Peroxynitrite, a product between nitric oxide and superoxide anion, plays a cytotoxic role in the development of post-bypass systemic inflammatory response

Abstract

Objective: Cardiopulmonary bypass (CPB) is known to induce post-bypass systemic inflammatory response. Peroxynitrite (ONOO⁻) is a potent oxidant formed by a rapid reaction between nitric oxide (NO) and superoxide anion. We hypothesized that ONOO⁻ plays a role in the development of post-bypass systemic inflammatory response and examined the efficacy of ONOO⁻ scavenger in a rat-CPB model. Methods: Adult Sprague–Dawley rats underwent 60 min of CPB (100 ml/kg per min, 34 °C). Group-P (n=10) received 50 mg/kg of ONOO⁻ scavenger, quercetin, intraperitoneally 24 h before the initiation of CPB, and Group-C (n=10) served as controls. Results: There were significant time-dependent changes in plasma nitrate+nitrite (NO_x), the percentage ratio of nitrotyrosine to tyrosine (%NO₂-Tyr: an indicator of ONOO⁻ formation), interleukin (IL)-6, IL-8, and respiratory index (RI). There were significant differences in %NO₂-Tyr between the groups both at CPB termination (Group-P vs C; 0.26±0.07 vs 0.55±0.11%, P<0.01) and 3 h after CPB termination (0.65±0.14 vs 1.46±0.25%, P<0.01); whereas there were no significant differences in NO_x between the groups at any sampling point ((at CPB termination) Group-P vs C; 31.6±4.3 vs 32.7±4.1 μmol/l, (3 h after CPB termination) Group-P vs C; 47.8±4.9 vs 51.7±5.3 μmol/l). Group-P showed significantly lower plasma IL-6 (176.8±44.3 vs 302.4±78.1 pg/ml, P<0.01), IL-8 (9.45±1.78 vs 16.42±2.53 ng/ml, P<0.01) and RI (1.07±0.19 vs 1.54±0.25, P<0.01) 3 h after CPB termination, though there were no significant differences between the groups at CPB termination. Conclusions: These results suggest that ONOO⁻ plays a crucial role in the development of post-bypass systemic inflammatory response and the pretreatment with quercetin has a potential benefit to avoid deleterious effects of ONOO⁻

Cardiopulmonary bypass, Nitric oxide, Peroxynitrite, Inflammatory response, Cytokines

1 Introduction

Peroxynitrite (ONOO⁻) is a potent oxidant formed by a rapid reaction between nitric oxide (NO) and superoxide anion, and plays various pathophysiological roles in the development of inflammation [1–4]. Tyrosine nitration is one of ONOO⁻-mediated cytotoxic effects [5], and nitrotyrosine formation is widely considered as an indicator of ONOO⁻ production in clinical situations [6,7]. We have clinically demonstrated that ONOO⁻ is produced from human myocardium after ischemia-reperfusion by the measurement of plasma nitrotyrosine [8,9].

On the other hand, cardiopulmonary bypass (CPB) is thought to induce a burst of chemotactic mediators through blood contact with artificial surfaces [10,11], which results in post-bypass systemic inflammatory response [10–12]. CPB has been reported to enhance the production of NO and superoxide anion [13,14]. ONOO⁻-mediated oxidization is considered 1000-fold greater than hydrogen peroxide-mediated one [15], and thus, subsequent ONOO⁻ formation may enhance the development of inflammation. In the present study, we hypothesized that ONOO⁻ plays a crucial role in the development of post-bypass systemic inflammatory response and examined the efficacy of ONOO⁻ scavenger in a rat-CPB model.

2 Methods and materials

2.1 Animal care

Adult male Sprague–Dawley (SD) rats weighing 400–450 g were used in the present study. All animals received humane care in compliance with the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the US National Institute of Health (NIH Publication No. 86-23, revised 1996).

2.2 Group classification

Twenty rats were randomly divided into two groups, according to the administration of glutamine prior to the initiation of CPB. Group-P (n=10) intraperitoneally received 50 mg/kg body-weight of quercetin (an ONOO⁻ scavenger; 3,3′,4′,5,7-pentahydroxyflavone; Sigma Chemical Co., St Louis, MO, USA) [16] 24 h before the initiation of CPB. Group-C (n=10) served as control and only saline was added in the same way.

2.3 Experimental protocol (surgical procedure for rat-CPB)

Experimental rat-CPB was instituted with the use of a roller pump and a membrane oxygenator, according to the method we previously described [17,18]. Anesthesia was introduced by intraperitoneal administration of sodium pentobarbital (50 mg/kg body-weight), and respiration was maintained by mandatory lung ventilation. The bypass circuit was primed with the following solution without blood components; 12 ml of plasma expander, 8 ml of lactate Ringer's solution, 2 ml of 7% sodium bicarbonate, 2 ml of mannitol, 100 units of heparin, and 1.5 mg of Tobramycin. After systemic heparinization (300 units/kg body-weight of heparin sulfate), siphon-dependent venous drainage was established with the use of two 16-G catheters, and oxygenated blood was returned using a 20-G catheter.

All rats underwent 60 min of CPB. Perfusion flow rate was maintained at 100 ml/kg per min, and perfusate temperature was set at 34 °C. Neither additional crystalloid solution nor blood component was infused throughout the experiment, and the CPB-remaining solution was infused gradually after the termination of CPB.

2.4 Blood analysis

Arterial blood (1.5 ml) was sampled at the following three times; (1) before the initiation of CPB; (2) at the termination of CPB, and (3) 3 h after the termination of CPB. Plasma levels of nitrate+nitrite (NO_x; as a marker of NO production), nitrotyrosine (as an indicator of ONOO⁻ formation), pro-inflammatory cytokines (interleukin (IL)-6 and IL-8; as markers of the development of inflammation) were measured [8,9,17,18]. Respiratory index (RI=AaDO₂/PaO₂) was calculated by arterial blood gas assay as a marker of post-bypass lung damage.  

Plasma NO_x was analyzed by an automated procedure based on the Griess reaction. Nitrotyrosine was measured by a high-pressure liquid chromatography method with a C-18 reverse-phase column as an index of nitration reaction of NO. Nitrotyrosine formation was expressed as the percentage ratio of nitrotyrosine to tyrosine (%NO₂-Tyr).  

Plasma level of IL-6 was measured by enzyme-linked immunosorbent assay with a commercially available kit (Rat ELISA Kits; Biosource International, Camarillo, CA), and the IL-8 level was measured by enzyme immunoassay with a commercially available kit (Rat IL-8 Kit; Panafarm Laboratory, Tokyo, Japan).

2.5 Statistical analysis

All data are expressed as mean±SD. Time-dependent changes and comparisons between the groups were analyzed by two-way repeated-measures ANOVA and unpaired Student's t-test. All analysis was performed using the StatView v5.0 statistical package (Abacus Concepts Inc., Berkeley, CA). A P-value of less than 0.05 was considered statistically significant.

3 Results

There were no technical failures or operative deaths in the 20 consecutive rats used in the present study. There were no significant differences in the hemoglobin level at any sampling point between the groups, and the degree of CPB-induced hemodilution was considered similar in the two groups.

3.1 Plasma nitrate+nitrite

There were time-dependent changes in plasma NO_x levels in both groups (P<0.0001, ANOVA, treatment effect). There was no significant difference in plasma NO_x level between the groups at any sampling point ((before CPB) Group-P vs C: 25.3±3.8 vs 24.2±4.0 μmol/l; (at CPB termination) Group-P vs C: 31.6±4.3 vs 32.7±4.1 μmol/l; (3 h after CPB termination) Group-P vs C: 47.8±4.9 vs 51.7±5.3 μmol/l) (Fig. 1a) .

Fig. 1

Changes in (a) plasma levels of nitrate+nitrite, and (b) the percentage ratio of nitrotyrosine to tyrosine before and after cardiopulmonary bypass (CPB). Data are expressed as mean±SD. pre-CPB, before the initiation of CPB; CPB-off, at the termination of CPB; after 3 hrs, 3 h after CPB termination.

3.2 Nitrotyrosine formation

Nitrotyrosine was not detected in the supernatant fluid obtained prior to CPB in either group. There were time-dependent changes in %NO₂-Tyr in both groups (P<0.0001, ANOVA, treatment effect). The %NO₂-Tyr in Group-P was significantly lower both at CPB termination (Group-P vs C; 0.26±0.07 vs 0.55±0.11%, P<0.01) and 3 h after CPB termination (Group-P vs C; 0.65±0.14 vs 1.46±0.25%, P<0.01)) (Fig. 1b).

3.3 Inflammatory cytokines

Before the initiation of CPB, plasma IL-6 level was below minimum detectable levels in both groups. After the termination of CPB, plasma IL-6 was detected and there were significant time-dependent changes in IL-6 in both groups (P<0.0001, ANOVA, treatment effect). Plasma IL-6 level was significantly lower in Group-P than in Group-C 3 h after the termination of CPB (Group-P vs C; 176.8±44.3 vs 302.4±78.1 pg/ml, P<0.01); whereas there was no significant difference between the groups at the termination of CPB (Group-P vs C; 72.3±14.1 vs 80.5±13.7 pg/ml) (Fig. 2a) .

Fig. 2

Changes in the plasma levels of pro-inflammatory cytokines; (a) interleukin-6 and (b) interleukin-8, before and after cardiopulmonary bypass (CPB). Data are expressed as mean±SD. pre-CPB, before the initiation of CPB; CPB-off, at the termination of CPB; after 3 hrs, 3 h after CPB termination.

Plasma IL-8 level before CPB was not significantly different between the groups (Group-P vs C; 0.56±0.10, vs Group-C: 0.54±0.13 ng/ml). Similar to the pattern of IL-6, there were significant time-dependent changes in plasma IL-8 in both groups (P<0.0001, ANOVA, treatment effect), and there were significant differences in plasma IL-8 level between the groups 3 h after CPB termination (Group-P vs C; 9.45±1.78 vs 16.42±2.53 ng/ml, P<0.01), not at CPB termination (Group-P vs C; 3.86±0.91 vs 4.51±0.86 ng/ml) (Fig. 2b).

3.4 Respiratory index

RI before CPB did not differ significantly between the groups (Group-P vs C: 0.32±0.09 vs 0.30±0.11), and there were significant time-dependent changes in RI value in both groups (P<0.0001, ANOVA, treatment effect). Group-P showed significantly lower RI values than did Group-C 3 h after CPB termination (1.07±0.19 vs 1.54±0.25, P<0.01); whereas there was no significant difference between the groups at the termination of CPB (Group-P vs C: 0.89±0.17 vs 0.92±0.16).

4 Discussion

The present study demonstrated that quercetin-mediated decrease in tyrosine nitration resulted in attenuating post-bypass inflammation in a rat-CPB model, as evidenced by smaller plasma level of IL-6 and IL-8. Although clinically-available parameters for evaluation were not included and a possible mechanism of attenuating inflammation was not elucidated, these results can indicate that ONOO⁻ plays a cytotoxic role in the development of post-bypass systemic inflammatory response.

The cytologic effect of ONOO⁻ remains controversial. Recent studies have suggested the protective effect of ONOO⁻ on the development of myocardial ischemia-reperfusion injury [19,20]. In the present study, the inflammatory response was mainly induced by blood contact with artificial surface of the bypass circuit. Hearts were not subjected to ischemic insults and cardioplegic solution was not used, which affects the production of NO and ONOO⁻. The amount of ONOO⁻ produced through bypass-induced blood activation is expected to be much smaller than that produced after myocardial ischemia-reperfusion. Therefore, the results of the present study are not essentially inconsistent with those of the above studies.

As for the mechanism of detoxifying ONOO⁻, Ronson et al. indicated that ONOO⁻ is converted to nitrosothiols under biologic conditions and this detoxification reaction contributes to regenerating NO through nitrosothiols as well as preventing toxic build up of ONOO⁻[20]. The present study demonstrated that quercetin-treated group showed significantly lower nitrotyrosine formation than quercetin-untreated group; while there was no significant difference in plasma NO_x level between the groups at ant sampling point. However, these results do not necessarily contradict the above possible mechanism. The end product of ONOO⁻ is also nitrate and the actual amount of nitrate derived from NO cannot be confirmed by plasma NO level alone.

The amounts of NO and subsequent ONOO⁻ are largely attributed to the activities of NO synthase (NOS). The NO production is regulated by endothelial-constitutive NOS (ecNOS) and inducible NOS (iNOS) in the development of CPB-induced inflammatory response [13,14], and the amount of NO is thought to be largely enhanced by the expression of iNOS in the late phase of inflammatory response [14]. There may be a relationship among iNOS activation, the amount of ONOO⁻ and the degree of inflammation. It is interesting that the present study showed significant differences in plasma pro-inflammatory cytokines and respiratory index between the groups only 3 h after CPB termination. These results indicate that ONOO⁻ may not participate in the development of CPB-induced inflammatory response in the early phase. The effect of ONOO⁻ scavenger on NOS activities remains to be examined to elucidate the detail mechanism of quercetin-mediated cytoprotective effects.

Quercetin, a natural flavonoid, is considered to be the most efficient ONOO⁻ scavenger [16,21]. The efficiency has been examined in various experimental studies [22–25], and some of them suggest that quercetin-mediated cytoprotective effect is probably associated with a decrease in the amount of NO which is removed by ONOO⁻ formation [22,25]. However, the scavenging activity is considered susceptible to environmental conditions [23]. Further studies should be needed to determine the most reliable delivery method such as optimal dose and additives.

In conclusion, the present study was first designed to elucidate a role of ONOO- in the development of CPB-induced inflammation in a rat-CPB model. Pretreatment with quercetin reduced nitrotyrosine formation and attenuated systemic inflammation after bypass-perfusion. Although there remain several issues to be elucidated for clinical application, ONOO⁻ plays a crucial role in the development of post-bypass systemic inflammatory response and quercetin has a potential benefit to avoid deleterious effects of ONOO⁻.