Main Research Findings

1) Administration of Thymogen was shown to inhibit lipid peroxidation and improve the reparative regeneration of hepatocytes.

2) Research found that different variations of Thymogen have the potential to further increase the regenerative capacity of hepatocytes.

Selected Data
1) This study performed by researchers Chulanova et al was designed to evaluate the hepatoprotective potential of thymogen and its structurally modified analogues in a rat model of acute toxic hepatopathy. The experiments were performed on 64 male Wistar rats, each weighing between 180 and 220 g, which were housed under standard laboratory conditions [1]
Acute hepatopathy was induced in the rats using hydrazine hydrochloride, a well-established hepatotropic toxin commonly employed in preclinical research on hepatoprotective agents. The animals received a single intraperitoneal injection of hydrazine hydrochloride at a dose of 50 mg/kg diluted in 0.1 ml of saline. Thymogen, a synthetic dipeptide with immunomodulatory and regenerative properties, was studied in both its original form and as two modified analogues that were synthesized in a specialized peptide synthesis laboratory: D-Ala-thymogen and thymogen-D-Ala.
Based on earlier research, dosing regimens were selected to include both low and high concentrations. Thymogen was administered at 10 and 100 μg/kg, whereas the analogues were given at 12 and 120 μg/kg, equimolar to the parent compound. The peptides were dissolved in saline to achieve the appropriate concentrations and injected intraperitoneally in a fixed volume of 0.1 ml. The treatment course consisted of once-daily injections administered over five consecutive days, beginning 24 hours after the initial hydrazine injection [1].
The animals were randomly allocated into eight groups of eight rats each. The control group received saline alone, while the pathological control group was treated with hydrazine followed by saline. The six experimental groups received hydrazine followed by either thymogen or one of its analogues at the low or high dose levels. Thus, comparisons could be made between untreated controls, hydrazine-only controls, and animals treated with thymogen or its modified derivatives.
At the end of the treatment course, animals were sacrificed by exsanguination under chloral hydrate anesthesia. Blood was collected from the right ventricle, and the livers were harvested for biochemical and histological analysis. To assess oxidative stress and antioxidant defenses, two markers were measured in both blood plasma and liver homogenates. Malondialdehyde (MDA) was quantified as an indicator of lipid peroxidation and pro-oxidant activity using the thiobarbituric acid reaction. Catalase activity, representing antioxidant defense, was measured by monitoring its reaction with hydrogen peroxide. All measurements were carried out using a PE-5300VI spectrophotometer and liver homogenates were prepared according to standard procedures [1].
Liver tissue was processed by fixation in 10% neutral formalin, embedded in paraffin, sectioned into slices 7–10 μm thick, and stained with hematoxylin and eosin. Histomorphological assessment included three quantitative parameters. The mitotic index was calculated to evaluate proliferative activity of hepatocytes. The binuclear hepatocyte index was determined as an indicator of cellular regenerative potential, while the surface area of hepatocyte nuclei was measured to provide insights into nuclear morphology and cell health [1].
In summary, this experimental framework involved inducing acute hepatotoxicity in rats with hydrazine hydrochloride, followed by treatment with Thymogen or its structural analogues at varying doses. Biochemical markers of oxidative stress and antioxidant defense, along with histological parameters of hepatocyte proliferation and regeneration, were assessed to evaluate potential hepatoprotective effects. The experimental design included multiple control groups, standardized dosing, and validated analytical methods while ensuring reliable assessment of whether Thymogen and its analogues could mitigate oxidative damage and support liver regeneration in chemically induced hepatopathy [1].
2) The study conducted by Chulanova et al investigated the hepatoprotective and antioxidant properties of the peptide thymogen and its structural analogues using a model of carbon tetrachloride (CCl4)-induced liver injury in rats. Male Wistar rats weighing between 180 and 220 g, were selected for the experiment and maintained under standard laboratory conditions with free access to food and water. To induce acute toxic hepatopathy, rats were administered CCl4 intragastrically for five consecutive days. The toxin was delivered as a 50% solution in sunflower oil at a dosage of 3 ml/kg body weight, with a 24-hour interval between each administration. This method is well established in experimental hepatology, as CCl4 reliably produces oxidative stress, lipid peroxidation, and hepatocellular damage that mimic toxic liver injury in humans [2].
For pharmacological intervention, the researchers evaluated Thymogen, a known immunomodulatory dipeptide, alongside two newly synthesized structural analogues: D-Ala-Thymogen and Thymogen-D-Ala. The peptides were administered intraperitoneally in low therapeutic doses, consistent with manufacturer recommendations and prior studies. Thymogen was injected at 1 μg/kg, while its structural analogues were delivered at equimolar doses of 1.2 μg/kg. All treatments were given in a constant volume of 0.1 ml and administered once daily for five days. Importantly, the peptide treatments were synchronized with CCl4 administration, ensuring that the compounds were tested under active conditions of chemical liver injury.
The experimental design divided the rats into five groups of eight animals each. The first was a control group, which received sunflower oil intragastrically along with intraperitoneal saline injections. The second, a pathological control group, received CCl4 to induce liver damage but was treated only with saline intraperitoneally. The remaining three groups were experimental groups that received CCl4 combined with either thymogen, D-Ala-Thymogen, or Thymogen-D-Ala. In both the control and pathological control groups, isotonic sodium chloride solution was administered intraperitoneally in equivalent volumes to maintain consistency across experimental conditions [2].
Twelve hours after the final peptide administration, all animals were euthanized under chloral hydrate anesthesia at a dosage of 300 mg/kg. Blood samples were collected from the right ventricle using heparinized syringes, and the livers were excised for further biochemical and histological analysis. Histological sections were prepared after fixation in 10% formalin solution buffered with 0.1 M phosphate, embedding in paraffin, and sectioning at 7–8 μm thickness. These sections were stained with hematoxylin and eosin and examined under microscope to assess the general morphology of liver tissue and regenerative hepatocyte activity. Three key parameters were quantified: the mitotic index, which measures hepatocyte division; the binuclear hepatocyte index, reflecting polyploidization and a regenerative reserve mechanism; and the surface of hepatocyte nuclei, an indicator of nuclear size and activity [2].
In addition to histological assessment, biochemical markers were used to evaluate oxidative stress and antioxidant defenses. Liver homogenates were prepared by homogenizing weighed portions of liver tissue in tenfold volumes of ice-cold saline, followed by centrifugation at 3000 rpm for ten minutes. The supernatants were analyzed for biochemical markers. MDA levels were measured as an indicator of lipid peroxidation and oxidative stress using the thiobarbituric acid (TBA) reaction, with optical density determined on a spectrophotometer. Catalase activity, a key component of the antioxidant defense system, was measured spectrophotometrically on the same device by monitoring its reaction with 3% hydrogen peroxide solution. These markers were assessed both in blood plasma and in liver homogenates, providing a systemic and organ-specific view of oxidative processes and antioxidant capacity.
Overall, this study established a controlled experimental framework to investigate the hepatoprotective and antioxidative roles of thymogen and its structural analogues against CCl4-induced acute liver injury. By combining histological indicators of regenerative activity with biochemical markers of oxidative stress and antioxidant defense, the research aimed to clarify whether these peptides can mitigate liver injury and promote recovery. Furthermore, the use of both Thymogen and its newly synthesized analogues allowed for direct comparisons of efficacy, providing insights into whether structural modification enhances biological activity [2].

Discussion
1) Researchers Chulanova et al examined the ability of Thymogen and its analogues to promote regeneration and protection of hepatocytes in models of induced liver injury. For the purpose of this study hydrazine-induced liver injury was characterized by the development of granular dystrophy and associated structural and biochemical alterations. Among these changes, a significant increase in the binuclear hepatocyte index and a reduction in the surface area of hepatocyte nuclei were observed. However, there were no notable changes in the mitotic index, suggesting that hepatocyte proliferation by mitosis was not prominently activated in the untreated toxic condition. These findings indicated that liver damage caused by hydrazine disrupts normal cellular morphology and function while initiating limited compensatory responses [1].
When Thymogen and its structural analogues were administered, all peptides demonstrated a capacity to stimulate reparative regeneration of the injured liver. Interestingly, the most pronounced hepatoprotective and regenerative effects were observed with lower doses of these compounds. For example, Thymogen administered at 10 μg/kg significantly increased the mitotic index, enhancing it by 7.2 times compared to the pathological control. This result highlighted the strong ability of Thymogen at low doses to trigger hepatocyte proliferation through mitosis.
In contrast, the modified analogues of thymogen administered at 12 μg/kg did not significantly increase the mitotic index. Instead, they primarily increased the proportion of binuclear hepatocytes. This pattern suggested that at low doses, the structural analogues promoted liver regeneration through polyploidization rather than mitosis. Since binuclear hepatocytes are widely recognized as a cellular reserve for regeneration, this finding pointed to a different but still effective reparative pathway. Furthermore, both thymogen and its analogues at lower doses were associated with an increase in hepatocyte nuclear surface area, reflecting improved cellular health and regenerative activity [1].
At higher doses, however, the effects of the peptides differed. Increasing the dose of thymogen to 100 μg/kg did not lead to a further rise in the mitotic index, suggesting a plateau in its proliferative effect. In contrast, the structural analogues at 120 μg/kg did stimulate mitotic activity: D-Ala-thymogen increased the mitotic index by 6.5 times, and thymogen-D-Ala by 7.5 times. Despite this stimulation of mitosis, neither analogue at this higher dose significantly increased the binuclear hepatocyte or surface area of hepatocyte nuclei. This outcome suggested that the mode of reparative regeneration is dependent on the peptide dose. Lower doses favored a broader regenerative response involving binuclear hepatocytes and nuclear enlargement, while higher doses primarily activated mitotic pathways without the same supportive morphological changes.
Biochemical analysis confirmed the protective effects of thymogen and its analogues on oxidative stress markers. Hydrazine intoxication markedly increased MDA concentrations, reflecting lipid peroxidation and oxidative stress, while simultaneously decreasing catalase activity in both blood plasma and liver homogenates. Administration of all peptides reduced free radical activity, thereby lowering MDA levels. At lower doses, the modified analogues at 12 μg/kg demonstrated a more pronounced reduction of MDA in liver homogenates compared to thymogen at 10 μg/kg. Conversely, thymogen at 100 μg/kg produced a greater reduction of plasma MDA, though concentrations in the liver homogenate were similar to those observed at the lower dose. The analogues at 120 μg/kg further decreased plasma MDA, but this effect was weaker in the liver homogenate, indicating some tissue-specific differences in their antioxidant actions [1].
With respect to catalase activity, both Thymogen and its analogues increased antioxidant defense capacity. However, increasing the dose of either thymogen or its structural analogues did not lead to greater catalase activity. This lack of dose-dependent enhancement was interpreted as a negative feedback response, a common phenomenon for regulatory peptides that often exhibit optimal effects within a narrow therapeutic range.
These findings were consistent with earlier studies, where very low doses of Thymogen and its analogues were shown to activate reparative and antioxidant processes in the same experimental model. Collectively, the results of the current study reinforced the idea that raising the dose of thymogen or its analogues does not result in a proportional increase in protective effects. Instead, low and medium doses proved to be the most effective, with optimal levels identified as 1 and 10 μg/kg for thymogen, and 1.2 and 12 μg/kg for its analogues [1].
In conclusion, the incorporation of D-Ala into the thymogen structure enhances both reparative and antioxidant activities. Among the analogues, thymogen-D-Ala showed slightly greater efficacy in certain parameters, suggesting that structural modifications can improve peptide performance. However, escalating doses to 100 μg/kg for thymogen and 120 μg/kg for its analogues did not produce superior hepatoprotective outcomes, underscoring the importance of precise dosing in maximizing therapeutic benefit [1].
2) The results of the study completed by the research team of Chulanova et al revealed that repeated administration of CCl₄ to experimental animals produced clear signs of toxic liver damage, as reflected in several measured parameters. Specifically, the five consecutive doses of CCl₄ significantly suppressed hepatocyte reparative regeneration. This was demonstrated by notable decreases in the mitotic index, the binuclear hepatocyte index, and the surface area of hepatocyte nuclei. At the same time, CCl₄ exposure also impaired the liver’s antioxidant defenses, as evidenced by reduced catalase activity, while simultaneously promoting oxidative stress, indicated by an increase in MDA levels in both blood plasma and liver homogenates. These findings confirmed that CCl₄ successfully induced acute hepatopathy and provided a reliable model for testing the corrective effects of thymogen and its structural analogues [2].
When Thymogen and its analogues were administered under conditions of CCl₄ intoxication, the peptides exerted a strong protective and restorative influence. Treatment with these peptides improved hepatocyte regenerative activity and partially normalized the laboratory parameters that had been altered by toxic exposure. Importantly, significant differences emerged between the effects of unmodified thymogen and its structurally modified versions.
Thymogen itself promoted hepatocyte division and regeneration, as reflected by an increase in mitotic index by a factor of 1.9 compared with the pathological control group, a change that was highly statistically significant (p < 0.001). However, the modified peptides exhibited much stronger effects. Administration of D-Ala-Thymogen elevated the mitotic index by 4.9 times, and Thymogen-D-Ala raised the mitotic index by 5.3 times. These findings indicated that structural modifications of thymogen substantially amplified its ability to stimulate mitotic activity in damaged hepatocytes. In addition to changes in the MI, both binuclear hepatocyte and surface area of hepatocyte nuclei were significantly elevated in all treatment groups, showing that all peptides contributed to nuclear growth and binucleation of hepatocytes. Nevertheless, the increases in these parameters were more pronounced with the use of thymogen analogues than with thymogen alone, suggesting that structural modification enhanced overall hepatocyte regeneration [2].
The antioxidant profile of the peptides also showed meaningful improvements. Both thymogen and its analogues lowered the concentrations of MDA in blood plasma and liver homogenate, which reflected reduced lipid peroxidation and free radical activity. Among the peptides, Thymogen-D-Ala demonstrated the strongest antioxidant activity. In blood plasma, this analogue reduced MDA levels by 3.9 times, and in liver homogenate it reduced MDA concentrations by 1.7 times. These results suggested that the C-terminal D-Ala modification conferred enhanced stability or functional activity that allowed the peptide to exert a stronger protective effect against oxidative stress [2].
In summary, structural modification of Thymogen by incorporating D-Ala at either the N- or C-terminal positions significantly increased the peptide’s biological activity under conditions of acute toxic hepatopathy induced by CCl₄. While Thymogen itself showed protective effects, both D-Ala-Thymogen and Thymogen-D-Ala were effective in stimulating hepatocyte mitotic activity and improving morphometric indices of liver regeneration. Of the two analogues, Thymogen-D-Ala displayed the most potent effects, particularly in reducing oxidative stress markers and enhancing antioxidant defenses. These findings support the therapeutic potential of designing peptide agents based on Thymogen but modified with structural components that extend their stability and functional efficacy. The results point to promising prospects for the development of new hepatoprotective drugs that can more effectively promote liver regeneration and counteract oxidative damage following toxic injury [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Chulanova AA, Smakhtina AM, Mal GS, et al. Hepatoprotective Effects of Thymogen Analogues in Hydrazine Hepatopathy in Rats. Bull Exp Biol Med. 2025;178(6):722-725. doi:10.1007/s10517-025-06405-y

[2] Chulanova AA, Smakhtin MY, Bobyntsev II, Mishina ES, Artyushkova EB, Smakhtina AM. Reparative and Antioxidant Effects of New Analogues of Immunomodulator Thymogen in Experimental Model of Liver Damage. Bull Exp Biol Med. 2023;175(5):700-703. doi:10.1007/s10517-023-05929-5

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