The Study Of Therapeutic Effort And Mechanism Of Alcohol Extract From Dahuang Mudan Decoction On Colitis Mice Inducted By Trinitrobenzene Sulfonic Acid

ObjectiveUlcerative colitis is a common nonspecific intestinal disease that is mainly provoked by the over-response of immune system to self-antigen though some pathogenic factors associated with the disease have been reported, so the disease is defined as an autoimmune disease. Experimental Colitis in mice induced by TNBS/ethanol solution shares a lot of similarities with human ulcerative colitis in pathological features and pathogenesis. The Chinese herbal formula, Dahuang Mudan Tang consists of 5 herbals including Dahuang 12g, Taoren 12g, Danpi 9g, Mangxiao 9g, Dongguazi 30g, is record in the classical Chinese medicine book of Jinkui Yaolue written by Zhang Zhongjing and is used to treat intestinal carbuncle because the formula has the function of activate blood and resolve stasis, clear heat, disperse swelling and nodules. The research of Dahuang MudanTang on experimental colitis in mice and the anti-inflammatory, anti-oxidant and regulation of the immune system effect of its two compounds rhein and paeonol have been reported. All the studies suggest that alcohol extraction of Dahuang MudanTang may also has therapeutic effect on colitis in mice. In order to find out whether the alcohol extraction of Dahuang Mudan Tang has therapeutic effect on colitis in mice and what the mechanism of the alcohol extraction works, behaviours and the colonic mucosa pathological changes were observed in experimental colitis mice, and the changes of Treg numbers in different tissues were detected by flow cytometry, the levels of TGF-β and IL-10 in peripheral blood plasma and sIgA in intestinal mucosa were assayed by ELISA, and proliferation index of lymphocytes in spleen was also observed. In addition, the effect of the alcohol extraction of Dahuang Mudan Tang on immune system in mice was observed to get a better understanding of its immunopharmacology.Methods1.Experimental colitis model in mice was induced by TNBS-ethanol solution, the disease activity index (DAI), macroscopic damage index (CMDI) and tissue damage index (TDI) were observed to evaluate whether the model was succeed.2.Experimental colitis mice were administrated the alcohol extraction of Dahuang Mudan Tang in low, middle and high dose for 6 days. In contral, PBS and SASP were administrated. After that, behaviours, mucosal damage and DAI changes were observed in experimental colitis mice, and TDI was also evaluated by light microscopy after the ulcerative tissue in colon stained by HE in histologic section.3.Then, proliferation index of lymphocytes in spleen was assayed by MTT, and mucosal slgA level was detected by ELISA.4.CD4+CD25+Foxp3+Treg numbers in peripheral blood, Peyer’s Patches (PPS), mesenteric lymph nodes (MLN) and spleen were analysed by flow cytometry. The levels of TGF-β and IL-10 in peripheral blood plasma were also detected by ELISA.5.In addition, Cyclophosphamide induced immunosuppressive mice model was established to discuss the immunopharmacologic action of the alcohol extraction of Dahuang Mudan Tang. Then, after the mice were administrated by alcohol extraction, the thymus index and spleen index were measured by immune organ weight method. Phagocytosis of mononuclear-macrophage in mice was measured by Carbon clearance method, and the humral immune function was determined by Serum hemolysin formation experiment. The cellular immune function was determined by DTH that the mice were induced by DNFB. Results1.DAI, CMDI and TDI elevated significantly after the mice induced by TNBS-ethanol solution, but reduced after treated by the alcohol extraction of Dahuang Mudan Tang. It suggested that the alcohol extraction has the therapeutic effect on experimental colitis in mice.2.Mucosal slgA level in experimental colitis mice elevated after the mice were administrated the alcohol extraction for 6 days in the all three doses. And the proliferation index of lymphocytes in spleen was also elevated significantly.3.We also found that CD4+CD25+Foxp3+Treg numbers of peripheral blood, PPS, MLN and spleen increased in experimental colitis mice administered by the alcohol extraction. Simultaneously, The levels of TGF-β and IL-10 in peripheral blood plasma increased.4.1n addition, after administration of the three dose alcohol extraction, the thymus and spleen index, Phagocytosis of mononuclear-macrophage and the humral and cellular immune function were all strengthened in immunosuppressive mice induced by cyclophosphamide. Conelusion1.The alcohol extraction of Dahuang Mudan Tang has the therapeutic effects on experimental colitis in mice,and the mechanism mechanism it works may associate with the function to elevate the number of CD4+CD25+Foxp3+Treg in peripheral blood, PPS, MLN and spleen, and the levels of TGF and IL-10 in peripheral blood plasma.2.The alcohol extraction of Dahuang Mudan Tang also have the functions to strengthen the innate and adaptive immunity in the immunosuppressive mice.