The in vitro and in vivo anti-inflammatory effect of osthole, the major natural coumarin from Cnidium monnieri (L.) Cuss, via the blocking of the activation of the NF-κB and MAPK/p38 pathways

Abstract

Background: Ulcerative colitis (UC) is a chronic inflammatory condition of the intestines and is difficult to cure once diagnosed. The efficacy of the current clinical treatment for UC is limited. Common anti-inflammatory drugs are prone to adverse effects, while novel biological agents are expensive, although tolerated by patients. Therefore, an urgency exists to find more safe and effective drugs to treat UC. Osthole is an active constituent isolated from the fruit of Cnidium monnieri (L.) Cuss. Osthole has anti-inflammatory activities and offers certain intestinal protection. These characteristics indicate that osthole has the potential to inhibit UC.

Purpose: The study was conducted to investigate the anti-inflammatory potential of osthole in LPS-induced RAW 264.7 cells and dextran sulphate sodium (DSS)-induced ulcerative colitis in mice.

Methods: In in vitro experiments, mouse monocyte-macrophage RAW 264.7 cells were stimulated by 1 μg/ml LPS to produce inflammatory mediators. Griess reagent was used to determine Nitric Oxide (NO) production, and ELISA kits were used to determine the levels of PGE_2, TNF-α, and IL-6. The anti-inflammatory mechanisms of osthole were detected using western blot. In in vivo experiments, UC was induced via the intragastric administration of 3.5% DSS to BALB/C mice for 7 days. During the experiment, clinical signs and body weight were monitored and recorded daily to calculate the DAI score. At the end of the experiment, the colon lengths were measured. The colonic histopathological lesions were evaluated. MPO activity and TNF-α levels were determined using the corresponding kits. The protein expression of TNF-α and NF-κB pathways were analysed using western blot.

Results: In an in vitro study, osthole inhibited the production of NO, PGE2, TNF-α, and IL-6 in LPS-induced RAW 264.7 cells. The results of western blot showed that osthole inhibited the expression of iNOS, COX-2, p38 MAPK and IκB α in RAW 264.7 cells. On this basis, in DSS-induced UC mice, it was found that osthole relieved the symptoms of UC by inhibiting weight loss, colon shortening and the DAI score, and simultaneously alleviating colon tissue lesions. It was also found that osthole reduced the levels of TNF-α in serum and colon tissues and effectively inhibited the activity of MPO. The western blot results showed that osthole reduced the expression of NF-κB p65 and p-IκB α and increased the content of IκB α in colon tissues.

Conclusion: Osthole exerted anti-inflammatory effects by blocking the activation of the NF-κB and MAPK/p38 pathways. Additionally, osthole possesses therapeutic potential in the treatment of UC.

Keywords: Anti-inflammatory; NF-κB; Osthole; RAW 264.7 cells; p38 MAPK; ulcerative colitis.