Effect Of Total Alkaloids Of Sophora Alopecuroides On Dextran Sulfate Sodium Induced-acute Ulcerative Colitis In Mice

General IntroductionUlcerative Colitis (UC) is a chronic and relapsing disease of the large intestine (rectum and colon) characterized by a series of gastrointestinal syndrome including abdominal pain, diarrhea and hemafecia. In the world, the incidence of UC is in the range from 0.5/105 to 24.5/105 while in recent years reaches 11.6/105 in China which is still increasing year by year. The pathological changes of UC are complex and can be divided into chronic and acuteness. If the patients with UC suffer from repeated attacks, the acute UC will change into chronic phase. Recent data suggests that 5-10% patients with UC are likely to develop malignant tumor and have cumulative risk of malignancy reach 40% after 25 years of having this disease. Therefore, there is an urgent need to seek for alternative remedies for UC. It is of significance to for the treatment of acute UC to reduce its chronic and malignant process. However, there has been no specific radical measures to cure this disease in clinic which is defined as one of persitence of a disease in the modern by the World Health Organization.Sophora alopecuroid,L a traditional Chinese herbal, is widely used for enteritis and bacillary dysentery for many years. Our previous studies have found that total alkaloid of Sophora alopecuroides(TASA) was its chief active components. Preliminary clinical studies have shown that total effective rate of TASA on UC reached 67.2%. Studies on animals have shown that TASA could significantly alleviate the general condition and symptoms of animals with experimental colitis by preventing loss of weight and reducing the mucosal inflammation and necrosis. It could increase the weight, spleen index and thymus index of immunosuppressed mice as well. It achieved its role by down-regulating high expressions of CD8-CD28-Tregs of colitis rats at chronic phase and reducing the expressions of IL-10, ICAM-1, MIF and TFF3. But there has been no information on whether TASA was therapeutic for DSS-induced acute UC in mice.The etiology and pathogenesis of UC still remain unclear, while more and more evidences show that the dysregulation of mucosal immunological function plays major roles in the initiation and propagation of this disease. The immunological dysfunction of intestine resulted in the abnormal recruitment and activation of lamina propria mononuclear cells (LPMC) such as T cells and macrophages. LPMC can make the intestinal inflammation sustain and produce numerous pro-inflammatory cytokines such as interleukin-1β(IL-1β), interferonγ(IFN-γ) and tumour necrosis factorα(TNF-α).These cytokines can further activate inflammatory signaling molecules such as NF-κB and STAT3 and then regulate the expression of cytokines. IL-6/STAT3 is an important signaling pathway, which has been demonstrated to take part in regulating immune reactions, inflammatory reactions and the growth and differentiation of cells. Previous investigation has showed that strongly activated state of STAT3 were found in patients with IBD and in animal models of colitis, therefore, it might play an important role in the pathogenesis of UC. However, the specific mechanism of STAT3 on UC remains unclear.Objective:l.To investigate the therapeutic effects of TASA on acute UC induced by dextran sulfate sodium (DSS) in mice and to explore its effects on the levels of IL-1β,IL-4 and IL-6 in serum.2. To investigate the expression and activation of IL-6 and STAT3 in colonic mucosa of mice with DSS-induced UC and explore the regulating mechanism of JAK-STAT signal pathway on pathogenesis of UC and the effects of TASA on this pathway.Methods:Sixty C57BL/6 mice were randomly divided into six groups:normal group, DSS group, SASP group (520 mg-kg-1), low dose, middle dose and high dose TASA group (TAL, TAM, TAH:20,40,80 mg-kg-1). The normal group was drinking distilled water freely, while the other groups were administrated 5% DSS solution as drinking water freely for 7 days to induce acute UC. At the same time, the mice in the treatment groups were administrated TASA or SASP and the mice in control and normal groups were given the same dose of normal sodium. The mice were sacrificed after 7 days.1. The general condition of the mice including appetite, drink, activity and coat color was observed daily during the experiment. Disease activity index (DAI) was evaluated. The general morphological and histopathological changes were observed and histological score was detected.2. The activity of serum IL-1β, IL-6 and IL-4 was determined by enzyme linked immunosorbent assay(ELISA).3. Expressions and their localization of IL-6, STAT3 and p-STAT3 were assayed by Western blotting and immunohistochemistry (HIC).Results:1. Body weight loss, diarrhea and bleeding from the anus, emaciation, laziness, poor appetite were observed in mice with colitis. Serious inflammation such as bowel wall thickening and hyperemia, edema and ulcers were observed in colon of mice and colon length was significantly decreased in the colitis mice because of mucosal inflammation. Administration of TASA and SASP significantly ameliorated the symptoms of colitis. Compared with normal group, the DAI score markedly increased in DSS group and the difference was significant (P=0.000). TASA and SASP decreased DAI significantly (P=0.000). Histological examination showed that typical inflammatory changes in colonic architecture were observed, such as mucosal congestion, the thickening of the colon wall, lost of epithelial cell, distorted glands, ulceration, crypt dilation, and goblet cell depletion, as well as infiltration of inflammatory cell composed mainly of macrophages, lymphocytes and plasma cells. While histological analysis of the colons from TASA-treated mice showed greatly reduced cell infiltration, mucosal injury and edema. The histological scores were higher than that in DSS group which was significant(P=0.000), while the treatment groups decreased histological scores significantly(P=0.000) and there was no significant difference among the treatment groups(P>0.05).2. The expressions of serum IL-1βand IL-6 in DSS group were higher than that in normal group and there was a significant difference (P=0.000). The levels of IL-1βand IL-6 in TAH, TAM, TAL and SASP group were lower than that in DSS group which were significant(P=0.000) but higher than normal group. In contrast, IL-4 level in serum in DSS group was reduced compared with normal group (P=0.006). Although the serum content of IL-4 in treatment groups was increased compared with DSS group, there were no significant difference among these groups (P>0.05).3. Immunohistochemstry indicated that the expressions of IL-6 and STAT3 were detected mainly in the cytoplasm of a few lamina propria mononuclear cells and pit cells in normal mucosa in normal group. In animals with DSS-induced colitis, intestinal mucosa was damaged and inflammatory cell was infiltrated in the mucosa. The number of IL-6-positive cells in DSS group was increased with stronger staining density compared with the treatment groups and the difference were significant(P=0.002,0.001,0.000,0.004). There was no significant difference among these treatment groups(P>0.05). Although STAT3 expression in DSS group were significantly higher than that in normal group (P=0.000), there was no significant difference among these treatment groups and DSS group (P>0.05). In normal group, p-STAT3 expression was mainly in mice and almost no or only rarely p-STAT3 positive cells were detected in the nucleus of scattered mononuclear cells in normal mucosa. Marked p-STAT3 immunoreactivity was evident throughout the nuclei of the inflammatory cell in intestinal mucosa in DSS group. The number of p-STAT3-positive cells in DSS group was increased with stronger staining density compared with normal group and the difference was significant(P=0.000), while the number of positive cells in SASP, TAH, TAM and TAL was significantly lower than that in DSS group(P=0.008,0.004,0.002,0.012). There were no significant difference among these treatment groups(P>0.05). Western blotting analysis showed that the expressions of IL-6, STAT3 and p-STAT3 protein were increased in DSS group compared with normal group and the difference was significant (P=0.000, 0.009,0.000). The expressions of IL-6 in SASP, TAH, TAM and TAL group were reduced compared with DSS group which were significant (P=0.021,0.005,0.016, 0.021) but there was no significant difference between these treatment groups(P>0.05). The expressions of STAT3 in treatment groups were lower than that in DSS group and only TAH and TAM group had significant difference compared with DSS group (P=0.026,0.018). There was no significant difference between these treatment groups(P>0.05). The expressions of p-STAT3 in SASP, TAH, TAM and TAL group were reduced compared with DSS group which were significant (P=0.000, 0.000,0.000,0.000) but there was no significant difference between these treatment groups(P>0.05).Conclusions:1.TASA could effectively alleviate the symptoms of DSS-induced acute UC, relieve mucosal inflammation, reduce DAI score and histological score.2. TASA significantly reduced the levels of IL-1βand IL-6 and improved the level of IL-4 in serum. TASA could significantly ameliorate DSS-induced colitis through regulating pro- and anti-inflammtory cytokine production.3. The expressions of IL-6, STAT3 and p-STAT3 were increased in intestinal mucosa of acute UC, with p-STAT3 for the most. The result indicates that STAT3 in acute phase of UC is phosphorylated by upstream IL-6 and active STAT3 (p-STAT3) translocates into nucleus and then regulate gene expression of cytokines. Treatment with TASA reduced the expressions of IL-6, STAT3 and p-STAT3, especially p-STAT3 expression. These data suggest that STAT3 may take important part in the pathogenesis of UC through IL-6/JAK/STAT3 signaling pathway and TASA can inhibit the activity of this pathway to regulate imbalance of inflammatory cytokines to recur UC.