Citrus Nobiletin Rebanlances Intestinal Dysmotility And Suppresses Trinitrobenzene Sulfonic Acid-induced Experimental Colitis

Background:Inflammatory bowel disease (IBD) is an inflammatory disease of the digestive tract with chronic or recurring immune response. The clinical symptoms of IBD conclude constipation, diarrhea, and blood stoolor. Risk of colitis transformed to colonic cancer is very high. There is no effective drug to treat IBD in clinic. Excessive immune response, increased intestinal permeability and abnormal intestinal motility contribute to the development of IBD. Nobiletin exerts suppressive effects on pneumonia and skin inflammation as well as resists cancer and relieves indigestion. However, the effects of nobiletin on intestinal dysmotility and inflammation remain unclear. In the present study, we aim to observe the effects of nobiletin on gastrointestinal dysmotility and therapeutic roles of nobiletin in treating IBD respectively.Aim:1. To measure the effects of nobiletin on the intestinal contractility in different contractile states and to clarify nobiletin-induced bidirectional regulation on gastrointestinal motility.2. To evaluate the suppressive effects of nobiletin on trinitrobenzene sulfonic acid (TNBS)-induced colitis.Methods:1. Bidirectional regulation exerted by nobiletin:(1) We determine the bidirectional regulation effects of nobiletin on jejunal contractility in 6 low and high contractile states respectively in vitro.(2) We measure the effects of nobiletin on excretion in constipation-prominent (CP) and diarrhea-prominent (DP) rats respectively; we also observe the phosphorylation level of myosin light chain, changes of myosin light chain kinase content and gut motility balance induced by nobiletin using western blotting and real time-PCR.(3) We observed the effects of calcium, cholinergic nerve, muscarinic acetylcholine receptor, adrenergic nerve and neurons containing nitric oxide, alpha adrenergic receptor and beta-adrenergic receptor in nobiletin-induced gut motility balance using corresponding blockers.2. Treatment of nobiletin on inflammatory bowel disease(1) IBD model was established by intracolonic administration trinitrobenzene sulfonic acid.(2) Observe changes of body weight, food intake and colonic pathological changes, intestinal epithelial barrier function in IBD rats after nobiletin treatment.(3) Observe the changes of TNF-α, IL-1β, and IL-6 using Elisa kit in IBD rats; Observe the effects of nobiletin on changes of Nuclear Factor-icB p65, inducible nitric oxide synthase and cyclooxygenase 2 using Western Blotting assay in IBDResults:1. Nobiletin-induced a gut motility balance:(1) Nobiletin exerts stimulatory effects on jejunal contractility in low concentration (1.25-5.0 μmol/L) while inhibitory effects in high concentration (10.0-40.0 μmol/L). At the concentration of 2.5 μmol/L, nobiletin induced bidirectional regulation on jejunal contractility; characterized as stimulatory effects in low contractile states while inhibitory effects in high contractile states.(2) Nobiletin-induced bidirectional regulation significantly was blocked by verapamil, TTX, atropine, phentolamine, propranolol, and L-NNA respectively, suggesting that the bidirectional regulation was related to the activation of cholinergic nerve, adrenergic nerve and neurons containing nitric oxide respectively.2. Suppressive effects of nobiletin on colitis:(1) Nobiletin reversed the decreased body weight/food intake in IBD rats; symptoms including neutrophils infiltration and mucosal barrier damage were alleviated after nobiletin treatment; nobiletin also reversed the decreased gross damage score, colon index and myeloperoxidase in colitis rats;(2) Nobiletin reversed the high levels of TNF-a, IL-1β, IL-6 in colitis rats; high expressions of claudin-2, myosin light chain (MLCK), NF-κB p65, PI3K/Akt, inducible nitric oxide synthase and cyclooxygenase 2 was reversed after nobiletin treatment in colitis rats.Discussion:1. Nobiletin-exerted bidirectional regulation on jejunal contractility was Ca2+-dependent, and was mediated through the regulation of enteric nervous system; in vivo, nobiletin reversed the abnormal expressions of MLCK in CP and DP rat jejunal tissue.2. Nobiletin exerted significant suppressive effects on colon inflammation in IBD rat through down-regulation of cytokines as well as inflammation mediators.3. Nobiletin decreased the intestinal epithelial permeability through down-regulation of Akt-NF-κB-MLCK pathway and restoration of barrier function.Novelty:1. To our knowledge, it is the first time to reveal that nobiletin exerted bidirectional regulation on jejunal contractility. The bidirectional regulation was contractile states-dependent, and was related to enteric nervous system, and myosin phosphorylation;2. To our knowledge, it is the first time to reveal that nobiletin significantly alleviated IBD symptoms induced by TNBS in rats through reducing inflammation, restored the barrier function and increasing gut motility, It is the first time to report to treat IBD using multi-target therapy including anti-inflammation, restoration of barrier function and alleviation of gastrointestinal dysmotility induced by nobiletin.Significance:1. Bidirectional regulation on jejunal contractility induced by nobiletin could maintain the balance of smooth muscle contraction states; suggesting nobiletin was capable of maintaining gut motility balance.2. Nobiletin significantly suppressed IBD symptoms induced by TNBS through decreasing inflammation, restoration of barrier function and increasing gastrointestinal motility, which provide some scientific information for the clinical treatment of IBD.