ABSTRACT
Background In the process of radiotherapy, when radiation kills tumor cells, it inevitably damages normal tissue cells. Objective To investigate the role of Toll-like receptor 4 (TLR4)/nuclear factor−kappa B (NF-κB) signaling pathway in the improvement of cognitive impairment induced by ionizing radiation by hydrogen-rich water before and after whole brain irradiation in rats. Methods Fifteen male SD rats were randomly divided into three groups: control group, irradiated group (IR group), and hydrogen-rich water intervention group (IR+HRW group), with 5 rats in each group. The control group was not irradiated, but was given purified water (20 mL·kg−1) by gavage every day, while the IR group and the IR+HRW group were irradiated with a single dose of 20 Gy. Three days before, 10 min before, and 30 days after irradiation, purified water/hydrogen-rich water (20 mL·kg−1) was given by continuous gavage every day. The general condition of the rats was observed every day, and the body weight were measured on the 7th, 14th, 21st, and 30th days after irradiation. On the 30th day after irradiation, the learning and memory ability of the rats was tested by Morris water maze; the pathological changes of hippocampus were detected by hematoxylin-eosin (HE) staining after sacrificing the rats; the contents of glutathione (GSH), malondialdehyde (MDA), interleukin-1β (IL-1β), and hydroxyl radicals in brain tissues were detected by enzyme linked immunosorbent assay (ELISA); the mRNA and protein expression levels of TLR4, NF-κB, NOD-like receptor pyrin domain 3 (NLRP3), and cysteinyl aspartate specific proteinase 1 (Caspase 1) were detected by quantitative real-time PCR (qRT-PCR) and Western blotting in the hippocampus of rats. Results After irradiation, the rats in the IR group showed symptoms such as head hair removal and salivation, while the symptoms of the rats in the IR+HRW group were milder. No animal died in the control and the IR+HRW groups, while one rat died in the IR group. From day 14 to day 30 after irradiation, the body weight of the rats in the IR+HRW group tended to be higher than that in the IR group, but the difference was not statistically significant (P>0.05). The Morris water maze results showed that the escape latency of the IR+HRW group was shortened compared with that of IR group from day 1 to day 5 except day 3, but the difference was not statistically significant (P>0.05). For the rats in the IR+HRW group, it took less time to reach the original location of the platform after removing the platform on day 6 and the number of crossing the platform and the residence time in the original platform quadrant increased (P<0.05). The HE staining showed that the number of hippocampal cells in the IR+HRW group was slightly reduced and arranged neatly, without obvious nuclear hyperchromatic and pyknotic phenomenon. The ELISA results showed that the MDA and hydroxyl radical levels were decreased in the IR+HRW group compared with the IR group (P<0.05), the GSH content was increased, and the IL-1β concentration was decreased, but the differences were not statistically significant (P>0.05). The results of qRT-PCR showed that the mRNA expression levels of TLR4 and Caspase 1 in the hippocampus of the IR+HRW group were decreased compared with the IR group (P<0.05), and the mRNA expression levels of NF-κB and NLRP3 were also decreased, but the differences were not statistically significant (P>0.05). The results of Western blotting showed that the expression levels of TLR4 and Caspase 1 protein in the hippocampus of the IR+HRW group were decreased compared with the IR group (P<0.05), and the expression levels of NF-κB p65 and NLRP3 protein were also decreased, but the differences were not statistically significant (P>0.05). Conclusion Hydrogen-rich water can improve cognitive impairment induced by ionizing radiation in rats, and its mechanism may be related to regulating TLR4/NF-κB signaling pathway, inhibiting inflammatory factors, and attenuating oxidative stress.
ABSTRACT
Background At present, radiation therapy is widely used in clinical treatment of tumors. However, while radiation therapy damages tumor cells, it also injures surrounding normal tissues. Studies have shown that hydrogen is a potential radiation-protective agent. Objective To investigate the neuroprotective mechanisms of hydrogen-rich water activating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/cysteinyl aspartate specificproteinase-9 (Caspase-9) signaling pathway in acute radiation-induced brain injury. Methods Forty male SD rats were randomly divided into four groups: control group, irradiation only group (IR), high-dose hydrogen-rich water intervention group (IR+HHRW), and low-dose hydrogen-rich water intervention group (IR+LHRW), 10 rats in each group. Except for the control group, animals in each group received a single 20 Gy whole brain irradiation. Animals in all groups were gavaged once a day from 3 d before irradiation to 7 d after irradiation, pure water (20 mL·kg−1) was given to the control and the IR groups, and hydrogen-rich water (20 mL·kg−1, 10 mL·kg−1) was given to the IR+HHRW and the IR+LHRW groups. After 7 d of intervention, 5 rats in each group were selected for the Morris water maze experiment for behavioral evaluation. Autopsies were conducted after anesthesia for the remaining animals and blood samples were collected for hematological analysis. Rat brains were harvested for TUNEL staining to observe neuronal apoptosis. HE staining was performed to observe histopathological changes, enzyme-linked immunosorbent assay was adopted to detect oxidative stress-related indicators, and real-time PCR and Western blotting were used to measure the expressions of PI3K/AKT/Caspase-9 pathway-related genes and proteins. Results The body weight of rats receiving irradiation decreased after 7 d of irradiation compared with the control group (P<0.05), and the symptoms such as arched back and malaise occurred to varying degrees, and the symptoms of rats in the IR+HHRW group were significantly milder than those in the IR group. The behavioral test results showed that the escape latency of rats in the IR+HHRW group or the IR+LHRW group was shorter than that in the IR group from day 2 to day 5 (P<0.05), and it took less time for rats in the IR+HHRW group to reach the original position after removing the platform on day 6 (P<0.05). The hematological test results showed that red blood cell (RBC) count, hemoglobin (HGB) level, and white blood cell (WBC) count were significantly decreased in the IR group (P<0.05), and the changes in the IR+HHRW group were improved (P<0.05). The HE staining results showed that the number of abnormal nerve cells, broken and dissolved nuclei, and the degree of damage in the IR+HHRW group were significantly reduced than those in the IR group. The results of oxidative stress evaluation showed that the ability of the IR group to inhibit free radicals decreased, the level of malondialdehyde (MDA) increased (P<0.01); the MDA level decreased after LHRW intervention (P<0.05); the SOD activity was elevated after HHRW intervention (P<0.05). The TUNEL staining results showed that the apoptosis signals in the IR+HHRW group were sparser than those in the IR group (P<0.05). The real-time PCR results showed that compared with the IR group, the mRNA expression levels of PI3K and AKT in the IR+HHRW group and the IR+LHRW group increased (P<0.05), while the mRNA expression levels of Cytc and Caspase-9 decreased (P<0.05). The Western blotting results showed that compared with the IR group, the phospho-AKT (pAKT) protein expression level in the IR+HHRW group increased significantly (P<0.05), while the expression of Caspase-9 and Cytc proteins decreased significantly (P<0.05). Conclusion Hydrogen-rich water can significantly reduce inflammation and oxidative stress caused by acute irradiation-induced brain injury, and decrease neuronal apoptosis. The mechanism may be related to the PI3K/AKT/Caspase-9 signaling pathway.
ABSTRACT
Objective To explore the protective effect of hydrogen-rich water on the oxidative stress injury of astrocytes in mice and its effect on phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signal pathway. Methods In vitro, mice astrocytes were cultured and the logarithmic growth period cells were taken for experiment. ① Experiment one: some cells were acted by 1.25, 2.50, 5.00, 10.00 μmol/L hydrogen peroxide (H2O2) for 20 minutes to determine the appropriate concentration required for astrocyte damage induced by H2O2; cultivating 3, 6, 9, and 12 hours with hydrogen-rich water of 25, 50, 100, and 200 μmol/L, respectively, to determine the concentration and time of hydrogen-rich water pretreatment; the 50 μmol/L hydrogen-rich water was cultured together with PI3K/Akt signal pathway inhibitors wortmannin (WM) 200 nmol/L or 400 nmol/L to determine the best inhibition concentration of wortmannin. Astrocyte activity was detected by methyl thiazolyl tetrazolium (MTT) colorimetry.② Experiment two: some cells were divided into blank control group, H2O2 injury group, hydrogen-rich water pretreatment group (HW+H2O2 group), and co-culture of hydrogen-rich water and wortmannin pretreatment group (HW+WM+H2O2 group). The mRNA expressions of PI3K and Akt were detected by reverse transcription-polymerase chain reaction (RT-PCR); the protein expressions of PI3K, Akt and phosphorylated Akt (p-Akt) were detected by Western Blot. Results ① Experiment one: the survival rate of the blank control group was 100%. Cell activity gradually decreased with the increase of H2O2 concentration, and the survival rate of the H2O2 action 20 minutes cells of 2.50 μmol/L was reduced to about 50%, so a cell injury model was established at this concentration. With the increase of hydrogen-rich water pretreatment concentration, and the duration of action, the cell survival rate increased first and then decreased. The cell survival rate was highest when 50 μmol/L hydrogen-rich water was pretreated with 9 hours, so a hydrogen-rich water pre-protection model was established. After 200 nmol/L or 400 nmol/L wortmannin was cultured together with hydrogen-rich water, cell activity was inhibited, and the cell survival rate of 200 nmol/L wortmannin group was no significantly different compared with that of H2O2 injury group, so the astrocyte suppression model was established. ② Experiment two: compared with the blank control group, the mRNA expressions of PI3K and Akt and the protein expressions of PI3K, Akt and p-Akt were significantly decreased in the H2O2 injury group. Compared with the H2O2 injury group, the PI3K, Akt mRNA expressions and PI3K, Akt, p-Akt protein expressions were significantly increased in the HW+H2O2 group [PI3K mRNA (2-ΔΔCT): 0.843±0.019 vs. 0.631±0.038, Akt mRNA (2-ΔΔCT): 0.591±0.025 vs. 0.558±0.037, PI3K/β-actin: 1.277±0.008 vs. 0.757±0.004, Akt/β-actin: 1.308±0.015 vs. 0.682±0.006, p-Akt/β-actin: 1.210±0.005 vs. 0.614±0.005, all P < 0.05]. The mRNA expressions of PI3K, Akt in the HW+WM+H2O2 group was 0.784±0.159 and 0.556±0.037, respectively, and the protein expressions of PI3K, Akt, p-Akt was 0.715±0.006, 0.686±0.005, and 0.606±0.004, respectively, both were significantly lower than those in HW+H2O2 group (all P < 0.05), and there was no significant difference with H2O2 injury group (all P >0.05). Conclusion Hydrogen-rich water activates the PI3K/Akt pathway, thereby mediates mice astrocytes to exert the biological function of antioxidant.
ABSTRACT
Aims: The aim of the paper was to study the effect and specific mechanism of hydrogen-rich water (HRW) improving cucumber (Cucumis sativus) seedlings growth. Study Design: 14-day-old cucumber seedlings were treated with different saturation hydrogen-rich water in root for three times every three days. After one day of the last treatment, growth parameters were determined and plants tissues were sampled for test photosynthetic characteristics. Place and Duration of Study: In 2017, cucumber (C. sativus ‘JinYan4’) were germinated in College of Bioscience and Biotechnology of Shenyang Agricultural University (Lab ChuangXin). Methodology: 20 cucumbers and 3 replicates were determined for growth parameters. The determination of total soluble sugar and soluble protein content are the methods of Anthrone colorimetry and Coomassic Brilliant Blue. Chlorophyll content was tested by using ethanol immersion extraction method. Gas exchange parameters were measured with LI-6400XT portable photosynthesis system. Chlorophyll fluorescence parameters of leaves were measured by a Handy PEA Fluorometer. Results: Our results showed that 50% saturation HRW significantly enhanced the growth and development of cucumber seedlings, including the improvement of fresh weight, plant height, stem diameter and leaf area. These responses were consistent with a significant increase of leaf water content, total soluble sugar content and soluble protein content, which was further confirmed by the determination of photosynthetic related parameters. Also, research results illustrated that HRW up-regulated chlorophyll content and changed chlorophyll fluorescence parameters of leaves. The increase of chlorophyll content promoted the absorption of light and enhanced plant photosynthesis. Furthermore, HRW changed the leaf stomata conductance and decreased transpiration so as to improve the photosynthetic rate. Conclusion: Taken together, these results suggest that the effect of HRW on cucumber seedling was associated with plant photosynthesis. Therefore, the application of HRW may be a promising strategy to improve cucumber growth.
ABSTRACT
Objective To investigate the protective effect of hydrogen-rich water on rat cognitive dysfunction induced by ionizing radiation.Methods A total of 20 SD rats were randomly divided into four groups with the ramdom number table method: control group(C),hydrogen-rich water group(HRW),irradiation group(IR)and hydrogen-rich water intervention group(HRW+IR),with 5 rats in each group.The spatial memory ability of rats was tested by a morris water maze.The expression of apoptosis-related genes was detected by real-time fluorescence quantitative PCR.The changes of glutathione(GSH),8-hydroxydeoxy guanosine(8-OHdG)and malondialdehyde(MDA)and SOD were also measured.Results The escape latency(F＝6.003,P＜0.05)and the swimming distances(F＝3.850,P＜0.05)of rats in four different groups had statistically significant differences.Compared with the IR group,the escape latency of the HRW+IR group was significantly decreased at 3,4,5 d after irradiation(P＜0.05),and the swimming distance of this group became much longer(P＜0.05).The levels of GSH,8-OHdG and MDA in these four groups had statistically significant differences(F＝6.450,5.033,4.113,P＜0.05).Compared with IR group,the concentration of GSH was increased(P＜0.05),but MDA and 8-OHdG decreased(P＜0.05)in the brain tissue of HRW+IR group,and the expressions of caspase-3,caspase-9 and bax genes were reduced(t＝2.956,3.087,5.246,P＜0.05),while the expression of bcl-2 gene was enhanced(t ＝-3.640,P ＜0.05)in the HRW+IR group.Conclusions Hydrogen-rich water attenuates the oxidative damage of ionizing radiation by neutralizing oxyhydrogen free radicals and thus protects brain from radiation damage.
ABSTRACT
Objective To investigate the effects of hydrogen-rich water (HRW) on brain edema and the expression of aquaporin 4 (AQP4) in brain tissue of rats after acute traumatic brain injury (TBI) and explor the mechanism of its action. Methods Ninety male adult Sprague-Dawley (SD) rats were randomly divided into three groups: Sham group, TBI model group (TBI group) and HRW intervention group (HRW group), each group n = 30 rats. The TBI rat models were replicated by craniocerebral collision, while the Sham group rats underwent only craniotomy without collision and the cranial opening was closed by suturing and bone wax. After successful modeling, in HRW group the rats received intraperitoneal injection of HRW 5 mL/kg, and in the TBI group and Sham group, equal amount of 5 mL/kg 0.9% normal saline was given by the same method, once a day for 5 days in all the groups. The neurological severity scores (NSS) and brain water contents were detected at postoperative 6, 12, 24, 48 hours and 5 days in the three groups;the cerebral cortex expression levels of AQP4 mRNA, positive expression levels of AQP4, the cerebral cortex AQP4 protein expression, protein kinase C (PKC) activity were detected in each group. Results ① The NSS of each time point in Sham group was 0; in TBI group, the rats levels of NSS and brain water contents showed a tendency of firstly rising and then decreasing, and after postoperative 24 hours, the levels reached the peak values and then gradually decreased; in HRW group, the levels of NSS and brain water contents were reduced significantly and at postoperative 24 hours when compared with those in TBI group, the levels of decreasing amplitude in HRW group were more significant [NSS score: 5.50±1.87 vs. 10.50±2.42, brain water content: (78.78±0.65)% vs. (79.98±0.61)% , all P ＜ 0.05].② The AQP4 mRNA expression levels of sham group at all time points were 1; compared with the Sham group, the AQP4 mRNA levels of TBI group and HRW group at the time points showed a tendency of firstly decreasing and then rising and reaching the valley level at 24 hours (2-ΔΔCt: 0.33±0.06, 0.36±0.12 vs. 1, both P ＞ 0.05). ③ The immunohistochemistry detection showed that the brain AQP4 mainly expressed in astrocytes in Sham group at various time points. However, in TBI group, the positive expression of AQP4 in astrocytes in injury area at each time point was decreased, at postoperative 24 hours being the most significant; the positive expression of AQP4 in HRW group was significantly higher than that in the TBI group at each time point and since postoperative 12 hours the statistical significant difference appeared in the comparison between the two groups [Absorbance (A) value: 0.206±0.010 vs. 0.170±0.014, P ＜ 0.05]. ④ The expressions of AQP4 protein at various time points were significantly lower in TBI group compared with those of Sham group, and reached the minimum at postoperative 24 hours (gray value: 0.282±0.100 vs. 1.281±0.115, P ＜ 0.05); but in HRW group, the expressions of AQP4 protein at various time points showed significantly higher than those of TBI group, and since postoperative 12 hours the statistical significant difference occurred (gray value:0.681±0.096 vs. 0.420±0.090, P ＜ 0.05). ⑤ The activity of PKC in TBI group at each time point was significantly decreased compared with that in Sham group, but it was significantly increased in HRW group at each time point and reached the maximum at postoperative 24 hours (A value: 2.67±0.52 vs. 1.51±0.42, P ＜ 0.05). ⑥ Correlation analysis:there was an obvious negative correlation between the brain activity of PKC and brain water content (r2= 0.209, P ＜ 0.001);but there was a significant positive correlation between the activity of PKC and the expression of AQP4 protein (r2= 0.406, P ＜0.001). Conclusion HRW can improve the brain edema in rats following TBI, and the mechanism may be associated with the up-regulation of expression of AQP4 protein and PKC activity in brain tissue.
ABSTRACT
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.
ABSTRACT
Objective To explore the protective effect and mechanism of hydrogen-rich water (HRW) on ethanol-induced acute gastric injury.Methods The experimental study was conducted.Forty kunming mice were divided into the 4 groups by random number table method:normal control group [0.01 mL/g normal saline (NS)+ 0.03 mL/g NS],HRW group (0.01 mL/g NS +0.03 mL/g HRW),ethanol model group (0.01 mL/g 56°alcoholic drinks +0.03 mL/g NS),HRW treated group (0.01 mL/g 56°alcoholic drinks +0.03 mL/g HRW).Ten mice in each group were administrated twice a day for 7 days.Testing indicators:(1) gastric ulcer index was measured,(2) pathological examination of gastric tissues,(3) activity of serum superoxide dismutase (SOD) and expressions of malondialdehyde (MDA),interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were measured,(4) expressions of SOD and MDA in gastric tissues were measured,(5) protein expressions of apoptosis related factors (caspase-3,bax,caspase-9,fas and caspase-8) in gastric tissues were measured by immunohistochemistry,(6) relative expressions of mRNA of apoptosis related factors (caspase-3,bax,caspase-9,fas and caspase-8) in gastric tissues were measured by realtime polymerase chain reaction (RT-PCR).Measurement data with normal distriburion were presented as (x)±s.Comparisons among groups were done using the one-way ANOVA and comparison between groups was done using the LSD-t test.Results (1) Gastric ulcer index was measured:gastric ulcer index of mice in the normal control group,HRW group,ethanol model group and HRW treated group were respectively 0,0,10.40± 1.64 and 3.92 ± 0.23,with statistically significant differences (F=175.050,P＜0.05).There was a statistically significant difference between the ethanol model group and normal control group or HRW treated group (t =19.835,12.352,P＜ 0.05).(2) Pathological examination pathological examination of gastric tissues:① Macropathology of gastric tissues:the surface of the gastric mucosa was normal and smooth in the normal control group and the HRW group,without ulcer,erosion and inflammation.The partial gastric mucosal erosion and ulcer in the ethanol model group was large and very severe.Compared with the ethanol model group,the area of gastric mucosal lesion was reduced in the HRW treated group.② Results of pathological examination of gastric tissues:gastric mucosa in the normal control group and HRW group were integrity.Compared with the normal control group,the partial gastric surface epithelium was degenerate and impaired in the ethanol model group.Compared with the ethanol model group,the gastric mucosal erosion and inflammatory cell infiltration were alleviated in the HRW treated group.(3) Expressions of serum SOD,MDA,IL-6 and TNF-α:expressions of serum SOD,MDA,SOD/MDA and IL-6 were respectively (70±6)U/mL,(7.52±0.23) μmol/L,9.40 ± 1.07,(6.3 ± 1.8) ng/L in the normal control group and (74 ± 4) U/mL,(7.61 ±0.91) μmol/L,9.91 ± 1.55,(5.1 ± 1.6)ng/ L in the HRW group and (101 ± 4) U/mL,(16.95 ± 0.66) μmol/L,5.99±0.17,(19.2±4.9) ng/L in the ethanol model group and (115±5) U/mL,(14.02±0.58) μmol/L,8.23±0.32,(7.1±1.8)ng/L in the HRW treated group,with statistically significant differences among the 4 groups (F=97.405,269.950,16.486,25.663,P＜0.05).The serum TNF-α levels were respectively (53± 14) ng/L,(67± 17) ng/L,(52± 13) ng/L,(58±21) ng/L in the above 4 groups,with no significant difference (F=0.862,P＞0.05).(4) Expressions of SOD and MDA in gastric tissues were measured:expressions of SOD and MDA and SOD/MDA were respectively (93 ± 18) U/mL,(7.90± 1.72) μmol/L,12.48±4.54 in the normal control group and (93±13) U/mL,(6.96± 1.49) μmol/L,13.83±3.40 in the HRW group and (121±31) U/mL,(17.10±4.88) μmoL/L,7.88± 3.70 in the ethanol model group and (143 ± 26) U/mL,(7.31 ± 1.58) μmoL/L,20.00±4.68 in the HRW treated group,with statistically significant differences among the 4 groups (F=5.463,15.051,7.388,P＜ 0.05).(5) The expressions of apoptosis related factors in gastric tissues:the results of immunohistochemistry showed that the levels of caspase-3,bax and fas were repectively 0.065 5± 0.003 7,0.065 7±0.003 0,0.225 4±0.024 3 in the normal control group and 0.065 7±0.002 7,0.064 9±0.003 0,0.246 0±0.022 3 in the HRW group and 0.330 7±0.017 3,0.335 4±0.033 3,0.397 0±0.028 5 in the ethanol model group and 0.096 7±0.003 0,0.084 8±0.001 7,0.375 0±0.035 6 in the HRW treated group,showing statistically significant differences among the 4 groups (F=1 004.222,309.171,48.555,P＜0.05).The levels of caspase-9 and caspase-8 were respectively 0.049 2±0.000 4,0.151 5±0.010 2 in the normal control group and 0.047 9±0.002 0,0.154 00.013 5 in the HRW group and 0.047 0±0.003 7,0.157 2±0.006 2 in the ethanol model group and 0.048 7±0.000 8,0.153 9±0.006 3 in the HRW treated group,with no statistically significant difference among the 4 groups (F=0.998,0.297,P＞0.05).(6) The mRNA expressions of apoptosis related factors in gastric tissues:resutls of RT-PCR showed that relative expressions of mRNA of caspase-3,bax,caspase-9 and fas were respectively 1.00±0.00,1.00±0.00,1.00±0.00,1.00±0.00 in the normal control group and 0.72±0.43,0.66±0.26,1.57±0.31,0.50±0.19 in the HRW group and 3.19±0.87,1.58±0.76,3.04± 1.15,2.84±0.98 in the ethanol model group and 0.49±0.16,0.69±0.25,2.98±0.85,0.53±0.24 in the HRW treated group,showing statistically significant differences among the 4 groups (F=32.106,5.038,9.706,23.387,P＜0.05).The mRNA levels of caspase-8 were respectively 1.00±0.00,1.50±0.60,1.36±0.34,1.32±0.43 in normal control group,HRW group,ethanol model group and HRW treated group,with no significant difference among the 4 groups (F=1.337,P＞0.05).Conclusions Hydrogen-rich water could alleviate ethanolinduced acute gastric injury by antioxidant,anti-inflammatory and anti-apoptosis.Hydrogen-rich water is safe and reliable,without toxic and side effects on the body.
ABSTRACT
Objective To explore the anti-oxidation stress effect of hydrogen-rich water (HW) on thyroid associated ophthalmopathy (TAO). Methods The orbital fibroblasts were derived from orbital adipose connective tissue of TAO patients and cultured in vitro. The cells were treated with different concentrations of hydrogen peroxide (H2O2) for 18 h, and the proliferation ability was detected by CCK-8 method to determine the appropriate H2O2 concentration. The cells were divided into four groups: blank group (normal culture), H2O2 group (treating cells with H2O2 for 18 h), HW+H2O2 group (culturing cells using culture media containing HW for 72 h in combination with H2O2 for 18 h), dexamethasone + H2O2 group (treating cells using dexamethasone 1 μmol/L for 72 h in combination with H2O2 for 18 h). After culturing for 72 h in each group, the fluorescence intensity of reactive oxygen species (ROS) was measured by flow cytometry, and the contents of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected by ELISA. Results We successfully cultured orbital fibroblasts derived from orbital adipose connective tissues of TAO patients. The higher concentration of H2O2, the greater inhibition effect on the proliferation ability of the orbital fibroblasts from TAO patients, and we finally chose 100 μmol/L H2O2. After 72 h of cell culture, the contents of MDA, SOD, and GSHPx and the fluorescence intensity of ROS were (1.63±0.29), (5.06±0.24), (3.94±0.29), and (2.34±0.24) nmol/mL, (10.51±0.32), (2.41±0.23), (5.58±0.29), and (7.98±0.15) U/mL, (107.79±1.06), (21.07±0.92), (49.19±6.75), and (76.33±4.70) U/mL and 18 275.82±521.05, 92 524.81±2 097.01, 54 460.87±572.64, and 35 961.37±540.61 in the blank, H2O2, HW+H2O2 and dexamethasone+H2O2 groups, respectively. Statistic analysis results showed that both HW and dexamethasone could significantly inhibit oxidative stress induced by H2O2 in orbital fibroblasts (all P<0.01), and the inhibitory effects of dexamethasone were significantly more obvious than those of HW (all P<0.01). Conclusio HW may be a treatment option for TAO through anti-oxidant stress.
ABSTRACT
Objective To investigate the protective effect of hydrogen-rich water (HRW) on radiation-induced hematopoietic stem and progenitor cells (HSPCs) injury.Methods Totally 32 C57BL/6 mice were randomly divided into four groups with 8 mice in each group,including control,HRW,radiation and radiation + HRW.Mice in HRW and radiation + HRW groups received 0.5 ml hydrogen-rich water per day by intragastric administration 5 min before irradiation until 7 d post-irradiation.Mice in other groups received 0.5 ml distilled water.Mice in radiation and radiation + HRW group were irradiated with 2 Gy of total body irradiation.Bone marrow cells were isolated at 15 d post-irradiation,and LSK cells were examined for the percentage of hematopoietic stem and progenitor cells,the ability of colony formation and reconstitution,reactive oxygen species (ROS) levels and cell apoptosis.Results Compared with radiation group,the percentages of hematopoietic progenitor cells and LSK cells,colony number of bone marrow cells were significantly increased in radiation + HRW group (t =-4.935,-7.898,5.488,P ＜ 0.05).An elevation of donor chimerism was also found in recipient mice administered HRW after competitive bone marrow transplantation (t =-12.769,P ＜ 0.05).Compared with radiation group,the ROS levels and cell apoptosis in LSK cells were significantly decreased (t =4.380,3.954,P ＜ 0.05).Conclusions Hydrogen-rich water exhibited a protective effect on radiation-induced HSPCs injury.
ABSTRACT
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 SpragueDawley (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 CD34was 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).Conclusion 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.
ABSTRACT
Objective To verify the efficiency and stability of hydrogen-rich water preparation with hydrogen-rich rods. Methods ①Seven firenew hydrogen-rich rods were separately placed in seven plastic bottles, each filled with distilled water and soaked for 6 h, before the hydrogen concentration of the water was measured.This process was repeated 10 times.②After the hydrogen-rich rods with the strongest and weakest hydrogen product capacity were removed, the remaining 5 hydrogen-rich rods were placed separately into 5 plastic bottles filled with distilled water,put in a water bath pot at 20,40 and 60℃, respectively, and kept for 2, 4, 6, 8 and 10 h, respectively.Then, the hydrogen concentration, oxidation-reduction potential(ORP),and dissolved oxygen concentration(DO) were measured at various time points.③In order to determine the hydrogen emission rate from the hydrogen-rich water, the hydrogen-rich rods were constantly kept in some samples and the others were removed.All the sample bottle caps were kept open during the experimental process, and the hydrogen concentration was measured at such time points as 0, 10 and 30 min, 1, 2, 5, 12, 24, 30, 48 and 72 h, respectively.Results ①The hydrogen-rich rods used in this study could well meet the requirements.②When the environment temperature was kept constant, the hydrogen concentration of the water was increased with the soaking time of the hydrogen-rich rods, and the ORP of the water was reduced.However, the DO of the water was decreased with the rise of the environment temperature.③When the hydrogen-rich water was kept in opened plastic bottles with a 25 mm oral diameter, the hydrogen concentration of the samples with the hydrogen-rich rods reserved was almost about 0.50 ppm until 72 h, and that of the others was reduced to almost 0 ppm.Conclusion Our results demonstrate that the hydrogen-rich rods test is a simple and effective method for preparing hydrogen-rich water, which will be an valuable and useful method for using hydrogen-rich water in health promotion and prevention of chronic diseases.
ABSTRACT
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 ratst 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.
ABSTRACT
Objective To investigate the protective effects of hydrogen-rich water postconditioning on glutamate (Glu) mediated ischemia/reperfusion (I/R) injury in isolated brain slices of neonatal rats and explore its mechanism of action.Methods The brains of Sprague-Dawley (SD) neonatal rats aged 7 days were cut into slices and cultured. And then the cultured slices were randomly divided into the normal control group, Glu injury group (1 mmol/L Glu for 30-minute injury), hydrogen-rich water postconditioning group (after Glu injury for 30 minutes, cultured with complete medium containing 100μmol/L of hydrogen-rich water), once per 3 hours to change the medium for totally 24 hours, each group having 12 holes. The brain slices were stained by hematoxylin-eosin (HE) staining to observe the changes of nerve cells. The lactate dehydrogenase (LDH) release rates, the numbers of nissl bodies and the basic fibroblast growth factor (bFGF) in each brain slice were determined to evaluate the degree of cerebral neuronal damage.Results Compared with the normal control group, the number of nerve cells was rare, and the structure not complete; the LDH release rate and the number of bFGF were increased significantly in Glu injury groups [LDH release rates: (50.66±4.93)% vs. (20.15±5.14)%, bFGF (cells/400 power field): 22.79±2.13 vs. 4.13±1.17, both P < 0.01); the Nissl body was decreased (cells/400 power field: 11.81±2.69 vs. 47.10±3.78,P < 0.01) in Glu injury group. Compared with Glu injury group, the morphological structure of brain nerve cells was restored, the LDH release rate was reduced [(39.13±3.66)% vs. (50.66±4.93)%]; bFGF was decreased (cells/400 power field: 14.22±1.22 vs. 22.79±2.13), and the Nissl body was increased (cells/400 power field: 23.25±6.05 vs. 11.81±2.69) in hydrogen-rich water postconditioning group (allP < 0.05).Conclusions Hydrogen-rich water postconditioning has protective effects on rat brain slices with I/R injury induced by Glu. Its mechanism was possibly related to the reduction of free radicals, calcium overload and inflammatory factors induced by excitatory amino acids toxicity, resulting in inhibition of cell apoptosis.
ABSTRACT
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 eosin (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.
ABSTRACT
Objective To observe the protective effects of different doses of hydrogen-rich water on radiation injury in mice,so as to provide scientific basis for the application of hydrogen-rich water.Methods The ICR mice were randomly divided into control group,irradiation group,amifostine group and hydrogen-rich water of low,medium and high dose groups.The 30 days survival rate,body weight,hematology parameters,serum biochemical parameters,organ weight and coefficient,bone marrow micronucleus rate,bone marrow nucleated cell count were observed after total body irradiation with 9.0 Gy gamma rays.Results After 30 d of irradiation,the hydrogen-rich water showed obvious protective effect on the survival rate and body weight in a dose dependent manner so that the survival was significantly higher than that of irradiation group (t =-2.67,P ＜ 0.05).The biochemical index,such as TP,ALB and CRE in the low dose group,TP,ALB,TBIL and CRE in the medium dose group,and TP,ALB,GLU,TBIL,BUN,GRE and UA in the high dose group also indicated the protective effects of hydrogen-rich water (t =-2.04--4.11,P ＜ 0.05).But the protective effect of hydrogen-rich water was not observed in hematology,organ weight and coefficient,and bone marrow micronucleus induction.Conclusions The hydrogen-rich water has anti-radiation effect,which may depend on the dose of hydrogen.
ABSTRACT
There are estimated to be about 700 species of bacteria in the oral cavity. Based on epidemiological investigations, some of these strains have been proposed as the pathogens responsible for oral diseases such as dental caries, gingivitis and periodontitis. Since electrolyzed hydrogen-rich water has been shown to have beneficial effects on human immunity, its use has increased. In our study, the antibacterial activity of hydrogen-rich water for oralagainst bacteria associated with oral disease was evaluated. The bacterial strains Streptococcus mutans, Fusobacterium nucleatum, Porphyromonas gingivalis and Tannerella forsythia were cultured in specific growth medium. S. mutans, F. nucleatum and P. gingivalis were soaked to thein both hydrogen water and tap water for 30 sec and then inoculated onto mitis-salivarius agar and brain heart infusion agar including supplemented withvitamin K and hemin, respectively. The numbers of bacterial colonies were then measured after cultivation for 48 hours. In the case of T. forsythia, which does not grow well on agar plates, inoculations into modified new oral spirochete (NOS) broth were performed and growth curve analysis was undertaken every day with a spectrophotometer. Hydrogen water showed antibacterial activity against all four bacterial strains in comparison with tap-water. We conclude from this that hydrogen water may have a positive impact on oral hygiene by helping to remove cariogenic bacteria and periodontopathogens.