Osthole Alleviates Experimental Colitis In Mice

Objective:Inflammatory bowel disease(IBD)is a complex group of chronic relapsing disorders affecting the gastrointestinal tract and is characterized by inflammation and tissue remodeling.IBD comprises two prototypes,ulcerative colitis and Crohn's disease,which can be distinguished by the portion of G1 tract that is affected and pathology.Although etiology remains largely unknown,recent research has suggested that genetic factors,environment,microbiota,and immune response are involved in the pathogenesis.Medical therapies for IBD are predominantly aminosalicylates and steroids,which are effective but are associated with a significant side-effect profile.Osthole(also known as osthol),7-methoxy-8-(3-methy1-2-butenyl)-2H-1-benzopyran-2-one,is a natural coumarin found in traditional Chinese medicinal plants.Osthole is shown to have multiple functions including activity of immunomodulation and anti inflammation.The pharmacological effects of osthole in inflammatory bowel disease remain elusive.In the present study,we induced colitis in mice and investigated the efficacy of osthole in the prevention of colitis and the underlying mechanism.Methods:Colitis was induced by infusing a 100 ?l enema of TNBS(2,4,6-trinitrobenzene sulfonic acid,2.8 mg)in 50%ethanol into the colonic lumen of mouse.To investigate the preventive effect of osthole on colitis,mice received osthole intraperitoneally at 100 mg/kg once daily starting from three days before exposed to TNBS till the end of the experiments.When the therapeutic effect of osthole was assessed,osthole started simultaneously with TNBS-treatment till the end of the experiments.For most studies,the animals were sacrificed and evaluated at 2 days after induction of colitis.For histopathological scoring,the time point chosen for sacrifice was 4 days after induction of colitis.Postmortem,the colon was removed and measured.The entire mouse colon was Swiss-rolled,formalin-fixed,and paraffin-embeded.Colon sections were stained with hematoxylin and eosin.Body weight and survival rate were examined at indicated time points.Colitis was quantified with a clinical score assessing weight loss,stool consistency,and bleeding as previously described.Expressions of cytokines and tight junction marker proteins were determined by realtime PCR and western blot(WB).The concentrations of cAMP in serum were determined with commercial enzyme immunoassay kits.In some experiments,H89,a specific protein kinase A(PKA)inhibitor,was intraperitoneally administered 1 h prior to TNBS as indicated.The phosphorylation levels of ERJK,JNK and P38 in colonic tissues were determined by WB.In vitro,mouse peritoneal macrophages and Raw264.7 cells were stimulated with LPS.The effect of osthole on LPS-induced upregulations of cytokines was determined by realtime PCR.To test whether cAMP/PKA and P38 phosphorylation mediated the anti inflammation activity of osthole,cells were treated with PKA inhibitors H89 or KT5720 or P38 inhibitor SB203580 2 h prior to LPS,expressions of cytokines were examined by realtime PCR analysis.Effect of osthole on LPS-induced phosphorylation of Erk?JNK and P38 was evaluated by WB.The involvement of cAMP/PKA pathway in MAPK activation regulated by osthole was also investigated.Results:Pretreatment with osthole ameliorated the weight loss and clinical score,dose-dependently reduced colon length shortening,improved the colonic histopathological changes,and decreased the expressions of inflammatory mediators in colonic tissue.When osthole started simultaneously with TNBS-treatment,no detectable protection was observed.Taken together,these results indicated a significant beneficial effect of osthole on the prevention of TNBS-induced colitis in mice.Significant reduction of serum cAMP levels was observed 2 days after TNBS treatment.Osthole led to a trend of elevation of cAMP(P=0.07)compared with TNBS group.However,H89 pretreatment failed to abolish osthole-induced alleviation of weight loss and colon length shortening.The phosphorylation levels of MAPK in colonic tissues were examined by WB.Prominent phosphorylation of p38 was observed in mice 2 days after TNBS.Osthole significantly inhibited TNBS-induced P38 activation.No detectable changes were observed in the phosphorylation levels of Erk and JNK.In vitro,we observed that osthole decreased the elevation of IL-1?,IL-6,COX2,MCP-1,and TNFa at the mRNA level in response to LPS.Inhibition of PKA with H89 or KT5720 completely reversed the effects of osthole on IL-lp,IL-6,COX2,and MCP-1,but not on TNFa.LPS-induced elevation of IL-1?,IL-6,COX2,CXCL10,and TNF? was strongly suppressed by SB203580.Inhibition of PKA with H89 or KT5720 failed to abolish the effect of SB203580 on these cytokines.In mouse peritoneal macrophages activated by LPS,the elevated expressions of IL-lp,IL-6,COX2,and MCP-1 induced by PKA inhibitors in osthole-treated cells was completely abolished by SB203580.Meanwhile,the expression of TNF? was also strongly inhibited by SB203580.The phosphorylation level of MAPK was examined in Raw264.7 cells by WB.Consistent with the findings in vivo,prominent phosphorylation of P38 was induced by stimulation with LPS.Pretreatment of osthole significantly reduced the phosphorylation level of P38.Inhibition of PKA with H89 or KT5720 partly reversed the suppressive effect of osthole on P38 phosphorylation.Conclusion:We demonstrated that osthole is effective in the prevention of TNBS-induced colitis.Osthole reduced the expression of inflammatory mediators,probably by inhibition of P38 phosphorylation via both cAMP/PKA-dependent and independent pathways,among which cAMP/PKA independent pathway plays a major role.Osthole can be considered as a potential natural resource to prevent IBD.