Inflammatory bowel disease (IBD) is a prevailing worldwide disease, especially in developing countries and its incidence has increased significantly in the past two decades . 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 related immune disorders. Both of them are incurable and are usually diagnosed at a young age with a significant morbidity .
It is currently accepted that multifactor, such as microbial flora, dysregulation of the immune response, genetic susceptibility, and environmental factors, involve in IBD, although it’s precise etiology remains elusive. In particular, a pronounced imbalance in the activation of pro-inflammatory and anti-inflammatory signaling pathways in the gut is thought to be an important contributor to the development and progression of IBD. S100A9 is an intracellular calcium-binding protein belonging to the S100 family, which was originally discovered as an immunogenic protein expressed and secreted by neutrophils . Besides neutrophils and monocytes, S100A8 and S100A9 can also be induced in keratinocytes and epithelial cells under inflammatory conditions [4, 5]. Growing evidence shows that S100A9 has a dual role in the inflammation response which is strongly upregulated in trauma, infections, heat, stress, and many other inflammations. S100A9 frequently interacts S100A8 to form a heterodimer (called calprotectin) and exhibits its biological functions . Extracellular S100A8/A9 functions by binding to different receptors, including pattern recognition receptors, like the receptor for advanced glycation end products (RAGE)  and toll-like receptor 4 (TLR4) . Then, the downstream signal of TLR4 is activated through the generation of the adaptor molecules containing the toll/interleukin-1 receptor (TIR) domains, such as TRIF-related adaptor molecule (TRAM)/toll/IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF) and myeloid differentiation factor 88 (MyD88), which causes the translocation of NF-κB and/or IRF3 into the nucleus . RAGE promoter contains NF-κB-binding sites and activates NF-κB or interacts high-mobility group box 1 (HMGB1), leading to the intracellular activation of NF-κB . S100A8/A9 is the proposed functional form in lesions associated with neutrophil influx, displaying antimicrobial activities against a variety of micro-organisms [11, 12]. S100A9 induces various inflammatory responses, such as cytokine secretion, chemotaxis, cell viability, and caspase-dependent or independent cell death [13, 14].
S100A9 is also upregulated in acute or chronic local inflammatory diseases. It is found that lungs of patients with airway inflammation including cystic fibrosis, asthma, acute respiratory distress disorder, and chronic obstructive pulmonary disease have a high expression of S100A8/A9 . Calprotectin complex of S100A8/A9 is present at high levels in the serum and corresponding intestinal tissues during inflammation and has been established as a valuable biomarker for a number of colonic inflammatory conditions . Several papers reported that S100A9 expression in myeloid cells is essential for the development of colon tumors, which activates the downstream genes promoting the occurrence, growth and metastasis of colon-associated tumors . A recent study found that dextran sulfate sodium sulfate (DSS)-induced colitis can be significantly improved by administration of a neutralizing anti-S100A9 antibody protein, accompanied with that the cellular infiltrate of innate immunity cells and production of pro-inflammatory cytokines is also significantly reduced. In addition, the inflammatory response, tumor cell proliferation and immune cell infiltration in colon tissue are suppressed in the mouse experimental model of azoxymethane (AOM)/DSS-induced colitis-associated cancer . Therefore, it is highly likely that S100A9 may be a therapeutic target for colitis and colitis-associated cancer.
Now 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 . However, these drugs are less effective for some patients and also frequently cause severe side effects, including opportunistic infections and malignancies . Therefore, no effective and safe clinical treatment strategy has been discovered for all IBD patients.
At present, the use of complementary and alternative medicine, including acupuncture, homeopathy and herbal medicines, for the treatment of patients with IBD is increasing worldwide. It is estimated that up to 70% of patients with IBD in North America and Europe were treated with complementary and alternative medicine [20, 21]. In China, several Chinese herbs are often used in the treatment of IBD, such as Pulsatilla chinensis (Bunge) Regel and Pulsatilla decoction, which were mainly used to treat UC in the traditional Chinese medicine for thousands of years . Pulsatilla decoction possesses a variety of pharmacological effects, and the active ingredients from these herbal plants and has shown hepatic protective, antiinflammatory, antibacterial, antitumor and antioxidant effects . Several studies have reported that Pulsatilla decoction could alleviate the release of inflammatory factors, such as IL-17, IL-1β, TNF-α in the serum of IBD patients, reduce syndromes and symptom scores, and suppress the activation of the NF-κB signaling pathway .
As the most important herb in Pulsatilla decoction, P. chinensis was widely used for the treatment of amoebic dysentery and malignant dysentery in China for two thousand years, which exhibits “blood-cooling” and detoxification activities . Accumulating evidence has demonstrated that P. chinensis and its ingredients have antioxidant, anti-inflammatory and antitumor effects. Our previous study reported that anemoside B4, the most abundant triterpenoid saponin isolated from P. chinensis, decreases inflammation and oxidant injury induced by cisplatin. It ameliorates LPS-induced kidney and lung damage, inhibits LPS-induced NF-κB activation in vivo [26, 27]. However, it is unclear whether anemoside B4 exhibits effective roles in treating IBD and it might be the molecular basis for the clinical effect of P. chinensis.
In the present study, we investigated the therapeutic effect of anemoside B4, on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rats. We firstly found that anemoside B4 exerts anti-inflammatory and anti-apoptotic properties through downregulating colonic inflammatory cytokine levels, and inhibiting colonic epithelium apoptosis. Furthermore, quantitative proteomics analyses indicated that S100A9-dependent pathway is involved in the therapeutic effects of anemoside B4. Our data suggest that anemoside B4 has the potential ability for the further therapy of colitis.