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Khamidova Ozoda J.., Institute of Biophysics and Biochemistry, National University of Uzbekistan Rustamova Sarvinoz I., Institute of Biophysics and Biochemistry, National University of Uzbekistan Kurbannazorova Ranokhon Sh., DSc., Institute of Biophysics and Biochemistry, National University of Uzbekistan Merzlyak Petr G., DSc., Institute of Biophysics and Biochemistry, National University of Uzbekistan Tashmukhamedov Bek A., Academician, Institute of Biophysics and Biochemistry, National University of Uzbekistan Sabirov Ravshan Z., Academician, Institute of Biophysics and Biochemistry, National University of Uzbekistan E-mail: zairovich@gmail.com
EFFECT OF ESCIN ON THE VOLUME REGULATION OF RAT THYMOCYTES
Abstract. It was demonstrated that a horse chestnut saponin, escin, stimulates the cell volume regulation system of thymocytes under hypoosmotic stress. However, this effect could be detected only at relatively low concentrations (1-5 ^M), whereas at higher doses (10-100 ^M) we observed multy-phase changes in the cellular volume and sustained swelling.
Keywords: escin, hypoosmotic stress, thymocytes, Donnan equilibrium, regulatory volume decrease.
Introduction (pH 7.4, 290 mOsm/kg-H2O). The H-buffer contained Escin (polyhydroxyolean-12-ene 3-O-monodesmosides) (mM): 5 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, 5 glucose, pH is a natural mixture of pentacyclic triterpenoid saponins de- 7.4 (40 mOsm/kg-H2O). Hypotonic solution was prepared rived from the seeds of the horse chestnut (Aesculus hip- by mixing the normal Ringer solution with H-buffer in a ratio pocastanum) [1; 2]. It exerts remarkable anti-inflammatory, of 3:4 (vol/vol). Cell isolation was performed as described anti-oedematous and vasoprotective properties. Recently, previously [4-8]. All animal experiments were conducted escin was found to induce apoptosis in cancer cells of various in accordance with the ARRIVE guidelines and approved origins, reduce the tumor size in vivo and is considered as a by the Bioethics Committee of the Institute of Biophysics perspective anti-cancer drug [1]. However, despite an increas- and Biochemistry. Briefly, the 6-8 weeks old rats, kept in a ing significance of escin in medicine, its molecular mechanism vivarium on an average diet, were anaesthetized with halo-of action and influence on the cellular membrane remains un- thane or diethyl ether and painlessly euthanized by cervical clear. In our previous study, we have demonstrated that escin dislocation. The thymi were dissected and carefully washed induced human red blood cell lysis by forming very large pores with an ice-cold normal Ringer solution. The thymi were then with a radius of app. 2.6 nm, which is comparable to the size minced using fine forceps and passed through a 100 ^m-nylon of hemoglobin [3]. In the present study, we show that escin mesh. The suspension was centrifuged at 1000 g for 5 min, greatly modulates the cell volume regulation of thymocyte the pellet was washed two times with the normal Ringer sounder hypoosmotic stress condition. lution and resuspended in this medium at a cell density of Materials and methods 100 x 106 cells/ml. The cell suspension was kept on ice for 1. The normal Ringer solution contained (mM): <5 h and contained no more than 5% of damaged cells as as-135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 11 HEPES, 5 glucose sayed by trypan blue exclusion. Cell volume changes under
Section 1. Biology
non-isoosmotic conditions were recorded by light transmit-tance measurement as described previously [4-7, 9]. Briefly, 900 ^l of the normal Ringer, or hypotonic solutions was added to the 1.5 cm3 glass cuvette thermo stated with a water jacket and equilibrated for 10 min. Escin (Sigma Cat No. E1378) was added from a stock solution in DMSO. The final solvent content never exceeded 0.1%, and at this dose it did not affect the measured parameters. An aliquot (100 ^l) of cell suspension was added to this medium to yield the final cell density of 10 x 106 cells/ml. The light transmittance was measured at 610 nm (band-pass filter) using a photometer MKMF-01 (Russia). The output signal was amplified by U5-11 amplifier (Russia), digitized at 100 Hz using a USB sensor interface GO! Link and recorded by Logger Lite software (Vernier, Beaverton, OR). The parameter RVD (Regulatory Volume Decrease) was calculated using the following equation:
RVD = (T -T J/(T -T)*100%
v max 15' v max iv
(1)
where T and T are the initial and maximal light transmit-
0 max o
tances, and T is the light transmittance measured 15 minutes after the onset of hypotonic stress. The RVD = 100 for complete recovery of the cell volume to the initial level, and RVD = = 0 when volume regulation is fully suppressed. Under control conditions, RVD usually had values of 60-90% depending on the cells condition, osmotic gradient, temperature and other experimental conditions.
Results and Discussion
In our research, we examined the influence different concentrations of escin on the cell volume regulation in rat thymocyte. In the isotonic medium (normal Ringer's solution), the volume of thymocytes remained unchanged for 15-20 minutes. When the thymocytes were placed in the hypotonic medium, they rapidly swelled (a passive response) and then started actively restore their initial volume (Fig. 1 A). In the control group, the parameter RVD averaged at 70.3 ± 4.5% (n = 5).
Figure 1. The effect of escin on regulation of thymocytes volume under hypoosmotic stress
Original recordings of the light transmittance change of the cell suspension (an indicator of the cell volume change, see Experimental section) of thymocytes in isotonic medium (Iso) and under hypoosmotic stress in Control hypotonic conditions (Hypo) and in the presence of escin at indicated concentrations (A, C, D) and respective mean values of the parameter RVD at 1 ^M and 5 ^M (B). Significantly different from the control values at P < 0.05, n is number of experiments
When escin was added to the hypoosmotic medium at a concentration of 1 ^M, we observed an up-regulation of the ability of cells to restore their volume, and parameter RVD was 85.3 ± 1.8% (n = 6). This effect was even more pronounced at a concentration of 5 ^M, at which RVD = 97.3 ± 5 (n = 6) (Fig. 1 A, B). The observed enhancement of the volume regulation is unusual and could be explained by the pore-forming properties of escin. We suppose that intracellular osmolytes exit the cells through the escin-formed pores thereby facilitating a decrease in the intracellular osmotic pressure.
When escin was applied at higher doses of 10-30 ^M, the time course of cell volume changes had a complex shape (Fig. 1 C). First, cells swelled and then started to decrease their volume. However, after app. 5 min cells again swelled to the volume which was lower than maximal level. Then, after app. 10 min from the beginning of osmotic stimulation, a second phase of volume decrease was recorded. Explanation of such a complex kinetics is difficult. We believe that, as the amount of substance increases, the number of escin-formed pores in the plasma membrane also increases. Since the size of the escin pore is very large [3], the system turns from a double-
Donnan equilibrium to a simple Donnan system resulting in the cellular swelling. The late phase of the light transmittance recordings could reflect cell lysis.
When we increased the dose of escin to 50 and 100 ^M, the shape of the time course was distorted even more (Fig. 1 D). We believe that recorded kinetics could largely reflect micelle formation process by the saponin, although initial swelling phase, regulatory volume decrease and secondary swelling we still observable.
In conclusion, we found a prominent stimulating effect of low doses of escin on the cell volume regulation of rat thymocytes, Since volume regulation machinery is deeply involved in the first phase of apoptosis - apoptotic volume decrease [10], we suppose that the effects of escin observed in our experiments are related to the recently explored anti-cancer properties of this drug.
This work was supported by the Ministry of Innovation-al Development of the Republic of Uzbekistan (under grant OA - 03 - T112, OT - 03-141, 0A-05-T080) and Academy of Sciences of Uzbekistan (under grant No. 123-06).
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