Abstract
BackgroundDespite the increased morbidity of ulcerative colitis (UC) in the developing countries, available treatments remain unsatisfactory. Therefore, it is urgent to discover more effective therapeutic strategies. Pulsatilla chinensis was widely used for the treatment of inflamed intestinal diseases including UC for thousands of years in China. Anemoside B4, the most abundant triterpenoid saponin isolated from P. chinensis, exerts anti-inflammatory and antioxidant effects and may be the most active compounds, which is responsible for the therapeutic effects. However, the mechanism how anemoside B4 executes its biological functions is still elusive.MethodsHere, we used the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rat model to evaluate the therapeutic effect of anemoside B4. Blood samples of colitis rats were collected for hematology analysis. The inflammation-associated factors were investigated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis was determined with EdU cell proliferation assay and TUNEL assay. The proteins regulated by anemoside B4 were identified by label-free quantitative proteomics. The significantly down-regulated proteins were verified by Western blotting analysis. mRNA expression was analyzed by quantitative real-time RT-PCR.ResultsThe results showed that anemoside B4 ameliorated TNBS-induced colitis symptoms, including tissue damage, inflammatory cell infiltration, and pro-inflammatory cytokine production, apoptosis and slowed proliferation in colon. Quantitative proteomic analyses discovered that 56 proteins were significantly altered by anemoside B4 in the TNBS-induced rats. These proteins mainly clustered in tricarboxylic acid (TCA) cycle and respiratory electron transport chain. Among the altered proteins, S100A9 is one of the most significantly down-regulated proteins and associated with NF-κB and MAPK signaling pathways in the pathogenesis of UC. Further experiments revealed that anemoside B4 suppressed the expression of S100A9 and its downstream genes including TLR4 and NF-κB in colon. In vitro, anemoside B4 could inhibit the NF-κB signaling pathway induced by recombinant S100A9 protein in human intestinal epithelial Caco-2 cells. Moreover, anemoside B4 inhibits neutrophils recruitment and activation in colon induced by TNBS.ConclusionsOur results demonstrate that anemoside B4 prevents TNBS-induced colitis by inhibiting the NF-κB signaling pathway through deactivating S100A9, suggesting that anemoside B4 is a promising therapeutic candidate for colitis.
Highlights
Inflammatory bowel disease (IBD) is a prevailing disease worldwide, especially in developing countries, and its incidence has increased significantly in the past two decades [1]
trinitrobenzene sulfonic acid (TNBS)-induced severe acute colitis model was used to determine the pharmacological activity of anemoside B4
Treatment with anemoside B4 ameliorated TNBS-induced colon length shortening and mesalazine was slightly less effective (Fig. 1c and d). These results demonstrated that inhibitory effect of anemoside B4 on colitis is slightly more effective than mesalazine
Summary
Inflammatory bowel disease (IBD) is a prevailing disease worldwide, especially in developing countries, and its incidence has increased significantly in the past two decades [1]. The two main forms of IBD are ulcerative colitis (UC) and Crohn’s disease (CD), which are characterized by abdominal pain, diarrhea, bowel obstruction, weight loss, and associated immune disorders Both of them are incurable and usually diagnosed at a young age with a significant morbidity [2]. Current clinical treatments for UC include several anti-inflammatory drugs, such as sulfasalazine, glucocorticoids, nonsteroid anti-inflammatory agents, inhibitors of pro-inflammatory pathways, tumor necrosis factor (TNF)-α, gut-homing α4β7 integrin, interleukin (IL)-12/IL-23, and Janus kinases [3]. These drugs are less effective for some patients and frequently cause severe side effects, including opportunistic infections and malignancies [4]. The mechanism how anemoside B4 executes its biological functions is still elusive
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