| Background:Crohn’s disease (CD), referred to as inflammatory bowel disease, emerged and dramatically increased for about a century. Its cause remains regarded as unknown despite abundant related researches. CD is thought to be the product of a combination of genetic and environmental factors that result in the abnormal regulation of immune responses. Acting via IL-10 and TGF-β，t regulatory cells (Tregs) expressing Forkhead Box P3 (Foxp3) have a vital role in maintaining gut immune homeostasis. Many studies have found association between changes in Tregs and CD. Tripterygium Wilfordii Glycoside (TWG), extracted from the root of the traditional Chinese medicine called Tripterygium wilfordii Hook F (TwHF), possess immunosuppressive and anti-inflammatory activity, as well as antineoplastic property. TWG has been widely used in China to treat a variety of autoimmune diseases such as rheumatoid arthritis, nephritis, and systemic lupus erythematosus with curative effect. The therapeutic effect of triptolide in experimental colitis model has been confirmed and attributable to the inhibition of TLRs/NF-κB and TNF-a/TNFR2 signaling pathways, suppression of IL-6/STAT3 signaling pathway and down-regulation of IL-17. TWG has been testified to be therapeutically effective in inducing remission of CD by our working team previously. However, its exact mechanism has not yet been established.Objective:We aimed to explore the effect of TWG on the in situ levels of inflammatory cytokines and the number of Foxp3+ Tregs in inflamed mucosa of CD..Methods:CD outpatients of the General Hospital of Nanjing Military Region, of which the disease activity were mild (CDAI,150-220) and moderate (CDAI,220-450), participated in this study. TWG was administrated orally at a dose of 60mg/day (Taizhou Pharmaceutical Co., Jiangsu Province, China). At baseline and 3 months after treatment of TWG, the intestinal biopsies of the inflamed tissue were taken in the same diseased bowels when the colonoscopy was performed. Biopsy specimens were fixed, processed to paraffin, then stained with haematoxylin and eosin and reviewed in a blinded fashion by a qualified pathologist according to a previously validated scoring system for histological abnormalities in CD mucosal biopsies. The changes of Foxp3+ Tregs as well as TNF-α and IL-10 in diseased tissue were visualized by immunochemistry. Western blot and ELISA were used to quantify levels of Foxp3 protein expression and inflammatory cytokines.Results:A total of 20 CD patients aged between 25 and 57 years were enrolled in this study. CD severity at entry was mild in 8 cases and moderate in 12. All patients received 3-month oral TWG medication and no one discontinued treatment because of adverse events. Histological features of CD before treatment showed ulceration, epithelial damage, reduction of tubular glands and inflammatory cell infiltration. TWG attenuated the intestinal inflammation and induced mucosal healing in CD patients. The pathological score decreased significantly 3 months after TWG treatment compared with baseline (8.1±.2 vs 6.8±2.6, P<0.05). Immunohistochemistry analysis was performed to investigate the change of Foxp3+Tregs in mucosal biopsies from CD patients. The number of Foxp3+ Tregs in mucosa increased significantly following TWG treatment. A significant up-regulation of Foxp3 protein was also detected by Western blot from CD patients treated with TWG. TWG treatment dramatically decreased the level of TNF-α in the mucosa from CD patients (189.6±17.4 vs 173.0±19.0, P<0.01). Production of the regulatory IL-10 significantly improved (45.7±16.3 vs 55.6±14.8, P< 0.05), in unison with the up-regulation of Foxp3+ Tregs.Conclusion:We confirmed the efficacy of TWG treatment in CD and showed that microscopic inflammation was attenuated by the modulation of in situ levels of inflammatory cytokines. The therapeutic mechanisms might involve the up-regulation of Foxp3+ Tregs. |
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