Main Research Findings

1) Administration of Vilon facilitates the progressive activation of facultative heterochromatin in cases of advanced age.

2) Subcutaneous injection of Vilon was shown to improve physical activity and endurance, lengthen lifespan, decrease body temperature, and prevent spontaneous tumor development in female CBA months starting at 6 months of age.

3) The effects of the peptide bioregulators Vilon and Epithalon were found to inhibit physiologically programmed cell death in splenic lymphocytes that underwent gamma irradiation in order to induce apoptosis.

 

Selected Data

1) The role of peptide bioregulators, such as Vilon, have been studied in geriatric practice in order to determine metabolic changes that may occur that relate to aging and “slowing down the biological clock” [1]. Current research indicates that these metabolic changes that take place are potentially regulated through genes in the distinct functional chromatin domains. Based on views suggesting that progressive heterochromatinization and gene inactivation occur during aging, the research team of Lezhava et al. examined chromosomes from lymphocyte cultures obtained from subjects aged 76-81 years, and compared them to cultures gathered from individuals of 26-35 years of age. Two separate samples were obtained from each subject; one was treated with 0.01 ug/ml of Vilon and the other was left intact. Treatment with the peptide was followed by 72 hours of incubation [2].

The researchers used differential scanning microcalorimetry (DSM) in order to study conformational changes in total heterochromatic fraction inside the cell. The process of denaturation of chromatin is typically characterized by defined heat absorption peaks. The mechanism of denaturation was identified through microcalorimetric investigations of the cellular nuclei of various tissue types. Additionally, nuclear chromatin and chromatin in solution were observed in order to observe thermal denaturation. This investigation revealed that membranes, cytoplasmic structures, and nuclear proteins typically denatured in temperatures of 40-70 ℃, while the chromatin denatured at more distinct points, including: 60, 76, 88, and 105 ℃. After gathering this initial data, samples of intact unstimulated lymphocytes and Vilon-treated unstimulated lymphocytes were assessed in order to determine the effects of Vilon on these different variables of chromatin denaturation [2].

2) The research team of Khavinson et al. examined the effects of Vilon on lifespan, physical and motor activity, reproductive function, body temperature, tumor formation, and free radical processes when administered subcutaneously to female CBA mice after 6 months of life. The age of the mice was important in the study considering that the immunological theory of aging has determined that age-related immune dysfunction results in an increased risk of oncological diseases and autoimmune disorders, as well as a loss of resistance against infections. While performing this study the research team took into account prior attempts to decelerate aging in mice by administering the polypeptide, thymalin. Based on previous findings, Vilon was chosen for this study due to its similar amino acid base to various thymus peptides and cytokines [3].

As it was previously mentioned, 120, 6 month old female CBA mice were divided into two groups with 60 subjects in each. The experimental group was administered a subcutaneous injection of 0.1 ug of Vilon for 5 days on a monthly basis. On the other hand, the control group was subcutaneously administered a physiological saline on the same schedule. Vaginal smears were taken daily for 2 weeks every 3 months in order to estimate estrous function through the cytology of the samples. Rectal temperature was also frequently measured using a TPEM-1 electronic thermometer. Additionally, motor activity was examined in an open field where the research team was able to record the number of crossed squares, rearings, and occurrences of face, body, and genital grooming.

In order to determine the long term effects of the peptide, the research team waited until 12 months after the initiation of the experiment to assess muscle power and fatigue. The subjects clung to a wire with their forepaws and were suspended at a height of 75-80 cm and the time spent hanging was recorded [3]. This test was performed twice with a 20 minute break between trials. Power recovery was then determined by finding the difference between the two trial times. Observation of the test subjects occurred until their natural deaths in order to calculate mean and maximum lifespans, as well as the age corresponding to 90% mortality rate [3]. Following natural death or euthanasia of the mice, each animal was autopsied and any tumors present were histologically examined by the research team.

3) In addition to causing immune system dysfunction, aging is also associated with cell loss. More specifically, this age-related cell loss is typically controlled by genetics and characterized by similar biomechanical manifestations leading to physiologically programmed cell death, or apoptosis. Apoptosis is different from necrosis and is induced through very low intensity endogenous and exogenous stimuli that are related to a specific program that promotes elimination of nonviable cells with DNA abnormalities. Apoptosis of cells in organs such as the pineal gland thymus, and gonads is directly related to age-related decline; in humans cellular apoptosis results in generalized premature aging but also has the potential to lead to severe diseases such as Alzheimer’s [4]. Similar to the previously discussed study, the research team of Khavinson et al. took into account prior research related to the use of peptide bioregulators isolated from the affected tissues. They applied that information to study the effects of synthetic peptide bioregulators, Epithalon, and Vilon on the apoptosis of lymphocytes.

The experiment was conducted using 50 male Wistar rats weighing between 120 grams and 140 grams. All animals were maintained under standard conditions and apoptosis was induced in the test subjects via irradiation. The animals were then randomly subdivided into a control group and three experimental treatment groups, each including 10 animals. The experimental rats underwent a gamma irradiation process using a GUB-2000 cobalt apparatus to deliver 200 rad/min [4]. The groups were labeled as follows: Group 1 rats served as the control and were not injected with a peptide. Group 2 rats were intraperitoneally injected with Epithalon. Group 3 rats were intraperitoneally injected with Vilon. Group 4 rats were injected with a physiological saline. Treatments with the peptides began 2 days after the gamma irradiation process took place; Epitalon and Vilon were administered to the test subjects daily for 5 days in doses of 0.5 ug mixed with 0.5 ml of physiological saline. Group 4 test subjects were intraperitoneally injected physiological saline following the same scheme as Groups 2 and 3 [4].

On day 8 following the gamma irradiation process, specimens were collected from all control and experimental rats. The research team decided to evaluate the amount of splenic lymphocytes as the test object due to their rapid renewal rate. All samples collected from the spleen were fixed in acid Bouin fixative over the course of 24 hours, followed by being embedded in paraffin. In order to identify the presence of apoptotic cells, the 5 μ samples were stained with hematoxylin and eosin. This procedure was followed by impregnation of the sections in order to reveal condensed chromatin in apoptotic cells.

The total number and the number of apoptotic cells in lymphoid follicles was determined through a morphometric study performed with an Imstar S.A. image analysis system, as well as the Morphostar-2 and Colquant softwares. The average reactive center area for each section was recorded as 0.06 mm^2. Additionally, an Axiophot-2 immersion microscope was used to count the cell in 60 fields (x900). Each area that was tested by the research team contained no less than 1000 lymphoid cell nuclei, and the overall percentage of apoptotic cells was calculated to determine the apoptotic index (AI) [4].

 

Discussion

1) Several different concentrations of Vilon, ranging from 0.1 to 0.01 ug/ml were tested in order to define the non-mutagenic dose of the peptide. In cultured lymphocytes that were obtained from young individuals, it was revealed that at concentrations of 0.01 ug/mL induced 2.0 +/- 0.8 chromosome aberrations per cell. For aged test subjects, administration of the peptide induced 3.60 +/- 0.6 chromosome aberrations per cell. For younger individuals, the amount of Vilon-induced chromosome aberrations did not exceed their own control values of 1.7 +/- 0.7. However, aberrations related to single and paired chromosomal fragments were more commonly seen in individuals aged from 78 to 81 years of age [2].

When examining the denaturation process in unstimulated lymphocytes and Vilon-treated lymphocytes from old donors, the research team observed complex profiles correlated with the heat absorption curves for each sample. It was previously determined that chromatin denatures at a temperature of 60-105 ℃ within the chromatin. That being said, the researchers assumed that heat absorption curves corresponding to endotherms I, II, and III, were related to the denaturation of the chromatin inside the lymphocyte samples. Additionally, the results of the study found that administering Vilon to the lymphocyte cultures results in alteration to the profiles of the various heart absorption curves.

The most significant change elicited by the peptide was the shift of endotherms II and III to lower temperatures. The heat distributions also exhibited significant variation as the heat increased at stage II but decreased at stage III. These findings allowed the research team to conclude that the endoterm transition between stages I and II were related to the unfolding of 10 and 30 nm fibers, while the endotherm transition to stage III was due to the unfolding of 30 nm fiber loops that are attached to the nuclear matrix. This transition between stages II and III after the administration of Vilon was determined to be due to the partial deheterochromatinization-decondensation of the aforementioned fiber loops [2].

Figure 1: Chromatin heat absorption in lymphocyte cultures from old donors (dashed line), cultures from young donors (dotted line), and Vilon-treated cultures from old donors (solid line).

When examining changes in the Ag-positive nucleolus organization region (NORs) in the lymphocyte cultures both intact and treated with Vilon, it was found that administration of the peptide significantly increased the frequency of Ag-positive NORs in acrocentric chromosomes. The index frequency of these chromosomal NORs in Vilon-treated samples from old donors corresponded to 2.39 per cell, which was considered significantly increased when comparing to the index frequency of the control culture cells that only reached 1.10. Overall, the research team found that treatment with Vilon resulted in an increase of acrocentric chromosome associations of all types [2].

Figure 2: the frequency of acrocentric chromosome associations in Vilon-treated cultures of aged individuals (a); the frequency of acrocentric chromosome associations in control and peptide-treated cultures (b); and the frequency of the Ag-poistive NORs in both control and Vilon-treated cultures.

2) The initial results of the study performed by Khavinson et al found that 18- and 21-month old mice treated with Vilon weighted in significantly higher than the subjects in the control group. Each 3 month measurement of food consumption did not show any differences in the amount of food consumed by the experimental group versus the control group. That being said, the researchers were able to infer that the higher body weight in the peptide-treated mice was not due to increased food consumption. Additionally, open field behavioral testing revealed that 6-9 month old mice in the control group were more active in the open field, however, as their age advanced these findings became insignificant [3].

In terms of muscle power, fatigue, and recovery, 18 month old mice exhibited the most individual variability in results. It was revealed that the ratio between body weight and hanging time were directly related as mice with a higher body weight had lower recorded hanging times. However, it was found that Vilon-treated mice with a higher body weight had longer recorded hanging times than control mice of a similar weight. These findings allowed the researchers to conclude that long-term administration of Vilon resulted in reduced muscular fatigue and increased muscular power.

Figure 3: Dynamics of motor activity of the control (1) and Vilon-treated (2) test subjects in an open field as it relates to age, estimated by (a) number of crossed squares, (b) number of rearings, and (c) time of grooming

When examining vaginal smear samples, the findings indicated that the overall duration of the estrous cycle in control mice increased with age while the relative number of short estrous cycles decreased, and the number of long cycles increased. However, these findings were not significant enough to conclude that prolonged administration of the peptide did not have specific effects of the age dynamics of estrous functioning. Furthermore, in control mice aged 9-15 months rectal temperatures increased during the diestrus phase, while no significant changes occurred after 18 months as the temperatures remained constant. There were no other changes observed in the control mice. Other notable changes included a decrease in average body temperature during the diestrus phase in 12-18 month old mice treated with Vilon when compared to control subjects [3].

Up to 21 months, there were no significant differences in survival rate between the control and experimental mice, however, after 21 months VIon was shown to significantly decrease mortality rates. Mice in the experimental group lived to 22 and 23 months which correlated to 1.24 and 2.57 times higher than the control subjects. Following natural death or euthanasia of the test subjects, an autopsy was performed to examine spontaneous tumor growth. The control group of test subjects had an incidence of spontaneous tumors of 30% with lung adenoma and breast tumors developed the most frequently with incidences of 20% and 8%, respectively [3]. Alternately, the mice treated with Vilon experienced inhibition of spontaneous carcinogenesis with a 1.5- and 1.8-fold decrease in all tumors and multiple tumors, respectively. Treatment with Vilon was also found to increase the lifespans of mice with tumors by more than a month, as well as elicit a 2-fold decrease in lung adenomas and an overall decrease in breast adenocarcinomas.

The researchers also noted that administration of Vilon had a positive effect on the physical activity and endurance of the test subjects. These effects directly corresponded to an increase in body weight and a decrease in body temperature. Decreased body temperature is important to mention in this context considering it relates to the prolongation of life through the deceleration of metabolic processes [3].

Figure 4: The effect of Vilon on the lifespan of CBA mice. (1) Control group; (2) Vilon-treated groups.

3) The results of the study conducted by Khavinson et al. revealed that all apoptotic lymphocytes were able to be identified by specific morphological signs including: chromatin margination through selective impregnation, cytoplasm condensation, and thickening with the formation of apoptotic bodies. Irradiation was shown to significantly stimulate the process of apoptosis as the apoptotic index of the intact rats was measured at 2.65 +/- .10%, which was 3.15 times higher than the baseline of 0.84 +/- 0.09%. Administration of the synthetic peptide bioregulator Epithalon was shown to decrease the apoptotic index by 2.12-fold to 1.25 +/- 0.15%. Administration of the synthetic peptide bioregulator Vilon was shown to decrease the apoptotic index by 3.4-fold and below the baseline levels to 0.78 +/- 0.12%. The researcher team noted that Vilon inhibited apoptosis in splenic lymphocytes to a greater degree than Epithalon. That being said, the apoptotic index decreased by 37.6% when comparing the scores of group 3 rats to group 2 rats [4].

While apoptosis is typically a conservative process that is controlled by genetics, there are various endogenous and exogenous stimuli that are able to affect cell viability and enhance the process of apoptosis in various tissues. However, the data collected by the research team of Khavinson et al. allowed them to conclude that synthetic peptide bioregulators such as Epithalon and Vilon are able to inhibit apoptosis and regulate cell renewal in a more effective manner, in comparison to previous studies related to the role of peptide bioregulators in the inhibition of age-related decline [4].

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 VK, Popovich IG, Linkova NS, Mironova ES, Ilina AR. Peptide Regulation of Gene Expression: A Systematic Review. Molecules. 2021 Nov 22;26(22):7053. doi: 10.3390/molecules26227053. PMID: 34834147; PMCID: PMC8619776.

[2] Lezhava T, Khavison V, Monaselidze J, Jokhadze T, Dvalishvili N, Bablishvili N, Barbakadze S. Bioregulator Vilon-induced reactivation of chromatin in cultured lymphocytes from old people. Biogerontology. 2004;5(2):73-9. doi: 10.1023/B:BGEN.0000025070.90330.7f. PMID: 15105581.

[3] Khavinson VK, Anisimov VN, Zavarzina NY, Zabezhinskii MA, Zimina OA, Popovich IG, Shtylik AV, Malinin VV, Morozov VG. Effect of vilon on biological age and lifespan in mice. Bull Exp Biol Med. 2000 Jul;130(7):687-90. doi: 10.1007/BF02682106. PMID: 11140587.

[4] Khavinson VK, Kvetnoii IM. Peptide bioregulators inhibit apoptosis. Bull Exp Biol Med. 2000 Dec;130(12):1175-6. PMID: 11276315.

 

PEPTIDES PREFER THE COLD
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