DhHP‑6 attenuates cerebral ischemia‑reperfusion injury in rats through the inhibition of apoptosis

Abstract

As a novel reactive oxygen species (ROS) scavenger, deuterohemin His peptide‑6 (DhHP‑6) has been demonstrated to prolong the lifespan of Caenorhabditis elegans and has also exhibited protective effects in myocardial ischemia‑reperfusion injury. Whether similar effects occur during cerebral ischemia‑reperfusion (CIR) injury remains to be elucidated. The present study evaluated the function of DhHP‑6 and its underlying mechanisms in a middle cerebral artery occlusion (MCAO) model in rats. The focal transient MCAO model was implemented using the Longa method of ischemia for 2 h followed by reperfusion for 22 h in male Wistar rats. DhHP‑6 was administered at the onset of reperfusion via intraperitoneal injection. The infarct volume, brain edema, brain apoptosis and neurological function were evaluated 24 h following stroke. To further determine the role of DhHP‑6 in CIR injury, the levels of ROS and malondialdehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH‑Px), and the protein expression levels of B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax), cleaved caspase‑3, cytochrome c, Bcl‑2 and phosphorylated‑Akt/Akt were measured in ischemic cortex tissues. The results demonstrated that DhHP‑6 significantly improved infarct volume, brain edema and neurological deficits, and reduced the percentage of TUNEL‑positive cells. The levels of ROS and MDA were decreased, whereas no significant changes in the activities of SOD, CAT and GSH‑Px were observed. The levels of Bax, cleaved caspase‑3, and cytochrome c were downregulated, whereas the levels of Bcl‑2 and p‑Akt/Akt were upregulated. The results of the present study indicated that DhHP‑6 may offer therapeutic potential for cerebral ischemia. The neuroprotective effects of DhHP‑6 maybe mediated by its anti‑oxidative properties, anti‑apoptotic activities, or activation of the phosphoinositide 3‑kinase/Akt survival pathway.

Figure 1.

Effect of DhHP-6 on cerebral ischemia induced by 2 h of middle cerebral artery occlusion and 22 h of reperfusion. (A) Triphenyltetrazolium chloride staining of coronal brain sections 22 h following reperfusion. Infarct brain tissues appeared unstained. Tissues in the I/R+DH group, but not the I/R+DL group, demonstrated significant reductions in (B) infarct volume, (C) neurological deficits and (D) brain edema, in a dose-dependent manner. Data are shown as the mean ± standard deviation. One-way analysis of variance and Tukey's post hoc test were performed. ^###P<0.001 and ^##P<0.01, vs. Sham group; *P<0.05, vs. I/R group (n=10). I/R, ischemia/reperfusion; DhHP-6, deuterohemin His peptide-6; DH, 1 mg/kg/day DhHP-6; DL, 0.1 mg/kg/day DhHP-6.

Figure 2.

Effect of DhHP-6 on oxidative stress induced by 2 h of middle cerebral artery occlusion and 22 h of reperfusion. (A) ROS levels in brain mitochondria are expressed as the percentage of fluorescence intensity relative to that in the Sham group. DhHP-6 attenuated ROS formation in a dose-dependent manner. Compared with the I/R group, the ROS level was significantly reduced in the I/R+DH group. (B) MDA content was significantly reduced in the I/R+DH group, compared with that in the I/R group. Data are shown as the mean ± standard deviation. One-way analysis of variance and Tukey's post hoc test were performed. ^##P<0.01, vs. Sham group; *P<0.05, vs. I/R group (n=10). I/R, ischemia/reperfusion; DhHP-6, deuterohemin His peptide-6; DH, 1 mg/kg/day DhHP-6; DL, 0.1 mg/kg/day DhHP-6, ROS, reactive oxygen species; MDA, malondialdehyde.

Figure 3.

Effect of DhHP-6 on neuronal apoptosis of the ischemic cortex in rats 22 h post-reperfusion. (A) TUNEL staining was used to identify apoptotic cells in the parietal cortex. Arrows indicate TUNEL-positive cells (magnification, ×200). (B) As demonstrated in the bar graphs, the apoptotic index indicates the percentage of TUNEL-positive cells in the ischemic cortex. The percentage of TUNEL-positive cells in the I/R+DH group was significantly lower, compared with that in the I/R group. Data are demonstrated as the mean ± standard deviation. One-way analysis of variance and Tukey's post hoc test were performed. ^###P<0.001, vs. Sham group; *P<0.05, vs. I/R group (n=5). DhHP-6, deuterohemin His peptide-6; DH, 1 mg/kg/day DhHP-6; DL, 0.1 mg/kg/day DhHP-6.

Figure 4.

Effects of DhHP-6 on expression levels of Bcl-2 and Bax in the rat brain 22 h post-reperfusion. The protein expression levels of Bcl-2 and Bax in subcellular fractions were examined using western blot analysis. Scanning and quantification of the intensity of protein bands were performed using image analysis software. Representative bands from the Sham, I/R, and DhHP-6 (I/R+DH and I/R+DL)-treated groups, and the corresponding β-actin bands (loading control) are demonstrated. Middle cerebral artery occlusion mediated a decrease in Bcl-2, and an increase in Bax led to a significantly decreased Bcl-2/Bax ratio in mitochondria, compared with the ratio in the Sham group. Compared with the I/R group, the ratio of Bcl-2/Bax in mitochondria was significantly higher in the I/R+DH group. Data are shown as the mean ± standard deviation. One-way analysis of variance and Tukey's post hoc test were used. ^###P<0.001, vs. Sham group; *P<0.05, vs. I/R group (n=3). DhHP-6, deuterohemin His peptide-6; DH, 1 mg/kg/day DhHP-6; DL, 0.1 mg/kg/day DhHP-6; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Figure 5.

Effects of DhHP-6 on the rate of cytochrome c release and the activation of caspase-3 22 h post-reperfusion. The contents of cytochrome c in the subcellular fractions were examined using western blot analysis. (A) The ratio of cytochrome c content in the cytosolic and mitochondrial fractions was increased significantly in the I/R group, but the ratio was decreased markedly in the I/R+DH group. (B) Additionally, the expression level of cleaved caspase-3 was increased in the I/R group at 22 h post-reperfusion, but significantly decreased in the I/R+DH group, compared with the I/R group. Data are shown as the mean ± standard deviation. One-way analysis of variance and Tukey's post hoc test were performed. ^###P<0.001 and ^##P<0.01, vs. Sham group; *P<0.05, vs. I/R group (n=3). DhHP-6, deuterohemin His peptide-6; DH, 1 mg/kg/day DhHP-6; DL, 0.1 mg/kg/day DhHP-6; mito, mitochondria; cyto, cytosol; I/R, ischemia/reperfusion.

Figure 6.

Effect of DhHP-6 on p-Akt/Akt 22 h post-reperfusion. To determine whether the activation of Akt contributes to DhHP-6-dependent protection against apoptosis induced by I/R injury, levels of p-Akt and Akt were examined using western blot analysis. The histograms demonstrate the level of p-Akt relative to that of Akt. Compared with the I/R group, the p-Akt/Akt ratio was significantly increased in the I/R+DH group. Data are shown as the mean ± standard deviation. One-way analysis of variance and Tukey's post hoc test were performed. ^##P<0.01, vs. Sham group; *P<0.05, vs. I/R group (n=3). DhHP-6, deuterohemin His peptide-6; DH, 1 mg/kg/day DhHP-6; DL, 0.1 mg/kg/day DhHP-6; p-, phosphorylated; I/R, ischemia/reperfusion; Akt, AKT serine/threonine kinase.