Osthole pretreatment alleviates TNBS-induced colitis in mice via both cAMP/PKA-dependent and independent pathways

Osthole, a natural coumarin found in traditional Chinese medicinal plants, has shown multiple biological activities. In the present study, we investigated the preventive effects of osthole on inflammatory bowel disease （IBD）. Colitis was induced in mice by infusing TNBS into the colonic lumen. Before TNBS treatment, the mice received osthole （100 mg·kg^-1·d^-1, ip） for 3 d. Pretreatment with osthole significantly ameliorated the clinical scores, colon length shortening, colonic histopathological changes and the expression of inflammatory mediators in TNBS-induced colitis. Pretreatment with osthole elevated serum cAMP levels; but treatment with the PKA inhibitor H89 （10 mg·kg^-1·d^-1, ip） did not abolish the beneficial effects of osthole on TNBS-induced colitis. In mouse peritoneal macrophages, pretreatment with osthole （50 pmol/L） significantly attenuated the LPS-induced elevation of cytokines at the mRNA level; inhibition of PKA completely reversed the inhibitory effects of osthole on IL-11% IL-6, COX2, and MCP-1 but not on TNFa. In Raw264.7 cells, the p38 inhibitor SB203580 markedly suppressed LPS-induced upregulation of the cytokines, whereas the PKA inhibitors H89 or KT5720 did not abolish the inhibitory effects of SB203580. Moreover, in LPS-stimulated mouse peritoneal macrophages, SB203580 strongly inhibited the restored expression of IL-113, IL-6, COX2, and MCP-1, which was achieved by abolishing the suppressive effects of osthole with the PKA inhibitors. Western blot analysis showed that osthole significantly suppressed the phosphorylation of p38, which was induced by TNBS in mice or by LPS in Raw264.7 cells. Inhibition of PKA partially reversed the suppressive effects of osthole on p38 phosphorylation in LPS-stimulated cells. Collectively, our results suggest that osthole is effective in the prevention of TNBS-induced colitis by reducing the expression of inflammatory mediators and attenuating p38 phosphorylation via both cAMP/PKA-dependent and independent pathways, among which the cAMP/PKA-independent pathway plays a major role.