The Role Of HSP70in Hyperbaric Oxygen Preconditioning On Decompression Sickness

Decompression sickness （DCS） is a multisystem condition due tobubbles formed when divers surface from depth, escape from a disabledsubmarine or astronauts perform space walk. The aeremia can result initching, arthralgia, paralysis and even death. Spinal cord and lung are twomarket organs of bubble injury. Hyperbaric oxygen （HBO） is an availableclinical therapy for carbon monoxide poisoning, newborn ischemia-hypoxiaencephalopathy and diabetic foot. HBO preconditioning has been proved toenhance tolerance to DCS via nitric oxide （from63.6%to30.3%）. Heat-shockprotein （HSP）70is the most widely studied proteins in the highly conservedHSP family in mammals. It can protection cell against injuries byanti-oxidation or anti-apoptosis. HSP70was also found to have protectiveeffects on DCS.We hypothesized that the beneficial effects of HBO precondition onDCS was related with HSP70and our experiments were performed aimingto study the mechanism of protective effect of HBO both in vivo and in vitro.Based on the above ideas, we took the following studies:1. The protective effect of HBO on DCS in vivo①HSPs expression after HBO exposure: HSPs （70,27and90）expressed in tissues of spinal cord and lung in rats was detected at differenttime points following HBO exposure by western blot. HSP27and HSP90showed a slight but not significant increase after HBO. HSP70increasedand reached highest at18h following exposure before decreasing. L-NAMEor quercetin counteracted the stimulation of HSP70by HBO.②Bubble formation in DCS rat models with different pre-treatments:The18h was selected that at this time point following HBO exposure, ratswere subject to simulated air dive and rapid decompression to induce DCS.Bubbles in pulmonary artery were detected using ultrasound imaging toobserve the possible effect of HBO preconditioning on DCS bubbleformation. The amounts of bubbles in rats pretreated with HBO or airshowed no difference.③Effects of HBO preconditioning on the incidence of DCS: The severity of DCS, oxidative and apoptotic parameters in the lung and spinalcord were compared among different groups of rats pretreated with HBO,HBO plus NG-nitro-L-arginine-methyl ester （L-NAME）, HBO plusquercetin, or normobaric air. HBO preconditioning significantly reduced themorbidity of DCS （from66.7%to36.7%）, the increased levels of oxidation（malondialdehyde,8-hydroxyguanine and hydrogen peroxide） and apoptosis（caspase-3and9activities and the number of apoptotic cells）. L-NAME orquercetin eliminated most of the beneficial effects of HBO on DCS.2. The protective effect of HBO on DCS in vitro①HSPs expression changes after HBO exposure: HSP27,32,70and90were detected at different time points after exposure in human umbilical veinendothelial cells （HUVEC）. HSP70reached highest at18h following HBOexposure before decreasing. L-NAME or quercetin counteracted thestimulation of HSP70by HBO.②HBO attenuates H₂O₂-suppressed cell injury in HUVEC: Cells werethen subject to H₂O₂to induce oxidative injury. Oxidative parameters and cellviability were detected in cells pretreated with HBO, HBO plus L-NAME orHBO plus quercetin. The increased levels of lactate dehydrogenase andmalondialdehyde induced by H₂O₂were reduced by HBO preconditioningsignificantly. HBO enhanced the cell viability and the activities of superoxidase dismutase and glutathione peroxidase decreased by H₂O₂. L-NAMEor quercetin eliminated most of the beneficial effects of HBO on oxidativeinjury.③HBO induces HSP70expression viaAKT and p38pathway inHUVEC: Cells were exposed to HBO and the cell signalling pathwayphoshoprotein array was detected0,6,12and18hours after HBO. Thephosphorylation Akt pathways increased12h after HBO and p38pathway6hafter HBO. P38inhibitor SB203580inhibited the expression of HSP70induced by HBO.In conclusion, these results indicated that HSP70was involved in thebeneficial effects of HBO on DCS, mainly by the anti-oxidation andanti-apoptosis effects. HBO also elicited preventive effect on the HUVECagainst oxidative injury. And Akt and p38pathway were involed in the HSP70expression induced by HBO. This study further revealed that HBOexposure is a promising modality to induce HSP70, and could be feasiblyapplied in diving practice in the prevention of DCS.