Mo2040 Tumor-Necrosis-Factor Alpha and Muramyl-Dipeptide Synergize in the Induction of Crohn's Disease Associated Intestinal Fistulae via Transcription Factor Ets-1

Current biological therapies for inflammatory bowel disease (IBD), aiming to block the production of key Th1 cytokines, have provided some benefits for patients. However, these agents are not universally applicable and often lose efficacy after long-term use. Chinese herbal therapies have been used to treat gastrointestinal (GI) symptoms for centuries, but the efficacy and underlying mechanisms are largely unknown. Huoxiang Zhengqi (HXZQ) is a 13-herb Chinese medicinal formula originated from the ancient Chinese pharmacy book “Formularies of the Bureau of the People's Welfare Pharmacies” and has been traditionally used to treat GI disorders including colitis. The proclaimed beneficial effects, however, are largely anectodotal and are not substantiated by scientific data. Aim: To determine whether HXZQ possess anti-inflammatory and intestinal function-modulating activities that contribute to its beneficial effects on GI disorders.Methods: Mouse bone marrow-derived macrophages (BMDM) were used for the studies on anti-inflammatory activities. T84 cells, grown on transwell plates to form fully differentiated monolayers, were used to establish the effect of HXZQ on epithelial barrier function. In addition, intestinal smooth muscle strips were placed in organ baths and mucosa were mounted in Ussing chambers to determine the effects of HXZQ in regulating smooth muscle contraction as well as epithelial secretion. Cytokine mRNA expression was measured by qPCR. Results: qPCR showed that HXZQ extracts potently inhibited LPS-stimulated mRNA expression of IL-12p40 and IL-1β, but had no effect on IL-6 (Table). GI inflammation is often accompanied by disrupted mucosal barrier due to the action of proinflammatory cytokines. As expected, IFNγ effectively decreased transepithelial electrical resistance (TER) and increased influx of cascade blue-dextran across T84 cell monolayer. The presence of HXZQ extracts, however, significantly attenuated this IFNγ-induced epithelial barrier dysfunction (TER changes from baseline in % after 72 hrs: vehicle 124±3 vs IFNγ 24±5 vs IFNγ plus HXZQ 93±15; influx of dextran in %: vehicle 0.3±0.0 vs IFNγ 3.0±0.4 vs IFNγ plusHXZQ1.2±0.1). GI inflammatory diseases are associated with abnormalities in gut function. HXZQ extracts significantly inhibited the intestinal smooth muscle responses to acetylcholine (ACH) and nerve stimulation (EFS), as well as the amplitude of spontaneous contraction (SC). Similarly, HXZQ extracts attenuated the ACH-induced chloride secretion of mucosal epithelial cells. Conclusions: HXZQ is able to inhibit the expression of proinflammatory cytokines from LPS-stimulated macrophages, attenuate the IFNγ-induced disruption of epithelial barrier, as well as to modulate gut function. These activities may contribute to the beneficial effect of HXZQ in GI inflammatory diseases.