Discovery of vitexin as a novel VDR agonist that mitigates the transition from chronic intestinal inflammation to colorectal cancer.

Colitis-associated colorectal cancer (CAC) frequently develops in patients with inflammatory bowel disease (IBD) who have been exposed to a prolonged state of chronic inflammation. The investigation of pharmacological agents and their mechanisms to prevent precancerous lesions and inhibit their progression remains a significant focus and challenge in CAC research. Previous studies have demonstrated that vitexin effectively mitigates CAC, however, its precise mechanism of action warrants further exploration. This study reveals that the absence of the Vitamin D receptor (VDR) accelerates the progression from chronic colitis to colorectal cancer. Our findings indicate that vitexin can specifically target the VDR protein, facilitating its translocation into the cell nucleus to exert transcriptional activity. Additionally, through a co-culture model of macrophages and cancer cells, we observed that vitexin promotes the polarization of macrophages towards the M1 phenotype, a process that is dependent on VDR. Furthermore, ChIP-seq analysis revealed that vitexin regulates the transcriptional activation of phenazine biosynthesis-like domain protein (PBLD) via VDR. ChIP assays and dual luciferase reporter assays were employed to identify the functional PBLD regulatory region, confirming that the VDR/PBLD pathway is critical for vitexin-mediated regulation of macrophage polarization. Finally, in a mouse model with myeloid VDR gene knockout, we found that the protective effects of vitexin were abolished in mid-stage CAC. In summary, our study establishes that vitexin targets VDR and modulates macrophage polarization through the VDR/PBLD pathway, thereby alleviating the transition from chronic colitis to colorectal cancer.

Esculin inhibits hepatic stellate cell activation and CCl4-induced liver fibrosis by activating the Nrf2/GPX4 signaling pathway.

BACKGROUND: Liver fibrosis (LF) is a pathological process of the liver that threatens human health. Currently, effective treatments are still lacking. Esculin, a prominent constituent found in the Fraxinus rhynchophylla. (bark), Aesculus hippocastanum. (bark), and Cichorium intybus. (herb), has been shown to possess significant anti-inflammatory, antioxidant, and antibacterial properties. However, to date, there have been no studies investigating its potential efficacy in the treatment of LF. OBJECTIVE: The study aims to investigate the therapeutic effect of esculin on LF and elucidate its potential molecular mechanism. METHODS: Carbon tetrachloride (CCl4) was injected intraperitoneally to induce LF in mice, and transforming growth factor ß1 (TGF-ß1) was injected to induce LX-2 cells to investigate the improvement effect of esculin on LF. Kit, histopathological staining, immunohistochemistry (IHC), immunofluorescence (IF), polymerase chain reaction (PCR), and western blot (WB) were used to detect the expression of fiber markers and nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling pathway in liver tissue and LX-2 cells. Finally, molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) were used to verify the targeting between Nrf2 and esculin. RESULTS: Esculin significantly inhibited CCl4-induced hepatic fibrosis and inflammation in mice. This was evidenced by the improvement of liver function indexes, fibrosis indicators, and histopathology. Additionally, esculin treatment prominently reduced the levels of pro-inflammatory factors, oxidative stress, and liver Fe2+ in CCl4-induced mice. In vitro studies also showed that esculin treatment significantly inhibited TGF-ß1-induced LX-2 cell activation and decreased alpha-smooth muscle actin (α-SMA) and collagen I expression. Mechanism experiments proved that esculin can activate the Nrf2/GPX4 signaling pathway and inhibit liver ferroptosis. However, when LX-2 cells were treated with the Nrf2 inhibitor (ML385), the therapeutic effect of esculin significantly decreased. CONCLUSION: This study is the first to demonstrate that esculin is a potential natural active ingredient in the treatment of LF, which can inhibit the activation of hepatic stellate cells (HSC) and improve LF. Its therapeutic effect is related to the activation of the Nrf2/GPX4 signaling pathway.

Monotropein inhibits colitis associated cancer through VDR/JAK1/STAT1 regulation of macrophage polarization.

Colorectal cancer (CRC) is a growing concern due to its high morbidity and mortality, and the search for effective and less toxic active substances against inflammatory bowel diseases has been a hot topic in the research and development of drugs against CRC. It is reported that monotropein isolated from the roots of Morinda officinalis, can improve Dextran Sodium Sulfate (DSS)-induced ulcerative colitis in mice, but its therapeutic effects and mechanisms for CRC treatment are still to be investigated. In the present study, we first used molecular docking, BLI, CESTA, and DARTS methods to detest whether monotropein targets VDR proteins. In addition, we used tumor cell conditioned co-culture and four models of macrophage polarisation to investigate the regulation of four macrophage polarisations by monotropein using RT-PCR, IF and western blot. Furthermore, we further validated the target of action of monotropein for the treatment of Azoxymethane (AOM)/DSS induced colitis associated cancer (CAC) using knockout animals. Meanwhile, we further explored the mechanism of action of monotropein in regulating polarisation by detecting JAK/STAT1-related genes and proteins. Molecular docking and biofilm interference techniques showed that monotropein bound to the VDR, and additional results from CESTA and DARTS suggested that VDR proteins are targets of monotropein. Furthermore, in tumor cell conditioned co-cultures or LPS + IFN-γ induced RAW264.7 cells, VDR translocation to the nucleus was reduced, JAK1/STAT1 signaling pathway proteins were up-regulated, and macrophages were polarised towards the M1-type after monotropein intervention. Animal models in which normal VDR or myeloid VDR was knocked out confirmed that JAK1 levels in intestinal tissues were increased after monotropein intervention, macrophages were polarised towards the M1 type, and CAC paracarcinomas were ameliorated. Taken together, the present study concluded that monotropein inhibited colitis-associated cancers through macrophage polarisation regulated by VDR/JAK1/STAT1.

High-fat diet and alcohol induced-mice could cause colonic injury through molecular mechanisms of endogenous toxins.

Due to the complexity and diverse causes, the pathological mechanism of diet-induced colonic injury and colitis remains unclear. In this study, we studied the effects of the combination of a high-fat diet (HFD) plus alcohol on colonic injury in mice. We found HFD plus alcohol treatment induced disturbance of the gut microbiota; increased the production of intestinal toxins lipopolysaccharide (LPS), indole, and skatole; destroyed the stability of the intestinal mucosa; and caused the colonic epithelial cells damage through the activation of nuclear factor (NF)-κB and aromatic hydrocarbon receptors (AhR) signaling pathways. To mimic the effect of HFD plus alcohol in vivo, NCM460 cells were stimulated with alcohol and oleic acid with/without intestinal toxins (LPS, indole, and skatole) in vitro. Combinative treatment of alcohol and oleic acid caused moderate damage on NCM460 cells, while combination with intestinal toxins induced serious cell apoptosis. Western blot data indicated that the activation of NF-κB and AhR pathways further augmented after intestinal toxins treatment in alcohol- and oleic acid-treated colonic cells. This study provided new evidence for the relationship between diet pattern and colonic inflammation, which might partly reveal the pathological development of diet-induced colon disease and the involvement of intestinal toxins.

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.