Role of mTOR/IκB-α/NF-κB pathway activation in ischemia/reperfusion-induced inflammatory response in rats

S. Sahan-Firat¹, Z. Kocak¹, M. Temiz-Resitoglu¹, D. S. Guden¹, O. Vezir², N. Sucu³, S. Balcı⁴, L. Tamer-Gumus⁴, B. Tunctan¹, K. U. Malik⁵. ¹Department of Pharmacology, Mersin University, Faculty of Pharmacy, Mersin, Turkey, ²Department of Cardiovascular Surgery, Mersin State Hospital, Mersin, Turkey, ³Department of Cardiovascular Surgery, Mersin University, Faculty of Medicine, Mersin, Turkey, ⁴Department of Biochemistry, Mersin University Faculty of Medicine, Mersin, Turkey, ⁵Department of Pharmacology, University of Tennessee College of Medicine, Memphis, TN, United States.

Introduction: Ischemia-reperfusion (I/R)-induced injury initiates complex inflammatory cascade by triggering a systemic inflammatory response that results in both target and distant organ injuries (1,2). Recent data indicate mTOR inhibitors significantly influence immune cells after I/R injury (3), but the underlying mechanism in hindlimb I/R remains unclear. This study was designed to investigate whether systemic inflammation following I/R is mediated by mTOR/IκB-α/NF-κB pathway activation in a rat model of hindlimb I/R.

Method: Wistar male rats were randomly divided into vehicle (n=8), I/R (n=8), rapamycin (n=8), and, I/R+rapamycin (n=8) groups. Experiments were performed in according to the NIH Guide and approved by Ethics Committee of Mersin University School of Medicine. Hindlimb tourniquet model was used to induce I/R-injury (4). After 4 h of ischemia, tourniquets were removed and the hindlimb was reperfused for 4 h. Vehicle and I/R groups received saline (4 ml/kg; i.p.), whereas rapamycin and I/R+rapamycin groups received rapamycin (1 mg/kg; i.p. dissolved in a mixture of ethanol:saline) 1 h before reperfusion. The solvent of rapamycin did not apply in any of groups because of the amount of ethanol is negligible. Rats were then anesthetized with ketamine (90 mg/kg, i.m.) and xylazine (10 mg/kg, i.m.) and gastrocnemius muscle, kidney, and blood were collected for the measurement of rpS6, IκB-α, NF-κB p65, iNOS, and COX-2 expression and/or activity by immunoblotting (5), TNF-α by ELISA kit, nitrite levels by Griess method (6), and MPO activity as described (7). Data were expressed as means±SEM and analysis were performed by one-way ANOVA followed by Student-Newman-Keuls test, Kruskal-Wallis test followed by Dunn\'s test and Student’s t or Mann-Whitney U tests as appropriate.

Results: Rapamycin prevented I/R-induced increase in rpS6, IκB-α, NF-κB p65, iNOS, and COX-2 expression and/or activity, nitrite and TNF-α levels as well as MPO activity (Table 1).

Table 1 Effect of rapamycin on I/R-induced changes related to inflammation

*( P < 0.05) vs vehicle group, ^# (P < 0.05) vs I/R group analysed by ANOVA followed by Student-Newman-Keuls test for multiple comparisons, Kruskal-Wallis test followed by Dunns test for multiple comparisons and Student’s t or Mann-Whitney U tests as appropriate.

Conclusion: Rapamycin protects against I/R-induced systemic inflammation with the target and distant organ injuries via inhibition of mTOR/IκB-α/NF-κB p65 pathway.

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