Magungunan anthelmintic N, N-diethyl-m-toluamide (DEET) an bayar da rahoton ya hana AChE (acetylcholinesterase) kuma yana da abubuwan da za su iya haifar da cututtuka na carcinogenic saboda wuce kima vascularization. A cikin wannan takarda, mun nuna cewa DEET musamman yana ƙarfafa ƙwayoyin endothelial waɗanda ke inganta angiogenesis, don haka ƙara girma girma. DEET yana kunna tsarin salula wanda ke haifar da angiogenesis, ciki har da yaduwa, ƙaura, da mannewa. Wannan yana da alaƙa da haɓakar NO samarwa da magana ta VEGF a cikin ƙwayoyin endothelial. Silencing na M3 ko yin amfani da magunguna M3 masu hanawa ya kawar da duk waɗannan tasirin, yana nuna cewa angiogenesis da DEET ta haifar da M3 mai hankali ne. Gwaje-gwajen da suka haɗa da siginar calcium a cikin ƙwayoyin endothelial da sel HEK masu wuce gona da iri na M3 masu karɓa, da kuma ɗaurewa da binciken docking, sun nuna cewa DEET yana aiki azaman mai daidaitawa na masu karɓar M3. Bugu da ƙari kuma, DEET yana hana AChE, don haka yana haɓaka bioavailability na acetylcholine da ɗaure shi ga masu karɓar M3, da haɓaka tasirin proangiogenic ta hanyar ƙa'idodin allosteric.
ECs na farko an ware su daga aorta na mice na Swiss. An daidaita hanyar cirewa daga ka'idar Kobayashi 26. An haɓaka Murine ECs a cikin matsakaicin EBM-2 wanda aka haɓaka tare da 5% FBS da ba a kunna zafi ba har zuwa nassi na huɗu.
An yi nazarin tasirin ƙididdiga guda biyu na DEET akan haɓakar HUVEC, U87MG, ko BF16F10 ta amfani da CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). A taƙaice, sel 5.103 a kowace rijiyar an shuka su a cikin farantin rijiyar 96, an ba su izinin haɗawa cikin dare, sannan a bi da su tare da DEET na sa'o'i 24. Bayan cire matsakaicin girma, ƙara maganin dauri ga kowane rijiyar microplate kuma sanya sel a 37 ° C na minti 30. An ƙaddara matakan fluorescence ta amfani da Mithras LB940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Jamus) sanye take da 485 nm tacewa da kuma 530 nm tacewa.
An shuka HUVEC a cikin faranti 96-rijiya a yawan sel 104 kowace rijiya. An yi amfani da kwayoyin halitta tare da DEET don 24 hours. An tantance iyawar salula ta amfani da gwajin MTT mai launi (Sigma-Aldrich, M5655). An sami ƙimar ƙimar gani a kan mai karanta microplate multimode (Mithras LB940) a tsawon 570 nm.
An yi nazarin tasirin DEET ta amfani da gwaje-gwajen angiogenesis na in vitro. Jiyya tare da 10-8 M ko 10-5 M DEET ya ƙãra samuwar tsawon capillary a cikin HUVECs (Fig. 1a, b, farin sanduna). Idan aka kwatanta da ƙungiyar kulawa, jiyya tare da ƙididdiga na DEET daga 10-14 zuwa 10-5 M ya nuna cewa tsayin capillary ya kai tudu a 10-8 M DEET (Ƙarin Hoton S2). Ba a sami wani gagarumin bambanci a cikin in vitro proangiogenic sakamako na HUVECs da aka bi da su tare da DEET a cikin kewayon maida hankali na 10-8 M da 10-5 M.
Don sanin tasirin DEET akan neovascularization, mun yi a vivo nazarin neovascularization. Bayan kwanaki 14, berayen da aka yi musu allura tare da sel na endothelial da aka riga aka tsara tare da 10-8 M ko 10-5 M DEET sun nuna karuwa mai yawa a cikin abun ciki na haemoglobin (Fig. 1c, farin sanduna).
Bugu da ƙari kuma, an yi nazarin neovascularization na DEET a cikin U87MG xenograft- bearing mice waɗanda aka yi musu allurar yau da kullum (ip) tare da DEET a wani kashi da aka sani don haifar da ƙwayar plasma na 10-5 M, wanda yake al'ada a cikin mutane da aka fallasa. a cikin 23. Ciwon daji da ake iya ganowa (watau ciwace-ciwacen daji> 100 mm3) an lura da su kwanaki 14 bayan allurar U87MG a cikin mice. A rana ta 28, an inganta haɓakar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta ta DEET idan aka kwatanta da mice mai kulawa (Fig. 1d, murabba'ai). Bugu da ƙari kuma, CD31 na ciwace-ciwacen ciwace-ciwacen daji ya nuna cewa DEET ya ƙaru sosai a yankin capillary amma ba ƙananan ƙwayoyin cuta ba. (Hoto na 1e-g).
Don ƙayyade rawar masu karɓa na muscarinic a cikin DETA-induced proliferation, 10-8 M ko 10-5 M DETA a gaban pFHHSiD (10-7 M, wani zaɓi na M3 antagonist). Maganin HUVEC. pFHHSiD gaba daya ya toshe kaddarorin DETA masu yaduwa a kowane taro (Table 1).
A ƙarƙashin waɗannan sharuɗɗan, mun kuma bincika ko DEET zai ƙara tsayin capillary a cikin ƙwayoyin HUVEC. Hakazalika, pFHHSiD ya hana tsayin capillary da DEET ya jawo (Hoto 1a, b, sanduna launin toka). Bugu da ƙari, an yi irin wannan gwaji tare da M3 siRNA. Kodayake siRNA mai sarrafawa ba ta da tasiri wajen haɓaka samuwar capillary, yin shiru na mai karɓar muscarin na M3 ya kawar da ikon DEET don ƙara tsayin capillary (Fig. 1a, b, black sanduna).
Bugu da ƙari kuma, duka 10-8 M ko 10-5 M DEET-induced vascularization in vitro da neovascularization a vivo an katange gaba daya ta pFHHSiD (Fig. 1c, d, Circles). Waɗannan sakamakon suna nuna cewa DEET yana haɓaka angiogenesis ta hanyar da ta dace da zaɓaɓɓun antagonists masu karɓa na M3 ko M3 siRNA.
AChE shine makasudin kwayoyin halitta na DEET. Magunguna irin su donedpezil, waɗanda ke aiki azaman masu hana AChE, na iya haɓaka EC angiogenesis a cikin vitro da kuma cikin ƙirar hindlimb ischemia na linzamin kwamfuta. Mun gwada tasirin nau'i biyu na DEET akan ayyukan enzyme AChE a cikin HUVEC. Ƙananan (10-8 M) da ƙananan (10-5 M) na DEET sun rage yawan aikin AChE na endothelial idan aka kwatanta da yanayin sarrafawa (Fig. 2).
Duk abubuwan da aka tattara na DEET (10-8 M da 10-5 M) sun rage ayyukan acetylcholinesterase akan HUVEC. An yi amfani da BW284c51 (10-5 M) azaman sarrafawa don masu hana acetylcholinesterase. An bayyana sakamakon a matsayin kashi na ayyukan AChE akan HUVEC da aka bi da su tare da adadin DEET guda biyu idan aka kwatanta da ƙwayoyin da aka yi wa abin hawa. Ana bayyana dabi'u azaman ma'anar ± SEM na gwaje-gwaje masu zaman kansu guda shida. *p <0.05 idan aka kwatanta da sarrafawa (Kruskal-Wallis da Dunn gwajin kwatancen mahara).
Nitric oxide (NO) yana da hannu a cikin tsarin angiogenic 33, sabili da haka, BABU samarwa a cikin HUVECs masu motsa jiki da aka yi nazarin. DEET da aka yi amfani da endothelial NO samarwa ya karu idan aka kwatanta da kwayoyin sarrafawa, amma ya kai mahimmanci kawai a kashi na 10-8 M (Fig. 3c). Don ƙayyade sauye-sauyen kwayoyin halitta masu sarrafa DEET-induced NO samarwa, eNOS magana da kunnawa an bincikar su ta hanyar lalatawar Yamma. Kodayake jiyya na DEET bai canza maganganun eNOS ba, yana ƙara ƙarar eNOS phosphorylation a wurin da yake kunnawa (Ser-1177) yayin da yake rage rukunin hanawa (Thr-495) idan aka kwatanta da ƙwayoyin da ba a kula da su ba a cikin eNOS phosphorylation (Fig. 3d). Bugu da ƙari kuma, an ƙididdige rabon eNOS na phosphorylated a wurin kunnawa da wurin hanawa bayan an daidaita adadin eNOS phosphorylated zuwa yawan adadin enzyme. Wannan rabo ya karu sosai a cikin HUVECs da aka bi da su tare da kowane maida hankali na DEET idan aka kwatanta da kwayoyin da ba a kula da su ba (Fig. 3d).
A ƙarshe, bayanin VEGF, ɗaya daga cikin manyan abubuwan proangiogenic, an bincika ta hanyar lalatawar Yamma. DEET yana ƙara haɓaka magana ta VEGF, yayin da pFHHSiD ya toshe wannan magana gaba ɗaya.
Tun da tasirin DEET yana da kula da toshewar magunguna da rage ka'idojin M3 masu karɓa, mun gwada hasashen cewa DEET na iya haɓaka siginar calcium. Abin mamaki, DEET ya kasa kara yawan calcium cytoplasmic a cikin HUVEC (bayanan da ba a nuna ba) da HEK / M3 (Fig. 4a, b) don duka abubuwan da aka yi amfani da su.
Lokacin aikawa: Dec-30-2024