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DYNAMICS OF INFLAMMATORY MEDIATORS IN CONDITIONS OF SUBSTANCE P BLOCK
Shevchenko O., Sych V.
Kharkiv National Medical University, Ukraine
Abstract
With the aim to determine the features of the concentration of tumor necrosis factor-a, interleukin-6 in the blood in carrageenan secondary chronic inflammation against the background of blockade of substance P an experimental prospective controlled randomized study was performed on 132 WAG rats in series of 6 rats each in the natural course and using a pharmacological aprepitant praparation - inhibitor of NK-1R in the control and simulated inflammation series for 6 hours, as well as 1, 2, 3, 5, 7, 10, 14, 21, 28 days. the peculiarities of the concentration of tumor necrosis factor-a, interleukin-6 in the blood in carrageenan secondary chronic inflammation on the background of blockade of substance P have been developed.
Keywords: interleukin-6, secondary chronic inflammation, substance P, tumor necrosis factor-a.
Background.
Inflammation is the fundamental pathological process underlying the majority of the diseases. The mechanisms of its development, progression, chronization are mediated by a plenty of humoral agents, i. e. "inflammatory mediators". Each of them while having multiple biological effect, very formally may be associated with predominantly stimulatory or, vice versa, depressor influence on inflammation (pro- or anti-inflammatory, accordingly). Tumor necrosis factor alpha, interlukin 6 are mostly associated with proinflammatory effect and serve as a reference mediators. In an inflammation, immune processes, the substance P, its neurokinin-1 receptor (NK-1R) play an integral role, apart of being involved in the regulation of a plenty of other pathophysiological processes - nociception, motility of gastrointestinal tract, cancerogenesis, heart failure etc. [1-4].
Thus, the substance P is involved in an initiation of T-helpers type 2 migration [5]; tachykininergic system is integrated with enkephalinergic [6]; substance P increases the expression and synthesis of such proin-flammatory mediators produced by macrophages as TNFa, endothelial vascular growth factor etc. The technological level of the laboratory procedures concerning substance P detection continuously undergo different improvements allowing more and more advanced studies [7-10].
While, there is a lack of evidence of a dynamics of inflammatory mediators, especially tumor necrosis factor alpha and interleukin 6, in conditions of substance P block.
Aim: to determine the features of the concentration of tumor necrosis factor-a, interleukin-6 in the blood in carrageenan secondary chronic inflammation against the background of blockade of substance P.
Material and methods.
An experimental prospective controlled randomized study was performed on 132 WAG rats in series of 6 rats each in the natural course and using a pharmacological aprepitant praparation - inhibitor of NK-1R in the control and simulated inflammation series for 6 hours, as well as 1, 2, 3, 5, 7, 10, 14, 21, 28 days.
The content of tumor necrosis factor alpha (TNF-a), interlukin 6 (IL-6) in the natural course of inflammation was estimated.
The rats were kept in the vivarium for 10-12 individuals in a cage under standard conditions on a normal diet with free access to water. To exclude the influence of natural circadian rhythms on the indicators, the experiment was performed in the autumn-winter period in a standardized way in the morning.
A carrageenan model of inflammation was selected, using 10 mg of a-carrageenan (Sigma, USA) in
1 ml of saline [11], which was injected intramuscularly into the rat thigh under thiopental anesthesia.
To inhibit the synthesis and effects of substance P NK-1R inhibitor aprepitant was used, which was administered intraperitoneally at a dose of 10 mg dissolved in 1 ml of isotonic sodium chloride solution, daily throughout the experiment [12].
Logical and statistical methods of analysis are applied taking into account the nature of the distribution.
All manipulations on animals were performed in accordance with national and international requirements. The study was approved by the Ethics and Bio-ethics Commission of Kharkiv National Medical University.
Results and discussion.
The content of TNF-a, IL-6 in the natural course of inflammation was estimated (Table 1).
Table 1
Dynamics of concentration of tumor necrosis factor a,
Series of animals TNF-a, pg/ml IL-6, pg/ml
Intact control 4.03 [3.96; 4.85] 7.93 [7.42; 8.11]
6 hours 5.27 [5.25; 5.31] 8.57 [8.49; 8.65]
1 day 6.11 [6.07; 6.22] 9.18 [9.12; 9.21]
2 days 8.65 [8.56; 8.79] 9.00 [8.93; 9.05]
3 days 9.91 [9.83; 10.02] 10.69 [10.53; 11.00]
5 days 8.08 [7.25; 8.24] 8.91 [8.47; 8.99]
7 days 7.00 [6.53; 7.05] 7.97 [7.89; 8.12]
10 days 4.15 [3.98; 4.21] 7.33 [7.25; 7.36]*
14 days 4.05 [3.96; 4.12] 7.97 [7.89; 8.11]
21 days 3.64 [3.57; 3.75] 7.72 [7.54; 7.88]
28 days 3.93 [3.88; 4.05] 8.05 [7.93; 8.11]
Note. * — the difference with control is significant at p<0.05; t — the difference with previous series is significant at p<0.05; i — the difference with the corresponding series for the blockade of substance P is significant at p<0.05.
In the natural course of inflammation, the general trend is a burst-like increase in the concentration of each of the studied humoral indicators of systemic inflammation for 1-3 days and a gradual further decrease (Figure. 1).
^/ml
12
10
8
6
4
2
0
Control 6 hours 1 day 2 days 3 days 5 days 7 days 10 days 14 days 21 days 28 days
---TNF-a -IL-6
Fig. 1. Dynamics of concentration of tumor necrosis factor a, interleukin 6 in the blood during the natural
course of inflammation
Regarding the control in the natural course of inflammation, statistically significant differences were found in the concentration of IL-6 - in 10 days (p=0.04).
Trends comparing with controls were identified
for:
- TNF-a concentration - in 6 hours (p=0.08), in 1 day (p=0.08), in 2 days (p=0.08), in 3 days (p=0.08), in 5 days (p=0.05), in 7 days (p=0.08), in 10 days (p=0.07), in 14 days (p=0.05);
- concentration of IL-6 - in 6 hours (p=0.08), in 1 day (p=0.08), in 3 days (p=0.08), in 5 days (p=0.05), in 7 days (p=0.08), in 14 days (p=0.05).
Comparing with the previous duration of modeling of inflammation in the natural course of inflammation, no statistically significant differences were found for any of the studied indicators (TNF-a, IL-6).
Table 2
Dynamics of the concentrations of tumor necrosis factor a, interleukin 6 _in the blood during blockade of substance P_
Series TNF-a, IL-6,
of animals ^/ml ^/ml
Control with preparation injection 4.11 [4.00; 4.29] 8.05 [7.88; 8.12]
6 hours 3.88 [3.79; 4.90] 6.81 [6.75; 7.00]
1 day 5.00 [4.97; 5.03] 8.00 [7.92; 8.07]
2 days 4.16 [4.00; 4.33] 7.83 [7.72; 9.00]
3 days 5.32 [5.29; 5.51] 7.05 [6.94; 7.12]
5 days 4.07 [4.05; 4.11] 7.34 [7.29; 7.42]
7 days 4.51 [4.49; 4.60] 5.59 [4.55; 5.65]
10 days 3.44 [3.42; 3.49]t 6.45 [6.39; 6.51]tt
14 days 3.01 [2.94; 3.07]t 6.98 [6.91; 7.01]
21 days 3.69 [3.51; 4.00] 7.63 [7.44; 7.93]
28 days 3.98 [3.95; 4.04] 7.96 [7.87; 8.04]
Trends are defined comparing the previous series of duration of inflammation in relation to:
- TNF-a concentration - between 6 hours and 1 day (p=0.08), between 1 and 2 days (p=0.08), between 2 and 3 days (p=0.08), between 3 and 5 days (p=0.05), between 7 and 10 days (p=0.08), between 14 and 21 days (p=0.08), between 21 and 28 days (p=0.05),
- concentration of IL-6 - between 6 hours and 1 day (p=0.08), between 1 and 2 days (p=0.08), between 2 and 3 days (p=0.08), between 3 and 5 days (p=0.05), between 7 and 10 days (p=0.08), between 10 and 14 days (p=0.08), between 14 and 21 days (p=0.08).
The blood concentrations of TNF-a, IL-6 in case of blockade of substance P was studied (Table 2).
Note. * - the difference comparing with control is significant at p<0.05; t - the difference comparing with the previous series is significant at p<0.05; i - the difference comparing with the corresponding series in the natural course of inflammation is significant at p<0.05.
Comparing with the control of in case of blockade of substance P, statistically significant differences were found in the concentration of IL-6 - in 10 days (p=0.04).
Trends in differences comparing with control are identified for:
- concentration of TNF-a - in 1 day (p=0.08), in 3 days (p=0.08), in 7 days (p=0.08), in 10 days (p=0.05), in 14 days (p=0.05);
- concentrations of IL-6 -in 6 hours (p=0.08), in 3 days (p=0.08), in 5 days (p=0.08), in 7 days (p=0.08), in 14 days (p=0.05).
Comparing with the previous duration of inflammation modeling in the natural course of inflammation, statistically significant differences were found in the concentration of TNF-a - between 7 and 10 days (p=0.04), between 10 and 14 days (p=0.02); concentrations of IL-6 - between 7 and 10 days (p=0.04).
Trends comparing with the previous series of duration of inflammation are defined in relation to:
- concentration of TNF-a - between 6 hours and 1 day (p=0.08), between 1 and 2 days (p=0.08), between
2 and 3 days (p=0.08), between 3 and 5 days (p=0.08), between 5 and 7 days (p=0.08), between 14 and 21 days (p=0.05),
- concentration of IL-6 - between 6 hours and 1 day (p=0.08), between 2 and 3 days (p=0.08), between
3 and 5 days (p=0.08), between 5 and 7 days (p=0.08), between 14 and 21 days (p=0.05).
The studied parameters (TNF-a, IL-6) in series with blocking of substance P in comparison with the natural course of inflammation were analyzed.
The curve of the dynamics of TNF-a concentration in series when substance P was blocked is more flattened in comparison with that during the natural course of inflammation (Fig. 2).
^/ml 12
10
8
6
4
2
0
Control 6 hours 1 day 2 days 3 days 5 days 7 days 10 days 14 days 21 days 28 days
Natural course --- Substance P block
Fig. 2. Dynamics of the concentration of tumor necrosis factor a in the blood during the natural course of inflammation and in the blockade of substance P
The tendency to a lower content of TNF-a during blockade of substance P was established in 6 hours (p=0.08), in 1 day (p=0.08), in 2 days (p=0.08), in 3 days (p=0.08), in 5 days (p=0.05), in 7 days (p=0.08), in 10 days (p=0.07), in 14 days (p=0.05).
Similarly, the curve of the dynamics of the concentration of IL-6 in the blood in case of the substance P block is located in the range of lower values compared to the natural course (Fig. 3).
^/ml 12
10
8 6 4 2 0
Control 6 hours 1 day 2 days 3 days 5 days 7 days 10 days 14 days 21 days 28 days
Natural course --- Substance P block
Fig. 3. Dynamics of concentration of interleukin 6 in blood at a natural course of an inflammation and at blockade of substance P
The tendency to a lower concentration of IL-6 during blockade of substance P was established in 6 hours (p=0.08), in 1 day (p=0.08), in 3 days (p=0.08), in 5 days (p=0.05), in 7 days (p=0.08), in 10 days (p=0.04), in 14 days (p=0.05).
Thus, the peculiarities of the concentration of tumor necrosis factor-a, interleukin-6 in the blood in car-
rageenan secondary chronic inflammation on the background of blockade of substance P have been developed.
Conclusions.
1. The blood concentrations of TNF-a, IL-6 in case of blockade of substance P vary, and statistically significant differences are found in the concentration of IL-6 - in 10 days (p=0.04).
2. There are the trends in differences comparing with control are identified for concentration of TNF-a - in 1 day (p=0.08), in 3 days (p=0.08), in 7 days (p=0.08), in 10 days (p=0.05), in 14 days (p=0.05); concentrations of IL-6 -in 6 hours (p=0.08), in 3 days (p=0.08), in 5 days (p=0.08), in 7 days (p=0.08), in 14 days (p=0.05). This finding requires more thorough research with higher statistical power.
Prospects of further research concern the development of a criterional complex of blood system reactions system forecasting in the conditions of inflammation and effects of substance P blocking.
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