Discovery of a New Anti-Inflammatory Agent from Anemoside B4 Derivatives and Its Therapeutic Effect on Colitis by Targeting Pyruvate Carboxylase.

Anemoside B4 (AB4), a triterpenoidal saponin from Pulsatilla chinensis, shows significant anti-inflammatory activity, and may be used for treating inflammatory bowel disease (IBD). Nevertheless, its application is limited due to its high molecular weight and pronounced water solubility. To discover new effective agents for treating IBD, we synthesized 28 AB4 derivatives and evaluated their cytotoxic and anti-inflammatory activities in vitro. Among them, A3-6 exhibited significantly superior anti-inflammatory activity compared to AB4. It showed a significant improvement in the symptoms of DSS-induced colitis in mice, with a notably lower oral effective dose compared to AB4. Furthermore, we discovered that A3-6 bound with pyruvate carboxylase (PC), then inhibited PC activity, reprogramming macrophage function, and alleviated colitis. These findings indicate that A3-6 is a promising therapeutic candidate for colitis, and PC may be a potential new target for treating colitis.

Rosamultin ameliorates radiation injury via promoting DNA injury repair and suppressing oxidative stress in vitro and in vivo.

Radiotherapy remains the preferred treatment option for cancer patients with the advantages of broad indications and significant therapeutic effects. However, ionizing radiation can also damage normal tissues. Unfortunately, there are few anti-radiation damage drugs available on the market for radiotherapy patients. Our previous study showed that rosamultin had antioxidant and hepatoprotective activities. However, its anti-radiation activity has not been evaluated. Irradiating small intestinal epithelial cells and mice with whole-body X-rays radiation were used to evaluate the in vitro and in vivo effects of rosamultin, respectively. Intragastric administration of rosamultin improved survival, limited leukocyte depletion, and reduced damage to the spleen and small intestine in irradiated mice. Rosamultin reversed the downregulation of the apoptotic protein BCL-2 and the upregulation of BAX in irradiated mouse small intestine tissue and in irradiation-induced small intestinal epithelial cells. DNA-PKcs antagonists reversed the promoting DNA repair effects of rosamulin. Detailed mechanistic studies revealed that rosamultin promoted Translin-associated protein X (TRAX) into the nucleus. Knockdown of TRAX reduced the protective effect of rosamultin against DNA damage. In addition, rosamultin reduced irradiation-induced oxidative stress through promoting Nrf2/HO-1 signaling pathway. To sum up, in vitro and in vivo experiments using genetic knockdown and pharmacological activation demonstrated that rosamultin exerts radioprotection via the TRAX/NHEJ and Nrf2/HO pathways.

Anemoside B4 inhibits SARS-CoV-2 replication in vitro and in vivo.

Objective: Anemoside B4 (AB4), the most abundant triterpenoidal saponin isolated from Pulsatilla chinensis, inhibited influenza virus FM1 or Klebsiella pneumoniae-induced pneumonia. However, the anti-SARS-CoV-2 effect of AB4 has not been unraveled. Therefore, this study aimed to determine the antiviral activity and potential mechanism of AB4 in inhibiting human coronavirus SARS-CoV-2 in vivo and in vitro. Methods: The cytotoxicity of AB4 was evaluated using the Cell Counting Kit-8 (CCK8) assay. SARS-CoV-2 infected HEK293T, HPAEpiC, and Vero E6 cells were used for in vitro assays. The antiviral effect of AB4 in vivo was evaluated by SARS-CoV-2-infected hACE2-IRES-luc transgenic mouse model. Furthermore, label-free quantitative proteomics and bioinformatic analysis were performed to explore the potential antiviral mechanism of action of AB4. Type I IFN signaling-associated proteins were assessed using Western blotting or immumohistochemical staining. Results: The data showed that AB4 reduced the propagation of SARS-CoV-2 along with the decreased Nucleocapsid protein (N), Spike protein (S), and 3C-like protease (3CLpro) in HEK293T cells. In vivo antiviral activity data revealed that AB4 inhibited viral replication and relieved pneumonia in a SARS-CoV-2 infected mouse model. We further disclosed that the antiviral activity of AB4 was associated with the enhanced interferon (IFN)-ß response via the activation of retinoic acid-inducible gene I (RIG-1) like receptor (RLP) pathways. Additionally, label-free quantitative proteomic analyses discovered that 17 proteins were significantly altered by AB4 in the SARS-CoV-2 coronavirus infections cells. These proteins mainly clustered in RNA metabolism. Conclusion: Our results indicated that AB4 inhibited SARS-CoV-2 replication through the RLR pathways and moderated the RNA metabolism, suggesting that it would be a potential lead compound for the development of anti-SARS-CoV-2 drugs.

Anemoside B4, a new pyruvate carboxylase inhibitor, alleviates colitis by reprogramming macrophage function.

OBJECTIVES: Colitis is a global disease usually accompanied by intestinal epithelial damage and intestinal inflammation, and an increasing number of studies have found natural products to be highly effective in treating colitis. Anemoside B4 (AB4), an abundant saponin isolated from Pulsatilla chinensis (Bunge), which was found to have strong anti-inflammatory activity. However, the exact molecular mechanisms and direct targets of AB4 in the treatment of colitis remain to be discovered. METHODS: The anti-inflammatory activities of AB4 were verified in LPS-induced cell models and 2, 4, 6-trinitrobenzene sulfonic (TNBS) or dextran sulfate sodium (DSS)-induced colitis mice and rat models. The molecular target of AB4 was identified by affinity chromatography analysis using chemical probes derived from AB4. Experiments including proteomics, molecular docking, biotin pull-down, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to confirm the binding of AB4 to its molecular target. Overexpression of pyruvate carboxylase (PC) and PC agonist were used to study the effects of PC on the anti-inflammatory and metabolic regulation of AB4 in vitro and in vivo. RESULTS: AB4 not only significantly inhibited LPS-induced NF-κB activation and increased ROS levels in THP-1 cells, but also suppressed TNBS/DSS-induced colonic inflammation in mice and rats. The molecular target of AB4 was identified as PC, a key enzyme related to fatty acid, amino acid and tricarboxylic acid (TCA) cycle. We next demonstrated that AB4 specifically bound to the His879 site of PC and altered the protein's spatial conformation, thereby affecting the enzymatic activity of PC. LPS activated NF-κB pathway and increased PC activity, which caused metabolic reprogramming, while AB4 reversed this phenomenon by inhibiting the PC activity. In vivo studies showed that diisopropylamine dichloroacetate (DADA), a PC agonist, eliminated the therapeutic effects of AB4 by changing the metabolic rearrangement of intestinal tissues in colitis mice. CONCLUSION: We identified PC as a direct cellular target of AB4 in the modulation of inflammation, especially colitis. Moreover, PC/pyruvate metabolism/NF-κB is crucial for LPS-driven inflammation and oxidative stress. These findings shed more light on the possibilities of PC as a potential new target for treating colitis.

Ephrin B3 exacerbates colitis and colitis-associated colorectal cancer.

Ephrin B3, a member of Eph/ephrin family, contributes to embryogenesis and carcinogenesis, but few studies have suggested whether this ligand has regulatory effect on colitis. This study was to determine whether ephrin B3 played a role in colitis and colonic carcinogenesis. Dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced colitis-associated carcinogenesis model was established in Efnb3-deficient (Efnb3-/-) mice. Label-free quantitative proteomics were performed to identify the Efnb3-regulated proteins. Our results showed that Efnb3 knock out reduced the symptoms of DSS-induced colitis, such as disease activity index (DAI), inflammatory factors release, and dysfunction of the intestinal barrier. Quantitative proteomics revealed that Efnb3 regulated 95 proteins which clustered in the platelet degranulation, response to elevated platelet cytosolic Ca2+, MAPK signaling for integrins such as ITGB4. Furthermore, ephrin B3 inactived ITGB4/AKT signal pathway and then promoted epithelial barrier dysfunction. Simultaneously, ephrin B3 promoted Gremlin-1/NF-κB signal pathway and thereby increased inflammatory factors release. In addition, the higher level of Efnb3 in colon cancer patients is correlated with worse survival. Efnb3-/- mice exhibited susceptibility to AOM/DSS-induced colorectal cancer. Our finding discovered that Efnb3 played an important role in the development of colitis and colitis-associated colorectal cancer. Efnb3 deficiency improved the intestinal barrier by ITGB4 and suppressed inflammation via Gremlin-1/NF-κB signal pathway, which may provide a novel therapeutic strategy for the treatment of colitis and colitis-associated colorectal cancer.