Ọgwụ anthelmintic N,N-diethyl-m-toluamide (DEETAkọpụtala na ọ na-egbochi AChE (acetylcholinesterase) ma nwee ike ibute kansa n'ihi oke vaskụla. N'akwụkwọ a, anyị na-egosi na DEET na-akpali mkpụrụ ndụ endothelial nke na-akwalite angiogenesis, si otú a na-eme ka uto etuto ahụ dịkwuo elu. DEET na-eme ka usoro mkpụrụ ndụ na-eduga na angiogenesis, gụnyere mmụba, mbugharị, na njikọta. Nke a na-ejikọta ya na mmụba mmepụta NO na ngosipụta VEGF na mkpụrụ ndụ endothelial. Ịgbachi nkịtị nke M3 ma ọ bụ iji ọgwụ mgbochi M3 kpochapụrụ mmetụta ndị a niile, na-egosi na angiogenesis nke DEET kpatara nwere mmetụta M3. Nnwale ndị metụtara akara calcium na mkpụrụ ndụ endothelial na HEK na-egosipụta oke ndị nnata M3, yana ọmụmụ njikọ na docking, na-egosi na DEET na-arụ ọrụ dị ka ihe na-eme ka ndị nnata M3 dịkwuo mma. Ọzọkwa, DEET na-egbochi AChE, si otú a na-amụba bioavailability nke acetylcholine na njikọ ya na ndị nnata M3, ma na-eme ka mmetụta proangiogenic dịkwuo mma site na nhazi allosteric.
E kewapụrụ ECs mbụ site na aorta nke òké Switzerland. E si na usoro Kobayashi 26 gbanwee usoro mwepụta ahụ. E tinyere Murine ECs na EBM-2 medium tinyere 5% FBS nke na-anaghị arụ ọrụ okpomọkụ ruo mgbe ọ ga-agafe.
E jiri CyQUANT Cell Proliferation Assess Kit (Molecular Probes, C7026) nyochaa mmetụta nke mkpokọta DEET abụọ nwere na mmụba nke HUVEC, U87MG, ma ọ bụ BF16F10. N'ụzọ dị mkpirikpi, a kụrụ mkpụrụ ndụ 5.103 n'ime otu olulu mmiri 96, hapụ ha ka ha tinye ha n'abalị, wee jiri DEET gwọọ ha ruo awa 24. Mgbe ewepụsịrị usoro uto ahụ, tinye ihe ngwọta agba na olulu mmiri ọ bụla nke microplate ahụ wee tinye mkpụrụ ndụ ndị ahụ na okpomọkụ 37 °C maka nkeji 30. E jiri ihe na-agụ microplate multimode Mithras LB940 (Berthold Technologies, Bad Wildbad, Germany) nke nwere ihe nzacha mkpali 485 nm na ihe nzacha mwepụ 530 nm.
A kụrụ HUVEC n'ime efere olulu 96 na njupụta nke mkpụrụ ndụ 104 n'otu olulu. E jiri DEET gwọọ mkpụrụ ndụ ahụ ruo awa 24. E jiri ule MTT agba agba (Sigma-Aldrich, M5655) chọpụta ike mkpụrụ ndụ. E nwetara ụkpụrụ njupụta anya na igwe na-agụ ihe na-agụ microplate multimode (Mithras LB940) na ogologo ebili mmiri nke 570 nm.
E jiri nyocha in vitro angiogenesis mụọ mmetụta DEET. Ọgwụgwọ ejiri 10-8 M ma ọ bụ 10-5 M DEET mụbaa nhazi ogologo capillary na HUVECs (Foto 1a, b, ogwe ọcha). Ma e jiri ya tụnyere otu njikwa ahụ, ọgwụgwọ ejiri DEET mee ihe site na 10-14 ruo 10-5 M gosiri na ogologo capillary ruru ọkwa dị elu na 10-8 M DEET (Foto mgbakwunye S2). A hụghị ọdịiche dị mkpa na mmetụta in vitro proangiogenic nke HUVECs ejiri DEET gwọọ na oke njupụta nke 10-8 M na 10-5 M.
Iji chọpụta mmetụta DEET nwere na neovascularization, anyị mere nnyocha in vivo neovascularization. Mgbe ụbọchị iri na anọ gachara, òké ndị a gbara mkpụrụ ndụ endothelial nke e ji 10-8 M ma ọ bụ 10-5 M DEET mee ihe gosiri mmụba dị ukwuu na ọdịnaya hemoglobin (Foto 1c, ogwe ọcha).
Ọzọkwa, a mụrụ DEET nke na-akpata neovascularization n'ime òké ndị nwere xenograft U87MG nke a na-agba DEET kwa ụbọchị (ip) na dose a maara na ọ na-akpali mkpokọta plasma nke 10-5 M, nke bụ ihe nkịtị n'ime ụmụ mmadụ ekpughere. na 23. A hụrụ etuto ndị a na-achọpụta (ya bụ, etuto >100 mm3) ụbọchị 14 ka a gbanyere mkpụrụ ndụ U87MG n'ime òké. Na ụbọchị 28, uto etuto ahụ mụbara nke ukwuu na òké ndị a gwọrọ DEET ma e jiri ya tụnyere òké ndị a na-achịkwa (Foto 1d, squares). Ọzọkwa, ntụpọ nke etuto CD31 gosiri na DEET mụbara nke ukwuu na mpaghara capillary mana ọ bụghị njupụta microvessel. (Foto 1e–g).
Iji chọpụta ọrụ nke ndị na-anabata muscarinic na mmụba nke DETA kpatara, ejiri 10-8 M ma ọ bụ 10-5 M DETA n'ihu pFHHSiD (10-7 M, onye na-emegide ndị na-anabata M3 ahọpụtara). Ọgwụgwọ nke HUVEC. pFHHSiD gbochiri ihe ndị na-agbasa nke DETA kpamkpam na mkpokọta niile (Tebụl 1).
N'okpuru ọnọdụ ndị a, anyị lere anya ma DEET ga-eme ka ogologo capillary dịkwuo elu na mkpụrụ ndụ HUVEC. N'otu aka ahụ, pFHHSiD gbochiri ogologo capillary nke DEET kpatara nke ukwuu (Foto 1a, b, ogwe isi awọ). Ọzọkwa, emere nnwale ndị yiri ya na M3 siRNA. Ọ bụ ezie na njikwa siRNA adịghị arụ ọrụ n'ịkwalite nhazi capillary, imechi ihe nnabata muscarinic M3 kwụsịrị ikike DEET ịbawanye ogologo capillary (Foto 1a, b, ogwe ojii).
Ọzọkwa, ma 10-8 M ma ọ bụ 10-5 M nke DEET kpatara vascularization in vitro na neovascularization in vivo ka pFHHSiD gbochiri kpamkpam (Foto 1c, d, okirikiri). Nsonaazụ ndị a na-egosi na DEET na-akwalite angiogenesis site na ụzọ na-emetụta ndị na-emegide ndị na-anabata M3 ma ọ bụ M3 siRNA ahọpụtara.
AChE bụ ihe mgbaru ọsọ nke DEET. Ọgwụ dịka donepezil, nke na-arụ ọrụ dị ka ihe mgbochi AChE, nwere ike ịkpalite angiogenesis EC in vitro na na ụdị ischemia hindlimb òké14. Anyị nwalere mmetụta nke mkpokọta DEET abụọ na ọrụ enzyme AChE na HUVEC. Ngụkọta DEET dị ala (10-8 M) na nke dị elu (10-5 M) belatara ọrụ endothelial AChE ma e jiri ya tụnyere ọnọdụ njikwa (Foto 2).
Mkpokọta DEET abụọ (10-8 M na 10-5 M) belatara ọrụ acetylcholinesterase na HUVEC. Ejiri BW284c51 (10-5 M) mee ihe dị ka njikwa maka ndị na-egbochi acetylcholinesterase. A na-egosipụta nsonaazụ dị ka pasent nke ọrụ AChE na HUVEC ejiri mkpokọta DEET abụọ gwọọ ma e jiri ya tụnyere mkpụrụ ndụ ejiri ụgbọala gwọọ. A na-egosipụta uru dị ka nkezi ± SEM nke nnwale isii nọọrọ onwe ha. *p < 0.05 ma e jiri ya tụnyere njikwa (ule ntụnyere ọtụtụ Kruskal-Wallis na Dunn).
Nitric oxide (NO) na-etinye aka na usoro angiogenic 33, ya mere, a mụrụ mmepụta NO na HUVECs nke DEET kpaliri. Mmepụta endothelial nke DEET gwọrọ NO mụbara ma e jiri ya tụnyere mkpụrụ ndụ njikwa, mana ọ ruru ihe dị mkpa naanị na dose nke 10-8 M (Foto 3c). Iji chọpụta mgbanwe molekul na-achịkwa mmepụta NO nke DEET kpalitere, Western blotting nyochachara ngosipụta na mkpali nke eNOS. Ọ bụ ezie na ọgwụgwọ DEET agbanweghị ngosipụta eNOS, ọ mụbara phosphorylation eNOS nke ukwuu na ebe ọ na-arụ ọrụ (Ser-1177) ebe ọ na-ebelata ebe mgbochi ya (Thr-495) ma e jiri ya tụnyere mkpụrụ ndụ ndị a na-agwọghị na phosphorylation eNOS (Foto 3d). Ọzọkwa, a gbakọrọ oke nke eNOS phosphorylated na ebe mmalite na ebe mgbochi mgbe emechara ka ọnụọgụ nke eNOS phosphorylated dị na mkpokọta enzyme. Oke a mụbara nke ukwuu na HUVECs ejiri mkpokọta DEET ọ bụla gwọọ ma e jiri ya tụnyere mkpụrụ ndụ ndị a na-agwọghị (Foto 3d).
N'ikpeazụ, Western blotting nyochachara ngosipụta nke VEGF, otu n'ime ihe ndị bụ isi na-akpata proangiogenic, DEET mụbara ngosipụta VEGF nke ukwuu, ebe pFHHSiD gbochiri ngosipụta a kpamkpam.
Ebe ọ bụ na mmetụta nke DEET na-emetụta ma mgbochi ọgwụ na mbelata nke ndị na-anabata M3, anyị nwalere echiche na DEET nwere ike ime ka mgbaàmà calcium dịkwuo mma. Ihe ijuanya bụ na DEET enweghị ike ịbawanye calcium cytoplasmic na HUVEC (data egosighi) na HEK/M3 (Fig. 4a, b) maka mkpokọta abụọ ejiri.
Oge ozi: Disemba-30-2024