ʻO ka lāʻau anthelmintic N,N-diethyl-m-toluamide (DEET) ua hōʻike ʻia e kāohi iā AChE (acetylcholinesterase) a he mau waiwai carcinogenic paha ma muli o ka vascularization nui. Ma kēia pepa, hōʻike mākou e hoʻoulu kūikawā ʻo DEET i nā cell endothelial e hoʻolaha i ka angiogenesis, no laila e hoʻonui ai i ka ulu ʻana o ka tumor. Hoʻoulu ʻo DEET i nā kaʻina hana cellular e alakaʻi ana i ka angiogenesis, me ka proliferation, migration, a me ka adhesion. Pili kēia me ka hoʻonui ʻia o ka hana NO a me ka hōʻike ʻana o VEGF i nā cell endothelial. ʻO ka hoʻopau ʻana o M3 a i ʻole ka hoʻohana ʻana i nā mea hoʻopaʻa pharmacological M3 i hoʻopau i kēia mau hopena āpau, e hōʻike ana he M3-sensitive ka angiogenesis i hoʻokomo ʻia e DEET. ʻO nā hoʻokolohua e pili ana i ka calcium signaling i loko o nā cell endothelial a me HEK e overexpressing M3 receptors, a me nā haʻawina hoʻopaʻa a me ka docking, e hōʻike ana e hana ʻo DEET ma ke ʻano he modulator allosteric o nā receptors M3. Eia kekahi, kāohi ʻo DEET iā AChE, no laila e hoʻonui ai i ka bioavailability o acetylcholine a me kona hoʻopaʻa ʻana i nā receptors M3, a me ka hoʻonui ʻana i nā hopena proangiogenic ma o ka hoʻoponopono allosteric.
Ua hoʻokaʻawale ʻia nā EC mua mai ka aorta o nā ʻiole Swiss. Ua hoʻololi ʻia ke ʻano unuhi mai ke kaʻina hana Kobayashi 26. Ua mahi ʻia nā Murine EC i loko o ka medium EBM-2 i hoʻohui ʻia me 5% FBS i hoʻopau ʻia i ka wela a hiki i ka hala ʻana o ka hā.
Ua kālailai ʻia ka hopena o ʻelua mau ʻano o DEET ma ka hoʻonui ʻana o HUVEC, U87MG, a i ʻole BF16F10 me ka hoʻohana ʻana i ka CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). I ka pōkole, ua lūlū ʻia he 5.103 mau cell i kēlā me kēia lua i loko o kahi pā 96-lua, i ʻae ʻia e hoʻopili i ka pō, a laila mālama ʻia me DEET no 24 hola. Ma hope o ka wehe ʻana i ka medium ulu, e hoʻohui i ka hopena hoʻopaʻa dye i kēlā me kēia lua o ka microplate a hoʻomoʻa i nā cell ma 37 °C no 30 min. Ua hoʻoholo ʻia nā pae fluorescence me ka hoʻohana ʻana i kahi mea heluhelu microplate multimode Mithras LB940 (Berthold Technologies, Bad Wildbad, Kelemānia) i lako me nā kānana hoʻonāukiuki 485 nm a me nā kānana hoʻokuʻu 530 nm.
Ua lūlū ʻia ʻo HUVEC i loko o nā pā 96-pūpū ma kahi mānoanoa o 104 mau cell i kēlā me kēia pūpū. Ua mālama ʻia nā cell me DEET no 24 hola. Ua loiloi ʻia ke ola o nā cell me ka hoʻohana ʻana i kahi colorimetric MTT assay (Sigma-Aldrich, M5655). Ua loaʻa nā waiwai optical density ma kahi mea heluhelu microplate multimode (Mithras LB940) ma kahi nalu o 570 nm.
Ua aʻo ʻia nā hopena o DEET me ka hoʻohana ʻana i nā hoʻāʻo angiogenesis in vitro. ʻO ka mālama ʻana me 10-8 M a i ʻole 10-5 M DEET i hoʻonui i ka hoʻokumu ʻia ʻana o ka lōʻihi capillary ma HUVECs (Kiʻi 1a, b, nā kaha keʻokeʻo). Ke hoʻohālikelike ʻia me ka hui kaohi, ua hōʻike ka mālama ʻana me nā ʻano DEET mai 10-14 a 10-5 M ua hiki ka lōʻihi capillary i kahi plateau ma 10-8 M DEET (Kiʻi Hoʻohui S2). ʻAʻohe ʻokoʻa koʻikoʻi i loaʻa i ka hopena proangiogenic in vitro o HUVECs i mālama ʻia me DEET ma ka pae ʻano o 10-8 M a me 10-5 M.
No ka hoʻoholo ʻana i ka hopena o DEET ma ka neovascularization, ua hana mākou i nā haʻawina neovascularization in vivo. Ma hope o 14 mau lā, ua hōʻike nā ʻiole i hoʻopili ʻia me nā cell endothelial i hoʻoulu mua ʻia me 10-8 M a i ʻole 10-5 M DEET i ka piʻi nui ʻana o ka nui o ka hemoglobin (Kiʻi 1c, nā kaha keʻokeʻo).
Eia kekahi, ua aʻo ʻia ka neovascularization i hoʻokomo ʻia e DEET i loko o nā ʻiole U87MG xenograft-bearing i hoʻokomo ʻia i kēlā me kēia lā (ip) me DEET ma kahi nui i ʻike ʻia e hoʻoulu i nā ʻano plasma o 10-5 M, he mea maʻamau ia i nā kānaka i hōʻike ʻia. ma 23. Ua ʻike ʻia nā puʻupuʻu i ʻike ʻia (ʻo ia hoʻi nā puʻupuʻu >100 mm3) 14 mau lā ma hope o ka hoʻokomo ʻana o nā pūnaewele U87MG i loko o nā ʻiole. I ka lā 28, ua hoʻonui nui ʻia ka ulu ʻana o ka puʻupuʻu i nā ʻiole i mālama ʻia e DEET i hoʻohālikelike ʻia me nā ʻiole kaohi (Kiʻi 1d, squares). Eia kekahi, ua hōʻike ka hoʻoluʻu ʻana o CD31 i nā puʻupuʻu ua hoʻonui nui ʻo DEET i ka ʻāpana capillary akā ʻaʻole ka nui o nā microvessel. (Kiʻi 1e-g).
No ka hoʻoholo ʻana i ke kuleana o nā mea loaʻa muscarinic i ka hoʻonui ʻia ʻana o DETA, ua hoʻohana ʻia ʻo 10-8 M a i ʻole 10-5 M DETA i ke alo o pFHHSiD (10-7 M, kahi antagonist receptor M3 koho). ʻO ka mālama ʻana iā HUVEC. Ua ālai loa ʻo pFHHSiD i nā waiwai hoʻonui o DETA ma nā ʻano āpau (Papa 1).
Ma lalo o kēia mau kūlana, ua nānā pū mākou inā e hoʻonui ʻo DEET i ka lōʻihi o ke capillary i loko o nā pūnaewele HUVEC. Pēlā nō, ua pale nui ʻo pFHHSiD i ka lōʻihi o ke capillary i hoʻokomo ʻia e DEET (Kiʻi 1a, b, nā kaha hina). Eia kekahi, ua hana ʻia nā hoʻokolohua like me M3 siRNA. ʻOiai ʻaʻole i kūpono ka siRNA kaohi i ka hoʻolaha ʻana i ka hoʻokumu ʻana o ke capillary, ʻo ka hoʻopau ʻana i ka mea hoʻokipa muscarinic M3 i hoʻopau i ka hiki o DEET ke hoʻonui i ka lōʻihi o ke capillary (Kiʻi 1a, b, nā kaha ʻeleʻele).
Eia kekahi, ua ālai loa ʻia ka vascularization i hoʻokomo ʻia e 10-8 M a i ʻole 10-5 M DEET i loko o vitro a me ka neovascularization i loko o vivo e pFHHSiD (Kiʻi 1c, d, mau pōʻai). Hōʻike kēia mau hopena e hoʻolaha ana ʻo DEET i ka angiogenesis ma o kahi ala i pili i nā antagonists receptor M3 koho a i ʻole M3 siRNA.
ʻO AChE ka pahuhopu molekala o DEET. Hiki i nā lāʻau lapaʻau e like me donepezil, e hana ana ma ke ʻano he mau mea hoʻopaʻa AChE, ke hoʻoulu i ka EC angiogenesis in vitro a ma nā hiʻohiʻona ischemia hindlimb mouse14. Ua hoʻāʻo mākou i ka hopena o ʻelua mau ʻano o DEET ma ka hana enzyme AChE ma HUVEC. ʻO ka haʻahaʻa (10-8 M) a me ke kiʻekiʻe (10-5 M) o DEET i hoʻemi i ka hana endothelial AChE i hoʻohālikelike ʻia me nā kūlana kaohi (Kiʻi 2).
Ua hoʻemi nā ʻano like ʻole o DEET (10-8 M a me 10-5 M) i ka hana acetylcholinesterase ma HUVEC. Ua hoʻohana ʻia ʻo BW284c51 (10-5 M) ma ke ʻano he kaohi no nā mea hoʻopaʻa acetylcholinesterase. Hōʻike ʻia nā hopena ma ke ʻano he pakeneka o ka hana AChE ma HUVEC i mālama ʻia me nā ʻano like ʻelua o DEET i hoʻohālikelike ʻia me nā cell i mālama ʻia me ke kaʻa. Hōʻike ʻia nā waiwai ma ke ʻano he awelika ± SEM o ʻeono mau hoʻokolohua kūʻokoʻa. *p < 0.05 i hoʻohālikelike ʻia me ka kaohi (hoʻāʻo hoʻohālikelike lehulehu ʻo Kruskal-Wallis lāua ʻo Dunn).
Pili ka Nitric oxide (NO) i ke kaʻina hana angiogenic 33, no laila, ua aʻo ʻia ka hana ʻana o NO i loko o nā HUVEC i hoʻoulu ʻia e DEET. Ua hoʻonui ʻia ka hana ʻana o ka endothelial NO i mālama ʻia e DEET i hoʻohālikelike ʻia me nā cell kaohi, akā ua hōʻea i ke koʻikoʻi ma kahi mahele o 10-8 M (Kiʻi 3c). No ka hoʻoholo ʻana i nā loli molekala e kāohi ana i ka hana ʻana o ka DEET-induced NO, ua kālailai ʻia ka hōʻike ʻana a me ka hoʻāla ʻana o eNOS e Western blotting. ʻOiai ʻaʻole i hoʻololi ka mālama ʻana o DEET i ka hōʻike ʻana o eNOS, ua hoʻonui nui ia i ka phosphorylation eNOS ma kona wahi hoʻāla (Ser-1177) ʻoiai e hoʻemi ana i kona wahi inhibitory (Thr-495) i hoʻohālikelike ʻia me nā cell i mālama ʻole ʻia ma ka phosphorylation eNOS (Kiʻi 3d). Eia kekahi, ua helu ʻia ka lakio o ka phosphorylated eNOS ma kahi hoʻāla a me kahi inhibitory ma hope o ka hoʻomaʻamaʻa ʻana i ka nui o ka phosphorylated eNOS i ka nui o ka enzyme. Ua hoʻonui nui ʻia kēia lakio ma nā HUVEC i mālama ʻia me kēlā me kēia ʻano o DEET i hoʻohālikelike ʻia me nā cell i mālama ʻole ʻia (Kiʻi 3d).
ʻO ka mea hope loa, ua kālailai ʻia ka hōʻike ʻana o VEGF, kekahi o nā kumu proangiogenic nui, e ka Western blotting. Ua hoʻonui nui ʻo DEET i ka hōʻike ʻana o VEGF, ʻoiai ua ālai loa ʻo pFHHSiD i kēia hōʻike.
ʻOiai ua pili nā hopena o DEET i ka pale ʻana i ka pharmacological a me ka hoʻohaʻahaʻa ʻana o nā mea loaʻa M3, ua hoʻāʻo mākou i ka kuhiakau e hiki iā DEET ke hoʻonui i ka hōʻailona calcium. ʻO ka mea kupanaha, ʻaʻole i hoʻonui ʻo DEET i ka calcium cytoplasmic ma HUVEC (ʻaʻole i hōʻike ʻia ka ʻikepili) a me HEK/M3 (Kiʻi 4a, b) no nā ʻano ʻelua i hoʻohana ʻia.
Ka manawa hoʻouna: Dec-30-2024