Mushonga weanthelmintic N, N-diethyl-m-toluamide (DEET) yakanzi inhibit AChE (acetylcholinesterase) uye ine zvinogona kukonzera kenza nekuda kwekuwedzera kwevascularization. Mupepa rino, tinoratidza kuti DEET inonyanya kumutsa masero endothelial anokurudzira angiogenesis, nekudaro kuwedzera bundu kukura. DEET inomutsa cellular maitiro anotungamira kune angiogenesis, kusanganisira kuwanda, kutama, uye kunamatira. Izvi zvinosanganiswa nekuwedzera NO kugadzirwa uye VEGF kutaura muendothelial masero. Kunyarara kweM3 kana kushandisa pharmacological M3 inhibitors kwakabvisa zvose izvi, zvichiratidza kuti DEET-induced angiogenesis ndeyeM3-sensitive. Miedzo inosanganisira calcium signing mu endothelial uye HEK masero akawandisa M3 receptors, pamwe nekusunga uye zvidzidzo zvedocking, zvinoratidza kuti DEET inoshanda seallosteric modulator yeM3 receptors. Uyezve, DEET inhibits AChE, nokudaro kuwedzera bioavailability yeacetylcholine uye kusungirirwa kwayo kuM3 receptors, uye kuwedzera proangiogenic migumisiro kuburikidza neallosteric regulation.
Primary ECs dzakaparadzaniswa kubva kune aorta yeSwitzerland mice. Nzira yekuchera yakagadziriswa kubva kuKobayashi protocol 26. Murine ECs dzakagadzirwa muEBM-2 yepakati yakawedzerwa ne5% kupisa-inactivated FBS kusvika pandima yechina.
Mhedzisiro yezvikamu zviviri zveDEET pakupararira kweHUVEC, U87MG, kana BF16F10 yakaongororwa uchishandisa CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Nenguva pfupi, 5.103 masero patsime akadyarwa mu 96-tsime ndiro, akabvumirwa kubatanidza usiku hwose, ndokuzobatwa neDEET kwe24 h. Mushure mekubvisa svikiro rekukura, wedzera dhayi inosunga mhinduro kune yega yega microplate uye incubate maseru pa37 ° C kwemaminetsi makumi matatu. Fluorescence mazinga akatemerwa kushandisa Mithras LB940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany) yakagadzirirwa 485 nm excitation filters uye 530 nm emission filters.
HUVEC yakadyarwa mumapureti e96-matsime pakuwanda kwemasero zana nemana patsime. Masero akabatwa neDEET kwe24 h. Kushanda kwesero kwakaongororwa uchishandisa colorimetric MTT assay (Sigma-Aldrich, M5655). Optical density values yakawanikwa pane multimode microplate muverengi (Mithras LB940) pane wavelength ye570 nm.
Migumisiro yeDEET yakadzidzwa uchishandisa in vitro angiogenesis assays. Kurapa ne 10-8 M kana 10-5 M DEET kwakawedzera kuumbwa kwe capillary urefu muHUVECs (Fig. 1a, b, white bars). Kuenzaniswa neboka rekutonga, kurapwa neDEET kutenderera kubva ku10-14 kusvika ku10-5 M kwakaratidza kuti urefu hwe capillary hwakasvika pakakwirira pa 10-8 M DEET (Supplementary Fig. S2). Hapana mutsauko wakakosha wakawanikwa mune in vitro proangiogenic mhedzisiro yeHUVECs inorapwa neDEET muhuwandu hwekusungwa hwe10-8 M uye 10-5 M.
Kuti tione mhedzisiro yeDEET pane neovascularization, isu takaita mu vivo neovascularization zvidzidzo. Mushure memazuva gumi nemana, mbeva dzakaiswa masero endothelial precultured ne 10-8 M kana 10-5 M DEET yakaratidza kuwedzera kukuru kwehuwandu hwe hemoglobin (Fig. 1c, mabheji machena).
Uyezve, DEET-induced neovascularization yakadzidzwa muU87MG xenograft-bearing mbeva dzakaiswa jekiseni zuva nezuva (ip) neDEET pamushonga unozivikanwa kuti unokonzera plasma concentrations ye10-5 M, inowanzoitika muvanhu vakafumurwa. mu 23. Mamota anocherechedzwa (kureva mapundu> 100 mm3) akaonekwa mazuva gumi nemana mushure mejekiseni remasero eU87MG mumakonzo. Pazuva 28, kukura kwebundu kwakawedzerwa zvakanyanya mumakonzo akabatwa neDEET akaenzaniswa nemakonzo ekudzora (Fig. 1d, squares). Uyezve, kusvibiswa kweCD31 kwemamota kwakaratidza kuti DEET yakawedzera zvakanyanya capillary nzvimbo asi kwete microvessel density. (Mufananidzo 1e–g).
Kuti uone basa remuscarinic receptors muDETA-induced proliferation, 10-8 M kana 10-5 M DETA pamberi pepFHHSiD (10-7 M, anosarudza M3 receptor antagonist) yakashandiswa. Kurapa kweHUVEC. pFHHSiD yakavharira zvachose iyo proliferative zvivakwa zveDETA pane ese mafungidziro (Table 1).
Pasi pemamiriro aya, takaongororawo kana DEET yaizowedzera capillary kureba mumaseru eHUVEC. Saizvozvowo, pFHHSiD yakadzivirira zvakanyanya DEET-induced capillary urefu (Fig. 1a, b, grey bars). Uyezve, zviedzo zvakafanana zvakaitwa neM3 siRNA. Kunyange zvazvo siRNA yekudzora yakanga isingabatsiri mukusimudzira kuumbwa kwecapillary, kunyarara kweM3 muscarinic receptor kwakabvisa kukwanisa kweDEET kuwedzera capillary urefu (Fig. 1a, b, black bars).
Uyezve, zvose 10-8 M kana 10-5 M DEET-induced vascularization in vitro uye neovascularization in vivo yakavharwa zvachose nepFHHSiD (Fig. 1c, d, denderedzwa). Mhedzisiro iyi inoratidza kuti DEET inosimudzira angiogenesis kuburikidza negwara rinonzwa kune yakasarudzika M3 receptor antagonists kana M3 siRNA.
AChE ndiyo molecular target yeDEET. Mishonga yakadai se donepezil, inoshanda se AChE inhibitors, inogona kukurudzira EC angiogenesis in vitro uye mu mouse hindlimb ischemia models14. Takaedza mhedzisiro yezvikamu zviviri zveDEET pane AChE enzyme chiitiko muHUVEC. Yakaderera (10-8 M) uye yakakwirira (10-5 M) mazinga eDEET yakaderera endothelial AChE basa kana ichienzaniswa nemamiriro ekudzora (Fig. 2).
Zvose zviri zviviri zveDEET (10-8 M uye 10-5 M) zvakaderedza acetylcholinesterase chiitiko paHUVEC. BW284c51 (10-5 M) yakashandiswa sekutonga kweacetylcholinesterase inhibitors. Mhedzisiro inoratidzwa sechikamu chechikamu cheAChE chiitiko paHUVEC inorapwa neiyo miviri yekumisikidza yeDEET zvichienzaniswa neseru-akabatwa nemotokari. Values inoratidzwa sezvinoreva ± SEM yezvitanhatu zvakazvimirira zviedzo. * p <0.05 ichienzaniswa nekutonga (Kruskal-Wallis uye Dunn yakawanda yekuenzanisa bvunzo).
Nitric oxide (NO) inobatanidzwa mune angiogenic process 33, saka, HAPANA kugadzirwa muDEET-inosimudzira HUVECs yakadzidzwa. DEET-yakarapwa endothelial NO kugadzirwa kwakawedzera kuenzaniswa nekudzora maseru, asi yakasvika kukosha chete pachiyero che10-8 M (Fig. 3c). Kuti uone shanduko yemamorekuru inodzora DEET-induced NO kugadzirwa, eNOS kutaura uye activation yakaongororwa neWestern blotting. Kunyange zvazvo kurapwa kweDEET kusina kuchinja eNOS kutaura, yakawedzera zvakanyanya eNOS phosphorylation panzvimbo yayo yekushanda (Ser-1177) ichideredza nzvimbo yayo yekudzivirira (Thr-495) kana ichienzaniswa nemasero asina kugadziriswa mu eNOS phosphorylation (Fig. 3d). Uyezve, chiyero che phosphorylated eNOS panzvimbo yekushanda uye inhibitory site yakaverengwa mushure mekugadzirisa huwandu hwephosphorylated eNOS kusvika kuhuwandu hwehuwandu hwe enzyme. Chiyero ichi chakawedzerwa zvakanyanya muHUVECs inobatwa nega yega yega yeDEET kana ichienzaniswa nemasero asina kugadziriswa (Fig. 3d).
Pakupedzisira, kutaura kweVEGF, chimwe chezvinhu zvikuru zveproangiogenic, kwakaongororwa neWestern blotting. DEET yakawedzera zvakanyanya VEGF kutaura, nepo pFHHSiD yakavhara zvachose kutaura uku.
Sezvo mhedzisiro yeDEET inonzwa kune ese ari maviri pharmacological blockade uye downregulation yeM3 receptors, takaedza iyo hypothesis yekuti DEET inogona kuwedzera calcium kusaina. Zvinoshamisa kuti, DEET yakakundikana kuwedzera cytoplasmic calcium muHUVEC (data isina kuratidzwa) uye HEK / M3 (Fig. 4a, b) yezvikamu zvose zvakashandiswa.
Nguva yekutumira: Zvita-30-2024