Studies Of DAAO Inhibitors On Neuroprotection And Analgesia

As a conservative protease,D-amino acid oxidase(DAAO)is widely distributed in the liver,kidney,spinal cord,and brain tissues of mammals such as rats,mice,and humans.It is a flavoprotein that contains flavin adenine dinucleotide(FAD)as a coenzyme,which can remove two hydrogen atoms from the D-amino acid to form an imino acid and a reduced FAD.Meanwhile,the reduced FAD will spontaneously reoxidize in the presence of oxygen to produce hydrogen peroxide(H₂O₂),whilst imino acid will spontaneously hydrolyze to produce alpha-keto acid and ammonia.Over the years,our laboratory had conducted in-depth research on the physiological functions of DAAO in various neuropathological models.Early studies had found that DAAO/H₂O₂ mediates the development and progression of pain behavior in neuropathic pain models.Intrathecal injection of DAAO inhibitor and siRNA/DAAO gene silencing could significantly inhibit pain,and injection of exogenous H₂O₂ would completely reverse its mediated analgesic effect.In addition,early studies also indicated that DAAO/H₂O₂ mediated morphine tolerance and its associated pain hypersensitivity.Both morphine tolerance and pain hypersensitivity were reversed and inhibited by administering siRNA/DAAO gene ablation and DAAO inhibitors.The first part aimed to explore the pharmacological effects of DAAO in a mouse model of ischemia/reperfusion-induced stroke.By immunofluorescence-laser scanning confocal technique,it was discovered that astrocytes in the peri-infarct area of the cerebral cortex are in a continuous activation process and that the cell morphology changes greatly during the days after ischemia-reperfusion brain injury.On the third day after surgery,the cell body hypertrophy and prolonged protuberance were emerged,and on the 5th day after surgery,astrocytic hypertrophic scars were formed.The level of DAAO protein specifically expressed by astrocytes and its related enzyme activity were increased relevantly.DAAO inhibitor(Compound SUN)could effectively inhibit its enzymatic activity,decreased the content of H₂O₂ in brain tissue,significantly reduced the infarct size of brain tissue,and improved the neurobehavioral score after brain injury.The results showed that the neuroprotective effect of Compound SUN was dose-related,and the best time window for the treatment of acute stroke was at 0h after operation.Silencing of siRNA/DAAO gene in lateral ventricle injection also confirmed that inhibition of DAAO protein expression could effectively reduce the area of cerebral infarction in MCAO model,and significantly improve the behavioral evaluation of nerve injury.In addition,multiple injection of Compound SUN had a protective effect on MCAO-induced delayed brain injury.The second part aimed to explore the specificity of C57 mice in the pathological model of neuropathic pain and morphine tolerance,was not pharmacologically mediated by DAAO/H₂O₂.The previous formalin-based chronic pain and morphine tolerance model based on Swiss and Balb/c mice demonstrated that DAAO inhibitors could effectively exert significant analgesic effects and reverse analgesic tolerance and pain hypersensitivity induced by long-term morphine treatment by inhibiting DAAO enzymatic activity and reducing the production of by-product hydrogen peroxide.Current behavioral studies had shown that C57mice were not mediated by DAAO/H₂O₂ in both models,which meant that DAAO inhibitors were not effective in relieving chronic pain and controlling morphine tolerance and pain hypersensitivity in pathological models of C57 mice.The results showed that under physiological and pathological conditions,the content of glutathione peroxidase and reduced glutathione in the spinal cord of C57 mice was significantly higher than that of Swiss mice,with the raise of 100%and 35%,respectively.Therefore,we hypothesized that C57 mice had stronger activities on free radical scavenging and antioxidant capacity in the spinal cord.In addition,the level of hydrogen perioxide in spinal cord was measured 1 hour after intrathecal injection of 100?g/10?L D-serine.The results showed that the responses of Swiss and C57mice were diametrically opposed.The level of H₂O₂ in Swiss mice was nearly doubled,while that of C57 was markedly decreased,indicating that the endogenous D-amino acid in C57 as the substrate of DAAO was not deleted or insufficient.The exogenous substrate failed to increase the level of H₂O₂,and it further confirmed that the response mechanism of H₂O₂clearance in the spinal cord of C57 mice was activated and the removal efficiency was extremely high.That was a reason that C57 could maintains a relatively low level of hydrogen peroxide in these two pathological models.In total,C57 mice were not mediated by DAAO/H₂O₂ in the pathological models of formalin-induced tonic pain and morphine tolerance,but might result in pathological responses through other bological pathways.