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Hydrogen-rich water attenuates oxidative stress in rats with traumatic brain injury via Nrf2 pathway
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Hydrogen-rich water attenuates oxidative stress in rats with traumatic brain injury via Nrf2 pathway Jia Yuan, MD, Difen Wang, MD,* Ying Liu, MD, Xianjun Chen, MD, Hailing Zhang, MD, Feng Shen, MD, Xu Liu, MD, PhD, and Jiangquan Fu, MD Department of Critical Care Medicine, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou Province, P.R. China
article info
abstract
Article history:
Background: Several studies have recently found that oxidative stress plays a pivotal role in
Received 27 November 2017
the pathogenesis of traumatic brain injury (TBI) and may represent a target in TBI treat-
Received in revised form
ment. Hydrogen-rich water was recently shown to exert neuroprotective effects in various
29 January 2018
neurological diseases through its antioxidant properties. However, the mechanisms un-
Accepted 14 March 2018
derlying its effects in TBI are not clearly understood. The purpose of our study was to
Available online xxx
evaluate the neuroprotective role of hydrogen-rich water in rats with TBI and to elucidate the possible mechanisms underlying its effects.
Keywords:
Materials and methods: The TBI model was constructed according to the modified Feeney
Hydrogen-rich water
weight-drop method. In part 1 of the experiment, we measured oxidative stress levels by
Nrf2 pathway
observing the changes in catalase (CAT), glutathione peroxidase (GPx), and malondialde-
Traumatic brain injury
hyde (MDA) expressions. We also evaluated nuclear factor erythroid 2erelated factor 2
Oxidative stress
(Nrf2) levels to determine the role of the protein in the neuroprotective effects against TBI. In part 2, we verified the neuroprotective effects of hydrogen-rich water in TBI and observed its effects on Nrf2. All the experimental rats were divided into sham group, TBI group, and TBI þ hydrogen-rich water-treated (TBI þ HW) group. We randomly chose 20 rats from each group and recorded their 7-d survival rates. Modified neurological severity scores were recorded from an additional six rats per group, which were then sacrificed 24 h after testing. Spectrophotometry was used to measure GPx, CAT, and MDA levels, whereas western blotting, reverse transcription polymerase chain reaction, and immunohistochemistry were used to measure the expression of Nrf2 and downstream factors like heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). Results: GPx and CAT activity was significantly decreased, and MDA content was increased in the TBI group compared with the sham group at 6 h after TBI. MDA content peaked at 24 h after TBI. Nrf2 nucleoprotein levels were upregulated in the TBI group compared with the sham group and peaked at 24 h after TBI; however, no significant changes in Nrf2 mRNA levels were noted after TBI. Hydrogen-rich water administration significantly increased 7-
* Corresponding author. Department of Critical Care Medicine, The Affiliated Hospital of Guizhou Medical University, Guiyi Road No. 28, Yunyan District, Guiyang 550004, Guizhou Province, P.R. China. Tel./fax: þ86 0851 86772049. E-mail address: [email protected] (D. Wang). 0022-4804/$ e see front matter ª 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2018.03.024
yuan et al hydrogen rich water protects traumatic brain injury via nrf2 pathway
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d survival rates, reduced neurologic deficits, and lowered intracellular oxidative stress levels. Moreover, hydrogen-rich water caused Nrf2 to enter the cell nucleus, which resulted in increases in the expression of downstream factors such as HO-1 and NQO1. Conclusions: Our results indicate that hydrogen-rich water has neuroprotective effects against TBI by reducing oxidative stress and activating the Nrf2 pathway. ª 2018 Elsevier Inc. All rights reserved.
Introduction Traumatic brain injury (TBI) remains a great health concern, as the condition imposes serious mental and economic burdens on both families and society.1 The pathogenesis of TBI encompasses primary and secondary injuries. Primary injury is the initial mechanical injury to which brain cells are subjected, and secondary injury develops within hours, days, or even months after the primary mechanical injury. Complicated pathophysiologic processes, including calcium overload, inflammatory responses, neuronal apoptosis, glutamate-mediated excitotoxicity, and oxidative stress, occur in secondary injury.2-5 Oxidative stress is of great importance in TBI-induced secondary injury.5 However, the evidence regarding the effects of antioxidant therapies in patients with TBI in clinical practice is limited.6 TBI induces a series of delayed secondary biochemical and cellular metabolic changes and eventually causes a considerable increase in reactive oxygen species (ROS) production, thereby accelerating cell damage and neuronal dysfunction.7,8 Enormous oxygen free radicals released after TBI caused the lipid peroxidation in the membranes of cells, resulting in an increasing of cellular malondialdehyde (MDA) content.9 Glutathione peroxidase (GPx) specifically catalyzes hydrogen peroxide (H2O2) and other organic hydroperoxides into water with the glutathione being used as reducing agent, by which interrupts the process of the lipid peroxidation chain reaction.10 Catalase (CAT) protects cellular membrane structure and functional integrity against harmful effects of H2O2 by facilitating its degradation to oxygen and water.11 GPx and CAT play crucial roles in regulating the oxidative status in the cell after TBI.12 The nuclear factor erythroid 2erelated factor 2 (Nrf2) pathway, a significant endogenous antioxidant system,13 regulates the expression of more than 200 encoded endogenous protection genes and phase II detoxification enzymes. Nrf2 is a key regulatory protein in the antioxidant system and has been widely studied in many central nervous system (CNS) diseases,14-17 including TBI.17 Under normal physiological conditions, Nrf2 is bound to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm to promote the proteasomal degradation of Nrf2. Once exposed to ROS, Nrf2 separates from Keap1, resulting in the translocation of Nrf2 from the cytoplasm to the nucleus, wherein the protein binds to the antioxidant response element,18 through which it may produce a series of endogenous enzymes, such as heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). These enzymes enable cells to survive the effects of oxidative stress and many other toxins.19 Hydrogen gas (H2) is a colorless and tasteless gas. In 2007, H2 was shown to exert antioxidant effects on rats subjected to
ischemia-reperfusion (I/R) injury by selectively scavenging the hydroxyl radical, one of the most cytotoxic ROS.20 Hydrogenrich water, which comprises a therapeutic dose of hydrogen dissolved in normal saline/pure water, is produced and sold commercially.21 Hydrogen-rich water exerts an antioxidant effect similar to that of hydrogen inhalation treatment and possesses a good safety profile, is portable, and is easily administered.22 Hydrogen-rich water has also been widely noticed for its ability to repress inflammatory reactions and to attenuate neuronal apoptosis.23,24 Moreover, our previous study found that hydrogen-rich water promotes angiogenesis by upregulating HIF-1a and VEGF expressions in a TBI model.25 The antioxidative effect of hydrogen is the most important of its many beneficial effects and has been demonstrated in many diseases, such as hyperoxic lung injury, renal I/R injury, acute pancreatitis, sepsis, and several CNS diseases, including TBI.21,26-31 Although hydrogen-rich water has been shown to play an antioxidant role in TBI,21,31 the mechanisms underlying its effects in TBI are not fully understood. Moreover, whether the antioxidative effects of hydrogen-rich water are associated with the Nrf2 pathway remains to be studied further. The objective of this study was to evaluate the role of hydrogenrich water in TBI rats and to elucidate the possible mechanisms underlying its effects.
Materials and methods Experimental animals All Sprague-Dawley rats, adult males weighing 250-300 g (Animal Certificate No: SCXK (Army) 2012-0011), were purchased from the Third Military Medical University (Chongqing, China). The rats were placed in air-filtered, temperature-controlled rooms and were allowed free access to food and water. The experiments were approved by the Animal Care Committee of Guizhou Medical University and were conducted in accordance with the guiding principles for animal research of Guizhou Medical University. We attempted to minimize animal suffering in all experiments.
Construction of the TBI model The TBI model was constructed according to Feeney’s weightdrop method, with some modifications.32 Briefly, the rats were anesthetized with 10% chloral hydrate (3.5 mL/kg intraperitoneally [i.p.]), after which they were fixed in the prone position on a brain stereotaxic frame (RWD Life Science Co, Shenzhen, China) by a weight-drop device. We exposed the skull at the midline and drilled a hole with a diameter of
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5.0 mm 5.0 mm in the right parietal bone. The rats were then subjected to TBI in the right hemisphere (Fig. 1) of the brain. Specifically, each rat was struck with a force of 40 g 40 cm (a 40-g weight was dropped from a height of 40 cm), resulting in a brain contusion involving the right parietal lobe. Suture material was used to close the scalp wound. After recovering from anesthesia, the rats were returned to their feeding cages, where they were allowed free access to water and food.
Experimental design Part 1: measurement of oxidative stress levels and elucidation of the protective effects of Nrf2 against TBI We observed the changes in CAT, GPx, MDA, and Nrf2 expressions in TBI. The following two groups were studied in the experiment: a sham group (n ¼ 6) and a TBI group (n ¼ 30). The rats in the TBI group were subjected to TBI and were subdivided into the following five groups: a 6-h group, a 12-h group, a 24-h group, a 48-h group, and a 5-d group. The animals in the sham group underwent an identical surgical procedure but were not subjected to the hemisphere strike.
Part 2: assessment of the neuroprotective effects of hydrogenrich water in TBI and its effects on Nrf2 A total of 78 male rats were randomly divided into sham group, TBI group, and hydrogen-rich water-treated TBI (TBI þ HW) group, with 26 rats in each group. The rats in the TBI and TBI þ HW groups were subjected to TBI. The experimental protocols used for this part of the experiment were the same as those used for part 1. The rats in the TBI þ HW group were all i.p. injected with 5-mL/kg hydrogen-rich water (Hydrovita Biotechnology Co, Beijing, China) at 5 min after TBI, whereas the rats in the sham and TBI groups were all i.p. administered with 5-mL/kg normal saline, which was used as
a control treatment. Finally, 20 rats from each group were randomly selected, and their 7-d survival rate was recorded. We also subjected an additional six rats from each group to modified neurological severity score (mNSS) testing and then sacrificed them 24 h after the test.
Neurological deficit evaluation The mNSS was determined to evaluate TBI-induced neurobehavioral deficits in the rats at 24 h following TBI or sham operation. The mNSS score was determined by tests of motor skills, sensory capabilities, reflexes, and balance. The highest possible mNSS is 18, and higher scores are indicative of more severe neurological damage.33 Neurological injury according to the mNSS was graded as follows: severe injury (score of 1318), moderate injury (score of 7-12), mild injury (score of 1-6), and no injury (score of zero).
Determination of GPx, CAT, and MDA levels The rats were sacrificed under deep anesthesia, and the ipsilateral cortex, that is, the cortex containing the injury site, was collected. A portion of the tissue was weighed, homogenized with iced normal saline (1/9, w/v), and then centrifuged for 15 min at 4 C (3000 g/min). The appropriate commercial kits (Nanjing Jiancheng, Nanjing, China) were used to measure antioxidant enzymatic (GPx and CAT) activity and oxidative product (MDA) levels. All the experiments were performed in strict accordance with the instructions provided with each kit. A BCA Protein Assay Reagent Kit (Beyotime Biotechnology, Shanghai, China) was used to measure the protein concentration. Absorbance values were determined by a spectrophotometer.
Immunohistochemistry The tissues were immersed in 10% paraformaldehyde overnight for immunohistochemistry assay. Coronal tissues were consecutively cut into 5-mm sections to collect the damaged cortex. Endogenous peroxidase activity was blocked by 3% H2O2 after routine deparaffinization. The sections were incubated in blocking buffer for 20 min to block nonspecific antibody binding, after which they were incubated with primary antibodies against Nrf2 (1:150; Abcam, Cambridge, MA) overnight at 4 C. The sections were subsequently washed with PBS (3 times, 5 min/wash) and incubated with a secondary antibody conjugated with IgGHRP (1:500; Shengda, Guangzhou, China) for 25 min at 37 C. We counted the number of positive cells in three fields and then used the average of this number as the final data for each sample. Specifically, three fields in each sample were randomly selected, and then the cells were counted under a 400 light microscope. The entire procedure was performed in a blinded manner by two pathologists.
Western blot analysis
Fig. 1 e The TBI site in the rat model. (Color version of figure is available online.)
The preserved brains were cut into pieces and lysed after freezing and thawing. The cytoplasmic and nuclear proteins were extracted according to the instructions of the
yuan et al hydrogen rich water protects traumatic brain injury via nrf2 pathway
appropriate kit (KeyGen Biotech, Nanjing, China), and a BCA Protein Assay Reagent Kit was used to measure the concentrations of these proteins. The proteins (40 mg) were separated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. TBST with 5% skim milk buffer was used to block nonspecific binding for 2 h at room temperature. The membranes were incubated with the appropriate primary antibodies at the appropriate dilutions overnight at 4 C (mouse anti-rat Nrf2 antibody, 1:3500; rabbit anti-rat NQOl antibody, 1:10,000; rabbit anti-rat HO-1 antibody, 1:10,000 [all from Abcam]; anti-histone 3 [H3, 1:5000; Abmart, Shanghai, China]; and anti-b-actin [1:3500; Abmart]). All the membranes were subsequently incubated with the appropriate secondary antibodies (1:5000, goat anti-mouse/anti-rabbit; Abmart) for 2 h at room temperature. Finally, an Amersham ECL Plus Western Blotting Detection Kit (Merck Millipore, Billerica, MA) was used to detect the immunoreactive bands through chemiluminescence. The expression levels of the proteins of interest were normalized to those of b-actin or H3. All the procedures were performed in strict accordance with the instructions of the appropriate kits.
Reverse transcription polymerase chain reaction Total RNA was extracted with Trizol reagent using an extraction kit (Merck Millipore). After DNase digestion, nucleic acid concentrations and purity were assessed at 260/280 nm. A first-strand cDNA synthesis kit (Thermo Scientific, Waltham, MA) was then used to reverse-transcribe the RNA. The sequences of the primers (Biological Engineering Co, Shanghai, China) used for the experiment were as follows: Nrf2, 50 CAAATCCCACCTTGAACACA-30 and 50 -TGACTAATGGCAGCAGAGGA-30 ; HO-1, 50 -CAGAGTTTCTTCGCCAGAGG-30 and 50 GAGTGTGAGGACCCATCG -30 ; NQO1, 50 -ATTCCAGCCGACAACCAGAT-30 and 50 -CCGTGGCAGAACTATCCAAA-30 ; and band 50 actin, 50 -TGTCACCAACTGGGACGATA-30 GGGGTGTTGAAGGTCTCAAA-30 . The PCR amplification procedure comprised the following steps: incubation at 95 C for 10 min, followed by denaturation at 95 C for 15 s, annealing at 60 C for 60 s, and extension at 60 C for 60 s (42 reaction cycles comprising denaturation, annealing, and extension steps). The specificities of the primers were detected by melting curve analysis, and b-actin was used as an internal reference.
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Statistical analysis SPSS 20.0 was used for the statistical analysis. Quantitative data are represented by the mean standard deviation (SD). One-way analysis of variance followed by the least significant difference test was used for statistical and intergroup comparisons, and unpaired t tests were used to compare neurological deficit severities between two groups. The differences in survival were estimated by KaplaneMeier survival plots and the log-rank test. P < 0.05 was considered statistically significant.
Results Antioxidant enzyme (GPx and CAT) and oxidative product (MDA) levels in TBI As reported previously, CAT and GPx are indicators of antioxidant activity, and MDA is an indicator of lipid peroxidation. The levels of these three indicators were examined in rat tissues to determine the levels of oxidative stress induced by TBI. The data showed that CAT and GPx activity began to decrease at 6 h after TBI (P < 0.05, Fig. 2A and B) and then increased markedly from 48 h to 5 d after TBI. The lowest GPx and CAT activity levels were noted at 24 h after TBI. In addition, MDA levels increased significantly in the TBI group compared with the sham group (P < 0.05, Fig. 2C). Specifically, MDA levels began to increase at 6 h after TBI and peaked at 24 h after TBI. These findings suggest that TBI leads to decreases in GPx and CAT activity and increases in MDA levels in injured ipsilateral cortical tissue.
Changes in Nrf2 expression after TBI Western blotting and reverse transcription polymerase chain reaction (RT-PCR) were performed to detect Nrf2 protein and mRNA levels, respectively, in brain tissue at 6 h, 12 h, 24 h, 48 h, and 5 d after TBI. We found that Nrf2 protein expression levels were upregulated in TBI brains compared with sham brains and that Nrf2 protein expression began to increase at 6 h after TBI and peaked at 24 h after TBI (P < 0.05, Fig. 3A and B). However, the RT-PCR results were inconsistent with the western blotting results, as Nrf2 mRNA expression did not
Fig. 2 e Antioxidant enzyme (GPx and CAT) and oxidative product (MDA) levels in TBI. Bar graph illustrating the changes in (A) CAT, (B) GPx, and (C) MDA levels. Data are represented by the mean ± SD (n [ 6, *P < 0.05, **P < 0.01 versus the sham group).
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Fig. 3 e (A) Representative photographs of western blots showing the expression of the nuclear protein Nrf2 and the control H3 in the rat brain after TBI. (B) Nrf2 nucleoprotein expression (n [ 3, *P < 0.05, **P < 0.01 versus the sham group). (C) Nrf2 mRNA expression did not differ between TBI and each time point after TBI (n [ 6, P > 0.05 versus the sham group). Data are represented by the mean ± SD.
change significantly between TBI and any of the above postTBI time points (P > 0.05, Fig. 3C).
TBI group after hydrogen-rich water treatment (P < 0.01, Fig. 4B).
Hydrogen-rich water increased the 7-d survival rate and reduced neurologic deficits
Hydrogen-rich water attenuated oxidative stress after TBI
Survival was analyzed up to 7 d after TBI. The 7-d survival rate increased to 65% in the TBI þ HW group and was significantly higher than that in the TBI group (30% versus 65%, P < 0.05, Fig. 4A). The mNSS results showed that the rats showed neurological deficits of different severities at different time points after trauma. These included hemiplegia, sensory disturbances, ataxia, and myoclonus. The mNSS in the TBI þ HW group was lower than that in the
CAT and GPx levels decreased significantly after TBI (P < 0.01) but were almost normalized by hydrogen-rich water treatment (P < 0.01, Fig. 5A and B). Similarly, MDA levels increased significantly after TBI (P < 0.01 versus the sham group) but decreased significantly after hydrogen-rich water treatment (P < 0.01 versus the TBI group, Fig. 5C).
Hydrogen-rich water promoted the translocation of Nrf2 from the cytoplasm to the nucleus We performed western blotting to evaluate Nrf2 activation after hydrogen-rich water treatment. The results demonstrated that hydrogen-rich water treatment increased Nrf2 expression and significantly decreased cytoplasmic Nrf2 expression (P < 0.05 Fig. 6A and B), indicating that hydrogenrich water administration promotes the translocation of Nrf2 from the cytoplasm to the nucleus. The immunohistochemistry assay results confirmed these findings (Fig. 6C).
Hydrogen-rich water enhanced NQO1 and HO-1 protein and mRNA expressions Fig. 4 e Hydrogen-rich water has protective effects against TBI. (A) KaplaneMeier analysis showed that the 7d survival rate was almost 100% in the sham group, and that the rate was significantly different between the TBI and TBI D HW groups (n [ 20, 30% versus 65%, P < 0.05). (B) Neurological deficits were evaluated by the mNSS at 24 h after TBI. The mNSS was significantly reduced in the TBI D HW group compared with the TBI group (n [ 6, ## P < 0.01). (Color version of figure is available online.)
We investigated the expression of NQO1 and HO-1, which are downstream factors in the Nrf2 pathway. The western blotting results indicated that NQO1 and HO-1 protein expressions were upregulated in the TBI group compared with the sham group following TBI (P < 0.05, Fig. 7A and B). Hydrogen-rich water treatment significantly enhanced NQO1 and HO-1 protein expressions in the TBI þ HW group compared with the TBI group (P < 0.05, Fig. 7A and B). Consistent with these results,
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Fig. 5 e Hydrogen-rich water attenuated TBI-induced oxidative stress. Oxidative stress was evaluated by the measurement of (A) CAT, (B) GPx, and (C) MDA levels at 24 h after surgery. Data are represented by the mean ± SD (n [ 6, **P < 0.01 versus the sham group; ##P < 0.01 versus the TBI group).
the PCR results showed that NQO1 and HO-1 mRNA expressions were also significantly upregulated in the TBI group compared with the sham group, and that treatment with hydrogen-rich water increased NQO1 and HO-1 mRNA expressions in the TBI þ HW group compared with the TBI group (P < 0.05 Fig. 7C and D).
Discussion We investigated the neuroprotective effects of hydrogen-rich water in brain injuries induced by TBI and explored the possible mechanisms underlying these effects. We found that (1) oxidative stress, which is represented mainly by
antioxidant enzyme (GPx and CAT) and oxidative product (MDA) levels, is of great importance in TBI. Moreover, we noted that Nrf2 protein expression levels were upregulated after TBI and peaked at 24 h after TBI, a change that coincided with increases in oxidative stress levels. (2) Hydrogen-rich water had neuroprotective effects in TBI rats. Specifically, hydrogen-rich water increased 7-d survival rates, reduced neurologic deficits, and lowered intracellular oxidative stress levels. In addition, hydrogen-rich water induced the translocation of Nrf2 from the cytoplasm to the nucleus, which resulted in increases in the expression of downstream factors (HO-1 and NQO1). Neuronal dysfunction and death following TBI are largely dependent on secondary brain injury, which comprises a
Fig. 6 e Hydrogen-rich water accelerated the translocation of Nrf2 from the cell cytoplasm to the nucleus. (A, B) Western blot analysis showed that hydrogen-rich water increased Nrf2 levels in the nucleus and decreased Nrf2 levels in the cytoplasm. H3 and b-actin were used as internal references (n [ 3). Data are represented by the mean ± SD (@P < 0.05 versus the sham group, &P < 0.05 for cytoplasmic Nrf2 versus the TBI group, and #P < 0.05 for Nrf2 versus TBI group). (C) Representative photomicrographs of the immunohistochemical analysis of Nrf2 expression (tissues from different groups). Morphological examination showed that the TBI group featured a higher nuclear concentration of Nrf2 than the sham group. Morphological examination also showed that the concentration of Nrf2 was higher in the TBI D HW group than in the TBI group. (Color version of figure is available online.)
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Fig. 7 e Hydrogen-rich water upregulated the protein and mRNA expressions of downstream factors in the Nrf2 pathway. (A, B) HO-1 and NQO1 protein expressions was upregulated after TBI, and hydrogen-rich water elicited further increases in HO1 and NQO1 protein expressions (n [ 3, *P < 0.05 for HO-1 and @P < 0.05 for NQO1 versus the sham group; #P < 0.05 for HO1 and &P < 0.05 for NQO1 versus the TBI group). (C, D) NQO1 and HO-1 mRNA expressions were elevated after TBI, and hydrogen-rich water treatment elicited further increases in HO-1 and NQO1 mRNA expressions (n [ 6, **P < 0.01 versus the sham group and ##P < 0.01 versus the TBI group for HO-1, @P < 0.05 versus the sham group and &&P < 0.01 versus the TBI group for NQO1). Data are represented by the mean ± SD.
series of consecutive pathological processes that occur following primary injury and may develop within a few hours to a few days or months after injury.2 Increasing evidence suggests that oxidative stress plays a pivotal role in the pathophysiology of secondary brain injury caused by TBI.5 The production of excessive ROS as a result of the metabolism of nucleic acids, lipids, and proteins may cause serious damage to the cell after TBI.8 Several oxidative markers, including GPx and CAT, which are responsible for scavenging free radicals and catalyzing peroxides into nontoxic forms,10 are present after TBI. MDA is a common indicator of lipid peroxidation and reflects the level of free radicaleinduced damage.34 In our study, both GPx and CAT activity were markedly decreased in the TBI group compared with the sham group at 6 h after operation. However, MDA content was significantly increased in the TBI group compared with the sham group at 6 h after operation and peaked at 24 h after TBI. These findings indicated that oxidative stress was induced after TBI. Nrf2 is a key regulator factor of cellular antioxidants that is involved in secondary brain injury after TBI.35 Moreover, Nrf2 activators, such as ursolic acid, sulforaphane, tertbutylhydroquinone, and melatonin, have been confirmed to have the ability to attenuate post-TBI pathophysiologic phenomena, including neuronal apoptosis, cerebral edema, cognitive deficits, and oxidative stress.16,34,36,37 Nrf2 knock-
out mice had worse outcomes than wild-type mice in a previous study. However, the knock-out mice also displayed improvements in post-TBI neurologic function after ursolic acid administration.16 Our study demonstrated the time course of Nrf2 expression in rat brains subjected to TBI. Nrf2 protein expression levels were upregulated at 6 h after TBI and peaked at 24 h after TBI. Nrf2 levels initially remained high but then gradually decreased from 48 h to 5 d after TBI. These changes in Nrf2 expression coincided with the above mentioned changes in oxidative stress levels. These findings suggest that Nrf2 is important in protecting neurons from oxidative stress after TBI, and that Nrf2 upregulation at an early stage after TBI may be a target in its treatment. H2 is the predominant atmospheric gas, and its selective antioxidant function alleviates oxidative stresseinduced damage in the brain, heart, lung, intestine, kidney, and pancreas, as well as other major organs.30 Solubilized hydrogen (hydrogen-rich water) is a more convenient means of delivering molecular hydrogen.22 A hydrogen-rich water treatment significantly improved outcomes in TBI rats in previous studies.21,31 Similar findings have also been noted in several CNS diseases, such as hemorrhagic stroke, cerebral I/R injury, Alzheimer’s disease, and Parkinson’s disease, and the effects are attributable to the antioxidant properties of the water.27,38-40 In our study, we chose to administer 5-mL/kg hydrogen-rich water i.p. to explore its neuroprotective role in
yuan et al hydrogen rich water protects traumatic brain injury via nrf2 pathway
TBI. The dose used in this study was determined based on the results of earlier studies.31 Given that oxidative stress and Nrf2 nucleoprotein levels peaked at 24 h after TBI in part 1, we selected 24 h as the post-TBI observation time for part 2. The data showed that hydrogen-rich water significantly increased the 7-d survival rate and reduced neurologic deficits. Moreover, hydrogen-rich water administration attenuated oxidative stress after TBI, a result consistent with those of previous studies.31 Several in vivo and in vitro experiments have confirmed that the antioxidant effect of hydrogen is dependent not only on its ability to scavenge oxygen free radicals but also on its ability to regulate the Nrf2 signaling pathway.26,39,41 NQO1 and HO-1 are two of the most important antioxidant enzymes downstream of Nrf2, and both enzymes are associated with intracellular redox equilibrium.19 Hydrogen promotes the upregulation of Nrf2 and phase II enzymes, such as HO-1 and NQO1, in hyperoxic lung injury.26 Experimental data demonstrated that Nrf2 translocates from the cytoplasm to the nucleus following TBI, and that hydrogen-rich water accelerates this process. Hydrogen-rich water administration also significantly elevated the pre- and post-transcriptional levels of HO1 and NQO1 in the TBI þ HW group compared with the TBI and sham groups following TBI. These results indicated that hydrogen-rich water had neuroprotective effects against TBI by inhibiting oxidative stress, and that the mechanisms underlying the effects of Nrf2 may be associated with the translocation of the protein from the cytoplasm to the nucleus and the activation of downstream proteins. Interestingly, we found that the changes in Nrf2 mRNA expression were inconsistent with those in Nrf2 protein expression, as Nrf2 mRNA expression did not change significantly between the control group and each time point after TBI. Studies have shown that the Nrf2-Keap1 interaction is disrupted by the modification of Keap1 at cysteine and the phosphorylation of Nrf2 at serine.42 Several signaling pathway proteins, such as activated protein kinase C, phosphatidylinositol 3-kinase, and some mitogen-activated protein kinases, including extracellular signal-regulated kinase and c-Jun Nterminal kinase, reportedly phosphorylate Nrf2 directly and affect its translocation to the nucleus.43 According to the above mentioned evidence, we speculated that the regulation of Nrf2 transcriptional activity after TBI may occur at the posttranscriptional level rather than at the gene transcriptional level. However, Yang et al. found that Nrf2 gene and protein levels are upregulated in a cerebral ischemia rat model.44 We surmised that the differences between the results of that study and those of our study may be related to between-study differences in animal models and detection methods. This phenomenon needs to be explored further in the future. The limitations of this study should be mentioned. First, hydrogen-rich water was administered only at 24 h after TBI; thus, we do not know how effective this treatment is at other time points. In addition, the 24-h therapeutic window used in the present study is relatively narrow. Second, hydrogen-rich water may have anti-inflammatory effects, anti-apoptotic effects, and other effects against TBI that were not discussed in our study. Finally, the neuroprotective effects of hydrogen-rich water were not verified in Nrf2/ TBI mice. The use of such mice would confirm whether the effects of
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hydrogen-rich water are associated with Nrf2 signaling pathway activation.
Conclusions Our data showed that hydrogen-rich water has neuroprotective effects in TBI by reducing oxidative stress, and that Nrf2 signaling pathway activation may be one of the mechanisms by which hydrogen-rich water exerts its effects.
Acknowledgment This work was supported by grants from the High-level Personnel of Special Support Program (No. TZJF-2011-25), the Science & Technology Department (No. SY [2010] 3079), and the Key Specialty Construction of Clinical Projects (No. [2011] 52) in Guizhou Province, as well as the National Key Specialty Construction of Clinical Projects (No. [2011] 170) in China. The authors thank the Clinical Research Center of the Affiliated Hospital of Guizhou Medical University for their support during the experiment. Authors’ contributions: J.Y., D.W., and Y.L. conceived and designed the experiments. J.Y. performed the experiments, analyzed the data, and wrote the article. Y.L. supervised the experiments and contributed reagents/materials/analysis tools. X.C. and H.Z. contributed to the data collection. F.S., X.L., and J.F. supervised the data analysis and critically reviewed the article.
Disclosure The authors report no proprietary or commercial interests with respect to any product mentioned or concept discussed in the article.
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biomarkers and detection of free radical intermediates. J Neurochem. 2000;75:2178e2189. Smith JA, Park S, Krause JS, et al. Oxidative stress, DNA damage, and the telomeric complex as therapeutic targets in acute neurodegeneration. Neurochem Int. 2013;62:764e775. Wang L, Wang L, Dai Z, et al. Lack of mitochondrial ferritin aggravated neurological deficits via enhancing oxidative stress in a traumatic brain injury murine model. Biosci Rep. 2017;37:1e10. Miller DM, Wang JA, Buchanan AK, et al. Temporal and spatial dynamics of Nrf2-antioxidant response elements mediated gene targets in cortex and hippocampus after controlled cortical impact traumatic brain injury in mice. J Neurotrauma. 2014;31:1194e1201. Dash B, Phillips TD. Molecular characterization of a catalase from Hydra vulgaris. Gene. 2012;501:144e152. Yilmaz N, Dulger H, Kiymaz N, et al. Activity of mannitol and hypertonic saline therapy on the oxidant and antioxidant system during the acute term after traumatic brain injury in the rats. Brain Res. 2007;1164:132e135. Kobayashi M, Yamamoto M. Nrf2eKeap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species. Adv Enzyme Regul. 2006;46:113e140. Tanaka N, Ikeda Y, Ohta Y, et al. Expression of Keap1-Nrf2 system and antioxidative proteins in mouse brain after transient middle cerebral artery occlusion. Brain Res. 2011;1370:246e253. Wang Z, Ji C, Wu L, et al. Tert-butylhydroquinone alleviates early brain injury and cognitive dysfunction after experimental subarachnoid hemorrhage: role of Keap1/Nrf2/ ARE pathway. PLoS One. 2014;9:e97685. Ding H, Wang H, Zhu L, et al. Ursolic acid ameliorates early brain injury after experimental traumatic brain injury in mice by activating the Nrf2 pathway. Neurochem Res. 2017;42:337e346. Sun Y, Yang T, Leak RK, et al. Preventive and protective roles of dietary Nrf2 activators against central nervous system diseases. CNS Neurol Disord Drug Targets. 2017;16:326e338. de Vries HE, Witte M, Hondius D, et al. Nrf2-induced antioxidant protection: a promising target to counteract ROSmediated damage in neurodegenerative disease? Free Radic Biol Med. 2008;45:1375e1383. Zhang M, An C, Gao Y, et al. Emerging roles of Nrf2 and phase II antioxidant enzymes in neuroprotection. Prog Neurobiol. 2013;100:30e47. Ohsawa I, Ishikawa M, Takahashi K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med. 2007;13:688e694. Dohi K, Kraemer BC, Erickson MA, et al. Molecular hydrogen in drinking water protects against neurodegenerative changes induced by traumatic brain injury. PLoS One. 2014;9:e108034. Nagata K, Nakashima-Kamimura N, Mikami T, et al. Consumption of molecular hydrogen prevents the stressinduced impairments in hippocampus-dependent learning tasks during chronic physical restraint in mice. Neuropsychopharmacology. 2009;34:501e508. Wang C, Li J, Liu Q, et al. Hydrogen-rich saline reduces oxidative stress and inflammation by inhibit of JNK and NF-kB activation in a rat model of amyloid-beta-induced Alzheimer’s disease. Neurosci Lett. 2011;491:127e132. Yang Y, Li B, Liu C, et al. Hydrogen-rich saline protects immunocytes from radiation-induced apoptosis. Med Sci Monit. 2012;18:BR144eBR148. Ying L, Difen W, Jia Y, et al. Effect of hydrogen-rich water on the angiogenesis in lesion boundary brain tissue of traumatic brain injury-challenged rats. Int J Clin Exp Pathol. 2017;10:3807e3815.
26. Kawamura T, Wakabayashi N, Shigemura N, et al. Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo. Am J Physiol Lung Cell Mol Physiol. 2013;304:L646eL656. 27. Wang F, Yu G, Liu SY, et al. Hydrogen-rich saline protects against renal ischemia/reperfusion injury in rats. J Surg Res. 2011;167:e339ee344. 28. Zhang DQ, Feng H, Chen WC. Effects of hydrogen-rich saline on taurocholate-induced acute pancreatitis in rat. Evid Based Complement Alternat Med. 2013;2013:731932. 29. Iketani M, Ohshiro J, Urushibara T, et al. Preadministration of hydrogen-rich water protects against lipopolysaccharideinduced sepsis and attenuates liver injury. Shock. 2017;48:85e93. 30. Shen M, Zhang H, Yu C, et al. A review of experimental studies of hydrogen as a new therapeutic agent in emergency and critical care medicine. Med Gas Res. 2014;4:17. 31. Ji X, Tian Y, Xie K, et al. Protective effects of hydrogen-rich saline in a rat model of traumatic brain injury via reducing oxidative stress. J Surg Res. 2012;178:e9ee16. 32. Feeney DM, Boyeson MG, Linn RT, et al. Responses to cortical injury: I. Methodology and local effects of contusions in the rat. Brain Res. 1981;211:67e77. 33. Gao Y, Zhuang Z, Gao S, et al. Tetrahydrocurcumin reduces oxidative stress-induced apoptosis via the mitochondrial apoptotic pathway by modulating autophagy in rats after traumatic brain injury. Am J Transl Res. 2017;9:887e899. 34. Lu XY, Wang HD, Xu JG, et al. Pretreatment with tertbutylhydroquinone attenuates cerebral oxidative stress in mice after traumatic brain injury. J Surg Res. 2014;188:206e212. 35. Wu G, Liu Z. Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates neuroprotection in traumatic brain injury at least in part by inactivating microglia. Med Sci Monit. 2016;22:2161e2166. 36. Hong Y, Yan W, Chen S, et al. The role of Nrf2 signaling in the regulation of antioxidants and detoxifying enzymes after traumatic brain injury in rats and mice. Acta Pharmacol Sin. 2010;31:1421e1430. 37. Ding K, Wang H, Xu J, et al. Melatonin stimulates antioxidant enzymes and reduces oxidative stress in experimental traumatic brain injury: the Nrf2eARE signaling pathway as a potential mechanism. Free Radic Biol Med. 2014;73:1e11. 38. Manaenko A, Lekic T, Ma Q, et al. Hydrogen inhalation ameliorated mast cell-mediated brain injury after intracerebral hemorrhage in mice. Crit Care Med. 2013;41:1266e1275. 39. Li J, Wang C, Zhang JH, et al. Hydrogen rich saline improves memory function in a rat model of amyloid-beta-induced Alzheimer’s disease by reduction of oxidative stress. Brain Res. 2010;1328:152e161. 40. Fujita K, Seike T, Yutsudo N, et al. Hydrogen in drinking water reduces dopaminergic neuronal loss in the 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease. PLoS One. 2009;4:e7247. 41. Xie Q, Li XX, Zhang P, et al. Hydrogen gas protects against serum and glucose deprivation-induced myocardial injury in H9c2 cells through activation of the NF-E2-related factor 2/ heme oxygenase 1 signaling pathway. Mol Med Rep. 2014;10:1143e1149. 42. Taguchi K, Motohashi H, Yamamoto M. Molecular mechanisms of the Keap1-Nrf2 pathway in stress response and cancer evolution. Genes Cells. 2011;16:123e140. 43. Baird L, Dinkova-Kostova AT. The cytoprotective role of the Keap1-Nrf2 pathway. Arch Toxicol. 2011;85:241e272. 44. Yang C, Zhang X, Fan H, et al. Curcumin upregulates transcription factor Nrf2, HO-1 expression and protects rat brains against focal ischemia. Brain Res. 2009;1282:133e141.
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Hydrogen-rich water attenuates oxidative stress in rats with traumatic brain injury via Nrf2 pathway Jia Yuan, MD, Difen Wang, MD,* Ying Liu, MD, Xianjun Chen, MD, Hailing Zhang, MD, Feng Shen, MD, Xu Liu, MD, PhD, and Jiangquan Fu, MD Department of Critical Care Medicine, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou Province, P.R. China
article info
abstract
Article history:
Background: Several studies have recently found that oxidative stress plays a pivotal role in
Received 27 November 2017
the pathogenesis of traumatic brain injury (TBI) and may represent a target in TBI treat-
Received in revised form
ment. Hydrogen-rich water was recently shown to exert neuroprotective effects in various
29 January 2018
neurological diseases through its antioxidant properties. However, the mechanisms un-
Accepted 14 March 2018
derlying its effects in TBI are not clearly understood. The purpose of our study was to
Available online xxx
evaluate the neuroprotective role of hydrogen-rich water in rats with TBI and to elucidate the possible mechanisms underlying its effects.
Keywords:
Materials and methods: The TBI model was constructed according to the modified Feeney
Hydrogen-rich water
weight-drop method. In part 1 of the experiment, we measured oxidative stress levels by
Nrf2 pathway
observing the changes in catalase (CAT), glutathione peroxidase (GPx), and malondialde-
Traumatic brain injury
hyde (MDA) expressions. We also evaluated nuclear factor erythroid 2erelated factor 2
Oxidative stress
(Nrf2) levels to determine the role of the protein in the neuroprotective effects against TBI. In part 2, we verified the neuroprotective effects of hydrogen-rich water in TBI and observed its effects on Nrf2. All the experimental rats were divided into sham group, TBI group, and TBI þ hydrogen-rich water-treated (TBI þ HW) group. We randomly chose 20 rats from each group and recorded their 7-d survival rates. Modified neurological severity scores were recorded from an additional six rats per group, which were then sacrificed 24 h after testing. Spectrophotometry was used to measure GPx, CAT, and MDA levels, whereas western blotting, reverse transcription polymerase chain reaction, and immunohistochemistry were used to measure the expression of Nrf2 and downstream factors like heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). Results: GPx and CAT activity was significantly decreased, and MDA content was increased in the TBI group compared with the sham group at 6 h after TBI. MDA content peaked at 24 h after TBI. Nrf2 nucleoprotein levels were upregulated in the TBI group compared with the sham group and peaked at 24 h after TBI; however, no significant changes in Nrf2 mRNA levels were noted after TBI. Hydrogen-rich water administration significantly increased 7-
* Corresponding author. Department of Critical Care Medicine, The Affiliated Hospital of Guizhou Medical University, Guiyi Road No. 28, Yunyan District, Guiyang 550004, Guizhou Province, P.R. China. Tel./fax: þ86 0851 86772049. E-mail address: [email protected] (D. Wang). 0022-4804/$ e see front matter ª 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2018.03.024
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d survival rates, reduced neurologic deficits, and lowered intracellular oxidative stress levels. Moreover, hydrogen-rich water caused Nrf2 to enter the cell nucleus, which resulted in increases in the expression of downstream factors such as HO-1 and NQO1. Conclusions: Our results indicate that hydrogen-rich water has neuroprotective effects against TBI by reducing oxidative stress and activating the Nrf2 pathway. ª 2018 Elsevier Inc. All rights reserved.
Introduction Traumatic brain injury (TBI) remains a great health concern, as the condition imposes serious mental and economic burdens on both families and society.1 The pathogenesis of TBI encompasses primary and secondary injuries. Primary injury is the initial mechanical injury to which brain cells are subjected, and secondary injury develops within hours, days, or even months after the primary mechanical injury. Complicated pathophysiologic processes, including calcium overload, inflammatory responses, neuronal apoptosis, glutamate-mediated excitotoxicity, and oxidative stress, occur in secondary injury.2-5 Oxidative stress is of great importance in TBI-induced secondary injury.5 However, the evidence regarding the effects of antioxidant therapies in patients with TBI in clinical practice is limited.6 TBI induces a series of delayed secondary biochemical and cellular metabolic changes and eventually causes a considerable increase in reactive oxygen species (ROS) production, thereby accelerating cell damage and neuronal dysfunction.7,8 Enormous oxygen free radicals released after TBI caused the lipid peroxidation in the membranes of cells, resulting in an increasing of cellular malondialdehyde (MDA) content.9 Glutathione peroxidase (GPx) specifically catalyzes hydrogen peroxide (H2O2) and other organic hydroperoxides into water with the glutathione being used as reducing agent, by which interrupts the process of the lipid peroxidation chain reaction.10 Catalase (CAT) protects cellular membrane structure and functional integrity against harmful effects of H2O2 by facilitating its degradation to oxygen and water.11 GPx and CAT play crucial roles in regulating the oxidative status in the cell after TBI.12 The nuclear factor erythroid 2erelated factor 2 (Nrf2) pathway, a significant endogenous antioxidant system,13 regulates the expression of more than 200 encoded endogenous protection genes and phase II detoxification enzymes. Nrf2 is a key regulatory protein in the antioxidant system and has been widely studied in many central nervous system (CNS) diseases,14-17 including TBI.17 Under normal physiological conditions, Nrf2 is bound to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm to promote the proteasomal degradation of Nrf2. Once exposed to ROS, Nrf2 separates from Keap1, resulting in the translocation of Nrf2 from the cytoplasm to the nucleus, wherein the protein binds to the antioxidant response element,18 through which it may produce a series of endogenous enzymes, such as heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). These enzymes enable cells to survive the effects of oxidative stress and many other toxins.19 Hydrogen gas (H2) is a colorless and tasteless gas. In 2007, H2 was shown to exert antioxidant effects on rats subjected to
ischemia-reperfusion (I/R) injury by selectively scavenging the hydroxyl radical, one of the most cytotoxic ROS.20 Hydrogenrich water, which comprises a therapeutic dose of hydrogen dissolved in normal saline/pure water, is produced and sold commercially.21 Hydrogen-rich water exerts an antioxidant effect similar to that of hydrogen inhalation treatment and possesses a good safety profile, is portable, and is easily administered.22 Hydrogen-rich water has also been widely noticed for its ability to repress inflammatory reactions and to attenuate neuronal apoptosis.23,24 Moreover, our previous study found that hydrogen-rich water promotes angiogenesis by upregulating HIF-1a and VEGF expressions in a TBI model.25 The antioxidative effect of hydrogen is the most important of its many beneficial effects and has been demonstrated in many diseases, such as hyperoxic lung injury, renal I/R injury, acute pancreatitis, sepsis, and several CNS diseases, including TBI.21,26-31 Although hydrogen-rich water has been shown to play an antioxidant role in TBI,21,31 the mechanisms underlying its effects in TBI are not fully understood. Moreover, whether the antioxidative effects of hydrogen-rich water are associated with the Nrf2 pathway remains to be studied further. The objective of this study was to evaluate the role of hydrogenrich water in TBI rats and to elucidate the possible mechanisms underlying its effects.
Materials and methods Experimental animals All Sprague-Dawley rats, adult males weighing 250-300 g (Animal Certificate No: SCXK (Army) 2012-0011), were purchased from the Third Military Medical University (Chongqing, China). The rats were placed in air-filtered, temperature-controlled rooms and were allowed free access to food and water. The experiments were approved by the Animal Care Committee of Guizhou Medical University and were conducted in accordance with the guiding principles for animal research of Guizhou Medical University. We attempted to minimize animal suffering in all experiments.
Construction of the TBI model The TBI model was constructed according to Feeney’s weightdrop method, with some modifications.32 Briefly, the rats were anesthetized with 10% chloral hydrate (3.5 mL/kg intraperitoneally [i.p.]), after which they were fixed in the prone position on a brain stereotaxic frame (RWD Life Science Co, Shenzhen, China) by a weight-drop device. We exposed the skull at the midline and drilled a hole with a diameter of
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5.0 mm 5.0 mm in the right parietal bone. The rats were then subjected to TBI in the right hemisphere (Fig. 1) of the brain. Specifically, each rat was struck with a force of 40 g 40 cm (a 40-g weight was dropped from a height of 40 cm), resulting in a brain contusion involving the right parietal lobe. Suture material was used to close the scalp wound. After recovering from anesthesia, the rats were returned to their feeding cages, where they were allowed free access to water and food.
Experimental design Part 1: measurement of oxidative stress levels and elucidation of the protective effects of Nrf2 against TBI We observed the changes in CAT, GPx, MDA, and Nrf2 expressions in TBI. The following two groups were studied in the experiment: a sham group (n ¼ 6) and a TBI group (n ¼ 30). The rats in the TBI group were subjected to TBI and were subdivided into the following five groups: a 6-h group, a 12-h group, a 24-h group, a 48-h group, and a 5-d group. The animals in the sham group underwent an identical surgical procedure but were not subjected to the hemisphere strike.
Part 2: assessment of the neuroprotective effects of hydrogenrich water in TBI and its effects on Nrf2 A total of 78 male rats were randomly divided into sham group, TBI group, and hydrogen-rich water-treated TBI (TBI þ HW) group, with 26 rats in each group. The rats in the TBI and TBI þ HW groups were subjected to TBI. The experimental protocols used for this part of the experiment were the same as those used for part 1. The rats in the TBI þ HW group were all i.p. injected with 5-mL/kg hydrogen-rich water (Hydrovita Biotechnology Co, Beijing, China) at 5 min after TBI, whereas the rats in the sham and TBI groups were all i.p. administered with 5-mL/kg normal saline, which was used as
a control treatment. Finally, 20 rats from each group were randomly selected, and their 7-d survival rate was recorded. We also subjected an additional six rats from each group to modified neurological severity score (mNSS) testing and then sacrificed them 24 h after the test.
Neurological deficit evaluation The mNSS was determined to evaluate TBI-induced neurobehavioral deficits in the rats at 24 h following TBI or sham operation. The mNSS score was determined by tests of motor skills, sensory capabilities, reflexes, and balance. The highest possible mNSS is 18, and higher scores are indicative of more severe neurological damage.33 Neurological injury according to the mNSS was graded as follows: severe injury (score of 1318), moderate injury (score of 7-12), mild injury (score of 1-6), and no injury (score of zero).
Determination of GPx, CAT, and MDA levels The rats were sacrificed under deep anesthesia, and the ipsilateral cortex, that is, the cortex containing the injury site, was collected. A portion of the tissue was weighed, homogenized with iced normal saline (1/9, w/v), and then centrifuged for 15 min at 4 C (3000 g/min). The appropriate commercial kits (Nanjing Jiancheng, Nanjing, China) were used to measure antioxidant enzymatic (GPx and CAT) activity and oxidative product (MDA) levels. All the experiments were performed in strict accordance with the instructions provided with each kit. A BCA Protein Assay Reagent Kit (Beyotime Biotechnology, Shanghai, China) was used to measure the protein concentration. Absorbance values were determined by a spectrophotometer.
Immunohistochemistry The tissues were immersed in 10% paraformaldehyde overnight for immunohistochemistry assay. Coronal tissues were consecutively cut into 5-mm sections to collect the damaged cortex. Endogenous peroxidase activity was blocked by 3% H2O2 after routine deparaffinization. The sections were incubated in blocking buffer for 20 min to block nonspecific antibody binding, after which they were incubated with primary antibodies against Nrf2 (1:150; Abcam, Cambridge, MA) overnight at 4 C. The sections were subsequently washed with PBS (3 times, 5 min/wash) and incubated with a secondary antibody conjugated with IgGHRP (1:500; Shengda, Guangzhou, China) for 25 min at 37 C. We counted the number of positive cells in three fields and then used the average of this number as the final data for each sample. Specifically, three fields in each sample were randomly selected, and then the cells were counted under a 400 light microscope. The entire procedure was performed in a blinded manner by two pathologists.
Western blot analysis
Fig. 1 e The TBI site in the rat model. (Color version of figure is available online.)
The preserved brains were cut into pieces and lysed after freezing and thawing. The cytoplasmic and nuclear proteins were extracted according to the instructions of the
yuan et al hydrogen rich water protects traumatic brain injury via nrf2 pathway
appropriate kit (KeyGen Biotech, Nanjing, China), and a BCA Protein Assay Reagent Kit was used to measure the concentrations of these proteins. The proteins (40 mg) were separated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. TBST with 5% skim milk buffer was used to block nonspecific binding for 2 h at room temperature. The membranes were incubated with the appropriate primary antibodies at the appropriate dilutions overnight at 4 C (mouse anti-rat Nrf2 antibody, 1:3500; rabbit anti-rat NQOl antibody, 1:10,000; rabbit anti-rat HO-1 antibody, 1:10,000 [all from Abcam]; anti-histone 3 [H3, 1:5000; Abmart, Shanghai, China]; and anti-b-actin [1:3500; Abmart]). All the membranes were subsequently incubated with the appropriate secondary antibodies (1:5000, goat anti-mouse/anti-rabbit; Abmart) for 2 h at room temperature. Finally, an Amersham ECL Plus Western Blotting Detection Kit (Merck Millipore, Billerica, MA) was used to detect the immunoreactive bands through chemiluminescence. The expression levels of the proteins of interest were normalized to those of b-actin or H3. All the procedures were performed in strict accordance with the instructions of the appropriate kits.
Reverse transcription polymerase chain reaction Total RNA was extracted with Trizol reagent using an extraction kit (Merck Millipore). After DNase digestion, nucleic acid concentrations and purity were assessed at 260/280 nm. A first-strand cDNA synthesis kit (Thermo Scientific, Waltham, MA) was then used to reverse-transcribe the RNA. The sequences of the primers (Biological Engineering Co, Shanghai, China) used for the experiment were as follows: Nrf2, 50 CAAATCCCACCTTGAACACA-30 and 50 -TGACTAATGGCAGCAGAGGA-30 ; HO-1, 50 -CAGAGTTTCTTCGCCAGAGG-30 and 50 GAGTGTGAGGACCCATCG -30 ; NQO1, 50 -ATTCCAGCCGACAACCAGAT-30 and 50 -CCGTGGCAGAACTATCCAAA-30 ; and band 50 actin, 50 -TGTCACCAACTGGGACGATA-30 GGGGTGTTGAAGGTCTCAAA-30 . The PCR amplification procedure comprised the following steps: incubation at 95 C for 10 min, followed by denaturation at 95 C for 15 s, annealing at 60 C for 60 s, and extension at 60 C for 60 s (42 reaction cycles comprising denaturation, annealing, and extension steps). The specificities of the primers were detected by melting curve analysis, and b-actin was used as an internal reference.
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Statistical analysis SPSS 20.0 was used for the statistical analysis. Quantitative data are represented by the mean standard deviation (SD). One-way analysis of variance followed by the least significant difference test was used for statistical and intergroup comparisons, and unpaired t tests were used to compare neurological deficit severities between two groups. The differences in survival were estimated by KaplaneMeier survival plots and the log-rank test. P < 0.05 was considered statistically significant.
Results Antioxidant enzyme (GPx and CAT) and oxidative product (MDA) levels in TBI As reported previously, CAT and GPx are indicators of antioxidant activity, and MDA is an indicator of lipid peroxidation. The levels of these three indicators were examined in rat tissues to determine the levels of oxidative stress induced by TBI. The data showed that CAT and GPx activity began to decrease at 6 h after TBI (P < 0.05, Fig. 2A and B) and then increased markedly from 48 h to 5 d after TBI. The lowest GPx and CAT activity levels were noted at 24 h after TBI. In addition, MDA levels increased significantly in the TBI group compared with the sham group (P < 0.05, Fig. 2C). Specifically, MDA levels began to increase at 6 h after TBI and peaked at 24 h after TBI. These findings suggest that TBI leads to decreases in GPx and CAT activity and increases in MDA levels in injured ipsilateral cortical tissue.
Changes in Nrf2 expression after TBI Western blotting and reverse transcription polymerase chain reaction (RT-PCR) were performed to detect Nrf2 protein and mRNA levels, respectively, in brain tissue at 6 h, 12 h, 24 h, 48 h, and 5 d after TBI. We found that Nrf2 protein expression levels were upregulated in TBI brains compared with sham brains and that Nrf2 protein expression began to increase at 6 h after TBI and peaked at 24 h after TBI (P < 0.05, Fig. 3A and B). However, the RT-PCR results were inconsistent with the western blotting results, as Nrf2 mRNA expression did not
Fig. 2 e Antioxidant enzyme (GPx and CAT) and oxidative product (MDA) levels in TBI. Bar graph illustrating the changes in (A) CAT, (B) GPx, and (C) MDA levels. Data are represented by the mean ± SD (n [ 6, *P < 0.05, **P < 0.01 versus the sham group).
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Fig. 3 e (A) Representative photographs of western blots showing the expression of the nuclear protein Nrf2 and the control H3 in the rat brain after TBI. (B) Nrf2 nucleoprotein expression (n [ 3, *P < 0.05, **P < 0.01 versus the sham group). (C) Nrf2 mRNA expression did not differ between TBI and each time point after TBI (n [ 6, P > 0.05 versus the sham group). Data are represented by the mean ± SD.
change significantly between TBI and any of the above postTBI time points (P > 0.05, Fig. 3C).
TBI group after hydrogen-rich water treatment (P < 0.01, Fig. 4B).
Hydrogen-rich water increased the 7-d survival rate and reduced neurologic deficits
Hydrogen-rich water attenuated oxidative stress after TBI
Survival was analyzed up to 7 d after TBI. The 7-d survival rate increased to 65% in the TBI þ HW group and was significantly higher than that in the TBI group (30% versus 65%, P < 0.05, Fig. 4A). The mNSS results showed that the rats showed neurological deficits of different severities at different time points after trauma. These included hemiplegia, sensory disturbances, ataxia, and myoclonus. The mNSS in the TBI þ HW group was lower than that in the
CAT and GPx levels decreased significantly after TBI (P < 0.01) but were almost normalized by hydrogen-rich water treatment (P < 0.01, Fig. 5A and B). Similarly, MDA levels increased significantly after TBI (P < 0.01 versus the sham group) but decreased significantly after hydrogen-rich water treatment (P < 0.01 versus the TBI group, Fig. 5C).
Hydrogen-rich water promoted the translocation of Nrf2 from the cytoplasm to the nucleus We performed western blotting to evaluate Nrf2 activation after hydrogen-rich water treatment. The results demonstrated that hydrogen-rich water treatment increased Nrf2 expression and significantly decreased cytoplasmic Nrf2 expression (P < 0.05 Fig. 6A and B), indicating that hydrogenrich water administration promotes the translocation of Nrf2 from the cytoplasm to the nucleus. The immunohistochemistry assay results confirmed these findings (Fig. 6C).
Hydrogen-rich water enhanced NQO1 and HO-1 protein and mRNA expressions Fig. 4 e Hydrogen-rich water has protective effects against TBI. (A) KaplaneMeier analysis showed that the 7d survival rate was almost 100% in the sham group, and that the rate was significantly different between the TBI and TBI D HW groups (n [ 20, 30% versus 65%, P < 0.05). (B) Neurological deficits were evaluated by the mNSS at 24 h after TBI. The mNSS was significantly reduced in the TBI D HW group compared with the TBI group (n [ 6, ## P < 0.01). (Color version of figure is available online.)
We investigated the expression of NQO1 and HO-1, which are downstream factors in the Nrf2 pathway. The western blotting results indicated that NQO1 and HO-1 protein expressions were upregulated in the TBI group compared with the sham group following TBI (P < 0.05, Fig. 7A and B). Hydrogen-rich water treatment significantly enhanced NQO1 and HO-1 protein expressions in the TBI þ HW group compared with the TBI group (P < 0.05, Fig. 7A and B). Consistent with these results,
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Fig. 5 e Hydrogen-rich water attenuated TBI-induced oxidative stress. Oxidative stress was evaluated by the measurement of (A) CAT, (B) GPx, and (C) MDA levels at 24 h after surgery. Data are represented by the mean ± SD (n [ 6, **P < 0.01 versus the sham group; ##P < 0.01 versus the TBI group).
the PCR results showed that NQO1 and HO-1 mRNA expressions were also significantly upregulated in the TBI group compared with the sham group, and that treatment with hydrogen-rich water increased NQO1 and HO-1 mRNA expressions in the TBI þ HW group compared with the TBI group (P < 0.05 Fig. 7C and D).
Discussion We investigated the neuroprotective effects of hydrogen-rich water in brain injuries induced by TBI and explored the possible mechanisms underlying these effects. We found that (1) oxidative stress, which is represented mainly by
antioxidant enzyme (GPx and CAT) and oxidative product (MDA) levels, is of great importance in TBI. Moreover, we noted that Nrf2 protein expression levels were upregulated after TBI and peaked at 24 h after TBI, a change that coincided with increases in oxidative stress levels. (2) Hydrogen-rich water had neuroprotective effects in TBI rats. Specifically, hydrogen-rich water increased 7-d survival rates, reduced neurologic deficits, and lowered intracellular oxidative stress levels. In addition, hydrogen-rich water induced the translocation of Nrf2 from the cytoplasm to the nucleus, which resulted in increases in the expression of downstream factors (HO-1 and NQO1). Neuronal dysfunction and death following TBI are largely dependent on secondary brain injury, which comprises a
Fig. 6 e Hydrogen-rich water accelerated the translocation of Nrf2 from the cell cytoplasm to the nucleus. (A, B) Western blot analysis showed that hydrogen-rich water increased Nrf2 levels in the nucleus and decreased Nrf2 levels in the cytoplasm. H3 and b-actin were used as internal references (n [ 3). Data are represented by the mean ± SD (@P < 0.05 versus the sham group, &P < 0.05 for cytoplasmic Nrf2 versus the TBI group, and #P < 0.05 for Nrf2 versus TBI group). (C) Representative photomicrographs of the immunohistochemical analysis of Nrf2 expression (tissues from different groups). Morphological examination showed that the TBI group featured a higher nuclear concentration of Nrf2 than the sham group. Morphological examination also showed that the concentration of Nrf2 was higher in the TBI D HW group than in the TBI group. (Color version of figure is available online.)
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Fig. 7 e Hydrogen-rich water upregulated the protein and mRNA expressions of downstream factors in the Nrf2 pathway. (A, B) HO-1 and NQO1 protein expressions was upregulated after TBI, and hydrogen-rich water elicited further increases in HO1 and NQO1 protein expressions (n [ 3, *P < 0.05 for HO-1 and @P < 0.05 for NQO1 versus the sham group; #P < 0.05 for HO1 and &P < 0.05 for NQO1 versus the TBI group). (C, D) NQO1 and HO-1 mRNA expressions were elevated after TBI, and hydrogen-rich water treatment elicited further increases in HO-1 and NQO1 mRNA expressions (n [ 6, **P < 0.01 versus the sham group and ##P < 0.01 versus the TBI group for HO-1, @P < 0.05 versus the sham group and &&P < 0.01 versus the TBI group for NQO1). Data are represented by the mean ± SD.
series of consecutive pathological processes that occur following primary injury and may develop within a few hours to a few days or months after injury.2 Increasing evidence suggests that oxidative stress plays a pivotal role in the pathophysiology of secondary brain injury caused by TBI.5 The production of excessive ROS as a result of the metabolism of nucleic acids, lipids, and proteins may cause serious damage to the cell after TBI.8 Several oxidative markers, including GPx and CAT, which are responsible for scavenging free radicals and catalyzing peroxides into nontoxic forms,10 are present after TBI. MDA is a common indicator of lipid peroxidation and reflects the level of free radicaleinduced damage.34 In our study, both GPx and CAT activity were markedly decreased in the TBI group compared with the sham group at 6 h after operation. However, MDA content was significantly increased in the TBI group compared with the sham group at 6 h after operation and peaked at 24 h after TBI. These findings indicated that oxidative stress was induced after TBI. Nrf2 is a key regulator factor of cellular antioxidants that is involved in secondary brain injury after TBI.35 Moreover, Nrf2 activators, such as ursolic acid, sulforaphane, tertbutylhydroquinone, and melatonin, have been confirmed to have the ability to attenuate post-TBI pathophysiologic phenomena, including neuronal apoptosis, cerebral edema, cognitive deficits, and oxidative stress.16,34,36,37 Nrf2 knock-
out mice had worse outcomes than wild-type mice in a previous study. However, the knock-out mice also displayed improvements in post-TBI neurologic function after ursolic acid administration.16 Our study demonstrated the time course of Nrf2 expression in rat brains subjected to TBI. Nrf2 protein expression levels were upregulated at 6 h after TBI and peaked at 24 h after TBI. Nrf2 levels initially remained high but then gradually decreased from 48 h to 5 d after TBI. These changes in Nrf2 expression coincided with the above mentioned changes in oxidative stress levels. These findings suggest that Nrf2 is important in protecting neurons from oxidative stress after TBI, and that Nrf2 upregulation at an early stage after TBI may be a target in its treatment. H2 is the predominant atmospheric gas, and its selective antioxidant function alleviates oxidative stresseinduced damage in the brain, heart, lung, intestine, kidney, and pancreas, as well as other major organs.30 Solubilized hydrogen (hydrogen-rich water) is a more convenient means of delivering molecular hydrogen.22 A hydrogen-rich water treatment significantly improved outcomes in TBI rats in previous studies.21,31 Similar findings have also been noted in several CNS diseases, such as hemorrhagic stroke, cerebral I/R injury, Alzheimer’s disease, and Parkinson’s disease, and the effects are attributable to the antioxidant properties of the water.27,38-40 In our study, we chose to administer 5-mL/kg hydrogen-rich water i.p. to explore its neuroprotective role in
yuan et al hydrogen rich water protects traumatic brain injury via nrf2 pathway
TBI. The dose used in this study was determined based on the results of earlier studies.31 Given that oxidative stress and Nrf2 nucleoprotein levels peaked at 24 h after TBI in part 1, we selected 24 h as the post-TBI observation time for part 2. The data showed that hydrogen-rich water significantly increased the 7-d survival rate and reduced neurologic deficits. Moreover, hydrogen-rich water administration attenuated oxidative stress after TBI, a result consistent with those of previous studies.31 Several in vivo and in vitro experiments have confirmed that the antioxidant effect of hydrogen is dependent not only on its ability to scavenge oxygen free radicals but also on its ability to regulate the Nrf2 signaling pathway.26,39,41 NQO1 and HO-1 are two of the most important antioxidant enzymes downstream of Nrf2, and both enzymes are associated with intracellular redox equilibrium.19 Hydrogen promotes the upregulation of Nrf2 and phase II enzymes, such as HO-1 and NQO1, in hyperoxic lung injury.26 Experimental data demonstrated that Nrf2 translocates from the cytoplasm to the nucleus following TBI, and that hydrogen-rich water accelerates this process. Hydrogen-rich water administration also significantly elevated the pre- and post-transcriptional levels of HO1 and NQO1 in the TBI þ HW group compared with the TBI and sham groups following TBI. These results indicated that hydrogen-rich water had neuroprotective effects against TBI by inhibiting oxidative stress, and that the mechanisms underlying the effects of Nrf2 may be associated with the translocation of the protein from the cytoplasm to the nucleus and the activation of downstream proteins. Interestingly, we found that the changes in Nrf2 mRNA expression were inconsistent with those in Nrf2 protein expression, as Nrf2 mRNA expression did not change significantly between the control group and each time point after TBI. Studies have shown that the Nrf2-Keap1 interaction is disrupted by the modification of Keap1 at cysteine and the phosphorylation of Nrf2 at serine.42 Several signaling pathway proteins, such as activated protein kinase C, phosphatidylinositol 3-kinase, and some mitogen-activated protein kinases, including extracellular signal-regulated kinase and c-Jun Nterminal kinase, reportedly phosphorylate Nrf2 directly and affect its translocation to the nucleus.43 According to the above mentioned evidence, we speculated that the regulation of Nrf2 transcriptional activity after TBI may occur at the posttranscriptional level rather than at the gene transcriptional level. However, Yang et al. found that Nrf2 gene and protein levels are upregulated in a cerebral ischemia rat model.44 We surmised that the differences between the results of that study and those of our study may be related to between-study differences in animal models and detection methods. This phenomenon needs to be explored further in the future. The limitations of this study should be mentioned. First, hydrogen-rich water was administered only at 24 h after TBI; thus, we do not know how effective this treatment is at other time points. In addition, the 24-h therapeutic window used in the present study is relatively narrow. Second, hydrogen-rich water may have anti-inflammatory effects, anti-apoptotic effects, and other effects against TBI that were not discussed in our study. Finally, the neuroprotective effects of hydrogen-rich water were not verified in Nrf2/ TBI mice. The use of such mice would confirm whether the effects of
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hydrogen-rich water are associated with Nrf2 signaling pathway activation.
Conclusions Our data showed that hydrogen-rich water has neuroprotective effects in TBI by reducing oxidative stress, and that Nrf2 signaling pathway activation may be one of the mechanisms by which hydrogen-rich water exerts its effects.
Acknowledgment This work was supported by grants from the High-level Personnel of Special Support Program (No. TZJF-2011-25), the Science & Technology Department (No. SY [2010] 3079), and the Key Specialty Construction of Clinical Projects (No. [2011] 52) in Guizhou Province, as well as the National Key Specialty Construction of Clinical Projects (No. [2011] 170) in China. The authors thank the Clinical Research Center of the Affiliated Hospital of Guizhou Medical University for their support during the experiment. Authors’ contributions: J.Y., D.W., and Y.L. conceived and designed the experiments. J.Y. performed the experiments, analyzed the data, and wrote the article. Y.L. supervised the experiments and contributed reagents/materials/analysis tools. X.C. and H.Z. contributed to the data collection. F.S., X.L., and J.F. supervised the data analysis and critically reviewed the article.
Disclosure The authors report no proprietary or commercial interests with respect to any product mentioned or concept discussed in the article.
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