The effect of dalargin on growth factors content in experimental ulcerative colitis

Introduction : The effectiveness of ulcerative colitis (UC) treatment is fairy moderate and it gives a rise to search for new ways of treating it. Considering the combination of dalargin effects, studying the dalargin influence on the UC development is of undoubted interest. The aim of the study was to evaluate the dalargin effect on the content of transforming growth factor-β (TGF-β ) and epidermal growth factor (EGF) in the colonic wall in mice with experimental ulcerative colitis. Materials and methods: UC was simulated by replacing water with a 5% solution of dextran sodium sulfate in boiled water for 5 days. The mice were killed on the 5 th , 7 th and 28 th days. The concentrations of TGF-β and EGF in the homogenate of the medial colon were determined by enzyme-linked immunosorbent assay using standard kits. Results and discussion: The dalargin daily subcutaneous administration (dose of 100 µg/kg) for 7 days led to a decrease in TGF-β levels on the 5 th and 7 th days compared to the control group. In chronic UC, the concentration of TGF-β was higher than in the control group. The EGF concentration was increased in mice with UC treated with dalargin throughout the experiment. There were no differences in dalargin and sulfasalazine effects on the content of TGF-β , and the concentration of EGF throughout the experiment was significantly higher in the animals treated with dalargin. Conclusion: Effect of dalargin on the TGF-β and EGF concentrations was explained by its stimulating action to opioid µ-receptors localized on immune cells of the colon.


Introduction
Ulcerative colitis (UC) is a chronic, relapsing, multifactorial disease of the colon; it has a bimodal pattern of incidence, with the main onset peak between ages 15 and 30 years, and a second smaller peak between ages 50 and 70 years, significantly worsening the quality of life of patients, leading to the development of severe complications and disability of patients (Du and Ha 2020).The pathogenesis of UC is associated with the disruption of the barrier function of the colon mucosa, penetration of luminal microflora into the submucosal layer of the colonic wall, pathological activation of neutrophils, macrophages, dendritic cells, T-and Blymphocytes and the subsequent development of immune inflammation, causing the ulcers formation and colonic crypts destruction (Le Berre et al. 2023).The effectiveness of modern methods of UC treatment is fairy moderate, which makes it urgent to search for new ways of treating it (Le Berre et al. 2023).Considering the important role of genetic factors in the development of UC, innovative methods, including gene therapy, have an undoubted future (Polikarpova et al. 2022); however, new directions for the use of the already known drugs are also important.
Dalargin was proposed as an antiulcer drug, but currently it is used mainly in the treatment of pancreatitis (Bulgakov 2018).Considering the dalargin effect in the treatment of gastrointestinal tract diseases, as well as the immunomodulatory and antioxidant effects of the drug (Bulgakov 2018;Platonova et al. 2018), studying the dalargin effect on the UC development is of undoubted interest.Previously, we established its medicinal effect in experimental UC in mice, manifested by a decrease in the index of disease activity, the reduction in ulcers and infiltrates in the colonic wall (Liashev et al. 2023).But the mechanism of the pharmacological dalargin effect in UC is unknown.
The participation of transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) in the UC development has been confirmed.TGF-β is a family of pleiotropic cytokines produced by various immune and epithelial cells, fibroblasts (Zhao et al. 2020;Chandiran and Cauley 2023).In the colon, TGF-β suppresses the development of the immune response to antigens of luminal microflora and takes part in the immunological tolerance formation (Yun et al. 2019;Triantafillidis et al. 2020).It was previously shown that TGF-β promotes reparative processes in the mucous membrane of the colon in UC (Tatiya-Aphiradee et al. 2018); however, its excess production can contribute to the fibrosis development in the colonic wall (Naghdalipour et al. 2022), leading to the formation of strictures requiring surgery.It has been established that functional activity of TGF-β in animals with experimental UC and patients with inflammatory bowel disease is impaired, and its correction can be considered as a potential way to treat UC (Tatiya-Aphiradee et al. 2022).
EGF is synthesized by intestinal epithelial cells during inflammation and binds to specific receptors (EGFR) (Lu et al. 2014).EGF has a pronounced stimulating effect on the migration and proliferation of fibroblasts, which helps to restore the colonic barrier.The cytokine suppresses the immune response to luminal microbiota antigens, reducing the severity of colon damage in UC.Activation of EGFR has an effect on the UC development, indicating the possibility of using EGF as a means of UC treating (Lu et al. 2014).
Thus, studying the dalargin effect, as a potential treatment for UC, on the content of TGF-β and EGF in the wall of the medial colon is of undoubted interest.
The aim of the study was to evaluate the dalargin effect on the content of transforming growth factor-β and epidermal growth factor in the colonic wall in mice with experimental ulcerative colitis.

Drugs
Dalargin (Tir-D-Ala-Gly-Phen-Leu-Arg) (NPO Microgen, Russia) was dissolved in a 0.9% sodium chloride solution, applied subcutaneously in a volume of 0.1 mL daily at a dose of 100 µg/kg body weight once a day for 7 days from the beginning of UC simulation.As shown earlier, dalargin manifested high pharmacological activity at the indicated dose (Lishmanov et al. 2012).Sulfasalazine (KRKA, Slovenia) was used as a reference drug and administered intragastrically to mice in the suspension form in physiological solution at a dose of 200 mg/kg body weight in a volume of 0.3 mL for 7 days from the beginning of UC simulation (Motov et al. 2021).The treatment of experimental UC in rats with intragastric sulfasalazine administration at a dose of 200 mg/kg decreased the disease activity index, the area of ulcers and hemorrhages in the rectum, and the content of induced NO-synthase, IL-1β, IL-6, TNFα in the rectal homogenate (Zhu et al. 2019;Motov et al. 2021).

Experimental design
The investigation was carried out on 67 male Balb/C mice, 4 of which were intact.UC was simulated in 63 remaining mice.All UC animals were randomly divided in 3 experimental groups: 1) control (UC+saline, n=21); 2) experimental No. 1 (UC+dalargin solution, n=21); 3) experimental No. 2 (UC+sulfasalazine, n=21).Seven mice from each group were killed by cervical dislocation under the chloral hydrate anesthesia (Macklin, China) on the 5 th , 7 th and 28 th days.Considering that dalargin and sulfasalazine were administered to animals in different ways, the control group included 12 mice treated with saline subcutaneously and 9 mice administered with intragastric saline, and therefore 4 mice treated with saline subcutaneously and 3 mice treated with saline intragastrically were killed on each days.It was established earlier there were neither clinical nor morphological differences between Balb/C mice with experimental UC treated with saline in the indicated ways at all stages of the experiment (Garo et al. 2019).Saline was administered once a day for 7 days from the beginning of UC simulation in a volume of 0.1 mL subcutaneously or 0.3 mL intragastrically.

Research methods
UC was simulated by replacing drinking water with a 5% solution of DSS in boiled water for 5 days (Naghdalipour et al. 2022).Earlier, the development of acute UC was shown on the 5 th and 7 th days, and chronic colitis on the 28 th day after the beginning of DSS solution drinking (Khomyakova et al. 2013).Early morphological studies confirmed UC development in animals after drinking a 5% DSS solution (Liashev et al. 2023).The mice were killed by cervical dislocation under chloral hydrate anesthesia on the 5 th , 7 th and 28 th days; the colon was removed, then opened with a longitudinal incision along the edge of the mesentery attachment, washed with phosphate-buffered saline (pH=7.4,0.01 M), and the medial section was isolated and tissue (50 mg) was homogenized in a Potter-Elweheim homogenizer for 10 minutes.The homogenate was centrifuged in an SL-16R centrifuge (Thermo Fisher Scientific, Germany) for 10 minutes at 3000 rpm.After centrifugation, the supernatant was collected in test tubes, frozen at t=-40 o C and stored for no longer than 2 months.The concentrations of TGFβ and EGF in the homogenate of the medial colon were determined by enzyme-linked immunosorbent assay (ELISA) using standard kits from Cloud-Clone Corp. (China) on a Lazurit automatic enzyme immunoassay analyzer (Dynex Technologies, USA), according to the instructions.

Statistical data processing
Statistical analysis of the biochemical results was carried out using Statistica software version 10 (USA).All samples were tested for the type of distribution using the Shapiro-Wilk W-test.The results were described as a median (Me), lower and upper quartiles (Q1 and Q3, respectively) due to the absence of a normal distribution in most ordered samples.Mann-Whitney U-test was used to determine the significance of the differences.The null hypothesis was rejected at the level of statistical significance p<0.05.

Results and discussion
UC simulation in male Balb/C mice led to an increase in the TGF-β content in the homogenate of the medial colon on the 5 th and 7 th days of the experiment (acute UC) by 4.3 and 3.9 times, respectively (P=0.0107), compared with the intact group (Table 1).There were no significant differences in the TGF-β content on the 28 th day (chronic UC), between the intact and control groups (P=0.2193).There were no significant differences between the intact and control groups in the EGF content on the 5 th of UC simulation in the homogenate of the medial colon (P=0.0890).The EGF concentration increased by 29.6% and 3.4 times (P=0.0107) on the 7 th and 28 th days of the experiment.
The dalargin administration led to a decrease in TGFβ levels on the 5 th and 7 th days of the experiment compared to the control group by 49.4% and 45.8% (P=0.0022),respectively.In chronic UC, the concentration of TGF-β was higher than in the control group -by 40.4% (P=0.0212).An increase in the EGF concentration was also found in mice with UC treated with dalargin throughout the experiment: on the 5 th dayby 91.7% (P=0.0022), on the 7 th day -by 2.4 times (P=0.0022),and on the 28 th -by 2.9 times (P=0.0022).
A decrease in TGF-β levels was shown on the 5 th and 7 th days after the beginning of UC simulation by 36.4% and 48.5%, respectively (P=0.0022) in the group treated with sulfasalazine, and in the level of EGF on the 7 thonly by 40.2% (P=0.0033).There were no significant differences in the concentrations of both growth factors in other periods.
Comparing the effects of dalargin and sulfasalazine on the content in TGF-β and EGF in the homogenate of the medial colon revealed the absence of significant differences in the TGF-β content, and the EGF concentration throughout the experiment was significantly higher in animals treated with dalargin.The EGF content was higher in the group treated with dalargin compared to animals treated with sulfasalazine by 95.75% (P=0.0049) on the 5 th day, by 69.7% -on the 7 th day (P=0.0022),by 3.3 times -on the 28 th day (P=0.0022).
The results obtained confirm the literature data on a TGF-β increase in both laboratory animals with experimental colitis and in patients with UC (Zhu et al. 2019;Naghdalipour et al. 2022).However, some studies showed a TGF-β decrease in the homogenate of the colon of mice with experimental UC (Luo et al. 2019;Liu et al. 2021).Such results can be explained by the development of UC of various severity in laboratory animals, since different murine strains have different resistance to factors provoking UC (Khomyakova et al. 2013).According to many researchers, TGF-β plays a key role in the development of inflammation in the colon (Yun et al. 2019).The TGF-β-Smad signaling pathway is the most important in the implementation of the anti-inflammatory TGF-β effect, since it regulates the Th17/Treg balance (Liu et al. 2021).It is known that activated Th17-cells accumulate in the colonic wall in UC and produce proinflammatory cytokines, including IL-17A, IL-17F, and IL-21.
In contrast, Tregs secrete IL-6 and TGF-β, suppressing Th17 activity (Liu et al. 2021).The TGF-β-Smad signaling pathway controls the differentiation of Th17 and Treg: TGF-β in low concentration enhances IL-6 and IL-17 effects, promoting Th17-cells differentiation, and TGF-β in high concentration enhances Treg differentiation (Liu et al. 2021).It was previously shown that a significant increase in the TGF-β concentration led to activation of the TGF-β-Smad signaling pathway, an increase in the activity of Smad7, which is accompanied by a decrease in the antiinflammatory TGF-β effect (Garo et al. 2019).In addition, it was found that Smad7 suppression inhibits the activity of epithelial myosin light chain kinase and causes a decrease in the permeability of the epithelial barrier of the colonic mucosa (Bai et al. 2022).
An increase in the EGF content was established on the 7 th and 28 th days of the experimental UC development.The medicinal effect of an EGF analogue on the UC development was shown earlier (Zhou et al. 2022).It has been established that activation of EGF receptors on colonic epithelial cells has a cytoprotective effect and also suppresses the production of nuclear factor kB, TNFα and interferon-γ by macrophages (Lu et al. 2014).There is evidence of the conjugation of the effects of TGF-β and EGF through the pro-inflammatory signaling pathway TGF-β-EGF receptor (El Mahdy et al. 2023), with drug effects associated, among other things, with inhibition of this mechanism.
The dalargin administration intramuscularly at a dose of 100 mcg/kg once a day for 7 days had a corrective effect on the TGF-β content in the homogenate of the medial colon in mice with experimental UC: the TGF-β concentration was significantly lower on the 5 th and 7 th days, and it was higher compared to the control group on the 28 th day.The EGF content increased throughout the experiment in UC+dalargin group.
Dalargin is a leu-enkephalin analogue manifesting affinity for opioid µ-and δ-receptors (Bulgakov 2018).Opioid µ-type receptors are present on neutrophils, macrophages, dendritic cells, T and B lymphocytes in the colonic wall (Raeeszadeh-Sarmazdeh et al. 2020).An increase in the expression of opioid µ-receptor mRNA has been established during the acute period of UC.The normal expression of opioid µ-receptor mRNA was observed in the chronic UC (Lashgari et al. 2021).These data indicate the involvement of opioid µ-receptors in the regulation of inflammation in the colon.The use of the enkephalinase inhibitor opiorphin in experimental UC in mice caused a decrease in the concentration of proinflammatory cytokines (IL-1β, IL-6, TNFα) and an Table 1.Effect of dalargin on the concentrations of transforming growth factor-β and epidermal growth factor in the homogenate of the medial colon increase in the content of anti-inflammatory IL-10, both in the colonic wall and in the plasma, as well as a decrease in the activity index disease and reduction in the area of ulcers of the colonic mucosa (Luo et al. 2022).
Thus, an increase in leu-and met-enkephalins content in the blood plasma, as well as an increase in the expression and activity of opioid µ-receptors in opiorphin administration, led to the suppression of the activity of nuclear factor kB, p65 protein, and Toll-like receptor (TLR-4), induced NO-synthase and cyclooxygenase type 2 (Raeeszadeh-Sarmazdeh et al. 2020).The involvement of opioid µ-receptors in the anti-inflammatory effect of opiorphin was confirmed by the fact that these effects were not observed in naloxone administration, which blocks the activity of opioid µreceptors (Luo et al. 2022).

Conclusion
Thus, the study established that the dalargin administration had a corrective effect on the TGF-β content and a stimulating effect on the EGF concentration in the homogenate of the medial colon in mice with experimental UC.Dalargin effect of on EGF was significantly higher than the effect of sulfasalazine throughout the experiment.No significant differences were established between the effects of dalargin and sulfasalazine on the TGF-β content.Apparently, the pharmacological dalargin effect on TGF-β and EGF concentrations was explained by its stimulating effect on opioid µ-receptors localized on macrophages, neutrophils, and lymphocytes of the colonic wall.
A decrease in TGF-β activity under dalargin influence inhibits the activity of Smad7, which contributes to the anti-inflammatory TGF-β effect.An increase in EGF activity in the group treated with dalargin enhanced reparative processes in the colon and corrected the permeability of the colonic barrier.The results obtained open up prospects for dalargin as a drug of choice for the UC treatment, including its combination with other drugs.Note: x -p<0.05 compared to the intact group; *-p<0.05compared to the control group; 1 -p<0.05compared to the ulcerative colitis + sulfasalazine group.
Male Balb/C mice weighing 20-23 g were purchased from the Stolbovaya branch of the Federal State Budgetary Institution of Science "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency".All mice were housed 7/cage and were fed standard laboratory chow in an animal room with 12 h dark/light cycles at a constant temperature of 20±5 o C. All animal experiments were conducted in the Laboratory of Preclinical trials of Drugs of the Research Institute of Experimental Medicine of Kursk State Medical University under guidelines of humane treatment of laboratory animals (Lipatov et al. 2019a, 2019b), the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 and the Rules of Good Laboratory Practice in the Russian Federation (order of the Ministry of Health of the Russian Federation No. 199n dated April 1, 2016.The experiments were approved by the Regional Ethics Committee (REC) (Minutes No. 1 of April 3 2023).