Intravenous high mobility group box 1 upregulates the expression of HIF-1α in the myocardium via a protein kinase B-dependent pathway in rats following acute myocardial ischemia

Abstract

The effects of intravenous high mobility group box 1 (HMGB1) on myocardial ischemia/reperfusion (I/R) injury remains to be elucidated. The purpose of the present study was to investigate the effects of intravenous HMGB1 on the expression of hypoxia inducible factor-1α (HIF-1α) in the myocardium of rats following acute myocardial ischemia, and to examine the effects of intravenous HMGB1 on myocardial I/R injury. Male Wistar rats were divided into the following groups: Sham operation group (n=10), a group exposed to ischemia for 30 min and reperfusion for 4 h (I/R group) as a control (n=10), an HMGB group, in which 100 ng/kg HMGB was administered intravenously 30 min prior to ischemia (n=10), an LY group, in which LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), was administered intravenously (0.3 mg/kg) 40 min prior to ischemia (n=10), and the HMGB1+LY group, in which HMGB1 (100 ng/kg) and LY294002 (0.3 mg/kg) were administered intravenously 30 min and 40 min prior to ischemia, respectively (n=10). The serum levels of cardiac troponin I (cTnI) and tumor necrosis factor-α (TNF-α), and myocardial infarct size were measured. The expression levels of phosphorylated Akt and HIF-1α were investigated using western blot analyses. The results showed that pre-treatment with HMGB1 significantly decreased serum levels of cTnI, and TNF-α, and reduced myocardial infarct size following 4 h reperfusion (all P<0.05). HMGB1 also increased the expression levels of HIF-1α and p-Akt induced by I/R (P<0.05). LY294002 was found to eliminate the effects of intravenous HMGB1 on myocardial I/R injury (P<0.05). These results suggest that intravenous pre-treatment with HMGB1 may exert its cardioprotective effects via the upregulation of the myocardial expression of HIF-1α, which may be regulated by the PI3K/Akt signaling pathway, in rats following acute myocardial I/R.

Figure 1

Histopathological changes in myocardial tissue, determined using hematoxylin and eosin staining (magnification, ×400). (A) Sham group; myocardial fibers were arranged regularly with clear boundaries and no inflammatory infiltration. (B) I/R group; focal myocardial damage with uneven staining was observed. Morphological changes in affected cardiomyocytes primarily comprised different degrees of swelling, necrosis, myocytolysis and myofibrillar loss. Myocardial fibers were disrupted and arranged irregularly (arrow 1). Infarction foci were infiltrated with numerous neutrophils (arrow 2). (C) HMGB group; myocardial fibers were arranged relatively regularly (arrow 1). Infiltration of numerous neutrophils was observed (arrow 2). (D) LY group; myocardial fibers were disrupted and dissolved, and stripes had disappeared (arrow 1). Numerous inflammatory cells infiltrated the area surrounding the infarction foci and capillaries (arrow 2). (E) HMGB1+LY group; myocardial fibers were arranged irregularly, a number being dissolved and disrupted. The number of cardiomyocytes were reduced and intermuscular spaces widened (arrow 1). Infarction foci and capillaries were surrounded by infiltration of inflammatory cells (arrow 2). I/R, ischemia/reperfusion;HMBG, high mobility group box 1; LY, LY294002.

Figure 2

IS in the treatment groups. 2,3,5-triphenyltetrazolium chloride-Evans blue staining was used to determine the area of the myocardial infarction. Normal myocardium was stained blue, ischemic myocardium was stained a brick red color and infarcted myocardium stained white. IS, infarct size; I/R, ischemia/reperfusion; LY, LY294002; HMGB1, high mobility group box 1. ^ΔP<0.05 vs. sham group; ^▽P<0.05 vs. I/R group; ^▲P<0.05 vs. HMGB1 group.

Figure 3

Protein expression of HMGB1. Data are expressed as the mean ± standard deviation. ^ΔP<0.05 vs. sham group; ^▽P<0.05 vs. I/R group; ^▲P<0.05 vs. HMGB1 group. I/R, ischemia/reperfusion; LY, LY294002; HMGB1, high mobility group box 1; HIF-1α, hypoxia inducible factor-1α.

Figure 4

Protein expression of p-Akt. Data are expressed as the mean ± standard deviation. ^ΔP<0.05 vs. sham group; ^▽P<0.05 vs. I/R group; ^▲P<0.05 vs. HMGB1 group. I/R, ischmia/reperfusion; LY, LY294002; HMGB1, high mobility group box 1; p-Akt, phosphorylated Akt; t-Akt, total Akt.