Hydrogen-rich water reduces cell damage by reducing excessive autophagy in mouse neuronal cells after oxygen glucose deprivation/reoxygenation

Objective: To investigate whether hydrogen-rich water exerts a protective effect against cellular injury by affecting the level of autophagy after oxygen glucose deprivation/reoxygenation (OGD/R) in a mouse hippocampal neuronal cell line (HT22 cells). Methods: HT22 cells in logarithmic growth phase were cultured in vitro. Cell viability was detected by cell counting kit-8 (CCK-8) assay to find the optimal concentration of Na2S2O4. HT22 cells were divided into control group (NC group), OGD/R group (sugar-free medium+10 mmol/L Na2S2O4 treated for 90 minutes and then changed to normal medium for 4 hours) and hydrogen-rich water treatment group (HW group, sugar-free medium+10 mmol/L Na2S2O4 treated for 90 minutes and then changed to medium containing hydrogen-rich water for 4 hours). The morphology of HT22 cells was observed by inverted microscopy; cell activity was detected by CCK-8 method; cell ultrastructure was observed by transmission electron microscopy; the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 was detected by immunofluorescence; the protein expression of LC3II/I and Beclin-1, markers of cellular autophagy, was detected by Western blotting. Results: Inverted microscopy showed that compared with the NC group, the OGD/R group had poor cell status, swollen cytosol, visible cell lysis fragments and significantly lower cell activity [(49.1±2.7)% vs. (100.0±9.7)%, P < 0.01]; compared with the OGD/R group, the HW group had improved cell status and remarkably higher cell activity [(63.3±1.8)% vs. (49.1±2.7)%, P < 0.01]. Transmission electron microscopy showed that the neuronal nuclear membrane of cells in the OGD/R group was lysed and a higher number of autophagic lysosomes were visible compared with the NC group; compared with the OGD/R group, the neuronal damage of cells in the HW group was reduced and the number of autophagic lysosomes was notably decreased. The results of immunofluorescence assay showed that the expressions of LC3 and Beclin-1 were outstandingly enhanced in the OGD/R group compared with the NC group, and the expressions of LC3 and Beclin-1 were markedly weakened in the HW group compared with the OGD/R group. Western blotting assay showed that the expressions were prominently higher in both LC3II/I and Beclin-1 in the OGD/R group compared with the NC group (LC3II/I: 1.44±0.05 vs. 0.37±0.03, Beclin-1/β-actin: 1.00±0.02 vs. 0.64±0.01, both P < 0.01); compared with the OGD/R group, the protein expression of both LC3II/I and Beclin-1 in the HW group cells were notably lower (LC3II/I: 0.54±0.02 vs. 1.44±0.05, Beclin-1/β-actin: 0.83±0.07 vs. 1.00±0.02, both P < 0.01). Conclusions: Hydrogen-rich water has a significant protective effect on OGD/R-causing HT22 cell injury, and the mechanism may be related to the inhibition of autophagy.

Protective effect of saturated hydrogen saline against cerebral ischemia-reperfusion injury in rats

Objective To study the protective effect of saturated hydrogen saline against cerebral ischemia-reperfusion injury and the related mechanism. Methods Rat middle cerebral artery occlusion (MCAO) models were established by thread ligation of the middle cerebral artery. The rats were sacrificed 24 h later. The cerebral infarction volume was determined by TTC staining, the water content in brain tissue by dry-wet weight method, the degree of cerebral cells by Nissl staining, and the levels of IL-1β and TNF-α in the ischemic cerebral tissues by ELISA. Results Compared with control group, hydrogen saline decreased the brain water content and cerebral infarction volume, and increased the quantity of nissel’s body in the cortex; meanwhile, it also significantly decreased the concentrations of IL-1β and TNF-α in brain tissue(P<0. 05). Conclusion Hydrogen saline can alleviate the cerebral ischemia-reperfusion injury, probably by inhibiting the inflammation response.

Hydrogen saline offers neuroprotection by reducing oxidative stress in a focal cerebral ischemia-reperfusion rat model

Hydrogen gas is neuroprotective in cerebral ischemia animal models. In this study, we tested the neuroprotective effects of hydrogen saline, which is safe and easy to use clinically, in a rat model of middle cerebral artery occlusion (MCAO). Sprague-Dawley male rats weighting 250-280 g were divided into sham, MCAO plus hydrogen saline and MCAO groups, and subjected to 90-min ischemia followed by 24 h of reperfusion. Hydrogen saline was injected intraperitoneally at 1 ml/100 g body weight. Infarct volume and brain water content were evaluated at different time points after reperfusion. Oxidative stress, inflammation, and apoptotic cell death markers were measured. Hydrogen saline significantly reduced the infarct volume and edema and improved the neurological function, when it was administered at 0, 3 and 6 h after reperfusion. Hydrogen saline decreased 8-hydroxyl-2′-deoxyguanosine (8-OHdG), reduced malondidehyde, interleukin-1β, tumor necrosis factor-α, and suppressed caspase 3 activity in the ischemic brain. These findings demonstrated hydrogen saline is neuroprotective when administered within 6 h after ischemia. Because hydrogen saline is safe and easy to use, it has clinical potentials to reduce neurological injuries.

Effects of hydrogen rich water on the expression of Nrf 2 and the oxidative stress in rats with traumatic brain injury

Objective: To investigate the effects of hydrogen rich water on the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and oxidative stress in rats with traumatic brain injury (TBI). Methods: Ninety healthy male Sprague-Dawley (SD) rats were randomly divided into sham operation group, TBI group and hydrogen rich water treatment group (HW group), with 30 rats in each group. TBI model was reproduced by the modified Feeney weight dropping method. The skulls of rats in sham operation group underwent only craniotomy without direct hit. The rats in HW group received brain injury by hitting after craniotomy, followed by injection of hydrogen rich water (5 mL/kg) intraperitoneally once a day for 5 days after successful reproduction of the model. The rats in sham operation group and TBI group were given an equal amount of normal saline in same manner. Six rats from each group were sacrificed at 6, 12, 24, 48 hours and 5 days after evaluating neurological severity scores (NSS). The brain tissue in injured ipsilateral cortex was harvested. The activity of catalase (CAT), glutathione peroxidase (GSH-Px), and content of malondialdehyde (MDA) were determined by spectrophotometry. The expressions of mRNA and nucleoprotein of Nrf2 were determined by quantitative real-time polymerase chain reaction (RT-qPCR) and Western Blot. The pathological changes were observed with microscopy after hematoxylin and cosin (HE) staining. Results: (1) NSS score: compared with TBI group, NSS in HW group at 12, 24, 48 hours and 5 days were significantly decreased (12 hours: 9.83 ± 2.32 vs. 13.17 ± 2.71, 24 hours: 9.83 ± 2.79 vs. 13.50 ± 2.43, 48 hours: 7.50 ± 2.07 vs. 11.83 ± 2.14, 5 days: 5.50 ± 1.87 vs. 10.50 ± 2.43, all P < 0.05). (2) Compared with sham operation group, the activity of GSH-Px and CAT in TBI group were markedly declined after operation, while the MDA content was elevated significantly, especially at 24 hours [CAT (kU/g): 1.080 ± 0.312 vs. 3.571 ± 0.758, GSH-Px (kU/g): 9.195 ± 3.173 vs. 32.385 ± 10.619; MDA (µmol/g): 12.282 ± 2.896 vs. 4.349 ± 1.511, all P < 0.01]. Compared with TBI group, the parameters in HW group were improved, and they were similar as sham operation group. (3) RT-qPCR: no significant difference was found in the expression of Nrf2 mRNA at each time point in three groups. (4) Western Blot: the expression of Nrf2 nucleoprotein (gray value) in TBI group was apparently higher than that in sham operation group, and peaked at 24 hours (0.703 ± 0.262 vs. 0.238 ± 0.120, P < 0.05), and the expression in HW group was obviously higher than that in TBI group, especially at 24 hours (1.110 ± 0.372 vs. 0.703 ± 0.262, P < 0.05). (5) HE staining: the brain structure in sham operation group was found to be intact. However, there were different degrees of pathological changes at each time in TBI group, especially at 24 hours. The pathological damage of brain tissue in HW group was significantly milder. Conclusions: Hydrogen rich water can up-regulate the expression of Nrf2, and reduce oxidative damage of traumatic brain injury in rats. Nrf2 can up-regulate the expression of its downstream antioxidant enzymes, which may be the mechanism of the upregulation expression of Nrf2 in the study.

Effects of hydrogen-rich water on the expression of aquaporin 1 in the cerebral cortex of rat with traumatic brain injury

Objective: To investigate the effect of hydrogen-rich water on cerebral edema and aquaporin 1 (AQP1) expression in rats with traumatic brain injury (TBI). Methods: Ninety male Sprague-Dawley (SD) rats were randomly divided into sham operation group, TBI model group, hydrogen-rich water treatment group (H group),with 30 rats in each group. TBI model was reproduced by weight dropping method. The skulls of rats in sham operation group underwent only craniotomy without direct hit and with bone wax sealed suture.5 mL/kg of hydrogen-rich water injection was given intraperitoneally after model reproduction in H group, and equal amount of normal saline was given in sham and TBI groups, once a day for both groups for 5 days. Six rats from each group were sacrificed at 6,12,24,48 hours and 5 days after evaluating neurological severity scores (NSS).The cerebral cortex was harvested, and the pathological changes in morphology of brain tissue were observed with light microscope. The positive expression of AQP1 in cerebral cortex was observed with immunohistochemistry by light microscopy, the AQP1 mRNA expression in cerebral cortex was determined by real-time fluorescent quantization reverse transcription-polymerase chain reaction (RT-PCR),and the AQP1 protein expression in cerebral cortex was determined by Western Blot. Results: ① All rats in sham operation group had a NSS of zero at each time point. NSS of TBI group was obviously raised with time prolongation, and peaked at 24 hours followed by a lower tendency, while the score in H group was significantly lower than that of TBI group, and the difference was the most obvious at 24 hours as compared with TBI group (9.83 ± 2.78 vs.13.50± 2.42,P < 0.05).② It was shown by light microscope that in the TBI group there were pathological changes in cerebral cortex, including obvious irregular arrangement of nerve cells, cerebral edema, obvious bleeding, especially at 24 hours, then the cerebral edema became vanished gradually; and the positive expression of AQP1 in the pia mater at all the time points in the TBI group was significantly increased, and it was most obvious at 24 hours. Compared with TBI group, the pathological changes at time points of 12 hours to 5 days in H group was significantly lessened, and the positive expression of AQP1 in the cerebral pia mater was reduced obviously.③ Compared with sham operation group, the mRNA and protein expressions of AQP1 in cerebral cortex in TBI group were significantly elevated, peaked at 24 hours [AQP1 mRNA (2-△△Ct):7.50±0.26 vs.1,AQP1 protein (gray value):1.986±0.110 vs.0.336±0.034, both P < 0.05], then they gradually declined. The mRNA and protein expressions of AQP1 in cerebral cortex were significantly decreased after hydrogen-rich water treatment [24-hour AQP1 mRNA (2-△△Ct):5.40±0.21 vs.7.50±0.26, 24-hour AQP1 protein (gray value): 1.246±0.137 vs.1.986±0.110, both P < 0.05]. Conclusions: The up-regulation of AQP1 mRNA and protein in rats’ cerebral cortex after TBI perhaps participates in edema formation which might be involved in the pathophysiology of cerebral edema in TBI. Early treatment with an intraperitoneally injection of hydrogen-rich water is capable of attenuating the extent of TBI-induced up-regulation of AQP1 mRNA and protein, alleviating cerebral edema, and achieving its protective effects.

Effect of hydrogen-rich water on the CD34 expression in lesion boundary brain tissue of rats with traumatic brain injury

Objective: To observe the effect of hydrogen-rich water on the CD34 expression and angiogenesis in lesion boundary brain tissue of rats with traumatic brain injury (TBI). Methods: A total of 54 adult male Sprague-Dawley (SD) rats were divided into three groups by random number table: namely sham-operated group (sham group), trauma group (TBI group), and trauma + hydrogen-rich water group (TBI+HW group), the rats in each group were subdivided into 1, 3 and 7 days subgroups according to the time points after trauma, with 6 rats in each subgroup. The TBI model was reproduced by using a modified Feency method for free fall impact, and the rats in sham group were not given brain impact after craniotomy. The rats in TBI+HW group were given intraperitoneal injection of hydrogen-rich water (5 mL/kg) after TBI model reproduction, and then once a day until being sacrificed, and the rats in sham group and TBI group were given the same amount of normal saline. The neurological severity scores (NSS) for neurologic deficits were calculated at corresponding time points, and then the rats were sacrificed for brain tissue at 3 mm around lesion boundary. After hematoxylin-eosin (HE) staining, the pathological changes in lesion boundary brain tissue were observed under light microscope. The expression of CD34+ cells was observed by immunohistochemical analysis, which markers were used to count the newborn blood capillary sprouts around the traumatic brain tissue. The protein expression of CD34 was determined by Western Blot. Results: NSS scores at all time points in sham group were 0. NSS scores in TBI and TBI+HW groups showed a decreased tendency with time prolongation after TBI, which showed more significant in TBI+HW group, NSS scores at 3 days and 7 days were significantly lower than those of TBI group (3 day: 8.67±0.52 vs. 11.56±1.94, 7 days: 7.33±0.52 vs. 8.17±0.98, both P < 0.05). Under light microscope, the brain tissue of rats in sham group was normal. After injury, pathological changes in lesion boundary brain tissue in TBI group were characterized by obvious hemorrhagic necrosis, severe brain edema, a large number of degeneration and necrosis of nerve cells and inflammatory cell infiltration, and the pathological changes were more obvious at 3 days. The edema area in TBI+HW group was slightly smaller than that of TBI group, and the surrounding edema was slightly reduced. It was shown by immunohistochemistry that only a very small number of neoformative capillaries were found in sham group. The number of neoformative capillaries in lesion boundary brain tissue was gradually increased with time prolongation in TBI group. The number of neoformative capillaries in TBI+HW group was more significantly, which was significantly higher than that of TBI group at 3 days and 7 days after injury (cells/HP: 10.59±1.88 vs. 8.61±1.22 at 3 days, 23.20±3.16 vs. 17.01±2.64 at 7 days, both P < 0.05). It was shown by Western Blot that the expression of CD34 protein at all time points in TBI group was significantly increased as compared with that of sham group. The expression of CD34 protein at 1 day and 3 days in TBI+HW group was slightly increased as compared with that of TBI group without significant difference, but it was significantly up-regulated at 7 days after injury, which was significantly higher than that of TBI group (gray value: 1.36±0.36 vs. 0.74±0.08, P < 0.05). Conclusions: Hydrogen-rich water promote CD34+ cells home to the site of injured tissue in rats with TBI, is involved in angiogenesis, and improve clinical outcomes during brain functional recovery.

Hydrogen-rich water attenuates oxidative stress in rats with traumatic brain injury via Nrf2 pathway

Background: Several studies have recently found that oxidative stress plays a pivotal role in the pathogenesis of traumatic brain injury (TBI) and may represent a target in TBI treatment. Hydrogen-rich water was recently shown to exert neuroprotective effects in various neurological diseases through its antioxidant properties. However, the mechanisms underlying its effects in TBI are not clearly understood. The purpose of our study was to evaluate the neuroprotective role of hydrogen-rich water in rats with TBI and to elucidate the possible mechanisms underlying its effects. Materials and methods: The TBI model was constructed according to the modified Feeney weight-drop method. In part 1 of the experiment, we measured oxidative stress levels by observing the changes in catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) expressions. We also evaluated nuclear factor erythroid 2-related factor 2 (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 + hydrogen-rich water-treated (TBI + 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-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.

Effect of hydrogen-rich water on the chondriosome damage and cytokines in brain tissue of rats with traumatic brain injury

Objective: To observe the effect of hydrogen-rich water on the chondriosome damage and cytokines change in brain tissue of rats with traumatic brain injury (TBI). Methods: Fifty-four health male Sprague-Dawley (SD) rats were divided into three groups by random number table: sham group, trauma group (TBI group), and trauma+hydrogen-rich water group (TBI+HW group), the rats in each group were subdivided into 1, 3 and 7 days subgroups according to the time points after trauma, with 6 rats in each subgroup. The TBI model was reproduced by using a modified Feency method for free fall impact, and the rats in sham group were not given brain impact after craniotomy. The rats in TBI+HW group were given intraperitoneal injection of hydrogen-rich water (5 mL/kg) after TBI model reproduction, and then once a day until being sacrificed; and the rats in sham group and TBI group were given the same amount of normal saline. The neurological severity scores (NSS) for neurologic deficits were calculated at corresponding time points, and then the rats were sacrificed to harvest brain tissue at 3 mm around lesion boundary. The cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were determined by enzyme linked immunosorbent assay (ELISA); the protein expressions of Bax, Bcl-2 were determined by Western Blot; the RFU of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and mitochondrial membrane permeability (MPTP) were determined by fluorescence and enzyme sign method. Results: TBI and TBI+HW groups appeared obvious neurologic damage after injury in rats. NSS scores in TBI and TBI+HW groups showed a decreased tendency with time prolongation after TBI. NSS scores in TBI+HW group at 3 days and 7 days were significantly lower than those of TBI group (NSS score: 9.67±0.82 vs. 11.17±1.17, 6.83±0.75 vs. 8.50±1.04, both P < 0.05). Compared with sham group, the expressions of TNF-α, IL-1β, RFU of ROS in chondriosome, protein expression of Bax in brain tissue in TBI group and TBI+HW group were significantly increased, peaked at 1 day, then they gradually declined. Each time point of RFU of MMP, MPTP in chondriosome and protein expression of Bcl-2 were significantly decreased, and gradually increased after one-day valley value. Compared with TBI group, the expressions of TNF-α, IL-1β, RFU of ROS in chondriosome and protein expression of Bax in brain tissue were all declined at corresponding time points [TNF-α (ng/L): 54.14±1.11 vs. 81.49±2.76, IL-1β (ng/L): 74.53±1.75 vs. 119.44±3.56, ROS (RFU): 92.30±2.46 vs. 121.33±6.57, Bax: 0.89±0.01 vs. 1.10±0.01, all P < 0.01]; RFU of MMP, MPTP in chondriosome and the protein expression of Bcl-2 were all increased at corresponding time points [MMP (RFU): 99.28±3.97 vs. 74.72±3.00, MPTP (RFU): 188.82±4.44 vs. 160.01±2.04, Bcl-2: 0.52±0.02 vs. 0.30±0.02, all P < 0.01]. Conclusions: The high expressions of cytokines and chondriosome damage were involved in the early TBI. Early treatment with an intraperitoneally injection of hydrogen-rich water can reduce chondriosome damage and inflammation factor release, reduce the nerve cell apoptosis after TBI, and protect brain function.