| Diabetic neuropathy(DN) is one of the major complications among these other chronic diabetic complications. The oxidative stress, inflammatory cytokines and apoptosis have been strongly implicated in the pathogenesis in the development of diabetic neuropathy. However, to date,there is no effective treatment for diabetic peripheral neuropathy. Recently, more and more studies confirmed that hydrogen gas(H2 <4%) and hydrogen-rich saline could exert a therapeutic antioxidant activity by selectively reducing hydroxyl radicals(the most cytotoxic ROS) and effectively protect against transient cerebral ischemia/reperfusion damage, sepsis,neurodegeneration disease. Furthermore, at present, increasing researchers have shown that H2 is a new therapeutic medical gas. This study, using the different animal models,was designed to investigate the protective effects of H2 in diabetic peripheral neuropathy and possible mechanisms for the clinic and the patients.Expirement one Effect of hydrogen-rich saline on the injury of Schwann cells induced by high glucoseObjective: Oxidative stress induced by high glucose is known to play important roles in the development of diabetic neuropathy. To investigate the effect of different concentration high glucose on the apoptosis of rat Schwann cells(SCs) and its possible mechanism.Methods: The RSC96 were cultured with DMEM medium containing 10% FBS, and seeded in 6 or 96-well plates, at 3 m L/well or 200μL /well and 2×106 cell/m L.Part 1:The effect of different concentration of high glucoses on the apoptosis of rat Schwann cells. Cultured rat Schwann cells(SCs) were randomly divided into five groups(n=30): normal control group(5.6mmol/L Con), high glucose groups(HG+25mmol/L, 50mmol/L, 100mmol/L) and mannitol group(MANNITOL). At the end of24 h, 48 h, and 72 h, cell growth conditions were observed under inverted microscope and the cell viability was measured by MTT assay, meanwhile inflammatory cytokines and the content of MDA and 8-hydroxy-2 deoxyguanosine(8-OHDG) were measured by specific ELISA kits.Part 2: 1.The effect of H2-rich(HS) medium on cell viability. The part experiment was divided into six groups(n=6): control group; control HS group; HG group and HG+H2 group. The SCs were cultured into normal or different concentration(0.15mmol/L; 0.30mmol/L; 0.60mmol/L) H2-rich medium to continue to cultute, at the same time HG(50mmol/L) or same dose PBS was added into medium for 48 h,cell viability was processed with MTT. 2. To investigate the effect of hydrogen-rich saline on the oxidative stress, inflammatory cytokines and the apoptosis of rat Schwann cells(SCs) induced by high glucose. The part experiment was divided into three groups: control group; HG group and HG+H2 group. Schwann cells were treated with primary DMEM(5.6m M) as control group(Control), with 0.6m M hydrogen-rich medium as hydrogen group(H2), with 44.4m M of glucose plus primary medium(50m M of glucose in the complete medium) as high glucose group(HG), with high glucose and accompanied by 0.6m M of hydrogen-rich medium as treatment group(HG+H2) for 48 h, respectively. At the end of 48 h, cell growth conditions were observed under inverted microscope and the cell viability was measured by MTT assay, inflammatory cytokines and the content of MDA and SOD were measured by Coomassie blue method, the expression of active caspase-3(17k Da) protein was assessed by Western blot, meanwhile the apoptosis was also detected by flow cytometry.Part 1:Results: Compared with other time points(24h and 72h), the cell survival rate was significantly decreased, the content of MDA and 8-OHDG was increased significantly(P < 0.05) in HG groups at the end of 48 h. At the end of 48 h, compared with Con group, the cell survival rate was significantly decreased; the content of MDA and 8-OHDG were increased significantly(P < 0.05) in HG groups, in which at the dose of 50mmol/L HG was significant compared with other HG groups. No significant difference was found between Con group and MANNITOL group.Part 2: Results : Hydrogen had no harmful effect on SCs by MTT(P>0.05). HG induced excessive cell viability decreased. But after hydrogen treatment, cell viability was improved(P<0.05) and showed a dose-dependent effect(P<0.05).Part 3: Results:1. At the end of 48 h, compared with Con group, the cell survival rate was significantly decreased; the expression of active caspase-3,apoptosis index, the content of MDA and 8-OHDG and the levels of inflammatory cytokines were increased significantly(P<0.05) in HG group and HG+HS group; 2. Compared with HG group, the cell survival rate, the level of inflammatory cytokine(IL-10) were significantly increased, while the expression of active caspase-3, the apoptosis index,the content of MDA and 8-OHDG and the level of inflammatory cytokines(TNF-α,HMGB1, IL-1β) were decreased significantly(P < 0.05) in HG+HS group.Conclusion: HS can effectively attenuate high glucose-induced apoptosis to rat Schwann cells though inhibiting oxidative stress, modulation of inflammatory cytokine and expression of caspase-3 protein.Expirement two Protective effect of hydrogen-rich saline on strepotozotocin –induced diabetic peripheral neuropathy model in ratsObjective: Hydrogen-rich saline(H2 was dissolved in normal saline for 6h under0.4MPa to a supersaturated level) has the anti-oxidant, anti-inflammatory and anti-apoptotic effects in vivo and in vitro. DPN is a common and intractable complication in patients with diabetes which leads to a poor quality of life secondary to pain. The aim of this study was to investigate the protective effect of hydrogen-rich saline on diabetic peripheral neuropathy induced by strepotozotocin.Methods: A single dose of 45 mg/kg STZ dissolved in citrate buffer was injected intra-peritoneally to induce diabetes in rats.Part 1:(1) 40 rats were randomly divided into five group(n=8 each group): normal control group(Con group),diabetic model group(DM group) and HS groups(DM+5ml/10ml/20 ml groups). Intra-peritoneal injection of HS(5 ml, 10 ml and20ml/kg/day, respectively) was performed for 4 weeks starting from the 4th week of STZ injection in HS groups. The rats in Con group and DM group received the equal amounts of normal saline. Behavioral tests were employed to assess nociceptive threshold at difference time points(4th,5th,6th,7th,8th). Detection of motor nerve conduction velocity(MNCV) of sciatic nerve was carried out after last behavioral test.The level of oxidative stress and inflammatory cytokines in sciatic were measured in the eighth week. Moreover, glucose levels, body weight and AGEs content in blood were measured before and at the end of the experiment to observe the effect of HS on these parameters.Part 2: Based on experiment 1, 24 rats were randomly divided into 3 groups(n=8each group): normal control group,diabetic model group and HS group. According to the results of experiment 1, treatment with 10 ml/kg HS was chosen in this experiment. Intraperitoneal injection of normal saline(10ml/kg/day, respectively) was performed for 4 weeks in normal control group and diabetic model group,meanwhile HS group received the equal volume of HS intraperitoneally daily. The expression of caspase-3, Bax and Bcl-2, and the apoptosis in sciatic were assessed in the eighth week.Results:Part 1: At the end of 8thweek, diabetic rats exhibited increased blood glucose levels and decreased body weight compared with normal control rats, the blood contents of AGEs in diabetic rats significantly increased as compared with those in the normal control rats(P< 0.00). The reduction of the blood contents of AGEs were observed in HS-treated groups, of which only HS at dose of 10ml/kg decreased the blood contents of AGEs significantly compared with diabetic model group. Chronic treatment with HS for 4 weeks significantly attenuated behavioral(thermal and mechanical pain thresholds, MNCV), biochemical(the levels of oxidative stress( SOD and MDA) and inflammatory cytokine(TNF-α, HMGB1,IL-1β, and IL-10), and molecular(apoptotic cell) changes associated with diabetic neuropathy(P<0.05), of which HS treated group at dose of 10 ml /kg significantly compared with the other HS-treated groups(P<0.05),without affecting the rats in control group(P>0.05).Part 2: Compared with Con group, the expression of caspase-3, Bax and Bcl-2 was increased in DM group and HG+HS group. Compared with DM group, the expression of caspase-3 and Bax was decreased, while the expression of Bcl-2 was increased(P﹤0.05).Conclusion: Treatment with HS not only attenuated the diabetic condition but also reversed neuropathic pain through modulation of oxidative stress, inflammatory cytokines and apoptosis in diabetic rats.Expirement three Beneficial effects of hydrogen-rich saline in experimental diabetic peripheral neuropathy via the mitochondrial KATP channelObjective: The activation of mitochondrial ATP sensitive potassium channels can effectively alleviate the damage of nerve cells. The aim of the present study was to investigate the effects of hydrogen-rich saline(HS) against on diabetic peripheral neuropathy and whether the effect depends on mitochondrial potassium ATP-dependent channel(mito-KATP) in strepotozotocin-induced experimental diabetes.Methods: A single dose of 45 mg/kg STZ dissolved in citrate buffer was injected intraperitoneally to induce diabetes in rats.(1) 32 rats were randomly divided into five group(n=8 each group): normal control group(Con group),diabetic model group(DM group), HS groups(DM + HS10 ml group) and HS+5-HD group. Intraperitoneal injection of HS(10ml/kg/day, respectively) and 5-HD was performed for 4 weeks starting from the 4th week of STZ injection in HS and HS+5-HD groups daily. 5-HD(a selective mito-KATP blocker, 20mg/kg) was injected 30 min before HS administration. Con group and DM group received the equal volume of normal saline intraperitoneally daily. Behavioral tests were employed to assess nociceptive threshold at 5th W, 6th W, 7th W, 8th W. Detection of motor nerve conduction velocity(MNCV) of sciatic nerve was carried out after last behavioral test. The level of oxidative stress and inflammatory cytokines in sciatic were measured at the end of8 th week. Moreover, the expression and the activity of caspase-3 was assessed by Western blot, meanwhile apoptosis in sciatic nerve was detected by TUNEL method.Results : At the end of 8thweek, compared with the Con group, the thermal and mechanical pain thresholds, MNCV of sciatic nerve, the activity of SOD and the level of IL-10 were decreased significantly, while the expression and the activity of caspase-3, the content of MDA, the number of TUNEL positive cells and the levels of TNF-α, HMGB1, IL-1β were increased significantly in DM group, HS group and HS+5-HD group. Compared with the DM group, the thermal and mechanical pain thresholds, MNCV of sciatic nerve, the activity of SOD and the level of IL-10 were increased significantly, while the expression and the activity of caspase-3, the content of MDA, the number of TUNEL positive cells and the levels of TNF-α, HMGB1,IL-1β were decreased significantly in HS group after chronic treatment with HS for 4weeks.Conclusion: HS may be a novel strategy for the treatment of diabetic peripheral neuropathy via activation of mito-KATP channels as well as modulation of inflammatory cytokines, reduction of oxidative stress, and apoptosis.Summary1. The protective effect of hydrogen-rich saline on the apoptosis of rat Schwann cells induced by high glucose was related to modulation of inflammatory cytokines,reduction of oxidative stress and apoptosis.2. Beneficial effects of hydrogen-rich saline in experimental diabetic peripheral neuropathy was related to modulation of inflammatory cytokines, reduction of oxidative stress and apoptosis in vivo.3. The anti-oxidant, anti-inflammatory and anti-apoptotic effects of hydrogen-rich saline could reverse the diabetic peripheral neuropathy induced by STZ partly via the activation of the mitochondrial KATP channel, moreover, HS may be a novel strategy for the treatment of diabetic peripheral neuropathy because there is no any side effects in the process of treatment. |
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