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

doi:10.21203/rs.3.rs-3953710/v1

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

https://doi.org/10.21203/rs.3.rs-3953710/v1

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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 (CCl₄) 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 CCl₄ + 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 CCl₄ + 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

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 CCl₄ 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.

2.1 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 (CCl₄ + ethanol) according to the following scheme: 5% oil solution of CCl₄ at the dose of 2 ml/kg was administered orally to the animals twice a week. To potentiate the effect of CCl₄, 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 CCl₄ + 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 CCl₄ + 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.

3.1 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 CCl₄ + 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 CCl₄ + 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 CCl₄ + 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 CCl₄ + 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 CCl₄ + 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 CCl₄ + 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 CCl₄ + 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 CCl₄ + 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 CCl₄ + 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 CCl₄ + 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 CCl₄ + 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 CCl₄ + 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).

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 CCl₄-induced liver fibrosis models [28], and the administration of COX-2 inhibitors has been shown to reduce the development of CCl₄-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 CCl₄ + 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 CCl₄ + ethanol withdrawal. Parenchymal dystrophic foci were present at 2 weeks but had been attenuated by 4 weeks post-cessation of CCl₄ + ethanol.

Biochemical markers ALT, AST, ALP and GGT returned to the level of healthy animals after 2 weeks of CCl₄ + ethanol withdrawal. Notably, the immediate increase in COX-2, indicating the inflammatory reaction, was observed after 2 months of fibrosis induction with CCl₄ + ethanol. However, COX-2 levels decreased to the level of the intact control within two weeks post-cessation of CCl₄ + 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 CCl₄ + 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 CCl₄ + 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 CCl₄ on cells is oxidative stress and lipid peroxidation [36]. In combination with alcohol, the effect of CCl₄ 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 CCl₄ + 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 CCl₄ + ethanol.

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).

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».

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

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Compound synthesis

2.2 Experiment scheme

2.3 Histopathology assessment

2.4 Biochemical assessment

2.5 Western blot analysis

2.6 Statistics

3 Results

3.1 Estimation of liver mass coefficient

3.2 Histological assessment of liver tissue

3.2.1 Assessment of overall liver tissue morphology

3.2.2 Quantification of liver fibrosis

3.3 Biochemical parameters of blood serum assessment

3.4 Assessment of COX-2 expression level

4 Discussion

5 Conclusion

Declarations

References

Additional Declarations

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