Ko te rongoā anthelmintic N,N-diethyl-m-toluamide (DEET) kua pūrongohia hei aukati i te AChE (acetylcholinesterase) ā, he āhuatanga mate pukupuku pea nā te nui rawa o te vascularization. I roto i tēnei pepa, ka whakaaturia e mātou ko te DEET e whakaoho ana i ngā pūtau endothelial e whakatairanga ana i te angiogenesis, ā, ka piki ake te tipu o te pukupuku. Ka whakahohe te DEET i ngā tukanga pūtau e arahi ana ki te angiogenesis, tae atu ki te tipu, te hekenga, me te piri. E pā ana tēnei ki te pikinga o te hanga NO me te whakaaturanga VEGF i roto i ngā pūtau endothelial. Ko te whakamōkito i te M3, te whakamahi rānei i ngā aukati M3 rongoā ka whakakore i ēnei pānga katoa, e kī ana ko te angiogenesis i puta mai i te DEET he tairongo ki te M3. Ko ngā whakamātautau e uru ana ki te tohu konupūmā i roto i ngā pūtau endothelial me te HEK e whakanui ana i ngā kaiwhakawhiwhi M3, me ngā rangahau here me te hononga, e tohu ana ko te DEET he kaiwhakahaere allosteric o ngā kaiwhakawhiwhi M3. I tua atu, ka aukati te DEET i te AChE, ā, ka piki ake te bioavailability o te acetylcholine me tōna herenga ki ngā kaiwhakawhiwhi M3, me te whakarei ake i ngā pānga proangiogenic mā te whakahaere allosteric.
I wehea ngā EC tuatahi mai i te aorta o ngā kiore Swiss. I ahu mai te tikanga tango i te kawa Kobayashi 26. I whakatōngia ngā EC murine ki roto i te reo EBM-2 i tāpirihia ki te 5% FBS whakakore-wera tae noa ki te putanga tuawhā.
I tātarihia te pānga o ngā kukū DEET e rua ki te horapa o HUVEC, U87MG, BF16F10 rānei mā te whakamahi i te CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). I roto i te poto, i whakatōkia ngā pūtau 5.103 mō ia poka ki roto i tētahi pereti 96-poka, i tukuna kia piri mō te pō, kātahi ka rongoatia ki te DEET mō te 24 hāora. I muri i te tango i te reo tipu, tāpirihia he otinga here tae ki ia poka o te pereti iti, ā, whakatōkia ngā pūtau ki te 37 °C mō te 30 meneti. I whakatauhia ngā taumata fluorescence mā te whakamahi i tētahi kaipānui pereti iti maha-aratau Mithras LB940 (Berthold Technologies, Bad Wildbad, Tiamana) me ngā tātari whakaoho 485 nm me ngā tātari tukunga 530 nm.
I whakatōkia ngā HUVEC ki roto i ngā pereti 96-pū i te kiato o te 104 pūtau mō ia pū. I rongoatia ngā pūtau ki te DEET mō te 24 hāora. I aromatawaitia te oranga o ngā pūtau mā te whakamahi i te whakamātautau MTT tae (Sigma-Aldrich, M5655). I whiwhihia ngā uara kiato whatu i runga i te kaipānui pereti iti maha-aratau (Mithras LB940) i te roanga ngaru o te 570 nm.
I tirotirohia ngā pānga o te DEET mā te whakamahi i ngā whakamātautau angiogenesis in vitro. Nā te maimoatanga ki te 10-8 M, 10-5 M DEET rānei i whakanui ake te hanganga o te roa o te capillary i roto i ngā HUVEC (Pikitia 1a, b, pae mā). Ki te whakatauritea ki te rōpū whakahaere, i whakaatuhia e te maimoatanga ki ngā kukū DEET mai i te 10-14 ki te 10-5 M kua eke te roa o te capillary ki te taumata papatahi i te 10-8 M DEET (Pikitia Tāpiri S2). Kāore i kitea he rerekētanga nui i roto i te pānga proangiogenic in vitro o ngā HUVEC i rongoatia ki te DEET i te awhe kukū o te 10-8 M me te 10-5 M.
Hei whakatau i te pānga o te DEET ki te neovascularization, i whakahaerehia e mātou he rangahau neovascularization in vivo. I muri i ngā rā 14, ko ngā kiore i werohia ki ngā pūtau endothelial i whakatōngia ki te 10-8 M, 10-5 M rānei DEET i whakaatu i te pikinga nui o te ihirangi hemoglobin (Pikitia 1c, ngā pae mā).
I tua atu, i rangahauhia te neovascularization i puta mai i te DEET i roto i ngā kiore U87MG xenograft-bearing i werohia i ia rā (ip) ki te DEET i te horopeta e mōhiotia ana ka puta he kukū plasma o te 10-5 M, he mea noa i roto i ngā tāngata kua pāngia. i roto i te 23. I kitea ngā pukupuku kitea (arā, ngā pukupuku >100 mm3) i ngā rā 14 i muri i te werohanga o ngā pūtau U87MG ki roto i ngā kiore. I te rā 28, i tino piki ake te tipu o te pukupuku i roto i ngā kiore i rongoatia ki te DEET ki te whakataurite ki ngā kiore mana (Pikitia 1d, tapawhā). I tua atu, i whakaatuhia e te tae CD31 o ngā pukupuku he nui ake te horahanga o te capillary i te DEET engari kāore i te kiato o ngā oko toto iti. (Pikitia 1e–g).
Hei whakatau i te mahi a ngā kaiwhakawhiwhi muscarinic i roto i te tipu i puta mai i te DETA, i whakamahia te 10-8 M, te 10-5 M rānei o te DETA i te aroaro o te pFHHSiD (10-7 M, he kaitukino kaiwhakawhiwhi M3 whiriwhiri). Te maimoatanga o te HUVEC. I ārai katoatia e te pFHHSiD ngā āhuatanga tipu o te DETA i ngā kukū katoa (Ripanga 1).
I raro i ēnei āhuatanga, i tirotirohia hoki e mātou mēnā ka whakanui ake a DEET i te roa o ngā uaua o te roro i roto i ngā pūtau HUVEC. Waihoki, i ārai nui a pFHHSiD i te roa o ngā uaua o te roro i puta mai i te DEET (Pikitia 1a, b, ngā pae hina). I tua atu, i mahia ngā whakamātautau rite ki te M3 siRNA. Ahakoa kāore i whai hua te siRNA whakahaere ki te whakatairanga i te hanganga uaua o te roro, ko te whakamōkito i te kaiwhakawhiwhi muscarinic M3 i whakakore i te kaha o te DEET ki te whakanui ake i te roa o ngā uaua o te roro (Pikitia 1a, b, ngā pae pango).
I tua atu, ko te vascularization i puta mai i te 10-8 M, 10-5 M rānei i roto i te vitro me te neovascularization i roto i te vivo i āraia katoatia e te pFHHSiD (Pikitia 1c, d, porowhita). E whakaatu ana ēnei hua ka whakatairanga te DEET i te angiogenesis mā te ara e pā ana ki ngā kaitukino kaiwhiwhi M3 whiriwhiri, ki te M3 siRNA rānei.
Ko te AChE te whāinga ngota o te DEET. Ka taea e ngā rongoā pēnei i te donepezil, e mahi ana hei aukati AChE, te whakaoho i te angiogenesis EC i roto i te vitro me roto i ngā tauira ischemia waewae muri o te kiore14. I whakamatauria e mātou te pānga o ngā kukū e rua o te DEET ki te mahi whākōkī AChE i roto i te HUVEC. Ko ngā kukū iti (10-8 M) me ngā kukū teitei (10-5 M) o te DEET i whakaiti i te mahi endothelial AChE ki te whakataurite ki ngā āhuatanga mana (Pikitia 2).
I whakaitihia e ngā kukū e rua o te DEET (10-8 M me te 10-5 M) te mahi acetylcholinesterase i runga i te HUVEC. I whakamahia a BW284c51 (10-5 M) hei mana whakahaere mō ngā aukati acetylcholinesterase. Ka whakaatuhia ngā hua hei ōrau o te mahi AChE i runga i te HUVEC i rongoatia ki ngā kukū e rua o te DEET ki te whakataurite ki ngā pūtau i rongoatia ki te waka. Ka whakaatuhia ngā uara hei toharite ± SEM o ngā whakamātautau motuhake e ono. *p < 0.05 ki te whakataurite ki te mana whakahaere (whakamātautau whakataurite maha a Kruskal-Wallis me Dunn).
Kei roto te hauota hauota (NO) i te tukanga angiogenic 33, nō reira, i rangahauhia te hanga NO i roto i ngā HUVEC i whakaohokia e te DEET. I piki ake te hanga NO endothelial i rongoatia ki te DEET ki te whakataurite ki ngā pūtau mana, engari i eke ki te hiranga i te horopeta o te 10-8 M anake (Pikitia 3c). Hei whakatau i ngā huringa ngota e whakahaere ana i te hanga NO i whakaohokia e te DEET, i tātarihia te whakaaturanga me te whakahohenga o te eNOS mā te Western blotting. Ahakoa kāore te maimoatanga DEET i whakarerekē i te whakaaturanga eNOS, i tino whakanuia te phosphorylation o te eNOS i tōna pae whakahohe (Ser-1177) i te wā e whakaitihia ana tōna pae aukati (Thr-495) ki te whakataurite ki ngā pūtau kāore i rongoatia i roto i te phosphorylation o te eNOS (Pikitia 3d). I tua atu, i tatauhia te ōwehenga o ngā eNOS kua phosphorylated i te pae whakahohenga me te pae aukati i muri i te whakataurite i te nui o ngā eNOS kua phosphorylated ki te nui katoa o te whākōkī. I tino piki ake tēnei ōwehenga i roto i ngā HUVEC i rongoatia ki ia kukū o te DEET ki te whakataurite ki ngā pūtau kāore i rongoatia (Pikitia 3d).
Hei whakamutunga, i tātarihia te whakapuakanga o te VEGF, tētahi o ngā āhuatanga matua o te angiogenic, mā te Western blotting. Nā te DEET i whakanui ake te whakapuakanga o te VEGF, engari nā te pFHHSiD i ārai katoa tēnei whakapuakanga.
I te mea he tairongo ngā pānga o te DEET ki te aukati rongoā me te whakahekenga o ngā kaiwhakawhiwhi M3, i whakamatauria e mātou te whakapae ka whakarei ake pea te DEET i te tohu konupūmā. He mea miharo, kāore i taea e te DEET te whakanui ake i te konupūmā cytoplasmic i roto i te HUVEC (kāore i whakaaturia ngā raraunga) me te HEK/M3 (Pikitia 4a, b) mō ngā kukū e rua i whakamahia.
Wā tuku: Hakihea-30-2024