The Investigation Of The Pain Relieve Mechanisim Of The D-amino Acid Oxidase Inhibition Of The Spinal Cord

D-amino acid oxidase (DAO) is a peroxisomal enzyme with FAD as cofactor,which catalyzes the oxidization of D-amino acids to the correspondingimino acids, producing ammonia and hydrogen peroxide. Early studies haveshown that DAO may play a role in chronic pain. However, only Sodiumbenzoate, an inhibitor of DAO with limited activity, has been tested inprevious studies. To further investigate the potential of DAO as a newdrug target for chronic pain, I tested more DAO inhibitors with differentchemical structure and relatively higher activity in this study as below:To confirm the high expression level of DAO in spinal tissue, weinvestigated the expression level of mRNA of DAO in C6cells (glioma cellline from Wistar rats) and also in the lumbar enlargement of spinal andthe dorsal root ganglion of rats, using RT-PCR. In addition, we analyzedthe expression of DAO protein in C6cells, using Western blot. The resultsshowed that the mRNA of DAO is highly expressed in both C6cells and Wistarrat lumbar enlargement of spinal. However, no expression of DAO was shownin the dorsal root ganglion；The expression level of DAO protein was highin C6cells. After that, we analyzed the inhibitory effect of four chemical, sodiumbenzoate, AS057278, CBIO and4-Nitro-3-pyrazole-carboxylic acid, usingstandard D’Aniello method. These four chemicals are distinguished fromeach other in terms of chemical structure and inhibitory effect. Theresults showed that, the IC50value of these four inhibitors are10.2、0.2、0.1mM and no effect. Then, we tested these chemicals using improvedD’Aniello method. The results showed that the IC50value of these fourinhibitors are93.4、32.8、1.1μM and no effect. Although the values ofIC50are different from the published report, the ratio among the IC50valueof these four chemicals is consistent with the previous study.Last, to analyze the alleviating effect of chronic pain as a resultof the inhibition of DAO in vivo, we studied the inhibitory effect of these3chemicals on the animal model of chronic pain, established by hypodermicinjection of formaldehyde. We treated the rats with every chemical withdifferent doses by intrathecal way, using MK-801, the known competitiveinhibitor of NMDA receptor with an identified effect on chronic pain, ascontrol. The results showed a positive correlation between the inhibitoryeffect of DAO in vitro and the potency of pain alleviation in the animalmodel. In addition, the potency of the3tested chemical is similar toMK-801, at a level of approximately60%. However, no potency of CBIO onthe pain was shown in a Carrageenan model.Our study further showed that, DAO may be a new target of chronic pain.In addition, it suggested that the pathological mechanism of chronic paincan be classified in two parts by whether it is sensitive to the inhibitionof DAO. The ration between the sensitive and insensitive part may beapproximately6:4. These results will surely contribute to thedevelopment of new Analgesic.