Asperuloside inhibited epithelial-mesenchymal transition in colitis associated cancer via activation of vitamin D receptor.

BACKGROUND: Asperuloside is a natural compound extracted from various herbs with several bioactivities. Its effects on anti-inflammation and anti-tumor indicated that asperuloside might prevent colorectal cancer developing from inflammatory bowel diseases (IBD). But there were few reports about the efficacy and mechanism of asperuloside on improving colorectal cancer. It has been reported that vitamin D receptor (VDR) could regulate the expression of SMAD3. In previous study, asperuloside could significantly improve the expression of VDR and reduced Smad3 mRNA in IEC-6 cell. PURPOSE: The present study was aimed to investigate the potential mechanism of asperuloside on inhibiting epithelial-mesenchymal transition (EMT) in colitis associated cancer. STUDY DESIGN: First, in LPS-injured IEC-6 cell, asperuloside inhibited phosphorylated p65 (p-p65) level, improved VDR expression and reduced Smad3 mRNA. Second, we wonder the relationship between VDR signaling and nucleus factor-kappaB (NF-κB) signaling during asperuloside on reducing Smad3 mRNA. And then, the effect of asperuloside on inhibiting EMT development through VDR/Smad3 was investigated. Finally, we testified the effect of asperuloside on protecting against colitis associated cancer (CAC) by inhibiting EMT development through VDR/Smad3. METHODS: Pyrrolidinedithiocarbamate ammonium (PDTC) was used for established NF-κB-inhibited IEC-6 cell. This cell was applied for investigating the relationship between NF-κB and VDR of asperuloside on inhibiting Smad3. VDR-inhibited cell was established by small interfering RNA (siRNA) of VDR and was employed to investigate the role of VDR for asperuloside on decreasing Smad3. Transforming growth factor ß1 (TGFß1) was used for inducing EMT/fibrosis in IEC-6 cell. TGFß1-stimulated cell was used for testifying the effect of asperuloside on inhibiting EMT development. AOM/DSS-induced CAC was established to investigate the effect of asperuloside on suppressing cancer development. RESULTS: Asperuloside inhibited the level of p-p65 which was up-regulated by LPS. Asperuloside could up-regulate VDR signaling and reduce Smad3 mRNA in NF-κB-knockdown IEC-6 cells. Asperuloside failed to reduce Smad3 mRNA due to VDR knockdown, which implied that asperuloside might down-regulate Smad3 mRNA dependently on activation of VDR signaling and independently on inhibiting NF-κB signaling. Asperuloside exhibited significant prevention of EMT development in TGFß1-induced IEC-6 cell (EMT cell) and mice CAC. Asperuloside reduced the transform of epithelial phenotype into motile mesenchymal phenotype in EMT cell along with decreasing levels of EMT markers by inhibiting Smad3 mRNA via activation of VDR. In mice with CAC, expression of VDR in colon was improved by asperuloside. Symptoms of colitis, tumor number and tumor size were significantly inhibited by asperuloside. Suppressed EMT development was determined by reduced α-SMA expression and decreased mRNAs of several EMT markers. CONCLUSION: Asperuloside might prevent CAC through inhibiting EMT development via regulation of VDR/Smad3 pathway.

Combined Administration of Vitamin D3 and Geniposide Is Less Effective than Single Use of Vitamin D3 or Geniposide in the Treatment of Ulcerative Colitis.

With the increasing incidence of ulcerative colitis (UC) in China, Chinese medicinal herbs or relatively active compounds are widely applied in treating UC. These medicines may be combined with other therapeutic agents such as vitamin D3. Nevertheless, the efficacy of these combinations for UC is unclear. Geniposide is an active component in many Chinese herbal medicines. It could ameliorate dextran sulfate sodium (DSS)-induced colitis in mice. This study was designed to determine the efficacy and mechanism of the single use and combination of geniposide and vitamin D3 on a mouse model of acute colitis. Data showed that a single administration of geniposide (2 mg/kg) or vitamin D3 (4 IU/day) could significantly improve the symptoms of UC and relieve colon damage. Geniposide and vitamin D could significantly decrease the levels of TNF-α and IL-6 in serum and colon, and increase the level of IL-10 in the colon. However, the combined treatment of geniposide (2 mg/kg) and vitamin D3 (4 IU/day) exerted less beneficial effects on UC in mice, indicating by less improvement of UC symptoms, colon damage, and inflammatory infiltration. The combination only downregulated the level of TNF-α in serum and IL-6 in the colon. Our data further demonstrated that geniposide could inhibit the activation of p38 MAPK and then restrict the vitamin D receptor signaling stimulated by vitamin D3. These results implied that the combination of geniposide and vitamin D3 might not be an ideal combined treatment for acute colitis, and the combination of vitamin D supplementary and geniposide (or herbal medicines rich in geniposide) need more evaluation before being applied to treat UC in clinic.

Asperuloside suppressing oxidative stress and inflammation in DSS-induced chronic colitis and RAW 264.7 macrophages via Nrf2/HO-1 and NF-κB pathways.

BACKGROUND: Inflammatory bowel diseases (IBDs), which mainly include Crohn's disease (CD) and ulcerative colitis (UC), are chronic idiopathic inflammatory disease of the gastrointestinal tract for which effective pharmacological treatments are lacking or options are very limited. PURPOSE: Here, we aim to investigate the therapeutic effects of an iridoid glycoside, asperuloside (ASP) on mice experimental chronic colitis induced by dextran sulfate sodium (DSS) and further explore underlying mechanisms in vitro and in vivo. METHODS: LPS-treated RAW 264.7 cells showed inflammation and were assessed for various physiological, morphological and biochemical parameters in the absence or presence of ASP. Chronic colitis was induced by 2% DSS in mice, which were used as an animal model to explore the pharmacodynamics of ASP. We detected p65 and Nrf2 pathway proteins via Western blot and RT-PCR analysis, assessed the cytokines TNF-α and IL-6 via ELISA, tested p65 and Nrf2 nuclear translocation via fluorescence. In addition, the docking affinity of ASP and p65 or Nrf2 proteins in the MOE 2015 software. RESULTS: We found that ASP attenuated weight loss, disease activity index (DAI) and colonic pathological damage in colitis mice and restored the expressions of inflammatory cytokines in the colon. In addition, ASP restored antioxidant capacity in DSS-induced chronic colitis mice and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Furthermore, ASP suppressed oxidative stress through increasing Nrf2, HO-1 and NQO-1 proteins expressions, and down-regulated nuclear levels of p65 to inhibit DSS-induced colonic oxidative stress and inflammation. Validation of the molecular docking results also indicated that ASP interacts with Nrf2 or p65 proteins. In summary, ASP improved DSS-induced chronic colitis by alleviating inflammation and oxidative stress, activating Nrf2/HO-1 signaling and limiting NF-κB signaling pathway, which may be an effective candidate for the treatment of IBD.