INTRODUCTION:

Mitochondrial permeability transition pore (mPTP) refers to a huge rise in the permeability of the inner mitochondrial membrane (IMM)¹ to aqueous solutions, which in mammalian mitochondria is approximately 1500 Da. The occurrence of high membrane permeability and its prevention by adenine nucleotides has been known since the 1950s and this process is being studied in a number of laboratories²^,^3,4^,5.

In 1979 Haworth and Hunter introduced the term “permeability transition”⁶ into science, they detailed that its main properties are associated with the opening of the inner membrane channel mPTP in cardiac mitochondria⁷^,^8,9. To corfirm this hypothesis patch-clamp studies on mammalian mitoplasts was studied, which revealed the presence of a high-conductance (≈ 1 nS) channel, the mitochondrial megachannel^10,11. The structure of MPTP consists of 3 components: consisting of the protein cyclophilin D (CycD), adenine dinucleotide translocase (ANT), voltage-dependent anion channel (VDAC)¹². Recent studies have found that mPTP plays a vital role in the regulation of necrosis and apoptosis of cell¹³. mPTP is known as a nonselective ion channel^14,15that plays a vital role in calcium exchange between the the cytosol and matrix. The entry and exit of Ca²⁺ into mitochondria occurs in different ways. Thus, calcium ions enter the matrix through the Ca²⁺uniport system of the inner mitochondrial membrane (IMM)¹⁶ and are released from the matrix through the Na⁺/Ca²⁺ and H⁺/Ca²⁺ exchange pathways, or mPTP. The opening of MPTP is triggered by calcium ions inside mitochondria^17,18. MPTP can also act as a regulator of cellular adaptation to hypoxia. The functional activity of MPTP is to change the conformation of the proteins that make up its structure, thereby participating in the regulation of metabolic processes. Mitochondrial swelling (opening of mPTP) is observed in some pathological circumstances, such as hepatic encephalopathy, stroke, myocardial infarction, muscular dystrophy, neurodegenerative diseases, traumatic brain injury etc. It is known that during myocardial ischemia and its subsequent reperfusion, cardiomyocytes die as a result of apoptosis^19,20.

In addition, mitoK_ATP channels have been widely studied in research recently. A huge number of scientific studies have determined that there is a pivotal regulatory role of the mitoK_ATPchannel in the formation of adaptive reactions of the body under hypoxic conditions^21,22. Under ischemic conditions, the cardiac mitoK_ATP channel has it`s main role in cardioprotection of cardiomyocytes²³. An increase in mitochondrial swelling leads to an acceleration of lipid peroxidation processes in membranes. Lipids, especially phospholipids, have important functions in plasma membrane^24,25 formation, cell signaling pathways, and energy storage. Many age-related diseases, including rheumatoid arthritis, kidney disease, cardiovascular disease, neurodegenerative diseases and diabetes are associated with lipid abnormalities. As we mentioned above, mitochondrial dysfunction and huge rise of production of reactive oxygen species (ROS)²⁶ lead to changes in lipid metabolism and increased lipid oxidation^27,28. Polyphenols are widely used as pharmacological corrective substances for mitochondrial membrane dysfunction^29,30.

Natural polyphenols are widely used as pharmacological substances with antioxidant properties³¹. Providing a general anti-inflammatory, hemo- and cardioprotective effect, they protect mitochondria from pathological processes³²^,33. In this study, in vitro experiments examined the effect of polyphenol YaN-1, a diazoimino derivative of gossypol, on the swelling of rat heart mitochondria^34,35the activity of mitoK_ATP channels³⁶ and the process of membrane lipid peroxidation³⁷.

MATERIAL AND METHODS:

Experimental chemistry part:

1-step. An aqueous solution of NaNO₂ was added to a cooled (0-5°C) solution of 4-aminoantipyrine in water and concentrated HCl and neutralized with CH₃COONa to obtain a solution of diazonium salt of 4-aminoantipyrine. Following that, this solution was added to a cooled solution of gossypol in alcohol, the precipitate was filtered off, washed with hexane and dried in vacuum. The yield of the reaction product is 85%.

2-step. A solution of 2-aminoethylhydrosulfate in alcohol in a ratio of 1:2 was added to an alcohol solution of the gossypol azo mixture obtained with 4-aminoantipyrine. The reaction mixture was maintained at a temperature of 60-65°C for exactly 30 minutes. The mixture from the reaction was cooled to room temperature and stored in the refrigerator overnight. The precipitate was then filtered, washed with hexane, and allowed to dry in open air. The yield of the reaction product was 70%.

Figure-1. Scheme for preparing the imino compound of the azo derivative of gossypol with 4-aminoantipyrine

Method for isolating mitochondria from heart tissue. In the experiment, rat cardiac mitochondria were isolated using differential centrifugation. This research method was developed by Egorova and Afanasyev³⁸. The composition of the separation medium: 75 mM sucrose (C₁₂H₂₂O₁₁), 225 mM mannitol, 10 mM tris-chloride (C₄H₁₁NO₃-HCl), 0.1 mM EDTA (C₁₀H₁₆N₂O₈), 0.1 mg/ml albumin (BSA or "Fraction V"), pH 7.4. The rat heart was isolated and washed with a cold 0,9% KCl solution. The cardiac mass is weighed, crushed, homogenized and the separation medium is added in a ratio of 1:6. Centrifugation is carried out in two stages at 0-2°C. At the first stage, it lasts 7-8 minutes at a speed of 1500 rpm. During this period, heavy cell aggregates settle. The liquid supernatant was transferred to another clean tube and centrifuged for 20 minutes at 6000 rpm in the second step. The isolated mitochondria were stored in tubes on ice and functional experiments were carried out. The amount of protein in mitochondria was determined calorimetrically using the Biuret method³⁹.

Determination of PTP status in mitochondria.:

The composition of the mitochondrial Ca²⁺-dependent megapore incubation medium was as follows: 200 mM sucrose (C₁₂H₂₂O₁₁), 20 mM HEPES (C₈H₁₈N₂O₄S), 5 mM succinate (C₄H₄O₄^-2), 2 μM rotenone (C₂₃H₂₂O₆), 20 mM Tris, 1 μg/ml oligomycin (C₄₅H₇₄O₁₁), 20 μM EGTA and 1 mM KH₂PO₄. pH 7.4⁴⁰. Cardiac conductivity mPTP was measured photometrically in mitochondrial suspension (0,3–0,4 mg/ml) in 3 ml wells at 26°C with constant stirring in a V-5000 spectrophotometer at a wavelength of 540 nm.

Determination of the activity of ATP-dependent potassium channels in cardiac mitochondria:

The permeability of the mitoK_ATP channel (0,3-0,4 mg/ml) was determined by the change in optical density in 3 ml wells at a wavelength of 540 nm in a V-5000 spectrophotometer. The incubation medium for the MitoK_ATP channel was as follows: 125 mM potassium chloride (KCl), 10 mM Hepes (C₈H₁₈N₂O₄S), 2,5 mM KH₂PO₄, 5 mM succinate, 2,5 mM K₂HPO₄, 1 mM MgCl₂, 0,005 mM rotenone (C₂₃H₂₂O₆) and 0,001 mM oligomycin (C₄₅H₇₄O₁₁), pH 7.4⁴¹. The activity of the MitoK_ATP channel was determined in the presence of 10 mM ATP.

Determination of the lipid peroxidation (LPO) process in the cardiac mitochondrial membrane:

The Fe²⁺/citrate system was utilized to investigate the lipid peroxidation (LPO) process in the mitochondrial membrane. As a result, the organelle size increases, and the mitochondria swell. This change in volume was determined photometrically. The composition of the incubation medium: 125 mM sucrose (C₁₂H₂₂O₁₁), 65 mM potassium chloride (KCl) and 10 mM HEPES (C₈H₁₈N₂O₄S), pH 7.2. Incubated, adding FeSO₄ - 50 μM and citrate – 2 mM to the reaction medium; the number of mitochondria 0,5 mg/ml; The experiments were carried out at a standard temperature of 25°C²⁷.

Drugs and chemicals: The following chemical reagents were used: Tris-chloride (Serva, Germany), citrate, EGTA, sucrose (C₁₂H₂₂O₁₁), ferrous sulfate, KH₂PO₄, rotenone, calcium chloride (Chemreaktivsnab, Russia), succinate, EDTA (Sandoz, Switzerland), Hepes, oligomycin (C₄₅H₇₄O₁₁) and Cyclosporine (Selleck, USA). All reagents were p.a. grade.

RESULTS AND DISCUSSION:

During in vitro experiments the effect of polyphenol YaN-1, a diazoimino derivative of gossypol, on the swelling of rat heart mitochondria was studied at concentrations of 10 µM, 15 µM and 25 µM. Swelling of cardiac mitochondria was induced using CaCl₂, an mPTP inducer, at a concentration of 10 μM. In the incubation medium without the presence of Ca²⁺ ions, mitochondrial swelling was not observed. The optical density of heart mitochondria was 0,008 ΔA₅₄₀. In our next experiment, it was found that the optical density, which reflects the swelling of cardiac mitochondria, was 0,385 ΔA₅₄₀ in the presence of Ca²⁺ ions at a concentration of 10 μM in the incubation medium. Thus, Ca²⁺-dependent swelling of rat heart mitochondria indicates that mPTP enters an open state. In the presence of 10 μM Ca²⁺-dependent swelling of cardiac mitochondria in the incubation medium of the YaN-1 polyphenol, a diazoimino derivative of gossypol, the optical density was 0.338 ΔA₅₄₀. The swelling of mitochondria in the heart of healthy rats, caused in the presence of Ca²⁺ ions at a concentration of 10 μM, was taken as 100% as a control. It was found that polyphenol YaN-1 at a concentration of 10 μM prevented the swelling of cardiac mitochondria by 12.2% compared to the control. Therefore, a small concentration of the polyphenol YaN-1 induced mitochondrial permeabilization, in part by inhibiting cardiac mitochondrial swelling. In the presence of polyphenol YaN-1, a derivative of diazoimino gossypol, in the incubation medium at a concentration of 15 μM and 25 μM, the optical density of Ca²⁺-dependent swelling of cardiac mitochondria was 0.285 ΔA₅₄₀ and 0.251 ΔA₅₄₀, respectively. It was found that polyphenol YaN-1 inhibits the opening of mPTP by 26.1% at a concentration of 15 μM and by 34.8% at a concentration of 25 μM. Thus, polyphenol YaN -1, a diazoimino derivative of gossypol, had an inhibitory effect on Ca²⁺-dependent mPTP which is located in the inner membrane of rat heart mitochondria in the concentration range of 10-25 μM (Fig. 1, A and B).

А- representative absorbance plot.

B- graph of all samples examined.

Figure-1. Effect of diazoimino derivative gossypol polyphenol YaN-1 on the function of rat cardiac mitochondria (*P <0.05; **P <0.01; n=5).

The classical MPTP inhibitor, CsA, inhibits Ca²⁺ induced swelling, which means the mPTP enters a closed state. In the incubation medium without the presence of Ca²⁺ ions, mitochondrial swelling was not observed. At the same time, the optical density of heart mitochondria was 0.009 ΔA₅₄₀. It was found that the optical density indicator, reflecting the swelling of cardiac mitochondria, was 0.294 ΔA₅₄₀ in the condition where the presence of Ca²⁺ ions at a concentration of 10 μM in the incubation medium. In the presence of CsA at a concentration of 0.50 μM, the optical density indicator, reflecting the swelling of cardiac mitochondria, was 0.084 ΔA₅₄₀. It was found that a CsA concentration of 0.50 μM inhibited the swelling of cardiac mitochondria by 71.1% compared to the In the presence of 20 µM polyphenol YaN-1, a diazoimino derivative of gossypol, and 0.50 µM concentration of CsA, the optical density index, reflecting the swelling of cardiac mitochondria, was 0.034 ΔA₅₄₀. As a result of the complex effect of the diazoimino derivative gossypol YaN-1 at a concentration of 20 μM and CsA at a concentration of 0.50 μM, it was found that PTP in cardiac mitochondria was inhibited by 88.3% compared to the control.

А- representative absorbance plot.

B- graph of all samples examined.

Figure-2. Effect of CsA (0.50 µM) and polyphenol YaN-1 on the swelling of rat cardiac mitochondria (*P <0.05; **P <0.01; n=5).

In the next experiment, the effect of polyphenol YaN-1, a derivative of diazoimino gossypol, on the activity of cardiac mitoK_ATP channels was studied. According to the obtained results, the permeability of the mitoK_ATP channel in the absence of ATP in the incubation medium was taken as the control (100%). The optical density of heart mitochondria was 0.394 ΔA₅₄₀. In the presence of 200 μM ATP in the incubation medium, the conductance of the mitoK_ATP channel was an optical density of 0.035 ΔA₅₄₀, respectively, and it was found that the activity of the mitoK_ATP channel was inhibited by 91.1% compared to the controlIn the presence of 10 and 20 μM YaN-1 polyphenol in the incubation medium, the conductance of the cardiac mitoK_ATP channel was 0.076 ΔA₅₄₀ and 0.178 ΔA₅₄₀ optical density, respectively. It was found that a concentration of YaN-1 polyphenol at a concentration of 10 μM activates the activity of cardiac mitoK_ATP channels by 19.3% compared to the control, and at a concentration of 20 μM - by 45.2%. In the presence of the YaN-1 polyphenol, a diazoimino derivative of gossypol, at 30 and 40 μM, the permeability of cardiac mitoK_ATP channels was 0.248 ΔA₅₄₀ and 0.330 ΔA₅₄₀ optical density. Incubation of cardiac mitochondria with 30 and 40 concentrations of YaN-1 polyphenol increased the activity of mitoK_ATP channels by 62.9 and 83.7%, respectively, compared to the control (Fig. 3, A and B).

А- representative absorbance plot.

B- graph of all samples examined.

Figure-3. Effect of polyphenol YaN-1, a diazoimino derivative of gossypol, on the mitoK_ATP channel of the rat cardiac (*P <0.05; **P <0.01; n=5).

Diazoxide, a classic MitoK_ATF channel activator, increases potassium ion permeability. During our next experiment, the effect of diazoxide and the diazoimino derivative of gossypol polyphenol YaN-1 on the activity of mitoK_ATP channels was studied by comparing. According to the results obtained, the permeability of the mitoK_ATP channel in the absence of ATP in the incubation medium was taken as the control (100%). The optical density of heart mitochondria was 0.349 ΔA₅₄₀. In the presence of 200 μM ATP in the incubation medium, the optical density of the mitoK_ATP channel permeability was 0.155 ΔA₅₄₀, and it was found that the activity of the mitoK_ATP channel was inhibited by 55.6% compared to the control. The concentration of 20 µM of the polyphenolic substance gossypol diazoimino derivative YaN-1 increases the activity of cardiac mitoK_ATP-channel compared to the condition in the presence of ATF with an optical density of 0.245 ΔA₅₄₀, and the optical density of 50 µM diazoxide substance was 0.272 ΔA₅₄₀, in the presence of 50 μM diazoxide and 20 μM YaN-1 polyphenol, the optical density was 0.287 ΔA₅₄₀. According to the results obtained, it was found that 20 μM concentration of the polyphenolic substance YaN-1 - diazoimino derivative gossypol - increased the permeability of cardiac mitoK_ATP channels by 70.2%, 50 μM concentration of diazoxide substance - by 77.9%, 50 μM diazoxide substance and YaN -1 polyphenol in 20 μM - by 82.2%, respectively (Fig. 4, A and B).

А- representative absorbance plot.

B- graph of all samples examined.

Figure-4. Effect of diazoxide and polyphenol YaN-1 on the mitoK_ATP channel of the rat cardiac (*P <0.05; **P <0.01; n=5).

Thus, it was established that the studied diazoimino derivative of gossypol YaN-1 polyphenol has an activating effect on the cardiac mitoK_ATP channel. It was found that this substance has an activator effect on the activity of mitoK_ATP channels in high concentrations.

Fe²⁺/citrate was used as an inducer of lipid peroxidation in the mitochondrial membrane of heart. In the presence of Fe²⁺/citrate in the incubation medium, lipid peroxidation of the mitochondrial membrane is accelerated and observed. In the incubation medium in the absence of Fe²⁺/citrate, mitochondrial swelling was not observed. The optical density of heart mitochondria was 0.019 ΔA₅₄₀. In the next experiment, it was found that the optical density index, reflecting the swelling of cardiac mitochondria in the presence of Fe²⁺/citrate in the incubation medium, was 0.331 ΔA₅₄₀. Fe²⁺/citrate-induced lipid peroxidation of cardiac mitochondria was taken as control as 100%. When cardiac mitochondria were incubated with polyphenol YaN-1, a diazoimino derivative of gossypol, at concentrations of 5, 10 and 20 μM, Fe²⁺/citrate-induced lipid peroxidation of the mitochondrial membrane was 0.290 ΔA₅₄₀, 0.194 ΔA₅₄₀ and 0.078 ΔA₅₄₀ optical density. It was found that the diazoimino derivative of gossypol YaN-1 inhibits lipid oxidation in the mitochondrial membrane of the heart by 12.4, 41.4 and 76.4%, respectively, at concentrations of 5, 10 and 20 μM (Fig. 5, A and B). ). Thus, the diazoimino derivative of gossypol - polyphenol YaN-1 - inhibited the process of lipid peroxidation of the heart mitochondrial membrane and had an antioxidant effect. The inhibitory effect of a polyphenolic compound on the mitochondrial membrane of lipid oxidation can be explained by the presence of a hydroxyl group in the rings of its structure.

А- representative absorbance plot.

B- graph of all samples examined.

Figure-5. Effect of diazoimino derivative gossypol polyphenol YaN-1 on the lipid peroxidation of the mitochondrial membrane of the rat cardiac (*P <0.05; **P <0.01; n=5).

CONCLUSION:

According to the results of the above experiments, the studied diazoimino derivative of gossypol YaN-1 polyphenol had a concentration-dependent inhibitory effect on PTP of cardiac mitochondria. It was established that the diazoimino derivative of gossypol YaN-1 has an activator-like effect on the activity of mitoK_ATP channels at high concentrations of polyphenols. When comparing the polyphenol YaN-1 with diazoxide, a classical activator of the MitoK_ATF channel, the polyphenolic compound had a strong activating effect on the permeability of diazoxide and potassium ions. Polyphenol YaN-1, a diazoimino derivative of gossypol, reduced the rate of lipid oxidation in the membranes of cardiac mitochondria and enhanced the antioxidant defense system. It has been established that this compound does not have pro-oxidant properties; it has a protective effect, correcting functional disorders at the level of mitochondria. Approaches to its membrane-active properties may serve as the basis and an important factor for the creation of a pharmacological drug in the near future.

ACKNOWLEDGEMENT:

This research was supported by grant F-OT-2021-465 of the Ministry of Innovative Development of the Republic of Uzbekistan.

CONFLICT OF INTEREST:

The authors have declared that no conﬂict of interest exists.

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Received on 04.10.2024 Revised on 19.02.2025

Accepted on 23.04.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2575-2581.

DOI: 10.52711/0974-360X.2025.00368

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