EFFECT OF SESQUITERPENE Оі-LACTONE ACHILLIN ON LEVEL OF LIPIDS AND EXPRESSI ON mRNA OF KEY GENES OF LIPID MET ABOLISM IN HEPATOMA TISSUE CULTURE (HTC)
Iu.A. Pfarger1, V.V. Ivanov1, A.V. Ratkin1, O.A. Kaidash1, M.M. Tsyganov2,3, V.S. Chuchalin1, S.M. Adekenov4, V.V. Novitsky1, N.V. Ryazantseva5
1Siberian State Medical University, 2 Moskovsky trakt, Tomsk, 634050
2Tomsk State University, 36 Lenina av., Tomsk, 634050
3Tomsk Cancer Research Institute, 5 Kooperativny st., Tomsk, 634050
4JSC “International scientific- industrial holding” Phytochemistry”, Karaganda, Kazakhstan, st. M. Gazaliyev 4
5Krasnoyarsk State Medical University named after Prof. V.F. Voino-Yasenetsky, 1 Partisan Zheleznyak, Krasnoyarsk, 660022
Keywords: сесквитерпеновый Оі-лактон ахиллин, гемфиброзил, культура гепатомы линии HTC, МТТ-тест, Nile Red, экспрессия мРНК, гены метаболизма липидов, sesquiterpene Оі-lactone aсhillin, gemfibrozil, hepatoma tissue culture (HTC), MTT assay, Nile Red, mRNA expression, genes of lipid metabolism
Abstract
Objective. To study the effect of sesquiterpene γ-lactone achillin on the lipid content and mRNA expression of key genes of lipid metabolism in the hepatoma tissue cell line HTC in the experimental hyperlipidemia. Materials and methods. The experimental hyperlipidaemia in HTC cells simulated by adding fat emulsion Lipofundin MCT/LCT at a final concentration 0.05 % to the incubation medium. After 48 h of incubating cell culture with achillin at the final concentration 500 μM the cell viability was investigated by using the MTT assay and assessed level of lipids by fluorescent vital stain Nile Red and the content of triacylglycerols (TAG) and cholesterol by enzymatic method. RNA was isolated from cells using the set Illustra RNAspin Mini RNA Isolation Kit («GE Healthcare»). The level of mRNA expression of key genes in lipid metabolism was assessed using a real-time quantitative polymerase chain reaction with reverse transcription by TaqMan technology. Results. Incubation cells with Lipofundin MCT/LCT (0.05%) resulted in an increase in the fluorescence intensity of Nile Red and increase the levels of TAG in cells. Achill (500 µM) had no cytotoxic effect on HTC cell and led to reduction of cholesterol and TAG in cells with hyperlipidemia induced by Lipofundin. This was accompanied by a decrease in fluorescence intensity of Nile Red dye in the cells. In the hepatoma culture achillin increased gene expression of carnitine palmitoyltransferase 1 (Cpt1a) and 2 (Cpt2) , 7-alpha-hydroxylase (Cyp7a1) and 3-hydroxy-3-methylglutaryl CoA reductase (Hmgcr) . On expression of gene acetyl-CoA cholesterol acyltransferase (Soat1) achill had an inhibitory effect. Conclusion. Lowering of cholesterol, TAG and Nile Red fluorescence intensity in hepatoma cells in experimental hyperlipidemia under the action of achillin in the final concentration 500 µM could be caused by increase of gene expression carnitine palmitoyltransferase 1 and 2; 7-alpha-hydroxylase, which contributes to increase the transport of fatty acids into the mitochondria, and synthesis of bile acids from cholesterol. In addition, inhibition of acetyl-CoA cholesterol acyltransferase helps to reduce the formation of cholesterol esters and their accumulation in the hepatocytes.
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