Influence of Xymedon and its conjugate with L-ascorbic acid on collagen remodeling in the liver fibrosis rat model

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Belyaev, Alexandra B. Vyshtakalyuk, Andrey A. Parfenov, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3953710/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jun, 2025 Read the published version in Uchenye Zapiski Kazanskogo Universiteta Seriya Estestvennye Nauki → Version 1 posted You are reading this latest preprint version Abstract Fibrosis of the liver is a chronic inflammatory process with activation of hepatic stellate cells and abnormal accumulation of proteins in the extracellular matrix. However, it is known that pyrimidine derivatives have a beneficial effect on the condition of various organs with the ongoing process of fibrosis. Therefore, the aim of this work was to investigate the effect of the drug Xymedon (1,2-dihydro-4,6-dimethyl-1-N-(2-hydroxyethyl)pyrimidine-2-one, (compound 1 ) and its conjugate with L -ascorbic acid (compound 2 ) on collagen remodeling in rat liver tissue. For this purpose, female Wistar rats were used to model fibrosis by oral administration of carbon tetrachloride (CCl 4 ) and ethanol for 8 weeks. Then the rats were treated with the studied compounds for 2 or 4 weeks. Histological analysis by hematoxylin-eosin and Van Gizon’s staining of liver slices, biochemical analysis of blood serum and Western blot analysis of COX-2 level in rat liver homogenates were performed. It has been shown that in the control group without treatment, after 2 weeks of withdrawal of CCl 4 + ethanol, collagen remodeling occurs to the certain chronic level. At the same time, compound 2 reduces the level of collagen fibers by 41% compared to the control group, while native compound 1 has no such effect. Also, in all groups studied, there was the decrease in the inflammatory marker COX-2 both after 2 weeks of CCl 4 + ethanol withdrawal and after treatment with studied compounds 1 and 2 . Thus, compound 2 (conjugate of Xymedon with L -ascorbic acid) has the greater antifibrotic effect on the rat liver fibrosis model compared to the native molecule of compound 1 (Xymedon). At the same time, this effect is not associated with the level of COX-2. Xymedon fibrosis carbon tetrachloride pyrimidines inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction The liver is one of the most important organs in the human body and performs various functions, including participating in the biotransformation of substances. Despite its high regenerative potential, chronic exposure to toxic agents can prevent the liver from recovering properly, leading to the development of hepatic fibrosis and cirrhosis (the last stage of fibrosis) [ 1 ]. Liver fibrosis and cirrhosis remain the leading cause of morbidity and mortality worldwide, and fibrosis is increasingly recognized as an important problem in modern healthcare [ 2 , 3 ]. Moreover, organ transplantation is often the only way to treat cirrhosis, which is difficult to manage for most patients [ 4 ]. Therefore, finding new ways of anti-fibrotic therapy is an important task [ 5 ]. Liver fibrosis is characterized by the chronic damage and the inflammatory processes in the liver tissue [ 22 ]. Inflammatory agents, including the enzyme cyclooxygenase-2 (COX-2), initiate inflammation, which is an important inducer of fibrosis [ 6 ]. Under the influence of inflammatory factors, hepatic stellate cells are activated, leading to the accumulation of extracellular matrix (ECM) components that results in liver dysfunction [ 22 ]. In this study, we used the CCl 4 and ethanol-induced fibrosis model, which is one of the most widely used animal models [ 1 ]. The Russian drug Xymedon (4,6-dimethyl-1,2-dihydro-1-(2-hydroxyethyl)pyrimidin-2-one) and its conjugate with L -ascorbic acid were used to treat the fibrotic changes (Fig. 1 ). Pyrimidines and their derivatives are known to have high biological activity and antihepatotoxic potential [ 7 , 8 ]. In addition, antifibrotic activity has also been demonstrated for some pyrimidine derivatives, such as a tyrosine kinase inhibitor targeting platelet-derived growth factor receptors (PDGFR) in the liver [ 9 ], guanylate cyclase stimulator in the kidney [ 10 ], selective DCN1-UBC12 inhibitor in the heart [ 11 ], autotoxin inhibitors in the heart and liver [ 12 ] and a dipeptidyl peptidase-4 (DPP-4) inhibitor in the kidney [ 13 ]. Our previous work with Xymedon and its conjugate with L -ascorbic acid has already demonstrated hepatoprotective activity in acute models of liver injury [ 14 , 15 ] and primary antifibrotic properties [ 16 ]. Thus, the aim of this work was to study the antifibrotic effect of Xymedon and its conjugate with L-ascorbic acid during the therapeutic scheme of compounds administration, as well as to study the effect of these compounds on the inflammatory marker COX-2 in the rat liver fibrosis model. 2 Materials and methods 2.1 Compound synthesis Xymedon (4,6-dimethyl-1,2-dihydro-1-(2-hydroxyethyl)pyrimidin-2-one) and its conjugate with L -ascorbic acid were synthesized by the previously described methods [ 15 , 17 ]. 2.2 Experiment scheme The experiment was conducted on 48 adults female Wistar rats weighing 220–280 g, obtained from the Research and Production Enterprise Laboratory Animal Farm based at the Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Pushchino). The animals were kept in accordance with References [ 18 , 19 ] in standard conditions in a vivarium with 12-h daylight and free access to food and water. The animals were fed with complete feed made according to specification (protein, 22%; fiber, 4% max.; fat, 5% max.; ash, 9% max.; humidity, 13.5% max.; caloric value, 295 kcal/100 g). The Local Ethics Committee of Kazan Federal University approved all animal experiments and protocols (Protocol No. 4, dated: May 18, 2017). Initially, the rats were randomly divided into two groups: the intact control group (n = 6) and a group of rats with modeled fibrosis (n = 42). Fibrosis was induced for 8 weeks by the administration of carbon tetrachloride and ethanol (CCl 4 + ethanol) according to the following scheme: 5% oil solution of CCl 4 at the dose of 2 ml/kg was administered orally to the animals twice a week. To potentiate the effect of CCl 4 , rats were given the 5% aqueous solution of ethanol in the drinking water with free access for 8 weeks. At week 9, rats with modeled fibrosis were withdrawn from CCl 4 + ethanol treatment and randomly divided into 6 groups of 6 rats each. Some rats received 2 weeks of fibrosis treatment: control group (n = 6), compound 1 group (n = 6), compound 2 group (n = 6); some rats received 4 weeks of fibrosis treatment: control group (n = 6), compound 1 group (n = 6), compound 2 group (n = 6). In addition, to control the model, biomaterial was collected from rats immediately after modeling fibrosis with CCl 4 + ethanol for 2 months (n = 6). Compounds 1 and 2 were administered intraperitoneally at a dose of 0.24 mg/kg and 0.5 mg/kg, respectively. Solutions of compounds for injection were prepared with physiological saline immediately prior to administration. The control group received an equivalent volume of physiological saline. The intact control group remained unaffected throughout the experiment. After administration of the compounds, the animals were euthanized, and material was collected after 2 and 4 weeks of fibrosis treatment. 2.3 Histopathology assessment Liver tissues were fixed into 10% buffered formalin, embedded in paraffin, deparaffinized, and rehydrated with distilled water. Liver sections of 5 µm thickness were stained with hematoxylin-eosin (H&E) and Van Gizon’s picrofuchsin method using a routine protocol. Morphometric analysis of liver sections was performed using the Nikon H550S microscope with NIS-Elements Basic Research software. Quantitative assessment of fibrosis (% fibrosis) was performed as the ratio of collagen area to visible area of liver tissue using methods of digital image analysis as described in our previous work [ 16 ] and in [ 20 ]. 2.4 Biochemical assessment Blood samples were collected posthumously. The samples were centrifuged at 4°C at 3000 rpm for 10 min. Then the serum samples were tested on the automated biochemical analyzer (ARD, Russia). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) were evaluated. 2.5 Western blot analysis Liver tissue samples were lysed according to the MicroRotofor Lysis Kit protocol (BioRad, USA). Total protein content was determined by the Bradford’s method using the Quick Start Bradford Protein Assay Kit (BioRad, USA) and the Epoch microplate spectrophotometer (BioTech, USA). Equal amounts of proteins were separated by 12.5% SDS polyacrylamide electrophoresis gels and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% skim milk for 1 hour and then incubated overnight at 4°C with the primary anticyclooxygenase-2 (COX-2) and anti-β-actin antibodies (SAB5500087 and ZRB1312 respectively, Sigma-Aldrich, USA) (diluted 1:1000 with 5% skim milk). The membranes were then washed with Tris-Buffered saline Tween-20 (TBST) and then incubated with horseradish peroxidase-conjugated secondary antibodies (A0545, Sigma-Aldrich, USA) (diluted 1:10000 with TBST) on the second day. Finally, the membranes were visualized using the Clarity Western ECL Substrate Kit (Bio-Rad, USA) and ChemiDoc Imaging Systems (Bio-Rad, USA) with Image Lab Touch software (Bio-Rad, USA). 2.6 Statistics All data in the article are presented as mean ± SEM. The normal distribution was determined using the Kolmogorov-Smirnov criterion. In the case of normal distribution, one-way ANOVA with Tukey’s post-hoc test was used, and statistical significance was considered as p ≤ 0.05. In the case of non-normal distribution, Kruskal-Wallis test with Mann-Whitney’s test and Bonferroni correction for multiple comparisons was used. The statistical tests used are presented in the text of the article in the captions of the figures. Statistical analysis was performed with the SPSS Statistics program. 3 Results 3.1 Estimation of liver mass coefficient During the study of the mass coefficient of the liver of rats after 2 weeks of fibrosis treatment (Fig. 2 ), it is shown that after exposure to CCl 4 + ethanol in the control group, there is the increase (p = 0.014) in the mass coefficient compared to the intact control group (2.75 ± 0.04% and 2.37 ± 0.04%, respectively). However, under the influence of compound 2 , there is the decrease (p = 0.005) in the liver mass coefficient (2.48 ± 0.07%), and under the influence of compound 1 , there is only the tendency to decrease the mass coefficient (2.66 ± 0.04%). When studying the mass coefficient of the liver of rats after 4 weeks of fibrosis treatment (Fig. 2 ), we see similar results. It can be seen that after exposure to CCl 4 + ethanol in the control group, there is the increase (p = 0.006) in the liver mass coefficient compared to the intact control group (2.88 ± 0.11% and 2.37 ± 0.04%, respectively). However, under the influence of compound 2 , there is the decrease (p = 0.033) in the liver mass coefficient (2.52 ± 0.05%), and under the influence of compound 1 , there is the tendency to decrease the mass coefficient (2.60 ± 0.09%). 3.2 Histological assessment of liver tissue 3.2.1 Assessment of overall liver tissue morphology The overall morphology of the liver tissue, the presence of steatosis and parenchymal dystrophies after 2 weeks of fibrosis treatment were evaluated by staining with hematoxylin and eosin (Fig. 3 -a). In the intact control group, the morphology of the liver tissue was normal and its structure was completely healthy. However, relatively large areas of hydropic and ballooning dystrophy predominate in the control group after two weeks of CCl 4 + ethanol withdrawal, indicating impaired hepatocyte function. Cases of steatosis are rare. However, according to the biochemical data (shown below), liver function is not compromised, which may be due to the more intense work of the remaining hepatocytes. In the experimental groups treated with compounds 1 and 2 , a decrease in the occurrence of parenchymal dystrophies and the absence of steatosis were observed, indicating better organ functioning. In addition, compound 2 was shown to reduce the liver's mass coefficient, unlike the control, indicating normal tissue functioning. After 4 weeks of fibrosis treatment (Fig. 3 -b), small foci of hydropic dystrophy of hepatocytes are still present in the liver tissue of the control group after CCl 4 + ethanol withdrawal compared to the intact control group. However, balloon dystrophy, like steatosis, is rare. In the experimental groups, which were administered the tested compounds, hepatocytes of regular shape with homogeneous and rarely granular cytoplasm are observed, indicating almost complete restoration of liver tissue structure. It should be noted that in the liver tissue, areas of hydropic dystrophy are more frequent in the group exposed to compound 1 than in the group exposed to compound 2 . 3.2.2 Quantification of liver fibrosis As the result of quantitative assessment of liver tissue fibrosis (Fig. 3 -c, d, e), it was shown that after 2 months of fibrosis modeling in rat liver tissue, there was the significant increase (p < 0.0001) in the expression level of collagen fibers, which was 5.7 times higher compared to the intact control (6.3 ± 0.9% and 1.05 ± 0.08%, respectively). However, after 2 weeks of CCl 4 + ethanol withdrawal, collagen fiber remodeling was observed in the control group at the level (2.91 ± 0.17%) that was 2.8 times higher (p < 0.0001) than in the intact control group, indicating the presence of chronic changes in liver tissue. The administration of compound 1 for 2 weeks had no significant effect on reducing the amount of collagen fibers (2.79 ± 0.1%) compared to the control group. However, with the administration of compound 2 , there was the significant reduction (p < 0.0001) of the detected collagen fibers by 1.7 times (1.72 ± 0.09%), approaching the levels of the intact group, indicating the more effective resolution of fibrosis. Quantitative assessment of fibrosis after 4 weeks of treatment showed the similar result to that after 2 weeks of treatment (Fig. 3 -e). In the control group, after 4 weeks of CCl 4 + ethanol withdrawal, the level of collagen remained 3 times higher (p < 0.0001) compared to the intact control group (3.10 ± 0.32% and 1.05 ± 0.08%, respectively). In the case of compound 1 administration in liver tissue, the relatively high level of collagen fibers was observed (2.94 ± 0.38%), while with compound 2 administration, there was the tendency (p = 0.02) to reduce collagen fibers by 1.5 times (2.03 ± 0.13%) compared to the control group. In conclusion, it can be stated that the main stage of collagen remodeling occurred during the first two weeks of treatment and cessation of CCl 4 + ethanol, and then fibrotic changes were preserved at the certain chronic level. 3.3 Biochemical parameters of blood serum assessment Our findings indicate that modeling of rat liver fibrosis resulted in changes in serum biochemical indices (Fig. 4 ). At the week 8 of fibrosis modeling and exposure to CCl 4 + ethanol, the biochemical indicators of liver tissue damage demonstrated changes compared to the intact control group. The 7-fold increase (p = 0.0001) in the ALT index compared to the intact control group (312.8 ± 46.7 and 44.0 ± 1.8 U/L, respectively) and the 4-fold increase (p = 0.002) in the AST index compared to the intact control group (538.5 ± 129.0 and 123.8 ± 7.8 U/L, respectively), were observed. In addition, changes in markers of cholestatic injury in liver tissue were noted at the 8th week of CCl 4 + ethanol exposure. The levels of GGT and ALP increased by 2-fold and 2.7-fold (p < 0.0001), respectively, compared to the intact control group (2.7 ± 0.7 and 1.3 ± 0.3 U/L for GGT and 409.8 ± 50.1 and 152.8 ± 19.5 U/L for ALP, respectively). However, restoration of serum biochemical indices to their initial values was observed in the control group after 2 weeks of CCl 4 + ethanol withdrawal and in the experimental groups with administration of compounds 1 and 2 after 2 weeks of treatment (Fig. 4 ). This observation indicates the restoration of liver tissue functional activity. The similar picture was observed after 4 weeks of fibrosis treatment (data not shown). 3.4 Assessment of COX-2 expression level In this study, we examined the expression level of the inflammatory mediator COX-2 only after 2 weeks of fibrosis treatment (Fig. 5 ), because signs of liver tissue recovery are observed within the first 2 weeks, as described above. Western blot analysis of COX-2 expression showed that the level of COX-2 was significantly increased (p = 0.0003) when CCl 4 + ethanol-induced fibrosis was modeled for 2 months compared to the intact control group (0.24 ± 0.03 versus 0.11 ± 0.01 COX-2/β-actin ratio). However, after 2 weeks of CCl 4 + ethanol withdrawal, the level of COX-2 in the control group (0.09 ± 0.01 COX-2/β-actin ratio) decreased almost to the level of the intact control group (p = 0.3713). This effect was also observed in the groups receiving compound 1 (p = 0.2397) and compound 2 (p = 0.016) (0.08 ± 0.01 and 0.05 ± 0.01 COX-2/β-actin ratio, respectively). 4 Discussion Liver cirrhosis, the final stage of fibrosis, is one of the main causes of death worldwide. The most common cause of this disease is non-alcoholic fatty liver disease [ 21 ]. The pathogenesis of liver fibrosis is understood to involve the accumulation of extracellular matrix, including collagen, in response to injury. The presence of ECM is the primary histologic marker of disease progression [ 22 ]. Injury to liver tissue induces an inflammatory response that promotes the expression of inflammatory mediators and the transdifferentiation of stellate cells into myofibroblasts. Then the myofibroblasts secrete extracellular matrix components [ 23 – 25 ]. In this experiment, we examined the inflammatory marker COX-2, because it is known that pyrimidine derivatives are inhibitors of this enzyme [ 26 ]. COX-2 is an enzyme involved in the biosynthesis of prostaglandins, which maintain chronic inflammation [ 27 ]. COX-2 levels are increased in CCl 4 -induced liver fibrosis models [ 28 ], and the administration of COX-2 inhibitors has been shown to reduce the development of CCl 4 -induced rat liver fibrosis [ 29 ]. In addition, stellate cells activated express COX-2 [ 30 ]. Thus, COX-2 expression may serve as an indirect marker of the attenuation or activation of extracellular matrix deposition by activated stellate cells. Thus, in our work, liver fibrosis was modeled in rats and treated for 2 or 4 weeks. It was shown that after the induction of fibrosis for 2 months followed by the cessation of CCl 4 + ethanol, collagen remodeling occurred within 2 weeks and maintained at the chronic elevated level for up to 4 weeks post-cessation compared to the intact control group. The elevated liver mass coefficient persisted for 4 weeks after of CCl 4 + ethanol withdrawal. Parenchymal dystrophic foci were present at 2 weeks but had been attenuated by 4 weeks post-cessation of CCl 4 + ethanol. Biochemical markers ALT, AST, ALP and GGT returned to the level of healthy animals after 2 weeks of CCl 4 + ethanol withdrawal. Notably, the immediate increase in COX-2, indicating the inflammatory reaction, was observed after 2 months of fibrosis induction with CCl 4 + ethanol. However, COX-2 levels decreased to the level of the intact control within two weeks post-cessation of CCl 4 + ethanol. These findings indicate the liver’s capacity for self-repair after the removal of the damaging agent, consistent with prior research [ 31 , 32 ]. Moreover, it is established that ECM self-degradation and reversibility of liver fibrosis are achievable upon removal of the damaging agent. It is noteworthy that after removal of the cause of fibrosis, the liver tissue can adapt to a new structure to ensure its normal functioning, making fibrosis clinically but not morphologically reversible [ 33 , 34 ]. While compound 1 treatment led to the faster restoration of rat liver tissue and reduced detection of parenchymal dystrophies within the second week of fibrosis treatment, it did not accelerate the collagen remodeling processes compared to the CCl 4 + ethanol control group. Conversely, the treatment of compound 2 not only restored liver tissue structure but also significantly increased collagen fiber remodeling, decreased liver mass coefficient, and showed the trend of reduced COX-2 expression compared to the CCl 4 + ethanol control group. Thus, this study, as well as our previous work, demonstrated the higher efficacy of the compound 2 compared to the compound 1 in the treatment of both acute toxic liver injury [ 35 ] and chronic liver injury models [ 16 ]. What is the relationship between accelerated collagen remodeling and the effects of compound 2 ? The main damaging mechanism of CCl 4 on cells is oxidative stress and lipid peroxidation [ 36 ]. In combination with alcohol, the effect of CCl 4 is enhanced due to the exacerbation of oxidative stress and increased load on cytochrome P-450 [ 31 ]. This leads to chronic cell damage, activation of the inflammatory process, and initiation of fibrotic changes in liver tissue. However, compound 1 is known to affect the levels of adenylyl cyclase and cyclic adenosine monophosphate (cAMP) in immunocompetent cells [ 37 ]. This may indicate the putative effect of compound 1 on cell receptors associated with G-protein and adenylyl cyclase activity, such as adrenergic [ 38 ] or P2Y receptors [ 39 ]. In turn, cAMP as a secondary messenger can play a variety of biological roles [ 40 ], including the role of a regulator of the inflammatory process [ 41 ], cell proliferation processes [ 42 ], and activation of hepatic stellate cells [ 43 ]. It has been shown that an increase in the level of cAMP, including due to phosphodiesterase inhibitors, reduces the proliferation of hepatic stellate cells, inhibits their transdifferentiation into myofibroblasts, and thus reduces fibrosis signs [ 44 ]. Thus, the influence of compound 1 on the intracellular level of cAMP can simulate various cell states, which may explain the ability of compound 1 to accelerate liver tissue repair, as shown in the description of the overall liver tissue morphology above. In our previous work [ 45 ], we also showed that closing the putative active site of the compound 1 molecule reduced its biological activity, indicating its putative effect on cell receptors. However, conjugation of compound 1 with L -ascorbic acid enhanced the antifibrotic effect of the native compound 1 in this study, which can be explained by several reasons. It has been shown that conjugation of compound 1 molecule with L -ascorbic acid can increase their bioavailability [ 46 ] as well as enhance their biological effect [ 47 ]. Moreover, L -ascorbic acid has been shown to have hepatoprotective activity [ 48 ] and is known for its antioxidant activity [ 49 ], which could positively affect the reduction of oxidative stress induced by CCl 4 + ethanol. In addition, in our previous work [ 50 ], compound 2 showed antiradical activity and antioxidant properties that are less pronounced in the native compound 1 , what could also affect the reduction of oxidative stress after the effect of CCl 4 + ethanol. 5 Conclusion The conjugation of the pyrimidine derivative of the Russian drug Xymedon with L -ascorbic acid has been found to enhance its antifibrotic properties in the rat liver fibrosis treatment model. Administration of the Xymedon- L -ascorbic acid conjugate resulted in a 41% faster collagen remodeling when compared to the control group. However, the antifibrotic effect of this conjugate is not associated with its influence on the expression level of the inflammatory enzyme cyclooxygenase-2 (COX-2). Declarations This study was performed within the framework of the state assignment of the Federal Research Center «Kazan Scientific Center of the Russian Academy of Sciences». The local ethical committee of the Kazan (Volga Region) Federal University (records of May 18, 2017, No. 4) approved all studies and protocols for handling animals. The authors have no competing interests to declare that are relevant to the content of this article. Contributions Methodology: Belyaev Grigory, Vyshtakalyuk Alexandra, Parfenov Andrey, Semenov Vyacheslav; design and synthesis of test compounds: Semenov Vyacheslav, Galyametdinova Irina; formal analysis and investigation: Belyaev Grigory; writing-original draft preparation: Belyaev Grigory; writing-review and editing: Vyshtakalyuk Alexandra, Semenov Vyacheslav, Zobov Vladimir. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments This study was performed within the framework of the state assignment of the Federal Research Center «Kazan Scientific Center of the Russian Academy of Sciences». References Faccioli LAP, Dias ML, Paranhos BA, dos Santos Goldenberg RC (2022) Liver cirrhosis: An overview of experimental models in rodents. Life Sci 301:120615. https://doi.org/10.1016/j.lfs.2022.120615 Devarbhavi H, Asrani SK, Arab JP, Nartey Y.A, Pose E, Kamath PS (2023) Global burden of liver disease: 2023 update. J Hepatol 79:516-537. https://doi.org/10.1016/j.jhep.2023.03.017 Dees C, Chakraborty D, Distler JHW (2021) Cellular and molecular mechanisms in fibrosis. Exp Dermatol 30:121-131. https://doi.org/10.1111/exd.14193 Ginès P, Krag A, Abraldes JG, Solà E, Fabrellas N, Kamath PS (2021) Liver cirrhosis. Lancet 398:1359-1376. https://doi.org/10.1016/s0140-6736(21)01374-x Tang JT, Mao YM (2020) Development of new drugs for the treatment of nonalcoholic steatohepatitis. J Dig Dis 21:3-11. https://doi.org/10.1111/1751-2980.12830 Yang H, Xuefeng Y, Shandong W, Jianhua X (2020) COX-2 in liver fibrosis. Clin Chim Acta 506:196-203. https://doi.org/10.1016/j.cca.2020.03.024 Khasimbi S, Ali F, Manda K, Sharma A, Chauhan G, Wakode S (2021) Dihydropyrimidinones scaffold as a promising nucleus for synthetic profile and various therapeutic targets: A Review. Curr Org Synth 18:270-293. https://doi.org/10.2174/1570179417666201207215710 Bhat MA, Al-Omar MA, Khan AA, Alanazi AM, Naglah AM (2019) Synthesis and antihepatotoxic activity of dihydropyrimidinone derivatives linked with 1, 4-benzodioxane. Drug Des Devel Ther 13:2393-2404. https://doi.org/10.2147/dddt.s198865 Karimi J, Mohammadalipour A, Sheikh N, Khodadadi I, Hashemnia M, Goudarzi F, et al (2020) Protective effects of combined Losartan and Nilotinib on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Drug Chem Toxicol 43:468-478. https://doi.org/10.1080/01480545.2018.1504960 Sravani S, Saifi MA, Godugu C (2020) Riociguat ameliorates kidney injury and fibrosis in an animal model. Biochem Biophys Res Commun 530:706-712. https://doi.org/10.1016/j.bbrc.2020.07.128 He ZX, An Q, Wei B, Zhou W J, Wei BF, Gong YP, et al (2021) Discovery of Potent and Selective 2-(Benzylthio) pyrimidine-based DCN1-UBC12 Inhibitors for Anticardiac Fibrotic Effects. J Med Chem 65:163-190. https://doi.org/10.1021/acs.jmedchem.1c01207 Jiang N, Zhou Y, Zhu M, Zhang J, Cao M, Lei H, et al (2020) Optimization and evaluation of novel tetrahydropyrido [4, 3-d] pyrimidine derivatives as ATX inhibitors for cardiac and hepatic fibrosis. Eur J Med Chem 187:111904. https://doi.org/10.1016/j.ejmech.2019.111904 Seo JB, Choi YK, Woo HI, Jung YA, Lee S, Lee S, Park M, Lee IK, Jung GS, Park KG (2019) Gemigliptin attenuates renal fibrosis through down-regulation of the NLRP3 inflammasome. Diabetes Metab J 43:830-839. https://doi.org/10.4093/dmj.2018.0181 Vyshtakalyuk A, Nazarov N, Zueva I, Lantsova A, Minnekhanova O, Busygin D, et al (2013) Study of hepatoprotective effects of xymedon. Bull Exp Biol Med 155:643-646. https://doi.org/10.1007/s10517-013-2215-0 Vyshtakalyuk AB, Semenov VE, Zobov VV, Galyametdinova IV, Gumarova LF, Parfenov AA, et al (2017) Synthesis and primary evaluation of the hepatoprotective properties of novel pyrimidine derivatives. Russ J Bioorg Chem 43:604-611. https://doi.org/10.1007/s12668-017-0461-8 Belyaev GP, Vyshtakalyuk AB, Parfenov AA, Galyametdinova IV, Semenov VE, Zobov VV (2023) Antifibrotic effect of pyrimidine derivatives of Xymedon and its conjugate with L-ascorbic acid. Uch Zap Kazan Univ, Ser Estestv Nauki 165:175–189. https://doi.org/10.26907/2542-064x.2023.2.175-189 (In Russian) Reznik VS, Pashkurov NG (1966) Reactions of pyrimidinols and pyrimidinethiols with 2-chloroethanol and with 2-chloro-1-propanol. Bull Acad Sci USSR, Div Chem Sci 15:1554-1557. https://doi.org/10.1007/bf00848915 Mironov, A. N. et al. (2012). Rukovodstvo po provedeniyu doklinicheskih issledovanij lekarstvennyh sredstv. Chast` pervaya. Grif i K: Moskva, Rossiya. (In Russian). European Commission. Commission Recommendation of 18 June 2007 on guidelines for the accommodation and care of animals used for experimental and other scientific purposes. OJEU 2007;L197:1-89. Arjmand A, Tsipouras MG, Tzallas AT, Forlano R, Manousou P, Giannakeas N (2020) Quantification of liver fibrosis—A comparative study. Appl Sci 10:447. https://doi.org/10.3390/app10020447 Cheemerla S, Balakrishnan M (2021) Global epidemiology of chronic liver disease. Clin Liver Dis 17:365-370. https://doi.org/10.1002/cld.1061 Zhang M, Serna-Salas S, Damba T, Borghesan M, Demaria M, Moshage H (2021) Hepatic stellate cell senescence in liver fibrosis: Characteristics, mechanisms and perspectives. Mech Ageing Dev 199:111572. https://doi.org/10.1016/j.mad.2021.111572 Li J, Wang T, Liu P, Yang F, Wang X, Zheng W, et al (2021) Hesperetin ameliorates hepatic oxidative stress and inflammation via the PI3K/AKT-Nrf2-ARE pathway in oleic acid-induced HepG2 cells and a rat model of high-fat diet-induced NAFLD. Food Funct 12:3898-3918. https://doi.org/10.1039/d0fo02736g Zoubek ME, Trautwein C, Strnad P (2017) Reversal of liver fibrosis: From fiction to reality. Best Pract Res Clin Gastroenterol 31:129-141. https://doi.org/10.1016/j.bpg.2017.04.005 Tsuchida T, Friedman SL (2017) Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol 14:397-411. https://doi.org/10.1038/nrgastro.2017.38 Abdelgawad MA, Bakr RB, Azouz AA (2018) Novel pyrimidine-pyridine hybrids: synthesis, cyclooxygenase inhibition, anti-inflammatory activity and ulcerogenic liability. Bioorg Chem 77:339-348. https://doi.org/10.1016/j.bioorg.2018.01.028 Kaur B, Singh P (2022) Inflammation: biochemistry, cellular targets, anti-inflammatory agents and challenges with special emphasis on cyclooxygenase-2. Bioorg Chem 121:105663. https://doi.org/10.1016/j.bioorg.2022.105663 Ning C, Gao X, Wang C, Huo X, Liu Z, Sun H, et al (2018) Hepatoprotective effect of ginsenoside Rg1 from Panax ginseng on carbon tetrachloride‐induced acute liver injury by activating Nrf2 signaling pathway in mice. Environ Toxicol 33:1050-1060. https://doi.org/10.1002/tox.22616 Planagumà A, Clària J, Miquel R, López‐Parra M, Titos E, Masferrer JL, et al (2005) The selective cyclooxygenase‐2 inhibitor SC‐236 reduces liver fibrosis by mechanisms involving non‐parenchymal cell apoptosis and PPARγ activation. FASEB J 19:1120-1122. https://doi.org/10.1096/fj.04-2753fje Ftahy MM, Latif NSA, Alalkamy EF, El-Batrawi FA, Galal AH, Khatab HM (2013) Antifibrotic potential of a selective COX-2 inhibitor (celecoxib) on liver fibrosis in rats. Comp Clin Pathol 22:425-430. https://doi.org/10.1007/s00580-012-1427-4 Thomes PG, Rasineni K, Yang L, Donohue Jr TM, Kubik JL, McNiven MA, et al (2019) Ethanol withdrawal mitigates fatty liver by normalizing lipid catabolism. Am J Physiol Gastrointest Liver Physiol 316:G509-G518. https://doi.org/10.1152/ajpgi.00376.2018 Peugnet-González I, Martínez-Hernández SL, Ávila-Blanco ME, Hernández-Marín DA, Macias-Pérez JR, Aldaba-Muruato LR, et al (2023) Hepatoprotective and antifibrotic activity of watercress extract in a model of CCl4-induced liver fibrosis in Wistar rats. J Funct Foods 109:105760. https://doi.org/10.1016/j.jff.2023.105760 Di Vinicius I, Baptista AP, Barbosa Jr AA, Andrade ZA (2005) Morphological signs of cirrhosis regression: Experimental observations on carbon tetrachloride-induced liver cirrhosis of rats. Pathol, Res Pract 201:449-456. https://doi.org/10.1016/j.prp.2005.05.009 Ortiz C, Schierwagen R, Schaefer L, Klein S, Trepat X, Trebicka J (2021) Extracellular matrix remodeling in chronic liver disease. Curr Tissue Microenviron Rep 2:41-52. https://doi.org/10.1007/s43152-021-00030-3 Vyshtakalyuk AB, Parfenov AA, Galyametdinova IV, Semenov VE, Zobov VV (2022) Antiapoptotic mechanism for the implementation of the hepato-protective effect of pyrimidine derivatives. Int J Pharm Sci Res 13:3922-3931. https://doi.org/10.13040/IJPSR.0975-8232.13(10).39231 Aramjoo H, Mohammadparast-Tabas P, Farkhondeh T, Zardast M, Makhdoumi M, Samarghandian S, et al (2022) Protective effect of Sophora pachycarpa seed extract on carbon tetrachloride-induced toxicity in rats. BMC Complement Med Ther 22:76. https://doi.org/10.1186/s12906-022-03554-9 Slabnov YuD, Cherepnev GV, Karimova FG, Garaev RS (1998) Effect of pyrimidine derivatives on adenylate cyclase system of immunocompetent cell regulation in vitro. Bull Exp Biol Med 125:588-590. https://doi.org/10.1007/bf02445248 Alcántara-Hernández R, Hernández-Méndez A (2018) Adrenergic signaling molecular complexes. Gac Med Mex 154:223-235. https://doi.org/10.24875/gmm.m18000135 Communi D, Horckmans M, Boeynaems JM (2021) P2Y4, P2Y6 and P2Y11 receptors: From the early days of cloning to their function. Biochem Pharmacol 187:114347. https://doi.org/10.1016/j.bcp.2020.114347 Beavo JA, Brunton LL (2002) Cyclic nucleotide research—still expanding after half a century. Nat Rev Mol Cell Biol 3:710-718. https://doi.org/10.1038/nrm911 Pacini ESA, Satori NA, Jackson EK, Godinho RO (2022) Extracellular cAMP-Adenosine Pathway Signaling: A Potential Therapeutic Target in Chronic Inflammatory Airway Diseases. Front Immunol 13:866097. https://doi.org/10.3389/fimmu.2022.866097 Guo R, Liu T, Shasaltaneh MD, Wang X, Imani S, Wen Q (2022) Targeting adenylate cyclase family: New concept of targeted cancer therapy. Front Oncol 12:829212. https://doi.org/10.3389/fonc.2022.829212 Insel PA, Murray F, Yokoyama U, Romano S, Yun H, Brown L, et al (2012) cAMP and Epac in the regulation of tissue fibrosis. Br J Pharmacol 166:447-456. https://doi.org/10.1111/j.1476-5381.2012.01847.x El Awdan SA, Abdel Rahman RF, Ibrahim HM, Hegazy RR, El Marasy SA, Badawi M, et al (2019) Regression of fibrosis by cilostazol in a rat model of thioacetamide-induced liver fibrosis: Up regulation of hepatic cAMP, and modulation of inflammatory, oxidative stress and apoptotic biomarkers. PloS One 14:e0216301. https://doi.org/10.1371/journal.pone.0216301 Belyaev GP, Vyshtakalyuk AB, Parfenov AA, Shashin MS, Galyametdinova IV, Semenov VE, et al (2022) Comparative assessment of hepatoprotective properties of some “doubled” 4, 6-dimethyl-1, 2-dihydro-1-(2-hydroxyethyl) pyrimidin-2-one derivatives. Russ Chem Bull 71:2701-2710. https://doi.org/10.1007/s11172-022-3699-4 Harrison FE, May JM (2009) Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2. Free Radic Biol Med 46:719-730. https://doi.org/10.1016/j.freeradbiomed.2008.12.018 Moteki H, Kimura M, Sunaga K, Tsuda T, Ogihara M (2013) Signal transduction mechanism for potentiation by α1-and β2-adrenoceptor agonists of l-ascorbic acid-induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Eur J Pharmacol 700:2-12. https://doi.org/10.1016/j.ejphar.2012.12.010 Kimura M, Moteki H, Uchida M, Natsume H, Ogihara M (2014) L-ascorbic acid-and L-ascorbic acid 2-glucoside accelerate in vivo liver regeneration and lower serum alanine aminotransaminase activity in 70% partially hepatectomized rats. Biol Pharm Bull 37:597-603. https://doi.org/10.1248/bpb.b13-00839 Njus D, Kelley PM, Tu YJ, Schlegel HB (2020) Ascorbic acid: The chemistry underlying its antioxidant properties. Free Radic Biol Med 159:37-43. https://doi.org/10.1016/j.freeradbiomed.2020.07.013 Vyshtakalyuk AB, Semenov VE, Sudakov IA, Bushmeleva KN, Gumarova LF, Parfenov AA, et al (2018) Xymedon conjugate with biogenic acids. Antioxidant properties of a conjugate of Xymedon with L-ascorbic acid. Russ Chem Bull 67:705-711. https://doi.org/10.1007/s11172-018-2126-3 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 12 Jun, 2025 Read the published version in Uchenye Zapiski Kazanskogo Universiteta Seriya Estestvennye Nauki → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3953710","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273361297,"identity":"51729e49-36c9-47ce-97c1-0d6ff9e10c33","order_by":0,"name":"Grigory P. Belyaev","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYNACAwZmfhCdUECCFnbJBpAWAxLs4Tc4ANFLGMhHHz784kPBPWnj86sTPzwwYJDnFzuAX4vhubQ0yxkGxcZmN95ulgA6zHDm7AQCWnp4zIx5DBKSzW6c3QDSkmBwm6AW/m8gLfWbZ5zd/IMoLfI8PMyPgVqYDfh7txFniwEPmxnjDKAWiRu82ywSDCQI+0W+h/nxhw9/Epj5+89uvvmjwkaeX5qQLQcY2CTALAmwSgn8ysG2NDAwfwCz+A8QVj0KRsEoGAUjEwAAwJ5Ap5gOUMgAAAAASUVORK5CYII=","orcid":"","institution":"A.E. Arbuzov Institute of Organic and Physical Chemistry","correspondingAuthor":true,"prefix":"","firstName":"Grigory","middleName":"P.","lastName":"Belyaev","suffix":""},{"id":273361298,"identity":"31d81b9c-6803-4016-88e2-36c6ccd2bde5","order_by":1,"name":"Alexandra B. Vyshtakalyuk","email":"","orcid":"","institution":"A.E. Arbuzov Institute of Organic and Physical Chemistry","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"B.","lastName":"Vyshtakalyuk","suffix":""},{"id":273361299,"identity":"dac3bb81-fc91-40d6-a07c-c8a880c97675","order_by":2,"name":"Andrey A. Parfenov","email":"","orcid":"","institution":"A.E. Arbuzov Institute of Organic and Physical Chemistry","correspondingAuthor":false,"prefix":"","firstName":"Andrey","middleName":"A.","lastName":"Parfenov","suffix":""},{"id":273361300,"identity":"9d751404-83fa-40fc-80bd-e2fb9e0f9f79","order_by":3,"name":"Irina V. Galyametdinova","email":"","orcid":"","institution":"A.E. Arbuzov Institute of Organic and Physical Chemistry","correspondingAuthor":false,"prefix":"","firstName":"Irina","middleName":"V.","lastName":"Galyametdinova","suffix":""},{"id":273361301,"identity":"a5db20da-c01a-482b-add0-b60ee1f40dc4","order_by":4,"name":"Vyacheslav E. Semenov","email":"","orcid":"","institution":"A.E. Arbuzov Institute of Organic and Physical Chemistry","correspondingAuthor":false,"prefix":"","firstName":"Vyacheslav","middleName":"E.","lastName":"Semenov","suffix":""},{"id":273361302,"identity":"061b9fb7-def3-43f6-93ac-d075a6504b1d","order_by":5,"name":"Vladimir V. Zobov","email":"","orcid":"","institution":"A.E. Arbuzov Institute of Organic and Physical Chemistry","correspondingAuthor":false,"prefix":"","firstName":"Vladimir","middleName":"V.","lastName":"Zobov","suffix":""}],"badges":[],"createdAt":"2024-02-13 14:30:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3953710/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3953710/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.26907/2542-064X.2025.2.276-296","type":"published","date":"2025-06-13T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51331029,"identity":"daaed0bb-791f-4c55-abed-af3a825ea57c","added_by":"auto","created_at":"2024-02-19 17:51:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9669,"visible":true,"origin":"","legend":"\u003cp\u003eStructural formula of studied compounds. 1 – Xymedon (compound \u003cstrong\u003e1\u003c/strong\u003e), 2 – Xymedon conjugate with \u003cem\u003eL\u003c/em\u003e-ascorbic acid (compound \u003cstrong\u003e2\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3953710/v1/100f9f4c1098e8abe1817586.png"},{"id":51331030,"identity":"e6b8cf7a-787b-446b-9daf-3e1c217bf09d","added_by":"auto","created_at":"2024-02-19 17:51:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":194628,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of compounds on rat body/liver mass ratio. Statistical analysis was performed using ANOVA with Tukey’s post hoc test at p ≤ 0.05.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3953710/v1/ab556553fef4f29baf2e36d6.png"},{"id":51331032,"identity":"61cdeadc-7dc5-4d20-a08d-0c9f15b0f9f6","added_by":"auto","created_at":"2024-02-19 17:51:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9276042,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e), (\u003cstrong\u003eb\u003c/strong\u003e) – The effect of compounds on the morphology of rat liver tissue, hematoxylin-eosin staining: (\u003cstrong\u003ea\u003c/strong\u003e) – after 2 weeks of treatment, (\u003cstrong\u003eb\u003c/strong\u003e) – after 4 weeks of treatment. (\u003cstrong\u003ec\u003c/strong\u003e), (\u003cstrong\u003ed\u003c/strong\u003e) – The effect of compounds on the development of collagen fibers (red color) in rat liver tissue, Van Gizon’s staining: (\u003cstrong\u003ec\u003c/strong\u003e) – after 2 weeks of treatment, (\u003cstrong\u003ed\u003c/strong\u003e) – after 4 weeks of treatment. (\u003cstrong\u003ee\u003c/strong\u003e) – The effect of compounds on changes in collagen area in rat liver tissue. Statistical analysis was performed using ANOVA with Tukey’s post hoc test at p ≤ 0.05.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3953710/v1/6c0aac81ef914efbd74b2f97.png"},{"id":51331028,"identity":"798d6a1b-3ac5-4105-a7bb-538db0090ff2","added_by":"auto","created_at":"2024-02-19 17:51:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":321076,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of compounds on serum biochemical parameters of rats after 2 weeks of fibrosis treatment. (a) – aspartate aminotransferase (AST) activity. (b) – alanine aminotransferase (ALT) activity. (c) – lactate dehydrogenase (LDH) activity. (d) – alkaline phosphotase (ALP) activity. Statistical analysis was performed using ANOVA with Tukey’s post hoc test at p ≤ 0.05.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3953710/v1/8cb1255a3f0700c0cfc8cbb7.png"},{"id":51331031,"identity":"05f5149a-96e8-4c8f-83ed-85be6a456c10","added_by":"auto","created_at":"2024-02-19 17:51:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":186131,"visible":true,"origin":"","legend":"\u003cp\u003eThe Western blot analysis for the inflammatory marker COX-2 in rat liver homogenates after 2 weeks of fibrosis treatment. Statistical analysis was performed using Kruskal-Wallis’s test with Mann-Whitney’s test and Bonferroni correction at p \u0026lt; 0.0051.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3953710/v1/83c620cf6d73ba4b887027e9.png"},{"id":86366758,"identity":"60c7b962-015f-4421-a77a-e66be8cf7d82","added_by":"auto","created_at":"2025-07-09 21:53:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12894875,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3953710/v1/4932e77c-5c9a-462e-9bc2-2c15ecd6fd84.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of Xymedon and its conjugate with L-ascorbic acid on collagen remodeling in the liver fibrosis rat model","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe liver is one of the most important organs in the human body and performs various functions, including participating in the biotransformation of substances. Despite its high regenerative potential, chronic exposure to toxic agents can prevent the liver from recovering properly, leading to the development of hepatic fibrosis and cirrhosis (the last stage of fibrosis) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Liver fibrosis and cirrhosis remain the leading cause of morbidity and mortality worldwide, and fibrosis is increasingly recognized as an important problem in modern healthcare [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Moreover, organ transplantation is often the only way to treat cirrhosis, which is difficult to manage for most patients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, finding new ways of anti-fibrotic therapy is an important task [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLiver fibrosis is characterized by the chronic damage and the inflammatory processes in the liver tissue [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Inflammatory agents, including the enzyme cyclooxygenase-2 (COX-2), initiate inflammation, which is an important inducer of fibrosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Under the influence of inflammatory factors, hepatic stellate cells are activated, leading to the accumulation of extracellular matrix (ECM) components that results in liver dysfunction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we used the CCl\u003csub\u003e4\u003c/sub\u003e and ethanol-induced fibrosis model, which is one of the most widely used animal models [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The Russian drug Xymedon (4,6-dimethyl-1,2-dihydro-1-(2-hydroxyethyl)pyrimidin-2-one) and its conjugate with \u003cem\u003eL\u003c/em\u003e-ascorbic acid were used to treat the fibrotic changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Pyrimidines and their derivatives are known to have high biological activity and antihepatotoxic potential [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition, antifibrotic activity has also been demonstrated for some pyrimidine derivatives, such as a tyrosine kinase inhibitor targeting platelet-derived growth factor receptors (PDGFR) in the liver [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], guanylate cyclase stimulator in the kidney [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], selective DCN1-UBC12 inhibitor in the heart [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], autotoxin inhibitors in the heart and liver [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and a dipeptidyl peptidase-4 (DPP-4) inhibitor in the kidney [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Our previous work with Xymedon and its conjugate with \u003cem\u003eL\u003c/em\u003e-ascorbic acid has already demonstrated hepatoprotective activity in acute models of liver injury [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and primary antifibrotic properties [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThus, the aim of this work was to study the antifibrotic effect of Xymedon and its conjugate with L-ascorbic acid during the therapeutic scheme of compounds administration, as well as to study the effect of these compounds on the inflammatory marker COX-2 in the rat liver fibrosis model.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Compound synthesis\u003c/h2\u003e \u003cp\u003eXymedon (4,6-dimethyl-1,2-dihydro-1-(2-hydroxyethyl)pyrimidin-2-one) and its conjugate with \u003cem\u003eL\u003c/em\u003e-ascorbic acid were synthesized by the previously described methods [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experiment scheme\u003c/h2\u003e \u003cp\u003eThe experiment was conducted on 48 adults female Wistar rats weighing 220\u0026ndash;280 g, obtained from the Research and Production Enterprise Laboratory Animal Farm based at the Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Pushchino). The animals were kept in accordance with References [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] in standard conditions in a vivarium with 12-h daylight and free access to food and water. The animals were fed with complete feed made according to specification (protein, 22%; fiber, 4% max.; fat, 5% max.; ash, 9% max.; humidity, 13.5% max.; caloric value, 295 kcal/100 g). The Local Ethics Committee of Kazan Federal University approved all animal experiments and protocols (Protocol No. 4, dated: May 18, 2017).\u003c/p\u003e \u003cp\u003eInitially, the rats were randomly divided into two groups: the intact control group (n\u0026thinsp;=\u0026thinsp;6) and a group of rats with modeled fibrosis (n\u0026thinsp;=\u0026thinsp;42). Fibrosis was induced for 8 weeks by the administration of carbon tetrachloride and ethanol (CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol) according to the following scheme: 5% oil solution of CCl\u003csub\u003e4\u003c/sub\u003e at the dose of 2 ml/kg was administered orally to the animals twice a week. To potentiate the effect of CCl\u003csub\u003e4\u003c/sub\u003e, rats were given the 5% aqueous solution of ethanol in the drinking water with free access for 8 weeks. At week 9, rats with modeled fibrosis were withdrawn from CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol treatment and randomly divided into 6 groups of 6 rats each. Some rats received 2 weeks of fibrosis treatment: control group (n\u0026thinsp;=\u0026thinsp;6), compound \u003cb\u003e1\u003c/b\u003e group (n\u0026thinsp;=\u0026thinsp;6), compound \u003cb\u003e2\u003c/b\u003e group (n\u0026thinsp;=\u0026thinsp;6); some rats received 4 weeks of fibrosis treatment: control group (n\u0026thinsp;=\u0026thinsp;6), compound \u003cb\u003e1\u003c/b\u003e group (n\u0026thinsp;=\u0026thinsp;6), compound \u003cb\u003e2\u003c/b\u003e group (n\u0026thinsp;=\u0026thinsp;6). In addition, to control the model, biomaterial was collected from rats immediately after modeling fibrosis with CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol for 2 months (n\u0026thinsp;=\u0026thinsp;6). Compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e were administered intraperitoneally at a dose of 0.24 mg/kg and 0.5 mg/kg, respectively. Solutions of compounds for injection were prepared with physiological saline immediately prior to administration. The control group received an equivalent volume of physiological saline. The intact control group remained unaffected throughout the experiment. After administration of the compounds, the animals were euthanized, and material was collected after 2 and 4 weeks of fibrosis treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Histopathology assessment\u003c/h2\u003e \u003cp\u003eLiver tissues were fixed into 10% buffered formalin, embedded in paraffin, deparaffinized, and rehydrated with distilled water. Liver sections of 5 \u0026micro;m thickness were stained with hematoxylin-eosin (H\u0026amp;E) and Van Gizon\u0026rsquo;s picrofuchsin method using a routine protocol. Morphometric analysis of liver sections was performed using the Nikon H550S microscope with NIS-Elements Basic Research software. Quantitative assessment of fibrosis (% fibrosis) was performed as the ratio of collagen area to visible area of liver tissue using methods of digital image analysis as described in our previous work [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and in [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Biochemical assessment\u003c/h2\u003e \u003cp\u003eBlood samples were collected posthumously. The samples were centrifuged at 4\u0026deg;C at 3000 rpm for 10 min. Then the serum samples were tested on the automated biochemical analyzer (ARD, Russia). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) were evaluated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Western blot analysis\u003c/h2\u003e \u003cp\u003eLiver tissue samples were lysed according to the MicroRotofor Lysis Kit protocol (BioRad, USA). Total protein content was determined by the Bradford\u0026rsquo;s method using the Quick Start Bradford Protein Assay Kit (BioRad, USA) and the Epoch microplate spectrophotometer (BioTech, USA). Equal amounts of proteins were separated by 12.5% SDS polyacrylamide electrophoresis gels and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% skim milk for 1 hour and then incubated overnight at 4\u0026deg;C with the primary anticyclooxygenase-2 (COX-2) and anti-β-actin antibodies (SAB5500087 and ZRB1312 respectively, Sigma-Aldrich, USA) (diluted 1:1000 with 5% skim milk). The membranes were then washed with Tris-Buffered saline Tween-20 (TBST) and then incubated with horseradish peroxidase-conjugated secondary antibodies (A0545, Sigma-Aldrich, USA) (diluted 1:10000 with TBST) on the second day. Finally, the membranes were visualized using the Clarity Western ECL Substrate Kit (Bio-Rad, USA) and ChemiDoc Imaging Systems (Bio-Rad, USA) with Image Lab Touch software (Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistics\u003c/h2\u003e \u003cp\u003eAll data in the article are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The normal distribution was determined using the Kolmogorov-Smirnov criterion. In the case of normal distribution, one-way ANOVA with Tukey\u0026rsquo;s post-hoc test was used, and statistical significance was considered as p\u0026thinsp;\u0026le;\u0026thinsp;0.05. In the case of non-normal distribution, Kruskal-Wallis test with Mann-Whitney\u0026rsquo;s test and Bonferroni correction for multiple comparisons was used. The statistical tests used are presented in the text of the article in the captions of the figures. Statistical analysis was performed with the SPSS Statistics program.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Estimation of liver mass coefficient\u003c/h2\u003e \u003cp\u003eDuring the study of the mass coefficient of the liver of rats after 2 weeks of fibrosis treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), it is shown that after exposure to CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol in the control group, there is the increase (p\u0026thinsp;=\u0026thinsp;0.014) in the mass coefficient compared to the intact control group (2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04% and 2.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04%, respectively). However, under the influence of compound \u003cb\u003e2\u003c/b\u003e, there is the decrease (p\u0026thinsp;=\u0026thinsp;0.005) in the liver mass coefficient (2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07%), and under the influence of compound \u003cb\u003e1\u003c/b\u003e, there is only the tendency to decrease the mass coefficient (2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04%).\u003c/p\u003e \u003cp\u003eWhen studying the mass coefficient of the liver of rats after 4 weeks of fibrosis treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we see similar results. It can be seen that after exposure to CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol in the control group, there is the increase (p\u0026thinsp;=\u0026thinsp;0.006) in the liver mass coefficient compared to the intact control group (2.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11% and 2.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04%, respectively). However, under the influence of compound \u003cb\u003e2\u003c/b\u003e, there is the decrease (p\u0026thinsp;=\u0026thinsp;0.033) in the liver mass coefficient (2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%), and under the influence of compound \u003cb\u003e1\u003c/b\u003e, there is the tendency to decrease the mass coefficient (2.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Histological assessment of liver tissue\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Assessment of overall liver tissue morphology\u003c/h2\u003e \u003cp\u003eThe overall morphology of the liver tissue, the presence of steatosis and parenchymal dystrophies after 2 weeks of fibrosis treatment were evaluated by staining with hematoxylin and eosin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-a). In the intact control group, the morphology of the liver tissue was normal and its structure was completely healthy. However, relatively large areas of hydropic and ballooning dystrophy predominate in the control group after two weeks of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol withdrawal, indicating impaired hepatocyte function. Cases of steatosis are rare. However, according to the biochemical data (shown below), liver function is not compromised, which may be due to the more intense work of the remaining hepatocytes. In the experimental groups treated with compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e, a decrease in the occurrence of parenchymal dystrophies and the absence of steatosis were observed, indicating better organ functioning. In addition, compound \u003cb\u003e2\u003c/b\u003e was shown to reduce the liver's mass coefficient, unlike the control, indicating normal tissue functioning.\u003c/p\u003e \u003cp\u003eAfter 4 weeks of fibrosis treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-b), small foci of hydropic dystrophy of hepatocytes are still present in the liver tissue of the control group after CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol withdrawal compared to the intact control group. However, balloon dystrophy, like steatosis, is rare. In the experimental groups, which were administered the tested compounds, hepatocytes of regular shape with homogeneous and rarely granular cytoplasm are observed, indicating almost complete restoration of liver tissue structure. It should be noted that in the liver tissue, areas of hydropic dystrophy are more frequent in the group exposed to compound \u003cb\u003e1\u003c/b\u003e than in the group exposed to compound \u003cb\u003e2\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Quantification of liver fibrosis\u003c/h2\u003e \u003cp\u003eAs the result of quantitative assessment of liver tissue fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-c, d, e), it was shown that after 2 months of fibrosis modeling in rat liver tissue, there was the significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the expression level of collagen fibers, which was 5.7 times higher compared to the intact control (6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9% and 1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08%, respectively). However, after 2 weeks of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol withdrawal, collagen fiber remodeling was observed in the control group at the level (2.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17%) that was 2.8 times higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) than in the intact control group, indicating the presence of chronic changes in liver tissue. The administration of compound \u003cb\u003e1\u003c/b\u003e for 2 weeks had no significant effect on reducing the amount of collagen fibers (2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%) compared to the control group. However, with the administration of compound \u003cb\u003e2\u003c/b\u003e, there was the significant reduction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) of the detected collagen fibers by 1.7 times (1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09%), approaching the levels of the intact group, indicating the more effective resolution of fibrosis.\u003c/p\u003e \u003cp\u003eQuantitative assessment of fibrosis after 4 weeks of treatment showed the similar result to that after 2 weeks of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-e). In the control group, after 4 weeks of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol withdrawal, the level of collagen remained 3 times higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to the intact control group (3.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32% and 1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08%, respectively). In the case of compound \u003cb\u003e1\u003c/b\u003e administration in liver tissue, the relatively high level of collagen fibers was observed (2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38%), while with compound \u003cb\u003e2\u003c/b\u003e administration, there was the tendency (p\u0026thinsp;=\u0026thinsp;0.02) to reduce collagen fibers by 1.5 times (2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13%) compared to the control group. In conclusion, it can be stated that the main stage of collagen remodeling occurred during the first two weeks of treatment and cessation of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol, and then fibrotic changes were preserved at the certain chronic level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Biochemical parameters of blood serum assessment\u003c/h2\u003e \u003cp\u003eOur findings indicate that modeling of rat liver fibrosis resulted in changes in serum biochemical indices (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At the week 8 of fibrosis modeling and exposure to CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol, the biochemical indicators of liver tissue damage demonstrated changes compared to the intact control group. The 7-fold increase (p\u0026thinsp;=\u0026thinsp;0.0001) in the ALT index compared to the intact control group (312.8\u0026thinsp;\u0026plusmn;\u0026thinsp;46.7 and 44.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 U/L, respectively) and the 4-fold increase (p\u0026thinsp;=\u0026thinsp;0.002) in the AST index compared to the intact control group (538.5\u0026thinsp;\u0026plusmn;\u0026thinsp;129.0 and 123.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8 U/L, respectively), were observed. In addition, changes in markers of cholestatic injury in liver tissue were noted at the 8th week of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol exposure. The levels of GGT and ALP increased by 2-fold and 2.7-fold (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), respectively, compared to the intact control group (2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 and 1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 U/L for GGT and 409.8\u0026thinsp;\u0026plusmn;\u0026thinsp;50.1 and 152.8\u0026thinsp;\u0026plusmn;\u0026thinsp;19.5 U/L for ALP, respectively). However, restoration of serum biochemical indices to their initial values was observed in the control group after 2 weeks of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol withdrawal and in the experimental groups with administration of compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e after 2 weeks of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This observation indicates the restoration of liver tissue functional activity. The similar picture was observed after 4 weeks of fibrosis treatment (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Assessment of COX-2 expression level\u003c/h2\u003e \u003cp\u003eIn this study, we examined the expression level of the inflammatory mediator COX-2 only after 2 weeks of fibrosis treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), because signs of liver tissue recovery are observed within the first 2 weeks, as described above. Western blot analysis of COX-2 expression showed that the level of COX-2 was significantly increased (p\u0026thinsp;=\u0026thinsp;0.0003) when CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol-induced fibrosis was modeled for 2 months compared to the intact control group (0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 versus 0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 COX-2/β-actin ratio). However, after 2 weeks of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol withdrawal, the level of COX-2 in the control group (0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 COX-2/β-actin ratio) decreased almost to the level of the intact control group (p\u0026thinsp;=\u0026thinsp;0.3713). This effect was also observed in the groups receiving compound \u003cb\u003e1\u003c/b\u003e (p\u0026thinsp;=\u0026thinsp;0.2397) and compound \u003cb\u003e2\u003c/b\u003e (p\u0026thinsp;=\u0026thinsp;0.016) (0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and 0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 COX-2/β-actin ratio, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eLiver cirrhosis, the final stage of fibrosis, is one of the main causes of death worldwide. The most common cause of this disease is non-alcoholic fatty liver disease [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The pathogenesis of liver fibrosis is understood to involve the accumulation of extracellular matrix, including collagen, in response to injury. The presence of ECM is the primary histologic marker of disease progression [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Injury to liver tissue induces an inflammatory response that promotes the expression of inflammatory mediators and the transdifferentiation of stellate cells into myofibroblasts. Then the myofibroblasts secrete extracellular matrix components [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this experiment, we examined the inflammatory marker COX-2, because it is known that pyrimidine derivatives are inhibitors of this enzyme [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. COX-2 is an enzyme involved in the biosynthesis of prostaglandins, which maintain chronic inflammation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. COX-2 levels are increased in CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis models [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and the administration of COX-2 inhibitors has been shown to reduce the development of CCl\u003csub\u003e4\u003c/sub\u003e-induced rat liver fibrosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In addition, stellate cells activated express COX-2 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Thus, COX-2 expression may serve as an indirect marker of the attenuation or activation of extracellular matrix deposition by activated stellate cells.\u003c/p\u003e \u003cp\u003eThus, in our work, liver fibrosis was modeled in rats and treated for 2 or 4 weeks. It was shown that after the induction of fibrosis for 2 months followed by the cessation of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol, collagen remodeling occurred within 2 weeks and maintained at the chronic elevated level for up to 4 weeks post-cessation compared to the intact control group. The elevated liver mass coefficient persisted for 4 weeks after of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol withdrawal. Parenchymal dystrophic foci were present at 2 weeks but had been attenuated by 4 weeks post-cessation of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol.\u003c/p\u003e \u003cp\u003eBiochemical markers ALT, AST, ALP and GGT returned to the level of healthy animals after 2 weeks of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol withdrawal. Notably, the immediate increase in COX-2, indicating the inflammatory reaction, was observed after 2 months of fibrosis induction with CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol. However, COX-2 levels decreased to the level of the intact control within two weeks post-cessation of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol. These findings indicate the liver\u0026rsquo;s capacity for self-repair after the removal of the damaging agent, consistent with prior research [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Moreover, it is established that ECM self-degradation and reversibility of liver fibrosis are achievable upon removal of the damaging agent. It is noteworthy that after removal of the cause of fibrosis, the liver tissue can adapt to a new structure to ensure its normal functioning, making fibrosis clinically but not morphologically reversible [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile compound \u003cb\u003e1\u003c/b\u003e treatment led to the faster restoration of rat liver tissue and reduced detection of parenchymal dystrophies within the second week of fibrosis treatment, it did not accelerate the collagen remodeling processes compared to the CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol control group. Conversely, the treatment of compound \u003cb\u003e2\u003c/b\u003e not only restored liver tissue structure but also significantly increased collagen fiber remodeling, decreased liver mass coefficient, and showed the trend of reduced COX-2 expression compared to the CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol control group. Thus, this study, as well as our previous work, demonstrated the higher efficacy of the compound \u003cb\u003e2\u003c/b\u003e compared to the compound \u003cb\u003e1\u003c/b\u003e in the treatment of both acute toxic liver injury [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and chronic liver injury models [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhat is the relationship between accelerated collagen remodeling and the effects of compound \u003cb\u003e2\u003c/b\u003e? The main damaging mechanism of CCl\u003csub\u003e4\u003c/sub\u003e on cells is oxidative stress and lipid peroxidation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In combination with alcohol, the effect of CCl\u003csub\u003e4\u003c/sub\u003e is enhanced due to the exacerbation of oxidative stress and increased load on cytochrome P-450 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This leads to chronic cell damage, activation of the inflammatory process, and initiation of fibrotic changes in liver tissue. However, compound \u003cb\u003e1\u003c/b\u003e is known to affect the levels of adenylyl cyclase and cyclic adenosine monophosphate (cAMP) in immunocompetent cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This may indicate the putative effect of compound \u003cb\u003e1\u003c/b\u003e on cell receptors associated with G-protein and adenylyl cyclase activity, such as adrenergic [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] or P2Y receptors [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In turn, cAMP as a secondary messenger can play a variety of biological roles [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], including the role of a regulator of the inflammatory process [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], cell proliferation processes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and activation of hepatic stellate cells [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. It has been shown that an increase in the level of cAMP, including due to phosphodiesterase inhibitors, reduces the proliferation of hepatic stellate cells, inhibits their transdifferentiation into myofibroblasts, and thus reduces fibrosis signs [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Thus, the influence of compound \u003cb\u003e1\u003c/b\u003e on the intracellular level of cAMP can simulate various cell states, which may explain the ability of compound \u003cb\u003e1\u003c/b\u003e to accelerate liver tissue repair, as shown in the description of the overall liver tissue morphology above. In our previous work [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], we also showed that closing the putative active site of the compound \u003cb\u003e1\u003c/b\u003e molecule reduced its biological activity, indicating its putative effect on cell receptors. However, conjugation of compound \u003cb\u003e1\u003c/b\u003e with \u003cem\u003eL\u003c/em\u003e-ascorbic acid enhanced the antifibrotic effect of the native compound \u003cb\u003e1\u003c/b\u003e in this study, which can be explained by several reasons. It has been shown that conjugation of compound \u003cb\u003e1\u003c/b\u003e molecule with \u003cem\u003eL\u003c/em\u003e-ascorbic acid can increase their bioavailability [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] as well as enhance their biological effect [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Moreover, \u003cem\u003eL\u003c/em\u003e-ascorbic acid has been shown to have hepatoprotective activity [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and is known for its antioxidant activity [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], which could positively affect the reduction of oxidative stress induced by CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol. In addition, in our previous work [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], compound \u003cb\u003e2\u003c/b\u003e showed antiradical activity and antioxidant properties that are less pronounced in the native compound \u003cb\u003e1\u003c/b\u003e, what could also affect the reduction of oxidative stress after the effect of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThe conjugation of the pyrimidine derivative of the Russian drug Xymedon with \u003cem\u003eL\u003c/em\u003e-ascorbic acid has been found to enhance its antifibrotic properties in the rat liver fibrosis treatment model. Administration of the Xymedon-\u003cem\u003eL\u003c/em\u003e-ascorbic acid conjugate resulted in a 41% faster collagen remodeling when compared to the control group. However, the antifibrotic effect of this conjugate is not associated with its influence on the expression level of the inflammatory enzyme cyclooxygenase-2 (COX-2).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis study was performed within the framework of the state assignment of the Federal Research Center \u0026laquo;Kazan Scientific Center of the Russian Academy of Sciences\u0026raquo;.\u003c/p\u003e\n\u003cp\u003eThe local ethical committee of the Kazan (Volga Region) Federal University (records of May 18, 2017, No. 4) approved all studies and protocols for handling animals.\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMethodology: Belyaev Grigory, Vyshtakalyuk Alexandra, Parfenov Andrey, Semenov Vyacheslav; design and synthesis of test compounds: Semenov Vyacheslav, Galyametdinova Irina; formal analysis and investigation: Belyaev Grigory; writing-original draft preparation: Belyaev Grigory; writing-review and editing: Vyshtakalyuk Alexandra, Semenov Vyacheslav, Zobov Vladimir.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed within the framework of the state assignment of the Federal Research Center \u0026laquo;Kazan Scientific Center of the Russian Academy of Sciences\u0026raquo;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFaccioli LAP, Dias ML, Paranhos BA, dos Santos Goldenberg RC (2022) Liver cirrhosis: An overview of experimental models in rodents. Life Sci 301:120615. https://doi.org/10.1016/j.lfs.2022.120615 \u003c/li\u003e\n\u003cli\u003eDevarbhavi H, Asrani SK, Arab JP, Nartey Y.A, Pose E, Kamath PS (2023) Global burden of liver disease: 2023 update. J Hepatol 79:516-537. https://doi.org/10.1016/j.jhep.2023.03.017 \u003c/li\u003e\n\u003cli\u003eDees C, Chakraborty D, Distler JHW (2021) Cellular and molecular mechanisms in fibrosis. Exp Dermatol 30:121-131. https://doi.org/10.1111/exd.14193\u003c/li\u003e\n\u003cli\u003eGin\u0026egrave;s P, Krag A, Abraldes JG, Sol\u0026agrave; E, Fabrellas N, Kamath PS (2021) Liver cirrhosis. Lancet 398:1359-1376. https://doi.org/10.1016/s0140-6736(21)01374-x \u003c/li\u003e\n\u003cli\u003eTang JT, Mao YM (2020) Development of new drugs for the treatment of nonalcoholic steatohepatitis. J Dig Dis 21:3-11. https://doi.org/10.1111/1751-2980.12830\u003c/li\u003e\n\u003cli\u003eYang H, Xuefeng Y, Shandong W, Jianhua X (2020) COX-2 in liver fibrosis. Clin Chim Acta 506:196-203. https://doi.org/10.1016/j.cca.2020.03.024 \u003c/li\u003e\n\u003cli\u003eKhasimbi S, Ali F, Manda K, Sharma A, Chauhan G, Wakode S (2021) Dihydropyrimidinones scaffold as a promising nucleus for synthetic profile and various therapeutic targets: A Review. Curr Org Synth 18:270-293. https://doi.org/10.2174/1570179417666201207215710 \u003c/li\u003e\n\u003cli\u003eBhat MA, Al-Omar MA, Khan AA, Alanazi AM, Naglah AM (2019) Synthesis and antihepatotoxic activity of dihydropyrimidinone derivatives linked with 1, 4-benzodioxane. Drug Des Devel Ther 13:2393-2404. https://doi.org/10.2147/dddt.s198865 \u003c/li\u003e\n\u003cli\u003eKarimi J, Mohammadalipour A, Sheikh N, Khodadadi I, Hashemnia M, Goudarzi F, et al (2020) Protective effects of combined Losartan and Nilotinib on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Drug Chem Toxicol 43:468-478. https://doi.org/10.1080/01480545.2018.1504960 \u003c/li\u003e\n\u003cli\u003eSravani S, Saifi MA, Godugu C (2020) Riociguat ameliorates kidney injury and fibrosis in an animal model. Biochem Biophys Res Commun 530:706-712. https://doi.org/10.1016/j.bbrc.2020.07.128 \u003c/li\u003e\n\u003cli\u003eHe ZX, An Q, Wei B, Zhou W J, Wei BF, Gong YP, et al (2021) Discovery of Potent and Selective 2-(Benzylthio) pyrimidine-based DCN1-UBC12 Inhibitors for Anticardiac Fibrotic Effects. J Med Chem 65:163-190. https://doi.org/10.1021/acs.jmedchem.1c01207 \u003c/li\u003e\n\u003cli\u003eJiang N, Zhou Y, Zhu M, Zhang J, Cao M, Lei H, et al (2020) Optimization and evaluation of novel tetrahydropyrido [4, 3-d] pyrimidine derivatives as ATX inhibitors for cardiac and hepatic fibrosis. Eur J Med Chem 187:111904. https://doi.org/10.1016/j.ejmech.2019.111904 \u003c/li\u003e\n\u003cli\u003eSeo JB, Choi YK, Woo HI, Jung YA, Lee S, Lee S, Park M, Lee IK, Jung GS, Park KG (2019) Gemigliptin attenuates renal fibrosis through down-regulation of the NLRP3 inflammasome. Diabetes Metab J 43:830-839. https://doi.org/10.4093/dmj.2018.0181\u003c/li\u003e\n\u003cli\u003eVyshtakalyuk A, Nazarov N, Zueva I, Lantsova A, Minnekhanova O, Busygin D, et al (2013) Study of hepatoprotective effects of xymedon. Bull Exp Biol Med 155:643-646. https://doi.org/10.1007/s10517-013-2215-0 \u003c/li\u003e\n\u003cli\u003eVyshtakalyuk AB, Semenov VE, Zobov VV, Galyametdinova IV, Gumarova LF, Parfenov AA, et al (2017) Synthesis and primary evaluation of the hepatoprotective properties of novel pyrimidine derivatives. Russ J Bioorg Chem 43:604-611. https://doi.org/10.1007/s12668-017-0461-8\u003c/li\u003e\n\u003cli\u003eBelyaev GP, Vyshtakalyuk AB, Parfenov AA, Galyametdinova IV, Semenov VE, Zobov VV (2023) Antifibrotic effect of pyrimidine derivatives of Xymedon and its conjugate with L-ascorbic acid. Uch Zap Kazan Univ, Ser Estestv Nauki 165:175\u0026ndash;189. https://doi.org/10.26907/2542-064x.2023.2.175-189 (In Russian)\u003c/li\u003e\n\u003cli\u003eReznik VS, Pashkurov NG (1966) Reactions of pyrimidinols and pyrimidinethiols with 2-chloroethanol and with 2-chloro-1-propanol. Bull Acad Sci USSR, Div Chem Sci 15:1554-1557. https://doi.org/10.1007/bf00848915 \u003c/li\u003e\n\u003cli\u003eMironov, A. N. et al. (2012). Rukovodstvo po provedeniyu doklinicheskih issledovanij lekarstvennyh sredstv. Chast` pervaya. Grif i K: Moskva, Rossiya. (In Russian).\u003c/li\u003e\n\u003cli\u003eEuropean Commission. Commission Recommendation of 18 June 2007 on guidelines for the accommodation and care of animals used for experimental and other scientific purposes. OJEU 2007;L197:1-89.\u003c/li\u003e\n\u003cli\u003eArjmand A, Tsipouras MG, Tzallas AT, Forlano R, Manousou P, Giannakeas N (2020) Quantification of liver fibrosis\u0026mdash;A comparative study. Appl Sci 10:447. https://doi.org/10.3390/app10020447 \u003c/li\u003e\n\u003cli\u003eCheemerla S, Balakrishnan M (2021) Global epidemiology of chronic liver disease. Clin Liver Dis 17:365-370. https://doi.org/10.1002/cld.1061\u003c/li\u003e\n\u003cli\u003eZhang M, Serna-Salas S, Damba T, Borghesan M, Demaria M, Moshage H (2021) Hepatic stellate cell senescence in liver fibrosis: Characteristics, mechanisms and perspectives. Mech Ageing Dev 199:111572. https://doi.org/10.1016/j.mad.2021.111572 \u003c/li\u003e\n\u003cli\u003eLi J, Wang T, Liu P, Yang F, Wang X, Zheng W, et al (2021) Hesperetin ameliorates hepatic oxidative stress and inflammation via the PI3K/AKT-Nrf2-ARE pathway in oleic acid-induced HepG2 cells and a rat model of high-fat diet-induced NAFLD. Food Funct 12:3898-3918. https://doi.org/10.1039/d0fo02736g \u003c/li\u003e\n\u003cli\u003eZoubek ME, Trautwein C, Strnad P (2017) Reversal of liver fibrosis: From fiction to reality. Best Pract Res Clin Gastroenterol 31:129-141. https://doi.org/10.1016/j.bpg.2017.04.005 \u003c/li\u003e\n\u003cli\u003eTsuchida T, Friedman SL (2017) Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol 14:397-411. https://doi.org/10.1038/nrgastro.2017.38\u003c/li\u003e\n\u003cli\u003eAbdelgawad MA, Bakr RB, Azouz AA (2018) Novel pyrimidine-pyridine hybrids: synthesis, cyclooxygenase inhibition, anti-inflammatory activity and ulcerogenic liability. Bioorg Chem 77:339-348. https://doi.org/10.1016/j.bioorg.2018.01.028 \u003c/li\u003e\n\u003cli\u003eKaur B, Singh P (2022) Inflammation: biochemistry, cellular targets, anti-inflammatory agents and challenges with special emphasis on cyclooxygenase-2. Bioorg Chem 121:105663. https://doi.org/10.1016/j.bioorg.2022.105663 \u003c/li\u003e\n\u003cli\u003eNing C, Gao X, Wang C, Huo X, Liu Z, Sun H, et al (2018) Hepatoprotective effect of ginsenoside Rg1 from Panax ginseng on carbon tetrachloride‐induced acute liver injury by activating Nrf2 signaling pathway in mice. Environ Toxicol 33:1050-1060. https://doi.org/10.1002/tox.22616 \u003c/li\u003e\n\u003cli\u003ePlanagum\u0026agrave; A, Cl\u0026agrave;ria J, Miquel R, L\u0026oacute;pez‐Parra M, Titos E, Masferrer JL, et al (2005) The selective cyclooxygenase‐2 inhibitor SC‐236 reduces liver fibrosis by mechanisms involving non‐parenchymal cell apoptosis and PPAR\u0026gamma; activation. FASEB J 19:1120-1122. https://doi.org/10.1096/fj.04-2753fje\u003c/li\u003e\n\u003cli\u003eFtahy MM, Latif NSA, Alalkamy EF, El-Batrawi FA, Galal AH, Khatab HM (2013) Antifibrotic potential of a selective COX-2 inhibitor (celecoxib) on liver fibrosis in rats. Comp Clin Pathol 22:425-430. https://doi.org/10.1007/s00580-012-1427-4 \u003c/li\u003e\n\u003cli\u003eThomes PG, Rasineni K, Yang L, Donohue Jr TM, Kubik JL, McNiven MA, et al (2019) Ethanol withdrawal mitigates fatty liver by normalizing lipid catabolism. Am J Physiol Gastrointest Liver Physiol 316:G509-G518. https://doi.org/10.1152/ajpgi.00376.2018 \u003c/li\u003e\n\u003cli\u003ePeugnet-Gonz\u0026aacute;lez I, Mart\u0026iacute;nez-Hern\u0026aacute;ndez SL, \u0026Aacute;vila-Blanco ME, Hern\u0026aacute;ndez-Mar\u0026iacute;n DA, Macias-P\u0026eacute;rez JR, Aldaba-Muruato LR, et al (2023) Hepatoprotective and antifibrotic activity of watercress extract in a model of CCl4-induced liver fibrosis in Wistar rats. J Funct Foods 109:105760. https://doi.org/10.1016/j.jff.2023.105760 \u003c/li\u003e\n\u003cli\u003eDi Vinicius I, Baptista AP, Barbosa Jr AA, Andrade ZA (2005) Morphological signs of cirrhosis regression: Experimental observations on carbon tetrachloride-induced liver cirrhosis of rats. Pathol, Res Pract 201:449-456. https://doi.org/10.1016/j.prp.2005.05.009\u003c/li\u003e\n\u003cli\u003eOrtiz C, Schierwagen R, Schaefer L, Klein S, Trepat X, Trebicka J (2021) Extracellular matrix remodeling in chronic liver disease. Curr Tissue Microenviron Rep 2:41-52. https://doi.org/10.1007/s43152-021-00030-3 \u003c/li\u003e\n\u003cli\u003eVyshtakalyuk AB, Parfenov AA, Galyametdinova IV, Semenov VE, Zobov VV (2022) Antiapoptotic mechanism for the implementation of the hepato-protective effect of pyrimidine derivatives. Int J Pharm Sci Res 13:3922-3931. https://doi.org/10.13040/IJPSR.0975-8232.13(10).39231 \u003c/li\u003e\n\u003cli\u003eAramjoo H, Mohammadparast-Tabas P, Farkhondeh T, Zardast M, Makhdoumi M, Samarghandian S, et al (2022) Protective effect of Sophora pachycarpa seed extract on carbon tetrachloride-induced toxicity in rats. BMC Complement Med Ther 22:76. https://doi.org/10.1186/s12906-022-03554-9\u003c/li\u003e\n\u003cli\u003eSlabnov YuD, Cherepnev GV, Karimova FG, Garaev RS (1998) Effect of pyrimidine derivatives on adenylate cyclase system of immunocompetent cell regulation in vitro. Bull Exp Biol Med 125:588-590. https://doi.org/10.1007/bf02445248 \u003c/li\u003e\n\u003cli\u003eAlc\u0026aacute;ntara-Hern\u0026aacute;ndez R, Hern\u0026aacute;ndez-M\u0026eacute;ndez A (2018) Adrenergic signaling molecular complexes. Gac Med Mex 154:223-235. https://doi.org/10.24875/gmm.m18000135 \u003c/li\u003e\n\u003cli\u003eCommuni D, Horckmans M, Boeynaems JM (2021) P2Y4, P2Y6 and P2Y11 receptors: From the early days of cloning to their function. Biochem Pharmacol 187:114347. https://doi.org/10.1016/j.bcp.2020.114347 \u003c/li\u003e\n\u003cli\u003eBeavo JA, Brunton LL (2002) Cyclic nucleotide research\u0026mdash;still expanding after half a century. Nat Rev Mol Cell Biol 3:710-718. https://doi.org/10.1038/nrm911\u003c/li\u003e\n\u003cli\u003ePacini ESA, Satori NA, Jackson EK, Godinho RO (2022) Extracellular cAMP-Adenosine Pathway Signaling: A Potential Therapeutic Target in Chronic Inflammatory Airway Diseases. Front Immunol 13:866097. https://doi.org/10.3389/fimmu.2022.866097 \u003c/li\u003e\n\u003cli\u003eGuo R, Liu T, Shasaltaneh MD, Wang X, Imani S, Wen Q (2022) Targeting adenylate cyclase family: New concept of targeted cancer therapy. Front Oncol 12:829212. https://doi.org/10.3389/fonc.2022.829212 \u003c/li\u003e\n\u003cli\u003eInsel PA, Murray F, Yokoyama U, Romano S, Yun H, Brown L, et al (2012) cAMP and Epac in the regulation of tissue fibrosis. Br J Pharmacol 166:447-456. https://doi.org/10.1111/j.1476-5381.2012.01847.x \u003c/li\u003e\n\u003cli\u003eEl Awdan SA, Abdel Rahman RF, Ibrahim HM, Hegazy RR, El Marasy SA, Badawi M, et al (2019) Regression of fibrosis by cilostazol in a rat model of thioacetamide-induced liver fibrosis: Up regulation of hepatic cAMP, and modulation of inflammatory, oxidative stress and apoptotic biomarkers. PloS One 14:e0216301. https://doi.org/10.1371/journal.pone.0216301 \u003c/li\u003e\n\u003cli\u003eBelyaev GP, Vyshtakalyuk AB, Parfenov AA, Shashin MS, Galyametdinova IV, Semenov VE, et al (2022) Comparative assessment of hepatoprotective properties of some \u0026ldquo;doubled\u0026rdquo; 4, 6-dimethyl-1, 2-dihydro-1-(2-hydroxyethyl) pyrimidin-2-one derivatives. Russ Chem Bull 71:2701-2710. https://doi.org/10.1007/s11172-022-3699-4 \u003c/li\u003e\n\u003cli\u003eHarrison FE, May JM (2009) Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2. Free Radic Biol Med 46:719-730. https://doi.org/10.1016/j.freeradbiomed.2008.12.018 \u003c/li\u003e\n\u003cli\u003eMoteki H, Kimura M, Sunaga K, Tsuda T, Ogihara M (2013) Signal transduction mechanism for potentiation by \u0026alpha;1-and \u0026beta;2-adrenoceptor agonists of l-ascorbic acid-induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Eur J Pharmacol 700:2-12. https://doi.org/10.1016/j.ejphar.2012.12.010 \u003c/li\u003e\n\u003cli\u003eKimura M, Moteki H, Uchida M, Natsume H, Ogihara M (2014) L-ascorbic acid-and L-ascorbic acid 2-glucoside accelerate in vivo liver regeneration and lower serum alanine aminotransaminase activity in 70% partially hepatectomized rats. Biol Pharm Bull 37:597-603. https://doi.org/10.1248/bpb.b13-00839 \u003c/li\u003e\n\u003cli\u003eNjus D, Kelley PM, Tu YJ, Schlegel HB (2020) Ascorbic acid: The chemistry underlying its antioxidant properties. Free Radic Biol Med 159:37-43. https://doi.org/10.1016/j.freeradbiomed.2020.07.013 \u003c/li\u003e\n\u003cli\u003eVyshtakalyuk AB, Semenov VE, Sudakov IA, Bushmeleva KN, Gumarova LF, Parfenov AA, et al (2018) Xymedon conjugate with biogenic acids. Antioxidant properties of a conjugate of Xymedon with L-ascorbic acid. Russ Chem Bull 67:705-711. https://doi.org/10.1007/s11172-018-2126-3 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Xymedon, fibrosis, carbon tetrachloride, pyrimidines, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-3953710/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3953710/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFibrosis of the liver is a chronic inflammatory process with activation of hepatic stellate cells and abnormal accumulation of proteins in the extracellular matrix. However, it is known that pyrimidine derivatives have a beneficial effect on the condition of various organs with the ongoing process of fibrosis. Therefore, the aim of this work was to investigate the effect of the drug Xymedon (1,2-dihydro-4,6-dimethyl-1-N-(2-hydroxyethyl)pyrimidine-2-one, (compound \u003cb\u003e1\u003c/b\u003e) and its conjugate with \u003cem\u003eL\u003c/em\u003e-ascorbic acid (compound \u003cb\u003e2\u003c/b\u003e) on collagen remodeling in rat liver tissue. For this purpose, female Wistar rats were used to model fibrosis by oral administration of carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e) and ethanol for 8 weeks. Then the rats were treated with the studied compounds for 2 or 4 weeks. Histological analysis by hematoxylin-eosin and Van Gizon\u0026rsquo;s staining of liver slices, biochemical analysis of blood serum and Western blot analysis of COX-2 level in rat liver homogenates were performed. It has been shown that in the control group without treatment, after 2 weeks of withdrawal of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol, collagen remodeling occurs to the certain chronic level. At the same time, compound \u003cb\u003e2\u003c/b\u003e reduces the level of collagen fibers by 41% compared to the control group, while native compound \u003cb\u003e1\u003c/b\u003e has no such effect. Also, in all groups studied, there was the decrease in the inflammatory marker COX-2 both after 2 weeks of CCl\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ethanol withdrawal and after treatment with studied compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e. Thus, compound \u003cb\u003e2\u003c/b\u003e (conjugate of Xymedon with \u003cem\u003eL\u003c/em\u003e-ascorbic acid) has the greater antifibrotic effect on the rat liver fibrosis model compared to the native molecule of compound \u003cb\u003e1\u003c/b\u003e (Xymedon). At the same time, this effect is not associated with the level of COX-2.\u003c/p\u003e","manuscriptTitle":"Influence of Xymedon and its conjugate with L-ascorbic acid on collagen remodeling in the liver fibrosis rat model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-19 17:51:21","doi":"10.21203/rs.3.rs-3953710/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12f05173-f78c-44fb-a7f5-6209f3fa4f1f","owner":[],"postedDate":"February 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-09T21:52:56+00:00","versionOfRecord":{"articleIdentity":"rs-3953710","link":"https://doi.org/10.26907/2542-064X.2025.2.276-296","journal":{"identity":"uchenye-zapiski-kazanskogo-universiteta-seriya-estestvennye-nauki","isVorOnly":true,"title":"Uchenye Zapiski Kazanskogo Universiteta Seriya Estestvennye Nauki"},"publishedOn":"2025-06-13 00:00:00","publishedOnDateReadable":"June 13th, 2025"},"versionCreatedAt":"2024-02-19 17:51:21","video":"","vorDoi":"10.26907/2542-064X.2025.2.276-296","vorDoiUrl":"https://doi.org/10.26907/2542-064X.2025.2.276-296","workflowStages":[]},"version":"v1","identity":"rs-3953710","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3953710","identity":"rs-3953710","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}