P38 MAPK-dependent Targets of Gaseous Transmitters Proapoptotic Action in Jurkat Cells

Background: p38 MAPK participates in gases-induced apoptosis signal transmission though it hasn’t molecular structure necessary for interaction with gaseotransmitters.Methodology: annexin positive cells and cells with decreased mitochondrial transmembrane potential were detected by flow cytometry. Caspase 3 and 9 activity was investigated via spectrophotometry. Protein contents of Bcl-2 family members were measured with western blot analysis.Results: Caspase-3 and -9 activity and the number of cells with decreased mitochondrial membrane potential showed the apoptotic reaction vector when p38 MAPK-dependent pathways of NO and CO action inhibited. Inhibition of p38 kinase in H2S treated cells led to caspase-3 activation accompanied with the decrease of the number of apoptotic cells. Proteins Bcl-2 and Bad were the p38 MAPK-dependent targets of all three gases action. P38 MAPK influenced on protein Aven when intracellular H2S concentration increased and on Bcl-xl protein in CO-treated cells. In both cases p38 kinase was the negative regulator of these proteins and promoted the proapoptotic action of gases abolishing the gases-mediated increase of the Aven and Bcl-xl content.    Conclusion: p38 MAPK acted proapoptotically in the cases of intracellular increase of NO and H2S and antiapoptotically in CO-treated cells.

ЦИТОТОКСИЧЕСКИЕ И ГИПОЛИПИДЕМИЧЕСКИЕ СВОЙСТВА АРГЛАБИНА НА КУЛЬТУРЕ КЛЕТОК ГЕПАТОМЫ КРЫС

Investigated toxicity and lipid-lowering activity (in vitro) of sesquiterpene γ-lactone arglabin as a potential lipid-lowering drugs.Material and methods. Was studied the effect of Arglabin and gemfibrozil (comparison drug) on the lipid level of the rat hepatoma tissue culture (HTC) with fluorescent method with vital dye Nile Red and staining cells with the dye Oil Red O. The cell viability was assessed by using MTT-test and staining with trypan blue.Results. Cultivation of HTC cell cultures with arglabin and gemfibrozil in concentrations up to 50 μm and 0.5 mm, respectively, resulted in dose-dependent decrease in the fluorescence intensity of Nile Red, which is associated with a reduction in lipid level in the cells. In these concentrations the drugs did not exert cytotoxic effect, and HTC viability did not change compared with the corresponding rate in the control culture.Conclusion. Arglabin and gemfibrozil does not affect the viability of hepatoma cells in rats at a concentration up to 50 μm and 0.5 mm, respectively. Arglabin and gemfibrozil dose-dependently reduce the content of lipids in the HTC culture, as evidenced by the decrease in Nile Red fluorescence and decrease lipid droplets stained with Oil Red O in the cytosol.Цель исследования. Оценка in vitro токсичности и гиполипидемической активности сесквитерпенового γ-лактона арглабина в качестве потенциального гиполипидемического средства.Материал и методы. В работе изучали влияние арглабина и гемфиброзила (препарат сравнения) на содержание липидов в клеточной культуре крысиной гепатомы (HTC) флуоресцентным методом с витальным красителем Nile Red и окрашиванием клеток с красителем Oil Red O. Жизнеспособность клеток оценивали с помощью МТТ-теста и окрашиванием с трипановым синим.Результаты. Культивирование клеточной культуры HTC с арглабином и гемфиброзилом в концентрациях до 50 мкМ и 0,5 мМ соответственно приводило к дозозависимому уменьшению интенсивности флуоресценции Nile Red, что связано со снижением содержания липидов в клетках. В этих концентрациях препараты не оказывали цитотоксического действия, и жизнеспособность клеток НТС не изменялась по сравнению с соответствующим показателем контрольной культуры.Заключение. Арглабин и гемфиброзил не влияют на жизнеспособность клеток гепатомы крыс НТС при концентрации до 50 мкМ и до 0,5 мМ соответственно. Арглабин и гемфиброзил дозозависимо снижают содержание липидов в культуре НТС, что подтверждается уменьшением флуоресценции Nile Red и снижением содержания окрашенных Oil Red O липидных капель в цитозоле