The Negative Regulation Of Hyperbaric Oxygen On Heat Shock Protein 32 In Spinal Cord Neurons And The Prevention Against Decompression Sickness

Decompression sickness(DCS)is a pivotal medical concern in diving and hyperbaric operations that depends mainly on prevention.Spinal cord is involved in DCS.And the onset of spinal cord injury in DCS is rapid and serious,and it may cause serious sequelae.However,there is still a lack of preventive strategies.Hyperbaric oxygen(HBO)pretreament is a prevention method can enhance damage resistance by mobilizing endogenous protective mechanism.Our previous studies found that HBO pretreatment increase internal reactive oxygen species(ROS)moderately,and mobilize endogenous protective mechanisms like heat shock protein 32(HSP32)up-regulation in spinal cord neurons and exert DCS spinal cord injury resistace.However,HBO pretreatment not only upregulates the expression of HSP32 through ROS/p38 MAPK/Nrf2 pathway,but also inhibits the overexpression of HSP32 by activating the MEK1/2/Bach1 pathway in spinal cord neurons.Studies indicate that although HSP32 play anti-oxidant,anti-inflammatory and anti-apoptosis roles by decomposing free heme and producing protective metabolites,the overexpression of HSP32 may have harmful effects.That suggest the physiological role of the HSP32 negative regulation which indeuced by HBO is to inhibit the harmful effects which come with HSP32 overexpression in neurons.While,how do the HBO manage both of the positive and negative regulation of HSP32? What effects could HSP32 overexpression exert in neurons? There are no relevant studies at present.Moreover,the application of HBO pretreament in practice may be logistically limited by the need for hyperbaric chambers and trained physicians.And wether a normobaric oxygen pretreatment conbined with the inhibition of negative regulation could induce HSP32 expression moderately and prevent DCS spinal cord injury? The purpose of this study is to further explore the mechanism of HBO on the positive and negative regulation of HSP32 in spinal cord neurons,reveal the physiological role of HSP32 negative regulation,and explore the application of HSP32 negative regulation in the prevention of DCS spinal cord injury.And provide foundations for HBO and normobaric oxygen pretreatment and DCS prevention researches.Part I: The Negative Regulation of Hyperbaric Oxygen on HSP32 in Spinal Cord NeuronsObjective: To further explore the positive and negative regulatory mechanisms of HSP32 expression which induced by HBO in spinal cord neurons.Methods: This study was conducted in primary rat spinal cord neurons.Firstly,by using phosphorylated protein mass spectrometry and western blot to screen the phosphorylation signaling molecules activtion during 280 k Pa-60 min HBO in neurons,and revealing their activation process during HBO exposure.Secondly,the upstream and downstream relationships among activated signaling molecules,p38 MAPK and MEK1/2were measured by using specific inhibitors and RNA interference.And at last,the relationships between activated signaling molecules,ROS and HSP32 expression were observed during HBO exposure?Results: HBO exposure promoted the phosphorylation of MEKK4,TAK1 and B-Raf in rat spinal cord neurons.The phosphorylation level of these signaling molecules increased significantly after 40 min,and peak at 60 min in HBO exposure procedure.Then the phosphorylation decreased significantly post HBO exposure.Inhibiting TAK1 phosphorylation or interfering TAK1 expression could significantly inhibit the phosphorylation of MEK1/2,and partially inhibit p38 phosphorylation and promotes HSP32 expression at last.After interfering the MEKK4 expression,the phosphorylation of p38 and the expression of HSP32 were significantly inhibited,but had no significant effect on the MEK1/2 phosphorylation.Inhibition of B-Raf had no significant effects on the phosphorylation of p38,MEK1/2 and the expression of HSP32.After eliminating mitochondrial ROS during HBO,the phosphorylation of TAK1,p38,MEK1/2 and the expression of HSP32 were significantly inhibited.Conclusion: HBO exposure activates TAK1 by increasing mitochondrial ROS and then activate the MEK1/2 pathway to inhibite the expression of HSP32,while MEKK4 can also be activated and conbined with TAK1 activate p38 pathway to up-regulate HSP32 expression.Part II: The Physiological Role of Hyperbaric Oxygen induced HSP32 Negative RegulationObjective: To explore the effect of HSP32 overexpression which induced by MEK1/2inhibition during HBO exposure,and the interference of damage resistance in spinal cord neurons,clarify the physiological role of the negative regulation of HSP32 after HBO exposure.Methods: This study was conducted in primary rat spinal cord neurons.Firstly,by setting a gradient HBO exposure while inhibiting MEK1/2 activation,to establish a model of HSP32 overexpression.Secondly,oxidative stress and oxygen glucose deprivation were used to simulate DCS spinal cord injury neural damage in the HSP32 overexpression neurons.The neuronal activity,oxidative stress,inflammatory and apoptotic indicators were measured to evaluate neural damage.At last,the content of free iron,carbon monoxide and biliverdin/bilirubin were measured after HSP32 overexpression,and their roles in neural damage resistance were observed by using specific inhibitors or RNA interference.Results: HBO exposure moderately increased HSP32 levels(2.5 folds of control)in spinal cord neurons,and promote the survival of neurons,improve the deterioration of oxidative,inflammatory and apoptosis parameters.After inhibiting the MEK1/2 pathway,overexpressed HSP32(4.5 folds of control)completely reversed these protective effects.After HSP32 overexpression,the levels of free iron and biliverdin/bilirubin in neurons were significantly increased.Reducing free iron could attenuate the adverse effects on the damage resistance in spinal cord neurons,while inhibiting biliverdin/bilirubin could improve the adverse effects.Conclusions: The overexpression of HSP32 can attenuate damage resistance in rat spinal cord neurons by increasing free iron.The physiological role of HSP32 negative regulation is to inhibit the overexpression of HSP32 and avoid its adverse effect.Part III: The Role of HSP32 Negative Regulation in the Prevention of Spinal Cord Injury in Decompression SicknessObjective: To observe the effect of normobaric oxygen(NBO)exposure combined with MEK1/2 inhibitors on the expression of HSP32 in rat spinal cord,and the effect in DCS spinal cord injury prevention in rats.Methods: This study was conducted in SD rats.Firstly,the rats were treated with 100 k Pa-60 min NBO combined with MEK1/2 inhibitor U0126,the expression of HSP32 in spinal cord were detect post HBO exposure.Secondly,at the peaked time of HSP32 expression,the rats were treated with a 700 k Pa-90 min simulated air diving and 200 k Pa/min rapid decompression for DCS modeling.the sign of DCS,hindlimb motor function,spinal cord and serum injury biomarkers were measured to evaluate the effects of NBO-U0126 combined pretreatment.Results: The expression of HSP32 in rat spinal cord increased significantly and peaked at 12 h after NBO-U0126 combined treatment.Compared with Air group,NBO-U0126 pretreatment significantly decreased the incidence of DCS(42.8% vs.71.4%),improved motor function,attenuated oxidative stress,inflammatory response and apoptosis in spinal cord and serum after DCS modeling.HSP32 antagonist zinc porphyrin can partially inhibit the protective effect of NBO-U0126 combined pretreatment.While neither NBO nor U0126 had significant effect on the incidence of DCS and injury biomarkers in surviving rats.Conclusion: The combined pretreatment of NBO and MEK1/2 inhibitor U0126 effectively reduced the incidence of DCS and alleviate DCS spinal cord injury in rats by up-regulating HSP32 expression,which provide a much more feasible approach using non-pressurized NBO instead of HBO in DCS prevention.