| Hyperbaric oxygen (HBO) has been used widely as a primary therapy in patients with carbon monoxide poisoning, decompression sickness, and arterial gas embolism, and as an adjunctive therapy for the treatment of various diseases accompanied by impaired oxygen delivery. Recent studies have shown that HBO preconditioning (HBO-PC) has neuroprotective effects against focal and global cerebral ischemia, and induces ischemic tolerance in other organs including spinal cord, myocardium and liver. It is well accepted that breathing greater than1ATA of oxygen will increase production of reactive oxygen species. This is critically important as it is the molecular basis for a number of therapeutic mechanisms. Reactive oxygen species and also reactive nitrogen species serve as signaling molecules in transduction cascades, or pathways, for a variety of growth factors, cytokines, and hormones. Reactive oxygen species are generated as part of normal metabolism by mitochondria, endoplasmic reticulum, peroxisomes, various oxidases, and phospholipid metabolism. Reactive oxygen species act in conjunction with several redox systems involving glutathione, thioredoxin, and pyridine nucleotides and play central roles in coordinating cell signaling and also antioxidant, protective pathways. Antioxidant systems combat reactive oxygen species. Because clinical hyperbaric oxygen protocols are relatively brief, studies show that antioxidant defenses are adequate so that biochemical stresses are reversible. This point is central to the ensuing discussion-oxidative stress is not synonymous with oxygen toxicity.An elaborate network of highly inducible proteins protects aerobic cells against the cumulative damaging effects of reactive oxygen intermediates and toxic electrophiles, which are the major causes of neoplastic and chronic degenerative diseases. These cytoprotective proteins share common transcriptional regulation, through the Keapl-Nrf2pathway, which can be activated by various exogenous and endogenous small molecules (inducers). Inducers chemically react with critical cysteine residues of the sensor protein Keap1, leading to stabilisation and nuclear translocation of transcription factor Nrf2, and ultimately to coordinate enhanced expression of genes coding for cytoprotective proteins. In addition, inducers inhibit pro-inflammatory responses, and there is a linear correlation spanning more than six orders of magnitude of concentrations between inducer and anti-inflammatory activity. Genetic deletion of transcription factor Nrf2renders cells and animals much more sensitive to the damaging effects of electrophiles, oxidants and inflammatory agents in comparison with their wild-type counterparts. Conversely, activation of the Keapl-Nrf2pathway allows survival and adaptation under various conditions of stress and has protective effects in many animal models. Crosstalks with other signalling pathways broadens the role of the Keap1-Nrf2pathway in determining the fate of the cell, impacting fundamental biological processes such as proliferation, apoptosis, angiogenesis and metastasis.The expression of phase-Ⅱ detoxification and antioxidant enzymes is governed by a cis-acting regulatory element named the antioxidant response element (ARE). ARE-containing genes are regulated by Nrf2. ARE-regulated genes are preferentially activated in astrocytes, which consequently have more efficient detoxification and antioxidant defences than neurons. Astrocytes closely interact with neurons to provide structural, metabolic and trophic support, as well as actively participating in the modulation of neuronal excitability and neurotransmission. Therefore, functional alterations in astrocytes can shape the interaction with surrounding cells, such as neurons and microglia. Activation of Nrf2in astrocytes protects neurons from a wide array of insults in different in vitro and in vivo paradigms, confirming the role of astrocytes in determining the vulnerability of neurons to noxious stimuli.Therefore, we believe that neuroprotective effect of HBO pretreatment may be mainly realized by the antioxidant response in astrocytes. To explore the critical role of astrocytes’ Nrf2activation on HBO pretreatment induced ischemic tolerance in the nervous system, the subject was based on the mouse spinal cord ischemia-reperfusion injury model, focus on two levels of the whole animal and cellular level, we designed the following experiment:The first part:Protective effects of HBO pretreatment on mouse spinal cord ischemia-reperfusion (SCIR) injuryObjective:To establish a stable mouse SCIR damage model and confirm the protective effect of HBO pretreatment on SCIR damage. Methods:At24h after HBO exposure (2.5ATA,100%O2,90min), a Mouse Model of SCIR caused by aortic cross-clamping. The animals were divided into Sham group, HBO+SCIR group and SCIR group. The Basso mouse scale for locomotion, spinal cord water content, motor-evoked potentials (MEPs), TUNEL assay were used to detect changes in spinal cord function and morphology.Results:Basso scores were significantly higher in HBO pretreatment group as compared with SCIR group without HBO at24h,36h and48h after aortic cross clamping (7.2±0.8vs5.3±0.5at24h,5.2±0.8vs2.5±0.5at36h,5.0±0.6vs2.0±0.6, at48h,*P<0.01), retain relatively intact hind limb motor function; At48h after SCIR, the ratio of wet/dry weight was significantly lower in HBO+SCIR group as compared with SCIR group (HBO+SCIR70.21±1.79%vs SCIR75.69±1.28%,**P<0.01). At1week after SCIR, the latency of MEPs in SCIR group was significantly prolonged as compared with HBO+SCIR group (SCIR4.4±0.38ms vs. HBO+SCIR3.25±0.3ms,**P<0.01); the amplitude was reduced at1week after SCI and was smaller in SCIR mice than HBO+SCIR mice (SCIR5.2±0.29μV vs. HBO+SCIR8.88±0.74μV,**P<0.01). At1week after injury, the numbers of TUNEL-positive cells in the spinal cords with SCIR were significantly increased compared with Sham group (##P<0.01vs Sham). Treatment with HBO significantly reduced the SCIR-induced increase in TUNEL-positive cells (**P<0.01vs SCIR). The enzymatic activity of caspase-9or caspase-3at1week was reduced in the spinal cord of HBO-PC+SCIR group and SCIR group as compared to Sham group (HBO+SCIR or SCIR vs. Sham,##P<0.01). Moreover, the enzymatic activity of caspase-9or caspase-3at1week after SCIR was reduced in HBO-PC+SCIR group as compared to SCIR group (**P<0.01vs SCIR).Conclusion:HBO pretreatment reduces the SCIR injury, such as improve hindlimb motor function missing, reduce secondary spinal cord edema, ameliorate the reactivity of the MEPs, inhibit or slow down the process of apoptosis after injury, and thus play a protective effect on the SCIR damage.The second part:Effect of HBO pretreatment on Nrf2/ARE pathway in spinal cordObjective:To observe the change of Nrf2activity after HBO in spinal cord.Methods:At12h and24h after HBO, Western blot, ELISA, realtime-PCR were used to detect the changes of Nrf2activity in spinal cord.Results:At12h and24h after HBO pretreatment. The Nrf2nuclear protein expression of spinal cord was significantly increased when compared with CON group (**P<0.01vs CON group). The DNA-binding activity of nuclear Nrf2significantly increased at12h (8.22±1.1fold) and24h (5.03±1.09fold) after HBO pretreatment. glutamate cysteine ligase (GCL), γ-glutamyltransferase (yGT), multidrugresistance protein1(MRP1)(Nrf2target gene) protein level of spinal cord were significantly increased when compared with CON group (5.93±0.99,3.18±0.69,7.42±1.09at12h;4.28±0.86,2.47±0.49,4.43±1.24at24h,*P<0.01vs CON). At12h,24h,36h,48h after SCIR, the glutathione (GSH) level of spinal cord was significantly lower in HBO+SCIR group and SCIR group when compared with Sham group, but it was much higher in HBO+SCIR group as compared with SCIR group.Conclusion:HBO treatment not only significantly improved the Nrf2nuclear protein content in the spinal cord tissue, and also increased its DNA binding activity. Therefore, we confirmed that Nrf2transcriptional activity was enhanced in spinal cord tissue after HBO treatment. Nrf2downstream target genes (GCL, yGT, MRP1) were all key proteins for intracellular GSH synthesis and transit, mRNA and protein content? also increased significantly after HBO. At all time point after SCIR, GSH content in spinal cord significantly increased, to obtain more tissue antioxidant capacity.The third part:HBO-PC-induced Nrf2activation in astrocytes protects neurons from ischemia-reperfusion insultsWe examined the hypothesis that the HBO-PC plays a role in the protection of neurons by astrocytes Nrf2activation.Methods:Establish spinal cord astrocytes and neurons co-culture system, simulate ischemia-reperfusion injury in vitro by oxygen and glucose deprivation (OGD). MTT assay, Western blot, ELISA, realtime PCR were used to detect Nrf2activity in cells after HBO pretreatment.Results:In astrocytes and neurons co-culture system, astrocytes survival rates have no significant change after OGD; under the condition of astrocytes existing, HBO can significantly improve neuronal survival rates and reduced the nerve damage induced by OGD. HBO-PC can significantly increase Nrf2nuclear protein content and DNA binding activity in astrocytes of co-culture system, and simultaneous increased the expression of Nrf2downstream target genes GCL, yGT and MRP1. But the Nrf2nuclear protein content of neurons did not change significantly. HBO-PC significantly increased the GSH content in culture medium of co-culture system. While the HBO-PC was subject to the culture system with only neurons, the GSH content of the culture medium did not significantly change.Conclusion:Nrf2activation in astrocytes has played an important role on HBO-induced spinal cord ischemic tolerance. |
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