Selective COX-2 inhibitor with anti-inflammatory effect of 2,6-Bis(4-methylphenylsulfonamido)-benzoic acid through suppression of NF-κB and MAPK pathways in LPS-induced Raw 264.7 cells

다국어 초록 (Multilingual Abstract)

Most non-steroidal anti-inflammatory drugs (NSAIDs) cause mucosal injury and ulceration throughout the gastrointestinal tract by inhibiting both cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2). Anthranilic acid derivatives have been widely used in pharmacological therapy because of their various activities such as anti-inflammatory activity, cardiovascular activity, antimicrobial activity, and analgesic activity. In the example, N-aryl substituted derivatives of anthranilic acid including mefenamic acid, tolfenamic acid, and meclofenamic acid have been used as NSAIDs. In this study, we focused on finding a compound which has selective COX-2 inhibitory activity and anti-inflammatory effects among N-sulfonylated anthranilic acids. All synthetic compounds were examined COX inhibitory activities and anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. Notably, 2,6-Bis(4-methylphenylsulfonamido)-benzoic acid inhibited the expression of COX-2 but did not affect that of COX-1. In addition, 2,6-Bis(4-methylphenylsulfonamido)-benzoic acid suppressed NF-κB and MAPK pathways. We also proved that pretreatment of cells with 2,6-Bis(4-methylphenylsulfonamido)-benzoic acid attenuated the ROS and NO production. Consequently, these results suggest that 2,6-Bis(4-methylphenylsulfonamido)-benzoic acid has selective COX-2 inhibitory activity and anti-inflammatory activities and can be a potential therapeutic agent for inflammatory disorders.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials and Methods 4
• 2.1. Chemicals 4
• 2.2. Cell lines and cell culture conditions 4
• 2.3. General procedure for the C–H sulfonyl amidation of benzoic acids to sulfonyl azides (3a-3m and 4b–4h) 5
• 2.4. Treatment of synthesis compounds and positive control 5
• 2.5. MTT assay for Cell viability 6
• 2.6. Western blot analysis 6
• 2.7. Measurement of COX enzyme inhibition 7
• 2.8. RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) 7
• 2.9. 2’,7’-dichlorofluorescein diacetate (DCF-DA) assay 8
• 2.10. Measurement of Nitric Oxide production 8
• 2.11. Statistical analysis 9
• 3. Results 10
• 3.1. Cell viability of synthesis compounds and mefenamic acid in Raw 264.7 cells 10
• 3.2. The expression of inflammatory mediators in LPS-induced Raw 264.7 cells 10
• 3.3. Screening of N-sulfonylated anthranilic acids and mefenamic acid for anti-inflammatory activity in LPS-induced Raw 264.7 cells 11
• 3.4. Inhibition of COX-1 and COX-2 enzymes of N-sulfonyl anthranilic acids and mefenamic acid 11
• 3.5. 2,6-Bis(4-MPSA)-BA decreased protein and mRNA levels of pro-inflammatory cytokines in LPS-induced Raw 264.7 cells 12
• 3.6. Effects of LPS-induced MAPK pathway by 2,6-Bis(4-MPSA)-BA in Raw 264.7 cells 12
• 3.7. Inhibition of LPS-induced NF-κB activation by 2,6-Bis(4-MPSA)-BA in Raw 264.7 cells 13
• 3.8. Suppression of intracellular ROS generation by 2,6-Bis(4-MPSA)-BA in LPS-induced Raw 264.7 cells 13
• 3.9. Inhibition of NO production by 2,6-Bis(4-MPSA)-BA in LPS-induced Raw 264.7 cells 14
• 4. Discussion 15
• 5. References 17
• 6. 국문요약 76