Effects of GHK peptide and its structural analogues on dynamics of healing and bacterial contamination of infected wound

Full Text

LIST OF ABBREVIATIONS

CFU — colony forming unit

D-Ala — D-Alanine

GHK — Glycyl-Histidyl-Lysine

HR — healing rate

RWHC — relative wound healing coefficient

INTRODUCTION

Increasing the effectiveness of the regenerative mechanisms in wound processes is an actual trend in the modern biomedical research. It has now been established that the main sequentially developing regeneration stages in tissue damages are inflammatory reaction, wound healing, extracellular matrix remodeling, anti-inflammatory and anti-fibrotic response; as well as tissue remodeling [1–3]. These processes are provided by rather complex and diverse mechanisms involving all regulatory processes of an organism [4–6]. A possible way to realize this task may be investigation of peptidergic mechanisms of activating wound healing processes. Up to date, a significant amount of information has been accumulated on the use of peptide molecules and pharmacological drugs on their basis in the correction of various pathological processes [6]. Therefore, the use of tripeptide Glycyl-Histidyl-Lysine (GHK) seems promising in accelerating the resolution of wound process.

This peptide is known to act on tissue regeneration processes [7, 8], and also possesses antioxidant, immunotropic and anti-inflammatory effects [9, 10]. Upon that, the main disadvantage of all regulatory peptides is their rapid degradation by proteolytic enzymes.

One of possible ways to increase the stability of peptide molecules, and, consequently, the duration of their biological effect, is incorporation of D-isomers of amino acids in their structure. In the previous studies we found that GHK-D-Ala (D-Alanine) produced a more prominent effect on regenerative processes in the wound than GHK and facilitated an increase in the number of fibroblast cells, macrophages with the underlying decrease in granulocytes and lymphocytes [10]. A significant effect of GHK and its structural analogues D-Ala-GHK and GHK-D-Ala on the parameters of the innate immunity and lipid peroxidation in infected skin wound was also shown. The most prominent and stable effects were also observed with GHK-D-Ala [111]. The data obtained in these works necessitated the study of the integrative wound healing parameters of GHK and its structural analogues D-Ala-GHK и GHK-D-Ala, used for clinical assessment of the effectiveness of regenerative processes.

The aim of this study to assess the dynamics of healing and of bacterial contamination of infected wound with use of GHK peptide and its structural modifications with D-Ala.

MATERIALS AND METHODS

The experiments were conducted on 150 male Wistar rats weighing 180 g–240 g aged 6–8 months (Stolbovaya branch of the Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency). The animals were kept in standard vivarium conditions with free access to water and food, 12-hour light regime and air temperature 22 ± 2°С. Each experimental group included 10 rats.

The study used GHK peptide and its structural analogues D-Ala-GHK and GHK-D-Ala (synthesized at the Research Institute of Chemistry of St. Petersburg State University), which were dissolved in saline and administered intracutaneously (at two points around the wound, changing the injection areas daily by 90° clockwise) at doses of 0.5 μg/kg and 1.5 μg/kg in 0.1 ml 24 hours after modeling an infected wound, followed by administration of the drug at the same dose every 24 hours for 3^rd, 7^th, or 10^th days. In the control series, animals were administered equivalent volumes of saline at similar intervals at a rate of 1 ml per 1 kg of body weight. The last administration of the drugs was carried out 24 hours before the animals were withdrawn from the experiment.

The animals were withdrawn from the experiment by taking blood from the right ventricle of the heart under ether narcosis.

All studies were conducted in compliance with the principles of the European Convention for Protection of Vertebrate Animals used in experimental research; ‘Guidelines on the conduct of preclinical trials of medical drugs’ (Moscow, 2012) and in accordance with the decision of the Regional Ethics Committee at Kursk State Medical University (Protocol No. 1 of January 16, 2014).

The infected (natural infection) open wound was modeled by inflicting full-thickness wounds of a standard size (250 mm²) on a shaven portion on the back of an anesthetized animal.

Immediately after modeling of the wound process, and also on the first, third, seventh and tenth day, the wounds were photographed with a Canon A410 digital camera using a millimeter grid with subsequent measurement of wound areas at these control periods. The wound areas were calculated in the ImageJ program (National Institutes of Health, USA).

For objective planimetric assessment of wound healing by the change in its area, the relative wound healing coefficient (RWHC) was used a decrease in the wound area from the initial size expressed as a percentage, calculated from the formula:

RWHC = (S₀ — S) / S₀ × 100%,

where S₀ — initial, and S — final wound surface (mm²) [12].

The wound healing rate (a decrease in the wound area per day in %) was also calculated using the formula:

HR = (RWHC₁ — RWHC₀) / Т,

where HR — healing rate, RWHC₁ — coefficient at the time of measurement (final), RWHC₀ — coefficient at the previous measurement, T — the number of days between measurements.

On visual examination, the clinical wound healing parameters were assessed: the time of disappearance of perifocal edema, wound cleansing, appearance of granulations and beginning of the marginal epithelialization.

For quantitative determination of the microbial contamination, the wound was pre-treated with isotonic sodium chloride solution, then with 70% ethyl alcohol to remove vegetating microflora, pus and detritus from its surface. The material was taken after withdrawal of the animal from the experiment, in a sterile box by excising a part of wound of 0.1 g–0.5 g to the full wound depth. Then the biopsy material was weighted to calculate the conversion coefficient (K) for 1 g of tissue, a suspension was prepared in the isotonic sodium chloride solution at a ratio of 1:10, and the material was inoculated on the dense nutrient medium (meat-peptone agar) by Drigalsky method. The cultures were incubated in a thermostat (37°С) for 20 hours, then at room temperature for 24 hours, after which colonies were counted with conversion to 1 g of tissue.

The colonies were counted on dishes, in which they grew isolated from each other, and their number did not exceed 300. The number of microbes in 1 g of tissue was calculated using the formula:

N = n × 10 × 10 (or × 100, or × 1000) × К,

where N — number of microbes in 1 g of bioptate, n — number of microbes grown in a Petri dish, 10 — recalculation to 1 g of suspension, 10, 100 or 1000 — dilution of the material inoculated in the Petri dish, in which colonies were calculated, K — conversion coefficient of the weighted quantity per 1 g of bioptate.

Statistical processing of the obtained data was performed using the R version 4.1.0 programming language [13] in the integrated development environment RStudio Desktop ver. 1.4.1717 (RStudio, PBC, USA). To check the normality of the distribution, the Shapiro–Wilk test (the shapiro.test() function from the standard package) was used, and the Levene test (the levene.test() function from the lawstat package) was used to check the equality of variances. In case of confirmation of the hypotheses, the data were presented as ‘mean ± standard deviation’ (M ± SD), M and SD were calculated using the mean() and sd() functions from the standard package; in case of deviation — as ‘Median [lower quartile; upper quartile]’ (Me [Q1; Q3]), which were calculated using the median() and quantile() functions from the standard package. To determine the differences between the groups, Kruskal–Wallis test (the kruskal.test() function from the standard package) with Dunn's post hoc test with Benjamini–Hochberg correction (the dunnTest() function from the FSA package) was used. Dunn's test was performed only in the presence of significant intergroup differences: the p-value for the calculated Kruskal–Wallis test with 6 intergroup degrees of freedom and 70 intragroup degrees of freedom (H_6,70) was less than 0.05. The z-value and the degree of its significance (p-value) were calculated for it. Differences were considered significant at p < 0.05.

RESULTS

As follows from Table 1, on day 3^rd, no significant differences were observed in the wound area and wound healing coefficient between experimental and control groups of animals.

 

Table 1. Dynamics of Change in Planimetric Parameters of Wound Healing (n = 10) with Use of D-Ala-GHK and GHK-D-Ala Peptides, M ± SD/Me [Q1; Q3]

Groups	Timing
	1st day	3rd day	7th day	10th day
Wound area, mm2
Control	250.00 [249.25; 250.00]	216.60 ± 39.88	165.50 ± 47.43	85.50 [72; 155.75]
GHK 0.5 mkg/kg	250.00 [248.50; 250.00]	211.50 ± 63.93	63.40 ± 28.32***	26.50 [19.5; 29.00]**
GHK 1.5 mkg/kg	249.00 [248.00; 250.00]	209.00 ± 35.05	72.60 ± 36.41***	14.50 [8.5; 23.25]***
D-Ala-GHK 0.5 mkg/kg	249.50 [248.25; 250.00]	174.60 ± 28.78	66.60 ± 23.33***	36.00 [23.5; 43.50]*
D-Ala-GHK 1.5 mkg/kg	250.00 [249.25; 250.00]	174.30 ± 37.85	97.10 ± 17.99***	35.50 [24.25; 59.25]*
GHK-D-Ala 0.5 mkg/kg	249.50 [248.00; 250.00]	169.00 ± 47.01	96.70 ± 34.08***	6.30 [5.1; 8.60]***
GHK-D-Ala 1.5 mkg/kg	249.50 [248.00; 250.00]	172.00 ± 47.86	113.80 ± 42.07**	7.20 [5.7; 16.00]***
Kruskal–Wallis test	H6,70 = 2.71, p = 0.84	H6,70 = 12.47, p = 0.052	H6,70 = 32.96, p = 0.00001	H6,70 = 51.85, p = 0.00001
Relative wound healing coefficient, %
Control	0.40 [0.00; 0.79]	6.80 [2.30; 25.88]	21.32 ± 27.97	36.22 [14.02; 45.85]
GHK 0.5 mkg/kg	0.20 [-0.30; 0.70]	17.56 [2.21; 35.27]	66.68 ± 21.11***	62.55 [33.68; 71.87]
GHK 1.5 mkg/kg	0.40 [0.00; 0.80]	13.39 [10.18; 22.30]	65.21 ± 18.06***	77.98 [60.72; 86.80]**
D-Ala-GHK 0.5 mkg/kg	0.40 [0.10; 0.70]	27.33 [20.42; 38.63]	59.79 ± 19.41***	48.17 [36.42; 55.03]
D-Ala-GHK 1.5 mkg/kg	0.20 [0.00; 0.80]	25.74 [19.97; 39.93]	43.16 ± 9.00	61.57 [45.01; 74.50]
GHK-D-Ala 0.5 mkg/kg	0.80 [0.10; 0.80]	36.97 [15.32; 45.22]	40.31 ± 24.89	92.00 [89.80; 94.59]***
GHK-D-Ala 1.5 mkg/kg	0.80 [0.10; 1.09]	22.01 [19.27; 47.91]	33.04 ± 21.96	91.98 [89.79; 94.64]***
Kruskal–Wallis test	H6,70 = 2.78, p = 0.83	H6,70 = 12.47, p = 0.052	H6,70 = 27.85, p = 0.0001	H6,70 = 47.39, p = 0.00003

Notes: * — p < 0.05; ** — p < 0.01; *** — p < 0.001 in comparison with the control group (by post hoc Dunn’s test)

 

On day 7^th, the reduction in the wound area reached reliable significant differences in all the experimental groups. The most prominent changes were observed after the introduction of GHK at doses of 0.5 (by 62%, z = -4.53, p = 0.000005) and 1.5 μg/kg (by 56%, z = -4.11, p = 0.00003), as well as D-Alа-GHK at a dose of 0.5 μg/kg (by 60%, z = -4.4, p = 0.00001), which was reflected in a reliably significant increase in the wound healing coefficient in these groups by 2.37 (z = 3.78, p = 0.00015), 2.17 (z = 3.74, p = 0.00018) and 1.94 (z = 3.13, p = 0.0017) times, respectively. The increase in this parameter after the introduction of GHK-D-Ala was not statistically significant.

By day 10^th of the experiment, the use of peptides was accompanied by a significant decrease in the wound area in all groups. The most pronounced differences relative to the control values were noted after the introduction of GHK-D-Ala peptide at doses of 0.5 (by 93%, z = -5.91, p = 0.000000004) and 1.5 μg/kg (by 92%, z = -5.33, p = 0.00000007). After the introduction of D-Alа-GHK peptide, the decrease in this parameter was least pronounced: by 58% (z = -2.19, p = 0.0028) at a dose of 0.5 μg/kg and by 59% (z = -2.03, p = 0.0042) at a dose of 1.5 μg/kg. It is noteworthy that the effect of these peptides did not depend on the dose. Against the background use of GHK, the wound area was smaller than the control values after administration at doses of 0.5 μg/kg (by 69%, z = -2.91, p = 0.00036) and 1.5 μg/kg (by 83%, z = -4.36, p = 0.000015).

Within 1–3 days, the wound healing rate in rats given GHK did not significantly differ from the control group (Table 2). With the introduction of D-Ala-GHK and GHK-D-Ala, healing accelerated in equal measure but did not reach statistically significant differences. Over the period of 3–7 days, GHK at a dose of 0.5 µg/kg increased the healing rate 2.7 times (z = 2.44, p = 0.015), and at a dose ща 1.5 µg/kg —1.4 times (z = 2.68, p = 0.007). However, on the contrary, with the introduction of GHK-D-Ala, a tendency to a decrease in this parameter was noted without reaching a reliably significant level. The use of D-Ala-GHK was accompanied by multidirectional unreliable changes in the experimental groups. At the final stage of the experiment (7–10 days), with the introduction of GHK-D-Ala, a significant acceleration of wound healing was noted. Thus, at a dose of 0.5 µg/kg, the peptide accelerated the process 4.7 times (z = 2.57, p = 0.001) and at a dose of 1.5 µg/kg — 5.3 times (z = 3.09, p = 0.0002). At the same time, no significant changes in the healing rate were noted after administration of GHK and D-Ala-GHK.

 

Table 2. Dynamics of Wound Healing Rate (n = 10) with Use of D-Ala-GHK and GHK-D-Ala Peptides, M ± SD/Me [Q1; Q3], %

Groups	Timing
	1st–3rd days	3rd–7th days	7th–10th days
Control	6.43 ± 8.04	5.85 [-3.12; 8.19]	3.68 ± 8.15
GHK 0.5 mkg/kg	7.52 ± 12.66	15.87 [3.64; 18.86]**	-5.10 ± 14.53
GHK 1.5 mkg/kg	7.85 ± 7.27	14.20 [11.03; 15.38]***	2.83 ± 10.81
D-Ala-GHK 0.5 mkg/kg	14.64 ± 5.91	8.74 [6.36; 12.33]	-5.34 ± 12.78
D-Ala-GHK 1.5 mkg/kg	14.89 ± 7.91	4.54 [-1.91; 7.56]	5.62 ± 6.82
GHK-D-Ala 0.5 mkg/kg	15.89 ± 9.61	3.90 [-5.54; 9.74]	17.24 ± 8.86*
GHK-D-Ala 1.5 mkg/kg	15.16 ± 9.70	1.56 [-1.43; 6.70]	19.62 ± 7.63**
Kruskal–Wallis test	H6,70 = 12.01, p = 0.0616	H6,70 = 20.04, p = 0.0027	H6,70 = 34.49, p = 0.00001

Notes: * — p < 0.05; ** — p < 0.01; *** — p < 0.001 in comparison with the control group (by post hoc Dunn’s test)

 

The established increase in the wound healing rate with the introduction of GHK-D-Ala also manifested in the clinical parameters (Table 3). Thus, at both doses, the peptide facilitated the earliest disappearance of perifocal edema, wound cleansing, appearance of granulations and beginning of marginal epithelialization. The effects of GHK and D-Ala-GHK were less pronounced, without any statistically significant differences between them. To note, the changes in the periods of appearance of the clinical signs of wound healing were reliable with all peptides.

 

Table 3. Dynamics of Clinical Parameters of Wound Healing (n = 10) with Use of Peptides D-Ala-GHK and GHK-D-Ala, M ± SD/Me [Q1; Q3]

Parametrs Groups	Disappearance of perifocal edema, day	Wound cleansing, day	Appearance of granulations, day	Beginning of marginal epithelialization, day
Control	8 [8; 8, 75]	9 [8, 25; 9]	7 [7; 8]	9 [8; 9]
GHK 0.5 mkg/kg	6 [5; 6]***	6 [6; 6]***	5 [5; 5]***	5 [5; 5]***
GHK 1.5 mkg/kg	6 [6; 6]***	6,00 [6; 6, 75]***	5 [5; 6]**	6 [5; 6]**
D-Ala-GHK 0.5 mkg/kg	6 [6; 7]*	7 [6; 7]*	5,50 [5; 6]*	6 [5; 6]**
D-Ala-GHK 1.5 mkg/kg	6 [6; 6]**	6 [6; 7]**	5 [5; 6]**	6 [5; 6]**
GHK-D-Ala 0.5 mkg/kg	5 [4; 5]***	5 [4, 25; 5]***	3 [3; 3, 75]***	4 [3; 4]***
GHK-D-Ala 1.5 mkg/kg	4 [4; 5]***	5 [5; 5, 75]***	4 [3; 4]***	5 [4; 5]***
Kruskal–Wallis test	H6,70 = 56,85, p = 0,00001	H6,70 = 49,93, p = 0,00001	H6,70 = 56,97, p = 0,00001	H6,70 = 50,96, p = 0,00001

Notes: * — p < 0.05; ** — p < 0.01; *** — p < 0.001 in comparison with the control group (by post hoc Dunn’s test)

 

It is known that the microbial contamination plays an important role in the healing process of an infected wound. As can be seen from Table 4, on day 3^rd this parameter had close values across all groups of animals. However, on day 7^th it significantly decreased after the introduction of both GHK and its analogues. Upon that, the greatest decrease in microbial contamination (32%, z = 2.64, p = 0.0017) was noted with GHK-D-Ala at a dose of 0.5 μg/kg. A similar pattern of changes in this parameter, but more strongly expressed, persisted on day 10^th of the experiment. Thus, the greatest decrease in microbial contamination (37%, z = 2.77, p = 0.0011) was also observed with GHK-D-Ala at a dose of 0.5 μg/kg, and the lowest effect was noted after the administration of GHK at a similar dose (8%, z = 1.56, p = 0.042).

 

Table 4. Parameters of Microbial Contamination of Wound (n = 10) with Use of Peptides D-Ala-GHK and GHK-D-Ala, M ± SD/Me [Q1; Q3], DlgCFU per 1g of Autoptate

Groups	Timing
	3rd day	7th day	10th day
Control	6.48 [6.47; 6.48]	5.98 ± 5.06	5.86 [5.79; 5.90]
GHK 0.5 mkg/kg	6.48 [6.44; 6.48]	5.83 ± 5.23***	5.41 [5.30; 5.50] *
GHK 1.5 mkg/kg	6.47 [6.46; 6.48]	5.33 ± 4.46***	4.28 [4.18; 4.37] ***
D-Ala-GHK 0.5 mkg/kg	6.48 [6.44; 6.48]	5.44 ± 4.79***	4.30 [4.26; 4.34] *
D-Ala-GHK 1.5 mkg/kg	6.47 [6.44; 6.48]	5.29 ± 4.24***	4.10 [4.01; 4.17] ***
GHK-D-Ala 0.5 mkg/kg	6.48 [6.47; 6.48]	4.08 ± 3.45***	3.68 [3.51; 3.83] ***
GHK-D-Ala 1.5 mkg/kg	6.47 [6.43; 6.48]	5.81 ± 4.94***	4.47 [4.33; 4.48] ***
Kruskal–Wallis test	H6,70 = 2.78, p = 0.8355	H6,70 = 12.47, p = 0.032	H6,70 = 56.97, p = 0.00001

Notes: * — p < 0.05; ** — p < 0.01; *** — p < 0.001 in comparison with the control group (by post hoc Dunn’s test); CFU — colony forming unit

 

DISCUSSION

Thus, GHK and its structural analogues D-Ala-GHK and GHK-D-Ala promoted acceleration of wound healing and reduction of its bacterial contamination. To note, the obtained data are in line with the results of our previous studies, and their comparison to a large extent contributes to elucidation of the mechanisms of the identified effects. It is known that divalent copper cations are necessary for production of hydroxyl radicals, which are an important component in oxygen-dependent bactericidal mechanisms of phagocytic cells. Complete wound cleansing from necrotized elements and microbes is the main condition for the effectiveness of reparative processes. However, increased production of active oxygen species, which are important factors of secondary alteration in the inflammation focus, may be accompanied by a high activation of lipid peroxidation and an increase in the levels of malondialdehyde and acyl hydroperoxides. Upon that, the dynamics of change in the activity of these processes and their ratio may be quite complex [11].

GHK interacts with copper ions to form GHK-Cu complex that activates proliferative processes in skin wounds and positive chemotaxis of macrophages to the damaged area [14–16]. Previously, it was shown by us on a similar experimental model of a skin wound with the same observation periods that GHK and GHK-D-Ala reduced the number of granulocytes and lymphocytes and increased the number of fibroblast cells, macrophages and cellular index with higher activity of GHK-D-Ala [9]. The dynamics of these morphometric parameters obtained in histological examination of the wound bioptate, could underlie the changes in the planimetric parameters established in the present work. An early and more prominent, as compared to the control group, reduction of bacterial contamination of the wound could be due to earlier activation of free radical oxidation processes in phagocytic cells [10]. A rapid decrease in bacterial contamination could be a probable cause of an earlier noted decrease in some parameters of phagocytic activity at later observation periods [10] in result of a complete wound cleansing, which also contributed to a positive dynamic of the integrative planimetric parameters of wound healing.

Upon that, this effect can also be due to the antibacterial action of GHK-Cu²⁺. In particular, such an effect has been shown with local application of these complexes in the form of nanoparticles [17]. In this case, the C-terminus remains free in the structure of the GHK-Cu²⁺ molecule [9, 16] enabling attachment of D-Ala.

The increase in the effectiveness of GHK after its structural modification with D-Ala can not only be due to increase in the resistance to proteases, but also to the effect of amino acids produced in degradation of the peptide molecule. In particular, D-Ala maintains the potential of the mitochondrial membrane and prevents the formation of reactive oxygen species that damage cellular structures and nucleic acids [18–20].

CONCLUSION

Based on the data obtained, it can be concluded that the use of GHK and its structural analogues D-Ala-GHK and GHK-D-Ala accelerate healing of infected skin wound with the underlying reduction of bacterial contamination. Upon that, the most prominent changes of these parameters were observed after the introduction of GHK-D-Ala peptide, which points to the importance of protecting the GHK molecule from the action of carboxypeptidases.

A promising continuation of research in this field may be the development of a local agent for stimulating regeneration processes in the wound with an antibacterial effect based on the complex of GHK-D-Ala peptide and Cu²⁺ used in the form of nanoparticles.

ADDITIОNALLY

Funding. This study was not supported by any external sources of funding.

Conflict of interests. The authors declare no conflicts of interests.

Contribution of the authors: K. K. Rakhmetova — processing of material, writing the text; I. I. Bobyntsev — concept and design of study, general guidance, editing; L. V. Zhilyayeva — concept of study, editing; A. I. Bezhin — concept and design of study, general guidance, editing; A. O. Vorvul’ — processing of material, writing the text. The authors confirm the correspondence of their authorship to the ICMJE International Criteria. All authors made a substantial contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, final approval of the version to be published and agree to be accountable for all aspects of the work.

About the authors

Kamila K. Rakhmetova

Kursk State Medical University

Email: muminovak@mail.ru
ORCID iD: 0000-0001-5511-5962
SPIN-code: 5852-8840
Russian Federation, Kursk

Igor' I. Bobyntsev

Kursk State Medical University

Email: bobig@mail.ru
ORCID iD: 0000-0001-7745-2599
SPIN-code: 3947-0114

MD, Dr. Sci. (Med.), Professor

Russian Federation, Kursk

Lyudmila V. Zhilyayeva

Kursk State Medical University

Email: zhiljaevalv@kursksmu.net
ORCID iD: 0000-0003-0390-4155
SPIN-code: 1726-8119

MD, Cand. Sci. (Med.)

Russian Federation, Kursk

Aleksandr I. Bezhin

Kursk State Medical University

Email: bezhinai@kursksmu.net
ORCID iD: 0000-0003-3776-9449
SPIN-code: 1250-5549

MD, Dr. Sci. (Med.), Professor

Russian Federation, Kursk

Anton O. Vorvul'

Kursk State Medical University

Author for correspondence.
Email: vorvul1996@mail.ru
ORCID iD: 0000-0002-1529-6014
SPIN-code: 8398-9376

MD, Cand. Sci. (Med.)

Russian Federation, Kursk

References

Supplementary files