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Section 1. Biology
Pozilov Mamurjon Komiljonovich, Junior research, A. S. Sadykov Institute of Bioorganic Chemistry,
Academy of Sciences of Uzbekistan, E-mail: [email protected] Asrarov Muzaffar Islamovich, Professor, A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, E-mail: [email protected] Urmanova Gulbakhor Urunboevna, Senior lecturer Tashkent pediatric medical Institute Eshbakova Komila Alibekovna, Senior research, S.Yu. Yunusov Institute of Chemistry of Plant Substances, Academy of Sciences of Uzbekistan
Protective effect of salvifolin on liver mitochondrial function in rats with experimental diabetes
Abstract: The influence of diterpenoid salvifolin on mitochondrial function was investigated. It was shown that in streptozotocin-induced diabetes damaged functional systems of rat liver mitochondria: respiration and oxidative phosphorylation, mitochondrial permeability transition pore and ATP -dependent potassium channel. Pharmacotherapy with salvifolin (intraperitoneally in dose of 3,5 mg/kg body weight) for 8 days has a protective effect on mitochondria in experimental diabetes, correction membrane disorders.
Keywords: diabetes, mitochondria, respiration, oxidation phosphorylation, permeability transition pore, salvifolin.
Introduction potassium channel of the mitochondria (mitoKATF channel)
Studying the mechanisms of damage to cellular struc- in diabetes, as well as modulators of their data available. It was
tures and functions in various pathologies and ways to treat found that open ATP-dependent potassium channel (mito-
these injuries with pharmacological agents is a priority of KATP channel) protects the heart from ischemic contractures
modern biomedical research. Despite the variety of treat- and improve its post ischemic functional recovery [4, 2463-
ments for diabetes remains popular search for new pharma- 2469; 5, 51-57], play an important cardioprotective role dur-
cological agents and their "targets". In the cell, such "targets" ing all phases of the ischemia-reperfusion myocardial injury
are the mitochondrial membrane and localized in their struc- [6, 1-21]. The literature also little information on potassium
ture, firstly mitochondrial respiratory chain, megapora (mito- channel modulators of plant origin, having the property of
chondrial permeability transition pore, mPTP) [1, 101-127]. lowering blood sugar.
The formation of reactive oxygen species in cells and excessive Clerodane-type salvifolin isolated from the Pulicaria salvi-
activation of free radical oxidation processes underlie the de- ifolia, having a hypoglycemic effect, it normalizes the metabol-
velopment of diabetes [2, 1405-1423; 3, 1986-2001]. It is ic processes ofthe body in experimental diabetes [7, 86-91; 8,
also known that the mitochondria is disrupted the functioning 161-163]. However, salvifolin influence of on mitochondrial
of the citric acid cycle, there is a partial uncoupling of oxida- function is not investigated and therefore the aim was to study
tion and phosphorylation. These processes are considered as the effect of salvifolin on respiration and oxidative phosphory-
a universal mechanism that combines the basic biochemical lation, state of mPTP and mitoKATP channel in the rat liver
pathways of the toxic effect of hyperglycemia on the body. mitochondria of streptozotocin (STZ)-induced diabetic rats.
The development of experimental diabetes also involves Methods
ATP-dependent potassium channels in the plasma mem- For screening and detailed study of the mechanism of ac-
brane-localized (5-cells of the pancreas are opened. To date, tion of pharmacological agents are widely used various experi-
known inhibitors and activators of potassium channels. How- mental models of diabetes caused by administration of alloxan
ever, in the literature on the role and status ofATP-dependent and STZ al., cytotoxic activity on (-cells of the pancreas. We
have in this study used an experimental model of diabetes induced by STZ.
Experiments were performed on 90 white mongrel male rats weighing 180-200 g The animals were divided into three groups: I group — intact, II group — the animals with experimental diabetes, which once were injected intraperitoneally with an STZ (50 mg/kg body weight intraperitoneally in a 0,1 mol/L citrate buffer, pH 4,5) (control) and III group — STZ-induced diabetes + salvifolin (intraperitoneally dose of 3,5 mg/kg body weight) for 8 days starting from 12 days after administration of STZ and reaching a predetermined level of hyperglycemia. Blood glucose was determined using glucose oxidase method set «Glucose — enzymatic-colorimetric test» (Cypress diagnostic, Belgium).
The content ofmitochondrial protein was determined by the Lowry method in the modification ofthe Peterson [9, 346-356].
Mitochondria isolated from rat liver by differential centrifu-gation according to Schneider [10, 619-635]. Nuclei and cellular fragments were removed by centrifugation at 600 g for 7 minutes in a centrifuge. The mitochondria are pelleted at 10000 g for 15 minutes at the same temperature. The mitochondrial pellet was washed twice in the isolation EDTA-free medium.
mPTP condition assessed by the speed of Ca 2+ -dependent swelling of mitochondria, the mitochondrial suspension recording light scattering at 540 nm. Experiments at room temperature 25 °C in a medium containing 200 mM sucrose, 20 ^M EGTA, 5 mM succinate, 2 ^M rotenone, 1 ^g/ml oli-gomycin, 20 mM Tris, 20 mM HEPES, and 1 mM KH2PO4, pH 7,2 [11, 16755-16760]. The concentration of mitochondria in the swelling experiments was 0,5 mg protein/ml.
Mitochondrial respiration and oxidative phosphorylation was measured polarography method (polyarograph 0H-105, Hungary) at 25°C. The assay medium contained 100 mM sucrose, 75 mM KC1, 10 mM Tris-HCl, 2,5 mM K2HP04, pH 7,4 and 10 mM succinate or 5 mM glutamate and 1 mM malate as respiratory substrates. Protein concentration of mi-
tochondria corresponded to 3 mg/ml of the reaction medium ADP (200 ^M) was added as a respiratory stimulant. Calculated the rate of mitochondrial respiration in different metabolic states: V3 -respiration rate after making ADP, V4 — respiration rate after spending listed ADP. The indices characterizing pair of oxidation and phosphorylation in mitochondria: respiratory control (RC) ratio (RC=V3/V4) and the coefficient of phosphorylation ofADP/O. Mitochondrial respiration rate in different metabolic states are expressed in nanograms of consumed oxygen atoms per 1 minute per 1 mg of mitochondrial protein. The respiratory control and ADF/O ratio was calculated according to the method of Chance [12, 409-427].
The rate of swelling of the mitochondria was studied using a photometer LMF-69. Mitochondrial swelling induced activation KATP Mg 2+ channel was recorded using a change in light scattering at a wavelength of540 nm. Mitochondria were added to the standard incubation medium of the following composition: 125 mM KCl, 10 mM Hepes, 5 mM succinate, 1 mM MgCl2 2,5 mM K2HPO4, 2,5 mM KH2PO4, rotenone 1 ^M/ml, oligomycin 1 ^/ml, pH 7,4.
Static analysis of data was performed using the program features Origine 7.5 (Microcal Software Inc., Northampton MA). The data were expressed as means ± S. E. M. Paired Student's t-test was used for estimation of significance; minimum accepted level of significance was p < 0.05.
Results and discussion
Figure 1 shows the results of a study of respiratory and phosphorylating activity of rat liver mitochondria from STZ-induced diabetic and salvifolin action. The results showed that in STZ-induced diabetes, the rate of mitochondrial respiration is stimulated by the oxidation of substrates — glutamate + malate and succinate. Intoxication with STZ in rat respiration rate in the rat liver mitochondria state V3 is increased by 37,4±2,5% in the oxidation of malate+glutamate and succinate in the oxidation — by 34,8±3,0%, compared with that of mitochondria liver of intact animals (Fig.1A).
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Streptozotocin A
I I Streptozotocin+salvifolin
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3 '4
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V
V
Succinate
4,0n
3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
I I Kontrol
Streptozotocin I I Streptozotocin+salvfolin
B
RC ADP/O RC ADP/O
Glutamate+Malate
Succinate
Fig. 1. The effect of salvifolin on mitochondrial respiration with glutamate+malat and succinate in the liver of rats with STZ-indused diabetes. (A — mitochondrial respiration in states V3 and V4; B — RC and ADP/O ratio) *Р<0,05; **P<0,01; n=5
Under these conditions, the rate of mitochondrial respiration in a state V4, during the oxidation of malate+glutamate and succinate increased by 124,4±3,5% and 78,0±4,2% respectively, compared to controls (Fig.1A).
In experimental diabetes conditions, a decrease of the coefficient of RC to 38,8±1,3% during oxidation glutamate malate and 24,9±1,1% at oxidation of succinate compared to the control (Fig.1B). Such a decrease is due to increased RC mitochondrial respiration in state V4 compared with stat-eV3 Also, when reduced STZ-induced diabetes coefficient ADP/O mitochondria compared to intact group 24,3±1,6% when the oxidation and malate+glutamate 36,0±2,1% when compared succinate oxidation control (Fig.1B).
The studies the effect of pharmacotherapy salvifolin on respiration and oxidative phosphorylation of rat liver mitochondria was investigate. The rate of respiration of mitochondria isolated from rat liver III-group (pharmacotherapy salvifolin) was lower than the rate of respiration of the rat liver mitochondria II-group. Rat liver mitochondria respiration in the group III at state V3 of oxidation glutamate + malate and succinate inhibited by 16,4 ± 3,9% and 24,8 ± 4,5% respectively compared control (Fig.1A). Respira-
Ca2+ \
tion rat liver mitochondria in the groups III reduced state at glutamate+malat V4 90,2±2,8% succinate and 56,4±2,2% as compared with the mitochondrial respiration II-group (Fig.lA). In terms of pharmacotherapy salvifolin, RC coefficient increased the oxidation of glutamate+malate and succinate to 28,9±1,4% and 14,7±1,2%, respectively, compared indicator STZ-induced diabetes (Fig.lB). Pharmacotherapy with salvifolin index increases ADP/O at 14,6±1,7% in the oxidation of glutamate and malate 21,7±1,9% during succinate oxidation as compared with the II-group rats (Fig.1B). The findings suggest that pharmacotherapy with salvifolin increases the coupling of oxidation and phosphorylation in the mitochondria. The results indicate the activation of the respiratory V3 and V4 in the oxidation of substrates liver mitochondria STZ-induced diabetes rat, which is partially removed sal-vifolin pharmacotherapy.
The results of our research on models of STZ-induced diabetes rats showed a significant hypoglycemic effect in in-traperitoneal application salvifolin. Fig. 2 shows the results of experiments on the effect of experimental diabetes and salvifolin effect on the permeability of rat liver mitochondria.
STZ+sahdfolin+CyclA 1 Comtrol+CycLA
Fig. 2. Effects of the pharmacotherapy of salvifolin on mPTP opening rat liver mitochondria at STZ-induced diabetes
(Changing of calcium-induced mitochondrial swelling in liver mitochondria isolated from intact rats, STZ-induced diabetes rats and salvifolin treated rats (STZ-induced diabetes+salvifolin). Mitochondrial increase in volume was determined by fallowing the decrease in the absorbance at 540 nm of the mitochondrial suspension. The small increase in absorbance is due to the formation of calcium complexes in the mitochondrial matrix. Addition of cyclosporine A 4 ^M, P<0,05. n=4.)
In our experimental conditions used (incubation medium containing Ca-EGTA buffer) swelling of mitochondria can be seen as the result of an open state of mPTP, and the suppression of swelling — as a closed, ie, using this technique, you can assess the condition of mPTP in the STZ-induced diabetes and action salvifolin. Adding to the incubation medium +50 ^M CaCl2 leads to swelling of mitochondria and liver of rats in group I (Fig. 2). This swelling rate of liver mitochondria was 0,19 AE540/ 5min, respectively. In those conditions, the rate of swelling of mitochondria isolated from rat liver group II (STZ-induced diabetes), was equal to 0,49 AE540/ 5min, which is 157,8±4,7% higher than in the control group (Fig. 2).
Since we used conditions the swelling of mitochondria can be regarded as the opening of mPTP, the results indicate that in STZ-induced diabetes mPTP liver are in the open state. Pharmacotherapy salvifolin rats with STZ-induced diabetes is associated with marked contact inhibition of liver mitochondrial swelling. Thus, the rate of swelling of mitochondria isolated from the liver of rats of group III STZ-induced diabetes+salvifolin) was 0.27AEJ40/5min that 116,0±4,3% less than the rate of swelling of the rat liver mitochondrial group II (Fig. 2). Under these conditions, the classic mPTP inhibitor cyclosporin-A at the concentration 4 ^M full inhibited the mitochondrial swelling, isolated from liver I and III groups of rats, in contrast to II groups.
Thus, the STZ-induced diabetes causes, including the development of mitochondrial dysfunction, manifested opening mPTP. Pharmacotherapy rats with STZ-induced diabetes salvifolin corrects mitochondrial dysfunction, effectively influencing the state of mPTP.
Our experimental results indicate that diabetes with STZ seriously impairs the function mitoKATP channel rat liver mito-
chondria (Fig. 3). Experiments have shown that in the absence ofATP in the incubation medium, with STZ-induced diabetes activity mitoKATp channel is increased by 20±4,5% as compared to that of an intact rat group. In the presence of ATP, in experimental diabetes mitoKATp channel liver becomes more open state, ie, the rate of swelling of mitochondria in rat liver group II up 92,1±4,8% than the group intact mitochondria (Fig. 3). In diabetes, liver mitoKATP channel becomes more open state, ie, the swelling rate of the rat liver mitochondrial group II is higher by 88,5±2,5%, than the control group mitochondria (Fig. 3).
Pharmacotherapy salvifolinom corrects pathological change in function mitoKATp channel: wherein the rate ofswelling of rat liver mitochondria group III was inhibited in the absence ofATP to 9,4±0,6% and in the presence ofATP to 65,7±4,4% in comparison with swelling rate ofmitochondrial group II (Fig. 3).
Thus, at the STZ-induced diabetes occurs the opening the mitoKATp channel which leads to increased transport of K+ ions into the mitochondrial matrix, changes in potassium homeostasis cytosol and mitochondrial membrane potential, as well as uncoupling of oxidation and phosphorylation. The pathological process, ie, mitoKATp channel open in experimental diabetes may develop as a result of reducing the concentration of adenine nucleotides.
It is known that the protein components mitoKATp channel — mitoSUR mitoKIR and regulates the state channel inhibitors mitoKAKATP channel acts on these components [1, 101-127]. It is possible we studied diterpenoid salvifolin also interaction regulatory sites of the channel — mitoSUR, as a result of the channel is inhibited.
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3
Fig 3. The effect of salvifolin on mitoKATP channel of the rat liver mitochondria with normal and STZ- diabetes
1 — intact, 2 — STZ-diabetes (control), 3 — STZ-diabetes+salvifolin. *P<0,05; **P<0,01; n=4
Antidibetic drugs sulfonylurea have an inhibitory effect on the receptors mitoSUR, resulting mitoKATP been closed channel [13, 961-965; 14, 13578-13582]. However, there is no data in the literature about the mechanisms of action of modulators mitoKATP channel liver in experimental diabetes. Obviously, in a diabetes affects not only pancreatic tissue, heart, kidney and brain [15, 807-812; 16, 1-9; 17, 31333147], but other tissues of the body.
Thus, at the experimental diabetes respiration and coupled phosphorylation of rat liver mitochondria are seriously damaged. In this accelerated rate of oxygen consumption in states V3 and preferably V4, RC coefficient decreases when the oxidation of glutamate+malate and succinate compared with analogical mitochondrial indices of I group rat liver.
Under the influence of the body salvifolina STZ-induced diabetic rats a decrease in mitochondrial respiration rate noted in both states. However, despite pharmacotherapy by salvifolin, these rates of respiration were higher than the control groups. At the pharmacotherapy salvifolin also observed an increase in the coefficients of RC and ADP/O, indicating that phosphorylation of effective function of liver mitochondria at effects of salvifolin. Thus, the identified corrective
action salvifolin to functional impairment of mitochondrial STZ-induced diabetes rat, which is reflected in the restoration which is reflected in the restoration of the state of coupling of respiration and phosphorylation.
We have also revealed that at the STZ-induced diabetes mPTP switches to state of high permeability, which first found authors [18, 519-523; 19, 231-237]. The observed swelling of mitochondria us, ie mPTP high permeability transition in the state in experimental diabetes, may be a result of the membrane are interconnected processes: lipid peroxidation, oxidation of the thiol groups mPTP, decrease in membrane potential, the formation of fatty acids and free radicals. It is possible the mechanism of inhibition mPTP salvifolin, is the reduction of the above processes or interaction with cyclophilin D, which is located in the matrix of mitochondria.
We have shown, that at the STZ-induced diabetes occurs the opening the mitoKATP channel. Perhaps, the opening mitoKATP channel leads to increased transport of K+ ions into the mitochondrial matrix, changes in potassium homeostasis cytosol and mitochondrial membrane potential, as well as uncoupling of oxidation and phosphorylation. The pathological process, ie, mitoKATP channel open in experimental diabetes
may develop as a result of reducing the concentration of adenine nucleotides.
It is known that the protein components mitoKATP channel — mitoSUR mitoKIR and regulates the state channel inhibitors mitoKATP channel acts on these components [1, 101-127]. It is possible we studied salvifolin also interaction regulatory sites of the channel — mitoSUR, as a result of the channel is inhibited.
Conclusions
Thus, in STZ-induced diabetes impairs the function of rat liver mitochondria as the mitochondrial respiration, the functional state of mPTP and mitoKATP channel. Pharmacotherapy diterpenoid salvifolinom has a protective effect on mitochondria in experimental diabetes, correction membrane disorders.
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