Protective Effects Of Hyperbaric Oxygen On Neuronal Injury By Down-Regulating The ROS/PKC-Regulated Drpl Pathway In Heatstroke Rats

Central nervous system(CNS)damage is the main feature of severe HS and often results in neurological sequelae even if HS treatment is timely and effective.Hyperbaric oxygen(HBO)therapy is a noninvasive treatment for acute ischemichypoxic encephalopathy,traumatic brain injury and neurological rehabilitation.Although HBO has been found to reduce HS-induced brain injury by extenuating oxidative injury,the pathophysiological and molecular biological mechanisms of HBO treatment remain unclear.Dynamin-related protein 1(Drpl)is a major regulator of mitochondrial fission and plays an important role in cellular apoptosis.Reactive oxygen species(ROS)can act as an upstream signaling molecule to regulate protein kinase C(PKC)δ protein kinases to promote the phosphorylation of Drp1 ser616(p-Drp 1 ser616)during ischemia-reperfusion injury.We hypothesized that HBO may protect against HS-induced brain injury by inhibiting Drpl and the ROS/PKC pathway.Our experiments were performed aiming to study the mechanism of protective effect of HBO both in vivo and in vitro.According to the above hypothesis,we took the following studies:1.The protective effect of HBO on HS-induced brain injury in ratsRats were exposed to HBO(250 kPa-60 min)after the onset of HS,or the same pressure but normal air as a control.The mortality,survival time,thermoregulatory and neurological function of rats were examined.Histopathologic changes and oxidative and apoptotic parameters of the hippocampus,hypothalamus and brain stem were also detected.Drpl expression and phosphorylation were determined by Western blot and immunohistochemical staining.As the results,HBO decreased the lethality,thermoregulatory dysfunction and neurological dysfunction including cognitive dysfunction in the MWM test by attenuating oxidative injury(Lower levels of MDA and 8-OHDG and higher activities of SOD and GSH/GSSH)and apoptosis(lower activities of Caspase-3 and 6 and fewer numbers of TUNEL positive apoptotic neurons)in brains of rats following HS.Expression of Drp1 and dephosphorylation of DrplSer637 was increased by HS and was not influenced by HBO.P-Drp1Ser616 activity was increased by HS but inhibited by HBO.These results indicated that HBO may decrease HS-induced brain injury by attenuating oxidative injury and apoptosis through down-regulating the phosphorylation of Drp1ser616.2.The protective effect of HBO on heat stress(HS)in primary rat hippocampal neurons by regulating the phosphorylation of Drp1 through ROS/PKC δ pathway①To detect the phosphorylation of Drp1 in neurons at different time points after HS,cell cultures were randomized into the control group,the 0,1.5,3,6,9 and 12 h post-HS group.The expression of p-Drplser616 reached highest at 3 h following HS.Therefore,the 3-h time point post HS was selected for observations of the effects of HBO on HS.②To examine the role of Drp1 in the protective effect of HBO against HS,rat hippocampal neurons were used as a HS cellular model to examine the effects of HBO and Drp1 inhibitor(Mdivi-1).The cellular viability,oxidative state,apoptosis and mitochondrial membrane potential were measured.Expression and phosphorylation of Drp1 and protein kinase C(PKC)δ were detected by Western blot.HBO decreased HSinduced cytotoxicity,oxidative injury,ROS generation and apoptosis.Mdivi-1 treatment produced the same effects and had a trend to decrease oxidative injury at 3 h after HS.But the difference is not statistically significant.HBO and Mdivi-1 decreased the phosphorylation of Drp1Ser616 induced by HS and HBO decreased the phosphorylation of PKCδ induced by HS.These results indicated that HBO alleviating HS-induced neuronal injury by decreasing the expression of p-Drp1ser616.③To examine the pathway of HBO on regulating the phosphorylation of Drpl.PKC inhibitor(Go6983)and ROS scavenger(NAC)were used before HS on neurons to determine their specific regulatory relationship during this process.Both Go6983 and NAC inhibited HS-activated p-DrpSer616 and p-PKC δ by Western blot.The results indicated that HBO decreased the phosphorylation of Drp1ser616 by downregulating the ROS/PKC δ pathway in cellular HS models.In conclusion,HBO attenuated oxidative injury and neuronal apoptosis by decreasing the phosphorylation of Drp1ser616 through inhibiting ROS/PKC δ pathway,thus alleviating HS-induced brain injury.