1. RESULTS and DISSCUSSION
EVALUATION OF METABOLIC EFFECTS OF TWO TRITERPENIC DERIVATES OBTAINED FROM SALVIA SP. ON ENDOTHELIAL PRIMARY CELL CULTURE HUVEC AUTHORS: M. CONSTANTINOVICI, L. ZGLIMBEA, N. DOCIU, A. NETOIU, L. OLARIU INSTITUTION: S.C. BIOTEHNOS S.A., OTOPENI, ROMANIA author’s
RESULTS and DISSCUSSION
Metabolic effects at cellular level were correlatively evaluated through studying the influence on cell proliferative capacity and release of lactate dehydrogenase in extracellular medium as response to cellular stress conditions. The brute results obtained from the direct measurement of probes’ absorbance intensities were statistically processed and reported to blanks and negative controls in order to obtain the real effect of the tested substances, without basal interferences.
The triterpenic acid effect on proliferation and viability status of HUVEC cells was positive stimulating them in a dose dependent manner; citotoxicity was moderate until 8 µM, afterthat doubling its value (Fig. 1, 2). The triterpenoid compound exhibited a more drastic LDH release and thus was tested just in the range of 0.5 to 5 µM, but the proliferative pathways were enhanced comparing with the acid effect. (Fig. 3).
OBJECTIVE
The endothelial cells form a monolayer in every single blood vessel in the circulation and are actively involved in several regulatory processes in the body. The most important one is the angiogenesis, or the formation of new blood vessels out of pre-existing capillaries, which is a sequence of events that is fundamental to many physiologic and pathologic processes. Recent studies demonstrated the implications of angiogenesis (endothelial cells’ growth and proliferation) in the protection against ischemia–reperfusion injury of the heart [1, 2, 3]. Thus, new developed vegetal compounds with pharmacological properties mostly the same as statins, but without their adverse side effects, could be used as angiogenesis enhancers towards stimulation of endothelial cells proliferation as one of the therapeutical way to protect heart against ischemia injuries.
Our research in this paper was straightened towards biological effect evaluation of two triterpenic derivates, obtained from Salviae sp. The screening of their biological effects was done in vitro using cell biology specific protocols: viability and citotoxicity evaluation, using ELISA technique. The biological material used was HUVEC, human umbilical vein endothelial cell line, which permits to study in vitro the biological effect of these derivates on angiogenesis process.
MATERIALS and METHODS
HUVEC, human umbilical vein endothelial cell line, ATCC catalog number PCS ;
CellTiter 96AQueous One Solution Cell Proliferation Assay, Promega G3580;
CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega G1780;
HUVEC cells, passages between 4-8, were cultured on 96 wells plates for 24h in growth medium containing RPMI and 10% (v ⁄ v) fetal bovine serum in a 90–95% humidified atmosphere of 5% (v ⁄ v) CO2 at 370 C.
The tested substances were diluted in culture medium in working concentration ranged between 0.5 to 10 µM for triterpenic acid and 0.5 to 5 µM for triterpenoid derivate and left in contact with HUVEC endothelium for another 24 h.
Their effects were first evaluated by assaying the metabolic activity of viable cells using the chemical reduction of (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) into formazan, which is soluble in tissue culture medium). The measurement of the absorbance of the formazan was carried out in 96 well microplates at 492nm [4]. This assay measures dehydrogenase enzyme activity found in metabolically active cells, thus being a direct index of their viability correlated with the concentrations of tested substances.
The cytotoxic effects were evaluated measuring lactate dehydrogenase (LDH), a stable cytosolic enzyme released upon cell lysis. Released LDH in culture supernatants was measured with a 30-minute coupled enzymatic assay, which results in the conversion of a tetrazolium salt (INT) into a red formazan product [5]. The amount of color formed, measured at 490 nm, was proportional to the number of lysed cells as response to applied concentrations from the tested substances.
The results are the average of n = 3 experiments performed with 6 concentrations of the compounds, along with its carrier DMSO, all in triplicate; the exposure time was 24h for all the probes.
EQUIPMENTS AND SOFTWARE:
Multimode Reader LB941, produced by Berthold Technologies, for ELISA technique.
Fig. 1. Triterpenic acid (0.5-4 µM) effect on endothelial cells metabolism.
Fig. 2. Triterpenic acid effect (5-10 µM) on endothelial cells metabolism .
Fig. 3. Triterpenoid effect on endothelial cells metabolism.
Refferences
1.Zhu X-Y. et al., Life Sciences 83 (2008), 801–809.
2. Wayman, N.S., Ellis, B.L., Thiemermann, C., Medical Sci. Monitor 9 (5), BR155–BR159.
3. Wolfrum S. et al., J. Cardiovascular Pharm. 44 (3), 348–355.
4. CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay, Part# TB169, PRINTED IN USA, Revised 5/09.
5. CytoTox 96® Non-Radioactive Cytotoxicity Assay, Part# TB163, Printed in USA, Revised 3/09.
Conclusions: probably for both triterpenic compounds LDH enhanced activity was a basal metabolic effect related to their presence in culture medium, but not affecting the viability and proliferative status of cells. These results could be used as a starting point for more profound evaluation of metabolic implications of triterpenic derivates angiogenic effect as therapeutical agents of cardiovascular problems.