Mushonga wekurwisa marwadzo N,N-diethyl-m-toluamide (DEET) zvakanzi zvinodzivisa AChE (acetylcholinesterase) uye zvinogona kukonzera kenza nekuda kwekuwanda kweropa. Mupepa rino, tinoratidza kuti DEET inonyanya kukurudzira masero e endothelial anokurudzira angiogenesis, nokudaro zvichiwedzera kukura kwebundu. DEET inomutsa maitiro emasero anotungamira ku angiogenesis, kusanganisira kuwanda, kufamba, uye kunamira. Izvi zvine chekuita nekuwedzera kwekugadzirwa kweNO uye kuratidzwa kweVEGF mumasero e endothelial. Kunyarara kweM3 kana kushandisa mishonga yeM3 inhibitors kwakabvisa mhedzisiro iyi yese, zvichiratidza kuti DEET-induced angiogenesis inonzwa M3. Kuedza kunosanganisira calcium signaling mumasero e endothelial neHEK anoburitsa M3 receptors zvakanyanya, pamwe nezvidzidzo zvekusunga nekuisa, zvinoratidza kuti DEET inoshanda se allosteric modulator yeM3 receptors. Uyezve, DEET inodzivisa AChE, nokudaro ichiwedzera bioavailability yeacetylcholine uye kubatana kwayo neM3 receptors, uye inowedzera proangiogenic effects kuburikidza ne allosteric regulation.
MaEC ekutanga akaparadzaniswa kubva mutsinga yeropa yemakonzo eSwiss. Nzira yekuburitsa yakatorwa kubva kuKobayashi protocol 26. MaEC emurine akarimwa muEBM-2 medium yakawedzerwa ne5% yeFBS isina kupisa kusvika padanho rechina.
Mhedzisiro yekuwanda kweDEET kaviri pakuwanda kweHUVEC, U87MG, kana BF16F10 yakaongororwa uchishandisa CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Muchidimbu, masero 5.103 patsime rimwe nerimwe akadyarwa mundiro ine matsime 96, akabvumirwa kunamirwa husiku hwese, uye ozorapwa neDEET kwemaawa 24. Mushure mekubvisa nzvimbo yekukura, wedzera dye binding solution kune rimwe nerimwe tsime re microplate uye isa masero pa37 °C kwemaminitsi 30. Mazinga eFluorescence akaonekwa uchishandisa Mithras LB940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany) ine mafirita e485 nm excitation uye mafirita e530 nm emission.
HUVEC yakadyarwa mumaplate emvura 96 ane density yemasero 104 patsime rega rega. Masero akarapwa neDEET kwemaawa makumi maviri nemana. Kushanda kwemasero kwakaongororwa uchishandisa colorimetric MTT assay (Sigma-Aldrich, M5655). Optical density values yakawanikwa pa multimode microplate reader (Mithras LB940) pa wavelength ye570 nm.
Mhedzisiro yeDEET yakadzidzwa uchishandisa bvunzo dze in vitro angiogenesis. Kurapa ne 10-8 M kana 10-5 M DEET kwakawedzera kuumbwa kwehurefu hwe capillary muHUVECs (Mufananidzo 1a, b, white bars). Zvichienzaniswa neboka rekudzora, kurapwa ne DEET concentrations kubva pa 10-14 kusvika 10-5 M kwakaratidza kuti hurefu hwe capillary hwakasvika pa plateau pa 10-8 M DEET (Supplementary Fig. S2). Hapana musiyano wakakosha wakawanikwa mu in vitro proangiogenic effect yeHUVECs dzakarapwa neDEET muhuwandu hwe 10-8 M uye 10-5 M.
Kuti tione kuti DEET inobatsira sei pakukura kwemasero eropa, takaita zvidzidzo zvekuongorora kukura kwemasero eropa mumuviri. Mushure memazuva gumi nemana, makonzo akabayiwa maendothelial cells akakura kare aine 10-8 M kana 10-5 M DEET akaratidza kuwedzera kukuru kwehuwandu hwehemoglobin (Mufananidzo 1c, white bars).
Uyezve, DEET-induced neovascularization yakaongororwa mumbeva dzine U87MG xenograft dzaibayiwa jekiseni zuva nezuva (ip) neDEET pachiyero chinozivikanwa kuti chinokonzera kuwanda kweplasma kwe10-5 M, izvo zvakajairika muvanhu vakasangana nedambudziko. muna 23. Matumbu anoonekwa (kureva matumbu >100 mm3) akaonekwa mazuva gumi nemana mushure mekubayiwa masero eU87MG mumakonzo. Pazuva rechi28, kukura kwetumbu kwakawedzera zvakanyanya mumbeva dzakarapwa neDEET zvichienzaniswa nembeva dzinodzora (Mufananidzo 1d, squares). Uyezve, kusvibiswa kweCD31 kwematumbu kwakaratidza kuti DEET yakawedzera zvakanyanya nzvimbo ye capillary asi kwete microvessel density. (Mufananidzo 1e–g).
Kuti vaone basa remuscarinic receptors mukupararira kweDETA, 10-8 M kana 10-5 M DETA paine pFHHSiD (10-7 M, antagonist yeM3 receptor selective) yakashandiswa. Kurapa HUVEC. pFHHSiD yakavhara zvachose properties yeDETA pahuwandu hwese (Tafura 1).
Pasi pemamiriro ezvinhu aya, takaongororawo kana DEET yaizowedzera urefu hwema capillary mumasero eHUVEC. Saizvozvowo, pFHHSiD yakadzivirira zvakanyanya urefu hwema capillary hunokonzerwa neDEET (Mufananidzo 1a, b, grey bars). Uyezve, kuyedza kwakafanana kwakaitwa neM3 siRNA. Kunyangwe control siRNA isina kushanda mukusimudzira kuumbwa kwema capillary, kunyaradzwa kweM3 muscarinic receptor kwakabvisa kugona kweDEET kuwedzera urefu hwema capillary (Mufananidzo 1a, b, black bars).
Uyezve, zvese 10-8 M kana 10-5 M DEET-induced vascularization in vitro uye neovascularization in vivo zvakavharirwa zvachose ne pFHHSiD (Mufananidzo 1c, d, madenderedzwa). Izvi zvinoratidza kuti DEET inokurudzira angiogenesis kuburikidza nenzira inonzwa kune selective M3 receptor antagonists kana M3 siRNA.
AChE ndiyo inonyanya kurwiswa neDEET. Mishonga yakaita sedonepezil, inoshanda seAChE inhibitors, inogona kukurudzira EC angiogenesis in vitro uye mu mouse hindlimb ischemia models14. Takaedza mhedzisiro yehuwandu huviri hweDEET pabasa reAChE enzyme muHUVEC. Huwandu hwakaderera (10-8 M) uye hwakakwira (10-5 M) hweDEET hwakaderedza basa reAChE re endothelial zvichienzaniswa nemamiriro ekudzora (Mufananidzo 2).
Kuwanda kweDEET (10-8 M uye 10-5 M) kwakaderedza kushanda kweacetylcholinesterase paHUVEC. BW284c51 (10-5 M) yakashandiswa senzira yekudzora mishonga inodzivirira acetylcholinesterase. Mhedzisiro yacho inoratidzwa sechikamu chebasa reAChE paHUVEC rakarapwa nekuwanda kweDEET kaviri zvichienzaniswa nemasero akarapwa nemotokari. Makoshero acho anoratidzwa seavhareji ± SEM yezviedzo zvitanhatu zvakazvimiririra. *p < 0.05 zvichienzaniswa nekutonga (Kruskal-Wallis naDunn multiple comparison test).
Nitric oxide (NO) inobatanidzwa muangiogenic process 33, saka, NO production muDEET-stimulated HUVECs yakaongororwa. DEET-treated endothelial NO production yakawedzerwa zvichienzaniswa ne control cells, asi yakasvika pakukosha chete pa dose ye 10-8 M (Mufananidzo 3c). Kuti vaone shanduko dzemamorekuru dzinodzora DEET-induced NO production, eNOS expression uye activation zvakaongororwa neWestern blotting. Kunyangwe kurapwa kweDEET kusina kuchinja eNOS expression, kwakawedzera zvakanyanya eNOS phosphorylation panzvimbo yayo ye activation (Ser-1177) ukuwo kuchideredza inhibitory site yayo (Thr-495) zvichienzaniswa ne cells dzisina kurapwa mu eNOS phosphorylation (Mufananidzo 3d). Uyezve, ratio ye phosphorylated eNOS panzvimbo ye activation uye inhibitory site yakaverengerwa mushure mekururamisa huwandu hwe phosphorylated eNOS kune huwandu hwese hwe enzyme. Iyi ratio yakawedzerwa zvakanyanya muHUVECs dzakarapwa ne concentration yega yega yeDEET zvichienzaniswa ne cells dzisina kurapwa (Mufananidzo 3d).
Pakupedzisira, kuratidzwa kweVEGF, chimwe chezvinhu zvikuru zvinokonzera angiogenesis, kwakaongororwa neWestern blotting. DEET yakawedzera zvakanyanya kuratidzwa kweVEGF, nepo pFHHSiD yakavhara zvachose kuratidzwa uku.
Sezvo migumisiro yeDEET ichinyanya kukanganiswa nekuvharirwa kwemishonga uye kudzikiswa kweM3 receptors, takaedza fungidziro yekuti DEET inogona kuwedzera calcium signaling. Zvinoshamisa kuti DEET yakakundikana kuwedzera cytoplasmic calcium muHUVEC (data harina kuratidzwa) uye HEK/M3 (Mufananidzo 4a, b) yehuwandu hwese hwakashandiswa.
Nguva yekutumira: Zvita-30-2024