Introduction

Exposure to hypobaric hypoxia (HH) at high altitude, characterized by low barometric pressure and low oxygen level, exerts detrimental effects on many organs [1], especially the brain due to its high oxygen consumption [2]. Numerous studies have reported that HH exposure induces a drop in the oxygen saturation in the brain, which leading to deficit in cognitive processes, such as spatial learning, memory, language, concentration, mood, fear, and reaction time [3,4,5,6,7]. With the increasing number of people travelling to plateau for working or traveling, the cognitive impairments induced by HH are of major public health importance and have economic consequences [8].

The mechanisms of HH-induced cognitive impairment are very complex and have not been fully understood. Researchers have found several potential mechanisms such as DNA methylation [9], mitochondrial dysfunction [10], acetylcholinesterase (AChE) activation [11], dendritic atrophy in the hippocampus [12] and dysfunction of cholinergic system [13]. The most recognized mechanisms are oxidative stress [14], activation of pro-inflammatory cytokines [15], and apoptosis [16]. In recent years, abundant investigations have proved that use of agents possessing antioxidant, anti-inflammatory and/or anti-apoptotic activities are useful for alleviation of impaired cognitive functions induced by HH. For example, crocin administration ameliorated the hypoxia-induced impairments in spatial working memory function by decreasing oxidative stress, keeping a high level of GSH, GSH-Px, and SOD and reducing MDA and GSSG content [17]. In addition, Epigallocatechin-3-Gallate treatment prevented HH-induced cognitive deficit by attenuating oxidative stress, iron accumulation, and apoptosis [7]. Echinacoside also could decrease of memory impairment following exposure to HH by suppressing oxidative stress via regulation of Keap1-Nrf2-ARE pathway [18]. Moreover, our recent study also indicated that 5, 6, 7, 8-Tetrahydroxyflavone, a flavone with excellent antioxidant activity, could ameliorate HH-induced cognitive deficit [19].

Nitronyl nitroxide (NIT) radicals (Fig. 1A) are a kind of novel free radical scavenger with remarkable pharmacological activities, including antioxidant, anti-ischemia-reperfusion injury [20], anti-nociceptive [21], anti-apoptosis [22], anti-virus [23], anti-tumor [24], anti-inflammatory and radioprotective [25] activities. Unlike traditional antioxidants that interact with lipid peroxyl radicals stochiometrically, NITs act as catalytic antioxidants and degrade free radicals similar to the ability of endogenous antioxidants (Fig. 1B), which increase efficacy and reduce the dosage required for protection. Furthermore, NITs are able to cross the blood-brain barrier (BBB) and permeate the cell membrane. Some appealing studies show that NITs possess neuroprotective effect and improve cognitive dysfunction. For example, Wang et al. found that NIT derivative NRbt exhibited excellent protective effects against infrasound-induced learning and memory impairments by inhibiting oxidative stress and neuronal apoptosis [26]. Shi et al. also reported that the NIT derivative L-NNNBP could improve spatial learning and memory through anti-oxidation and anti-apoptosis in an Alzheimer’s disease (AD) model of APP/PS1 mice [27].

Fig. 1
figure 1

Introduction of nitronyl nitroxide radicals (NITs). A Chemical structures of NIT and Tempol. B The redox transformation of the various oxidation states of NITs. C Chemistry structure of HPN

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Our previous study proved that 4′-hydroxyl-2-subsitiuted phenylnitronyl nitroxide (HPN, Fig. 1) could protect against hypoxia induced damage in PC12 cells [28] and attenuate brain injury induced by HH in mice [29]. Nevertheless, it remained unclear whether HPN was useful in preventing and treating HH-induced cognitive impairment. Therefore, the aim of present study was to detect, for the first time, the protective effect and underlying mechanism of HPN on HH-induced memory injury in mice.

Materials and Methods

Reagents and Chemicals

HPN was prepared according to our previous reported method [29]. The commercial test kits for hydrogen peroxide (H2O2), malondialdelyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and reduced glutathione (GSH) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Bicinchoninic acid (BCA) protein assay kit was purchased from Solarbio Science & Technology Co., Ltd (Beijing, China). The enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) as well as the primary antibodies including nuclear factor kappa-B (NF-κB, ab16502; 1: 1000), TNF-α (ab34674; 1: 1000), B-cell lymphoma protein 2-associated X (Bax, ab32503; 1: 1000), B-cell lymphoma protein 2 (Bcl-2, ab196495; 1: 1000), and Cleaved caspase-3 (ab214430; 1: 1000) were from purchased Abcam (Cambridge Science Park, UK). Secondary antibodies such as HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Proteintech Group, Inc (Chicago, USA).

Experimental Animals

Male Balb/c mice aged 4–6 weeks old and weighed 20 g ± 2 g were obtained from Hunan SJA Laboratory Animal Co., Ltd (Changsha, China). All mice were housed in temperature-regulated animal facility at 22 ± 2 °C and humidity 55 ± 5% with 12 h light-dark cycle. The mice had free access to food and water. While the diet of the mice will be limited to 80% of the free intake for ensuring the experiment performed smoothly and keeping the mice healthy during eight-arm radical maze training and testing.

Eight-Arm Radical Maze Training

After 3 days of adaptive care, the mice underwent eight-arm radical maze training according to previous reported method in our lab [30], see Fig. 2A and B. In brief, mice were placed in the maze twice a day to adapt to the environment for a few days before training. During this period, baits were placed at the end of each arm. The mice could freely consume the baits in all eight arms for 10 min. When starting the training, the baits were only placed at the end of four of the eight arms (arms 1, 4, 5, 7). Mice were placed in the center of the labyrinth at the beginning of each trial and allowed to consume the baits within 5 min. A training session would be end when the mice ate all four cereal baits or once 5 min had elapsed. The maze was wiped with ethanol between each training session. The number of working memory errors (WME: entries into arm from which the baits had already been eaten), reference memory errors (RME: entries into arm that was never baited), total errors (TE: the total number of errors within 5 min, and the total reaction times (TT: the time taken for rats to eat all baits) were recorded using RM-200 radical maze analysis test system (Chengdu TME Technology Co, Ltd). The training was once daily for 21d and could be regarded as a success when five consecutive TE was less than 1 and the WME is 0.

Fig. 2
figure 2

Experimental procedures. A The schematic diagram of the experimental protocols used in this study. B eight-arm radical maze test. C The change of body weight of mouse during HH exposure. The results were expressed as the mean ± SD of ten independent experiments. *P < 0.5, ** P < 0.01 vs. Normoxic group

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Hypobaric Hypoxia Exposure

Fifty mice from the successfully trained ones were randomly divided into five groups, including normoxic group, HH group, and three HH + HPN groups [Low (25 mg/kg), Medium (50 mg/kg), and High (100 mg/kg)]. The mice were intraperitonally (i.p) administered with HPN or vehicle (equal volume of distilled water with 0.5% tween 80) 0.5 h before HH exposure. The doses of the HPN used in this study was determined according to our previous reported research [29]. Besides the normoxic group, the mice in other groups were exposed to HH (corresponding to 8000 m altitude) in a large decompression chamber (Guizhou Fenglei Aviation Ordnance Co., Ltd. model DYC-3070) for 3 days with free access to food and water. Each day the mice were brought to a high altitude at 4000 m for supplementation of HPN at 9.00 am ∼ 9.30 am and weight the body using electronic balance. After that, the altitude was reraised to 8,000 m. The mice in control group were maintained outside the chamber at the local altitude of 1,400 m (Lanzhou, China).

Determination of the Ability of Learning and Memory After HH Exposure

The eight-arm radical maze test was performed on mice in each group following HH exposure. The WME, RME, TE, and TT were recorded in each group of mice and each test was performed twice and averaged.

Nissl Staining and TUNEL Staining

Following behavior test, three mice of each group were anesthetized by intraperitoneal injection of 5% chloral hydrate (4 mg/kg) and killed by cervical dislocation. The brains were surgically dissected and then postfixed in 4% paraformaldehyde for 48 h, after which they were embedded in paraffin for staining. A series of 5-µm-thick sections were cut and stained with toluidine blue for Nissl staining or TUNEL reaction mixture for TUNEL staining.

Prepared of Serum and Hippocampus

After behavior test, blood samples of six mice in each group were collected by cardiac puncture under anesthesia and then the brain tissues were surgically removed from mice and washed in cold PBS. Hippocampus tissue was separated from the brain, rinsed with cold PBS, and kept at -80 °C for subsequent assays. Serum was separated by centrifugation at 3,000 rpm for 15 min at 4 °C and used for subsequent measurement of the levels of TNF-α, IL-1β, and IL-6.

Determination of Oxidative Stress Parameters in the Hippocampus

The hippocampus tissues six mice in each group were homogenized 1:10 with phosphate buffer saline (pH = 7.4) and centrifuged at 12,000 rpm for 10 min. The supernatant was collected and used for determining the concentration of H2O2, MDA, GSH and the activities of SOD, CAT, GSH-Px according to the manufacturer’s instructions. The protein content in the hippocampus homogenate was determined by BCA method.

Measurements of Cytokines in Serum and Hippocampus

The contents of IL-6, IL-1β, and TNF-α in serum and hippocampus were detected using related ELISA kit, according to the manufacturer’s instructions. The results of the levels of inflammatory cytokines in serum and hippocampus were expressed as pg/ml and pg/mg protein, respectively.

Western Blot Analysis

The hippocampus tissues of mice were homogenized in ice cold RIPA buffer and centrifuged at 14,000 × g for 30 min at 4 °C. Total protein concentrations were determined using BCA protein assay kits. Equal amounts of protein were loaded on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h in skim milk at room temperature (RT), followed by incubation with the primary antibodies against β-actin (1:2000), NF-kB (p65) (1:1000), TNF-α (1:1000), Bax (1:1000), Bcl-2 (1:1000), cleaved caspase-3 (1:1000), Nrf2(1:1000) and HO-1(1:1000) overnight at 4 °C. Thereafter, PVDF membranes were washed three times with TBST and incubated with appropriate secondary antibodies at RT for 2 h. The immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) kit (Amersham Bioscience, GE Healthcare, UK) and quantified by Image-Pro Plus 6.0 software for data analysis. β-actin was as the internal reference.

Statistical Analysis

The data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test were performed to examine the significant differences among the groups. P-values of < 0.05 was considered to be significant.

Results

Effects of HPN on Body Weight of Mice Under HH Condition

As shown in Fig. 2C, the initial weights were similar in all groups, while the body weights of mice in normoxic group showed an upward trend at the end of experiment. However, after HH exposure for 3 days the body weight of mice markedly decreased. Treatment with HPN slightly but not significantly reduced the weight loss of mice following HH exposure.

Effects of HPN on Learning and Memory of Mice Under HH Condition

The eight-arm radical maze, which has been widely used to examine the learning and spatial memory disorders, was adopted to evaluate the protective effect of HPN on the HH-induced cognitive impairment. As shown in Fig. 3, WME, RME, TE, and TT in HH group were significantly greater than those in normoxic group (P < 0.05 or P < 0.01), indicating that the HH exposure caused spatial memory impairment in mice. Treatment with HPN remarkable rescued the WME, RME, TE, and TT compared with HH group (P < 0.01), suggesting that HPN could ameliorate the memory impairment induced by HH.

Fig. 3
figure 3

Effects of HPN administration on HH-induced cognitive impairment in mice. A Representative track plots of mice in all experimental groups. Graphical representation of B working memory error (WME), C reference memory error (RME), D total errors (TE), and E total time (TT). The results were expressed as the mean ± SD of ten independent experiments. *P < 0.5, **P < 0.01 vs. Normoxic group, #P < 0.05, ##P < 0.01 vs. HH group

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Histopathological Analysis

Nissl staining was performed to explore the role of HPN in pathological changes of hippocampus in mice. As shown in Fig. 4, the cornu ammonis area 1 (CA1) region neurons in normoxic group were arranged neatly. However, the arrangement of neurons was disordered and sparse in the HH group. Treatment with HPN improved the pathological changes of neurons in the hippocampus in a dose dependent manner.

Fig. 4
figure 4

Effects of HPN administration on histopathological changes induced by HH in the hippocampus of mice. Representative images of Nissl staining of the hippocampus CA1 sections

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Effects of HPN on the Oxidative Status in the Hippocampus of Mice Following HH Exposure

The oxidative status was evaluated by assessing the levels of H2O2 and MDA in the hippocampus. As shown in Fig. 5A and B, H2O2 and MDA content were significantly elevated in the HH group compared to the normoxic group (P < 0.01), whereas they were significantly decreased after HPN treatment in the M-HPN and H-HPN groups in contrast to the HH group (P < 0.05 or P < 0.01, respectively). HPN at the dose of 25 mg/kg in the L-HPN group did not cause any significant decrease in the H2O2 and MDA content compared to the HH group (P > 0.05).

Fig. 5
figure 5

Effects of HPN on the parameters of oxidative stress and the antioxidant status in the hippocampus of mice following HH exposure. The levels of A H2O2, B MDA, C SOD, D CAT, E GSH-Px and F GSH in the hippocampus. The results were expressed as the mean ± SD of six independent experiments. *P < 0.5, **P < 0.01 vs. Normoxic group, #P < 0.05, ##P < 0.01 vs. HH group

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Effects of HPN on the Antioxidant Status in the Hippocampus of Mice Following HH Exposure

The influence of HPN on HH-induced change in the hippocampus antioxidant status was illustrated in Fig. 5C–F. Compared with the normoxic group, the SOD, CAT and GSH-Px activities and GSH content in the hippocampus of mice in the HH group were significantly decreased (P < 0.01). While treatment with HPN (50 and 100 mg/kg) could significantly increase the SOD, CAT and GSH-Px activities and GSH content (P < 0.05 or P < 0.01) as compared to HH group. No significant differences were observed on the SOD, CAT and GSH-Px activities and GSH content between L-HPN group and HH group (P > 0.05).

Effects of HPN on the Expression Levels of Nrf-2 and HO-1 in the Hippocampus of Mice Following HH Exposure

Activation of Nrf2 and downstream genes has been linked to strengthening antioxidant defense and cytoprotection. As illustrated in Fig. 6, compared with the normoxic group, HH exposure markedly upregulated the expression levels of Nrf-2 in nucleus and HO-1 in cytosol (P < 0.01). While the expression of Nrf-2 and HO-1 in the hippocampus of mice in the M-HPN and H-HPN groups further significantly increased (P < 0.05) compared with the HH group. No significant differences were observed on the expression of Nrf-2 and HO-1 proteins in the hippocampus of mice between L-HPN group and HH group (P > 0.05). These results indicated that HPN ameliorated HH-induced oxidative stress via activating of Nrf2/HO-1 pathway.

Fig. 6
figure 6

Effects of HPN on the expression of Nrf2 and HO-1 in the hippocampus of mice following HH exposure. A Representative western blots of Nrf2 and HO-1 expression. B Quantitative analysis of Nrf2 expression. C Quantitative analysis of HO-1expression. The band intensity was quantified by Image-Pro Plus 6.0 software. The results were expressed as the mean ± SD of four independent experiments. *P < 0.5, **P < 0.01 vs. Normoxic group, #P < 0.05, ##P < 0.01 vs. HH group

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Effects of HPN on Pro-inflammatory Cytokine Induced by HH in Serum and Hippocampus

HPN attenuated HH-induced increase in the serum and hippocampus pro-inflammatory cytokines. As shown in Fig. 7, compared to the normoxic group, the HH group showed a significant increase in serum IL-1β (25.23%), IL-6 (21.91%) and TNF-α (37.38%) (P < 0.01). While treatment with HPN (50 and 100 mg/kg) showed a significant reduction in serum IL-1β by 6.6% and 10.4%, in serum IL-6 by 9.5% and 12.3% and in serum TNF-α by 12.3% and 17.0%, respectively as compared to HH group (P < 0.05 or P < 0.01). Same as the trends in serum, HH exposure also significantly elevated the levels of IL-1β, IL-6 and TNF-α in the hippocampus as compared to normoxic group (P < 0.01). As expected, compared with those in HH group, the middle and high dose group of HPN could notably decrease the contents of IL-1β, IL-6 and TNF-α in the hippocampus (P < 0.05 or P < 0.01). The levels of IL-1β, IL-6 and TNF-α in L-HPN group were not significantly changed in the serum and hippocampus compared with the HH group (P > 0.05).

Fig. 7
figure 7

Effects of HPN on the production of proinflammatory cytokines in serum and hippocampus of mice following HH exposure. The contents of A and D IL-1β, B and E IL-6, C and F TNF-α in serum and hippocampus. The results were expressed as the mean ± SD of six independent experiments. *P < 0.5, **P < 0.01 vs. Normoxic group, #P < 0.05, ##P < 0.01 vs. HH group

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Effects of HPN on Inflammation Related Proteins in the Hippocampus of Mice Following HH Exposure

As shown in Fig. 8, compared with the normoxic group, the expression of NF-κB and TNF-α protein in the hippocampus of mice in the HH group increased significantly by 1.6 and 1.5 fold, respectively (P < 0.01). By contrast, NF-κB protein in the hippocampus of mice in the M-HPN and H-HPN groups significantly decreased by 30.9% and 58.3%, respectively (P < 0.01); while the expression of TNF-α protein in the hippocampus of mice in the M-HPN and H-HPN groups significantly increased by 38.9% and 50.1% (P < 0.05), respectively. No significant differences were observed on the expression of NF-κB and TNF-α protein in the hippocampus of mice between L-HPN group and HH group (P > 0.05).

Fig. 8
figure 8

Effects of HPN on the expression of inflammation related proteins in the hippocampus of mice following HH exposure. A Representative western blots of NF-κB and TNF-α expression. B Quantitative analysis of NF-κB expression. C Quantitative analysis of TNF-α expression. The band intensity was quantified by Image-Pro Plus 6.0 software. The results were expressed as the mean ± SD of four independent experiments. *P < 0.5, **P < 0.01 vs. Normoxic group, #P < 0.05, ##P < 0.01 vs. HH group

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Effects of HPN on Apoptosis in the Hippocampus of Mice Following HH Exposure

As revealed in Fig. 9, TUNEL staining showed that there were significantly more apoptotic positive cells in the HH group than in normoxic group. By contrast, administrations of HPN (50 and 100 mg/kg) effectively decreased the apoptotic positive cells.

Fig. 9
figure 9

Effects of HPN administration on cell apoptosis induced by HH in the hippocampus of mice. Representative images of TUNEL staining of the hippocampus CA1 sections. The green cells indicate the TUNEL-positive apoptotic cells

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Effects of HPN on Apoptosis Pathway-Related Proteins in the Hippocampus of Mice Following HH Exposure

As revealed in Fig. 10, HH exposure contributed to increase the expression of Bax, and cleaved-caspase-3 and the ratio of Bax/Bcl-2 as well as decreased the expression of Bcl-2 compared with normoxic group (P < 0.01). By contrast, administrations of HPN (50 and 100 mg/kg) effectively decreased the expressions of Bax, cleaved-caspase-3, and the ratio of Bax/Bcl-2, as well as increased the expressions of Bcl-2 in the hippocampus (P < 0.01 or P < 0.05). HPN at the dose of 25 mg/kg in the L-HPN group did not cause any significant changes in the expression of apoptosis pathway-related proteins compared to the HH group (P > 0.05).

Fig. 10
figure 10

Effects of HPN on the expression of apoptosis-related proteins in the hippocampus of mice after following exposure. A Representative western blots of Bcl-2, Bax and Caspase-3 expression. B Quantitative analysis of Bcl-2 expression. C Quantitative analysis of Bax expression. D Quantitative analysis of the ratio of Bax/Bcl-2. E Quantitative analysis of Caspase-3 expression. The band intensity was quantified by Image-Pro Plus 6.0 software. The results were expressed as the mean ± SD of four independent experiments. *P < 0.5, **P < 0.01 vs. Normoxic group, #P < 0.05, ##P < 0.01 vs. HH group

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Discussion

It has been clearly demonstrated that HH impaired cognitive function specifically affecting spatial learning and memory, which may incapacitate or compromise an individual’s performance of highly demanding mental functions [31]. The present study, for the first time, explored the protective effect of HPN on cognitive impairments under HH.

It is well accepted that HH causes weight loss, which might be attributed to the reduced food intake and increases energy expenditure induced by altitude-induced anorexia [32]. Similarly, we also found that mice exposed to HH showed significant body weight loss. However, treatment with HPN failed to reversed this change indicating that HPN had no impact on the energy intake.

In the present study, eight-arm radical maze test, which has been wildly used for evaluating spatial memory in rodent model [33, 34], was used to investigate the effect of HPN on cognitive function after HH exposure. In the current study, we found that HH group displayed learning and memory deficits with increased WME, RME, TE, and TT, which is in agreement with previous studies [17, 35, 36]. In contrast, the HPN group exhibited much smaller WME, RME, TE, and TT compared with the HH group, which indicated HH-induced impairment of spatial memory was rescued by HPN administration.

Animal studies have suggested that that CA1 region in the hippocampus, which is associated with learning and memory deficits, is the most vulnerable to HH exposure [11, 12, 37]. In according with these reports, we also found that HH exposure affected the neurons of CA1 region of hippocampus, where cells were irregularly arranged with reduced Nissl bodies when compared to the cells were in order with numerous Nissl bodies found in normoxic group. Treatment with HPN could alleviate HH-induced alterations.

Abundant investigations have proved that oxidative stress significantly increases under HH condition and plays an important role in the progression of cognitive impairments. HH exposure can induce the excessive generation of reactive oxygen species (ROS), which will lead to neurotoxicity and endanger the cellular function and viability by changing the neural fat composition, inducing membrane lipid peroxidation to generate a large amount of MDA, and altering the membrane integrity, fluidity, and permeability [38]. Besides, HH also inhibits the endogenous enzymatic (SOD, CAT, GSH-Px) and nonenzymatic (GSH, Vit. C) antioxidant systems, resulting in the accumulation of free radicals and aggravation of oxidative stress [14]. The brain is very vulnerable to oxidative stress injury due to high content of unsaturated fatty acids, and deficiency of antioxidant enzymes. In the current study, HH resulted in a significant increased oxidative stress markers (H2O2 and MDA) along with decreased effectiveness of antioxidant system (SOD, CAT, GSH-Px and GSH). HPN treatment significantly reduced the content of H2O2 and MDA, prevented reduction in GSH level and activities of SOD, CAT and GSH-Px in the hippocampus of mice following HH exposure. Numerous studies have shown that activation of the Nrf2/HO-1 antioxidant pathway exerts pivotal protective role against oxidative stress induced by HH [39, 40]. In current study, HH promoted Nrf2/HO-1 signaling as evidenced by the increased levels of Nrf2 and HO-1 in the cytosol or nuclei, which might be regarded as a compensatory effect to augment the ROS induced by HH. HPN treatment further promoted Nrf2/HO-1 signaling, as shown by the upregulated expressions of Nrf2 in nucleus and HO-1 in cytosol. These results indicate that HPN plays a positive protective role in HH-induced cognitive dysfunction, and its possible mechanism is to improve hippocampus antioxidant ability via activating the Nrf2/HO-1 pathway.

Generally, inflammation response, which characterized by the accumulation of inflammatory cells and the release of pro-inflammatory responses (TNF-α, IL-1β and IL-6), is often accompanied by oxidative stress under HH condition. Inflammatory cells can release of ROS and exaggerate oxidative damage. On the other hand, oxidative stress products enhance pro-inflammatory responses. Extensive studies have reported that the levels of proinflammatory cytokines are elevated in individuals exposed to high altitude [41,42,43]. Animal studies also indicate that HH significant increases the levels of IL-1β, IL-6, and TNF-α in serum and brain [44, 45]. High level of proinflammatory mediators is known to worsen HH induced injury. All these researches indicated that neuroinflammation had significant contributions to the cognitive impairments at high altitude. Accordingly, our results implied that HH exposure could induce hippocampal neuroinflammation, featuring as the increase of proinflammatory mediators, including IL-1β, IL-6, and TNF-α both in the serum and hippocampus of mice. Conversely, we found that HPN administration notably suppressed the production of these proinflammatory mediators both in serum and hippocampus of mice. It is known that NF-B signaling pathway plays an important role in inflammation. NF-κB is an important transcription factor widely distributed in cells and tissues and initiates and regulates inflammatory response [46]. Under normal conditions, NF-κB is sequestered by bounding to IκBα (NF-κB inhibitor protein) in the cytosol. While in some stress situations, such as oxidative stress [47], IκBα is phosphorylated and degraded, thus resulting in the phosphorylation and translocation of NF-κB into the nucleus to promote transcription of target genes, including TNF-α, IL-1β, and ICAM-1. Several studies have suggested that the cyclic nitroxide Tempol (Fig. 1A) exerted anti-inflammatory properties by reducing the activation of NF-κB in many animal models [48, 49]. HPN owned the same functional group (N-O) with tempol in the structure. It is reasonable to hypothesis that HPN might exhibit similar anti-inflammatory mechanism. Therefore, the expression of NF-κB and TNF-α in the NF-κB signaling pathway were investigated in the present study. Our experimental results confirmed that HH exposure significantly upregulated the expression of NF-κB and TNF-α protein in the hippocampus of mice. Similar to our research, Luan et al. reported that HH could activate NF-κB signaling pathway and increase the protein expression levels of IL-1β and TNF-α in the brain [45]. Treatment with HPN obviously attenuated the expression of NF-κB and TNF-α in the hippocampus of mice following HH exposure. These results indicate that HPN exerts an anti-inflammatory activity via inhibiting the activation of NF-κB pathway.

It has been demonstrated that abnormal neuron loss due to cell apoptosis is one of the reasons for cognitive impairment after exposed to HH [16, 50]. Thus, suppression of apoptosis is considered a therapeutic strategy to protect against HH mediated cognitive deficits. Previous studies have indicated that HPN exhibited anti-apoptotic activity in vitro [28] and in vivo [51]. In the current study, we also found that apoptotic neural cells in the hippocampus were significantly increased in the HH group and HPN treatment significantly inhibited the apoptosis of neural cells. In order to understand the underlying mechanisms of apoptosis modulation with HH exposure and HPN treatment, the expression of apoptotic-related proteins in the hippocampus was investigated in current study. Bcl-2 family is known to play a key role in promoting or inhibiting intrinsic apoptotic pathway triggered by mitochondrial dysfunction. Bcl-2 and Bax, two important members of the Bcl-2 gene family, are known as anti-apoptotic and pro-apoptotic proteins, respectively. The unbalance of Bcl-2 and Bax leads to abnormal neuron loss and ultimately results in cognitive impairment. Caspase-3 is the most important initiator and performer of terminal cleavage enzymes and apoptosis. In the present study, we demonstrated that HH exposure dramatically down-regulated the expression of Bcl-2 and up-regulated the expression of Bax and cleaved caspase-3 in the hippocampus of mice. However, treatment with HPN suppressed the up-regulation of Bax and cleaved-Caspase-3 and increased the Bcl-2 protein expression. These results suggest that HPN may protect mice from HH induced cognitive deficits via reducing the apoptosis of hippocampal cells.

Conclusion

The results obtained in this study demonstrate that HPN can improve HH-induced cognitive impairment in mice, possibly through multiple mechanisms including the alleviation the oxidants accumulation, suppression of the inflammatory response and inhibition of neural apoptosis. These findings indicate that HPN may be effective as a novel therapeutic agent for prevention and treatment of HH-induced memory impairment. Study of the safety and pharmacokinetics of HPN will be performed in the future.