Down-Regulation Of Spinal D-Amino Acid Oxidase Expression Blocks Chronic Pain

D-amino acid oxidase (DAAO) is a peroxisomal enzyme with FAD as cofactor, which catalyzes the oxidization of D-amino acids to the corresponding imino acids, producing ammonia and hydrogen peroxide. Early studies have shown that DAAO may play a role in chronic pain by inhibitors of DAAO and gene mutation mice. To further investigate the potential of DAAO as a new drug target for chronic pain, I applied RNA interference as a more special tool to this study as below:In this article, we describe a method for construction of recombinant advenovirus shRNA expression vector targeting rat DAAO gene.the expression vector can transcribe to shRNA, and shRNA can degrade to siRNA with the help of Dicer as the Dicer can recognize the structure of"loop"we designed specially according some principles. Then the oligonucleotides were synthesized cloned into a plasmid pDC316-EGFP-U6 which was one type of recombinant adenovirus expression vectors.We employed NRK-52E rat kidney tubule epithelial cells to test the efficacy of Ad-shDAAO to knock down DAAO expression. Expressions of mRNA and protein of DAAO were tested by Real-time PCR and Western blotting at 24 and 48 hrs after transfection, respectively. Transfection with Ad-shDAAO significantly inhibited DAAO mRNA by 51.7% or 49.1%, respectively, compared to the saline or nonspecific Ad-shControl group (P<0.05 by ANOVA followed by post-hoc Student-Newman-Keuls test). Furthermore, Ad-shDAAO significantly inhibited DAAO protein expression by 50.2% or 51.2%, respectively, compared to the saline or nonspecific Ad-shControl group (P<0.05) Multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7 days significantly inhibited spinal DAAO expression by 50-80% as measured by real-time quantitative PCR and Western blot, and inhibited spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60% but not acute phase nociception.In addition, multi-daily intrathecal injections of the DAAO gene silencer siRNA/DAAO in the regimen to effectively block spinal DAAO expression also yielded anti-allodynic effects by approximately 40% and the analgesia remained for at least 6 days after stopping administrations of drugs.In order to test whether the anti-allodynic effect of CBIO is specifically via inhibition of the spinal DAAO, we further examined the effect of CBIO on bone cancer pain in the above two groups of rats on the 10th day postdose of oligonucleotides. Both groups of rats received intrathecal single bolus injection of CBIO (10μg) and their mechanical withdrawal thresholds were measured for 4 hours. As shown in Fig. 4B, CBIO blocked mechanical allodynia in a time-dependent manner in ipsilateral paws by approximately 40% maximum possible effect (peak) compared to the pre-injection control in nonspecific oligonucleotide-treated rats However, CBIO was not effective in reducing mechanical withdrawal thresholds in rats multi-daily pretreated with siRNA/DAAO. The results indicated that CBIO produced bone cancer allodynia specifically via inhibition of spinal DAAO.In the spinal cord, DAAO is almost restrictively found in astrocytes. This study tested whether intrathecal administration of siRNA/DAAO or Ad-shDAAO blocked activation of astrocytes marked by GFAP (glial fibrillary acidic protein) in the spinal cord. GFAP gene expression in the homogenates of the spinal cord was determined by Real-time quantitative PCR. Multiple-daily intrathecal injections of siRNA/DAAO (5μg/kg/day) but not nonspecific oligonucleotide for 7 days significantly reduced GFAP gene expression by approximately 40.6%, compared to the PEI-treated rats (P<0.05 by ANOVA followed by post-hoc Student-Newman-Keuls test). Spinal GFAP protein expression was also detected by Western blot and its ratio scanned withβ-actin was calculated. siRNA/DAAO but not nonspecific oligonucleotide significantly reduced GFAP protein expression by approximately 32.0%, compared to the PEI-treated rats (P<0.05). The same inhibitory effect on GFAP expression of astrocytes was confirmed by application of Ad-shDAAO.These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord.