Protective Mechanism Of Hydrogen-rich Water On Retinal Oxidative Stress Injury

ObjectiveOxidative stress is the main component of the pathogenesis of AMD,and the researches have shown that HRW can prevent from oxidation and cure many kinds of ophthalmopathy,but the effect of HRW on AMD is still lack of research.Our study explored the protection mechanism of HRW on retinal injury induced by oxidative stress.Methods150 healthy male C57BL/6J mice aged 6-8 weeks were randomly divided into Control group,NaIO₃ group,NaIO₃+HRW group,NaIO₃+AICAR group and NaIO₃+AICAR+HRW group.NaIO₃+HRW group,NaIO₃+AICAR+HRW mice were given intragastric administration with HRW（10mg/kg/day）7days for prophylactic treatment,while Control group,NaIO₃ group and NaIO₃+AICAR group were given distilled water for the same time and dose.On the7th day（A day before NaIO₃ injection）,mice in NaIO₃+AICAR group and NaIO₃+AICAR+HRW group were intraperitoneally injected with AICAR at500 mg/kg,while mice in Control group,NaIO₃ group and NaIO₃+HRW group were intraperitoneally injected with 0.9%normal saline at the same dose.Next NaIO₃ group,NaIO₃+HRW group,NaIO₃+AICAR group,NaIO₃+AICAR+HRW group mice were injected with NaIO₃ by tail vein at 20 mg/kg dose,while Control group mice were injected with 0.9%normal saline at the same dose.After NaIO₃ injection,mice in each group continued to be given intragastric administration as before for 5 days and then drew materials.After HE staining,the retinal morphology was observed.Detecting the content of ROS in the retina by using DHE staining.The SA-β-gal staining was used to detect the aging of the retina.The following is the molecular mechanism analysis.Retina was harvested,and then used Western-blot to detect the oxidative stress hallmark OGG1 protein;In addition,we detected the aging related proteins including Sirt3,p53,p21 and p16 proteins;We also detected the DNA damage response protein ATM,cyclinD1,NF-κB,as well as the High mobility group protein HMGB1 which related to DNA repair.ResultsHE staining of retinal histomorphology showed that RPE layer of NaIO₃mice became thinner and discontinuous,and a large number of black deposits similar to verruca vitreosa could be seen.HRW reduced the thinning of RPE layer and the number and size of black deposits.The results of DHE staining showed that the red fluorescence intensity of retina in NaIO₃+HRW group（31144±574.8）was significantly lower than that in NaIO₃ group（39909±133.5）,and the difference was statistically significant（P<0.01）.In addition,Western-blot showed that the relative expression of oxidative stress marker protein OGG1 in NaIO₃+HRW group（0.44±0.004）was significantly lower than that in NaIO₃ group（0.60±0.005）,and the difference was statistically significant（P<0.01）.SA-β-gal staining showed that HRW significantly reduced the blue-green precipitation in retina compared with NaIO₃ group.Western-blot showed that HRW decreased the expression of Sirt3（0.32±0.004）,p53（0.27±0.03）,p21（0.20±0.01）,and p16（0.36±0.01）in mice retina comparing with NaIO₃ group,and the difference was statistically significant（P<0.01）.In addition,compared with NaIO₃ group,HRW decreased the expression of ATM（0.06±0.009）,cyclinD1（0.50±0.002）,NF-κB（0.55±0.01）,and increased the expression of DNA repair-related protein HMGB1（1.232±0.03）,with significant differences.After treatment with AICAR,the expression of OGG1protein decreased,the expression of Sirt3,p53,p21 and p16 decreased significantly,the expression of ATM,cyclinD1 and NF-κB decreased significantly,and the expression of HMGB1 increased obviously.The changes of the above protein expression after AICAR+HRW were more significant than that after AICAR only.ConclusionHRW can protect the retina from oxidative stress injury by regulating the expression of Sirt3 protein and attenuating senescence.