hlab ntsha hauv HUVECs ntau ntxiv (Daim Duab 1a, b, cov kab dawb). Piv nrog pawg tswj hwm, kev kho mob nrog DEET concentration ntawm 10-14 txog 10-5 M qhia tau tias qhov ntev ntawm cov hlab ntsha tau mus txog qhov siab tshaj plaws ntawm 10-8 M DEET (Daim Duab Ntxiv S2). Tsis muaj qhov sib txawv tseem ceeb pom nyob rau hauv cov teebmeem proangiogenic hauv vitro ntawm HUVECs kho nrog DEET hauv qhov ntau ntawm 10-8 M thiab 10-5 M.
Yuav kom paub txog qhov cuam tshuam ntawm DEET rau kev tsim cov hlab ntsha tshiab, peb tau ua kev tshawb fawb txog kev tsim cov hlab ntsha tshiab hauv vivo. Tom qab 14 hnub, cov nas uas tau txhaj tshuaj nrog cov hlwb endothelial uas tau cog ua ntej nrog 10-8 M lossis 10-5 M DEET tau qhia tias muaj kev nce ntxiv ntawm cov hemoglobin (Daim Duab 1c, cov kab dawb).
Ntxiv mus, DEET-induced neovascularization tau kawm hauv cov nas uas muaj U87MG xenograft uas tau txhaj tshuaj txhua hnub (ip) nrog DEET ntawm qhov koob tshuaj uas paub tias ua rau muaj cov plasma concentration ntawm 10-5 M, uas yog qhov ib txwm muaj rau cov neeg raug. hauv 23. Cov qog nqaij hlav uas pom tau (piv txwv li cov qog nqaij hlav >100 mm3) tau pom 14 hnub tom qab txhaj tshuaj U87MG rau hauv nas. Thaum hnub 28, kev loj hlob ntawm cov qog nqaij hlav tau nce ntxiv ntau hauv cov nas uas tau kho nrog DEET piv rau cov nas tswj (Daim duab 1d, squares). Ntxiv mus, CD31 staining ntawm cov qog nqaij hlav tau qhia tias DEET ua rau thaj chaw capillary nce ntxiv ntau tab sis tsis yog microvessel density. (Daim duab 1e-g).
Txhawm rau txiav txim siab lub luag haujlwm ntawm cov receptors muscarinic hauv DETA-induced proliferation, 10-8 M lossis 10-5 M DETA nyob rau hauv lub xub ntiag ntawm pFHHSiD (10-7 M, ib qho selective M3 receptor antagonist) tau siv. Kev kho mob ntawm HUVEC. pFHHSiD thaiv tag nrho cov khoom proliferative ntawm DETA ntawm txhua qhov concentration (Table 1).
Nyob rau hauv cov xwm txheej no, peb kuj tau tshuaj xyuas seb DEET puas yuav ua rau cov hlab ntsha ntev dua hauv HUVEC hlwb. Ib yam li ntawd, pFHHSiD tiv thaiv tau DEET-induced capillary ntev (Daim duab 1a, b, grey bars). Ntxiv mus, cov kev sim zoo sib xws tau ua nrog M3 siRNA. Txawm hais tias kev tswj siRNA tsis zoo hauv kev txhawb nqa capillary tsim, kev ntsiag to ntawm M3 muscarinic receptor tshem tawm lub peev xwm ntawm DEET los ua kom cov hlab ntsha ntev dua (Daim duab 1a, b, black bars).
Ntxiv mus, ob qho tib si 10-8 M lossis 10-5 M DEET-induced vascularization hauv vitro thiab neovascularization hauv vivo raug thaiv tag nrho los ntawm pFHHSiD (Daim duab 1c, d, voj voog). Cov txiaj ntsig no qhia tau tias DEET txhawb nqa angiogenesis los ntawm txoj hauv kev rhiab rau cov M3 receptor antagonists lossis M3 siRNA.
AChE yog lub hom phiaj molecular ntawm DEET. Cov tshuaj xws li donepezil, uas ua haujlwm ua AChE inhibitors, tuaj yeem txhawb EC angiogenesis hauv vitro thiab hauv nas hindlimb ischemia qauv14. Peb tau sim cov nyhuv ntawm ob qhov concentration ntawm DEET rau AChE enzyme kev ua haujlwm hauv HUVEC. Qis (10-8 M) thiab siab (10-5 M) concentration ntawm DEET txo qis endothelial AChE kev ua haujlwm piv rau cov xwm txheej tswj (Daim duab 2).
Ob qho kev sib xyaw ntawm DEET (10-8 M thiab 10-5 M) txo cov haujlwm acetylcholinesterase ntawm HUVEC. BW284c51 (10-5 M) tau siv los ua kev tswj hwm rau acetylcholinesterase inhibitors. Cov txiaj ntsig tau qhia ua feem pua ntawm AChE kev ua haujlwm ntawm HUVEC kho nrog ob qhov kev sib xyaw ntawm DEET piv rau cov hlwb kho tsheb. Cov nqi tau qhia ua qhov nruab nrab ± SEM ntawm rau qhov kev sim ywj pheej. * p <0.05 piv rau kev tswj hwm (Kruskal-Wallis thiab Dunn ntau qhov kev sib piv xeem).
Nitric oxide (NO) koom nrog rau hauv cov txheej txheem angiogenic 33, yog li ntawd, NO ntau lawm hauv DEET-stimulated HUVECs tau kawm. DEET-kho endothelial NO ntau lawm tau nce ntxiv piv rau cov hlwb tswj, tab sis tsuas yog ncav cuag qhov tseem ceeb ntawm 10-8 M (Daim duab 3c). Txhawm rau txiav txim siab qhov kev hloov pauv molecular uas tswj DEET-induced NO ntau lawm, eNOS kev qhia thiab kev ua haujlwm tau soj ntsuam los ntawm Western blotting. Txawm hais tias kev kho DEET tsis hloov eNOS kev qhia, nws ua rau eNOS phosphorylation ntau ntxiv ntawm nws qhov chaw ua haujlwm (Ser-1177) thaum txo nws qhov chaw inhibitory (Thr-495) piv rau cov hlwb tsis kho hauv eNOS phosphorylation (Daim duab 3d). Ntxiv mus, qhov piv ntawm phosphorylated eNOS ntawm qhov chaw ua haujlwm thiab qhov chaw inhibitory tau suav tom qab normalizing qhov ntau ntawm phosphorylated eNOS rau tag nrho cov nyiaj ntawm enzyme. Qhov piv no tau nce ntxiv hauv HUVECs kho nrog txhua qhov concentration ntawm DEET piv rau cov hlwb tsis kho (Daim duab 3d).
Thaum kawg, qhov kev qhia tawm ntawm VEGF, ib qho ntawm cov yam tseem ceeb proangiogenic, tau raug soj ntsuam los ntawm Western blotting. DEET ua rau VEGF qhia tawm ntau ntxiv, thaum pFHHSiD thaiv qhov kev qhia tawm no tag nrho.
Vim tias cov teebmeem ntawm DEET yog qhov rhiab heev rau ob qho kev thaiv tshuaj thiab kev txo qis ntawm M3 receptors, peb tau sim qhov kev xav tias DEET yuav txhim kho cov calcium signaling. Qhov xav tsis thoob, DEET tsis ua tiav qhov nce cytoplasmic calcium hauv HUVEC (cov ntaub ntawv tsis tau qhia) thiab HEK / M3 (Daim duab 4a, b) rau ob qho kev siv.
Lub sijhawm tshaj tawm: Lub Kaum Ob Hlis-30-2024