Protective effect of molecular hydrogen against H 2 O 2 -induced oxidative injury in cultured rat spinal cord neurons

Abstract

Objective To investigate the protective effect of molecular hydrogen against hydrogen peroxide(H2O2)-induced oxidative injury in primary cultures of rat spinal cord neurons and the related mechanism.Methods Rat spinal cord neurons were cultured for 7days and were randomly assigned to four groups with different treatments-(1)Normal medium(NM)control:treated with NM;(2)Hydrogen-rich medium(HM):treated with HM;(3)NM+H2O2:treated with 100 μmol/L H2O2and 15μmol/L ferrous chloride(FeCl2)after pretreatment with NM for 2h;and(4)HM+H2O2:treated with 100μmol/L H2O2and 15μmol/L FeCl2after pretreatment with HM for 2h.The respective media were changed every 6hin each group,and 12hlater the cells were collected for assays of reactive oxygen species(ROS),hydroxyl radical(HO·), apoptosis,and the expression of glycogen synthase kinase-3β(GSK-3β)and p-GSK-3β.Results Compared with NM,HM significantly reduced the H2O2-induced intracellular production of ROS and HO·in purified neurons(P0.01),significantly decreased the number of apoptotic neurons(P0.01)and expression of caspase-3(P0.01),and significantly promoted phosphorylation of GSK-3β(P0.01).Conclusion Molecular hydrogen has protective effect against H2O2-induced oxidative injury in primary cultured neurons.The mechanisms might be related to reduction of intracellular production of ROS and HO·,inhibition of apoptosis in neurons and promotion of GSK-3βphosphorylation.