EFFECT OF ACTH 6-9 -PRO-GLY-PRO PEPTIDE ON SPECTRAL PARAMETERS OF HEART RATE VARIABILITY IN WISTAR RATS DURING PHYSICAL EXERTION (PILOT STUDY)

2074-0581-2023-25-1-71-83


Цель исследования Introduction
Regulatory peptides are biologically active substances with a wide range of biological and physiological effects that can be realized both at the level of an individual cell and at the tissue and/or organ levels.Of great interest are N-terminal analogs of adrenocorticotropic hormone (ACTH), the advantage of which is the absence of hormonal activity [1].The most studied representative of this group is the ACTH 4-7 -Pro-Gly-Pro (Semax) peptide, which is a Met-Glu-His-Phe peptide sequence corresponding to the ACTH region from 4 to 7 amino acid residues, protected from the action of carboxypeptidases by attachment of glyproline Pro-Gly-Pro (PGP) to its C-terminus [1].Its effect on the level of anxiety, depression, cognitive activity, and pain sensitivity, was demonstrated, it also increases adaptive capabilities both with a single and course administration [1].Semax has been shown to have an effect on autonomic regulation in laboratory animals [2].
ACTH 6-9 -PGP is peptide structurally and functionally related to ACTH 4-7 -PGP.The His-Phe-Arg-Trp sequence (ACTH 6-9 ) is the active center of ACTH, which interacts with MC1R, MC3R, and MC4R [3].ACTH 6-9 -PGP has a wide range of neurotropic effects and, in comparable doses, exhibits more pronounced activity than ACTH 4-7 -PGP in relation to anxiety and depressive reactions, the development and consolidation of conditioned reflexes, and pain sensitivity [4][5][6].At the same time, despite the extensive study of the effect of the peptide on the functioning of the somatic nervous system, its effect on the autonomic regulation of the body has not been studied enough.Indirectly, the effects of the peptide on the autonomous system can be assessed by its action on the entero-cerebral axis under conditions of chronic stress [7].One of the main ways of communication between the nervous system and the gastrointestinal tract is provided by the vagus nerve [8].In this regard, it is of interest to investigate the effects of ACTH 6-9 -PGP on autonomic regulation, the study of which is possible through the analysis of HRV [9].

Purpose of the study
Study of the effect of the ACTH 6-9 -Pro-Gly-Pro peptide on the spectral parameters of HRV in Wistar rats after a short-term exercise.
Статистическая обработка результатов проведена с использованием программы Statistica 13 (TIBCO Software Inc., USA), а также языка программирования R v. 4 HRV indicators were recorded using the Physiobelt 2.5.1 software and hardware complex (Neurobotics, Russia), which was fixed on the animal with a vest and allowed the rat to move freely.For the adaptation to this device, before the start of the experiment, rats were subjected to test recordings for 5 minutes 3 times a week.The analysis of HRV was carried out according to spectral parameters: TP is the total power of the HRV spectrum, HF (ms 2 ) is the total power of the high-frequency component of HRV, LF (ms 2 ) is the total power of the low-frequency component of HRV, VLF (ms 2 ) is the total power of the very low-frequency component of HRV, HF (%) is the power of the spectrum of the high-frequency component of the variability in % of the total power of the oscillations, LF (%) is the power of the spectrum of the low-frequency component of the variability in % of the total power of the oscillations, VLF (%) is the power of the spectrum of the very low-frequency component of the variability in % of the total power fluctuations, LF/HF -index of vagosympathetic interaction, IC -index of centralization [10,11].
The study used ACTH 6-9 -PGP, synthesized at the Institute of Molecular Genetics, National Research Center "Kurchatov Institute".Before use, the peptide was dissolved in normal saline and administered intraperitoneally to laboratory animals at doses of 5, 50, and 500 μg/kg once in a volume of 1 ml/kg.Animals of the control group were injected with normal saline in an equivalent volume.Each group included 6 males and 6 females.
The experiments were carried out in the daytime, from 9 am to 3 pm.The first recording (record 1) of the cardiac signal started after 15 minutes of adaptation of the rat to the Physiobelt device in a clean empty plastic cage similar to the one in which the animals were initially kept.Next, the animal was intraperitoneally injected with a peptide/normal saline, and after 15 minutes, the cardio signal was recorded (record 2).Then the rat was exposed to a two-minute run on a treadmill (Treadmill LE8710, Panlab, Spain) at a speed of 15 m/min, the angle of the treadmill was 15°.Immediately after the end of the exercise, the animal's ECG signal was recorded (record 3); the rat was returned to the cage in which the animal was resting, and after 15 minutes of rest, the fourth recording was performed (record 4).

Results and discussion
when analyzing the initial values of HRV indicators, attention was drawn to the pronounced shift of spectral characteristics towards the VLF component in all the studied groups, which indicated the predominance of the mechanisms of humoral and suprasegmental regulation of heart rate (Table 1) [12].There was no statistically significant difference between the values of all studied HRV parameters between the groups.However, the mean values of TP, LF (ms 2 ), VLF (ms 2 ), and IC in the control group were significantly higher than in the other groups, and the absence of a statistical difference allowed us to consider the groups homogeneous.
The next step in the analysis of the obtained results was the study of the dynamics of the magnitude of HRV spectral parameters within the studied groups.As can be seen from Table 2, in the control group, 15 minutes after the injection of saline, an increase in VLF (%) by 21.6% and a decrease in LF (%) by 12.3% were recorded compared with the initial level.This fact indicates the activation of heart rate (HR) regulation at the suprasegmental level associated with the activation of the hypothalamus, against the background of a decreased activity of the sympathetic nervous system [13].Moderate physical activity contributed to a statistically significant, more than double increase in the HF, characterizing parasympathetic regulation, compared with the value of this indicator at 1 st and 2 nd recording.Based on literature data regarding changes in the HF component of HRV due to the active release of catecholamines and cytokines as a result of stimulation [14,15], in our work, the role of the stimulus was played by moderate physical activity [16].At the same time, the value of the indicator VLF decreased by 24.4% while LF increased by 17.5%, which reflected the increased role of sympathetic mechanisms of HR regulation [13].The decrease in LF/HF from baseline might be due to the mobilization of energy and metabolic reserves through autonomic regulation of the HR.It is noteworthy that the ratio of the frequency components VLF>LF>HF did not change at all stages of observation, which proved the dominant role of the ergotropic region of the hypothalamus in the control group [17].In the group of animals that received 5 μg/kg of the peptide, the analysis of spectral parameters revealed a stable condition of the regulatory mechanisms at all stages of the study.Statistically significant differences were established only in relation to the LF/HF value (Fig. 1), however, the low variability of this parameter confirmed that in this group the regulatory mechanisms remained close to the initial level of the functional condition even under the influence of physical activity, which might be associated with adaptive the action of the peptide at a dose of 5 µg/kg.

Таблица 1 Исходные показатели ВСР в исследуемых группах (M±SD, n=12)
Table 3 shows the results of HRV in the group of animals treated with the peptide at a dose of 50 μg/kg.Moderate physical activity after administration of the peptide at this dose was accompanied by a statistically significant increase in the power of the HF and LF spectra by 2.5-2.6 times compared to the values of the indicated parameters in records 1 and 2, as well as a redistribution of the frequency characteristics of the spectrum (%) as LF>VLF>HF.The established features of HRV indicators showed a pronounced activity of the sympathetic nervous system against the background of high adaptation of the body, supported by the mobilization of reserves of the hypothalamus-pituitary-adrenal axis [18].In the recovery period (record 4), there were statistically significant differences with the results of record 3 in terms of HF (ms 2 , %), LF (ms 2 , %), and VLF (%), which reflected the restoration of the initial level of the functional condition of the studied animals.Attention was drawn to the statistically significantly high value of IC compared with the value of record 3, which indicated Примечания: р -статистическая значимость различий показателей между всеми записями (по критерию Фридмана); post-hoc: р 1 -статистическая значимость различий по сравнению с записью 1; р 2 -по сравнению с записью 2; р 3 -по сравнению с записью 3 (post-hoc по критерию Коновера) Notes: p -statistical significance of differences in indicators between all records (according to the Friedman criterion); post-hoc: р 1 -the statistical significance of differences compared with record 1; p 2 -compared with record 2; p 3 -compared with record 3 (post-hoc according to the Conover criterion)
An analysis of the dynamics of HRV parameters after the administration of the peptide at a dose of 500 μg/kg made it possible to establish that moderate physical activity was accompanied by an increase in the power of HF waves compared to the results of recordings 1 and 2 (Table 4).A percentage ratio of the frequency component LF>HF>VLF, which had been uncommon for the previous groups, indicated the suppression of the influence of the central neurohumoral mechanisms of HR control and a pronounced tension of both the sympathetic and parasympathetic nervous systems [19].The dynamics of the LF/HF indicator confirm the presence of an autonomic balance with a moderate prevalence of sympathetic influences in the study groups after physical activity.At the same time, IC decreased by 6.6 times compared to the previous level (record 2), which reflected the leading role in the regulation of the autonomous HR control circuit.The recovery period was characterized by a statistically significant decrease in the parasympathetic activity (a decrease in the HF value), the restoration of the percentage of frequency indicators in the VLF>LF>HF spectrum, an increase in the LF/HF and Примечания: р -статистическая значимость различий показателей между всеми записями (по критерию Фридмана); post-hoc: р 1 -статистическая значимость различий по сравнению с записью 1; р 2 -по сравнению с записью 2; р 3 -по сравнению с записью 3 (post-hoc по критерию Коновера) Notes: p -statistical significance of differences in indicators between all records (according to the Friedman criterion); post-hoc: р 1 -the statistical significance of differences compared with record 1; p 2 -compared with record 2; p 3 -compared with record 3 (post-hoc according to the Conover criterion)  [14].В дальнейшем сравнительный анализ результатов ВСР позволил установить статистически значимые различия между изучаемыми группами на каждом из этапов исследования.В табл.5 представлены результаты показателей ВСР между группами через 15 минут после введения пептида/физиологического раствора (запись 2), согласно которым у животных после введения пептида в дозе 5 мкг/кг регистрируется наибольшее значение показателей ТР и VLF (мс 2 ).Данные факты свидетельствуют о максимальных энергетических и метаболических резервах в IC values compared to the results of recording 3.These changes indicate the maintenance of the original level of the functional condition in the studied animals through the activity of the central mechanisms of neurohumoral regulation [14].
Subsequently, a comparative analysis of the results of HRV made it possible to establish statistically significant differences between the studied groups at each stage of the study.Table 5 shows the results of HRV values in the groups 15 minutes after the administration of the peptide/saline (record 2), according to which the animals getting a dose of 5 μg/kg of the peptide recorded the highest values of TP and VLF (ms 2 ).These facts indicated the maximum energy and metabolic reserves in the 5 µg/kg group, which provided resistance to changing conditions.
The values of HRV after moderate physical activity (record 3) in the studied groups are presented in Table 6.It was found that in the 5 µg/kg group the absolute power of HF waves and LF waves was significantly lower than in other groups.At the same time, the power of VLF waves was higher than in the 50 and 500 µg/kg groups, but lower than in the control group.The distribution of frequency components within the groups also had statistically significant differences: in the control and 5 µg/kg groups, the initial gradation VLF>LF>HF was maintained, while in the 50 Примечания: р -статистическая значимость различий показателей между всеми записями (по критерию Фридмана); post-hoc: р 1 -статистическая значимость различий по сравнению с записью 1; р 2 -по сравнению с записью 2; р 3 -по сравнению с записью 3 (post-hoc по критерию Коновера) Notes: p -statistical significance of differences in indicators between all records (according to the Friedman criterion); post-hoc: р1 -the statistical significance of differences compared with record 1; p 2 -compared with record 2; p 3 -compared with record 3 (post-hoc according to the Conover criterion)
Анализ величины показателей ВСР на последнем этапе исследования (запись 4) между группами позволил установить, µg/kg group, the distribution LF>VLF>HF was established, and in group 500 µg/kg it constituted LF>HF>VLF.It should be noted that the IC in the 5 µg/kg group was 5.8 and 7.3 times higher than in the 50 and 500 µg/kg groups, respectively.The results obtained suggest that the peptide in different doses caused the activation of various mechanisms of neurohumoral regulation.In particular, a dose of 5 μg/kg stabilized the functional condition of the studied animals at the initial level and, despite the moderate physical activity performed by the studied animals, the dominant influence of suprasegmental regulation mechanisms and centralization of HR control were noted.At doses of 50 and 500 μg/kg, the effects of the peptide were similar to each other, however, they have a number of differences from the results in the control group and 5 μg/kg groups, which were manifested by the sympathetic-parasympathetic balance of autonomic regulation and the activity of the peripheral contour of the HR regulation.It should be noted that doses of 50 and 500 μg/kg sharply increased the relative power of LF waves characterizing the activity of the sympathetic nervous system.
An analysis of the HRV values at the last stage of the study (record 4) between the groups made it possible to establish that the 5 mcg/kg group was characterized by statistically significant differences in the value of TP, HF (ms 2 ), and LF (ms 2 ) compared with the control group, as well as LF (ms 2 ) and LF/HF compared to the 500 μg/kg group (Table 7).These features of HRV indicators in the 5 µg/kg group once again proved a pronounced adaptogenic effect and stability of the functional condition of the animals.In the 50 μg/kg group, the minimum value of the absolute power of the high frequencies of the spectrum was noted, which indicated a reduced effect of parasympathetic regulation of the HR.[20,21], а MC4R были обнаружены в паравентрикулярных ядрах гипоталамуса [22].Поэтому АКТГ 6-9 -ПГП, являющийся фармакором MCRs, может изменять ак-The adaptogenic effect of the peptide in the form of activation of suprasegmental structures might be associated with the activation of MCRs in them.It is known that MC3Rs are expressed in the cortex, ventromedial nuclei, medial preoptic zone of the hypothalamus, hippocampus, and anterior amygdala [20,21], while MC4Rs were found in the paraventricular nuclei of the hypothalamus [22].Therefore, ACTH 6-9 -PGP, which is the pharmacophore of MCRs, can change the activity of these brain structures due to interaction with MC3R and MC4R.

Conclusion
Thus, our pilot study of the effects of the ACTH 6-9 -PGP peptide on HRV in Wistar rats showed that its single intraperitoneal administration at a dose of 5 μg/kg promotes the adaptation of laboratory animals to moderate physical activity due to the activation of suprasegmental structures, while in doses of 50 μg/kg and 500 μg/kg -activation of the peripheral parts of the sympathetic and parasympathetic systems.The results of this work and data from other studies of the effects of N-terminal ACTH analogs indicate the prospects for further study of the effects of the peptide on neurohumoral regulation in experimental animals.In addition, the heterogeneous results of this work indicate the need to expand the study by increasing the number of animals and investigating, along with spectral characteristics, the statistical and geometric indicators of HRV.

Table 1
Baseline HRV indicators in the study groups(M±SD, n=12)

Table 2
HRV parameters of Wistar rats in the control group(M±SD, n=12)

Table 3
Effect of the peptide at a dose of 50 µg/kg on the HRV parameters of Wistar rats(M±SD, n=12)

Table 4
Effect of the peptide at a dose of 500 μg/kg on the HRV parameters of Wistar rats(M±SD, n=12)

Table 6
Indicators of HRV after exercise in the study groups(M±SD, n=12)