ʻO ka lāʻau anthelmintic N,N-diethyl-m-toluamide (DEET) ua hōʻike ʻia e kāohi iā AChE (acetylcholinesterase) a loaʻa iā ia nā waiwai carcinogenic ma muli o ka nui o ka vascularization. Ma kēia pepa, hōʻike mākou i ka hoʻoulu ʻana o DEET i nā cell endothelial e hoʻoikaika ana i ka angiogenesis, a laila e hoʻonui ai i ka ulu ʻana o ka tumora. Hoʻoulu ʻo DEET i nā kaʻina cellular e alakaʻi ana i ka angiogenesis, me ka hoʻonui, ka neʻe ʻana, a me ka adhesion. Hoʻopili ʻia kēia me ka hoʻonui ʻana i ka hana NO a me ka hōʻike VEGF i nā cell endothelial. ʻO ka hoʻopanee ʻana o M3 a i ʻole ka hoʻohana ʻana i nā mea hoʻopiʻi pharmacological M3 i hoʻopau i kēia mau hopena a pau, e hōʻike ana i ka DEET-induced angiogenesis he M3-sensitive. ʻO nā hoʻokolohua e pili ana i ka hōʻailona calcium i nā endothelial a me nā pūnae HEK overexpressing M3 receptors, a me ka hoʻopaʻa ʻana a me ka hoʻopaʻa ʻana i nā haʻawina, e hōʻike ana e hana ana ʻo DEET ma ke ʻano he modulator allosteric o nā mea loaʻa M3. Eia kekahi, ke kāohi nei ʻo DEET iā AChE, a laila e hoʻonui ai i ka bioavailability o ka acetylcholine a me kona hoʻopaʻa ʻana i nā mea hoʻokipa M3, a hoʻonui 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 ka protocol Kobayashi 26. Ua hoʻoulu ʻia nā Murine EC ma EBM-2 medium i hoʻohui ʻia me 5% FBS wela-inactivated a hiki i ka ʻehā o ka pauku.
Ua nānā ʻia ka hopena o ʻelua mau manaʻo 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). ʻO ka pōkole, 5.103 mau pūnāwai no ka pūnāwai i hua ʻia i loko o kahi pā 96-punawai, ʻae ʻia e hoʻopili i ka pō, a laila mālama ʻia me DEET no 24 mau hola. Ma hope o ka wehe ʻana i ka mea ulu, e hoʻohui i ka wai hoʻopaʻa paʻa i kēlā me kēia pūnāwai o ka microplate a hoʻomoʻi i nā cell ma 37 ° C no 30 min. Hoʻoholo ʻia nā pae fluorescence me ka Mithras LB940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Kelemānia) i hoʻolako ʻia me nā kānana excitation 485 nm a me nā kānana emission 530 nm.
Ua kanu ʻia ʻo HUVEC i loko o nā papa 96-well ma kahi mānoanoa o 104 cell no ka pūnāwai. Ua mālama ʻia nā kelepona me DEET no 24 mau hola. Ua loiloi ʻia ke ola o ka cell me ka hoʻohana ʻana i kahi colorimetric MTT assay (Sigma-Aldrich, M5655). Loaʻa ʻia nā waiwai kikoʻī kikoʻī ma kahi mea heluhelu microplate multimode (Mithras LB940) ma kahi hawewe o 570 nm.
Ua aʻo ʻia nā hopena o DEET me ka hoʻohana ʻana i ka in vitro angiogenesis assays. Hoʻonui ka lāʻau lapaʻau me 10-8 M a i ʻole 10-5 M DEET i ka hoʻokumu ʻana o ka lōʻihi capillary ma HUVECs (Fig. 1a, b, nā kī keʻokeʻo). Ke hoʻohālikelikeʻia me ka pūʻulu hoʻomalu,ʻo ka mālamaʻana me ka DEET nā manaʻo mai ka 10-14 a hiki i ka 10-5 M i hōʻike i ka lōʻihi o ka capillary i hiki i kahi pāpū ma 10-8 M DEET (Supplementary Fig. S2). ʻAʻohe ʻokoʻa koʻikoʻi i loaʻa i ka hopena in vitro proangiogenic o HUVEC i mālama ʻia me DEET ma ka pae hoʻopaʻa ʻana 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 vivo. Ma hope o nā lā 14, ua hōʻike ʻia nā ʻiole i hoʻopaʻa ʻia me nā cell endothelial precultureed me 10-8 M a i ʻole 10-5 M DEET i ka piʻi nui ʻana o ka hemoglobin content (Fig. 1c, nā kī keʻokeʻo).
Eia kekahi, ua aʻo ʻia ka neovascularization DEET-induced i ka U87MG xenograft-bearing mice i hoʻopaʻa ʻia i kēlā me kēia lā (ip) me DEET ma kahi ʻano i ʻike ʻia e hoʻoulu i ka plasma concentrations o 10-5 M, he mea maʻamau i nā kānaka i hōʻike ʻia. i 23. Ua ʻike ʻia nā ʻōpū hiki ke ʻike ʻia (ʻo ia hoʻi nā maʻi maʻi >100 mm3) i 14 mau lā ma hope o ka hoʻokomo ʻia ʻana o nā pūnaewele U87MG i nā ʻiole. I ka lā 28, ua hoʻonui nui ʻia ka ulu ʻana o ka tumora i nā ʻiole i mālama ʻia e DEET i hoʻohālikelike ʻia me nā ʻiole mana (Fig. 1d, squares). Eia kekahi, ua hōʻike ʻia ka hoʻopaʻa ʻana o CD31 o nā maʻi maʻi i ka DEET i hoʻonui nui i ka ʻāpana capillary akā ʻaʻole ka microvessel density. (Fig. 1e–g).
No ka hoʻoholoʻana i ka hana o nā muscarinic receptors i ka DETA-induced proliferation, 10-8 M a iʻole 10-5 M DETA i mua o ka pFHHSiD (10-7 M, kahi koho M3 receptor antagonist). Lapaau o HUVEC. Ua ālai loa ka pFHHSiD i nā waiwai proliferative o DETA ma nā manaʻo a pau (Table 1).
Ma lalo o kēia mau kūlana, ua nānā pū mākou inā e hoʻonui ʻo DEET i ka lōʻihi o ka capillary i loko o nā keʻena HUVEC. Pēlā nō, ua pale nui ka pFHHSiD i ka lōʻihi o ka capillary i hoʻokomo ʻia e DEET (Fig. 1a, b, nā pahu hina). Eia kekahi, ua hana ʻia nā hoʻokolohua like me M3 siRNA. ʻOiai ʻaʻole maikaʻi ka mana siRNA i ka hoʻoikaika ʻana i ka hoʻokumu ʻana o ka capillary, ʻo ka silencing o ka M3 muscarinic receptor i hoʻopau i ka hiki o DEET e hoʻonui i ka lōʻihi o ka capillary (Fig. 1a, b, ʻeleʻele bars).
Eia kekahi, ʻo ka 10-8 M a i ʻole 10-5 M DEET i hoʻokomo ʻia i ka vascularization in vitro a me ka neovascularization in vivo ua paʻa loa ʻia e pFHHSiD (Fig. 1c, d, nā pōʻai). Hōʻike kēia mau hopena i ka hoʻolaha ʻana o DEET i ka angiogenesis ma o ke ala e pili ana i nā antagonists M3 receptor a i ʻole M3 siRNA.
ʻO AChE ka pahuhopu molekula o DEET. ʻO nā lāʻau lapaʻau e like me donepezil, e hana ana ma ke ʻano he AChE inhibitors, hiki ke hoʻoulu i ka EC angiogenesis in vitro a i loko o nā hiʻohiʻona ischemia hindlimb iole14. Ua hoʻāʻo mākou i ka hopena o ʻelua mau ʻāpana DEET ma ka hana enzyme AChE ma HUVEC. ʻO nā haʻahaʻa haʻahaʻa (10-8 M) a me nā kiʻekiʻe (10-5 M) o DEET i hoʻemi i ka hana endothelial AChE i hoʻohālikelike ʻia i nā kūlana mana (Fig. 2).
ʻO nā manaʻo ʻelua o DEET (10-8 M a me 10-5 M) i hoʻemi i ka hana acetylcholinesterase ma HUVEC. Ua hoʻohana ʻia ʻo BW284c51 (10-5 M) ma ke ʻano he mana no ka acetylcholinesterase inhibitors. Hōʻike ʻia nā hopena ma ke ʻano he pākēneka o ka hana AChE ma HUVEC i mālama ʻia me nā ʻāpana ʻelua o DEET i hoʻohālikelike ʻia me nā cell i mālama ʻia i ka kaʻa. Hōʻike ʻia nā waiwai e like me ka mean ± SEM o nā hoʻokolohua kūʻokoʻa ʻeono. *p <0.05 i hoʻohālikelike ʻia me ka mana (Kruskal-Wallis a me Dunn hoʻohālikelike hoʻohālikelike lehulehu).
Hoʻokomo ʻia ka Nitric oxide (NO) i ke kaʻina angiogenic 33, no laila, NO ka hana ʻana ma DEET-hoʻoulu ʻia HUVEC i aʻo ʻia. Ua hoʻonuiʻia ka hana endothelial NO DEET i hoʻohālikelikeʻia me nā pūnaewele mana, akā ua hiki i ke koʻikoʻi wale nō ma kahi o 10-8 M (Fig. 3c). No ka hoʻoholo ʻana i nā hoʻololi molekala e hoʻokele ana i ka hana NO DEET-induced, ua ʻike ʻia ka ʻōlelo eNOS a me ka hoʻōla ʻana e ka Western blotting. ʻOiai ʻaʻole i hoʻololi ka lāʻau DEET i ka hōʻike eNOS, ua hoʻonui nui ia i ka phosphorylation eNOS ma kona kahua hoʻāla (Ser-1177) ʻoiai e hoʻemi ana i kāna kahua paʻa (Thr-495) i hoʻohālikelike ʻia me nā cell untreated i ka eNOS phosphorylation (Fig. 3d). Eia kekahi, ua helu ʻia ka lakio o ka eNOS phosphorylated ma ke kahua hoʻāla a me ka pae inhibitory ma hope o ka hoʻohālikelike ʻana i ka nui o ka eNOS phosphorylated i ka nui o ka enzyme. Ua hoʻonui nui ʻia kēia lakio i nā HUVEC i mālama ʻia me kēlā me kēia neʻe ʻana o DEET i hoʻohālikelike ʻia me nā cell untreated (Fig. 3d).
ʻO ka mea hope loa, ua nānā ʻia ka ʻōlelo o VEGF, kekahi o nā kumu proangiogenic nui, e ka Western blotting. Ua hoʻonui nui ʻo DEET i ka hōʻike VEGF, ʻoiai ua pale ʻo pFHHSiD i kēia ʻōlelo.
Ma muli o ka paʻakikī o ka hopena o DEET i ka blockade pharmacological a me ka hoʻohaʻahaʻa ʻana i nā mea hoʻokipa M3, ua hoʻāʻo mākou i ke kuhiakau e hiki ai i ka DEET ke hoʻonui i ka hōʻailona calcium. ʻO ka mea kupanaha,ʻaʻole i hoʻonui ka DEET i ka calcium cytoplasmic i HUVEC (ʻikeʻole i hōʻikeʻia) a me HEK / M3 (Fig. 4a, b) no nā manaʻoʻelua i hoʻohanaʻia.
Ka manawa hoʻouna: Dec-30-2024