ANTIAPOPTOTIC MECHANISM FOR THE IMPLEMENTATION OF THE HEPATOPROTECTIVE EFFECT OF PYRIMIDINE DERIVATIVES

ANTIAPOPTOTIC MECHANISM FOR THE IMPLEMENTATION OF THE HEPATOPROTECTIVE EFFECT OF PYRIMIDINE DERIVATIVES

A. Parfenov , A. B. Vyshtakalyuk *, I. V. Galyametdinova, V. E. Semenov and V. V. Zobov

Institute of Fundamental Medicine and Biology of Kazan Federal University, 5 Tovarisheskaya Str., Kazan, Russia.

ABSTRACT: The present study aimed to study the effect of the drug Xymedon and its conjugate with L-ascorbic acid exhibiting hepatoprotective activity on the apoptosis of rat liver cells against the background of the influence of hepatotoxic agent tetrachloromethane. Though the general effects of Xymedon and its conjugates with various biogenic acids have been studied, the molecular markers affected by the compounds have not yet been established. Experimental methods of investigation included modeling of toxic liver damage by oral administration of carbon tetrachloride to laboratory Wistar rats. To establish the molecular mechanisms of the antiapoptotic effect involved in the hepatoprotective activity of Xymedon and its conjugate with L-ascorbic acid, the level of early apoptosis markers Akt, BAD, BCL-2, p53, Active Caspase-8 Active Caspase-9 in rat liver homogenates was determined by multiplex analysis method using the MagPix system («MerkMillipore», USA). The study showed that Xymedon and its derivative with L-ascorbic acid exhibited an antiapoptotic effect, significantly reducing the number of early apoptosis markers BAD, Active Caspase-9. Also, the derivative with L-ascorbic acid reduces the expression of p53. Moreover, it was shown that some biochemical markers are normalized under the influence of the studied compounds. The results obtained have both scientific value, bringing us closer to the true mechanism of action of pyrimidine derivatives, and practical value - the possibility to create an effective hepatoprotective drug with a strong evidence base and degree of study.

Keywords: Pyrimidine derivatives, Apoptosis, Carbon tetrachloride, Toxic liver damage

INTRODUCTION: The liver is the body that plays a key role in the detoxification of exogenous and endogenous toxic substances. Late treatment of mild forms of liver pathologies caused by hepatotoxic substances can lead to more serious up to necrotic damage ¹.

he objects of the study were a drug based on the pyrimidine derivative Xymedon (compound 1), and it’s conjugate with L-ascorbic acid (compound 2). The structural formula of compounds 1 and 2 is shown in Fig. 1.

Xymedon (1, 2 – dihydro - 4, 6 – dimethy L – 1 - (2 - hydroxyethyl) - pyrimidin- 2 -one) is an original Russian drug compound a tissue regeneration stimulant. The chemical compound was synthesized at the Institute of Organic and Physical Chemistry in 1966. This compound has many activities, including membrane stabilizing, regenerating, immunostimulating, etc. ².

Earlier data have shown that conjugation of Xymedon with some bioactive acids leads to an increase in the protective effect of the Xymedon molecule itself ^3-5. As a result of exploratory studies, Xymedon conjugate with L-ascorbic acid, among other studied conjugates, has a more pronounced and stable hepatoprotective effect. It has been shown in vivo that the conjugate of xymedon with L-ascorbic acid helps to reduce the severity of morphological signs of liver damage induced by carbon tetrachloride (CCl₄) ⁶. It was also found that Xymedon conjugate with L-ascorbic acid contributes to the normalization of biochemical markers of liver tissue damage in the blood of animals against the background of liver damage induced by CCl₄ ⁶. Moreover, a more pronounced hepatoprotective effect was shown for Xymedon conjugate with L-ascorbic acid than for the comparison drug – Tiotriazolin ¹⁰. Thus, understanding the fact that we know the hepatoprotective potential of Xymedon conjugate with L-ascorbic acid, we attempted to establish the mechanism by which the protective effect of the studied conjugate is carried out. This study aimed to study the effect of Xymedon and its conjugate with L-ascorbic acid, which has hepatoprotective activity, on apoptosis of rat liver cells against the background of injuries caused by CCl₄.

FIG. 1: STRUCTURAL FORMULA OF THE STUDIED COMPOUNDS. 1: xymedon; 2: conjugate of xymedon with l-ascorbic acid

MATERIALS AND METHODS:

Synthesis of the Studied Compounds: Xymedon (1, 2-dihydro-4, 6-dimethyl-1-(2-hydroxyethyl)-pyrimidin-2-one) and the conjugate of Xymedon with L-ascorbic acid were synthesized by previously described methods from 2-dihydro-4, 6-dimethylpyrimidin-2-one and 2- chloroethanol ^{7, 8}.

Design of the Experiment Involving the Experimental Animals: The experiments were performed on adult male Wistar rats weighing 300-350 g obtained from the Laboratory Animal Breeding Station of FIBH RAS (Pushchino, Russia). Animals were kept in accordance with the provisions of preclinical test guidelines ⁹ and European Community guidelines for the housing and care of animals used for experiments and other research purposes (EC Committee guidance on management and care of animals used for experiments and other research purposes, 2007/526/EC, of June 18th 2007.) in standard vivarium conditions with a 12-hour light day and unrestricted access to food and water. Animals were fed a complex feed made according to the Specification (protein 22%, fiber no more than 4%, fat no more than 5%, ash no more than 9%, moisture no more than 13.5%, Caloric value 295 kcal per 100 g. All animal studies and protocols were approved by the local ethical committee of Kazan Federal University (Protocol № 4 of May 18, 2017). In total, 24 animals participated in the experiment (6 animals in each of the four experimental groups). The experimental scheme was as follows: administration of CCl₄ for 3 days, then administration of drugs for 6 days (a total of 9 days). Material (blood for biochemical studies, liver samples) was taken the day after the last CCl₄ injection (day 4 of the experiment), 3 days after stopping CCl₄ injection and 3 days of drug administration (day 7 of the experiment) and 6 days after stopping CCl₄ administration and 6 days of drug administration (day 10 of the experiment), when animals were euthanized.

Carbon tetrachloride was administered orally as a 35% oil solution at a 1.5 ml/kg dose. Investigated compounds 1 and 2 were injected intraperitoneally at 0.24 and 0.5 mg/kg doses. The drug solutions were prepared every day immediately before administration to the animals. Serum was prepared by double centrifugation of blood at 3000 rpm and +4°C. Serum was stored in a freezer at -25°C before analysis. Biochemical parameters were studied using an automatic biochemical analyzer ARD 200 (ARD, Russia) and reagent kits.

Histological Studies: Histological processing - preparation of paraffin sections, staining with Garris hematoxylin (BioVitrum, Russia), alcohol eosin (BioVitrum, Russia) were performed according to standard protocols described in more detail in our previous works ^3-7. Frozen sections were also prepared and stained with Sudan 4 (BioVitrum, Russia) according to standard protocols. For microscopy and microphotography the Nikon (Nikon, Japan) microscope, digital camera (Nikon, Japan) and NIS Elements software (Nikon, Japan) were used.

Study of Molecular Markers of Apoptosis: Markers were studied in rat liver lysates according to the protocol of Early Apoptosis Magnetic Bead Kit (Merck / Millipore Sigma, USA) with measurement on Mag Pix device (Merk / Millipore, USA). To study apoptosis markers, 5 mg of liver tissue taken from animals during euthanasia was lysed beforehand according to the instructions of Micro Rotofor Lysis Kit. (BioRad, USA) with Protease Inhibitor Cocktail (Merck/Sigma-Aldrich, USA) added. Next, all samples were aligned for total protein concentration to 100 µg/ml by measuring the protein concentration in the samples by the Bradford method using the Quick Start Bradford Protein Assay Kit (BioRad, USA) and an Epoch microplate spectrophotometer (BioTech, USA). Bovine serum albumin (Amresco, USA) was used to construct a calibration chart.

Statistical Analysis: All experimental data obtained underwent statistical analysis. We calculated the mean values and standard errors of the mean. Figures contain mean values for each standard parameter and standard error. For each sample the normality of the distribution was evaluated using the Kolmogorov-Smirnov criterion. In cases of normal distribution, samples were compared using Student's t-criterion. In cases of non-normal distribution, the comparison was done using Mann-Whitney U-test. Statistical processing was performed using SPSS 13.0. Data visualization was performed in Origin Lab 2018, the layout of figures in Adobe Photoshop 18.00.

RESULTS:

Evaluation of the Clinical Condition of the Animals: After a three-day course of CCl₄ injection, the animals showed a picture of acute toxic damage to the liver, confirmed on histological sections, where foci of steatosis, hepatocyte dystrophy and necrotic changes were visible Fig. 2.

FIG. 2: MICROPHOTOGRAPHS OF RAT LIVER SECTIONS AFTER THREE-DAY EXPOSURE TO CCL₄. a: hematoxylin and eosin staining (1-hydropic dystrophy, 2-caunselmain cell, 3-large dropletadipose degeneration, 4-balloon dystrophy, 5-small droplet adipose degeneration); b: sudan iv staining (orange drops are lipid droplets); lens 20x. zoom 300x

Among the changes in the biochemical blood parameters, we observed a twofold increase in the activity of ASAT (aspartate aminotransferase) Fig. 3A, while ALAT activity decreased in comparison with the reference values of the intact group of animals Fig. 3B. By day 10 of the experiment, the level of ALAT (alanine aminotransferase) activity is practically unchanged, but ASAT activity by day 10 sharply decreases more than twofold compared with the reference values of the intact group of animals. Against the background of introduction of compounds 1 and 2, the changes in the activity of aminotransferases were not as pronounced as in the control animals. Thus, the activity of ALAT when compounds 1 and 2 were injected on day 7 decreased less significantly relative to intact animals, but its level was significantly higher than in the control group on the same day of the experiment. On day 10 of the experiment, ALAT activity in the experimental groups remained at the level of day 7, but did not statistically differ from the control group on the same day Fig. 3B. ASAT activity against the background of administration of compounds 1 and 2 practically did not differ from the reference values of the intact group of animals. However, administration of compound 2 on day 10 of the experiment resulted in a decrease in ASAT activity compared to the intact group of animals, but its activity was significantly higher than that in the control group on the same day Fig. 3A.

FIG. 3: EFFECT OF COMPOUNDS 1 AND 2 ON CELL CYTOLYSIS MARKERS. a: aspartate aminotransferase (asat) activity, b: alanine aminotransferase (alat) activity; 1 differences from intact group are significant by t-test, p < 0.05; 2 differences from control group of the same day are significant by t-test, p < 0.05.

After a three-day course of CCl₄ administration, we observed signs of biliary excretion and bilirubin excretion in animals. Namely, accumulation of alkaline phosphatase and bilirubin in blood plasma. The increase in total bilirubin is mainly associated with an increase in the fraction of conjugated bilirubin Fig. 4A-D. On day 7 of the experiment, compounds 1 and 2 showed no significant improvement in any of the indices. However, while the control group on day 10 of the experiment showed a fairly strong decrease in the ALP activity (alkaline phosphatase) Fig. 4A and concentration of all bilirubin fractions Fig. 4B-D, the use of compounds 1 and 2 on day 10 of the experiment showed their stabilization in comparison with the indices of the intact group of animals. ALP activity and concentration of bilirubin fractions are significantly higher than those of the control group on the same day but still remain lower than in the intact group of animals Fig. 4A-D.

FIG. 4: EFFECT OF COMPOUNDS 1 AND 2 ON MARKERS OF BILIARY EXCRETION AND BILIRUBIN EXCRETION. a: alkaline phosphatase (alp) activity; b: total bilirubin concentration; c: unconjugated bilirubin concentration; d: conjugated bilirubin concentration; 1 difference from the intact group are reliable by t-test, p < 0.05; 2 differences from the control group of the same day are reliable by t-test, p < 0.05.

Disruption of albumin synthesis and a decrease in its concentration in blood serum in animals injected with CCl₄ is observed immediately after the cancellation of the toxicant on day 4 of the experiment. With each subsequent day after the cancellation of CCl₄, the albumin concentration in the blood serum decreased and reached its minimum on the last, tenth day of observation. Administration of compounds 1 and 2 for 6 days after cancellation of CCl₄ contributed to the fact that albumin concentration decreased to a lesser extent than in the control group, and on day 10 of the experiment, this index in the experimental groups was statistically significantly higher than in the control group Fig. 5A. The concentration of total serum protein Fig. 5B in the control group of animals also decreased; however, on days 4 and 7 of the experiment, not as critical as the albumin concentration. The minimum protein concentration was also recorded on the last, 10th day of the experiment. The use of compounds 1 and 2 against the background of CCl₄-induced toxic damage resulted in a smaller decrease in protein concentration than the control (the differences with the control group on day 10 are statistically reliable, with the intact group - not reliable). At the same time, the character of changes in protein concentration under the influence of substances was similar to that of albumin concentration Fig. 5B. The serum glucose concentration was not significantly affected by CCl₄ on days 4 and 7 of the experiment Fig. 5B. However, on day 10 of the experiment, the blood glucose concentration reached minimum values. Quite interesting results were obtained using compounds 1 and 2 against the background of toxic liver damage. If against the background of CCl₄ exposure, by day 10 of the experiment, a natural decrease in this index was observed in control; no such decrease was observed with compounds 1 and 2. Moreover, we noted that the compounds used led to a significant increase in glucose concentration in the blood serum compared to the intact group of animals in all control points of the experiment Fig. 5B.

FIG. 5: EFFECT OF COMPOUNDS 1 AND 2 ON MARKERS OF PROTEIN METABOLISM AND GLUCOSE. a: albumin concentration; b: total protein concentration; c: glucose concentration; 1 differences from the intact group are significant by t-test, p < 0.05; 2 differences from the control group of the same day are significant by t-test, p < 0.05.

Molecular Markers of Early Apoptosis: The study of markers of early apoptosis in liver lysates allowed us to see how a three-day CCl₄ injection promotes the emergence of such a trend as an increase in MFI (Mean intensity of fluorescence) of the proapoptotic BAD protein of BCL family on days 7 and 10 of the experiment Fig. 6A. At the same time, the more days pass since the cessation of the introduction of the toxicant, the greater the MFI of BAD is detected in the liver homogenates of the control group rats. In turn, administration of compounds 1 and 2 contributed to a decrease in the MFI of this marker of early apoptosis. Compound 1 was found to have no significant effect on the 7th day of the experiment Fig. 6A. But on day 10, compound 1 produced a sharp, significant decrease in BAD-protein MFI both in comparison to the same parameters in the control group and in comparison to the intact group of animals Fig. 6A. When compound 2 was used, a significant decrease in the BAD-protein MFI was already observed on day 7 of the experiment, both compared to the control group of animals on the same day and compared to the intact animals Fig. 5A. On day 10 of the experiment, when compound 2 was injected, a significant decrease in this MFI BAD protein was also observed Fig. 6A. Considering the antiapoptotic markers BCL-2 and Akt in this study, we cannot speak about any reliable effect of CCl₄ and compounds 1 and 2 on them. However, interesting, quite logical correlations between proapoptotic BAD and antiapoptotic BCL-2 and Akt are observed. A decrease in the MFI of antiapoptotic BCL-2 and Akt markers increases the MFI of proapoptotic BAD. It is worth noting that this statement is not supported by statistical analysis Fig. 6 B, C.

FIG. 6: EFFECT OF COMPOUNDS 1 AND 2 ON MEAN FLUORESCENCE INTENSITY (MFI) OF EARLY APOPTOSIS MARKERS. a: MFI BAD; b: MFI BCL-2; c: MFI Akt; 1 differences from intact group are significant by U-test, p < 0.05; 2 differences from control group of the same day are significant by U-test, p < 0.05.

FIG. 7: EFFECT OF COMPOUNDS 1 AND 2 ON THE MEAN FLUORESCENCE INTENSITY (MFI) OF ACTIVATED CASPASES 9 AND 8. a: MFI of activated caspase 9 (CASP9); b: MFI of activated caspase 8 (CASP8); 1 differences from intact group are significant by U-test, p < 0.05; 2 differences from control group of the same day are significant by U-test, p < 0.05.

Three-day injection of CCl₄ contributed to a significant increase in the MFI of activated CASP9 on days 7 and 10 of the experiment relative to the reference values of the intact group of animals Fig. 7A. Administration of compound 1 contributed to a significant decrease in CASP9 activation on day 10 of the experiment relative to the control group on the same day of the study Fig. 7A. As early as day 7 of the experiment, administration of compound 2 contributed to a significant decrease in CASP9 activation Fig. 7A. On day 10 of the experiment, compound 2 also contributed to a decrease in CASP9 activation relative to the animal control group (Fig. 7A. In turn, administration of CCl₄ and drugs did not result in a significant change in the MFI of activated CASP8 Fig. 7B. We also observed a significant decrease in the MFI of p53 protein when using compound 2 relative to controls on similar days of the study Fig. 8.

FIG. 8: EFFECT OF COMPOUNDS 1 AND 2 ON THE AVERAGE FLUORESCENCE INTENSITY OF MFI P53; 2 DIFFERENCES FROM THE CONTROL GROUP OF THE SAME DAY ARE SIGNIFICANT BY U-TEST, P < 0.05.

DISCUSSION: The mechanism of action of the Xymedon molecule is presumably related to the regulation of adenylate cyclase (AC) activity since there is evidence that Xymedon reduces the activity of this signaling molecule in immunocompetent cells ¹⁰. Speaking about further consequences of AC activation or inhibition, it is worth mentioning that there are different AC isoforms whose regulation is mediated by G-proteins ¹¹. The regulation of AC activity through G-protein subunits is top-specific, i.e., it depends on the AC isoform and cell type ¹¹. AC activity is associated with cAMP production (cyclic adenosine monophosphate) in cell ¹². cAMP, in its turn, activates regulatory processes in a cell, depending on its type ¹³. For example, there is evidence that high cAMP concentrations stimulate the proliferation of epithelial cells, hepatocytes, adipocytes and pancreatic β-cells ¹⁴. But at the same time, high cAMP production by fibroblast, smooth muscle, and neoplastic B-cells, on the contrary, suppresses proliferation ¹⁴. After a three-day course of CCl₄ injection in animals, there is a picture of acute toxic damage to the liver, expressed in a change in the activity of ASAT and ALAT enzymes. The sharp increase in ASAT level after a course of toxicant injection can be explained because ASAT, like ALAT, is contained in large quantities in liver cells ¹⁵. Its highest activity is observed in the mitochondria of the peripheral zone of the acini ¹⁶. Consequently, acutely toxic damage to the cells of this zone will cause a sharper increase in serum ASAT activity than ALAT.

A further decrease in ASAT activity is because the half-life from the bloodstream is about 17 h for total ASAT and an average of 87 hours for mitochondrial ASAT ¹⁷. While pathological changes in ALAT and ASAT activities were observed in the control group, Xymedon compounds and their conjugate with L-ascorbic acid resulted in more stable preservation of the activities of these aminotransferases. This indicates less necrotic damage to the liver parenchyma. Increased necrotic damage on day 4 of the experiment led to the occurrence of stagnation. It increased levels of ALP, as well as impaired excretion of bilirubin from the blood, which is a sign of jaundice ¹⁸.

A sharp decrease in ALP activity and the level of total bilirubin and its fractions on day 10 in the control group, in all probability, is caused by the decrease of functional activity of hepatocytes as a result of toxic damage. More stable ALP activity and concentrations of bilirubin fractions when using Xymedon and its conjugate with L-ascorbic acid, especially on day 10 of the experiment, testify to the fact that under the influence of these compounds, either the activity of undamaged hepatocytes increased or the necrotic damage to the organ in general decreased, so the metabolism of bilirubin passes in the liver ¹⁸. The decrease in the concentrations of total protein and albumin noted in our experiment against the background of exposure to a toxicant looks quite logical since a decrease in synthetic liver function is observed in liver parenchyma damage ¹⁹. As follows from the data obtained, when using Xymedon and its conjugate with L-ascorbic acid, the preservation of synthetic liver function or intensification of such processes in undamaged hepatocytes is observed. In favor of the theory about the intensification of hepatocyte metabolism processes, there are data on blood glucose concentration. It is known that the liver is the main depository of the main energy source for eukaryotic cells - glucose in the form of glycogen ²⁰. From liver glycogen, under the influence of the enzyme system of hepatocytes and external signaling factors, glucose is formed again, which covers the energy deficit of cells of the whole organism ²¹.

In our case, however, after a course of drug administration, the level of glucose rises considerably above the reference values obtained in intact animals. This may be evidence of glycogenolysis activation, which allows supporting the theory about the intensification of metabolic processes in liver cells. Activation of glycogenolysis can also be explained by an increase in cAMP under the influence of the molecule Xymedon. cAMP in a hepatocyte can increase glucose production by increasing the transcription of gluconeogenic enzymes ^{22, 23}. The increase in glucose production after AC activation and the increase in cAMP concentration in hepatocytes after exposure to Xymedon conjugate with L-ascorbic acid can explain the earlier data that this compound leads to a 440% increase in the performance of rats without a decrease in blood glucose levels ²⁴.

Our studies of early apoptosis markers revealed that Xymedon (on day 10 of the experiment) and its conjugate with L-ascorbic acid (on days 7 and 10 of the experiment) reduced the MFI of the proapoptotic protein BAD. Protein containing only BH3, BAD inhibits antiapoptotic BCL-2 and BCL-xL and promotes BAX/BAK activation in response to apoptotic stimuli ²⁵. This is evidence that these compounds reduce the initiation of apoptotic processes in cells. Thus, compensating not only for the effect of CCl₄, but also leading to a decrease in the expression of the proapoptotic protein BAD below the values of the intact group of animals. However, in this case, the decrease in MFI of the proapoptotic protein BAD is not related to MFI Akt, which is considered an inhibitor of apoptosis because of its ability to inactivate proapoptotic molecules, including caspase-9 (CASP9) and BAD protein ²⁶. Thus, the decrease in BAD MFI under the influence of Xymedon and its conjugate with L-ascorbic acid is most likely related not to its Akt phosphorylation but a decrease in the expression of this protein in the cell. We also found no significant increase in MFI of the antiapoptotic protein BCL-2. This protein prevents apoptosis either by sequestering the proformas of caspases that cause cell death or by preventing the release of mitochondrial apoptogenic factors such as cytochrome C and AIF (apoptosis-inducing factor) into the cytoplasm ²⁸.

In the experiment conducted, CCl₄ promoted activation of predominantly internal liver cell apoptosis pathway related to CASP9 activation ²⁸. The observed significant decrease in fluorescence intensity of both BAD protein and activated caspase-9 when injecting Xymedon on day 10 of the experiment and the conjugate of Xymedon with L-ascorbic acid on days 7 and 10 of the experiment may be evidence of less damage of mitochondrial membranes and less release of cytochrome C that trigger activation of apoptotic processes ²⁹. Enhanced antiapoptotic properties of the conjugate of Xymedon with L-ascorbic acid, expressed in the fact that these properties of the conjugate occur earlier in comparison with Xymedon, firstly, probably due to the high bioavailability of ascorbic acid for cells, which probably contributes to the bioavailability of the Xymedon molecule as well.

Secondly, there is evidence that L-ascorbic acid can act synergistically with other substances. For example, ascorbic acid, together with phenylephrine and metaproterenol, can enhance the synthesis and proliferation of primary hepatocyte culture DNA ³⁰. Phenylephrine and metaproterenol themselves do not accelerate the synthesis and proliferation of primary hepatocyte culture DNA ³⁰. Besides, it was shown that L-ascorbic acid itself can accelerate the regeneration of rat liver tissue ²¹.

CONCLUSIONS: It was shown that Xymedon and its conjugate with L-ascorbic acid, against the background of damage caused by three-day administration of CCl4, contribute to the preservation of liver cell function, which is manifested in the reduction of the activity of cytolysis markers ASAT and ALAT, normalization of synthetic liver function, preservation of function of bilirubin metabolism in the liver. Also, these compounds cause a significant increase in blood glucose. As a result of this study, Xymedon and its conjugate with L-ascorbic acid against the background of exposure to CCl₄ contribute to a decrease in the lysates of rat liver tissue proapoptotic markers BAD and CASP9.

The antiapoptotic activity of Xymedon conjugate with L-ascorbic acid always occurred earlier (on day 7 of the experiment) than that of Xymedon (manifestation of activity on day 10 of the experiment). The derivative of Xymedon with L-ascorbic acid was found to significantly decrease the expression of the proapoptotic protein p53, while Xymedon, in turn, does not affect this marker.

ACKNOWLEDGEMENTS: Nil

Funding: This work was supported financially by the Russian Foundation for Basic Research No.20-315-90039; A. Vyshtakalyuk, I. Galyametdimova, V. Semenov and V. Zobov were partially supported by State Assignment (FRC Kazan Scientific Center of RAS).

Ethical Compliance: All animal studies and protocols were approved by the local ethical committee of Kazan Federal University (Protocol № 4 of May 18, 2017).

CONFLICTS OF INTEREST: The author declares no conflict of interest.

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    Vyshtakalyuk AB, Parfenov AA, Galyametdinova IV, Semenov VE and Zobov VV: Antiapoptotic mechanism for the implementation of the hepato-protective effect of pyrimidine derivatives. Int J Pharm Sci & Res 2022; 13(10): 3922-31. doi: 10.13040/IJPSR.0975-8232.13(10).3922-31.

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