The Therapeutic and Immunoregulatory Effects of Astragalus Polysaccharides on TNBS-induced Colitis in Rats
|School||Beijing Union Medical College|
|Keywords||trinitrobenzene sulfonic acid inflammatory bowel disease astragalus polysaccharides T-bet GATA-3 Th1/Th2 balance|
Background:Inflammatory bowel disease (IBD) is a major chronic inflammatory disease of the gastrointestinal tract in human。Its incidence has been increasing for the past several decades in Chinese people. The etiology of IBD is considered to be multifactorial, which includes genetic factors, dysfunction of immunoregulation, disruption of intestinal mucosal barrier and intestinal flora. Immune system is thought to play a critical role in the development and persistence of IBD. The traditional therapeutic agents such as 5-ASA and glucocorticoid as well as new biotherapeutic ones such as Infliximab have good therapeutic effect, but there is a small portion of patients who can not benefit from these therapies. In addition, to remain remission is another tough problem. Recently, basic and clinical research in the field of traditional Chinese medicine have developed dramatically. Studies showed that traditional Chinese medicine was a promising therapy in IBD. Astragalus membranaceus is one of the most frequently used traditional Chinese medicine, which is famous for" bu qi " and "ulcer cure". Studies showed that the extract of astragalus membranaceus possessed both preventive and therapeutic potential in experimental colitis. But the therapeutic and immunoregulatory effects of Astragalus polysaccarides (APS) on TNBS-induced colitis in rats has not been reported.Objective:To investigate the therapeutic and immunoregulatory effects of APS on TNBS-induced colitis in rats.Methods: 1. Establishment of experimental colitis. A total of 40 SD rats were randomly divided into five groups (n=8, each group):normal control, TNBS group (treated with water, the same volume with the intervention groups), lower dosage group (treated with APS 0.5g/kg.d), higher dosage group (treated with APS 1.0g/kg.d) and prednisolone group. Experimental colitis was induced in rats by enema administration of TNBS. Rats were treated by lavage everyday for the following 14 days.2. Evaluation of therapeutic effect. Rats were sacrificed on d16. Macroscopic lesion and histological damage were determined, and the activity of myeloperoxidase (MPO) was measured in the excised colonic tissues.3. Investigation of immunoregulatory mechanism. Cytokine levels (IL-4, IL-10) were determined by enzyme-linked immunosorbent assay. T-box transcription factor (T-bet) and GATA-binding protein-3 (GATA-3) expression were determined by immunohistochemisty and immunoblot analysis.Results:1. Both macroscopic lesion and histological colonic damage scores were elevated in TNBS group, the activity of MPO was higher than normal control significantly (P=0.01). APS (0.5g/Kg.d) treatment reduced both macroscopic lesion and histological colonic damage, and this was accompanied by reduction of MPO activity significantly (P=0.03). The macroscopic lesion and histological colonic damage scores of APS (1.0g/Kg.d) group were near to TNBS group, but the activity of MPO was significantly higher than normal control and APS (0.5g/Kg.d) group (P=0.003,0.008). There was no significant difference between Prednisone group and the other groups.2. The levels of IL-4 and IL-10 decreased in TNBS group comparing with normal control, especially the level of IL-10 decreased significantly (P=0.007). APS (0.5g/Kg.d)treatment increased the levels of IL-4 and IL-10 with no significance. APS (1.0g/Kg.d) treatment further reduced the level of IL-4 but not IL-10. Prednisone treatment also decreased the levels of these two kinds of cytokines. The levels of IL-4 and IL-10 in higher dosage group and Prednisone group were lower than normal control significantly (APS 1.0g vs normal control, P=0.046, 0.035; PREDNISONE group vs normal control, P=0.049,0.001).3. GATA-3 expression was higher than T-bet in normal control. Both GATA-3 and T-bet expression increased in TNBS group comparing with normal control, but T-bet increased more, resulting in significant reduction of GATA-3/T-bet ratio (P=0.025). APS (0.5g/Kg.d) treatment encouraged the expression of both T-bet and GATA-3 with significance (P=0.04,0.019), but GATA-3 increased more, resulting in raise of GATA-3/T-bet ratio. PREDNISONE inhibited the expression of both two transcriptions, and T-bet decreased more, resulting in raise of the GATA-3/T-bet ratio too. GATA-3/T-bet ratio was higher in Prednisone group than APS lower dosage group.Summary:1. Treatment with different dosage of APS showed different effects on TNBS-induced colitis. Lower dosage treatment possesed therapeutic potential in experimental colitis, while higher dosage treatment might aggravate the colitis.2. Treatment of lower dosage APS increased the levels of Th2 type cytokines IL-4 and IL-10, while higher dosage treatment decreased the level of IL-4, but increased that of IL-10. The immunoregulation mechanism of lower dosage APS was associated with encouragement of both GATA-3 and T-bet expression, especially for GATA-3.3. Prednisone possesed immunosuppression effect. It decreased the levels of IL-4 and IL-10, and inhibited the expression of both GATA-3 and T-bet, especially for T-bet.4. Treatment with lower dosage APS and prednisone could raise GATA-3/T-bet ratio, and help to restore Th1/Th2 balance.This immunoregulatory effect of prednisone was better than that of lower dosage APS. Conclusion:APS possesed bilateral immunoregulatory effects on TNBS-induced colitis. Lower dosage APS could restore Th1/Th2 balance by encouraging the expression of anti-inflammatory cytokines and transcriptions. This may explain the immunoregulation mechanism of its anti-inflammatory and intestinal protective effects.