Studies On The Antinociceptive Effects Of Hyperbaric Oxygen Treatments And The Protective Effects Of Hydrogen On Lung Oxidative Injury

Study I: 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 HBO has not been used in clinical pain control, there are a number of clinical observations on the antinociception of HBO, including complex regional pain syndrome, fibromyalgia, chronic osteomyelitis, migraine and cluster headache. Thus, this study focuses on the antinociceptions of HBO and the related mechanisms.In the first part of this study, we found that a single HBO treatment can produce acute antinociception, and there was a significant linear relationship between analgesia and oxygen pressure. We further studied the time course of the antinociception of a single HBO treatment. The result showed that the antinociception of HBO can be maintained at 80 % immediately, 10 min, 30 min, 1 ?? and 90 min after exposure. It decreased to less than 50% at 120 minutes and 20% at 180 minutes. More significantly, four daily 1 ?? utes of HBO treatment induce D a unique biphasic antinociceptions:(1) early analgesia: appeared at one hour after HBO treatment, which lasted about 8 hours;(2) the late analgesia: appeared at one day after the early analgesia, which lasted for nearly three weeks.In the second part of the study, we focused on the role of NOS in the antinociceptions of HBO. First, we treated mice with non-specific NOS inhibitor, n NOS inhibitor, e NOS and i NOS inhibitor, then observed their impacts on the antinociception of a single HBO treatment. It was found that L-NAME and SMTC significantly inhibited the antinociception of HBO. A single dose of L-NAME and SMTC significantly inhibited the early antinociception induced by repeated HBO treatment, while continue administration of L-NAME and SMTC during HBO treatments significantly inhibited the late antinociceptions. In the NOS knockout mice, only the antinociception of n NOS knockout mice was significantly inhibited. In addition, we found that one hour after HBO exposure, the NO level in the cortex, brainstem and spinal cord was significantly increased. While mice were exposed repeatedly HBO and then rested for 7 days, the NO level was not altered. Finally, Western blot showed that after exposed to HBO, n NOS content in the cerebellum, brainstem and spinal was significantly increased.In the third part of the study, we focused on the roles of GABA receptors and 5-HT1 A receptor. We first studied the GABA receptor antagonists on the antinociception of a single HBO treatment. Intracerebroventricular injection of GABAA receptor antagonist caused a dose-dependent antinociception change, but GABAB receptor antagonist had little impact on the antinociception. For early antinociception of repeated HBO treatment, intracerebroventricular injection of GABAA receptor antagonist and GABAB receptor antagonist three hours after HBO treatments had no significant effect on the antinociception, but the intracerebroventricular injection of GABAB receptor antagonist 7 days after HBO treatments caused a significant decline in the late antinociceptions of HBO, indicating that:(1) The GABAA receptor is involved in the antinociception caused by a single HBO exposure;(2) The GABAA receptor or GABAB receptor is not involved in the early antinociception of repeated HBO exposures;(3) The GABAB receptor is involved in the late antinociception of repeated HBO exposures. In addition, intracerebroventricular injection of different doses of 5-HT1 A receptor antagonist three hours after a single HBO treatment had no effect on the antinociception of a single HBO treatment. 5-HT1 A receptor antagonist also had no significant impact on the early antinociception of repeated HBO treatment, but intracerebroventricular injection of 5-HT1 A receptor antagonist seven days after repeated HBO treatments significantly inhibited the late antinociception of repeated HBO treatments. These results indicate that 5-HT1 A receptor was not involved in the single HBO induced antinociception or repeated HBO-induced early antinociception, but it may be involved in the late antinociception induced by repeated HBO treatments.In the fourth part, we used fluorescence method to observe the number of neurons. The results showed that repeated HBO treatments induced a significant increase of 5-HT1 A receptor-positive neurons in the PAG, but in n NOS knockout mice, the number was significantly reduced, suggesting the regulation of n NOS on the 5-HT1 A receptor. In GAD65 knockout mice, repeated HBO treatment significantly reduced the 5-HT1A receptor-positive neurons in the PAG, suggesting the regulation of GAD65 on the 5-HT1 A receptor. In addition, we did not observe n NOS + neurons and 5-HT1 A receptor positive neurons were co-localized, but the co-localization of n NOS + neurons and GAD-positive neurons in the PAG was observed, indicating that there may be deirect interaction between n NOS and GAD. Finally, Western Blot results showed that 7 days after repeated HBO treatment, the 5-HT1 A receptor and GABAB receptor protein were significantly increased in the PAG, but in the n NOS knockout mice, 5-HT1 A receptor and GABAB receptor expression were significantly inhibited. In GAD65 knockout mice, 5-HT1 A receptor protein expression was significantly inhibited.In summary, the present study demonstrated that the mechanism of acute antinociception of a single HBO treatment involved the activation of n NOS and GABAA receptors; the activation of n NOS was involved in the early antinociception of repeated HBO treatments and the late antinociception of repeated HBO treatments involved the activation of n NOS, GABAB receptors and 5-HT1 A receptor. Compared with other analgesics method, HBO has irreplaceable advantages, such as relatively low cost, no addiction and good tolerance. It has great clinical value. This study confirmed the short and long-acting antinociceptions of HBO and explored the mechanism from the central nerve system. It provided a theoretical basis for the clinical application of HBO. This study was the first one to choose the HBO-n NOS-GABA-5-HT signaling pathway to study the mechanism of the antinociceptions of HBO. A complete research pattern to study the analgesic mechanism of HBO may be developed by this study. With the discovery of the key target of antinociception, it can provide a new strategy to develop new analgesic drugs.Study II: 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, and 60-70% 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, H2 has long been limited to deep diving and was generally regarded as physiologic inert gas. In 1975, Malcolm Dole reported for the first time that hyperbaric H2(8 ATA) could be a possible treatment for cancer by decreasing ·OH. In 2001, Bouchra Gharib found that hyperbaric H2(8 ATA) attenuated parasite-induced liver inflammation, and the mechanism also involved eliminating ·OH increased by inflammation. But probably because it's hard to apply H2 at such a high pressure in clinical situation, these researches didn't draw widespread attention. Until 2007, when Ohsawa et al reported for the first time that 2% H2 at 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 that 2% H2 inhalation 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?diabetes, nephrotoxicity induced by cisplatin, Parkinson's disease, atherosclerosis in apolipoprotein and so on. All these evidence showed that H2 have 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-, H2O2 and ·OH. Since H2 can 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.In Part I, we investigated whether consumption of saturated hydrogen saline protects rats against paraquat-induced acute lung injury. Adult male 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 and then provided pure water or hydrogen water ad libitum for 72 h, 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, demonstrating that hydrogen water alleviated paraquat-induced acute lung injury possibly by inhibition of oxidative damage.In part II, we investigated whether molecular hydrogen could protect A549 cells from H2O2 induced oxidative damage. We determined the influences of different concentration of H2(0.16 m M, 0.32 m M, 0.48 m M, 0.64 m M and 0.8 m M) on A549 cell viability, lactate dehydrogenase leakage, SOD and GSH. The results showed after H2O2 treatment, the viability of A549 cells was siginificantly decreased. Cellular oxidative products MDA and 8-OHd G levels were greatly increased, but the anti-oxidative enzymes SOD and GSH were greatly comsumed, indicating that the anti-oxidative capacity was impaired. Molecular hydrogen could effectively prevent A549 cells from necrosis induced by H2O2, attenuate cellular lipid peroxidation and DNA damage, and prevent SOD and GSH decline. Combining with our previous researches in vivo, our results suggested that molecular hydrogen could prevent lung oxidative damage and might be a potential therapy to lung oxidative injury.