Cov tshuaj anthelmintic N, N-diethyl-m-toluamide (DEET) tau tshaj tawm tias inhibit AChE (acetylcholinesterase) thiab muaj peev xwm carcinogenic zog vim vascularization ntau dhau. Hauv daim ntawv no, peb qhia tias DEET tshwj xeeb txhawb cov hlwb endothelial uas txhawb nqa angiogenesis, yog li ua rau cov qog loj hlob tuaj. DEET activates cellular processes ua rau angiogenesis, nrog rau kev loj hlob, tsiv teb tsaws, thiab adhesion. Qhov no yog txuam nrog nce NO ntau lawm thiab VEGF qhia hauv endothelial hlwb. Silencing ntawm M3 los yog siv 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 nyob rau hauv endothelial thiab HEK hlwb overexpressing M3 receptors, raws li zoo raws li khi thiab docking kev tshawb fawb, qhia tias DEET ua raws li ib tug allosteric modulator ntawm M3 receptors. Tsis tas li ntawd, DEET inhibits AChE, yog li ua kom cov bioavailability ntawm acetylcholine thiab nws txoj kev khi rau M3 receptors, thiab txhim kho cov teebmeem proangiogenic los ntawm kev tswj hwm allosteric.
Thawj ECs raug cais tawm ntawm aorta ntawm Swiss nas. Txoj kev rho tawm tau hloov kho los ntawm Kobayashi raws tu qauv 26. Murine ECs tau coj mus rau hauv EBM-2 nruab nrab ntxiv nrog 5% cua sov-inactivated FBS kom txog rau thaum thib plaub.
Cov nyhuv ntawm ob qhov ntau ntawm DEET ntawm kev loj hlob ntawm HUVEC, U87MG, los yog BF16F10 tau soj ntsuam siv CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Luv luv, 5.103 hlwb ntawm ib lub qhov dej tau muab noob rau hauv 96-zoo phaj, tso cai rau xa mus rau hmo ntuj, thiab tom qab ntawd kho nrog DEET rau 24 teev. Tom qab tshem tawm qhov kev loj hlob nruab nrab, ntxiv dye binding tov rau txhua qhov dej ntawm microplate thiab incubate lub hlwb ntawm 37 ° C rau 30 min. Fluorescence qib tau txiav txim siab siv Mithras LB940 multimode microplate nyeem ntawv (Berthold Technologies, Bad Wildbad, Lub teb chaws Yelemees) nruab nrog 485 nm excitation lim thiab 530 nm emission lim.
HUVEC tau muab noob rau hauv 96-zoo daim hlau ntawm qhov ntom ntawm 104 hlwb ntawm ib qhov dej. Cells tau kho nrog DEET rau 24 teev. Cell viability raug soj ntsuam siv ib tug colorimetric MTT assay (Sigma-Aldrich, M5655). Optical density values tau txais los ntawm tus nyeem ntawv multimode microplate (Mithras LB940) ntawm lub wavelength ntawm 570 nm.
Cov teebmeem ntawm DEET tau kawm siv hauv vitro angiogenesis kev soj ntsuam. Kev kho mob nrog 10-8 M lossis 10-5 M DEET nce qhov tsim ntawm capillary ntev hauv HUVECs (Daim duab 1a, b, cov kab dawb). Piv nrog rau pawg tswj hwm, kev kho mob nrog DEET ntau npaum li ntawm 10-14 txog 10-5 M tau pom tias lub capillary ntev mus txog lub toj siab ntawm 10-8 M DEET (Supplementary Fig. S2). Tsis muaj qhov sib txawv tseem ceeb nyob rau hauv vitro proangiogenic nyhuv ntawm HUVECs kho nrog DEET nyob rau hauv cov concentration ntau ntawm 10-8 M thiab 10-5 M.
Txhawm rau txiav txim siab qhov cuam tshuam ntawm DEET ntawm neovascularization, peb tau ua hauv vivo neovascularization kev tshawb fawb. Tom qab 14 hnub, nas txhaj tshuaj endothelial hlwb precultured nrog 10-8 M lossis 10-5 M DEET pom tias muaj cov ntsiab lus hemoglobin nce ntxiv (Daim duab 1c, cov kab dawb).
Tsis tas li ntawd, DEET-induced neovascularization tau kawm hauv U87MG xenograft-cov kabmob nas uas tau txhaj tshuaj txhua hnub (ip) nrog DEET ntawm ib koob uas paub tias ua rau cov ntshav ntshav ntau ntawm 10-5 M, uas yog ib txwm muaj rau tib neeg raug. hauv 23. Cov qog uas kuaj tau (xws li cov qog> 100 mm3) tau pom 14 hnub tom qab txhaj tshuaj U87MG hlwb rau cov nas. Hnub tim 28, cov qog loj hlob zuj zus ntxiv hauv DEET-kho nas piv nrog cov nas tswj (Daim duab 1d, squares). Tsis tas li ntawd, CD31 staining ntawm cov qog pom tau hais tias DEET tau nce qhov chaw capillary tab sis tsis yog microvessel ntom. (Fig. 1e–g).
Txhawm rau txiav txim siab lub luag haujlwm ntawm muscarinic receptors hauv DETA-vim kev loj hlob, 10-8 M lossis 10-5 M DETA nyob rau hauv muaj pFHHSiD (10-7 M, xaiv M3 receptor antagonist) tau siv. Kev kho mob ntawm HUVEC. pFHHSiD tau thaiv tag nrho cov khoom muaj zog ntawm DETA ntawm txhua qhov ntau (Table 1).
Raws li cov xwm txheej no, peb kuj tau tshuaj xyuas seb DEET puas yuav ua kom ntev capillary hauv HUVEC hlwb. Ib yam li ntawd, pFHHSiD tseem tiv thaiv DEET-induced capillary ntev (Fig. 1a, b, grey bars). Tsis tas li ntawd, cov kev sim zoo sib xws tau ua nrog M3 siRNA. Txawm hais tias kev tswj siRNA tsis ua haujlwm zoo hauv kev txhawb nqa kev tsim cov capillary, kev ntsiag to ntawm M3 muscarinic receptor tshem tawm lub peev xwm ntawm DEET kom nce capillary ntev (Fig. 1a, b, dub bars).
Tsis tas li ntawd, 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 (Fig. 1c, d, voj voog). Cov txiaj ntsig no qhia tau tias DEET txhawb nqa angiogenesis los ntawm txoj hauv kev nkag siab rau xaiv M3 receptor antagonists lossis M3 siRNA.
AChE yog lub hom phiaj molecular ntawm DEET. Cov tshuaj xws li donepezil, uas ua raws li AChE inhibitors, tuaj yeem txhawb nqa EC angiogenesis hauv vitro thiab hauv nas hindlimb ischemia qauv14. Peb tau sim cov txiaj ntsig ntawm ob qhov ntau ntawm DEET ntawm AChE enzyme kev ua hauv HUVEC. Tsawg (10-8 M) thiab siab (10-5 M) cov ntsiab lus ntawm DEET txo qis kev ua haujlwm ntawm endothelial AChE piv rau kev tswj xyuas (Daim duab 2).
Ob qho kev sib xyaw ntawm DEET (10-8 M thiab 10-5 M) txo cov kev ua acetylcholinesterase ntawm HUVEC. BW284c51 (10-5 M) tau siv los ua kev tswj hwm rau acetylcholinesterase inhibitors. Cov txiaj ntsig tau nthuav tawm raws li feem pua ntawm AChE cov haujlwm ntawm HUVEC kho nrog ob qhov ntau ntawm DEET piv rau lub tsheb kho cov hlwb. Cov txiaj ntsig tau qhia tias txhais tau tias ± SEM ntawm rau qhov kev sim ywj pheej. *p <0.05 piv rau kev tswj (Kruskal-Wallis thiab Dunn ntau qhov kev sib piv).
Nitric oxide (NO) koom nrog cov txheej txheem angiogenic 33, yog li ntawd, TSIS MUAJ ntau lawm hauv DEET-stimulated HUVECs tau kawm. DEET-kho endothelial TSIS tau ntau lawm piv rau kev tswj cov hlwb, tab sis mus txog qhov tseem ceeb ntawm ib koob ntawm 10-8 M (Fig. 3c). Txhawm rau txiav txim siab cov kev hloov pauv molecular tswj DEET-induced TSIS MUAJ ntau lawm, eNOS qhia thiab ua kom tau raug tshuaj xyuas los ntawm Western blotting. Txawm hais tias kev kho DEET tsis hloov eNOS qhia, nws nce eNOS phosphorylation 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 (Fig. 3d). Tsis tas li ntawd, qhov piv ntawm phosphorylated eNOS ntawm qhov chaw ua haujlwm thiab qhov chaw inhibitory raug xam tom qab normalizing tus nqi ntawm phosphorylated eNOS rau tag nrho cov enzyme. Qhov piv txwv no tau nce ntxiv hauv HUVECs kho nrog txhua qhov concentration ntawm DEET piv rau cov hlwb tsis kho (Fig. 3d).
Thaum kawg, qhov kev qhia ntawm VEGF, yog ib qho tseem ceeb ntawm proangiogenic yam, tau soj ntsuam los ntawm Western blotting. DEET tau nce VEGF kev qhia ntau ntxiv, thaum pFHHSiD tau thaiv qhov kev qhia no.
Txij li thaum cov teebmeem ntawm DEET yog rhiab heev rau ob qho tib si pharmacological blockade thiab downregulation ntawm M3 receptors, peb tau sim cov kev xav tias DEET yuav txhim khu cov calcium signaling. Kuj ceeb tias, DEET ua tsis tau kom nce cytoplasmic calcium nyob rau hauv HUVEC (cov ntaub ntawv tsis qhia) thiab HEK/M3 (Fig. 4a, b) rau ob qho tib si concentrations siv.
Post lub sij hawm: Dec-30-2024