Studies On The Prevention Of Lung Oxidative Injury And Hyperoxic Seizure And The Role Of GABA Receptors In The Antinociceptive Effects Of Hyperbaric Oxygen Treatments

Study I:The effects of hydrogen gas on lung oxidative injuryLung oxidative damage happens in many situations. Many environmental factors, such as hyperoxia, air pollution, smoking, ion radiation, and heavy metal ion can cause lung oxidative damage. Some severe patients would suffer lung oxidative damage when they need long-term oxygen therapy. Air pollution, such as flue dust, airborne dust, NO2, SO2, O3, asbestos, has been proved to cause lung oxidative damage. It is generally accepted that oxidative damage is the dominating cellular damage caused by ion radiation, and60-70%of it is caused by hydroxyl radical (·OH). Furthermore, it is suggested that lung injury induced by smoking and some heavy metal ion, including cadmium and chromium, is also related to oxidative damage. Lung oxidative damage is so common that it would be greatly valuable to find a therapy for effective prevention.In medical field, H2has long been limited to deep diving and was generally regarded as physiologic inert gas. In1975, Malcolm Dole reported for the first time that hyperbaric H2(8atm) could be a possible treatment for cancer by decreasing·OH. In2001, Bouchra Gharib found that hyperbaric H2(8atm) attenuated parasite-induced liver inflammation, and the mechanism also involved eliminating·OH increased by inflammation. But probably because it’s hard to apply H2at such a high pressure in clinical situation, these researches didn’t draw widespread attention. Until2007, when Ohsawa et al reported for the first time that2%H2at atmosphere pressure could also act as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals, its importance to prevent oxidative damage raised widespread concern. Subsequently, it was reported that2%H2inhalation was effective to hepatic and cardiac hypoxia-ischemia injury, inflammation injury caused by small intestine transplantation, and neonatal hypoxia-ischemia injury. Besides, H2-saturated water drinking has also been proved to be effective to many disorders caused by oxidative stress, such as cerebral hypoxia-ischemia injury, human type II diabetes, nephrotoxicity induced by cisplatin, Parkinson’s disease, atherosclerosis in apolipoprotein and so on. All these evidence showed that H2have the ability to attenuate different kinds of oxidative damage.The mechanism of lung oxidative damage is complicated and remains unclear, but it’s certain that it is closely related to O2-, H2O2and·OH. Since H2can attenuate various oxidative damage by selectively decreasing·OH and peroxynitrite (·ONOO-), we hypothesize that it might prevent lung oxidative damage. In the present study we induced three different models of lung oxidative injury and applied three different ways to administrate H2. The results show that H2could effectively prevent lung from oxidative injury and indicate H2could be a potential treatment to clinical lung oxidative injury.In part I, we We investigated whether molecular hydrogen could protect alveolar type II epithelial (AT Ⅱ) cell line A549, from H2O2induced oxidative damage. We determined the influences of different concentration of H2(0.16mM,0.32mM,0.48mM,0.64mM and0.8mM) on A549cell viability, lactate dehydrogenase leakage, SOD, GSH and production of hydroxyl radical. The results showed that molecular hydrogen could effectively prevent A549cells from necrosis induced by H2O2, attenuate cellular lipid peroxidation and DNA damage, and prevent SOD and GSH decline. In addition, we proved that molecular hydrogen molecular hydrogen could enhance A549cell’s ability to eliminate·OH, indicating that molecular hydrogen molecular hydrogen could reduce·OH in A549cells produced by Fenton reaction initiated by H2O2. Combining with our previous researches in vivo, our results suggested that molecular hydrogen molecular hydrogen could prevent lung oxidative damage and might be a potential therapy to lung oxidative injury.In Part II, we investigated whether consumption of saturated hydrogen saline protects rats against paraquat-induced acute lung injury. Adult male Sprague-Dawley (SD) rats were randomly divided into four groups:control group; hydrogen water-only group (HW group); paraquat-only group (PQ group); paraquat and hydrogen water group (PQ+HW group). The rats in control group and HW group drank pure water or hydrogen water; the rats in PQ group and PQ+HW group were intraperitonealy injected with paraquat (35mg/kg) and then provided pure water or hydrogen water ad libitum for72h, respectively. The pleural effusion, lung wet to dry weight ratio (W/d ratio), the concentration of protein and total cell counts in bronchoalveolar lavage fluid (BALF) were measured. Lactate dehydrogenases (LDH) in BALF and malondialdehyde (MDA) level in lung tissue were measured to examine the oxidative damage. Hematoxylin and eosin (H&E) and TUNEL staining were used to examine histological changes and apoptosis. The obtained results obtained showed that hydrogen water ameliorated these biochemical and histological lung alterations induced by PQ in rats at the end of72hours, demonstrating that hydrogen water alleviated paraquat-induced acute lung injury possibly by inhibition of oxidative damage. Study Ⅱ:The effects of Edavarone and Teprenone on hyperoxic seizureHyperoxic seizure is a disease which occurs when human inhales hyperbaric oxygen (greater than200kPa) for a certain time. Its clinical manifestation is increased excitability of nerve cells which causes epilepsy. Hyperoxic seizure can occur during diving activities. When it occurs, divers may have systemic onset tonic contraction, paroxysmal spasms, loss of consciousness, which is accompanied by urinary incontinence. Divers tend to lose control and have drowning, floating or other diving accidents, which seriously impact divers’safety. Therefore, it is utmost important to effectively prevent hyperoxic seizure and maximize safety with hyperbaric oxygen in military and civilian diving and hyperbaric oxygen therapy.Part I:The potential to increase the risk of central nervous system oxygen toxicity (CNS-OT) under hyperbaric oxygen (HBO2) conditions of perfluorocarbon (PFC) has been hypothesized, but little is known about the effects, mechanism and prevention. A rat model of CNS-OT was used to evaluate the effects of PFC emulsion administered intravenously. The electroencephalogram (EEG) was recorded during6ATA HBO2exposure, and the levels of malondialdehyde (MDA), nitric oxide (NO) and hydrogen perioxide (H2O2), the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and NO synthases (NOS) in the brain cortex and hippocampus were examined after exposure. Edaravone, an effective antioxidant, was employed to prevent PFC related CNS-OT facilitating. The results showed that after PFC administration, the latency to first electrical discharge in EEG was significantly shortened, the MDA, H2O2, NO levels and NOS activity increased, the SOD, GPx and CAT activities decreased. Edaravone effectively protected against CNS-OT and the adverse effects of PFC. The results clearly demonstrated that PFC administered before HBO2would promote the occurrence of CNS-OT, and edaravone could serve as a promising chemical to prevent CNS-OT.Part II:In HSP family, HSP70is the most conservative, important and abundant one. It has antioxidant, anti-apoptotic and other functions. HSP70is a nonspecific cytoprotective protein, which can be classified into stress-induced HSP70(also known as Hsp72or Hsp70I), structure Hsc70(Hsp73), mitochondrial Hsp75(mtHsp75) and Grp78which is located in the endoplasmic reticulum (BiP). Recent studies have shown that HSP70induced by a variety of factors has a preventive effect on epilepsy. Hyperoxic seizure is a special type of epilepsy which shares the general pathology. Arieli et al have found adequate thermal stress can significantly inhibit hyperoxic seizure and the level of inhibition has a linear correlation with HSP70. In the present study, we found that a single orally dose of teprenone (HSP70inducer) significantly increased HSP70levels in rat brain tissue and inhibited hyperoxic seizure in rats. However, when quercetin (HSP70antagonist) was given, HSP70levels in rat brain tissue droped to normal levels, and hyperoxic seizure in rats returned to normal levels. The present study shows the effects of teprenone on hyperoxic seizure and reveals the important role of HSP70in the mechanism. Study III:The antinoceciptive effects of hyperbaric oxygen treatmentsPain, a complex physiological and mental activity, is one of the most common clinical symptoms which may lead to organ dysfunction. Severe and long-term pain will affect the function of human organs systems, such as sleep disorders, inhibiting digestive function, high blood pressure, accelerated heart rate (may cause abnormal heart rhythms to those who have heart disease), shortness of breath (ventilation is limited with chest or abdomen pain), autonomic dysfunction, decreased joint function and psychological barriers, causing drug abuse, suicide and other social problems. How to safely and effectively control the pain is of great significance for relieving the suffering of patients and improving patients’ life quality. Eventhough HBO2has not been used in clinical pain control, there are a number of clinical observations on the analgesic effect of HBO2, including complex regional pain syndrome, fibromyalgia, chronic osteomyelitis, migraine and cluster headache. Thus, this study focuses on the effects of HBO2on the acute and chronic pain and the related mechanisms.Exposure to hyperbaric oxygen (HBO2) causes an antinociceptive response in mice. However, breathing oxygen (O2) at an elevated pressure can potentially cause oxygen toxicity. The aim of this study was to identify the determinants of HBO2antinociception and the toxicity profile of HBO2. Male NIH Swiss mice were assessed for acute antinociceptive responsiveness under room air or100%02at1.0or3.5atmospheres absolute (ATA), using the acetic acid-induced abdominal constriction test. For the oxygen toxicity test, mice were exposed to3.5ATA oxygen for11min,60min,60min daily for2days (120min) or60min daily for4days (240min), then assessed by analyzing the levels of two oxidative stress markers, MDA (malondialdehyde) and protein carbonyl in brain, spinal cord and lung. The results show that only the combination of100%O2and3.5ATA caused significant antinociception. The antinociceptive effect of100%O2was pressure-dependent up to3.5ATA. In the oxygen toxicity test, mice exposed to HBO2for different time intervals had levels of brain, spinal cord and lung MDA and protein carbonyl that were comparable to that of control animals exposed to room air. The result indicates that treatment with100%O2evokes a pressure-dependent antinociceptive effect. Since there was no significant increase in levels of the oxidative stress markers in the tested tissues, it is concluded HBO2at3.5ATA produces antinociception in the absence of oxidative stress in mice. We determined whether gamma-Aminobutyric acid (GABA) receptors might be involved in the antinociceptive effects of the medical gases HBO2. Antinociceptive responsiveness to HBO2was assessed using the acetic acid-induced abdominal constriction test. Results show that for the short-term HBO2treatment, intracerebroventricular or intrathecal pretreatment with the selective GABAA receptor antagonist SR95531and GABAB receptor antagonist CGP35348antagonized the antinociceptive effect of HBO2, while GABA uptake inhibitor nipecotic acid facilitated the antinociceptive effect of HBO2. For the long-term HBO2treatment, intracerebroventricular pretreatment with SR95531and CGP35348didn’t antagonize the antinociceptive effect of HBO23hours after HBO2, but the intracerebroventricular pretreatment with CGP35348antagonized the antinociceptive effect7days after HBO2. This finding suggests different roles of GABA receptors in mediation of short-term and long-term hyperbaric oxygen treatment induced antinociceptive effect.11-min exposure fast activate the GABAA receptor both in brain and spinal cord to produce antinociceptive effects, while4daily HBO2exposures activated the GABAB receptor7days later to produce late phase antinociceptive effects.