The Effects And Mechanisms Underlie The Hydrogen Saline Was Involved In Deep Hypothermic Circulatory Arrest-Induced Oxidative Stress Injury In The Hippocampus

Background&Aims:Deep hypothermic circulatory arrest?DHCA?has been an important supplementary means for the treatment of complex cardiovascular and cerebrovascular diseases since 1950s,but prolonged DHCA contributes significantly to neurological deficit which remains a major cause of postoperative morbidity and mortality.It thus requires understanding the pathophysiology of DHCA prior to limit its shortcomings.The recent studies reported that the ischemia/reperfusion?I/R?injury associated with circulatory is a main factor that involved in pathophysiology of DHCA.From the accumulated data,it has become clearer that I/R injury is,in part,related to an increase in the generation of reactive oxygen species?ROS?after reperfusion.Therefore,in theory,agents against oxidative activity may alleviate the DHCA-mediate brain injury.Hydrogen is a novel anti-oxidant with certain unique properties including that is permeable to cell membranes and can target organelles and specifically quenches·OH and ONOO-exclusively while maintaining the metabolic oxidation.There is substantial evidence that hydrogen provides protection of oxidative stress-induced damage in different diseases or models such as intestinal I/R model,myocardial ischemia model,renal I/R injury and acute liver injury.MicroRNAs?miRNAs?are small non-coding RNAs of about 21-23nucleotides in length and can regulate gene expression at the posttranscriptional level by either degradation or translational repression of target message mRNAs?mRNAs?.There are several studies recently demonstrated that the expression of miRNAs could be altered by oxidative stress.Indeed,the miR-200 family?miR-200a/b/c/141 and-429?was induced significantly in response to oxidative stress in ovarian cancer cells and endothelial cells.In our previous study,we have showed that the expression of miR-29 family was altered in the model of DHCA.In the present study,we sought to establish an in vitro experimental condition of hypoxia/reoxygenation?H/R?injury in HT22 cells and used it to examine the effects of hydrogen on H/R-induced cell death.In addition,we would further study the potential functions of miRNA by which hydrogen exerts its effects on the H/R-induced cell death.Methods:The model of H/R was established using an airtight culture container and the anaeropack.Measurement of mitochondrial membrane potential?MMP?and reactive oxygen species?ROS?production was used H₂DCFDA and JC-1 staining.Western blot was used for the quantification of Akt,p-Akt,Bcl-2,Bax and cleaved caspase-3 proteins.The microRNA?miRNA?profile in hippocampal neurons from rat model of DHCA was determined by miRNA deep sequencing.MiR-200s and miR-29 family expression in HT-22 cells was analyzied by quantitative realtime PCR.Results:We established model of hypoxia/reoxygenation?H/R?with hypoxia for 18hours followed by reoxygenation for 6 hours?H18/R6?on HT-22 cells.The cell death was significant induced by H18/R6 treatment.At the same time,the elevation of ROS and reduction of MMP were significantly induced by the H18/R6 treatment.Hydrogen saline treatment significantly reduced H18/R6-caused cell death,increased ROS and decreased MMP.The levels of p-Akt?Ser 473?and Bcl-2 were significantly increased while Bax and cleaved caspase-3 were decreased by hydrogen treatment on the model of H18/R6.The expression of miR-200 family was significantly elevated in model of DHCA and H/R.Hydrogen administration inhibited the H/R-induced expression of miR-200 family in HT-22 cells.In addition,inhibition of miR-200 family suppressed H/R-caused cell death through inhibiting ROS production and MMP decreased.In addition,the levels of p-Akt?Ser 473?and Bcl-2 were significantly increased while Bax and cleaved caspase-3 were decreased by miR-200 family inhibition the model of H18/R6.Here,we also showed that the expression of the miR-29 family?miR-29a/b/c?was significantly reduced in model of DHCA and I/R.Overexpression of the miR-29 family inhibited the I/R-induced elevation of ROS and reduction of MMP in HT-22 cells.In addition,administration of the miR-29 family suppressed proteins of Keap1,Bax and PUMA and increased Nrf2 expression.We further demonstrated that the miR-29 family targeted the PUMA by luciferase reporter assay and Western blot analysis.Conclusions:These results suggest that H/R causes oxidative stress-induced cell death and that the hydrogen protects against H/R-induced cell death in HT22 cells,in part,due to reducing expression of miR-200 family.Our data suggest that by targeting a pro-apoptotic BCL2 family member PUMA,the miR-29 family might emerge as a strategy for protection against DHCA-mediated neural cell injury.In conclusion,our results provided the new theory for limited the DHCA-induced neural injury.