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ìÑä 577.125.085:547.913-314 ГИПОЛИПИДЕМИЧЕСКОЕ ДЕЙСТВИЕ СЕСКВИТЕРПЕНОВОГО γ-ЛАКТОНА АХИЛЛИНА НА КЛЕТОЧНОЙ КУЛЬТУРЕ КРЫСИНОЙ ГЕПАТОМЫ Иванов В.В.1, Ратькин А.В.1, Пфаргер Ю.А.1, Кайдаш О.А.1, Рязанцева Н.В.1, Адекенов С.М.2, Чучалин В.С.1 1 2 Ñèáèðñêèé ãîñóäàðñòâåííûé ìåäèöèíñêèé óíèâåðñèòåò, Òîìñê ÀÎ «Ìåæäóíàðîäíûé íàó÷íî-ïðîèçâîäñòâåííûé õîëäèíã «Ôèòîõèìèÿ», ã. Êàðàãàíäà, Ðåñïóáëèêà Êàçàõñòàí РЕЗЮМЕ Öåëü èññëåäîâàíèÿ – îöåíèòü in vitro ôàðìàêîëîãè÷åñêèå ýôôåêòû ñåñêâèòåðïåíîâîãî γ-ëàêòîíà àõèëëèíà â êà÷åñòâå ïîòåíöèàëüíîãî ãèïîëèïèäåìè÷åñêîãî ñðåäñòâà. å‡ÚÂË‡Î Ë ÏÂÚÓ‰˚. Èçó÷åíî âëèÿíèå ñåñêâèòåðïåíîâîãî γ-ëàêòîíà àõèëëèíà è ãåìôèáðîçèëà (ïðåïàðàò ñðàâíåíèÿ) íà ñîäåðæàíèå ëèïèäîâ â êëåòî÷íîé êóëüòóðå êðûñèíîé ãåïàòîìû (HTC) ôëóîðåñöåíòíûì ìåòîäîì ñ âèòàëüíûì êðàñèòåëåì Nile Red è îêðàøèâàíèåì êëåòîê êðàñèòåëåì Oil Red O ïðè èõ èíêóáàöèè ñ æèðîâîé ýìóëüñèåé – ëèïîôóíäèíîì. Æèçíåñïîñîáíîñòü êëåòîê îöåíèâàëè ñ ïîìîùüþ ÌÒÒ-òåñòà è îêðàøèâàíèåì ñ òðèïàíîâûì ñèíèì. êÂÁÛθڇÚ˚. Êóëüòèâèðîâàíèå êëåòî÷íîé êóëüòóðû HTC ñ àõèëëèíîì è ãåìôèáðîçèëîì â êîíöåíòðàöèÿõ îò 0,5 äî 1,5 è îò 0,25 äî 0,5 ììîëü ñîîòâåòñòâåííî ïðèâîäèëî ê äîçîçàâèñèìîìó óìåíüøåíèþ èíòåíñèâíîñòè ôëóîðåñöåíöèè Nile Red, ÷òî ñâÿçàíî ñî ñíèæåíèåì ñîäåðæàíèÿ ëèïèäîâ â êëåòêàõ.  ýòèõ êîíöåíòðàöèÿõ ïðåïàðàòû íå îêàçûâàëè öèòîòîêñè÷åñêîãî äåéñòâèÿ, è æèçíåñïîñîáíîñòü êëåòîê ÍÒÑ íå èçìåíÿëàñü ïî ñðàâíåíèþ ñ ñîîòâåòñòâóþùèì ïîêàçàòåëåì êîíòðîëüíîé êóëüòóðû. Ýêñïåðèìåíòàëüíóþ ãèïåðëèïèäåìèþ â êóëüòóðå ãåïàòîìû èíäóöèðîâàëè äîáàâëåíèåì â èíêóáàöèîííóþ ñðåäó æèðîâîé ýìóëüñèè ëèïîôóíäèíà â êîíå÷íîé êîíöåíòðàöèè 0,05%. Èíòåíñèâíîñòü ôëóîðåñöåíöèè Nile Red â êëåòêàõ âîçðàñòàëà â 4 ðàçà (p < 0,05), ÷òî ñâèäåòåëüñòâóåò î ñóùåñòâåííîì íàêîïëåíèè ëèïèäîâ â öèòîçîëå êëåòîê è ïîäòâåðæäàåòñÿ äàííûìè ìèêðîñêîïèè ïîñëå îêðàøèâàíèÿ íåéòðàëüíûõ ëèïèäîâ â êëåòêàõ êðàñèòåëåì Oil Red O. ýòèõ óñëîâèÿõ àõèëëèí è ãåìôèáðîçèë â êîíöåíòðàöèÿõ 0,5 è 0,25 ììîëü ñîîòâåòñòâåííî ñíèæàëè ñîäåðæàíèå ëèïèäîâ â êëåòêàõ. á‡Íβ˜ÂÌËÂ. Íà ìîäåëè ãèïåðëèïèäåìèè, èíäóöèðîâàííîé ëèïîôóíäèíîì, äîáàâëåíèå ñåñêâèòåðïåíîâîãî γ-ëàêòîíà àõèëëèíà â èíêóáàöèîííóþ ñðåäó ïðåïÿòñòâóåò íàêîïëåíèþ ëèïèäîâ â êëåòêàõ ÍÒÑ, î ÷åì ñâèäåòåëüñòâóåò óìåíüøåíèå ôëóîðåñöåíöèè Nile Red è ñíèæåíèå ñîäåðæàíèÿ îêðàøåííûõ Oil Red O ëèïèäíûõ êàïåëü â öèòîçîëå. Äëÿ óñòàíîâëåíèÿ ìîëåêóëÿðíûõ ìèøåíåé äåéñòâèÿ àõèëëèíà íà ìåòàáîëèçì ëèïèäîâ â êóëüòóðå ÍÒÑ â äàëüíåéøåì íåîáõîäèìî èññëåäîâàíèå ýêñïðåññèè ãåíîâ êëþ÷åâûõ ôåðìåíòîâ ëèïèäíîãî îáìåíà. КЛЮЧЕВЫЕ СЛОВА: ñåñêâèòåðïåíîâûé γ-ëàêòîí àõèëëèí, ãåìôèáðîçèë, êðûñèíàÿ ãåïàòîìà (HTC), ãèïîëèïèäåìè÷åñêîå äåéñòâèå. Введение Êîðîíàðíûå çàáîëåâàíèÿ ñåðäöà, îáóñëîâëåííûå àòåðîñêëåðîòè÷åñêèì ïîðàæåíèåì êîðîíàðíûõ àðòåðèé, ëèäèðóþò ïî ñìåðòíîñòè êàê â Ðîññèè, òàê è â áîëüøèíñòâå ñòðàí ìèðà [1, 2].  îñíîâå èõ ðàçâèòèÿ è Ðàòüêèí Àëåêñàíäð Âàëåíòèíîâè÷, òåë.: 8-903-915-3991, 8 (3822) 42-09-50; e-mail: [email protected] 28 ïðîãðåññèðîâàíèÿ ëåæèò íå òîëüêî ëîêàëüíîå ñóæåíèå ïðîñâåòà àðòåðèé àòåðîñêëåðîòè÷åñêèìè áëÿøêàìè, íî è àòåðîãåííàÿ äèñëèïèäåìèÿ [2–4]. Íàèáîëåå ÷àñòûì ïðîÿâëåíèåì àòåðîãåííîé äèñëèïèäåìèè ÿâëÿåòñÿ ñî÷åòàíèå ãèïåðòðèàöèëãëèöåðèäåìèè, íèçêîãî óðîâíÿ õîëåñòåðîëà â ëèïîïðîòåèíàõ âûñîêîé ïëîòíîñòè (ËÏÂÏ) è ïîâûøåííîãî ñîäåðæàíèÿ àòåðîãåííûõ ëèïîïðîòåèíîâ íèçêîé ïëîòíîñòè (ËÏÍÏ) [2]. Бюллетень сибирской медицины, 2014, том 13, № 5, с. 28–35 Оригинальные статьи Íåñìîòðÿ íà áîëüøîå ðàçíîîáðàçèå ñèíòåòè÷åñêèõ ãèïîëèïèäåìè÷åñêèõ ïðåïàðàòîâ íà ôàðìàöåâòè÷åñêîì ðûíêå, ïðîáëåìà òåðàïèè àòåðîñêëåðîçà è åãî ïðîÿâëåíèé åùå ïîëíîñòüþ íå ðåøåíà [4]. Ïåðñïåêòèâíûì èñòî÷íèêîì ëåêàðñòâåííûõ ñðåäñòâ ýòîé ãðóïïû ÿâëÿþòñÿ âåùåñòâà ðàñòèòåëüíîãî ïðîèñõîæäåíèÿ, õàðàêòåðèçóþùèåñÿ äîñòàòî÷íî âûñîêèì ôàðìàêîòåðàïåâòè÷åñêèì ïîòåíöèàëîì, íèçêèì óðîâíåì ïîáî÷íûõ ýôôåêòîâ è õîðîøåé ïåðåíîñèìîñòüþ [5]. Ñðåäè âåùåñòâ, ïðîäóöèðóåìûõ ðàñòåíèÿìè, àêòèâíî èçó÷àþòñÿ ñåñêâèòåðïåíîâûå ëàêòîíû, äëÿ êîòîðûõ ïîêàçàíû ïðîòèâîîïóõîëåâàÿ, àíòèèíâàçèâíàÿ, ïðîòèâîâîñïàëèòåëüíàÿ, àíòèìàëÿðèéíàÿ, ïðîòèâîâèðóñíàÿ, ïðîòèâîáàêòåðèàëüíàÿ, ïðîòèâîãðèáêîâàÿ àêòèâíîñòè [6–8]. Ñåñêâèòåðïåíîâûå γ-ëàêòîíû ïðåäñòàâëÿþò ñîáîé êëàññ õèìè÷åñêèõ ñîåäèíåíèé òåðïåíîèäíîé ñòðóêòóðû [8, 9] è ïîäîáíî ãèïîëèïèäåìè÷åñêèì ïðåïàðàòàì ãðóïïû ñòàòèíîâ ñîäåðæàò ëàêòîííîå êîëüöî â ñâîåé ñòðóêòóðå [9].  ÷àñòíîñòè, ãèïîëèïèäåìè÷åñêîå äåéñòâèå ïîêàçàíî äëÿ ñåñêâèòåðïåíîâîãî γ-ëàêòîíà ãâàéàíîâîãî ðÿäà ëåóêîìèçèíà, âûäåëåííîãî èç òûñÿ÷åëèñòíèêà ìåëêîöâåòêîâîãî Achillea micrantha Willd è ïîëûíè áåëîâàòîé Artemisia leucodes Schrenk [1, 9, 10]. Öåëü èññëåäîâàíèÿ – îöåíêà in vitro ôàðìàêîëîãè÷åñêèå ýôôåêòû ñåñêâèòåðïåíîâîãî γ-ëàêòîíà àõèëëèíà (1) â êà÷åñòâå ïîòåíöèàëüíîãî ãèïîëèïèäåìè÷åñêîãî ñðåäñòâà. O (1) H O O  íàñòîÿùåå âðåìÿ äëÿ ñêðèíèíãîâûõ èññëåäîâàíèé øèðîêî èñïîëüçóþòñÿ êóëüòóðû êëåòîê, ïîñêîëüêó îíè ñàìîâîñïðîèçâîäÿòñÿ, òðåáóþò ìåíüøèõ ìàòåðèàëüíûõ çàòðàò è ðàáîòà ñ íèìè íå îáðåìåíåíà ýòè÷åñêèìè îãðàíè÷åíèÿìè [11].  ñâÿçè ñ ýòèì â êà÷åñòâå áèîëîãè÷åñêîé ìîäåëè äëÿ èçó÷åíèÿ ãèïîëèïèäåìè÷åñêîãî äåéñòâèÿ ñåñêâèòåðïåíîâîãî γ-ëàêòîíà àõèëëèíà èñïîëüçîâàëè êëåòî÷íóþ êóëüòóðó ÍÒÑ. Материал и методы Àõèëëèí (1) – ñåñêâèòåðïåíîâûé ëàêòîí ãâàéàíîâîãî òèïà, âûäåëåííûé â ÀÎ «Ìåæäóíàðîäíûé íàó÷íî-ïðîèçâîäñòâåííûé õîëäèíã «Ôèòîõèìèÿ» èç Achillea micrantha Willd è Artemisia leucodes Schrenk [10]. Ñóáñòàíöèÿ çàðåãèñòðèðîâàíà íà òåððèòîðèè Ðåñïóáëèêè Êàçàõñòàí, èññëåäóåìûé îáðàçåö ñîîòâåòñòâóåò òðåáîâàíèÿì ÔÑ ÐÊ 42-1909-08, êîëè÷åñòâåííîå ñîäåðæàíèå – 99,7%.  êà÷åñòâå ïðåïàðàòà ñðàâíåíèÿ èñïîëüçîâàëè ãåìôèáðîçèë (Sigma-Aldrich, ÑØÀ). Àõèëëèí è ãåìôèáðîçèë ðàñòâîðÿëè â äèìåòèëñóëüôîêñèäå (ÄÌÑÎ) (ÏàíÝêî, Ðîññèÿ) è èñïîëüçîâàëè â êîíå÷íûõ êîíöåíòðàöèÿõ îò 0,25 äî 5,0 ììîëü.  ýêñïåðèìåíòå èñïîëüçîâàëè ïåðåâèâàåìóþ êëåòî÷íóþ êóëüòóðó êðûñèíîé ãåïàòîìû (HTC), ïîëó÷åííóþ èç Èíñòèòóòà öèòîëîãèè ÐÀÍ (ã. ÑàíêòÏåòåðáóðã). Êëåòî÷íóþ êóëüòóðó HTC êóëüòèâèðîâàëè â 6-ëóíî÷íûõ ïëàíøåòàõ (SPL life science, Êîðåÿ) äî 70–80% êîíôëþýíòíîãî ìîíîñëîÿ (0,5 ⋅ 106 êëåòîê â 1 ìë ñðåäû â òå÷åíèå äâóõ ñóòîê) â ñðåäå DMEM ñ Lãëóòàìèíîì («Áèîëîò», Ðîññèÿ) è äîáàâëåíèåì 10% ýìáðèîíàëüíîé òåëÿ÷üåé ñûâîðîòêè («PAA Laboratories», Àâñòðèÿ), 50 ìêã/ìë ãåíòàìèöèíà («ÏàíÝêî», Ðîññèÿ) (ïîëíàÿ ñðåäà DMEM) ïðè 37ºÑ â ÑÎ2èíêóáàòîðå ÌÑÎ-5ÀÑ («SANYO», ßïîíèÿ) â àòìîñôåðå 95% âîçäóõà è 5% ÑÎ2. ×åðåç 48 ÷ â èíêóáàöèîííóþ ñðåäó äîáàâëÿëè àõèëëèí è ãåìôèáðîçèë â ðàçëè÷íûõ êîíöåíòðàöèÿõ (â êîíòðîëüíóþ ïðîáó äîáàâëÿëè ñîîòâåòñòâóþùåå êîëè÷åñòâî ÄÌÑÎ) è êëåòêè êóëüòèâèðîâàëè 48 ÷. Ïîñëå äîñòèæåíèÿ êëåòêàìè 70–80% êîíôëþýíòíîãî ìîíîñëîÿ (÷åðåç 48 ÷) äëÿ ìîäåëèðîâàíèÿ ãèïåðëèïèäåìèè êëåòêè èíêóáèðîâàëè â ïðèñóòñòâèè æèðîâîé ýìóëüñèè – ëèïîôóíäèíà ÌÑÒ/ËÑÒ â êîíå÷íîé êîíöåíòðàöèè 0,05% (Á. Áðàóí Ìåäèêàë, Ðîññèÿ) – êàê îïèñàëè E. Ilan è ñîàâò. [12]. Ëèïîôóíäèí äîáàâëÿëè ïåðåä âíåñåíèåì ïðåïàðàòîâ, â êîíòðîëüíóþ ïðîáó âíîñèëè ñîîòâåòñòâóþùåå êîëè÷åñòâî ÄÌÑÎ. Æèçíåñïîñîáíîñòü êëåòî÷íîé ëèíèè HTC îöåíèâàëè ÷åðåç 48 ÷ ïîñëå âíåñåíèÿ ïðåïàðàòîâ â êóëüòóðàëüíóþ ñðåäó äâóìÿ ìåòîäàìè: ñ èñïîëüçîâàíèåì ÌÒÒ-òåñòà (ñòàíäàðòíûé êîëîðèìåòðè÷åñêèé ìåòîä) ñ ïðèìåíåíèåì â êà÷åñòâå ðåàãåíòà ñîëè òåòðàçîëèÿ – 3[4,5-äèìåòèëòèàçîëèë-2-åë]-2,5-äèôåíèëòåòðàçîëèóì áðîìèäà (ÌÒÒ-ðåàãåíò) (ÏàíÝêî, Ðîññèÿ) è ñ 0,1% òðèïàíîâûì ñèíèì (ÄÈÀÝÌ, Ãåðìàíèÿ). Ïîñëå èíêóáàöèè ñ ïðåïàðàòàìè êëåòêè ñíèìàëè ñ ïëàíøåòà ðàñòâîðîì òðèïñèíà (0,25%) è ÝÄÒÀ (0,02%) (ÏàíÝêî, Ðîññèÿ) è îòìûâàëè îäèí ðàç â 1õPBS (pH 7,4) (Ambion, ÑØÀ). Êîëè÷åñòâî âîññòàíîâëåííîãî ïðîäóêòà – ôîðìàçàíà – èçìåðÿëè íà ñïåêòðîôîòîìåòðå ÑÔ-2000 (ÎÊÁ-Ñïåêòð, Ðîññèÿ) ïðè äëèíå âîëíû 570 íì. Æèçíåñïîñîáíîñòü â êîíòðîëüíîé êóëüòóðå êëåòîê ïðèíèìàëè çà 100%. Êîëè÷åñòâî êëåòîê, îêðàøåííûõ òðèïàíîâûì ñèíèì, ïîäñ÷èòûâàëè â êàìåðå Ãîðÿåâà ïîä ñâåòîâûì ìèêðîñêîïîì ÌÈÊÌÅÄ-1 (Ðîññèÿ) ñ óâåëè÷åíèåì (îê. 7 õ îá. 9). Îïðåäåëÿëè ïðîöåíò íåîêðàøåííûõ (æèâûõ) êëåòîê ñðåäè 100% ïîñ÷èòàííûõ. Êàæäûé îïûò ïîâòîðÿëè 6–7 ðàç [1, 13, 14]. Бюллетень сибирской медицины, 2014, том 13, № 5, с. 28–35 29 Иванов В.В., Ратькин А.В., Пфаргер Ю.А. и др. Гиполипидемическое действие сесквитерпенового γ-лактона ахиллина… Ñîäåðæàíèå ëèïèäîâ â êëåòî÷íîé êóëüòóðå HTC îïðåäåëÿëè ôëóîðåñöåíòíûì ìåòîäîì ñ âèòàëüíûì ëèïîôèëüíûì êðàñèòåëåì Nile Red, êîòîðûé îêðàøèâàåò êàïëè ëèïèäîâ â öèòîçîëå ñîãëàñíî ïðîòîêîëó [14]. Äëÿ ýòîãî êëåòêè ñíèìàëè ñ ïëàñòèêà ðàñòâîðîì òðèïñèí-ÝÄÒÀ ÷åðåç 48 ÷ ïîñëå âíåñåíèÿ ïðåïàðàòîâ â êóëüòóðàëüíóþ ñðåäó è îòìûâàëè îäèí ðàç â 1õPBS (pH 7,4). Íàäîñàäî÷íóþ æèäêîñòü àêêóðàòíî àñïèðèðîâàëè è êëåòêè èíêóáèðîâàëè 30 ìèí ñ Nile Red (Sigma-Aldrich, ÑØÀ) â êîíå÷íîé êîíöåíòðàöèè 3 ìêìîëü. Çàòåì êëåòêè îòìûâàëè â 1õPBS (pH 7,4) è äåòåêòèðîâàëè ôëóîðåñöåíöèþ Nile Red íà ìèêðîïëàíøåòíîì ðèäåðå Infinite 200 PRO (Tecan, Øâåéöàðèÿ) ïðè äëèíå âîëíû âîçáóæäåíèÿ 580 íì è ýìèññèè 630 íì. Êàæäûé îïûò ïîâòîðÿëè 6 ðàç [1, 12, 14]. Îêðàøèâàíèå íåéòðàëüíûõ ëèïèäîâ ñ êðàñèòåëåì Oil Red O [15] òàêæå ïðîâîäèëè ÷åðåç 48 ÷ ïîñëå âíåñåíèÿ ïðåïàðàòîâ â êóëüòóðàëüíóþ ñðåäó. Êëåòêè ôèêñèðîâàëè â 10%-ì çàáóôåðåííîì ðàñòâîðå ôîðìàëèíà (Biovitrum, Ðîññèÿ) íà 10 ìèí. Çàòåì êëåòêè îòìûâàëè â 1õPBS (pH 7,4) â òå÷åíèå 1 ìèí è 60%-ì ðàñòâîðå èçîïðîïèëîâîãî ñïèðòà â òå÷åíèå 15 ñ. Êëåòêè îêðàøèâàëè Oil Red O (Sigma-Aldrich, ÑØÀ) ïðè 37 °Ñ â òå÷åíèå 1 ìèí â òåìíîòå. Íå ñâÿçàâøèéñÿ ñ êëåòêàìè êðàñèòåëü îòìûâàëè â 60%-ì èçîïðîïèëîâîì ñïèðòå â òå÷åíèå 15 ñ, 3 ðàçà ïî 3 ìèí â 1õPBS (pH 7,4) è ôîòîãðàôèðîâàëè íà ìèêðîñêîïå (Áèîìåä, Ðîññèÿ) (îá. 25 õ îê. 10), îñíàùåííîì öèôðîâîé êàìåðîé DCM 510 [15]. Ðåçóëüòàòû èññëåäîâàíèÿ îáðàáàòûâàëè ñ èñïîëüçîâàíèåì ïðîãðàììû Microsoft Exñel (2007), ñòàíäàðòíîãî ïàêåòà ïðîãðàìì SPSS 17.0 for Windows. Ðåçóëüòàòû ïðåäñòàâëåíû â âèäå âûáîðî÷íîãî ñðåäíåãî M è îøèáêè ñðåäíåãî m. Ðàâåíñòâî âûáîðî÷íûõ ñðåäíèõ ïðîâåðÿëè ñ ïðèìåíåíèåì t-êðèòåðèÿ Ñòüþäåíòà. Ñòàòèñòè÷åñêè çíà÷èìûìè ñ÷èòàëè ðàçëè÷èÿ ïðè óðîâíå çíà÷èìîñòè p < 0,05. âàíèè ìåòîäà îêðàñêè ñ òðèïàíîâûì ñèíèì íå ìåíåå (97,1 ± 1,5)%, à ÌÒÒ-òåñòà – íå ìåíåå (81,8 ± 2,2)% (ðèñ. 1, 2). Ïðè ïîâûøåíèè êîíöåíòðàöèè àõèëëèíà â êóëüòóðàëüíîé ñðåäå äî 5,0 ììîëü æèçíåñïîñîáíîñòü êëåòîê ñíèæàëàñü ïî ñðàâíåíèþ ñ êîíòðîëåì è ñîñòàâëÿëà (17,3 ± 2,6)% (p < 0,05) (ðèñ. 1). Ïðåïàðàò ñðàâíåíèÿ ãåìôèáðîçèë â êîíöåíòðàöèè 1,0 ììîëü öèòîòîêñè÷åñêè äåéñòâîâàë íà êëåòêè, ïðè ýòîì êîíöåíòðàöèÿ æèâûõ êëåòîê ñîñòàâëÿëà (16,0 ± 2,5)% (p < 0,05) (ðèñ. 2). Ðèñ. 1. Âëèÿíèå àõèëëèíà íà æèçíåñïîñîáíîñòü êëåòîê ñ ïðèìåíåíèåì ÌÒÒ-òåñòà è íà ôëóîðåñöåíöèþ Nile Red â êëåòî÷íîé êóëüòóðå êðûñèíîé ãåïàòîìû (ÍÒÑ), M ± m, n = 6. Æèçíåñïîñîáíîñòü êëåòîê è ôëóîðåñöåíöèþ Nile Red â êîíòðîëå ïðèíèìàëè çà 100%. * – ñòàòèñòè÷åñêè çíà÷èìûå ðàçëè÷èÿ ïî ñðàâíåíèþ ñ êîíòðîëåì ïðè p < 0,05 Результаты и обсуждение Èçâåñòíî, ÷òî ñåñêâèòåðïåíîâûå ëàêòîíû â çàâèñèìîñòè îò èõ ñòðóêòóðû îáëàäàþò öèòîòîêñè÷åñêèì äåéñòâèåì è èçó÷àþòñÿ êàê ïðîòèâîîïóõîëåâûå ïðåïàðàòû [8]. Ïîýòîìó äëÿ èññëåäîâàíèÿ in vitro ôàðìàêîëîãè÷åñêîé àêòèâíîñòè ïðåäâàðèòåëüíî ïðîâîäèëè îöåíêó æèçíåñïîñîáíîñòè êëåòîê ïðè èíêóáàöèè èõ â ñðåäå ñ ðàçëè÷íûìè êîíöåíòðàöèÿìè àõèëëèíà è ïðåïàðàòà ñðàâíåíèÿ.  ðåçóëüòàòå íàøèõ ýêñïåðèìåíòîâ óñòàíîâëåíî, ÷òî æèçíåñïîñîáíîñòü êëåòîê ÍÒÑ ïðè êóëüòèâèðîâàíèè ñ àõèëëèíîì â êîíöåíòðàöèè îò 0,5 äî 1,5 ììîëü è ãåìôèáðîçèëîì â êîíöåíòðàöèè îò 0,25 äî 0,5 ììîëü íå èçìåíÿëàñü ïî ñðàâíåíèþ ñ êîíòðîëüíîé êóëüòóðîé (p > 0,05), è ñîñòàâëÿëà ïðè èñïîëüçî30 Ðèñ. 2. Âëèÿíèå ãåìôèáðîçèëà íà æèçíåñïîñîáíîñòü êëåòîê ñ ïðèìåíåíèåì ÌÒÒ-òåñòà è íà ôëóîðåñöåíöèþ Nile Red â êëåòî÷íîé êóëüòóðå êðûñèíîé ãåïàòîìû (ÍÒÑ), M ± m, n = 6. Æèçíåñïîñîáíîñòü êëåòîê è ôëóîðåñöåíöèþ Nile Red â êîíòðîëå ïðèíèìàëè çà 100%. * – ñòàòèñòè÷åñêè çíà÷èìûå ðàçëè÷èÿ ïî ñðàâíåíèþ ñ êîíòðîëåì ïðè p < 0,05 Бюллетень сибирской медицины, 2014, том 13, № 5, с. 28–35 Оригинальные статьи Äîáàâëåíèå àõèëëèíà ê êóëüòóðå ÍÒÑ ïðèâîäèëî ê äîçîçàâèñèìîìó ñíèæåíèþ èíòåíñèâíîñòè ôëóîðåñöåíöèè Nile Red ñ (75,3 ± 1,3) äî (35,6 ± 2,8)% ïðè êîíöåíòðàöèè îò 0,5 äî 5,0 ììîëü (ðèñ. 1), ãåìôèáðîçèëà – ñ (99,2 ± 1,9) äî (57,9 ± 4,9)% ïðè êîíöåíòðàöèè îò 0,25 äî 1,0 ììîëü (p < 0,05) (ðèñ. 2). Ñòàòèñòè÷åñêè çíà÷èìîå óìåíüøåíèå èíòåíñèâíîñòè ôëóîðåñöåíöèè Nile Red â êëåòêàõ ÍÒÑ ðåãèñòðèðîâàëîñü ïðè êîíöåíòðàöèè àõèëëèíà 1,0 ììîëü è ãåìôèáðîçèëà – 0,5 ììîëü, ÷òî îòðàæàåò ñíèæåíèå ñîäåðæàíèÿ ëèïèäîâ â êëåòî÷íîé êóëüòóðå. Ïîëó÷åííûå ðåçóëüòàòû ïîäòâåðæäàþòñÿ äàííûìè ìèêðîñêîïèè êëåòîê ïîñëå îêðàøèâàíèÿ íåéòðàëüíûõ ëèïèäîâ â êëåòêàõ HTC êðàñèòåëåì Oil Red O. Ïîä äåéñòâèåì 1,0 ììîëü àõèëëèíà è 0,5 ììîëü ãåìôèáðîçèëà â êëåòêàõ ñíèæàëîñü ñîäåðæàíèå ëèïèäíûõ êàïåëü, îêðàøåííûõ Oil Red O, ïî ñðàâíåíèþ ñ êîíòðîëåì (ðèñ. 3, à, â, ã). Ñ ó÷åòîì ïîëó÷åííûõ ðåçóëüòàòîâ äëÿ èññëåäîâàíèÿ ãèïîëèïèäåìè÷åñêîãî äåéñòâèÿ íà ìîäåëè ãèïåðëèïèäåìèè in vitro àõèëëèí è ãåìôèáðîçèë èñïîëüçîâàíû â êîíöåíòðàöèÿõ îò 0,5 äî 1,5 ììîëü è îò 0,25 äî 0,5 ììîëü ñîîòâåòñòâåííî. Ïðè ýòèõ êîíöåíòðàöèÿõ æèçíåñïîñîáíîñòü êëåòîê çíà÷èìî íå èçìåíÿëàñü ïî ñðàâíåíèþ ñ êîíòðîëåì, è îòìå÷àëîñü ñíèæåíèå óðîâíÿ ëèïèäîâ â êëåòêàõ. Èññëåäîâàíèÿìè E. Ilan è ñîàâò. ïîêàçàíî, ÷òî êóëüòèâèðîâàíèå ïåðâè÷íûõ êðûñèíûõ ãåïàòîöèòîâ ñ ëèïîôóíäèíîì â òå÷åíèå 48 ÷ ïðèâîäèò ê íàêîïëåíèþ òðèàöèëãëèöåðîëîâ (ÒÀÃ) è æèðíûõ êèñëîò â êëåòêàõ [12]. Ïîýòîìó äëÿ ìîäåëèðîâàíèÿ ýêñïåðèìåíòàëüíîé ãèïåðëèïèäåìèè ìû äîáàâëÿëè â êóëüòóðàëüíóþ ñðåäó æèðîâóþ ýìóëüñèþ – ëèïîôóíäèí â êîíå÷íîé êîíöåíòðàöèè 0,05%. Ïðè èíêóáàöèè êëåòî÷íîé êóëüòóðû ÍÒÑ â ïðèñóòñòâèè ëèïîôóíäèíà â òå÷åíèå 48 ÷ èíòåíñèâíîñòü ôëóîðåñöåíöèè Nile Red â êëåòêàõ ïîâûøàëàñü â 4 ðàçà è ñîñòàâèëà (48 534,7 ± 1 924) åä. ôëóîðåñöåíöèè (â êîíòðîëå – (10 928,4 ± 967,3) åä. ôëóîðåñöåíöèè) (p < 0,05). Íàêîïëåíèå ëèïèäîâ â êóëüòóðå ÍÒÑ òàêæå ïîäòâåðäèëîñü äàííûìè ìèêðîñêîïèè êëåòîê ïîñëå îêðàøèâàíèÿ íåéòðàëüíûõ ëèïèäîâ êðàñèòåëåì Oil Red O (ðèñ. 3). Íà ðèñ. 3, á (èíêóáàöèÿ êëåòîê ñ ëèïîôóíäèíîì 0,05%) îòìå÷àëîñü áîëüøå ãðàíóë ëèïèäîâ, îêðàøåííûõ Oil Red O, ÷åì â êîíòðîëüíîé êóëüòóðå (ðèñ. 3, à). Íà ýêñïåðèìåíòàëüíîé ìîäåëè ãèïåðëèïèäåìèè àõèëëèí ïðîÿâëÿë ãèïîëèïèäåìè÷åñêîå äåéñòâèå è óæå â êîíöåíòðàöèè 0,5 ììîëü çíà÷èòåëüíî ñíèæàë èíòåíñèâíîñòü ôëóîðåñöåíöèè Nile Red ((72,5 ± 1,2)%) ïî ñðàâíåíèþ ñ êîíòðîëåì (p < 0,05) (ðèñ. 4). Ãåìôèáðîçèë óìåíüøàë ñîäåðæàíèå ëèïèäîâ â êëåòêàõ ÍÒÑ íà ýêñïåðèìåíòàëüíîé ìîäåëè ãèïåðëèïèäåìèè äî (36,5 ± 3,7)% ïðè êîíöåíòðàöèè 0,25 ììîëü (p < 0,05) (ðèñ. 5). à á â ã Ðèñ. 3. Ìèêðîôîòîãðàôèè êëåòî÷íîé êóëüòû HTC (îá. 25 õ îê. 10). Ãðàíóëû ëèïèäîâ, îêðàøåííûå êðàñèòåëåì Oil Red O, êðàñíîãî öâåòà: à – êîíòðîëü, á – 0,05% ëèïîôóíäèí, â – 1,0 ììîëü àõèëëèíà, ã – 0,5 ììîëü ãåìôèáðîçèëà Бюллетень сибирской медицины, 2014, том 13, № 5, с. 28–35 31 Иванов В.В., Ратькин А.В., Пфаргер Ю.А. и др. Гиполипидемическое действие сесквитерпенового γ-лактона ахиллина… Ðèñ. 4. Âëèÿíèå àõèëëèíà íà ôëóîðåñöåíöèþ Nile Red â êëåòî÷íîé êóëüòóðå êðûñèíîé ãåïàòîìû (ÍÒÑ) ïðè êóëüòèâèðîâàíèè êëåòîê ñ ëèïîôóíäèíîì â êîíöåíòðàöèè 0,05%, M ± m, n = 6. Ôëóîðåñöåíöèþ Nile Red â êîíòðîëå ïðèíèìàëè çà 100%. * – ñòàòèñòè÷åñêè çíà÷èìûå ðàçëè÷èÿ ïî ñðàâíåíèþ ñ ôëóîðåñöåíöèåé êîíòðîëÿ ïðè p < 0,05 ÒÀà è óâåëè÷åíèå èõ ãèäðîëèçà [17, 18]. Ãåìôèáðîçèë, áóäó÷è àãîíèñòîì ðåöåïòîðà, àêòèâèðóåìîãî ïåðîêñèñîìíûì ïðîëèôåðàòîðîì (PPAR), äåéñòâóåò íà ÿäåðíûå ðåöåïòîðû, óâåëè÷èâàÿ ýêñïðåññèþ ãåíîâ, êîòîðûå ðåãóëèðóþò ñèíòåç êëþ÷åâûõ ôåðìåíòîâ ëèïèäíîãî îáìåíà è áåëêîâ ìåòàáîëèçìà ëèïîïðîòåèíîâ [17]. Ñíèæåíèå ñèíòåçà ÒÀà ïðîèñõîäèò çà ñ÷åò èíãèáèðóþùåãî âëèÿíèÿ ãåìôèáðîçèëà íà àêòèâíîñòü ìèêðîñîìàëüíîé äèàöèëãëèöåðîëàöèëòðàíñôåðàçû. Ê òîìó æå ãåìôèáðîçèë àêòèâèðóåò äåãðàäàöèþ àïîëèïîïðîòåèíà  (ÀïîÂ), ÷òî ïðèâîäèò ê ñíèæåíèþ ñåêðåöèè ËÏÎÍÏ [19]. Ôëàâîíîèä íàðèíãåíèí, ïîëó÷åííûé èç öèòðóñîâûõ, ñíèæàåò ñåêðåöèþ ËÏÎÍÏ òàêæå çà ñ÷åò óâåëè÷åíèÿ âíóòðèêëåòî÷íîé äåãðàäàöèè Àïî [20]. Ïîëó÷åííûå íàìè äàííûå îá óìåíüøåíèè ôëóîðåñöåíöèè Nile Red è ñíèæåíèè ñîäåðæàíèÿ îêðàøåííûõ Oil Red O ëèïèäíûõ êàïåëü â öèòîçîëå êëåòîê ïîä âëèÿíèåì àõèëëèíà ìîãóò áûòü îáóñëîâëåíû èíãèáèðîâàíèåì ëèïîãåíåçà èëè ãèäðîëèçîì ÒÀÃ. Äëÿ óñòàíîâëåíèÿ ìîëåêóëÿðíûõ ìèøåíåé äåéñòâèÿ àõèëëèíà íà ìåòàáîëèçì ëèïèäîâ â êóëüòóðå ÍÒÑ íåîáõîäèìî â äàëüíåéøåì èññëåäîâàíèå ýêñïðåññèè ãåíîâ êëþ÷åâûõ ôåðìåíòîâ ëèïèäíîãî îáìåíà. Заключение Ðèñ. 5. Âëèÿíèå ãåìôèáðîçèëà íà ôëóîðåñöåíöèþ Nile Red â êëåòî÷íîé êóëüòóðå êðûñèíîé ãåïàòîìû (ÍÒÑ) ïðè êóëüòèâèðîâàíèè ñ 0,05% ëèïîôóíäèíîì, M ± m, n = 6. Ôëóîðåñöåíöèþ Nile Red â êîíòðîëå ïðèíèìàëè çà 100%. * – ñòàòèñòè÷åñêè çíà÷èìûå ðàçëè÷èÿ ïî ñðàâíåíèþ ñ ôëóîðåñöåíöèåé êîíòðîëÿ ïðè p < 0,05 Ñïîñîáíîñòü ëèïîôóíäèíà èíäóöèðîâàòü ãèïåðëèïèäåìèþ ïîêàçàíà íå òîëüêî íà êóëüòóðå ïåðâè÷íûõ ãåïàòîöèòîâ in vitro [13], íî è íà ýêñïåðèìåíòàõ in vivo [16]. Ëèïîôóíäèí ïðè ââåäåíèè ýêñïåðèìåíòàëüíûì æèâîòíûì âûçûâàë ïîâûøåíèå â êðîâè óðîâíÿ ÒÀÃ, îáùåãî õîëåñòåðîëà è õîëåñòåðîëà â ËÏÍÏ. Ýòî îáóñëîâëåíî òåì, ÷òî âûñîêèé óðîâåíü ýêçîãåííûõ ÒÀà èíäóöèðóåò ñèíòåç ÀïîÂ-100 è õîëåñòåðîëà â ïå÷åíè, ÷òî, â ñâîþ î÷åðåäü, ñïîñîáñòâóåò îáðàçîâàíèþ è ñåêðåöèè ëèïîïðîòåèíîâ î÷åíü íèçêîé ïëîòíîñòè (ËÏÎÍÏ) [16]. Ìåõàíèçì, ñ ïîìîùüþ êîòîðîãî àõèëëèí ñíèæàåò íàêîïëåíèå ëèïèäîâ â êëåòêàõ êðûñèíîé ãåïàòîìû, íå èçó÷åí.  òî æå âðåìÿ èçâåñòíî, ÷òî îñíîâíûìè ìåõàíèçìàìè ãèïîëèïèäåìè÷åñêîãî äåéñòâèÿ ôèáðàòîâ, â òîì ÷èñëå ãåìôèáðîçèëà, ÿâëÿþòñÿ ñíèæåíèå ñèíòåçà 32 Íà ìîäåëè ãèïåðëèïèäåìèè, èíäóöèðîâàííîé ëèïîôóíäèíîì, äîáàâëåíèå ñåñêâèòåðïåíîâîãî γ-ëàêòîíà àõèëëèíà â èíêóáàöèîííóþ ñðåäó ïðåïÿòñòâóåò íàêîïëåíèþ ëèïèäîâ â êëåòêàõ ÍÒÑ, î ÷åì ñâèäåòåëüñòâóåò óìåíüøåíèå ôëóîðåñöåíöèè Nile Red è ñíèæåíèå ñîäåðæàíèÿ îêðàøåííûõ Oil Red O ëèïèäíûõ êàïåëü â öèòîçîëå. Èññëåäîâàíèå âûïîëíåíî ïðè ôèíàíñîâîé ïîääåðæêå Ñîâåòà ïî ãðàíòàì Ïðåçèäåíòà Ðîññèéñêîé Ôåäåðàöèè, ¹ ÍØ-4184.2014.7 Литература 1. Ðàòüêèí À.Â., Àðûñòàí Ë.È., ßêîâëåâà Þ.À., Èâàíîâ Â.Â., Ðÿçàíöåâà Í.Â., ×ó÷àëèí Â.Ñ., Àäåêåíîâ Ñ.Ì. Âëèÿíèå ñåñêâèòåðïåíîâîãî γ-ëàêòîíà ëåóêîìèçèíà íà óðîâåíü òðèàöèëãëèöåðîëîâ â êëåòêàõ êðûñèíîé ãåïàòîìû ïðè ýêñïåðèìåíòàëüíîé ìîäåëè ãèïåðëèïèäåìèè // Ñèá. ìåä. îáîçðåíèå. 2014. Ò. 1. Ñ. 44–48. 2. Áóåâåðîâà Å.Ë., Äðàïêèíà Î.Ì., Èâàøêèí Â.Ò. Àòåðîãåííàÿ äèñëèïèäåìèÿ è ïå÷åíü // Ðîñ. ìåä. âåñòè. 2008. Ò. 13, ¹ 1.Ñ. 17–23. 3. Àìîñîâà Å.Í. Ãèïîëèïèäåìè÷åñêàÿ òåðàïèÿ ïðè èøåìè÷åñêîé áîëåçíè ñåðäöà // Óêð. êàðäèîë. æóðí. 2002. Ò. 6. Ñ. 13–18. 4. Ramsey S., Gold E.S., Aderem A. A systems biology approach to understanding atherosclerosis // EMBO Mol. Med. 2010. V. 2. P. 79–89. 5. Kumar D., Parcha V., Maithani A., Dhulia I. Effect and evaluation of antihyperlipidemic activity guided isolated fraction from total methanol extract of Salvadoraoleoides Бюллетень сибирской медицины, 2014, том 13, № 5, с. 28–35 Оригинальные статьи (Decne.) in Triton WR-1339 Induced hyperlipidemic rats // Pharmacogn. Mag. 2012. V. 8, ¹ 32. P. 314–318. 6. Wyrebska A., Gach K., Szemraj J., Szewczyk K., Hrabec E., Koszuk J., Janecki T., Janecka A. Comparison of antiinvasive activity of parthenolide and 3-isopropyl-2-methyl-4methyleneisoxazolidin-5-one (MZ-6) – a new compound with α-methylene-γ-lactone motif – on two breast cancer cell lines // Chem. Biol. Drug Des. 2013. V. 79. P. 112–120. 7. Wyrebska A., Szymanski J., Gach K., Piekielna J., Koszuk J., Janecki T., Janecka A. Apoptosis-mediated cytotoxic effects of parthenolide and the new synthetic analog MZ-6 on two breast cancer cell lines // Mol. Biol. Rep. 2013. V. 40. P. 1655–1663. 8. Chaturvedi D. Sesquiterpene lactones: structural diversity and their biological activities // Research signpost. 2011. P. 313–334. 9. Ðîäíîâà Å.À., Èâàíîâ Â.Â., Ëåäþêîâà Ñ.È., ×ó÷àëèí Â.Ñ., Ðàòüêèí À.Â., Ðàõèìîâà Á.Á., Õàáàðîâ È.À., Àäåêåíîâ Ñ.Ì. Ãèïîëèïèäåìè÷åñêîå äåéñòâèå ëåóêîìèçèíà íà ìîäåëè îñòðîé ãèïåðëèïèäåìèè, èíäóöèðîâàííîé ýòàíîëîì // Áþë. ñèá. ìåäèöèíû. 2013. Ò. 12, ¹ 1. Ñ. 43–48. 10. Àäåêåíîâ Ñ.Ì., Ãàôóðîâ Í.Ì., Òóðìóõàìáåòîâ À.Æ., Èâëåâ Â.È. Òåðïåíîèäû Achillea micrantha // Õèìèÿ ïðèðîä. ñîåäèíåíèé. 1987. ¹ 2. 305 ñ. 11. Ìîðîçîâ Ñ.Þ. Ïðèìåíåíèå ñèìâàñòàòèíà äëÿ ëå÷åíèÿ è ïðîôèëàêòèêè èøåìè÷åñêîé áîëåçíè ñåðäöà // Ðóñ. ìåä. æóðí., êàðäèîëîãèÿ. 2009. ¹ 8. Ñ. 606–609. 12. Ilan E., Tirosh Î., Madar Z. Triacylglycerol-mediated oxidative stress inhibits nitric oxide production in rat isolated hepatocytes // J. Nutr. 2005. V. 135, ¹ 9. P. 2090–2095. 13. Giri Sh., Nieber K. et al. Telomerase activity and hepatic functions of rat embryonic liver progenitor cell in nanoscaf- fold-coated model bioreactor // Mol. Cell. Biochem. 2010. V. 336. P. 137–149. 14. Shen Ch., Meng Q., Schmelzer E., Bader A. Gel entrapment culture of rat hepatocytes for investigation of tetracyclineinduced toxicity // Toxicol. Appl. Pharmacol. 2009. V. 238. P. 178–187. 15. Xu S., Huang Y., Xie Y. et al. Evaluation of foam cell formation in cultured macrophages: an improved method with Oil Red O staining and Dil-oxLDL uptake // Cytotechnology. 2010. V. 62. P. 473–481. 16. Roche L.D., Medina E.A., Perez A.F. et al. Lipofundininduced hyperlipidemia promotes oxidative stress and atherosclerotic lesions in New Zealand white rabbits // Int. J. Vasc. Med. 2012. V. 2012. P. 1–7. 17. Ozansoy G., Akin F.B. Effects of gemfibrozil treatment on vascular reactivity of streptozotocin-diabetic rat aorta // J. Pharm. Pharmacol. 2004. V. 56. P. 241–246. 18. Umeda Y., Kako Yu., Mizutani K. et al. Inhibitory action of gemfibrozil on cholesterol absorption in rat intestine // The journal of lipid research. 2001. V. 42. P. 1214–1219. 19. Zhu D., Ganji S.H., Kamanna V.S., Kashyap M.L. Effect of gemfibrozil of apolipoprotein B secretion and diacylglycerolacyltransferase activity in human hepatoblastoma (HepG2) cells // Atherosclerosis. 2002. V. 164, ¹ 2. P. 221–228. 20. Borradaile N.M., Dreu L.E. et al. Inhibition of hepatocyte apoB secretion by naringenin: enhanced rapid intracellular degradation independent of reduced microsomal cholesteryl esters // J. Lipid Res. 2002. V. 43, ¹ 9. P. 1544–1554. Ïîñòóïèëà â ðåäàêöèþ 19.05.2014 ã. Óòâåðæäåíà ê ïå÷àòè 09.10.2014 ã. à‚‡ÌÓ‚ Ç·‰ËÏË Ç·‰ËÏËӂ˘ – êàíä. áèîë. íàóê, äîöåíò êàôåäðû áèîõèìèè è ìîëåêóëÿðíîé áèîëîãèè ÑèáÃÌÓ, (ã. Òîìñê). ê‡Ú¸ÍËÌ ÄÎÂÍ҇̉ LJÎÂÌÚËÌӂ˘ () – êàíä. ôàðì. íàóê, äîöåíò êàôåäðû ôàðìàöåâòè÷åñêîé òåõíîëîãèè ÑèáÃÌÓ (ã. Òîìñê). èÙ‡„ ûÎËfl Ä̉‚̇ – àñïèðàíò êàôåäðû ïàòîôèçèîëîãèè ÑèáÃÌÓ (ã. Òîìñê). ä‡È‰‡¯ é脇 ÄÎÂÍ҇̉ӂ̇ – àñïèðàíò êàôåäðû ôàðìàêîëîãèè ÑèáÃÌÓ (ã. Òîìñê). êflÁ‡ÌˆÂ‚‡ ç‡Ú‡Î¸fl Ç·‰ËÏËӂ̇ – ä-ð ìåä. íàóê, ïðîôåññîð, ïðîðåêòîð ïî ñòðàòåãè÷åñêîìó ðàçâèòèþ, èííîâàöèîííîé ïîëèòèêå è íàóêå, çàâ. êàôåäðîé ìîëåêóëÿðíîé ìåäèöèíû è êëèíè÷åñêîé ëàáîðàòîðíîé äèàãíîñòèêè ÑèáÃÌÓ (ã. Òîìñê). ĉÂÍÂÌÓ‚ 넇Á˚ å˚Ìʇ҇ӂ˘ – ä-ð õèì. íàóê, àêàäåìèê ÍÀÍ ÐÊ, ïðåäñåäàòåëü ïðàâëåíèÿ ÀÎ «Ìåæäóíàðîäíûé íàó÷íî-ïðîèçâîäñòâåííûé õîëäèíã «Ôèòîõèìèÿ» (ã. Êàðàãàíäà, Ðåñïóáëèêà Êàçàõñòàí). óÛ˜‡ÎËÌ Ç·‰ËÏË ë„‚˘ – ä-ð ôàðì. íàóê, ïðîôåññîð, äåêàí ôàðìàöåâòè÷åñêîãî ôàêóëüòåòà, çàâ. êàôåäðîé ôàðìàöåâòè÷åñêîé òåõíîëîãèè ÑèáÃÌÓ (ã. Òîìñê). ê‡Ú¸ÍËÌ ÄÎÂÍ҇̉ LJÎÂÌÚËÌӂ˘, òåë.: 8-903-915-3991, 8 (3822) 42-09-50; e-mail: [email protected] Бюллетень сибирской медицины, 2014, том 13, № 5, с. 28–35 33 Иванов В.В., Ратькин А.В., Пфаргер Ю.А. и др. Гиполипидемическое действие сесквитерпенового γ-лактона ахиллина… INVESTIGATION OF HYPOLIPIDEMIC EFFECT OF SESQUITERPENE Γ-LACTONE AHILLIN IN HEPATOMA TISSUE CULTURE (HTC) CELLS Ivanov V.V.1, Ratkin A.V.1, Pfarger Yu.A.1, Kaidash O.A.1, Ryazantseva N.V.1, Adekenov S.M.2, Chuchalin V.S.1 1 2 Siberian State Medical University, Tomsk, Russian Federation International scientific- industrial holding “Phytochemistry”, Karaganda, Kazakhstan ABSTRACT Objective. Investigation of hypolipidemic effect of sesquiterpene γ-lactone ahillin in hepatoma tissue culture (HTC) cells. Material and methods. In this study we’ve evaluated the effect of γ-lactone sesquiterpene añhillin and gemfibrozil (comparator drug) on the lipid content in the hepatoma tissue culture (HTC) cell which were incubated with a fat emulsion lipofundin by fluorescent method with vital dye Nile Redand staining the cells with the dye Oil Red O. The cell viability was investigated using the MTT-test and staining with Trypan blue. Results. Cultivation cells HTC with añhillin and gemfibrozilat concentrations ranging from 0.5 to 1.5 mM and from 0.25 mM to 0.5 mM, respectively, resulted in dose-dependent decrease of the fluorescence’s intensity Nile Red. It reflects a decrease in lipid content in the cells. At these concentrations the drugs didn’t have cytotoxic effect and the cell viability didn’t change compared to the control culture. An experimental hyperlipidemia in the hepatoma culture cells was induced by adding to the incubation medium a fat emulsion lipofundin at a final concentration 0.05%. The intensity of fluorescence Nile Red in the cells was increased 4 fold (p < 0.05). This result suggests the significant accumulation of lipids in the cell’s cytosol and confirmed by microscopy after staining neutral lipids with the dye Oil Red O. Under these conditions añhillin and gemfibrozil reduced lipid content in cells and hadthe effect at concentrations of 0.5 mM and 0.25 mM respectively. Conclusion. In the lipofundin-mediated model of hyperlipidemia the sesquiterpene lactone añhillin prevents the lipid accumulation in cells. It confirms by decrease of fluorescence Nile Red and reduction lipid drops which were stained with Oil Red O in cytosol. To establish the molecular targets of añhillin’saction on lipid metabolism in cell culture HTC we need to investigate a gene expression of key enzymes of lipid metabolism. KEY WORDS: sesquiterpene γ-lactone añhillin, gemfibrozil, hepatoma tissue culture (HTC) cells, hypolipidemic effect. Bulletin of Siberian Medicine, 2014, vol. 13, no. 5, pp. 28–35 References 1. Ratkin A.V., Arystan LI, Yakovleva Y.A., Ivanov V.V., Ryazantseva N., Chuchalin V.S., Adekenov S.M. Influence of sesquiterpene g-lactone leucomelaena on the level of triacylglycerols in the cells of rat hepatoma in experimental models hyperlipidemia. Sibirskoye meditsinskoye obozrenie – Siberian Medical Review, 2014, no 1, pp. 44–48. 2. Buyeverov E.L., Drapkina O.M., Ivashkin V.T. Atherogenic dyslipidemia and liver. Rossiiskiye meditsinskiye vesti – Russian Medical News, 2008, vol. 13, no. 1, pp. 17–23. 3. Amosova E.N. Lipid-lowering therapy in coronary heart disease. Ukrainskiy kardiologicheskiy zhurnal – The Ukrainian Cardiological Journal, 2002, no. 6, pp. 13–18. 4. Ramsey S., Gold E.S., Aderem A. A systems biology approach to understanding atherosclerosis. EMBO Mol. Med., 2010, vol. 2, pp. 79–89. 34 5. Kumar D., Parcha V., Maithani A., Dhulia I. Effect and evaluation of antihyperlipidemic activity guided isolated fraction from total methanol extract of Salvadoraoleoides (Decne.) in Triton WR-1339 Induced hyperlipidemic rats. Pharmacogn. Mag., 2012, vol. 8, no. 32, pp. 314–318. 6. Wyrebska A., Gach K., Szemraj J., Szewczyk K., Hrabec E., Koszuk J., Janecki T., Janecka A. Comparison of antiinvasive activity of parthenolide and 3-isopropyl-2-methyl4-methyleneisoxazolidin-5-one (MZ-6) – a new compound with α-methylene-γ-lactone motif – on two breast cancer cell lines. Chem. Biol. Drug Des., 2013, vol. 79, pp. 112– 120. 7. Wyrebska A., Szymanski J., Gach K., Piekielna J., Koszuk J., Janecki T., Janecka A. Apoptosis-mediated cytotoxic effects of parthenolide and the new synthetic analog MZ-6 on two breast cancer cell lines. Mol. Biol. Rep., 2013, vol. 40, pp. 1655–1663. Бюллетень сибирской медицины, 2014, том 13, № 5, с. 28–35 Оригинальные статьи 8. Chaturvedi D. Sesquiterpene lactones: structural diversity and their biological activities. Research Signpost., 2011, pp. 313–334. 9. Rodnova E.A., Ivanov V.V., Lizukova S.R., Chuchalin V.S., Ratkin A.V., Rakhimov B.B., Khabarov I.A., Adekenov S.M. Hypolipidemic effect leucomelaena on the model of acute hyperlipidemia induced by ethanol. Bulleten sibirskoy meditsini – Bulletin of Siberian Medicine, 2013, vol. 12, no. 1, pp. 43–48. 10. Adekenov S.M., Gafurov N.M., Turmagambetov A.Z., Ivlev V.I. Terpenoids Achillea micrantha. Khimiya prirodnykh soyedineniy – Chemistry of Natural Compounds, 1987, no. 2, pp. 305. 11. Morozov H.E. Use of simvastatin for the treatment and prevention of coronary heart disease. Russkiy meditsikiy zhurnal, kardiologiya – Russian Journal of Medicine, Cardiology, 2009, no 8. pp. 606–609. 12. Ilan E., TiroshÎ., Madar Z. Triacylglycerol-mediated oxidative stress inhibits nitric oxide production in rat isolated hepatocytes. J. Nutr., 2005, vol. 135, no. 9, pp. 2090–2095. 13. 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Umeda Y., Kako Yu., Mizutani K. et al. Inhibitory action of gemfibrozil on cholesterol absorption in rat intestine. The journal of lipid research, 2001, vol. 42, pp. 1214–1219. 19. Zhu D., Ganji S.H., Kamanna V.S., Kashyap M.L. Effect of gemfibrozil of apolipoprotein B secretion and diacylglycerolacyltransferase activity in human hepatoblastoma (HepG2) cells. Atherosclerosis, 2002, vol. 164, no. 2, pp. 221–228. 20. Borradaile N.M., Dreu L.E. et al. Inhibition of hepatocyte apoB secretion by naringenin: enhanced rapid intracellular degradation independent of reduced microsomal cholesteryl esters. J. Lipid Res., 2002, vol. 43, no. 9, pp. 1544–1554. Ivanov Vladimir V., Siberian State Medical University, Tomsk, Russian Federation. Ratkin Aleksander V. (), Siberian State Medical University, Tomsk, Russian Federation. Pfarger Yulia A., Siberian State Medical University, Tomsk, Russian Federation. Kaidash Olga A., Siberian State Medical University, Tomsk, Russian Federation. 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