Медицина/6. Экпериментальная и клиническая фармакология

Candidate of Pharm. Sc. Butko Y. O., postgraduate student Tsulun O.V.

National University of Pharmacy, Ukraine

Effect of wound-healing drugs on ТNF-a and IL-1 levels in a rat burn-injury model

Burn injury is the most common traumatic injury in the world. Processes resulting from the burn injury involve nonspecific inflammatory response that transit to structural and metabolic disorders in tissues and systems [3, 4]. Cytokines play the key role in inflammation, in particular such pro-inflammatory interleukins as IL-1 and ТNF-a, which are produced in response to tissue damage and stimulate development of local inflammatory reaction leading to burn wound healing [2].

The aim of this study was to investigate the influence of wound-healing drugs containing ceramides on the dynamics of pro-inflammatory cytokines in blood of experimental animals with third-degree burn injury.

Materials and methods. A total of 60 male albino rats weighting 200-240 g were used in the experimental burn injury model. Burn wounds were inflicted in animals under anesthesia using electric soldering iron with 2.5-cm-diameter round metallic plate from one end. Time of exposure to 200⁰С heated plate was 10 s [1]. This method allowed creating third-degree burn wounds according to clinical classification of burns. Animals were divided into 5 groups: І – intact control (IC) (n = 6), ІІ – control pathology (untreated animals with burns) (n = 18), ІІІ – group treated with cream “Dexpantenol with ceramides” (n = 12), ІV – cream “Ceramides” (n = 12), V – cream “Bepanthen” (“bayer”, Germany) (n = 12). Immunoenzymatic assay was used for quantitative determination of cytokines IL-1 and TNF-a with the help of analyser “Libline-90” (Austria). All the interventions and euthanasia of animals were carried out according to principles of bioethics. Received results were processed by Statistica 6.0 program based on one way analysis of variance (ANOVA) using Mann-Whitney U test (р < 0.05).

Results and discussion. After reproducing third-degree burn injury in rats a dense dark brown scab was formed with a clear zone of necrosis and inflammatory changes in the surrounding tissues. On the 2^nd day of experiment the development of pathology in rats was followed by significant increase in levels of cytokines compared to IC: IL-1 increased by 32 times and TNF-a – by 15 times (Tabl. 1), indicating activation of immune response, hyperactivation of phagocytic cells, involvement of epithelial and endothelial cells associated with tissue destruction.

Table 1

Dynamics of IL-1 and TNF-a levels in blood of experimental animals with burn injury under treatment with study drugs.

Experimental groups	IL-1, ng/mL (days of treatment)		TNF-a, pg/mL (days of treatment)
	Day 9	Day 18	Day 9	Day 18
Intact control	2.22±0.27		16.23±1.56
Peak of pathology (Day 2)	71.23±1.63^*		236.51±11.76^*
Control pathology	46.88±1.80 ^*/@	13.25±1.01 ^*/@	147.14±6.69 ^*/@	28.17±1.56 ^*/@
Cream with ceramides	19.33±1.38 ^*/@/#/$	4.93±0.44 ^*/@/#/$	98.40±2.62 ^*/@/#	17.34±1.16 ^@/#
Cream with dexpantenol with ceramides	13.88±1.14 ^*/@/#/$	2.09±0.08 ^@/#/$	71.00±2.54 ^*/@/#/$	10.51±0.41 ^*/@/#/$
Cream “Bepanthen”	30.11±1.67 ^*/@/#	12.83±1.09 ^*/@	108.25±5.00 ^*/@/#	19.18±1.04 ^@/#

Notes. * – significant difference in comparison to intact control, р<0.05; ^@ – in comparison to peak of pathology, р<0.05; ^# – in comparison to control pathology, р<0.05; ^$ – in comparison to reference-drug, р<0.05.

The study of pro-inflammatory cytokines in dynamics revealed that on the 9^th day of experiment in control pathology group there was a decrease in IL-1 and TNF-a levels by 1.6 times, and on the 18^th day the level of IL-1 decreased by 5.4 times and TNF-a – by 8.4 times compared to the peak of pathology (PP) on the 2^nd day of experiment. Regardless of considerable decrease in serum levels of cytokines their rates were significantly higher compared to those of intact animals.

Immuno-biochemical analyses have shown that under treatment with creams “Ceramides” and “Dexpantenol with ceramides” the level of IL-1 decreased on the 9^th day by 3.7 and 5 times, on the 18^th day – by 14.5 and 34 times, while under treatment with cream “Bepanthen” on the 9^th day – by 2.4 times and on the 18^th day – by 5.6 times, compared to PP. The level of TNF-a decreased under influence of creams “Ceramides” and “Dexpantenol with ceramides” on the 9^th day – by 2.4 and 3.3 times, on the 18^th day – by 13.6 and 22.5 times, while under influence of cream “Bepanthen”: on the 9^th day – by 2.2 times and on the 18^th day - by 12.3 times, compared to the PP. Obtained data demonstrated that application of the study drugs throughout the whole surveillance period contributed to significant decrease in investigated pro-inflammatory cytokines levels compared to PP and reference-drug, confirming expressed efficacy of local burn wounds therapy.

Conclusion. Burn injury in rats is accompanied by a significant increase in pro-inflammatory cytokines. Significant decrease in IL-1 and TNF-a levels under treatment with creams containing ceramides confirms the important role of pro-inflammatory cytokines in burn wound healing and efficacy of local treatment. Wound-healing activity of preparations with ceramides exceeds the efficacy of reference-drug.

References

1. Експериментальне вивчення нових препаратів для місцевого лікування ран : Метод. рек. / Л. В. Яковлєва, О. В. Ткачова, Я. О. Бутко, Ю. Б. Лар’яновська – Харків: Вид-но НФаУ, 2013.

2. Коненков В.И., Макарова О.П., Багатова Н.П., Ракова И.Г. Динамика изменения активности цитокинов и функций нейтрофилов в крови крыс после термического ожога кожи // Цитокины и воспаление. – 2007. – № 3.

3. Velnar T., Bailey T., Smrkolj V. The wound healing process: an overview of the cellular and molecular mechanisms // J.Int.Med.Res. – 2009. – Vol. 37, № 5.

4. Dermal substitution with Matriderm in burns / H. Ryssel, G. Germann, O. Kloeters, E. Gazyakan et al. // Burns. – 2010. – Vol. 25, № 4.