The Mechanism Of Hyperbaric Oxygen Induced Reactive Oxygen Species Generation In Cultured Cells

Hyperbaric oxygen(HBO)is an effective therapy and has been widely used in the treatment of several diseases.The efficacy of HBO is positively correlated with exposure time and pressure within a certain range.However,excess HBO exposure can cause damage.Reactive oxygen species(ROS)is considered fundamental in HBO's effects.Moderate HBO exposure can induce ROS production moderately,and mobilize endogenous protective mechanisms against damage;excess HBO exposure lead to ROS accumulation,and causing serious oxidative stress.Therefore,in order to clarify the effect and mechanism of HBO on cellular ROS formation and optimize HBO treatment in practice,the following studies were carried out:Part ?: HBO induced endothelial ROS formation and the estimation of cellular ROS sourcesObjective: To investigate the origination of ROS in human umbilical vein endothelial cells(HUVECs)and establish an algorithm to calculate the proportion of ROS from each source.Methods: Whole-cell or mitochondria-targeted fluorescent probes were applied to mark superoxide anion,and the production of ROS by mitochondrial respiratory chain(MRC)complex II,NADPH oxidase(NOX)and xanthine oxidase(XO)were identified by flow cytometry,confocal imaging and microplate fluorometry with or without specific inhibitors after 280 kPa-60 min HBO exposure.Results: HBO increased ROS about 2.14-2.44 fold in mitochondria and 1.32-1.42 fold in whole cell,and was significantly decreased by MRC inhibition about 30% and 16%,respectively.NOX or XO inhibition did not affect HBO-induced ROS generation.Based on these data,it could be further estimated that mitochondrial ROS accounted for 32%-39% of basal whole-cell ROS including 3% from MRC complex II,and NOX accounted for at least 24%-29%.Following HBO treatment,almost all increased ROS originated from mitochondria,and MRC complex II contributed at least 45%-60%.Conclusions: This study provided a simple but effective method to estimate the origination of intracellular ROS and found that MRC was the main source of HBO-induced ROS generation in HUVECs.Part ?: ROS formation in rat spinal neurons under different scheduled HBO exposureObjective: To explore the ROS generation in primary rat spinal neurons under different scheduled HBO exposure,and verify ROS origination estimating algorithm.Methods: Superoxide anion or general ROS fluorescent probes were applied,and the production of ROS by MRC,NOX,XO and monoamine oxidase(MAO)were identified by microplate fluorometry with specific inhibitors after 21?100?145?190?235?280 kPa-60 min or 15?30?45?60?90?120 min-280 kPa HBO exposure.Results: Under basal state,MRC Complex II ROS accounted for 3%-4% of whole-cell ROS,NOX and MAO accounted for at least 35%-38% and 19%-20%,separately.Following 280 kPa-60 min HBO exposure,MRC complex II contributed at least 45%-60%,while the others were not participated.And the ROS levels raised with the increase of HBO exposure pressure and time duration,and remained after 235 kPa-60 min HBO exposure.Conclusion: This study proved that MRC was the main source of HBO induced ROS generation and the platform effect of ROS generation in rat spinal neurons under a certain range of HBO exposure.