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Niyazmetov Bahodir Allaberganovich, teacher, National University of Uzbekistan E-mail: [email protected] Abdullaev Gaffurjon Rakhimjonovich, doctor, of biological sciences, Namangan State University E-mail: [email protected] Babadjanova Sayyora Khushnutovna, candidate, of biological sciences, docent Urgench State University E-mail: [email protected] Almatov Karim Tajibayevich, doctor, of biological sciences, professor, National University of Uzbekistan
REGULATION OF THE ENERGETIC FUNCTION AND REACTIVE OXYGEN SPECIES FORMATION IN MITOCHONDRIA BY DAIDZIN
Abstract: In vitro assays, it was found that daidzin does not affect the respiratory function of mitochondria and the rate of Chance's respiratory control, but dose-dependent increases ADP/O ratio. This process is especially noticeable in the oxidation of succinate. Daidzin inhibits the processes of lipid peroxidation and the activity of lytic mitochondrial enzymes. This means that daidzin increasing bilayer areas of mitochondrial membranes, stabilizes the compactness of membranes. In this regard, the availability of exogenous phospholipase A2 cobra venom to the phospholipids of mitochondrial membranes is difficult.
Keywords: daidzin, liver, mitochondria, glutamate, succinate, ADP/O ratio, cobra venom, phospholipase A2.
1. Introduction The pharmacological action of these biologically active
Daidzin is a main isoflavone isolated from Pueraria substances is associated with antioxidant, antidipso-phaseoloides and is used to neutralize the side effects as- tropic and other effects [6]. In one study, it was found sociated with excessive drinking [1]. In in vitro studies that daidzin has a protective effect against carbon tetra-dadzin has proved to be a potent and selective inhibitor chloride, D-GalN - and t-butyl hydroperoxide - caused of mitochondrial aldehyde dehydrogenase (ALDH-2) by hepatotoxicity in vitro [7]. In this study, mice in the [2], which is the main isoform of ALDH catalyzing de- D-GalN / LPS-treated group began to die 6 hours lat-toxification ofethanol-dependent acetaldehyde. In recent er, and the death rate reached 73.3% (11 of 15 mice) studies, daidzin inhibited LPS-dependent production of after a day. Eight hours after the D-GalN / LPS injec-TNF-a in mice [3]. In addition, its antioxidant activity tion, severe necrosis was observed because of D-GalN / was investigated and established in various studies [4]. LPS-induced transient (acute) liver failure. It was found According to these studies, it can be assumed that dadzin that the level of serum aminotransferase significantly in-can have a protective function against the D-GalN / LPS creased. The results show that dadzin protects mice from caused hepatic insufficiency. D-GalN / LPS-induced acute hepatic insufficiency, the
In one research, it was studied a reducing activity evidence of which is a marked reduction in mortality and of daidzin on GalN / LPS-induced hepatic failure [5]. serum aminotransferase in a dose-dependent manner.
Undoubtedly, hepatic histopathology clearly showed that D-GalN/LPS-induced hemorrhage, necrosis and degeneration of hepatocytes, improved dramatically in mice pre-treated with daidzin. Although Hasumuma et al. in the study showed that daidzin does not have a protective effect against D-GalN/LPS-mortality [8].
Daidzin is nuclear receptor that is activated by fatty acids, prostaglandins, and functions as a transcription factor. Daidzin inhibits tumor necrosis factor by regulating positively the prostaglandins [9]. In contrast, the 6- and 8-chlorination of daidzin significantly reduced the agonist effect on prostaglandins [10]. In in vitro assays, it was found that the daidzin had the power to block the saturation of tyrosine with iodine [11]. Daidzin react with peroxisomes of proliferative receptors, activated with fatty acids [9].
Mitochondria are organelles, not only providing energy to the organism, but also supporting the dynamic interconnection of metabolism. Mitochondria possess all the basic functions of the cell: contractility, transport of ions, heredity, etc. [12, 13].
The aim of our research was to study the effect of daidzin on respiration and oxidative phosphorylation, the activity of oxidase systems and the formation of reactive oxygen species in liver mitochondria.
2. Materials and methods
The experiments were carried out on Wistar male rats with an initial body weight of 180-200 g. Mitochondria from rat liver cells were isolated according to the method of [14]. All procedures for the isolation of mitochondria were carried out at 0-2 ° C. The energy parameters of isolated mitochondria were studied by B. Chance, G. R. Williams [15].
The experiments were performed with the addition of daidzin into a polarographic cell. The study ofthe activity of rotenone sensitive and insensitive NADH-oxidase systems, as well as mitochondrial succinate oxidase, was carried out by K. T. Almatov et.al [16]. The process of lipid peroxidation of mitochondria were determined by the method [17]. Calculation of the content of products reacting with thiobarbituric acid was carried out taking into account the molar extinction of malonic dialdehyde equals to 1.56 x 106 mol cm-1 and expressed in nanomol malonaldialdehyde / min mg protein. Daidzin was used as a glycerin solution and was introduced into a polarographic cell (at a final concentration of 20, 40, 60 ^g /
mg of mitochondrial protein) and studied the features of the alteration in the functional state of mitochondria. The mitochondrial protein was determined by Lowry et al. [18]. The results are processed using the Student's t-test and its determination of the arithmetic mean M and its standard error m. Experiments were performed with the addition of daidzin into the polarographic cell. Daidzin was purchased by Shijiazhuang Zulei commerce Co., Ltd. (China).
3. Results and discussion
The data presented in (Table 1) show that daidzin in low concentrations does not affect the rate of glutamate oxidation. However, higher daidzin concentrations slightly decrease the oxidation of glutamate in various metabolic states. In this case, the values of the respiratory control of Chance do not change. With the introduction of daidzin in a dose of 20, 40 and 60 ^g/mg protein in a suspension of mitochondria, ADP/O ratio increases by 8.6; 11.5 and 12.3%, respectively, of the control level. This means that daidzin stimulates the exchange of adenine nucleotides (ATP4-/ADP3-) between the mitochondrial matrix and cytosol, carried out by a special transport system - translocase.
Dadzin does not affect the oxidation of succinate and the rate of the respiratory control of Chance, but the dose-dependent increase in the ADP/O quotient. Daidzin, at a dose of 20, 40 and 60 ^g/mg of mitochondrial protein increases the ADP/O quotient by 18.2; 35.1% and 52.7%, respectively, from the control.
In the presence ofdaidzin, the increase in the ADP/O quotient with succinate in the mitochondria of the liver, in our view, connects with the phenomenon of "reverse electron transfer" (according to the recovery of NAD+) [19]. A significant advantage of succinate in maintaining a high level of energy-dependent NADH as compared to NAD-dependent substrates as the main factor determining the special role of succinate in energy supply.
In our opinion, an increase in ATP content in mitochondria leads to the closure of the cyclosporin A sensitive pore.
Recently it has been shown that decrease of intracellular content of ATP level by only 15-20%, the intensity of all energy-dependent cell functions falls on 75-80% of the initial value, which leads to the development of multisystem pathologies: violations of the central nervous system, heart function, the synthetic
processes of the liver, kidneys, etc., Ability of the cell transmembrane potentials, synthetic processes, etc.) to support specific energy-dependent functions (neu- is located in close dependence on the intracellular con-rotransmitter, receptor, contractile, ion transport and tent of ATP.
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2 3 V4 Vdnp RC(V3:V4) ^DR/T
Figure 1. Influence of daidzin on respiration and oxidative phosphorylation in liver mitochondria (glutamate as a substrate)
t? 120-
iï
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II
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V„_ RC(V3:V4) ADP/f
Figure 2. Influence of daidzin on respiration and oxidative phosphorylation in liver mitochondria (succinate as a substrate)
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Figure 3. Influence of daidzin on activity of rotenone-sensitive and insensitive oxidases and succinic oxidase in mitochondria
60-
40-
20-
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80-
40
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V
V
2
3
4
Thus, daidzin does not affect the respiratory function, but it increases the ATP synthesizing function of mitochondria. Synthesis of ATP is particularly noticeable in the succinate pathway of the mitochondrial respiratory chain.
The effect of various biologically active substances on mitochondrial membranes can be determined by changes in the activities of the rotenone-sensitive and insensitive oxidases and succinic oxidase. The physiological significance of these enzymes lies not only in their participation in biotransformation of energy and transport of electrons through the respiratory chain to molecular oxygen, but also in regulating the rate of elimination of the arrival of substances and metabolites, as
well as changes in the conductivity and contractility of mitochondrial membranes [19].
4. Conclusion
To study the effect of daidzin on rotenone-sensitive and insensitive oxidases and succinic oxidase activities, 20, 40 and 60 ^g of daidzin were added to each mg of mitochondrial protein into the polarographic cell and the results of the oxidase activity change are shown in Fig. 2. From obtained results it is obvious that daidzin doesn't influence on activity of mitochondrial oxidases. Hence, daidzin enhances the synthesis of ATP without increasing the rate of oxygen and substrate consumption in the mitochondrial respiratory chain. In our opinion, daidzin increases the activity of adenenucleotide transferase.
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