The Effect of Ischemic Preconditioning on the Expression of Serum Cytokines after 2 Hours Ischemia and Timely Reperfusion in the Hindlimb of Mice

Jeong han Jin; Sung Jae Kim; Younkyoung Seo; Shinyoung Kim

doi:10.11637/kjpa.2016.29.2.53

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Jin, Kim, Seo, and Kim: The Effect of Ischemic Preconditioning on the Expression of Serum Cytokines after 2 Hours Ischemia and Timely Reperfusion in the Hindlimb of Mice

Original Article

Korean J Phys Anthropol 2016;29(2):53-60.

Published online: 20 January 2016

DOI: https://doi.org/10.11637/kjpa.2016.29.2.53

The Effect of Ischemic Preconditioning on the Expression of Serum Cytokines after 2 Hours Ischemia and Timely Reperfusion in the Hindlimb of Mice

Jeong han Jin¹, Sung Jae Kim¹, Younkyoung Seo², Shinyoung Kim^3,

¹Department of Anatomy and Cell Biology, College of Medicine, Hanyang University

²College of Nursing, Hanyang University

³Department of Physical Therapy, Ansan University

Correspondence to: Shinyoung Kim (Department of Physical Therapy, Ansan University) E-mail: sykim@ansan.ac.kr

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The large volume of reactive oxygen species are generated during reperfusion after transient or post-procedural ischemia, which leads to cell injury. This ischemia-reperfusion (IR) injury may cause local and even systemic injuries. Thus, the need to reduce the IR injury has been highlighted and in this regard studies have demonstrated the ischemic preconditioning (IP) in which short ischemia and reperfusion are repeated before ischemia. Such IP is known to protect the tissues from IR injury by reducing inflammation response during ischemia.

Thus, this study was based on IP known to protect the tissue with developing the mechanism of resistance to ischemia and reperfusion injury in cellular tissue. As the substance that plays an important role in the inflammatory response during IR injury is cytokines, this study was intended to review and discuss the methodologies of IP as well as to analyze the correlation of its effects on the expression of cytokines.

Left common iliac artery in male mice of which weight was from 40 g to 45 g, was treated for ischemia. The animal groups consisted of ischemia (IC) group receiving 2-hour ischemic treatment alone; IP group receiving short 5-minute ischemia and reperfusion treatments repeated three times; and, ischemic preconditioning-ischemia (IP-IC) group receiving IP treatment followed by 2-hour ischemic treatment. Following these treatments in each group, reperfusion for intergroup comparisons was carried out at 30 minutes, and 1, 2 and 4 hours. The results of this study were as follows:

First, the expression level of pro-inflammatory cytokines, IL-1β was the highest in IC group receiving 2-hour ischemic treatment alone (p<.001). Second, the expression level of anti-inflammatory cytokine, IL-4 was the highest in the IP group (p<.001). Third, the expression level of anti-inflammatory cytokine, IL-10 was the highest in the IP group (p<.001).

In conclusion, even though the results had the degree of difference, the expression level of pro-inflammation cytokine, IL-1β in IC group was significantly lower than that in IP group, and the expression levels of antiinflammatory cytokines, IL-4 and IL-10 in IP were significantly higher than those in IC group and IP-IC group.

Keywords: Mouse, Ischemic preconditioning, Ischemia-reperfusion injury, IL-1β, IL-4, IL-10

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Fig. 1.

The difference in the expression of IL-1β by reperfusion time. Data are presented as mean (pg/mL). IL-1β level decreased more significantly in IP group and IP-IC group than in ischemia group. At all the time points of reperfusion, IL-1β levels were lower in IP group than in IC group, and were the lowest in IP-IC group. Values are mean. ∗p<.01 versus control, ∗∗p<.001 versus control. ^#p<.001 versus IP-IC group. SC: Sham control; IC: Ischemia; IP: Ischemia preconditioning; IP-IC: Ischemia preconditioning and ischemia; 0.5H: Half an hour reperfusion after ischemia or ischemia preconditioning; 1H: 1 hour reperfusion after ischemia or ischemia preconditioning; 2H: 2 hours reperfusion after ischemia or ischemia preconditioning; 4H: 4 hours reperfusion after ischemia or ischemia preconditioning.

Fig. 2.

The difference in the expression of IL-4 by reperfusion time. Data are presented as mean (pg/mL). At 0.5H, IL-4 was highest in IP-IC group, At 1H and 4H, IL-4 were highest in IP group. At 2H, IL-4 in IC group was higher than in IP group. ∗p<.001 versus control, ^#p<.001 versus IP-IC group. SC: Sham control; IC: Ischemia; IP: Ischemia preconditioning; IP-IC: Ischemia preconditioning and ischemia; 0.5H: Half an hour reperfusion after ischemia or ischemia preconditioning; 1H: 1 hour reperfusion after ischemia or ischemia preconditioning; 2H: 2 hours reperfusion after ischemia or ischemia preconditioning; 4H: 4 hours reperfusion after ischemia or ischemia preconditioning

Fig. 3.

The difference in the expression of IL-10 by reperfusion time. Data are presented as mean (pg/mL). IL-10 showed the pattern of repeated increase and decrease in all the groups. At 0.5H and 1H, IL-10 was the highest in IP group. At 2H, IL-10 was the highest in IC group. At 4H, IL-10 was the highest in IP group. ∗p<.05 versus control at baseline,∗∗p<.01 versus control at baseline, ∗∗∗p<.001 versus control, ^#p<.05 versus IP-IC group, ^##p<.01 versus IP-IC group, ^†p<.05 versus IC group. SC: Sham control; IC: Ischemia; IP: Ischemia preconditioning; IP-IC: Ischemia preconditioning and ischemia; 0.5H: Half an hour reperfusion after ischemia or ischemia preconditioning; 1H: 1 hour reperfusion after ischemia or ischemia preconditioning; 2H: 2 hours reperfusion after ischemia or ischemia preconditioning; 4H: 4 hours reperfusion after ischemia or ischemia preconditioning.

Table 1.

The expression of cytokines IL-1β in reperfusion time point by the experimental groups. ∗p<.01 versus control at baseline, ∗∗p<.001 versus control at baseline.

			N	M	SD	F (p)
IL-1β	SC	–	8	167.00	25.026
	IC	0.5H	8	241.63	21.186	0.005 (∗)
		1H	8	278.38	12.070	0.000 (∗∗)
		2H	8	326.13	51.324	0.000 (∗∗)
		4H	7	273.14	35.793	0.001 (∗∗)
	IP	0.5H	8	207.25	30.626	0.005 (∗)
		1H	8	242.88	39.668	0.000 (∗∗)
		2H	6	221.83	73.725	0.000 (∗∗)
		4H	8	223.25	27.354	0.001 (∗∗)
	IP-IC	0.5H	8	192.88	27.300	0.005 (∗)
		1H	6	207.33	34.110	0.000 (∗∗)
		2H	8	171.88	39.223	0.000 (∗∗)
		4H	8	192.38	36.820	0.001 (∗∗)

SC: Sham control

IC: Ischemia

IP: Ischemia preconditioning IP-IC: Ischemia preconditioning and ischemia

0.5H: Half an hour reperfusion after ischemia or ischemia preconditioning

1H: 1 hour reperfusion after ischemia or ischemia preconditioning

2H: 2 hours reperfusion after ischemia or ischemia preconditioning

4H: 4 hours reperfusion after ischemia or ischemia preconditioning

Table 2.

The difference of the expression of IL-1β, IL-4, and IL-10 in reperfusion time point by the experimental groups. ∗p<05, ∗∗p<.01, ∗∗∗p<.001

		0.5H			1H			2H			4H
		M	SD	t (p)	M	SD	t (p)	M	SD	t (p)	M	SD	t (p)
IL-1β	IC IP-IC	241.63 192.88	21.186 27.300	3.990∗∗∗ (.001)	199.88 278.38	33.160 12.070	(.000) 6.292∗∗∗	171.88 326.13	39.223 51.324	(.000) 6.754∗∗∗	192.38 273.14	36.820 35.793	4.293∗∗∗ (.001)
IL-4	IC IP-IC	186.88 205.38	7.846 30.482	– 1.662 (.119)	203.25 201.88	17.442 19.335	.149 (.883)	300.75 220.63	38.321 32.147	4.531∗∗∗ (.000)	176.50 264.88	29.665 26.734	6.259∗∗∗ (.000)
IL-10	IC IP-IC	101.00 94.50	13.342 18.716	– 1.662 (.135)	96.13 111.33	7.530 16.848	– 2.289∗ (.041)	116.38 94.50	19.412 6.188	3.037∗∗ (.009)	79.13 96.75	14.397 9.618	2.879∗ (.012)

IC: Ischemia

IP-IC: Ischemia preconditioning and ischemia

0.5H: Half an hour reperfusion after ischemia or ischemia preconditioning

1H: 1 hour reperfusion after ischemia or ischemia preconditioning

2H: 2 hours reperfusion after ischemia or ischemia preconditioning

4H: 4 hours reperfusion after ischemia or ischemia preconditioning

Table 3.

The expression of cytokines IL-4 in reperfusion time point by the experimental groups. ∗p<.001 versus control at baseline.

			N	M	SD	F (p)
IL-4	SC	–	8	187.25	8.812
	IC	0.5H	8	186.88	7.846	0.130 ()
		1H	8	203.25	17.442	0.000 (∗)
		2H	8	300.75	38.321	0.000 (∗)
		4H	7	269.00	25.981	0.000 (∗)
	IP	0.5H	8	187.88	12.811	0.130 ()
		1H	8	278.88	42.650	0.000 (∗)
		2H	6	240.83	33.054	0.000 (∗)
		4H	8	278.25	39.176	0.000 (∗)
	IP-IC	0.5H	8	205.38	30.482	0.130 ()
		1H	6	202.33	22.853	0.000 (∗)
		2H	8	220.63	32.147	0.000 (∗)
		4H	8	176.50	29.665	0.000 (∗)

SC: Sham control

IC: Ischemia

IP: Ischemia preconditioning

IP-IC: Ischemia preconditioning and ischemia

0.5H: Half an hour reperfusion after ischemia or ischemia preconditioning

1H: 1 hour reperfusion after ischemia or ischemia preconditioning

2H: 2 hours reperfusion after ischemia or ischemia preconditioning

4H: 4 hours reperfusion after ischemia or ischemia preconditioning

Table 4.

The expression of cytokines IL-10 in reperfusion time point by the experimental groups. IL-10 showed the highest expression level in IP group. In IC group, IL-10 was the highest at 2H. In IP group, IL-10 was the highest at 1H. In IP-IC group, IL-10 was the highest at 1H. ∗p<.05 versus control at baseline, ∗∗p<.01 versus control at baseline, ∗∗∗p<.001 versus control at baseline.

			N	M	SD	F (p)
IL-10	SC	–	8	85.75	2.053
	IC	0.5H	8	101.00	13.342	0.005 (∗∗)
		1H	8	96.13	7.530	0.005 (∗∗)
		2H	8	116.38	19.412	0.047 (∗)
		4H	7	97.57	10.081	0.000 (∗∗∗)
	IP	0.5H	8	123.63	16.396	0.005 (∗∗)
		1H	8	135.25	30.089	0.005 (∗∗)
		2H	6	113.50	24.023	0.047 (∗)
		4H	8	121.63	18.213	0.000 (∗∗∗)
	IP-IC	0.5H	8	94.50	18.716	0.005 (∗∗)
		1H	6	111.33	16.848	0.005 (∗∗)
		2H	8	94.50	6.188	0.047 (∗)
		4H	8	79.13	14.397	0.000 (∗∗∗)

SC: Sham control

IC: Ischemia

IP: Ischemia preconditioning

IP-IC: Ischemia preconditioning and ischemia

0.5H: Half an hour reperfusion after ischemia or ischemia preconditioning

1H: 1 hour reperfusion after ischemia or ischemia preconditioning

2H: 2 hours reperfusion after ischemia or ischemia preconditioning

4H: 4 hours reperfusion after ischemia or ischemia preconditioning

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