Maganin anthelmintic N,N-diethyl-m-toluamide (DEETAn ruwaito cewa DEET yana hana AChE (acetylcholinesterase) kuma yana da yuwuwar haifar da cutar kansa saboda yawan jijiyoyin jini. A cikin wannan takarda, mun nuna cewa DEET musamman yana motsa ƙwayoyin endothelial waɗanda ke haɓaka angiogenesis, ta haka yana ƙara girman ƙari. DEET yana kunna hanyoyin ƙwayoyin halitta wanda ke haifar da angiogenesis, gami da yaduwa, ƙaura, da mannewa. Wannan yana da alaƙa da ƙaruwar samar da NO da bayyanar VEGF a cikin ƙwayoyin endothelial. Rufe M3 ko amfani da masu hana M3 na magunguna sun kawar da duk waɗannan tasirin, yana nuna cewa angiogenesis da DEET ke haifarwa yana da sauƙin amsawa ga M3. Gwaje-gwajen da suka haɗa da siginar calcium a cikin ƙwayoyin endothelial da HEK waɗanda ke nuna yawan masu karɓar M3, da kuma nazarin ɗaurewa da docking, sun nuna cewa DEET yana aiki azaman mai daidaita allosteric na masu karɓar M3. Bugu da ƙari, DEET yana hana AChE, ta haka yana ƙara yawan acetylcholine da ɗaure shi ga masu karɓar M3, da haɓaka tasirin proangiogenic ta hanyar daidaita allosteric.
An ware manyan ECs daga cikin aorta na beraye na Switzerland. An daidaita hanyar cirewa daga yarjejeniyar Kobayashi 26. An noma ECs na Murine a cikin matsakaicin EBM-2 tare da ƙarin kashi 5% na FBS wanda ba shi da aiki da zafi har zuwa wucewa ta huɗu.
An yi nazarin tasirin yawan DEET guda biyu kan yaduwar HUVEC, U87MG, ko BF16F10 ta amfani da Kit ɗin Gwaji na Ƙwayoyin Halitta na CyQUANT (Molecular Probes, C7026). A takaice, an shuka ƙwayoyin halitta 5.103 a kowace rijiya a cikin faranti mai rijiyoyi 96, an bar su su haɗu da dare ɗaya, sannan aka yi musu magani da DEET na tsawon awanni 24. Bayan cire hanyar girma, a ƙara maganin ɗaure rini a kowace rijiya ta microplate ɗin kuma a saka ƙwayoyin a zafin jiki na 37 °C na tsawon mintuna 30. An ƙayyade matakan haske ta amfani da na'urar karanta microplate mai yawan Mithras LB940 (Berthold Technologies, Bad Wildbad, Jamus) wacce aka sanye da matatun motsa jiki na 485 nm da matatun fitar da iska na 530 nm.
An shuka HUVEC a cikin faranti 96 masu rijiyoyi 96 a yawan ƙwayoyin halitta 104 a kowace rijiya. An yi wa ƙwayoyin magani da DEET na tsawon awanni 24. An tantance wanzuwar ƙwayoyin ta hanyar amfani da gwajin launi na MTT (Sigma-Aldrich, M5655). An sami ƙimar yawan gani akan na'urar karanta microplate mai yawa (Mithras LB940) a tsawon tsayin 570 nm.
An yi nazarin tasirin DEET ta amfani da gwaje-gwajen angiogenesis na in vitro. Yin magani da 10-8 M ko 10-5 M DEET ya ƙara samuwar tsawon capillary a cikin HUVECs (Hoto na 1a, b, sandunan fari). Idan aka kwatanta da ƙungiyar kulawa, magani da yawan DEET daga 10-14 zuwa 10-5 M ya nuna cewa tsawon capillary ya kai wani matsayi a 10-8 M DEET (Hoto na Ƙarin S2). Ba a sami wani bambanci mai mahimmanci ba a cikin tasirin in vitro na proangiogenic na HUVECs da aka yi wa magani da DEET a cikin kewayon yawan 10-8 M da 10-5 M.
Domin tantance tasirin DEET akan neovascularization, mun yi nazarin neovascularization in vivo. Bayan kwana 14, beraye da aka yi wa allurar ƙwayoyin endothelial waɗanda aka riga aka noma da 10-8 M ko 10-5 M DEET sun nuna ƙaruwa mai yawa a cikin adadin haemoglobin (Hoto na 1c, sandunan fari).
Bugu da ƙari, an yi nazarin neovascularization da DEET ta haifar a cikin beraye masu ɗauke da xenograft na U87MG waɗanda ake yi wa allurar DEET kowace rana (ip) a cikin allurar da aka sani tana haifar da yawan ƙwayoyin jini na 10-5 M, wanda al'ada ce a cikin mutane da aka fallasa. a cikin 23. An lura da ciwace-ciwacen da za a iya ganowa (watau ciwace-ciwacen da suka fi 100 mm3) kwanaki 14 bayan allurar ƙwayoyin U87MG a cikin beraye. A rana ta 28, haɓakar ciwace-ciwacen ya inganta sosai a cikin beraye da aka yi wa magani da DEET idan aka kwatanta da beraye masu sarrafawa (Hoto na 1d, murabba'ai). Bugu da ƙari, tabon CD31 na ciwace-ciwacen ya nuna cewa DEET ya ƙara yawan capillary amma ba yawan ƙwayoyin microvessel ba. (Hoto na 1e–g).
Domin tantance rawar da masu karɓar muscarinic ke takawa wajen yaɗuwar DETA, an yi amfani da DETA 10-8 M ko 10-5 M a gaban pFHHSiD (10-7 M, mai hana karɓar M3 zaɓi) wajen magance cutar. Maganin HUVEC. pFHHSiD ya toshe kaddarorin yaɗuwar DETA gaba ɗaya a duk yawan da aka samu (Tebur 1).
A ƙarƙashin waɗannan yanayi, mun kuma bincika ko DEET zai ƙara tsawon capillary a cikin ƙwayoyin HUVEC. Hakazalika, pFHHSiD ya hana tsayin capillary da DEET ya haifar sosai (Hoto na 1a, b, sandunan launin toka). Bugu da ƙari, an yi irin waɗannan gwaje-gwaje da M3 siRNA. Duk da cewa siRNA mai sarrafawa bai yi tasiri ba wajen haɓaka samuwar capillary, rufewar mai karɓar muscarinic na M3 ya kawar da ikon DEET na ƙara tsawon capillary (Hoto na 1a, b, sandunan baƙi).
Bugu da ƙari, duka jijiyoyin jini da DEET ta haifar da 10-8 M ko 10-5 M a cikin vitro da kuma neovascularization a cikin vivo an toshe su gaba ɗaya ta hanyar pFHHSiD (Hoto na 1c, d, da'irori). Waɗannan sakamakon sun nuna cewa DEET yana haɓaka angiogenesis ta hanyar hanyar da ke da alaƙa da zaɓin masu karɓar M3 ko M3 siRNA.
AChE shine burin kwayoyin halitta na DEET. Magunguna kamar donepezil, waɗanda ke aiki a matsayin masu hana AChE, na iya ƙarfafa angiogenesis na EC a cikin vitro da kuma a cikin samfuran ischemia na baya na linzamin kwamfuta14. Mun gwada tasirin yawan DEET guda biyu akan aikin enzyme na AChE a cikin HUVEC. Ƙarancin (10-8 M) da babban (10-5 M) na DEET ya rage aikin endothelial AChE idan aka kwatanta da yanayin sarrafawa (Hoto na 2).
Duk yawan DEET (10-8 M da 10-5 M) sun rage aikin acetylcholinesterase akan HUVEC. An yi amfani da BW284c51 (10-5 M) azaman iko ga masu hana acetylcholinesterase. An bayyana sakamakon a matsayin kashi na ayyukan AChE akan HUVEC da aka yi wa magani da yawan DEET guda biyu idan aka kwatanta da ƙwayoyin da aka yi wa magani da abin hawa. Ana bayyana ƙima a matsayin matsakaicin ± SEM na gwaje-gwaje shida masu zaman kansu. *p < 0.05 idan aka kwatanta da sarrafawa (gwajin kwatantawa da yawa na Kruskal-Wallis da Dunn).
Nitric oxide (NO) yana da hannu a cikin tsarin angiogenic 33, saboda haka, an yi nazarin samar da NO a cikin HUVECs da DEET ta motsa. An ƙara yawan samar da endothelial NO da DEET ta yi wa magani idan aka kwatanta da ƙwayoyin sarrafawa, amma ya kai ga mahimmanci kawai a kashi na 10-8 M (Hoto na 3c). Don tantance canje-canjen kwayoyin halitta da ke sarrafa samar da NO da DEET ta haifar, an yi nazarin bayyanar eNOS da kunnawa ta hanyar Western blotting. Kodayake maganin DEET bai canza bayyanar eNOS ba, ya ƙara yawan phosphorylation na eNOS sosai a wurin kunnawa (Ser-1177) yayin da yake rage wurin hana shi (Thr-495) idan aka kwatanta da ƙwayoyin da ba a yi wa magani ba a cikin eNOS phosphorylation (Hoto na 3d). Bugu da ƙari, an ƙididdige rabon eNOS da phosphorylated a wurin kunnawa da wurin hana bayan daidaita adadin eNOS da phosphorylated zuwa jimlar adadin enzyme. An ƙara wannan rabo sosai a cikin HUVECs da aka yi wa magani tare da kowane yawan DEET idan aka kwatanta da ƙwayoyin da ba a yi wa magani ba (Hoto na 3d).
A ƙarshe, an yi nazarin bayyanar VEGF, ɗaya daga cikin manyan abubuwan da ke haifar da cutar kansa, ta hanyar amfani da Western blotting. DEET ta ƙara yawan bayyanar VEGF sosai, yayin da pFHHSiD ta toshe wannan yanayin gaba ɗaya.
Tunda tasirin DEET yana da alaƙa da toshewar magunguna da raguwar masu karɓar M3, mun gwada hasashen cewa DEET na iya haɓaka siginar calcium. Abin mamaki, DEET ta kasa ƙara yawan sinadarin calcium a cikin HUVEC (bayanan da ba a nuna ba) da HEK/M3 (Hoto na 4a, b) ga duka yawan da aka yi amfani da su.
Lokacin Saƙo: Disamba-30-2024