Humanin Exerts Neuroprotection During Cardiac Ischemia-Reperfusion Injury

Abstract

Cardiac ischemia-reperfusion (I/R) injury has been shown to impair brain function. Humanin analogue (HNG) given prior to cardiac ischemia has been shown to attenuate both heart and brain mitochondrial dysfunction caused by cardiac I/R injury. In a clinical setting, patients received medical treatment for acute myocardial infarction either during or after the onset of myocardial ischemia; thus, in this study, we tested the hypothesis that the administration of HNG during cardiac I/R injury has therapeutic potential for brain protection. Thirty-six male Wistar rats were divided into two groups: a cardiac I/R group (n = 30), and a sham group (n = 6). The I/R rats were then divided into five subgroups to receive: 1) vehicle; 2) HNG (84 μg/kg); 3) HNG (168 μg/kg); 4) HNG (252 μg/kg) intravenously administered during the cardiac-ischemia; and 5) HNG at 252 μg/kg given at the onset of reperfusion. At the end of treatment, brains were removed for determination of blood-brain barrier (BBB) breakdown, oxidative stress, brain mitochondrial function, brain mitochondrial dynamics, p-tau, amyloid-β (Aβ) and apoptosis. HNG at a dose of 168 and 252 μg/kg administered during ischemia, and 252 μg/kg given at the onset of reperfusion effectively attenuated the brain mitochondrial dysfunction, tau hyperphosphorylation and Aβ accumulation, and apoptosis, without reducing BBB breakdown, brain oxidative stress, or mitochondrial dynamics, caused by cardiac I/R injury. In conclusion, humanin exerted neuroprotection during induced cardiac I/R injury via improvement in brain mitochondrial function, and the reduction of Alzheimer’s disease pathology and apoptosis.

Keywords

Alzheimer’s disease brain ischemia reperfusion injury mitochondria oxidative stress pathology

INTRODUCTION

Although myocardial reperfusion is the treatment of choice for the reduction of acute myocardial ischemic (AMI) injury and the limiting of infarct size in AMI patients [1, 2], myocardial reperfusion itself is complex and can cause deleterious effects to the ischemic myocardium, a process known as reperfusion injury [3, 4]. The burst of oxidative stress, which occurs within a few minutes of myocardial reperfusion, can lead to ventricular arrhythmias, myocardial stunning, microvascular obstruction, and cardiomyocyte death through a number of different mechanisms [3 –5]. When cardiac ischemia–reperfusion (I/R) occurs, it can cause I/R injury in the heart and other vital organs, particularly in the brain [4, 6]. As in injuries to the heart, previous studies showed that cardiac I/R events can also lead to brain I/R injury, resulting in a burst of oxidative stress which can lead to blood-brain barrier (BBB) breakdown, lipid peroxidation, mitochondrial swelling, brain apoptosis, and brain death [6, 7]. In addition, it has been shown that accumulation of amyloid-β (Aβ) and hyperphosphorylation of tau protein has been observed in the brain following cardiac I/R injury, resulting in the formation of Aβ plaques and neurofibrillary tangles, which are the pathological conditions associated with Alzheimer’s disease (AD) [8 –10]. Furthermore, the disruption of the BBB, indicated by decreased occludin expression, can lead to increased membrane lipid peroxidation and oxidative cell damage and contributes to further brain damage [11, 12].

Mitochondria play a critical role in the pathogenesis of cerebral I/R injury via reactive oxygen species (ROS) generation, mitochondrial failure or dysfunction, and mitochondrial apoptosis [6, 7]. It has been shown that mitochondrial dynamics, including mitochondrial fusion and fission, regulate mitochondrial function, mitochondrial shape control, and mitochondrial communication [13, 14]. Three mammalian proteins are required for mitochondrial fusion: Mfn1, Mfn2, and OPA [15, 16], whereas dynamin-like protein1 (Drp1), a predominantly cytosolic protein, facilitates mitochondrial fission [13]. In several neurodegenerative disorders including AD, Parkinson’s and Huntington’s diseases, disruption of mitochondrial dynamics has been observed [13, 14]. Although we recently demonstrated that cardiac I/R injury caused BBB breakdown, increased brain oxidative stress and resulted in brain mitochondrial dysfunction [17], the molecular mechanisms of brain injury following cardiac I/R condition, including AD pathology, mitochondrial dynamics, and apoptosis have not been elucidated.

Humanin is a neuroprotective and cytoprotective peptide which has anti-apoptotic, anti-oxidant, and anti-inflammatory properties [18, 19]. Humanin suppresses neuronal death induced by proteins related to AD or Aβ accumulation [20]. Humanin was also found to selectively bind to the pro-apoptotic protein, Bax, in the cytoplasm, preventing Bax translocation from the cytoplasm to the mitochondria, resulting in suppressed apoptosis [21]. More evidence has been accumulated from previous studies which showed that administration of the humanin analog (HNG) effectively protected the heart against cardiac I/R injury in a rodent model by decreasing infarct size and improving left ventricular function [22, 23]. Humanin has also been shown to exert neuroprotection in cases of cerebral I/R injury [24]. We have demonstrated previously that HNG administration prior to cardiac ischemia attenuated brain mitochondrial dysfunction caused by cardiac I/R injury [17]. However, in a clinical situation, patients seek medical treatment for acute myocardial infarction after the onset of myocardial ischemia; thus whether HNG administration after myocardial ischemia provides protective effects on the brain is not known.

In the present study, we investigated whether cardiac I/R injury could lead to BBB breakdown, lipid peroxidation, brain mitochondrial dysfunction, brain mitochondrial dynamics, AD pathology, and brain apoptosis. In addition, we determined the effects of HNG administration after myocardial ischemia on BBB breakdown, lipid peroxidation, brain mitochondrial function, brain mitochondrial dynamics, AD pathology, and brain apoptosis under conditions of cardiac I/R injury condition.

MATERIALS AND METHODS

Animals and experimental design

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Medicine, Chiang Mai University (Permit number: 26/2559), and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH guide, 8th edition, 2011). Adult male Wistar rats (300–350 g, n = 36) were obtained from the National Laboratory Animal Center, Mahidol University, Bangkok, Thailand. All animals were given ad libitum access to food and water. All animals were housed in controlled temperature and humidity rooms with 12-h light-dark cycles. HNG used in this study was synthesized by replaced amino acid at position 14 (Ser) to glycine ([Gly14]-humanin), there is an increase in activity of 1000-fold than humanin [20]. The structure of HNG (sHNG, [Gly14] – HN, [Gly14] – Humanin) is (AA: Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr- Gly-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala; MW: 2657.25; Cat No. SP-54838-1, Genemed Synthesis, Inc., San Antonio, TX, USA). The half-life of HNG in rats plasma appears to be greater than 4 h, which is much longer than the half-life measured in mice (<1 h) [25].

Our previous study showed that humanin treatment at 84 μg/kg only at before ischemia attenuated brain mitochondrial dysfunction but did not have neuroprotection when treatments during ischemia and in the reperfusion period in a cardiac ischemia–reperfusion injury model [17]. Therefore, 2 and 3 times of 84 μg/kg of HNG that are 168 and 252 μg/kg HNG were used during ischemia and at the reperfusion period to determine the neuroprotective effects in this study.

Male Wistar rats were subjected to a cardiac ischemia-reperfusion (I/R) injury protocol. Rats were randomly divided into 6 groups (n = 6/group) as follows: 1) sham group; 2) cardiac I/R group with normal saline (NSS) administration at 15 min after occlusion; 3) cardiac I/R group with HNG administration (84 μg/kg, IV, H84) [17] at 15 min after occlusion; 4) cardiac I/R group with HNG administration (168 μg/kg, IV, H168) at 15 min after occlusion; 5) cardiac I/R group with HNG administration (252 μg/kg, IV, H252) at 15 min after occlusion; 6) cardiac I/R group with HNG administration (252 μg/kg, IV, H252) at the onset of reperfusion. Then, animals were euthanized by an overdose injection of zoletil (100 mg/kg) prior to organ removal. Left hemisphere of the brain was removed rapidly for mitochondrial isolation and the right hemisphere was used for western blot and MDA analysis. Isolated brain mitochondria were used to investigate mitochondrial function. The experimental protocol is shown in Fig. 1.

Fig.1

Study protocol for experimental groups and timing of administration of humanin analog (HNG) or normal saline solution (NSS).

Cardiac ischemia-reperfusion injury protocol

Rats were anesthetized using an intramuscular injection of zoletil (50 mg/kg) and xylazine (0.15 ml/kg). The depth of anesthesia was checked by testing the absence of eyelid reflex, pedal withdrawal reflex, and the tail pinch reflex. A tracheostomy was performed and rats were ventilated with room air using a rodent ventilator at a volume of 200–250 μl, with the ventilation rate at 70–110 breaths/min to maintain the PCO₂, PO₂, and pH parameters within normal physiologic conditions [17 , 27]. A left-side thoracotomy was performed at the fourth intercostal space, and the pericardium was incised to expose the heart [17 , 27]. The left anterior descending (LAD) coronary artery was identified and ligated at approximately 2 mm distal to its origin. The end of a ligature was passed through a small vinyl tube, and was used to occlude the LAD by pulling the thread. The heart was monitored throughout the experiment, recordings being made through Lead II ECG. ST elevation was used to confirm ischemia. The heart was subjected to ischemia for 30 min, and then the ligature was loosened to allow reperfusion to the ischemic myocardium for 120 min [17 , 27].

Determination of brain oxidative stress by measuring malondialdehyde (MDA) level

The brain MDA concentrations were measured by using the HPLC method as described previously [17, 28]. The brain tissues were homogenized in a phosphate buffer and brain homogenate was mixed with 10% (w/v) trichloroacetic acid (TCA), heated at 90°C for 30 min and cooled down to room temperature. The mixture was centrifuged at 6,000 rpm for 10 min until a clear supernatant was achieved. The supernatant was mixed with 0.44 M H₃PO₄ and 0.6% (w/v) thiobarbituric acid (Sigma-Aldrich, Co., St. Louis, USA) solution and incubated at 90°C for 30 min to produce the significant, pink-colored products, thiobarbituric acid-reactive substances (TBARS). The solution was passed through a syringe filter (polysulfone type membrane, pore size 0.45 μm) and analyzed using the HPLC system at 532 nm. Data were collected and analyzed using the BDS software (BarSpec Ltd.). Tissue TBARS concentrations were determined directly from a standard curve and reported as MDA equivalent concentrations in μM/mg protein.

Isolation of brain mitochondria

Brain mitochondria were isolated using the differential centrifugation method [17, 28]. The rat brains were removed quickly and placed into ice-cold MSE solution (225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 5 mM HEPES, 1 mg/ml BSA, pH 7.4) to flush the blood out rapidly and they were then transferred into 5-ml cold MSE-nagarse solution. The brain tissue was finely minced and homogenized in the homogenizer at 600 rpm. The homogenate was then centrifuged at 2000 g for 4 min, and the supernatants were collected and centrifuged at 12,000 g for 9 min. Mitochondrial pellets were re-suspended in cold MSE-digitonin solution and centrifuged one more time at 12,000 g for 11 min. Mitochondrial pellets were collected and dissolved in cold respiration (RES) buffer (containing 150 mM KCl, 5 mM HEPES, 5 mM K₂HPO₄-3H₂O, 2 mM l-glutamate, 5 mM Pyruvate sodium salt). The protein concentration was determined according to the bicinchoninic acid (Sigma-Aldrich, Co., St. Louis, USA) assay as previously described [28].

Determination of brain mitochondrial ROS production

Brain mitochondrial ROS production was determined using the dichlorohydro-fluorescein diacetate (DCFDA; Sigma-Aldrich, Co., St. Louis, USA) dye [29]. Brain mitochondria were incubated with 2 μM DCFDA at 25°C for 20 min. ROS levels were determined using a fluorescent microplate reader at λ_ex 485 nm and λ_em 530 nm. The ROS level was represented as arbitrary units of fluorescence intensity of dichlorohydro-fluorescein (DCF) and fluorescence intensity in I/R groups and HNG treatment groups were normalized to the sham group.

Determination of brain mitochondrial membrane potential changes (ΔΨm)

Brain mitochondrial membrane potential changes were measured using the dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanineiodide (JC-1; Sigma-Aldrich, Co., St. Louis, MO, USA) [30]. Brain mitochondria were stained with JC-1 (310 nM) at 37°C for 15 min. The fluorescence intensity was determined using a fluorescent microplate reader. The fluorescence of the JC-1 monomer form (green) was excited at 488 nm and the emission was detected at 530 nm. JC-1 aggregate (red) fluorescence was excited at 488 nm and emission fluorescence was detected at 590 nm. Mitochondrial depolarization was indicated by a decreased red/green fluorescent intensity ratio (ΔΨm) and the ratio in I/R group and HNG treatment groups were normalized to the sham group.

Determination of brain mitochondrial swelling

Brain mitochondrial swelling was determined by changes in the absorbance of the mitochondrial suspensions at 540 nm using a microplate reader (Synergy HT, Bio Tek, Winooski, Vermont, USA) [31]. Mitochondria were incubated in RES buffer. Mitochondrial swelling was indicated by a decrease in absorbance, and absorbance in both the I/R groups and HNG treatment groups were normalized to the sham group.

Identification of brain mitochondria using electron microscopy

A transmission electron microscope was used to study brain mitochondrial morphology [17, 28]. Isolated brain mitochondria were fixed overnight in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 at 4°C. After this, brain mitochondria were post fixed with 1% cacodylate-buffered osmium tetroxide for 2 h at room temperature. The brain mitochondrial pellets were subsequently dehydrated in a graded series of ethanol and embedded in Epon-Araldite. Ultra-thin sections were cut with a diamond knife, placed on copper grids, stained with uranyl acetate and lead citrate, and observed using a transmission electron microscope.

Western blot analysis

At the end of experiment, the brain tissues were collected, frozen quickly in liquid nitrogen, and stored at –80°C for further analysis. Whole brain tissues were lysed with extraction buffer (20 mmol/L Tris–HCl, 1 mmol/L Na₃VO₄, 5 mmol/L NaF) and separated by electrophoresis on a 10 % sodium dodecyl sulfate–polyacrylamide gel and then transferred onto nitrocellulose membranes [17, 28]. Immunoblots were blocked for 1 h with 5% nonfat dry milk or 5% bovine serum albumin in Tris-buffer saline (pH 7.4) containing 0.1% Tween 20. They were then probed overnight at 4°C with the primary antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA, Merck, Kenilworth, NJ, USA, and Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) including the tight junction protein (occludin (H-279), sc-5562), pro-apoptotic protein (Bax (N-20), sc-493), anti-apoptotic protein (Bcl-2, #2876), apoptotic protein (Caspase-3, #9662), mitochondrial fusion protein (Mfn2 (D2D10), #9482), mitochondrial fission protein (DRP1 (D8H5), #5391), p-tau ((thr181), #12885), amyloid-β ((B-4), sc-28365), amyloid precursor protein (APP, Cat. No. 07-667), and a loading control (β-actin, sc-47778). This process was followed by incubation for 1 h at room temperature with a horseradish peroxidase–conjugated secondary antibody. The blots were then visualized with an ECL reagent. The film images of the western blots were scanned and subsequently analyzed using Image J (NIH image) analysis software.

Statistical analysis

All data were presented as mean±standard error of mean (SEM), and were processed using SPSS (Statistical Package for Social Sciences, Chicago, IL, USA) release 21.0. A one-way ANOVA followed by an LSD post hoc test was performed to compare the groups. p-value < 0.05 was considered statistically significant.

RESULTS

Effects of HNG treatment on BBB breakdown and brain MDA levels in rats with cardiac I/R injury

Cardiac I/R injury led to an increase in BBB breakdown as shown by a decrease in the expression of occludin, when compared to the sham group (Fig. 2A). Treatment with HNG either during ischemia or at the onset of reperfusion did not increase occludin expression (Fig. 2A), suggesting that HNG treatment did not improve BBB breakdown in cardiac I/R rats. Moreover, the I/R group showed increased brain MDA levels when compared with the sham group (Fig. 2B). All doses and periods of treatments with HNG did not decrease brain MDA levels (Fig. 2B), indicating that HNG administration after myocardial ischemia did not reduce brain oxidative stress in the I/R group.

Fig.2

The effects of HNG treatment on BBB breakdown (A) and brain MDA level (B). Sham: surgery without I/R; IR: vehicle group with cardiac I/R injury; H: humanin analog; H84: humanin analog at 84 μg/kg; H168: humanin analog at 168 μg/kg; H252: humanin analog at 252 μg/kg; Reper: reperfusion period. ^*p < 0.01 versus sham group.

Effects of HNG treatment on brain mitochondrial function

In the I/R group, brain mitochondrial ROS production (Fig. 3A), brain mitochondrial depolarization (Fig. 3B), and brain mitochondrial swelling (Fig. 3C, D) were significantly greater than those of the sham group. These findings suggested that cardiac I/R injury led to increased brain mitochondrial dysfunction. HNG at doses of 168 μg/kg and 252 μg/kg, administered during ischemia and at a dose of 252 μg/kg given at the onset of reperfusion, led to restoration of brain mitochondrial function as shown by decreased brain mitochondrial ROS levels (Fig. 3A), decreased brain mitochondrial depolarization (Fig. 3B), reduced brain mitochondrial swelling (Fig. 3C) and preserved brain mitochondrial morphology (Fig. 3D). However, treatment with HNG at a dose of 84 μg/kg during ischemia did not lead to any improvement in brain mitochondrial function in these cardiac I/R rats (Fig. 3A-D).

Fig.3

The effects of HNG treatment on mitochondrial function. Each panel represents: brain mitochondrial ROS production (A); brain mitochondrial depolarization (B); brain mitochondrial swelling (C); and brain mitochondrial morphology (D). Sham: surgery without I/R; IR: vehicle group with cardiac I/R injury; H: humanin analog; H84: humanin analog at 84 μg/kg; H168: humanin analog at 168 μg/kg; H252: humanin analog at 252 μg/kg; Reper: reperfusion period. ^*p < 0.05 versus sham group, ^†p < 0.05 versus IR group.

Effects of HNG treatment on brain mitochondrial fusion and fission

I/R rats showed a decrease in brain Mfn2 protein expression (Fig. 4A), whereas the expression of DRP1 was not altered in both the sham and the I/R group (Fig. 4B), suggesting that the I/R condition led to brain mitochondrial dynamic imbalance. Treatments with all doses of HNG during ischemia and at the onset of reperfusion altered neither the expression of Mfn2 nor DRP1 in rats with cardiac I/R (Fig. 4A, B). These findings suggested that HNG did not improve the brain mitochondrial dynamic balance in rats under conditions of cardiac I/R.

Fig.4

The effects of HNG treatment on mitochondrial dynamic balance and Alzheimer’s pathology. Each panel represents: mitochondrial fusion (Mfn2) protein expression (A); mitochondrial fission (DRP1) protein expression (B); amyloid-β protein precursor (AβPP) expression (C); amyloid-β (Aβ) protein expression (D); Aβ/AβPP ratio (E); and phospho-tau (p-tau) protein expression (F). Sham: surgery without I/R; IR: vehicle group with cardiac I/R injury; H: humanin analog; H84: humanin analog at 84 μg/kg; H168: humanin analog at 168 μg/kg; H252: humanin analog at 252 μg/kg; Reper: reperfusion period. *p < 0.05 versus sham group; ^†p < 0.05 versus IR group.

Effects of HNG treatment on brain AD pathology

In the present study, although the expression of AβPP protein was not significantly different among groups (Fig. 4C), cardiac I/R injury led to an increase in Aβ accumulation (Fig. 4D), Aβ/AβPP ratio (Fig. 4E), and tau hyperphosphorylation in the brain (Fig. 4F), when compared with the sham group. HNG at a dose of 168 μg/kg and 252 μg/kg administered during ischemia and at a dose of 252 μg/kg given at the onset of reperfusion effectively attenuated Aβ accumulation, Aβ/AβPP ratio and p-tau protein expression, when compared with the I/R group (Fig. 4D-F). However, treatment with HNG at a dose of 84 μg/kg during ischemia did not decrease the level of Aβ protein (Fig. 4D), Aβ/AβPP ratio (Fig. 4E), and p-tau expression (Fig. 4F).

Effects of HNG treatment on brain apoptosis

Cardiac I/R injury led to an increase in brain pro-apoptotic (Bax) protein expression (Fig. 5A, D) and apoptotic (Cleaved Caspase-3) protein expression (Fig. 5C, D), but had no effect on anti-apoptotic (Bcl-2) protein expression (Fig. 5B, D). These findings suggested that cardiac I/R injury led to increased brain apoptosis. HNG at a dose of 168 and 252 μg/kg administered during ischemia and 252 μg/kg at the onset of reperfusion significantly attenuated Bax protein expression (Fig. 5A, D) and decreased cleaved Caspase-3 (Fig. 5C, D) in rats with cardiac I/R. However, treatment with HNG at a dose of 84 μg/kg during ischemia did not cause a decrease in Bax protein expression (Fig. 5A, D) nor cleaved Caspase-3 (Fig. 5C, D). All treatments did not alter Bcl2 protein expression (Fig. 5B, D). These findings suggested that treatment with HNG at a dose of 168 μg/kg and 252 μg/kg administered during ischemia and at a dose of 252 μg/kg given at the onset of reperfusion significantly attenuated brain apoptosis under conditions of cardiac I/R injury in rats.

Fig.5

The effects of HNG treatment on brain apoptosis. Each panel represents pro-apoptotic Bax protein expression (A); anti-apoptotic Bcl-2 protein expression (B); Cleaved Caspase-3 (C); and western blot band (D). Sham: surgery without I/R; IR: vehicle group with cardiac I/R injury; H: humanin analog; H84: humanin analog at 84 μg/kg; H168: humanin analog at 168 μg/kg; H252: humanin analog at 252 μg/kg; Reper: reperfusion period. *p < 0.05 versus sham group, ^†p < 0.05 versus IR group.

DISCUSSION

The major findings of this study are as follows. First, cardiac I/R injury caused BBB breakdown, brain oxidative stress, brain mitochondrial dysfunction, brain mitochondrial dynamic imbalance, AD pathology and brain apoptosis. Second, HNG at a dose of 168 μg/kg and 252 μg/kg administered during ischemia and 252 μg/kg given at the onset of reperfusion, effectively attenuated brain mitochondrial dysfunction, AD pathology (tau hyperphosphorylation and Aβ accumulation) and apoptosis, but had no effect on BBB breakdown, brain oxidative stress and mitochondrial dynamics caused by cardiac I/R injury. Third, treatment with humanin at 84 μg/kg during ischemia did not improve any of the studied brain parameters in rats under cardiac I/R injury.

Previous studies have shown that cardiac I/R injury can lead to I/R injury in other vital organs, particularly in the brain [4, 6]. It has been shown that brain I/R injury leads to increased ROS production, BBB breakdown, mitochondrial swelling, brain apoptosis, and brain death [6, 7]. In this study, the I/R injury in the heart, but not in the brain, was found to also cause an increase in brain oxidative stress, BBB breakdown, and mitochondrial dynamic imbalance (as shown by decreased mitochondrial fusion protein Mfn-2) and led to brain mitochondrial dysfunction, which was similar to the findings of our previous study [17]. Moreover, the present study also showed increased brain apoptosis, indicated by increased Bax and cleaved caspase-3 protein expression in rat brains under cardiac I/R conditions. All of these findings suggest that cardiac I/R injury can lead to brain dysfunction and death. In addition, brain I/R injury caused by cardiac arrest or heart failure has been shown to lead to increased AD pathology as shown by increased tau hyperphosphorylation and Aβ accumulation in the brain [8, 10]. Consistent with those clinical results, cardiac I/R injury leads to increased tau hyperphosphorylation and Aβ accumulation in the brains of rats under conditions of cardiac I/R. These findings suggest that cardiac I/R injury may be a contributing factor to the development of AD pathology in the brain. In the present study, we determined only Aβ protein expression in rat brain tissue extract by western blot analysis. The limitation of the present study is that we did not confirm Aβ accumulation in the brain by the other methods such as immunohistochemistry or immunofluorescence. Moreover, the AβPP secretases that influenced in AβPP processing was not evaluated in the present study. Future studies are need to confirm these findings.

Humanin has been shown to exert neuroprotective and cytoprotective effects with its anti-apoptotic, anti-oxidant and anti-inflammatory properties [32, 33]. Our previous study [17] and the present study showed that 84 μg/kg of HNG treatment given prior to ischemia, during-ischemia and at the onset of reperfusion neither increased occludin expression nor decreased brain MDA levels after cardiac I/R injury. Consistent with these findings, the present study showed that treatment with all doses of HNG up to 252 μg/kg during-ischemia and at the onset of reperfusion did not increase occludin expression nor decrease brain MDA levels, indicating that it did not attenuate BBB breakdown or oxidative stress in the brain after cardiac I/R injury. However, it is possible that the HNG concentration used in this study (up to 252 μg/kg) might not have been sufficient to reduce oxidative stress in the brain after cardiac I/R injury. Although previous in vitro studies demonstrated that humanin can protect cells from oxidative stress in endothelial cells [34], cardiac myoblasts [32], cortical neuronal cultures [35], and endothelial cells of ApoE-deficient mice [36], our findings from in vivo models indicate otherwise. It is possible that the therapeutic dose of HNG applied directly to the in vitro cell cultures is not equivalent to the dose required for in vivo studies. Moreover, the effective dose of HNG (HNGF6A) used in ApoE-deficient mice was higher than the dose used in this study, the dosage being 1000 times more potent than the physiological level of humanin [36]. Future study and experimentation is needed to identify an optimal dose that suitably decreases brain oxidative stress following cardiac I/R injury. A previous study showed that only treatment with HNG at 84 μg/kg prior to ischemia decreased brain mitochondrial ROS levels [17]. Although HNG at 84 μg/kg during ischemia and after ischemia failed to decrease brain mitochondrial ROS levels [17], HNG at 168 μg/kg and 252 μg/kg administered during ischemia and 252 μg/kg given at the onset of reperfusion effectively decreased brain mitochondrial ROS levels. These findings suggest that HNG still exerted antioxidant properties in mitochondria during and after the ischemic period. The present study showed that treatment with all doses of HNG up to 252 μg/kg during-ischemia and at the onset of reperfusion did not reduce the mitochondrial dynamic imbalance caused by cardiac I/R injury, which was similar to the findings of our previous study [17]. There is no more data available to support the findings from our studies. Further studies in the future are needed to investigate the effects of HNG on the mitochondrial dynamics following cardiac I/R injury. In the present study, treatment with HNG at 168 μg/kg and 252 μg/kg administered during ischemia and 252 μg/kg given at the onset of reperfusion effectively reduced Bax protein expression, decreased cleaved caspase-3 and attenuated brain mitochondrial dysfunction in cardiac I/R rats. These findings suggest that humanin exerted an anti-apoptotic effect, which is consistent with a previous study which demonstrated that HNG prevents mitochondrial dysfunction by inhibiting Bax translocation from the cytoplasm to the mitochondria, thus suppressing apoptosis [17 , 37]. Moreover, HNG at 168 μg/kg and 252 μg/kg administered during ischemia and 252 μg/kg given at the onset of reperfusion effectively attenuated AD pathology as shown by decreased Aβ accumulation and tau hyperphosphorylation in cardiac I/R rats. These findings are supported by a previous study which found that humanin suppressed neuronal cell death induced by proteins related to AD or Aβ accumulation [20]. HNG decreased Aβ accumulation and tau hyperphosphorylation in the brain in response to cardiac I/R, suggesting that it may be able to prevent AD phenotype caused by cardiac I/R injury. However, more studies are needed to confirm that these changes can lead to the development of cognitive dysfunction in the future. All of the present findings indicate that humanin can exert neuroprotective effects in cases of cardiac I/R injury, both when administered during ischemia and at the onset of reperfusion.

In conclusion, the present study demonstrates the deleterious effects of cardiac I/R injury in the brain as indicated by BBB breakdown, brain oxidative stress, brain mitochondrial dysfunction, brain mitochondrial dynamic imbalance, AD pathology, and brain apoptosis. Treatment with humanin during ischemia and at the onset of reperfusion led to the attenuation of mitochondrial dysfunction and the reduction in AD pathology and brain apoptosis under conditions of cardiac I/R injury. These findings suggest that treatment with humanin during ischemia and at the onset of reperfusion could be useful in reducing the extent of brain damage after cardiac I/R injury.

Footnotes

ACKNOWLEDGMENTS

This work is supported by grants from the Thailand Research Fund grants MRG5980222 (SK) and RTA6080003 (SCC); the NSTDA Research Chair Grant from the National Science and Technology Development Agency Thailand (NC), and a Chiang Mai University Center of Excellence Award (NC).

Authors’ disclosures available online ().

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