More and more reports showed that ketamine abuse not only affected brain and cardiovascular system, but also damaged the urinary tract function. Ketamine-induced ulcerative cystitis (KIC) initially damaged the bladder mucosa and induced contracted bladder thereafter. We hypothesis that altered methylation of CpG sites in the COX-2 promoter region may contribute to the COX-2 transcriptional regulation in KIC diseases. The present study is to investigate whether the potential methylation of CpG sites in the COX-2 promoter region via the NF-κB transcriptional regulation and its effect on the transcriptional COX-2 expression in KIC animal model.
Experiments were performed on 36 female Sprague-Dawley (S-D) rats in three different groups: (a) the control group, which receive 0.9% saline (IP) injection, (b) the ketamine group, which receive ketamine (30 mg/kg/day, IP), and (c) the ketamine+COX-2 inhibitor group, which receive ketamine combined with COX-2 inhibitor injection (Parecoxib sodium, Dynastat®, Pfizer) (10 mg/kg/day, IP) for 3 months. The level of prostaglandin E (PGE2) was determined by enzyme-linked immunosorbent assay (ELISA). Double immunostaining and western blot analysis were performed for detecting the COX-2 expression coincided with NF-κB p65 expression. The methylation pattern in CpG sites encompassing COX-2 promoter region was determined by bisulfite methylation-specific PCR (MSP) and genomic sequencing. We also explore NF-κB transcriptional regulation and histone modification (H3K4, H3K 9, H3K27, H3K36, and H3K79) involved in the COX-2 promoter post-modification by chromatin immunoprecipation assay (ChIP).
Ketamine treatment induced the translocation of NF-κB p65 to nucleus and activated COX-2 expression and PGE2 production in bladder tissue, whereas COX-2 inhibitor suppressed the expression level. There are five potential binding sites for NF-κB transcriptional factor in the Cox-2 promoter region. Our data showed that DNA unmethylation in Cox-2 promoter region located from -830 to -71 bp. Moreover, the percentage of total six CpG sites in the Cox-2 promoter region located from -1522 to -1181 bp was 76.5 ± 10.5 % in the control group, 28.6 ± 6.0 % in ketamine group, and 41.4 ± 6.0 % in ketamine+COX-2 inhibitor group. Although the cloned sequences at different groups exhibit the heterogeneity in the methylation of CpG sites, we found that there is a hypo-methylation pattern of the Cox-2 promoter in association with the level of Cox-2 transcription in KIC.
DNA hypo-methylation of Cox-2 promoter may contributed to the Cox-2 transcriptional regulation as well as induced a pro-inflammatory response in KIC. COX-2 inhibitor treatment resulted in the methylation alternation of Cox-2 promoter and reduced Cox-2 transcriptional expression.