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ИНГИБИРУЮЩЕЕ ДЕЙСТВИЕ РАСТИТЕЛЬНОГО ФЛАВОНА ГИПОЛАЭТИНА-8-ГЛИКОЗИДА НА МИТОХОНДРИАЛЬНУЮ ПОРУ ПРОНИЦАЕМОСТИ ПЕЧЕНИ КРЫС ПРИ
АЛЛОКСАНОВОМ ДИБЕТЕ
М.И.Асраров1, Н.Б.Курбанова2, М.К.Позилов2 ^Институт биофизики и биохимии при Национальном университете
Узбекистана имени Мирзо Улугбека Национальный университет Узбекистана имени Мирзо Улугбека, Адрес: 100174, г.Ташкент, Алмазарский район, Вузгородок, ул. Университетская, 174.
Соответствующий автор email: asrarov54®>mail.ru Адреса электронной почты соавторов/ mamurion2281 &>mail.ru
Аннотация. Показано, что флавон гиполаэтин-8-глюкозид оказывает гипогликемический эффект у белых крыс с аллоксановым диабетом. При пероральиом введении гиполаэтин-8-глюкозида в течение 10 дней в дозе 10,0 мг/кг/день массы тела лабораторных животных, снижается индуцированное Ре2+/цитратом перекисное окисление липидов (ПОЛ) и ингибируется открытие митохондриальной поры проницаемости (шРТР) в митохондриях печени у диабетических крыс.
Ключевые слова: митохондрии, аллоксановый диабет, пора проницаемости, гиполатин-8-глюкозид, рутин.
THE INHIBITORY EFFECT OF PLANT FLAVON HYPOLAETIN-8-GLUCOSIDE ON THE LIVER MITOCHONDRIAL PERMEABILITY PORE IN ALLOXAN-INDUCED DIABETIC
RATS
M.I.Asrarov1, N.B.Kurbanova2, M.K.Pozilov2
'Institute of Biophysics and Biochemistry at the Mirzo Ulugbek National University of Uzbekistan 2National University of Uzbekistan named after Mirzo Ulugbek Tashkent, Uzbekistan Address: 100174, Tashkent city, Almazar district, Student's town, University St. 174 *Corresponding author email: asrarov54@mail.ru E-mail addresses of co-authors: mamurion2281 &>mail.ru
Annotation. The flavone hypolaetin-8-glucoside shown to have a hypoglycemic effect in white rats with alloxan diabetes. Oral administration of hypolaetin-8-glucoside for 10 days at a dose of 10.0 mg/kg/day of body weight in laboratory animals reduces Fe2+/citrate-induced lipid peroxidation (LPO) and inhibits the opening of the mitochondrial permeability pore (mPTP) in liver mitochondria in diabetic rats.
Keywords; mitochondria, permeability transition pore, alloxan diabetes, hypolaetin-8-glucoside, rutin.
Introduction
According to the latest data from the World Health Organization, the prevalence of diabetes is increasing. Determining the pathogenesis of diabetes,
searching for pharmacological drugs against them and researching mechanisms of hypoglycemic action remain one of the most urgent problems.
It is important to study the membrane disorders at the cell and organelle level and correct them with plant polyphenols in diabetes conditions.
Normally, insulin is secreted from ß-cells of pancreatic islets. Insulin increases glucose uptake in muscle and adipose tissue, while decreasing glucose production in the liver [1]. Insulin stimulates cell growth and differentiation, enhances lipogenesis, glycogen and protein synthesis. It also inhibits lipolysis, glycogenolysis and breakdown [2]. As a result of impaired utilization of glucose through the glucose transporter in the plasma membrane of the pancreas, there is a decrease in ATP synthesis and membrane depolarization does not take place. As a result, the Ca2+ channel is inhibited in the membrane, potassium channels are activated, and insulin secretion does not take place [3].An increase in the amount of glucose in the blood affects the activity of various organs as a factor of oxidative stress. In particular, it has a harmful effect on the enzymatic system of the liver, protein exchange and mitochondrial metabolic processes. The mitochondrial membrane regulates a variety of cellular functions, including Ca2+ signaling, lipid metabolism, production of reactive oxygen species, mitochondrial dynamics, and cell apoptosis. These functions are necessary to ensure the physiological function of the organelle, as well as to control homeostasis in cell metabolism. Mitochondrial dysfunction is responsible for the pathogenesis of many diseases [4]. Ca2+ ions are important for the maintenance of ion homeostasis in mitochondria, bioenergy production and the functional state of the cell (survival), in addition to participating in the signaling system in cells. This is because
several mitochondrial enzymes involved in the synthesis of ATP through the tricarboxylic acid cycle are controlled by Ca2+ ions. For example, a-ketoglutarate dehydrogenase, isocitrate dehydrogenase, pyruvate dehydrogenase and ATP synthase [5]. At the same time, prolonged or excessive accumulation of Ca2+ ions in the matrix can lead to the opening of the mitochondrial permeability transition pore (mPTP) and subsequent release of pro-apoptotic factors [6].
In diabetic conditions,
mitochondrial respiratory dysfunction and mPTP opening opening is determined [ As a result of energy deficiency, the overall functional activity of cells, tissues and organs is disturbed. This makes it necessary to search for biologically active substances that significantly reduce the amount of glucose in the blood and correct the functional state of tissues in diabetes.
One of these biologically active substances is rutin flavonoid, whose antidiabetic effect is manifested by reducing the absorption of carbohydrates from the small intestine, improving glucose absorption by tissues, suppressing gluconeogenesis, activating insulin secretion in p-cells and protecting the islets of Langerhans. Rutin significantly reduces the level of glucose in the blood plasma [8]. Oral administration of rutin to diabetes model animals activates liver hexokinase enzyme involved in gluconeogenesis and lipid metabolism [9]. Currently, the hypoglycemic activities of many flavonoid compounds studied and antidiabetic drugs are being prepared based on them.
At the experiments in vitro has been shown antioxidant and antidiabetic properties of the hypolaetin-8-glucoside and another flavonoids [10], wherein
presence of C-2-C-3 double bond and C-4 kenotic group are two essential structural features in the bioactivity of flavonoids especially for antidiabetic property. However, mechanisms of cytoprotective and membrane-active effects of flavonoid compounds at the level of membranes have not been fully explored. One such flavonoid compound is hypolatin-8-glucoside, whose effect on liver mitochondrial dysfunction in alloxan diabetes has not been studied.
The purpose of this work. The effect of hypolaetin-8-glucoside flavon on the case of mPTP and lipid peroxidation of rat liver mitochondria research comparatively with rutin flavonoid in condition of alloxan diabetes.
Research materials and methods. Experiments was researched in without breed, weight 180-200 gr male white rats. Experimental animals were consisted of IV groups: I group - healthy (control), II group - experimental (alloxan diabetes), III group - experimental (alloxan diabetes + hypolaetin-8-glucoside) and IV group - experimental (alloxan diabetes + rutin). Alloxan 150 mg/kg (0.2 ml) solution was injected once into the subcutaneous area of the abdomen in group II, III and IV laboratory animals after one day of fasting to induce diabetes.
After alloxan injection to rats, 12 days later, after the blood glucose level exceeded 11 mmol/1 once a day, animals of group II were given 0,2 ml of 0,9% NaCl solution, group III of the experiment hypolatin-8-glucoside flavonoid 10,0 mg/kg and rutin to group IV flavonoid in in the amount of 20 mg/kg was administered orally (per.os) for 10 days. The amount of glucose in the blood was determined by the glucose oxidase method ("Glucose - enzymatic-
colorimetric test", Cypress diagnostic, Belgium). Rat liver mitochondria were isolated by differential centrifugation.
Kinetics of mitochondrial swelling (0.3-0.4 mg/ml protein) was determined by spectrophotometer
(spectrophotometer V-5000) at 540 nm in an open cell (volume 3 ml) with constant stirring of the mitochondrial suspension at 26°C. was determined. The following incubation medium (IM) was used to determine mitochondrial PTP permeability: 200 mM sucrose, 20 |iM EGTA, 5 mM succinate, 2 |iM rotenone, 1 |ig/ml oligomycin, 20 mM Tris, 20 mM HEPES, and 1 mM KH2P04, pH 7.4 [11].
We used from Fe2+/citrate system to study the process of LPO in the mitochondrial membrane and the mitochondrial volume change was determined photometrically in the experiments. Incubation medium (mM): KCl - 125, KCl -65, HEPES -10, pH 7.2; The amount of mitochondria is 0.5 mg/ml; Mitochondria were incubated for 2 min in a medium containing 2 mM citrate before adding 50 [iM Fe2+ [12].
In the experiments, the kinetics of mitochondrial swelling was calculated as a percentage of the maximum, as the arithmetic mean value of 4-5 different experiments was calculated. Statistical processing of the results and drawing of pictures were performed using the Origin 6.1 (USA) computer program. Where *P<0.05 and **P<0.01 values represent statistical reliability.
The obtained results and their analysis. Several flavonoid compounds are strong antioxidants and antiradicals, and can neutralize free radicals produced during pathological processes. Flavonoid compounds in diabetic conditions increase ATP synthesis, increase resistance to insulin resistance and restore mitochondrial dysfunction [13]. It
has been extensively studied that these compounds have antidiabetic properties, but their effect on mitochondrial function is yet to be investigated. We compared hypolaetin-8-glucoside flavonoids
isolated from plants with rutin flavonoids on rat liver mitochondrial mPTP and LPO processes. Under alloxan diabetes
Ca2+
conditions initially examined the effects of hypolaetin-8-glucoside flavon on the contraction of rat liver mitochondria in the presence of CaCh. When a coconcentration of 20 [iM of CaCh is added to the incubation medium, mitochondrial inhibition is taken as a control (100%) (Fig. 1).
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Control (healthy)
Alloxan diabetes+ hypolatin-8-glucoside Alloxan diabetes+rutin
Alloxan diabetes
Figure 1. Effects of hypolaetin-8-glucoside and rutin on Ca2+ ion-induced swelling of rat liver mitochondria under alloxan diabetes conditions. Original recording of the recording of changes in the light transmittance of the mitochondrial suspension.
A comparison of the alloxan diabetes group with the control group (group I) revealed that the number of mitochondria isolated from rat liver increased by 79,9%. The glucose level in group 111 animals with alloxan diabetes decreased to a normal level as a result of administration of hypolaetin-8-glucoside flavonoid (orally once a day during 10
days). The inhibition of mitochondria isolated from the liver was significantly decreased by 52,7% as compared to alloxan diabetes when Ca2+ ions were present was found (Fig. 2). In group IV animals with alloxan diabetes, ruton flavonoid administration inhibited the throttling of liver mitochondria with Ca2+ ions by 34.5% (Fig. 2).
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4 -
2 -
O
**
□ Control (healthy) 3 Alloxan diabetes
3 Alloxan cliabetcsH hypolatin-8-glucoside 3 Alloxan diabetes+rutin
** -J—
* -Ï-
Figure 2. Effects of hypolaetin-8-glucoside and rutin flavonoids on Ca2+ ion-induced swelling of rat liver mitochondria under alloxan diabetes conditions. On the
ordinate axis - the division of mitochondria is shown based on the optical density change of DA540, on the abscissa axis - the experimental groups are presented; *P<0.05; **P<0.01; n=5.
The obtained results indicate that in alloxan diabetes, the conformation of rat liver mitochondria PTP in a highly conductive state may be the mechanism of disruption of cell and mitochondrial ion homeostasis and reduction of ATF synthesis. Thus, hypoglycemic activity hypolatin-8-glucoside flavonoid, alloxan, reduces mPTP permeabilization by reducing mitochondrial damage in diabetic conditions.
Diabetes results in a sharp increase in mitochondrial number and LPO of mitochondrial membranes in both the inner and outer membranes. As a result of mitochondrial damage, the LPO process in its membrane is accelerated, the membrane potential decreases, and ion permeability may change. The
formation of free radicals as a result of LPO is of great importance. Increased generation of ROS from the respiratory chain in mitochondria not only weakens the antioxidant system, but also damages the lipoprotein parts of membranes. In conditions of experimental diabetes and many other pathologies, LPO develops on the basis of oxidative stress. In the conditions of experimental diabetes, the role of biologically active substances in reducing the intensity of LPO is incomparable.
In our next experiment, the effects of hypolaetin-8-glucoside and rutin flavonoids on the inhibitory effect of alloxan on hepatic mitochondrial function in diabetes on the Fe2+/citrate-induced LPO process were investigated (Fig. 3).
Figure 3. Effect of hypolaetin-8-glucoside and rutin flavonoid on Fe2+/citrate-induced LPO process of rat liver mitochondria under alloxan diabetes condition.
Original recording of the recording of changes in the light transmittance of the mitochondrial suspension.
According to the experimental results, the induced LPO process, i.e., the rate of mitochondrial collapse was taken as 100% after the introduction of Fe2+/citrate into the IM. LPO was rised to 68% from control in mitochondria which alloxan diabetec rat. This indicates that the hydrolysis of phospholipids in the mitochondrial membrane is increased in diabetic conditions. As a result of
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administration of hypolaetin-8-glucoside flavonoid to group III rats with alloxan diabetes, it was found that the process of LPO induced by Fe2+/citrate in their liver mitochondria was inhibited by 43% compared to the indicators of group II (Fig. 4). It was found that under the influence of rutin flavonoid, the intensity of LPO was inhibited by 31% compared to group II.
Control (healthy) Alloxan diabetes
Alloxan diabetes+ hypolatin-8-glucoside Alloxan diabetes+i'utin
Figure 4. Effect of hypolatin-8-glucoside and rutin flavonoids on Fe2+/citrate-induced LPO process of rat liver mitochondria under alloxan diabetes conditions. Statistically (R<0.05) difference from control is marked (*).
These obtained results indicate that hypolaetin-8-glucoside flavonoid has antioxidant properties. The analysis of the results obtained from the experiments shows that the antioxidant property of hypolaethin-8-glucoside indicates that it has a stabilizing effect on the mitochondrial membrane because of the inhibition of the mitochondrial LPO process and mPTP.
Summary
Hypolaetin-8-glucoside flavon has hypoglycemic activ and alloxan normalizes blood glucose in diabetic conditions. Alloxan permeabilizes mPTP permeability by inhibiting mitochondrial dysfunction of the liver under diabetic conditions. Hypolaetin-8-glucoside
inhibited the intensity of LPO process induced by Fe2+/citrate in liver mitochondria under alloxan diabetes conditions and showed antioxidant properties.
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