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Section 6. Physiology
https://doi.org/10.29013/ELBLS-20-4-79-84
Pozilov Mamurjon Komiljonovich, Senior research, Institute of Biophysics and Biochemistry at the National University of Uzbekistan named after Mirzo Ulugbek E-mail: mamurjon2281@mail.ru Ernazarov Zafar Mamurovich, Junior research, Institute of Biophysics and Biochemistry at the National University of Uzbekistan named after Mirzo Ulugbek E-mail: Zafarbek1985@gmail.com Raximov Akmal Dilshod o'gli, Institute of Biophysics and Biochemistry at the National University of
Uzbekistan named after Mirzo Ulugbek Kukanova Nargiza Fahritdinovna, Institute of Biophysics and Biochemistry at the National University of
Uzbekistan named after Mirzo Ulugbek Asrarov Muzaffar Islamovich, Professor, Institute of Biophysics and Biochemistry at the National University of Uzbekistan named after Mirzo Ulugbek Makhmudov Rustamjon Rasuljonovich, A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan
INFLUENCE OF NATURAL POLYPHENOLIC COMPOUND GOSSITAN ON ION CHANNELS OF MITOCHONDRIA OF THE HEART AND PANCREAS IN STREPTOZOTOCIN-INDUCED DIABETES
Abstract. The influence of natural polyphenolic compound of gossitan on mitochondrial function was investigated. It was shown that in streptozotocin (STZ)-induced diabetes damaged functional systems of rat hearth and pancreas mitochondria: mitochondrial permeability transition pore (mPTP) and ATP-dependent potassium channel (mitoK -channel). Pharmacotherapy with gossitan (intraperitoneally in dose of10 mg/kg body weight) for 8 days has a protective effect on mitochondria in experimental diabetes, correction membrane disorders.
Keywords: Hearth, pancreas, mitochondria, mPTP, Streptozotocin-induced diabetes, gossitan.
The biological activity of polyphenol compounds isolated from plants is very high, and their mechanisms of action vary with each other [1, 1-3]. Quercetin, campherol, and epicatechins inhibit the formation of H2O2 in the mitochondria and the activity of the mitochondrial respiratory-chain complex 1 [2, 1562-1572]. Polyphenol compounds restore morphological changes in the mitochondrial matrix in experimental diabetic conditions. It also reduces the formation of free radicals, increases ATF synthesis and insulin resistance [3, 3135-3136].
Polyphenols are also effective in mitochondrial bioenergetics dysfunction, inhibition of uncoupling protein (UCP) activity, mPTP, and damage to proteins, and lipids that causes changes in various diseases [4, 67-78]. Although the antidiabetic activities of these compounds have been extensively studied, their effects on mitochondrial functional changes remain unexplored.
We know that mitochondria are significantly damaged in heart muscle injury in diabetes and ischemia. In diabetes, disruption of the homeostasis of calcium ions in the mitochondria occurs with the activation (or opening) of nonspecific pores in the inner membrane of the mitochondria, with the loss ofvarious substances and ions from the mitochondrial matrix, as well as changes in the outer membrane [5, 1009-1010].
These processes can result in decreased cellular ATF synthesis, significant changes in mitochondrial membranes, and consequent cell death. The formation of nonspecific pores associated with calcium ions in the mitochondria and the formation of reactive oxygen species (ROS) directly accelerates the process of cell death [6, 874-875] At present, although the role of cardiac mitochondria in the role of mitochondria in the pathogenesis of diabetes has been Despite extensive research, their correction with polyphenol compounds has not been adequately studied.
The aim of the study was to study the corrective effect of polyphenol gossitan [7, 109-110] iso-
lated from the Gossypium hirsutum L. plant on the dysfunction of mPTP and mitoKATP-channel of rat heart and pancreas mitochondria under conditions of STZ-diabetes.
Material and Methods. For screening and detailed study of the mechanism of action of pharmacological agents are widely used various experimental models of diabetes caused by administration of alloxan and STZ al., cytotoxic activity on (3-cells of the pancreas. We have in this study used an experimental model of diabetes induced by STZ.
Experiments were performed white mongrel male rats weighing 180-200 g. The animals were divided into three groups: I group - control, 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) and III group - STZ-induced diabetes+gossitan (intraperitoneally dose of10 mg/kg body weight) for 8 days starting from 12 days after administration of STZ and reaching a predetermined level ofhyperglycemia. Blood glucose was determined using glucose oxidase method set «Glucose - enzymatic-colorimetric test» (Cypress diagnostic, Belgium).
Mitochondria isolated from rat hearth and pancreas by differential centrifugation according to [8, 30-32; 9, 71-72]. Nuclei and cellular fragments were removed by centrifugation at 600 g for 7 minutes in a centrifuge. The mitochondria are pelleted at 7000 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 Ca2+ -dependent swelling of mitochondria, the mito-chondrial suspension recording light scattering at 540 nm. Experiments at 26 °C in a swelling medium of 200 mM sucrose, 20 ^M EGTA, 5 mM succinate, 2 ^M rotenone, 1 ^g/ml oligomycin, 20 mM Tris, 20 mM HEPES, and 1 mM KH2PO4, pH 7,2 [10, 16755-16760]. The concentration of mitochondria in the swelling experiments was 0,5 mg protein/ml.
The content of mitochondrial protein was determined by the Lowry method in the modification of the Peterson [11, 346-347].
Mitochondrial swelling induced activation mito-KATp-channel was recorded using a change in light scattering at a wavelength of 540 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 m M K2H-PO4, 2,5 mM KH2PO4, rotenone 1 ^M/ ml, oligomy-cin 1 ^/ml, pH 7,4.
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Figure 1. The effect of gositan on mitochondrial
As the result of diabetes the is increased Ca2+ retention capacity on cardiac mitochondria and a loss of membrane stability was observed, which provides a state of high permeability of mPTP. As a result, oral supplement of gossitan polyphenols (10.0 mg/kg) once daily for 8 days for STZ-diabetic rats was revealed and remarkable decrease can be seen on the swelling of mitochondria isolated from the heart. As a consequence, distend of mitochondria under the influence of gossitan was inhibited by 51.9±4.8% compared with STZ-diabetes (group II) was found (Fig. 1). The main causes for the opening of mPTP in STZ-diabetes include the development of oxidative stress, prooxidants, LPO induction, and oxidation of thiol groups in the mPTP complex. Due to the strong antiradical property of polyphenolic com-
The results were statistically processed using the Origin 6.1 program. The P value < 0.05 was considered as an indicator of significant differences.
Results and discussion. The investigation on the effect of hypoglycemic polyphenol gossitan on mitochondrial distend isolated from animal hearts, called the STZ-diabetes model, was studied. A concentration of 20 ^M Ca2+ ions was applied as an inducer for the induction of cardiac mitochondrial swelling. The results obtained, STZ-diabetes (group II), the distant of mitochondria increased by 86.1 ± 7.1% compared with control of (group I) was determined (fig. 1).
swelling in the heart of a rat with STZ-diabetes
pounds, it can reduce the amount of free radicals in mitochondria and possess capability of controlling the inhibitory properties of cyclosporin A by binding to the CyP-D matrix domain.
Through our next experiment the effect of the polyphenol gossitan on the pancreas mitochondria in the conditions of STZ-diabetic rats was also investigated. STZ is estimated as a specific target of pancreas, its plasma membrane can increased LPO both disrupt the activity of ion channels. On the other hand, it intensifies the generation of free radicals and causes impaired insulin secretion as a result of reduced ATF synthesis in the mitochondria.
The pharmacotherapy on animals with STZ-diabetes were per os with polyphenolic compounds. Once glucose levels approached towards normal position,
Group I control
Group III STZ-diabetes+gossitan Group II STZ-diabetes
they were decapitated and mitochondria were isolated pancreas mitochondria increased by 95.5±5.4% com-from pancreas. The results obtained, the distend of the pared with the control in STZ-diabetes (Fig. 2).
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STZ-diabetes STZ-diabetes+gossitan
Figure 2. The effect of gossitan on pancreas mitochondria swelling with STZ-induced diabetic rats (*p<0.05; **p<0.01; n=5)
After conducting pharmacotherapy on animals of group III with STZ-diabetes with gossitan polyphenols was apparent that their mitochondrial swelling pancreas was inhibited by 37.1±2.7% contrasted with group II. Polyphenolic compounds within possession of hypoglycemic properties which in experiments, can recreated mitochondrial dysfunction of pancreas in diabetes conditions.
Effect of gossitan on activity mitoKATP-channels in mitochondria of rat heart in experimental diabetes. According to modern researches, owing to priority
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of the functional significance of the mitoKATP-channel in ischemia and hypoxia it has been frequent studied in experiments. MitoKATP-channels is considered as a selective channel in mitochondria and plasma membrane, usually both channels exist common activators and inhibitors. Although such a pharmacological agents currently have been identified in a large scope, the selectivity of the modulator effectiveness actually depends on the type of cells which is investigated and the experimental conditions.
Control
STZ-diabetes
STZ-diabetes+gossitan
Figure 3. Effect of gossitan on rat heart mitoK -channel with STZ-induced diabetes (*p<0.05; n=6)
It can be hardly found data on modulators that affect the activity of the mitoKATP-channel of the liver and heart in experimental diabetes. It is stated only by a number of scientists that glibenclamide owe a productive effect on the activity of the mitoKATP-channel in diabetes [12, 485-488; 13, 101-105]. At present, according to the latest literature data, the mechanisms of inhibition of the KATP-channel of the plasma membrane of pancreatic cells in diabetes mellitus are widely studied [14, 161-162]. However, changes in the flow of K+ ions in the mitochondrial channel of the heart in diabetes and the polyphenolic compounds affecting them have not been sufficiently studied. In this regard, our experiments possess significant value in studying the effect of gossitan polyphenol in in vivo experiments in condition diabetes as well as pathological changes in the mitoK -channel.
It was obvious through the results defined that the mitoKATP-channel isolated from the hearts of animals with STZ-induced group II diabetes was inhibited by 21.6±1.8% in comparison group I (Fig 3.). Inhibition of the mitoKATP-channel channel of the heart, in contrast to the mitoKATP-channel channel of the liver, causes a deficiency energy supply in both normal and pathological conditions.
A decrease in the size of the cardiac mitochondrial matrix is estimated as the result of inhibition of
KATP-channels leads slowing down of respiration, and a sharp decrease in ATF synthesis could be noticed. When STZ-diabetic group III animals were treated with gossitan, the activation of the mitoKATP-channel separated from their heart was detected during the investigation. Consequently, the polyphenols of gossitan were productively effected in the mitochondrial channel of the heart in STZ-diabetes. Gossitan activates the reduction in the permeability of the channels as a result of diabetes. As a result, it can inhibit oxidative stress can emerge apoptosis in cardiomy-ocytes. In experiments modulators are exist within capability of various effects on plasma and mitoKATP-channels. They act as an activator of the KATP-channel of the plasma membrane of smooth muscles, as for mitoKATP-channel it functions as an inhibitor as well [15, 1183-1185]. It requires deep further investigation on the effect of polyphenolic compounds on the mitochondrial channel of the heart as an inhibitor and activator of condition of diabetes.
Conclusions: Gossitan polyphenol could afford to inhibite mPTP in rat liver and pancreas mitochondria in STZ-induced diabetic conditions.
By applying the polyphenols of gossitan to STZ-induced diabetic rats, their mitochondria which is seperated from their heart, the activeness of the mi-toKATP-channel was determined.
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