Main Research Findings

1) Intramuscular injections of Pancragen were found to normalize adhesion of mesenteric capillary endothelium, indicating the compound elicits homeostatic and endothelioprotective effects.

2) Treatment with Pancragen was found to promote differentiation of pancreatic gland cells, acting as a potential mechanism of action for the peptides anti-diabetic and anti-inflammatory effects.

 

Selected Data

1) The research team of Khavinson et al investigated the effects of the tetrapeptide Pancragen (Lys-Glu-Asp-Trp-NH2; PG) on blood glucose levels, capillary permeability, and adhesion in rats with experimental diabetes mellitus was conducted using male Wistar rats, aged 10-12 weeks and weighing between 170-210 g. These animals were housed under standard laboratory conditions with free access to food and water throughout the experimental period [1].

Experimental diabetes mellitus (DM) was induced in the rats through a single intraperitoneal injection of streptozotocin (ST) at a dose of 50 mg/kg, dissolved in 1 ml of 0.9% NaCl. Streptozotocin is known for its selective cytotoxic effect on pancreatic β-cells, and its administration was expected to lead to the development of DM, characterized by permanent hyperglycemia, polyuria, and glucosuria, typically within 2-3 weeks post-injection.

The experimental animals were divided into distinct groups, each comprising 20 rats, to assess the effects of Pancragen. Animals in group 1 received intramuscular injections of PG daily for 10 days. The dosage was 10 µg/ml, administered as a saline solution. Animals in group 2 received PG orally daily for 10 days. The dosage was 100 µg (as a pulverized tablet) suspended in 2 ml of solution, administered via a tube. Animals in group 3 served as a control for the ST-induced DM, receiving injections of saline according to the same protocol as the treatment groups. Finally, a separate group of healthy, untreated animals served as a baseline control [1].

Blood glucose levels were meticulously monitored using an Accu Chek glucometer. Measurements were taken on days 10, 15, and 20 following ST injection. Prior to blood collection, animals were subjected to an overnight fast, with fodder removed from their cages 14 hours beforehand, to ensure accurate baseline measurements. On day 20 after ST injection, additional urinary parameters were evaluated, specifically diuresis and urinary glucose, using the universal method on a COBAS biochemical analyzer.

A critical aspect of the study involved evaluating the function of the capillary endothelium, which is known to be significantly affected in diabetes. This was performed in narcotized animals using 50 mg/kg sodium thiopental delivered intraperitoneally through a complex life-time micromicroscopic technique. A segment of the small intestine was carefully mobilized to allow for the assessment of mesenteric microcirculation. The mesenteric mesentery, specifically the portion adjacent to the mesoappendix, was positioned on a warmed table, with the temperature meticulously maintained at 37.5-38.0°C. This region was continuously irrigated with warm saline at a rate of 0.5 ml/min. All endothelial function studies focused on venules with a diameter of 20-35 µm [1].

A sophisticated video-microscopic complex was utilized for these observations, comprising an MT-9 microscope, a CCD videocamera, an SLV-X55ME videotape recorder, and a KV-2185MT TV set. Data processing was conducted using specialized software, specifically AV Master analog digital transformer attached to an IBM-compatible PC, and Fast Cap 2.5.0 and VideoTest 5.0. This setup enabled dynamic geometrical, velocity, and quantitative analysis of the digital video images.

Capillary permeability was quantitatively assessed using Na-Fluorescein as a fluorochrome indicator. A dose of 2.5 mg/kg of Na-Fluorescein, prepared in saline immediately before the experiment, was injected intravenously (0.2 ml solution). The optical system incorporated appropriate photofilters to transform transmitted luminous flux into reflected light, allowing for videorecording of the time course of fluorescence in the selected area. The permeability coefficient (P), expressed in cm/sec, was calculated using a specific equation: P = 0.25 × dIi(t) × D × [Ii(0)/Iv(0)] / [Iv(t) – Ii(t) × dt]. Here, dIi(t) represents the increment in fluorochrome fluorescence intensity in the interstitium (luminance/sec units), D is the vessel diameter, Ii(0) and Iv(0) denote initial mean luminance outside and inside the capillary in transmitted luminous flux (luminance units), Iv(t) and Ii(t) represent fluorochrome fluorescence intensity inside the vessel and in the interstitial space (luminance units), respectively, and dt is time in seconds [1].

Finally, leukocyte adhesion to the vascular wall was evaluated by initially videorecording the microcirculation in transmitted light without photofilters. For enhanced contrast, the illumination luminous flux was transmitted through a photofilter with a wavelength of 360-560 nm. Leukocytes that remained immobilized for 5 seconds during the entire measurement period were classified as adherent. The ratio of adherent leukocytes to the vascular wall area was then calculated [1].

2) The study conducted by Khavinson et al aimed to elucidate the molecular mechanisms by which Pancragen influences the differentiation of pancreatic acinar and islet cells, particularly in the context of aging. To achieve this, experiments were conducted using in vitro cell cultures. The core of the experimental model involved using embryonic cultures of pancreatic acinar cells. Specifically, the MIA PaCa-2 cell line was chosen for these experiments [2].

The MIA PaCa-2 cells were utilized at two different passage numbers: Passage 1 and Passage 14 where cells were designated as “young” cultures or cells were designated as “aged” cultures, respectively. This distinction between “young” and “aged” cultures was based on the recommendations of the International Association of Cell Culture Studies, reflecting an in vitro model of cellular aging. Both “young” and “aged” cultures were subdivided into three experimental groups to assess the effects of Pancragen and a control peptide: Group 1 was a control group in which cultures received saline solution [2]. Cultures in group 2 were treated with bronchogen at a concentration of 20 ng/ml. Bronchogen served as a control peptide for comparison. Finally, cultures in group 3 were treated with Pancragen at a concentration of 20 ng/ml.

The cells for all groups were cultured in 25-cm² flasks containing 5 ml of DMEM (Dulbecco’s Modified Eagle Medium). The medium was further supplemented with L-glutamine, 15% fetal calf serum SC-BIOL, and 1% penicillin-streptomycin solution. The initial cell concentration at the start of culture was 10^6 cells/ml. All cultures were maintained at 37°C. To investigate the expression of key differentiation markers, immunocytochemistry was performed. The following primary monoclonal antibodies were used: Ptf1a (pancreatic transcription factor 1a), as a marker for acinar cells; Pdx1 (pancreatic and duodenal homeobox 1), as a key maturation marker for all pancreatic cell types; Pax6 (paired box gene 6), involved in the maturation of α-, β-, and δ-cells in the pancreas; Foxa2 (forkhead box protein A2), involved in the maturation of α-, β-, and δ-cells in the pancreas; NKx2.2 (NK2 homeobox 2.2), involved in the maturation of α-, β-, and δ-cells in the pancreas; and Pax4 (paired box gene 4), primarily involved in the maturation of δ-cells [2].

All primary antibodies were used at a dilution of 1:50. Secondary biotinylated anti-mouse antibodies were then applied. To facilitate antibody penetration, permeabilization of the cells was performed using 0.1% Triton-X100. The immunoreaction was visualized using horseradish peroxidase and diaminobenzidine (DAB) via the EnVision Detection System. The results of the immunocytochemical analysis were quantitatively evaluated morphometrically using a computer-assisted microscopic image analysis system. This system consisted of a Nikon Eclipse E400 microscope, a Nikon DXM1200 digital camera, and a Videotest-Morphology 5.0 software. For each analysis, 5 fields of view were examined at ×200 magnification. The primary parameter used for evaluation was the “area of expression,” calculated as the percentage of the total area of cells in the field of view that were occupied by immunopositive cells. This parameter effectively reflects the number of cells expressing the investigated differentiation factor [2].

 

Discussion

1) This study conducted by Khavinson et al investigated the effects of the tetrapeptide Pancragen on blood glucose levels, capillary permeability, and adhesion in Wistar rats with experimental ST-induced DM. The results indicate that Pancragen exerts homeostatic and endothelioprotective effects during the early stages of DM, with notable differences depending on the route of administration. Diabetes mellitus was successfully induced in the experimental animals, confirmed by biochemical analysis of blood and urine. By day 10 after ST injection, 30% of the animals exhibited hyperglycemia, and by day 20, the majority of animals showed clear signs of DM, consistent with the model’s characteristics. This established a reliable diabetic model for evaluating Pancragen’s therapeutic potential [1].

A key finding concerned Pancragen’s impact on blood glucose levels. Oral administration of Pancragen (Group 2) produced a “pronounced hypoglycemic effect” on day 10, meaning the number of animals with hyperglycemia in this group was halved compared to the untreated diabetic control group (Group 3). This suggests that oral Pancragen has a short-term, acute glucose-lowering effect during treatment. However, this hypoglycemic effect was not sustained after the cessation of Pancragen treatment, as blood glucose parameters in Group 2 by day 20 were similar to those of the control group (Group 3). In contrast, intramuscular administration (Group 1) did not demonstrate a significant hypoglycemic effect on day 10, with glucose levels remaining comparable to the control group.

 

Figure 1: Changes in ST-induced DM characterized by level of glycemia measured on A) day 10 and B) day 20, across the three treatment groups.

Regarding capillary permeability, the study found that ST-induced DM significantly altered endothelial function by day 20, leading to a decrease in vascular wall permeability in the majority of diabetic animals when compared to healthy controls. However, neither intramuscular nor oral administration of Pancragen modified this parameter of transcapillary exchange. This indicates that Pancragen, regardless of its administration route, did not directly impact the fundamental permeability characteristics of the capillaries in diabetic rats [1].

The most significant endothelioprotective effect of Pancragen was observed in capillary adhesion. In the diabetic control group (Group 3), vascular wall endothelial adhesion was reduced to 1.22 ± 0.32, a common pathological feature in DM. Intramuscular injection of Pancragen (Group 1) successfully “normalized” these endothelial adhesive characteristics, with values of 1.93 ± 0.31 becoming comparable to those observed in normal, healthy animals, and significantly different from Group 3. This protective effect was evident irrespective of the animal’s blood glucose level at that point. Interestingly, oral administration of Pancragen (Group 2) did not modify endothelial adhesion, suggesting that the route of administration is crucial for this particular endothelioprotective action.

In summary, the study concluded that long-term parenteral (intramuscular) administration of Pancragen yielded a long-lasting endothelium-protective effect by normalizing endothelial adhesion, a critical aspect of vascular health in diabetes. Conversely, oral treatment with Pancragen demonstrated a short-term hypoglycemic effect that was evident only during the treatment period, without a sustained impact on glucose levels or endothelial adhesion. These findings highlight the potential for Pancragen as a bioregulator for metabolic disorders and endothelial dysfunction in DM, with specific routes of administration conferring distinct therapeutic benefits [1].

2) This study completed by Khavinson et al investigated the effects of the tetrapeptide Pancragen on the differentiation of pancreatic acinar and islet cells, specifically observing its impact on “young” versus “aged” cell cultures. The researchers utilized immunocytochemistry to analyze the expression of key pancreatic differentiation markers and found significant age-related changes and a notable stimulatory effect of Pancragen [2].

A primary finding was that the expression of differentiation markers was generally reduced in aged pancreatic cells. For instance, the expression area of Ptf1a, a marker for acinar cells, decreased by 4.6 times in aged cultures compared to young ones. Similarly, Pdx1 expression, a key maturation marker for all pancreatic cell types, decreased by 2.72 times in aged cultures. The expression areas of Pax6, Foxa2, and Nkx2.2, involved in the maturation of α-, β-, and δ-cells, also showed a 2- to 4-fold reduction in aged cultures. These age-related reductions indicate a decline in the differentiation potential of pancreatic cells during cellular senescence.

Pancragen demonstrated a potent stimulatory effect on the expression of several differentiation markers. For Ptf1a, Pancragen increased its expression area by 1.5 times in young cultures and a remarkable 6 times in aged cultures compared to their respective controls. This suggests that Pancragen can significantly enhance the differentiation of acinar cells, particularly in aged tissues where expression is severely compromised. Similarly, Pancragen expanded the area of Pdx1 expression by 1.3-fold in young cultures and 2.6-fold in aged cultures, indicating a more pronounced stimulatory effect compared to bronchogen, a control peptide [2].

Pancragen also had a positive impact on markers associated with the maturation of various islet cells. In aged cultures, Pancragen increased the expression of Foxa2 by 1.6 times, Nkx2.2 by 2 times, and Pax6 by 2.3 times compared to control aged cultures. This suggests that Pancragen promotes the differentiation of multiple types of pancreatic islet cells (α-, β-, and δ-cells). Notably, the expression area of Pax4, a marker for δ-cell precursors, remained largely unchanged in aging cultures and after Pancragen introduction, consistent with other research findings [2].

In contrast, the control peptide, bronchogen, showed limited effects. It did not significantly alter Ptf1a expression in either young or aged cultures. While it increased Pdx1 expression by 10% in both young and aged cultures, its stimulatory effect was notably weaker than that of Pancragen. Bronchogen also had no effect on Foxa2 expression in young and aged cultures and only a weak stimulatory effect on Nkx2.2 and Pax6 in aged cultures [2].

The study highlighted that cell aging dramatically reduces the concentration of Pdx1 and Ptf1a proteins, which are essential for the maturation of acinar and islet cells. Pancragen effectively up-regulated Pdx1 and Ptf1a expression in aged cultures to levels comparable to those found in young cultures. This indicates Pancragen’s ability to restore critical differentiation processes in aging pancreatic cells. By increasing the expression of these differentiation factors, Pancragen stimulates the differentiation of various pancreatic islet cells, thereby promoting the synthesis of insulin, glucagon, somatostatin, and pancreatic polypeptide.

In conclusion, Pancragen peptide promotes the multi-directional differentiation of pancreatic cells, particularly reversing age-related declines in differentiation marker expression. Its inductive action on the differentiation and functional activity of acinar and islet cells positions it as a promising therapeutic tool for treating metabolic abnormalities such as diabetes mellitus and pancreatitis. The study underscores that transcription factors regulating pancreatic cell differentiation are pharmacological targets for Pancragen, providing molecular insights into its geroprotective and pancreas-protective actions [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] Khavinson VKh, Gavrisheva NA, Malinin VV, Chefu SG, Trofimov EL. Effect of pancragen on blood glucose level, capillary permeability and adhesion in rats with experimental diabetes mellitus. Bull Exp Biol Med. 2007;144(4):559-562. doi:10.1007/s10517-007-0377-3

[2] Khavinson VKh, Durnova AO, Polyakova VO, et al. Effects of pancragen on the differentiation of pancreatic cells during their ageing. Bull Exp Biol Med. 2013;154(4):501-504. doi:10.1007/s10517-013-1987-6

 

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