Ọgwụ anthelmintic N,N-diethyl-m-toluamide (DEET) A kọwo na ọ na-egbochi AChE (acetylcholinesterase) ma nwee ihe ndị nwere ike ịkpata carcinogenic n'ihi oke vaskụla. N'akwụkwọ a, anyị na-egosi na DEET na-akpali kpọmkwem sel endothelial nke na-akwalite angiogenesis, si otú ahụ na-amụba uto tumor. DEET na-arụ ọrụ usoro cellular nke na-eduga na angiogenesis, gụnyere mmụba, ọpụpụ, na adhesion. Ejikọtara nke a na mmụba NO mmepụta na okwu VEGF na sel endothelial. Nchichi nke M3 ma ọ bụ iji ọgwụ M3 inhibitors kagburu mmetụta ndị a niile, na-atụ aro na angiogenesis nke DEET kpatara bụ M3-sensitive. Nnwale metụtara calcium nrịbama na sel endothelial na HEK na-emebiga ihe ókè nke ndị na-anabata M3, yana njide na ọmụmụ ihe, na-egosi na DEET na-arụ ọrụ dị ka onye na-eme ihe na-ekpo ọkụ nke ndị na-anabata M3. Ọzọkwa, DEET na-egbochi AChE, si otú ahụ na-abawanye bioavailability nke acetylcholine na njikọ ya na ndị na-anabata M3, na ịkwalite mmetụta proangiogenic site na nhazi allosteric.
Ndị isi EC dịpụrụ adịpụ na aorta nke ụmụ oke Switzerland. Emebere usoro mmịpụta ahụ site na protocol Kobayashi 26 . Azụlitere Murine EC na EBM-2 ọkara gbakwunyere na 5% okpomọkụ anaghị arụ ọrụ FBS ruo nkeji nke anọ.
Achọpụtara mmetụta nke mkpokọta abụọ nke DEET na mmụba nke HUVEC, U87MG, ma ọ bụ BF16F10 site na iji CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Na nkenke, mkpụrụ ndụ 5.103 kwa olulu mmiri ka agbanyere na efere 96 nke ọma, kwere ka ọ tinye ya n'abali, wee mesoo ya na DEET maka awa 24. Mgbe ị wepụsịrị ihe na-eto eto, gbakwunye ihe ngwọta na-ejikọta agba na olulu mmiri ọ bụla nke microplate ma tinye mkpụrụ ndụ na 37 Celsius C maka 30 min. Achọpụtara ọkwa fluorescence site na iji Mithras LB940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany) nwere ihe nzacha mkpali 485 nm yana nzacha nm 530.
A na-akụ mkpụrụ HUVEC n'ime efere olulu mmiri 96 na njupụta nke sel 104 kwa olulu mmiri. Ejiri DEET gwọọ mkpụrụ ndụ maka awa 24. A tụlere ịdị ndụ cell site na iji nyocha MTT colorimetric (Sigma-Aldrich, M5655). Enwetara ụkpụrụ njupụta anya na onye na-agụ microplate multimode (Mithras LB940) na ogologo ogologo nke 570 nm.
A mụrụ mmetụta nke DEET site na iji in vitro angiogenesis assays. Ọgwụgwọ na 10-8 M ma ọ bụ 10-5 M DEET mụbara nguzobe ogologo capillary na HUVEC (Fig 1a, b, ogwe ọcha). E jiri ya tụnyere ndị na-ahụ maka njikwa, ọgwụgwọ na ntinye uche DEET sitere na 10-14 ruo 10-5 M gosiri na ogologo capillary ruru ala dị larịị na 10-8 M DEET (Nkọwa Fig. S2). Ọnweghị nnukwu ọdịiche dị na in vitro proangiogenic mmetụta nke HUVEC ndị a na-emeso ya na DEET na ntinye uche nke 10-8 M na 10-5 M.
Iji chọpụta mmetụta DEET na neovascularization, anyị mere na vivo neovascularization ọmụmụ. Mgbe ụbọchị iri na anọ gachara, ụmụ oke etinyere na mkpụrụ ndụ endothelial precultured na 10-8 M ma ọ bụ 10-5 M DEET gosipụtara mmụba dị ukwuu na ọdịnaya hemoglobin (Fig 1c, ogwe ọcha).
Ọzọkwa, a na-amụ neovascularization nke DEET na ụmụ oke U87MG xenograft nke a na-agbanye kwa ụbọchị (ip) na DEET na dose a maara iji mee ka ntinye uche nke plasma nke 10-5 M, nke bụ ihe nkịtị na ụmụ mmadụ ekpughere. na 23. Achọpụtara etuto ahụ (ya bụ etuto> 100 mm3) ụbọchị iri na anọ mgbe ịgbanye mkpụrụ ndụ U87MG n'ime ụmụ oke. N'ụbọchị 28, a na-akwalite uto tumor na ụmụ oke ndị a na-agwọ DEET ma e jiri ya tụnyere ụmụ oke nchịkwa (Fig 1d, squares). Ọzọkwa, ntụpọ CD31 nke etuto ahụ gosiri na DEET mụbara mpaghara capillary nke ukwuu mana ọ bụghị njupụta microvessel. (Fig 1e-g).
Iji chọpụta ọrụ nke ndị na-anabata muscarinic na mmụba nke DETA, 10-8 M ma ọ bụ 10-5 M DETA na ọnụnọ pFHHSiD (10-7 M, onye na-anabata M3 antagonist). Ọgwụgwọ HUVEC. pFHHSiD gbochiri akụrụngwa na-ebuwanye ibu nke DETA na mkpokọta niile (Table 1).
N'okpuru ọnọdụ ndị a, anyị nyochakwara ma DEET ọ ga-abawanye ogologo capillary na sel HUVEC. N'otu aka ahụ, pFHHSiD gbochiri nke ọma ogologo capillary DEET butere (Fig. 1a, b, bars gray). Ọzọkwa, ejiri M3 siRNA mee nnwale ndị yiri ya. Ọ bụ ezie na siRNA njikwa adịghị arụ ọrụ n'ịkwalite nhazi capillary, ịgbachi nkịtị nke onye na-anabata muscarin M3 kagburu ikike DEET iwelie ogologo ogologo capillary (Fig. 1a, b, black bars).
Ọzọkwa, ma 10-8 M ma ọ bụ 10-5 M DEET na-ebute vascularization in vitro na neovascularization na vivo bụ nke pFHHSiD (Fig. 1c, d, circles) na-egbochi kpamkpam. Nsonaazụ ndị a na-egosi na DEET na-akwalite angiogenesis site n'ụzọ nwere mmetụta maka ndị na-anabata M3 antagonists ma ọ bụ M3 siRNA.
AChE bụ ebumnuche molekụla nke DEET. Ọgwụ dị ka donepezil, nke na-eme ihe dị ka AChE inhibitors, nwere ike kpalie EC angiogenesis in vitro na òké hindlimb ischemia ụdị14. Anyị nwalere mmetụta nke mkpokọta abụọ nke DEET na ọrụ enzyme AChE na HUVEC. Obere (10-8 M) na elu (10-5 M) nke DEET na-ebelata ọrụ endothelial AChE ma e jiri ya tụnyere ọnọdụ nchịkwa (Fig 2).
Ntinye abụọ nke DEET (10-8 M na 10-5 M) belatara ọrụ acetylcholinesterase na HUVEC. A na-eji BW284c51 (10-5 M) mee ihe dị ka ihe nchịkwa maka ndị na-emechi acetylcholinesterase. A na-egosipụta nsonaazụ ya dị ka pasentị nke ọrụ AChE na HUVEC ejiri nchikota abụọ nke DEET emeso ya ma e jiri ya tụnyere sel ndị eji agwọ ụgbọ ala. A na-egosipụta ụkpụrụ dị ka pụtara ± SEM nke nnwale nnwere onwe isii. * p <0.05 atụnyere njikwa (Kruskal-Wallis na Dunn multiple ntụle ule).
Nitric oxide (NO) na-etinye aka na usoro angiogenic 33, ya mere, ọ dịghị mmepụta na DEET-stimulated HUVECs ka a mụrụ. DEET-mere endothelial NO mmepụta ka abawanyela ma e jiri ya tụnyere mkpụrụ ndụ na-achịkwa, ma ọ dị mkpa naanị na dose nke 10-8 M (Fig 3c). Iji chọpụta mgbanwe mkpụrụ ndụ ihe nketa na-achịkwa DEET-ebutere NO mmepụta, eNOS okwu na ịgbalite bụ nke Western blotting nyochara. Ọ bụ ezie na ọgwụgwọ DEET agbanweghị okwu eNOS, ọ na-abawanye ụbara eNOS phosphorylation na saịtị ya na-arụ ọrụ (Ser-1177) ebe ọ na-ebelata ebe nkwụsị ya (Thr-495) ma e jiri ya tụnyere mkpụrụ ndụ na-adịghị edozi na eNOS phosphorylation (Fig 3d). Ọzọkwa, a na-agbakọ nha nke eNOS phosphorylated phosphorylated na ebe a na-arụ ọrụ na ebe a na-egbochi ya ka emechara ka ọnụọgụ nke eNOS phosphorylated dị na ngụkọta nke enzyme. A na-abawanye oke nke a na HUVEC ndị a na-emeso ya na ntinye uche nke DEET ọ bụla ma e jiri ya tụnyere mkpụrụ ndụ anaghị agwọta (Fig 3d).
N'ikpeazụ, nkwupụta nke VEGF, otu n'ime ihe ndị bụ isi proangiogenic, bụ nke Western blotting nyochara. DEET mụbara okwu VEGF nke ukwuu, ebe pFHHSiD gbochiri okwu a kpamkpam.
Ebe ọ bụ na mmetụta nke DEET na-enwe mmetụta maka mgbochi ọgwụ ọgwụ na mbelata nke ndị na-anabata M3, anyị nwalere echiche ahụ na DEET nwere ike ịkwalite akara calcium. N'ụzọ dị ịtụnanya, DEET emeghị ka calcium cytoplasmic dị na HUVEC (data egosighi) na HEK / M3 (Fig. 4a, b) maka ntinye uche abụọ a na-eji.
Oge nzipu: Dec-30-2024