Preconditioning with high mobility group box 1 protein protects against myocardial ischemia-reperfusion injury.

OBJECTIVE: To investigate whether preconditioning with high mobility group box 1 protein (HMGB1) could reduce myocardial ischemia-reperfusion (I/R) injury. METHODS AND
RESULTS: Infarct size, lactate dehydrogenase (LDH), creatine kinase (CK), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were assessed. HMGB1 preconditioning reduced significantly the infarct size induced by I/R. The LDH, CK, TNF-α and IL-6 levels were significantly decreased by HMGB1 preconditioning compared to those in the I/R group.
CONCLUSION: The present study suggested that preconditioning with HMGB1 could induce HMGB1 tolerance and protect against myocardial I/R injury.

Methods

Experimental animal

Twenty six male Sprague-Dawley (SD) rats (250–300 g) were randomly assigned into 3 groups receiving the following treatments: Group 1: Sham-operated control (SO) (n  = 6): rats were subjected to surgical manipulation without the induction of myocardial ischemia. Group 2: Ischemia–reperfusion (I/R) (n  = 10): rats treated with PBS 24 h before ischemia, and then subjected to the left anterior descending coronary artery occlusion for 30 min followed by reperfusion for 4 h. Group 3: high mobility group box 1 protein (HMGB1) + I/R (n  = 10): rats treated with HMGB1 (200 µg/kg, ip, Sigma, USA) 24 h before ischemia, HMGB1 was dissolved in PBS.

Infarct size was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) method. Serum was taken to determine lactate dehydrogenase (LDH) and creatine kinase (CK) by using an Olympus AU 2700 Analyzer. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were measured using commercialized ELISA assay kits in myocardial tissue.

Statistical analysis

All values were expressed as mean ± SD. t-test was used for between-group comparisons. One-way ANOVA was used for comparisons among groups and the least-significant difference was used for post-hoc multiple comparisons. Statistical significance was defined as p  < 0.05.

Results

The infarct size was significantly reduced by HMGB1 preconditioning (30.5 ± 3.6%) compared to that in the I/R group (57.8 ± 4.1%) (p  < 0.05). Both LDH (2134.7 ± 172.6 U/L) and CK (2908.1 ± 192.8 U/L) were markedly increased in the I/R group compared to those in the SO group (789.4 ± 71.5 U/L and 1010.7 ± 79.2 U/L) (both p  < 0.05), respectively. Pretreatment with HMGB1 could prevent elevation of LDH (935.6 ± 91.9 U/L) and CK (1295.3 ± 99.2 U/L) in serum during myocardial I/R (both p  < 0.05). After 4 h reperfusion, the TNF-α (33.27 ± 2.84 pg/mg) and IL-6 (48.63 ± 3.75 pg/mg) levels of myocardial tissue in the I/R group were significantly increased compared to those in the SO group (16.85 ± 2.37 pg/mg and 25.74 ± 3.01 pg/mg) (both p  < 0.05), whereas pretreatment with HMGB1 could prevent elevation of the TNF-α (19.32 ± 2.07 pg/mg) and IL-6 (27.52 ± 2.32 pg/mg) levels of myocardial tissue (both p  < 0.05).

Discussion

Endotoxin tolerance was first described in 1946 as the reduced capacity of animals or humans or of cultured macrophage and monocytes to respond to lipopolysaccharide (LPS) activation following a previous exposure to a relatively low concentration of LPS [1]. Previous study showed that Gram-negative bacterial LPS induces myocardial protection [2]. Specifically, pretreatment with LPS for 24 h reduces myocardial I/R injury in mice [2]. This effect is not unique to LPS, since lipoteichoic acid, a cell wall component of Gram-positive bacteria, also reduces infarct size when administered to rats 8–24 h before myocardial ischemia [3]. Recently, Izuishi et al [4] reported that preconditioning with HMGB1 also could protect against hepatic I/R injury. However, whether preconditioning with HMGB1 could provide a cardioprotection during myocardial I/R remains unknown.

HMGB1, a non-chromosomal nuclear protein that maintains the nucleosome structure and regulates gene transcription, could be released by necrotic cell or activated innate immune cells (such as macrophages and monocytes) [5]. HMGB1 has been identified as a new proinflammatory cytokine and as late mediator of inflammation, sepsis, acute lung injury, severe acute respiratory syndrome, autoimmune disease and etc., and which has been found to play a pivotal role in the pathogenesis of the above inflammatory disorders [5], [6]. Recently, HMGB1 has been found that it acts as an early mediator of inflammation and cell injury during myocardial I/R [7]. These results suggest that HMGB1 play an important role in myocardial I/R injury. However, HMGB1 has been recognized as a member of endogenous compounds, called “alarmins,” which serve as danger signals to promote activation of the innate immune system in response to tissue injury as a result of trauma, I/R, or infection [8]. Izuishi et al [4] indicated that pretreatment of mice with HMGB1 protected against hepatic I/R injury and blunted the inflammatory response to this insult. And then, recently they found that preconditioning with HMGB1 could also induce LPS tolerance during hepatic I/R [8], indicating that preconditioning with HMGB1 may also protect against myocardial I/R injury as well as LPS.

In the study, we showed that preconditioning with HMGB1 could decrease myocardial injury (infarct size, LDH and CK). In addition, preconditioning with HMGB1 could also decrease the levels of TNF-α and IL-6 which were important pathophysiological components of myocardial I/R injury, indicating that it could blunt the inflammatory response. This is consistent with previous study, which demonstrated that there was a cross-talk between HMGB1 and inflammatory cytokines [5], [6]. These results suggested that preconditioning with HMGB1 could induce HMGB1 tolerance and protect against myocardial I/R injury.