Effects Of Hydrogen-rich Saline Research Articles

Effect of hydrogen-rich saline on melanopsin after acute blue light-induced retinal damage in rats.

Excessive exposure to blue light can cause retinal damage. Hydrogen-rich saline (HRS), one of the hydrogen therapies, has been demonstrated to be effective in eye photodamage, but the effect on the expression of melanopsin in intrinsically photosensitive retinal ganglion cells (ipRGCs) is unknown. In this study, we used a rat model of light-induced retinal injury to observe the expression of melanopsin after HRS treatment and to determine the effect of HRS on retinal ganglion cell protection. Adult SD rats were exposed to blue light (48 h) and treated with HRS for 0, 3, 7, and 14 days. Real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) were performed to find the expression of genes and proteins, respectively. The function of retinal ipRGCs was measured by pattern-evoked electroretinography (pERG). The number and morphological changes of melanopsin-positive ganglion cells in the retina were observed by immunofluorescence (IF). Acute blue light exposure caused a decrease in ipRGC function, decreased expression of melanopsin protein and the melanopsin-positive RGCs, and diminished immunoreactivity in dendrites. However, over time, melanopsin showed a tendency to self-recovery, with an increase in melanopsin protein expression and the number of melanopsin-positive RGCs, with incomplete recovery of function within two weeks. HRS treatment accelerated the recovery process, with a significant increase in melanopsin expression and the number of melanopsin-positive RGCs, and an improvement in the pERG waveform within two weeks.

Comparative study on protective effect of hydrogen rich saline and adipose-derived stem cells on hepatic ischemia-reperfusion and hepatectomy injury in swine

AimTo compare and evaluate the hepatoprotective effect of liver parenchyma injection of ADSCs and portal vein injection of HRS in laparoscopic hepatic ischemia reperfusion combined with hepatectomy injury in miniature pigs. MethodsEighteen miniature pigs were randomly assigned to IRI group, HRS group and ADSCs group. HRS was injected through the portal vein 10 min before reperfusion, 1 d, 2 d, and 3 d after surgery. ADSCs were injected into liver parenchyma after hepatectomy. The serum and liver tissue samples were collected at different time points (preoperative, and postoperative at 1 d, 3 d and 7 d). ResultsCompared with the IRI group, both ADSCs and HRS groups can promote liver function recovery, reduce oxidative stress, reduce inflammation, and promote liver regeneration. Compared with HRS, ALT and TBIL in ADSCs group were significantly decreased at 3 d, and AST was significantly reduced at 1 d. The activities of SOD and GSH-Px in ADSCs group were significantly higher than that in HRS group, but the MDA level in HRS group was markedly lower than that in ADSCs group at 1 d. IL-1β was significantly lower in the ADSCs group than in the HRS group at 1 day after operation. The expressions of HGF and PCNA were significantly higher than that in the HRS group at 3 day after surgery. ConclusionOur study has demonstrated that HRS and ADSCs have significant hepatoprotective effects in miniature pigs after HIRI and hepatectomy injury. However, liver parenchyma injection of ADSCs is more beneficial to the recovery of liver function than portal vein injection of HRS.

Effect of hydrogen-rich saline on apoptosis induced by hepatic ischemia reperfusion upon laparoscopic hepatectomy in miniature pigs

Hepatic ischemia reperfusion injury (HIRI) occurs commonly in liver surgery and liver transplantation. Hydrogen, a safe and effective antioxidant, exerts a protective effect against liver injury. In this study, we investigated the role of hydrogen-rich saline (HRS) in apoptosis in a miniature pig model of laparoscopic HIRI upon hepatectomy. Bama miniature pigs were randomly assigned to sham, I/R and HRS groups. The pigs received 10 mL/kg HRS by portal venous injection 10 min before reperfusion and at 1 d, 2 d, and 3 d after surgery. The results showed that HRS treatment significantly decreased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) activity and TUNEL-positive cells. Upon HRS treatment, the expression of P53 and Bax mRNA and protein by RT-qPCR and Western blot was markedly decreased, whereas the expression of bcl-2 mRNA and protein was significantly increased. Moreover, Caspase-3 and Caspase-9 activities were significantly decreased upon treatment with HRS. In conclusion, the results indicate that HRS could alleviate liver injury and improve liver function via inhibiting apoptosis after laparoscopic HIRI and hepatectomy injury in miniature pigs.

Protective effect of hydrogen-rich saline on pressure overload-induced cardiac hypertrophyin rats: possible role of JAK-STAT signaling

BackgroundMolecular hydrogen has been shown to have antioxidant effect and have been used to prevent oxidative stress-related diseases. The goal of this study was to explore if hydrogen-rich saline (HRS) plays a cardioprotective effect on abdominal aortic constriction (AAC) induced cardiac hypertrophy in rats. 60adult Sprague–Dawley rats received surgically the AAC for 6-week. After the surgery, the rats were randomly divided into 4 groups (15 for each):1: sham-operated (sham); 2: AAC-model; 3: AAC + Low HRS (LHRS); and 4: AAC + High HRS (HHRS). The rats in sham and AAC-model groups were treated with normal saline intraperitoneally, while rats in LHRS and HHRS groups were intraperitoneally treated with 3 or 6 mL/kg HRS daily, respectively, for 6-week.ResultsThe ratios of HW/BW and LVW/BW were shown in an order of Model > LHRS > HHRS > SHAM groups. The cardiac hypertrophy was also manifested with increased expressions of atrial natriuretic peptide (ANP), brain natriuretic peptides (BNP) and fibrosis of cardiac tissues in AAC-model group, which could likewise be restrained in LHRS and HHRS groups. Moreover, the JAK-STAT (Janus Kinase-Signal transducers and activators of transcription) signaling molecule expressions were decreased with HRS treatment.ConclusionsOur results showed a protective effect of HRS on pressure overload-induced cardiac hypertrophy in rats, which may be associated to a decreasing in JAK-STAT signaling pathway.

Prevention of ischemia-induced hearing loss by intravenous administration of hydrogen-rich saline in gerbil.

Hydrogen-rich water, which is a potent antioxidant agent, was investigated for its protective effects against ischemic damage of the cochlea in gerbils. The animals were subjected to transient cochlear ischemia by occluding the bilateral vertebral arteries for l5min. Five milliliters of hydrogen-rich saline was then intravenously administered immediately after the insult. Saline without hydrogen was used as a control. Effects of hydrogen were evaluated using the auditory brainstem response (ABR) and histological studies of the inner ear. In non-ischemia animals, ABR thresholds and histological findings of the cochlea did not change by administration of saline or hydrogen-rich saline. In contrast, transient cochlear ischemia caused a 24.2±3.8dB increase in the ABR threshold at 8kHz, and a decrease of 14.1%±1.8% in the number of inner hair cells (IHCs) at the basal turn on day 7. Ischemic damage was more severe at 16 and 32kHz. When the animals were treated with hydrogen-rich saline, cochlear damage was significantly reduced: the increase in ABR threshold was 11.7±2.6dB at 8kHz and the IHC loss was 7.5%±2.1% at the basal turn on day 7. The effects of hydrogen-rich saline were more prominent at higher frequencies. Intravenous administration of hydrogen-rich saline was effective in preventing acute hearing loss due to transient cochlear ischemia.

The Comparative Efficiency of Intraperitoneal and Intravitreous Injection of Hydrogen Rich Saline against N-Methyl-N-Nitrosourea Induced Retinal Degeneration: A Topographic Study.

Retinitis pigmentosa (RP) comprises a heterogeneous group of inherited retinal diseases leading to blindness. The present study explored the protective effects of hydrogen rich saline (HRS) against the photoreceptor degeneration in the N-Methyl-N-nitrosourea (MNU) administrated rat, a pharmacologically induced RP model. The therapeutic effects of intraperitoneal (IP) and intravitreous (IV) injections of HRS on regional retina was quantified via topographic measurements. The MNU administrated rats received IV or IP injections of HRS, and then they were subjected to electroretinography, multi electrode array, histological and immunohistochemistry examinations. The concentrations of the retinal malondialdehyde (MDA), superoxide dismutase (SOD), as well as the mRNA levels of apoptotic-associated genes were quantified. The IP and IV delivery pathways of HRS were both effective to ameliorate MNU induced photoreceptor degeneration. Moreover, the IV acted as a more efficient delivery method than the IP in terms of therapeutic effects. Particularly, the topographic measurements suggested that the IV delivery of HRS could alleviate MNU induced photoreceptor degeneration in the posterior retina. The immunostaining experiments also verified the comparative efficiency between IV and IP delivery of HRS on regional cone photoreceptors. Focal cone photoreceptors showed different susceptibilities to HRS and exhibited as a distinct spatial disequilibrium: cone photoreceptors in the ST quadrant were preferentially rescued; meanwhile, HRS induced protection was feeblest in the IN quadrant. Furthermore, the HRS treatment increased the level of retinal SOD, while reduce the level of retinal MDA in MNU administered rats. The expression levels of sever apoptotic -associated genes were significantly altered by HRS treatment. Collectively, these findings suggest that the IV space is an excellent target for HRS delivery. The IV delivery of HRS can efficiently alleviate the photoreceptors (especially these locate at the posterior retina) from MNU toxicity and act as a candidate treatment for RP.

Effect of hydrogen-rich saline on autophagy in spinal neurons of rats with neuropathic pain

Objective To evaluate the effect of hydrogen-rich saline on autophagy in the spinal neurons of rats with neuropathic pain. Methods Sixty pathogen-free male Sprague-Dawley rats, aged 8-10 weeks, weighing 220-250 g, were randomly assigned into 3 groups (n=20 each)using a random number table: sham operation group (S group), neuropathic pain group (NP group), and hydrogen-rich saline group (H group). Neuropathic pain was produced by chronic constriction injury of the sciatic nerve in the rats anesthetized with chloral hydrate.Hydrogen-rich saline 5 ml/kg was injected intraperitoneally once a day for 7 consecutive days in group H, while the equal volume of normal saline was given instead in S and NP groups.The mechanical paw withdrawal threshold (MWT)and thermal paw withdrawal latency (TWL)were measured at 1 day before operation (T0)and 1, 3, 5 and 7 days after operation (T1-4). The animals were sacrificed after measurement of pain threshold at day 7 after operation.L4-6 segments of the spinal cord were harvested to detect the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ), Beclin-1 and p62 by Western blot. Results Compared with group S, the MWT was significantly decreased, and the TWL was significantly shortened at T2-4, and the expression of LC3Ⅱ, Beclin-1 and p62 was significantly up-regulated at T4 in NP and H groups (P<0.05). Compared with group NP, the MWT was significantly increased, and the TWL was significantly prolonged at T2-4, and the expression of LC3Ⅱ and Beclin-1 was significantly up-regulated, and the expression of p62 was significantly down-regulated at T4 in group H (P <0.05). Conclusion The mechanism by which hydrogen-rich saline reduces neuropathic pain is related to induction of autophagy in the spinal neurons of rats. Key words: Hydrogen; Neuralgia; Autophagy; Spinal cord

Effects of Hydrogen-Rich Saline on Hepatectomy-Induced Postoperative Cognitive Dysfunction in Old Mice.

This study aims to investigate the protective effects and underlying mechanisms of hydrogen-rich saline on the cognitive functions of elder mice with partial hepatectomy-induced postoperative cognitive dysfunction (POCD). Ninety-six old male Kunming mice were randomly divided into 4 groups (n = 24 each): control group (group C), hydrogen-rich saline group (group H), POCD group (group P), and POCD + hydrogen-rich saline group (group PH). Cognitive function was subsequently assessed using Morris water-maze (MWM) test. TNF-α and IL-1β levels were measured by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, along with NF-κB activity determined by ELISA. The morphology of hippocampal tissues were further observed by HE staining. Learning and memory abilities of mice were significantly impaired at day 10 and day 14 post-surgery, as partial hepatectomy significantly prolonged the escape latency, decreased time at the original platform quadrant and frequency of crossing in group P when compared to group C (p < 0.05). The surgery also increased the contents of TNF-α, IL-1β, and NF-κB activity at all time points after surgery (p < 0.05). The introduction of hydrogen-rich saline (group PH) partially rescued spatial memory and learning as it shortened escape latency and increased time and crossing frequency of original platform compared to group P (p < 0.05). Moreover, such treatment also decreased TNF-α and IL-1β levels and NF-κB activity (p < 0.05). In addition, cell necrosis in the hippocampus induced by hepatectomy was also rescued by hydrogen-rich saline. Hydrogen-rich saline can alleviate POCD via inhibiting NF-κB activity in the hippocampus and reducing inflammatory response.

Effect of hydrogen-rich saline on TLR4/MyD88 signaling pathway in rats with endotoxin-induced acute lung injury

Objective To evaluate the effects of hydrogen-rich saline on Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)pathway in rats with endotoxin-induced acute lung injury (ALI). Methods Forty pathogen-free male Sprague-Dawley rats, aged 3 months, weighing 250-300 g, were randomly divided into 4 groups (n=10 each) using a random number table: control group (group C); ALI group; hydrogen-rich saline group (group H); ALI + hydrogen-rich saline group (ALI + H group). ALI was induced by intratracheal instillation of lipopolysaccharide (LPS) 5 mg/kg.At 1 and 6 h after LPS instillation, 0.6 mmol/L hydrogen-rich saline 10 ml/kg was injected intraperitoneally in H2 and ALI + H2 groups, while the equal volume of normal saline was given in the other two groups.At 12 h after LPS instillation, bronchoalveolar lavage fluid (BALF) was collected for determination of the concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 by enzyme-linked immunosorbent assay.The animals were then sacrificed, and lungs were removed for examination of the pathological changes which were scored and for determination of wet to dry lung weight ratio (W/D ratio), and expression of TLR4, MyD88 and nuclear factor kappa B (NF-κB) (by Western blot). Results Compared with group C, the concentrations of TNF-α, IL-1β and IL-6 in BALF, lung injury score and W/D ratio were significantly increased, and the expression of TLR4, MyD88 and NF-κB was up-regulated in ALI and ALI + H groups (P<0.05). Compared with group ALI, the concentrations of TNF-α, IL-1β and IL-6 in BALF, lung injury score and W/D ratio were significantly decreased, and the expression of TLR4, MyD88 and NF-κB was down-regulated in ALI + H group (P<0.05). Conclusion Hydrogen-rich saline can reduce endotoxin-induced ALI through inhibiting TLR4/MyD88 signaling pathway in the lung tissues of rats. Key words: Hydrogen; Toll-like receptor 4; Myeloid differentiation factor 88; Respiratory distress syndrome, adult; Endotoxemia

Effects of hydrogen-rich saline on aquaporin 1, 5 in septic rat lungs

Aquaporin 1(AQP1) and AQP5 have an important role in eliminating extravascular lung water, an increase of which contributes to lung injury in patients with sepsis and its consequent mortality. It has been reported that hydrogen-rich saline (HRS) has protective effects against sepsis-related lung injury. In this study, we hypothesized that the protective effect occurred by preserving the expression of AQP1 and AQP5. To test this hypothesis, male Sprague–Dawley rats received intratracheal administration of lipopolysaccharide (LPS) followed by intraperitoneal injection of HRS. Lung function, wet-to-dry weight ratio, and histopathology scores were determined. The expression of AQP1 and AQP5 at the messenger RNA and protein levels, as well as the involved pathways, was explored by quantitative polymerase chain reaction and Western blot. LPS significantly impaired lung function and downregulated the expression of AQP1 and AQP5 in the rat lung, all of which were attenuated by HRS treatment. Moreover, HRS treatment inhibited LPS-induced activation of p38 mitogen-activated protein kinase and jun N-terminal kinase, which is associated with LPS-induced downregulation of AQP1 and AQP5.

Effect of hydrogen-rich saline on acute kidney injury after orthotopic liver transplantation and the role of autophagy

Objective To investigate the effects of hydrogen-rich saline on acute kidney injury after orthotopic liver transplantation(OLT)in rats and the role of autophagy. Methods Fifty-six healthy adult male Sprague-Dawley rats, weighing 220–250 g, served as donors and recipients.Thirty-two recipient rats were randomly assigned into 4 groups using a random number table: sham operation group(group S), OLT group, hydrogen-rich saline group(group HS), and autophagy inhibitor chloroquine group(group CQ). In group S, laparotomy was performed, and the related blood vessels were isolated.The model of OLT was established in OLT, HS and CQ groups.In OLT and HS groups, normal saline and hydrogen-rich saline 6 ml/kg were injected, respectively, through the inferior vena cava at 5 min before anhepatic phase.In group CQ, chloroquine 60 mg/kg was injected intraperitoneally at 1 h before establishment of the model, and the other treatments were similar to those previously described in group HS.Blood samples were obtained at 6 h of neohepatic phase for determination of serum blood urea nitrogen(BUN)and creatinine(Cr)concentrations.After the end of blood sampling, the left kidneys were removed for determination of malondialdehyde(MDA)content(using thiobarbituric acid method), superoxide dismutase(SOD)activity(xanthine oxidase method), and cell apoptosis(using TUNEL), and for examination of the pathological changes of renal tissues.The damage to the renal tubules was scored.Apoptosis index(AI)was calculated.The expression of phosphorylated p53(p-p53), activated caspase-3, cytochrome c(Cyt c), microtubule-associated protein light chain 3Ⅱ(LC3Ⅱ), and Beclin-1 was detected by Western blot analysis. Results Compared with group S, the levels of BUN, Cr, and MDA, AI, and renal tubular damage score were significantly increased, the SOD activity was decreased, and the expression of activated caspase-3, Cyt c and LC3Ⅱ was up-regulated(P 0.05). Compared with group OLT, the levels of BUN, Cr, and MDA, AI, and renal tubular damage score were significantly decreased, the SOD activity was increased, the expression of activated caspase-3, Cyt c was down-regulated, and the expression of LC3Ⅱ, Beclin-1 and p-p53 was up-regulated in group HS(P 0.05). Conclusion Hydrogen-rich saline can attenuate acute kidney injury after OLT, and the mechanism is associated with activation of p53, enhancement of autophagy and inhibition of cell apoptosis in rats. Key words: Hydrogen; Liver transplantation; Kidney injury; Autophagy; Apoptosis

Effect ofhydrogen-rich saline on TLR4/NF-κB signaling pathway in sciatic nerve of rats with diabetic neuropathic pain

Objective To evaluate the effect of hydrogen-rich saline on Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway in the sciatic nerve of rats with diabetic neuropathic pain (DNP). Methods Pathogen-free male Sprague-Dawley rats, aged 8 weeks, weighing 180–210 g, were used in the study.DPN model was established by intraperitoneal injection of 1% streptozocin (STZ) 65 mg/kg.Twenty-four diabetic rats were randomly divided into 2 groups (n = 12 each) using a random number table: DPN group and hydrogen-rich saline group (HRS group). Another 12 normal rats were randomly selected and served as control group (group C). At 14 days after STZ injection, hydrogen-rich saline 5 ml/kg was injected intraperitoneally once a day for 14 consecutive days in group HRS, while the equal volume of normal saline was given in C and DNP groups.Mechanical paw withdrawal threshold to von Frey stimuli (MWT) and thermal paw withdrawal latency (TWL) were measured at 2 days before STZ injection (T0), and 7, 14, 21 and 28 days after STZ injection (T1-4). The motor nerve conduction velocity (MNCV) of the right hindlimb was measured after pain threshold was measured at T4.After measurement of neurological function was completed, the expression of TLR4 and NF-κB was detected in the sciatic nerve (by Western blot), the tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) contents in sciatic nerves were measured by enzyme-linked immunosorbent assay, and the neuronal apoptosis was detected by TUNEL.The apoptosis index was calculated. Results Compared with group C, the MWT was significantly decreased at T1-4, TWL was shortened at T2-4, and MNCV was decreased at T4, the expression of TLR4 and NF-κB, contents of TNF-α and IL-6, and apoptosis index were increased in HRS and DNP groups (P<0.05). Compared with group DNP, the MWT was significantly increased, and TWL was prolonged at T3, 4 MNCV was increased T4, and the expression of TLR4 and NF-κB, contents of TNF-α and IL-6, and apoptosis index were decreased in group HRS (P<0.05). Conclusion Hydrogen-rich saline can mitigate DNP through blocking TLR4/NF-κB signaling pathway in the sciatic nerve of rats. Key words: Hydrogen; Diabetes mellitus; Neuralgia; Toll-like receptor 4; NF-kappa B; Sciatica

The protective effect of hydrogen-rich saline on lung injury associated with severe acute pancreatitis and its impact on P38MAPK and NF-κB expression

Objective To investigate the protective effect of hydrogen-rich saline on lung injury associated with severe acute pancreatitis and its impact on P38MAPK and NF-κB expressions. Methods Fifty-four male Wistar rats were randomly (random number) divided into three groups: (1) hydrogen-rich saline treatment group (HRS group, n=18 ) , in which the rats were treated with hydrogen-rich saline (6 mL/kg) administered intravenously via tail vein and HRS (20 mL/kg) administered subcutaneously at 5 min after successful modeling. (2) Severe acute pancreatitis model group (SAP group, n=18) , in which rats received equivalent volume of normal saline instead of hydrogen-rich saline both intravenously and subcutaneously as in HRS group. (3) Sham operation group (SO group, n=18) , in which rats were treated with sham surgery, and received equivalent volume of normal saline as in SAP group. The model of severe acute pancreatitis (SAP) was made by retrograde injection of 5% sodium taurocholate (1 mL/kg) into cholepancreatic duct. All rats were sacrificed at 3 h, 12 h, and 24 h separately after the operation (n=6 at a time). The levels of serum amylase, lipase were measured. The ratio of wet and dry lung tissues was measured. The histopathological changes of lung tissues were observed under optic microscope. The expressions of P38MAPK, p-P38MAPK and NF-κB were measured by using immunohistochemistry method. Results Compared with SAP group, there were no significant differences in levels of serum amylase [12 h (5 306.7±909) vs. (5 435.0±441.2) ]and lipase [12 h (1 897.8±149.4) vs. (1 917.9±106.8) ]in HRS group (P >0.05) , but there were significant differences in the ratio of wet and dry lung tissues [12 h (3.12±0.58) vs. (1.87±0.25) ]and histopathology scores [12 h (2.14±0.38) vs. (3.58±0.32) ] (P <0.05). There was no significant difference in expression of P38MAPK in lung tissues among three groups at 12 h. Compared with SO group, the expressions of p-P38MAPK and NF-κB were significant increased in SAP group at 12 h, however, they were lower significantly in HRS group than those in SAP group. Conclusions Hydrogen-rich saline has a protective effect on lung injury associated with severe acute pancreatitis, and its mechanism may be likely related to the antioxidant effect and inhibiting the activation of P38MAPK and NF-κB. Key words: Hydrogen-rich saline; Pancreatitis; Lung injury; Protective effect; P38MAPK; NF-κB

Effects of hydrogen-rich saline on early acute kidney injury in severely burned rats by suppressing oxidative stress induced apoptosis and inflammation.

BackgroundEarly acute kidney injury (AKI) in severely burned patients predicts a high mortality that is multi-factorial. Hydrogen has been reported to alleviate organ injury via selective quenching of reactive oxygen species. This study investigated the potential protective effects of hydrogen against severe burn-induced early AKI in rats.MethodsSevere burn were induced via immersing the shaved back of rats into a 100°C bath for 15 s. Fifty-six Sprague–Dawley rats were randomly divided into Sham, Burn + saline, and Burn + hydrogen-rich saline (HS) groups, and renal function and the apoptotic index were measured. Kidney histopathology and immunofluorescence staining, quantitative real-time PCR, ELISA and western blotting were performed on the sera or renal tissues of burned rats to explore the underlying effects and mechanisms at varying time points post burn.ResultsRenal function and tubular apoptosis were improved by HS treatment. In addition, the oxidation–reduction potential and malondialdehyde levels were markedly reduced with HS treatment, whereas endogenous antioxidant enzyme activities were significantly increased. HS also decreased the myeloperoxidase levels and influenced the release of inflammatory mediators in the sera and renal tissues of the burned rats. The regulatory effects of HS included the inhibition of p38, JNK, ERK and NF-κB activation, and an increase in Akt phosphorylation.ConclusionHydrogen can attenuate severe burn-induced early AKI; the mechanisms of protection include the inhibition of oxidative stress induced apoptosis and inflammation, which may be mediated by regulation of the MAPKs, Akt and NF-κB signalling pathways.

Effects of hydrogen-rich saline on inflammatory responses in sciatic nerve and spinal cord of rats with neuropathic pain

Objective To evaluate the effects of hydrogen-rich saline on inflammatory responses in the sciatic nerve and spinal cord of rats with neuropathic pain (NP). Methods Ninety male adult Sprague-Dawley rats, weighing 180-220 g, were randomly divided into three groups (n=30 each) using a random number table: sham operation group (group S); NP group; hydrogen-rich saline group (group H). NP was induced by chronic constrictive injury.The animals were anesthetized with intraperitoneal 10% chloral hydrate 300 mg/kg.The left sciatic nerve was exposed and ligated with 4-0 silk at 1 mm intervals.After ligation of the sciatic nerve, hydrogen-rich saline (0.6 mmol/L) 10 ml/kg was injected intraperitoneally twice a day for 7 consecutive days in group H, and the equal volume of normal saline was given in S and NP groups.At 7, 10 and 14 days after ligation, the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.The rats were then sacrificed, and the left sciatic nerve and L4-6 segments of the spinal cords were removed for detection of the contents of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and high mobility group box-1 (HMGB1) at 7, 10 and 14 days after ligation. Results Compared with group S, the MWT and TWL were significantly decreased, and the contents of TNF-α, IL-1β and HMGB1 in the left sciatic nerve and spinal cords were increased at each time point in NP and H groups.Compared with group NP, the MWT and TWL were significantly increased, and the contents of TNF-α, IL-1β and HMGB1 in the left sciatic nerve and spinal cords were decreased at each time point in group H. Conclusion Hydrogen-rich saline alleviates NP through inhibiting inflammatory responses in the sciatic nerve and spinal cord of rats. Key words: Hydrogen; Neuralgia; Inflammation; Sciatic nerve; Spinal cord

Effects of hydrogen-rich saline on chronic inflammatory pain in rats

Objective To evaluate the effects of hydrogen-rich saline on chronic inflammatory pain in rats. Methods Thirty-two male Sprague-Dawley rats, aged 8-10 weeks, weighing 200-250 g, were randomly divided into 4 groups(n=8 each)using a random number table: control group(group C); complete Freund's adjuvant(CFA)group; hydrogen-rich saline group(group H2); CFA + hydrogen-rich saline group(CFA+ H2 group). Chronic inflammatory pain was induced by injecting CFA 100 μl into the plantar surface of the left hindpaw in CFA and CFA+ H2 groups.In H2 and CFA+ H2 groups, 0.6 mmol/L hydrogen-rich saline 5 ml/kg was injected intraperitoneally once a day for 7 consecutive days starting from 1 day after injection of CFA, while the equal volume of normal saline was given instead of hydrogen-rich saline in C and CFA groups.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured at 1 day before CFA injection and 1, 3 and 7 days after CFA injection.The rats were sacrificed after the last measurement of pain threshold on day 7 after CFA injection.The left lumbar segments(L4-5)of the spinal cord were removed for determination of nuclear factor E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)expression by Western blot. Results Compared with C group, no significant change was found in the MWT, TWL and expression of Nrf2 and HO-1 in H2 group, and the MWT was significantly decreased, the TWL was shortened, and the expression of Nrf2 and HO-1 was up-regulated in CFA and CFA+ H2 groups.Compared with CFA group, the MWT was significantly increased, the TWL was prolonged, and the expression of Nrf2 and HO-1 was up-regulated in CFA+ H2 group. Conclusion Hydrogen-rich saline can alleviate chronic inflammatory pain in rats, and activation of Nrf2/ARE signaling pathway in the spinal cord is involved in the mechanism. Key words: Hydrogen; Pain; Inflammation

Effects of hydrogen-rich saline on expression of phosphor-p38MAPK during cerebral ischemia-reperfusion in rats

Objective To evaluate the effect of hydrogen-rich saline on the expression of phosphor-p38 mitogen-activated protein kinase (p-p38MAPK) during cerebral ischemia-reperfusion (I/R) in rats. Methods Seventy-two adult male Sprague-Dawley rats, weighing 220-250 g, were randomly divided into 3 groups (n = 20 each) using a random number table: sham operation group (group S), I/R group and hydrogen-rich saline group (group I/RH). Cerebral ischemia was induced in chloral hydrate-anesthetized rats by 2 h middle cerebral artery occlusion in I/R and I/RH groups.The artery was only exposed but not occluded in group S. At 3 days before operation and immediately after onset of reperfusion, hydrogen-rich saline (0.6 mmol/L) 10 ml/kg was intraperitoneally injected in group I/RH, while the equal volume of normal saline was given in S and I/R groups.Neurological deficits were blindly assessed and scored at the end of 24 h reperfusion.The animals were then sacrificed, and brains were removed for microscopic examination and for determination of the cerebral infarct size (by TTC), brain water content, cell apoptosis (by TUNEL), and expression of p38MAPk and phosphor-p38MAPK (p-p38MAPK) (by immunohistochemistry and Western blot). Apoptosis index was calculated. Results Compared with group S, neurological deficit score, apoptosis index, brain water content and cerebral infarct size were significantly increased, and the expression of p38MAPK and p-p38MAPK was up-regulated in I/R and I/RH groups.Compared with group I/R, neurological deficit score, apoptosis index, brain water content and cerebral infarct size were significantly decreased, and the expression of p38MAPK and p-p38MAPK was down-regulated in group I/RH.The pathological changes of cerebral tissues were significantly attenuated in group I/RH as compared with group I/R. Conclusion Hydrogen-rich saline can reduce cell apoptosis through inhibiting p-p38MAPK expression, thus attenuating cerebral I/R injury in rats. Key words: Hydrogen; Reperfusion injury; Brain; Mitogen-activated protein kinases

Effects of hydrogen-rich saline on inflammatory responses during transient cerebral ischemia-reperfusion in rats

Objective To evaluate the effect of hydrogen-rich saline on inflammatory responses during transient cerebral ischemia-reperfusion (I/R) in rats. Methods Forty-five male Sprague-Dawley rats, aged 5-6 yr, weighing 180-250 g, were randomly divided into 3 groups (n=15 each) using a random number table: sham operation group (group S), group I/R and hydrogen-rich saline group (group H). The rats were anesthetized with intraperitoneal 10% chloral hydrate 0.3 ml/100 g. Transient cerebral ischemia was induced by bilateral common carotid artery occlusion with hypotension for 15 min, followed by reperfusion. Five rats were randomly chosen from each group, and Morris water maze was used to assess the cognitive function starting from 5 days before establishment of the model. Place navigation test lasted for 5 consecutive days. The escape latency, swimming speed and swimming distance were recorded. Spatial probe test was carried out on 1 and 3 days after establishment of the model. The time of staying at the target platform quadrant, frequency of crossing the original platform and swimming speed were recorded. The rats were sacrificed after the end of spatial probe test on 3 days after the model was established, and hippocampi were removed to examine the morphology of neurons in hippocampal CA1 region with light microscope. Five rats randomly chosen from each group were sacrificed on 1 day after the model was established, and hippocampi were removed to detect the contents of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β). The rest 5 rats in each group were sacrificed, and hippocampi were removed for determination of nuclear factor kappa B (NF-κB) activity (by immuno-histochemistry). Results In place navigation test before the model was established, the escape latency and swimming distance were gradually shortened with the prolonging training time, and no significant change was found in the swimming speed with the prolonging training time in the three groups. Compared with group S, the time of staying at the target platform quadrant was significantly shortened, and the frequency of crossing the original platform was reduced on 1 and 3 days after establishment of the model, and the contents of TNF-α and IL-1β and NF-κB activity were increased on 1 day after establishment of the model in group I/R. Compared with group I/R, the time of staying at the target platform quadrant was significantly prolonged, and the frequency of crossing the original platform was increased on 1 and 3 days after establishment of the model, and the contents of TNF-α and IL-1β and NF-κB activity were decreased on 1 day after establishment of the model in group H. There was no significant change in the swimming speed during spatial probe test on 1 and 3 days after establishment of the model. Conclusion The mechanism by which hydrogen-rich saline reduces transient cerebral I/R injury may be related to inhibition of inflammatory responses in rats. Key words: Hydrogen; Reperfusion injury; Brain; Inflammation

Effect of hydrogen-rich saline postconditioning on expression of vascular endothelial growth factor receptor-1 during myocardial ischemia-reperfusion injury in rats

Objective To investigate the effect of hydrogen-rich saline postconditioning on the expression of vascular endothelial growth factor receptor-1 (VEGFR1) during myocardial ischemia-reperfusion (I/R) injury in rats. Methods Thirty-six adult male Sprague-Dawley rats, weighing 250-280 g, were randomly divided into 3 groups (n=12 each) using a random number table: sham operation group (group S), group I/R and hydrogen-rich saline group (group H2). Myocardial I/R was induced by 30 min ligation of anterior descending branch of the left coronary artery followed by 2 h reperfusion.In group H2, hydrogen-rich saline 5 ml/kg was injected intravenously at 5 min before reperfusion, while the equal volume of normal saline was given in group I/R.Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and ± dp/dtmax were measured and recorded during reperfusion and at 120 min of reperfusion.The rats were sacrificed at 120 min of reperfusion, and myocardial specimens were obtained for determination of myocardial infarct size, contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (using ELISA), and expression of VEGFR1 (by Western blot). At 120 min of reperfusion, blood samples were collected from the common carotid artery to measure cardiac troponin I (cTnI) concentrations in serum. Results Compared with group S, LVSP and ± dp/dtmax were significantly decreased, LVEDP was increased, the myocardial infarct size was enlarged, cTnI concentrations in serum and contents of IL-6 and TNF-α were increased, and the expression of VEGFR1 was down-regulated at 120 min of reperfusion in H2 and I/R groups.Compared with group I/R, LVSP and ± dp/dtmax were significantly increased, LVEDP and myocardial infarct size were decreased, cTnI concentrations in serum and contents of IL-6 and TNF-α were decreased, and the expression of VEGFR1 was up-regulated at 120 min of reperfusion in group H2. Conclusion Hydrogen-rich saline postconditioning can reduce myocardial I/R injury possibly by up-regulating myocardial VEGFR1 expression and inhibiting inflammatory responses in the myocardium of rats. Key words: Hydrogen; Myocardial Reperfusion injury; Vascular endothelial growth factorreceptor-1; Ischemic postconditioning

Effects of hydrogen-rich saline on endoplasmic reticulum stress during myocardial ischemia-reperfusion in rats

To explore the effects of hydrogen-rich saline on endoplasmic reticulum stress during myocardial ischemia-reperfusion (I/R) in rats. A total of 150 healthy male Sprague-Dawley rats were selected and then randomly divided into 5 groups of normal control (I), sham operation (II), myocardial ischemia-reperfusion (III), hydrogen-rich saline (IV) and normal saline (V) (n = 30 each). Group I had no treatment. In group II, anterior descending branch was merely exposed but not ligated. Myocardial I/R was induced by an occlusion of anterior descending branch of left coronary artery for 30 min followed by 12 h and 24 h of reperfusion with bimbaum. Hydrogen-rich saline 1 ml/100 g was injected intraperitoneally 5 min before reperfusion in group IV. Normal saline 1 ml/100 g was injected intraperitoneally 5 min before reperfusion in group V. The rats were sacrificed at 12 h and 24 h of reperfusion and heart tissues harvested. The pathological changes of myocardial tissue were detected by hematoxylin & eosin staining. The apoptotic cardiomyocytes of myocardial tissue were tested by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). And the expressions of GRP78, Caspase-12, Bcl-2 and Bax in myocardial tissue were detected by Western blot. Compared with groups I and II, the numbers of apoptotic cardiomyocytes, the expression of GRP78, Caspase-12 and Bax in myocardial tissue significantly increased and the expression of Bcl-2 in myocardial tissue significantly decreased in groups III, IV and V. Compared with group III and V, the numbers of apoptotic cardiomyocytes, the expression of GRP78, Caspase-12 and Bax in myocardial tissue significantly decreased while the expression of Bcl-2 in myocardial tissue significantly increased in group IV. Hydrogen-rich saline may decrease cell apoptosis and attenuate myocardial reperfusion injury through inhibiting endoplasmic reticulum stress. The mechanism may be associated with decreasing the expression of GRP78, Caspase-12 and Bax and increasing the expression of Bcl-2 in myocardial tissue.