Identification of bioactive ingredients from Babaodan using UPLC-QTOF-MS analysis combined with network pharmacology guided bioassays.

Babaodan (BBD) is a traditional Chinese medicine (TCM) prescribed for various inflammatory diseases, including viral hepatitis and acute genitourinary tract infection. Like other TCMs, BBD is a multi-component formula whose chemical composition and mode of action are largely unknown. The current study identified the bioactive ingredients of BBD using ultrahigh-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) followed by mass spectrometry molecular networking analysis. Subsequently, network pharmacology analysis was performed to predict the potential targets and pathways regulated by BBD. Eventually, a panel of compounds was selected and examined for their anti-inflammatory effects using lipopolysaccharide-stimulated RAW264.7 cells. Eighty-six compounds, including saponins, bile acids, and fatty acids, were identified. Tumor necrosis factor-alpha was identified as a key molecule. Pathways in cancer, inflammatory bowel disease, and hepatitis were predicted to be the major regulatory pathways. The results from bioassays validated ginsenoside Rb1, ginsenoside Rd, deoxycholic acid, chenodeoxycholic acid, and taurochenodeoxycholic acid as novel bioactive ingredients in BBD with anti-inflammatory effects. In conclusion, our study explains the anti-inflammatory efficacy of BBD from both chemical and biological aspects, which provides a scientific basis for the clinical application of BBD in inflammation-related diseases.

Babao Dan Reverses Multiple-Drug Resistance in Gastric Cancer Cells via Triggering Apoptosis and Autophagy and Inhibiting PI3K/AKT/mTOR Signaling.

Multidrug resistance (MDR) is a critical reason for cancer chemotherapy failure. Babaodan (BBD) is a famous traditional Chinese patent medicine reported to have antigastric cancer activity. However, the roles and molecular mechanisms of the reversal of MDR of gastric cancer by BBD have not been well described until now. Therefore, the purpose of this study was to elucidate further the role of BBD in reversing the MDR of gastric cancer cells and its specific regulatory mechanism via in vitro experiments. To verify our results, MTT, Doxorubicin (DOX) staining, Rhodamin123 (Rho123) staining, DAPI staining, Annexin V-FITC, propidium iodide (PI), Cyto-ID, and western blot assays were performed. To determine whether BBD triggers apoptosis and autophagy through the PI3K/AKT/mTOR signaling, we also applied 3-methyladenine (3-MA), chloroquine (CQ), and 740Y-P (an activator of PI3K). The results showed that BBD reversed the MDR and induced apoptosis and autophagy of SGC7901/DDP cells. Pathway analyses suggested BBD inhibits PI3K/AKT/mTOR pathway activity and subsequent apoptosis-autophagy induction. Inhibition of autophagy with 3-MA and chloroquine (CQ) was performed to confirm that BBD promoted autophagy. PI3K agonist, 740Y-P, further verified BBD inhibition of PI3K/AKT/mTOR pathway activation. In conclusion, BBD may reverse the MDR of gastric cancer cells, induce apoptosis, and promote autophagy via inactivation of the PI3K/AKT/mTOR signaling pathway.

Xinhuang Tablets Improve Intestinal Barrier Function via Regulating Epithelial Tight Junctions in Dextran Sulfate Sodium-Induced Ulcerative Colitis Mice.

Intestinal mucosal barrier dysfunction is involved in the pathogenesis of inflammatory bowel disease, including ulcerative colitis (UC). Xinhuang tablets (XHTs) have been prescribed for several kinds of inflammatory diseases, including UC, whereas its possible underlying molecular mechanisms had never been explored. Mouse model of UC was constructed by DSS treatment and followed by XHT treatment. Disease activity index, histopathological of colonic tissue, tumor necrosis factor-alpha (TNF-α), and serum amyloid A (SAA) levels in serum were further assessed. The underlying mechanism was further explored by determination of the expression of epithelial tight junction-related protein. XHT administration ameliorated dextran sulfate sodium (DSS)-induced clinical symptoms, colonic histological injury, and decreased the circulating levels of TNF-α and SAA. Moreover, XHT treatment significantly increased the protein levels of zona occludens (ZO)-1, whereas decreased the levels of phosphorylation of Elk-1. In conclusion, this study confirmed the therapeutic effects of XHT treatment on UC in a DSS-induced mouse model, and indicated that by increasing expression of epithelial tight junctions and decreasing phosphorylation of Elk-1 might be one of the underlying mechanisms of XHT treatment on UC.

Effect of Babao Dan on angiogenesis of gastric cancer in vitro by regulating VEGFA/VEGFR2 signaling pathway.

BACKGROUND: To further elucidate the anti-angiogenesis effect of Babao Dan (BBD) in vitro, gastric cancer (GC) cells and human umbilical vein endothelial cells (HUVECs) were used to evaluate the regulation role of BBD by vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway. METHODS: After induced by VEGFA, GC cells (AGS, MGC80-3 and BGC823) were treated by different concentrations of BBD and then were detected cell viability, migration and VEGFA level. And the anti-angiogenesis effect of BBD was evaluated with HUVECs. To furtherly mimic the tumor microenvironment of angiogenesis, VEGFA as an inducer (10 ng/mL) was used to trigger a cascade of angiogenesis of HUVECs in vitro. RESULTS: The viability and migration of GC cells with VEGFA-induced or non-induced and VEGFA levels in GC cells were significantly inhibited by BBD with concentration-dependent manner (P<0.01). BBD significantly inhibited the HUVECs viability with concentration-dependent manner (P<0.01), which was consistent with the inhibitory action on augmentation of cell viability induced by VEGFA (P<0.01). BBD exhibited the similar inhibitory trend on cyto behavioral variability such as wound repairing (P<0.05), migration (P<0.01) and tube formation (P<0.01) and activation effect on cell apoptosis rate (P<0.01) with VEGFA-induced or non-induced. Moreover, BBD notably regulated the levels of VEGFA, VEGFR2, matrix metalloprotein 2 (MMP2) and matrix metalloprotein 9 (MMP9) of HUVECs on present or absent of VEGFA with dose-dependent manner. CONCLUSIONS: BBD inhibited GC growth against VEGFA-induced angiogenesis of HUVECs by VEGFA/VEGFR2 signaling pathway in vitro.

Babao Dan inhibits the migration and invasion of gastric cancer cells by suppressing epithelial-mesenchymal transition through the TGF-ß/Smad pathway.

OBJECTIVE: To investigate the anti-metastatic effects of Babao Dan (BBD) on gastric cancer (GC) cells (AGS and MGC80-3) and explore the underlying molecular mechanisms by which it inhibits epithelial-mesenchymal transition (EMT). METHODS: AGS and MGC80-3 cells were treated with BBD. In addition, cells were treated with the EMT inducer transforming growth factor-ß1 (TGF-ß1). Cell viability was determined using the MTT assay, and the live cell ratio was calculated via cell counting. Cell invasion and migration were evaluated using the Transwell assay. Western blotting was performed to measure the protein expression of EMT biomarkers and related genes. RESULTS: BBD inhibited the viability, migration, and invasion of AGS and MGC80-3 cells, but it did not reduce the live cell ratio. Furthermore, BBD inhibited the expression of N-cadherin, vimentin, zinc finger E-box binding homeobox (ZEB)1, ZEB2, Twist1, matrix metalloproteinase (MMP)2, MMP9, TGF-ß1, and p-Smad2/3, whereas E-cadherin expression was increased in AGS and MGC80-3 cells to different degrees. Using a GC cell model of EMT induced by TGF-ß1, we proved that BBD inhibited p-Smad2/3 and N-cadherin expression, cell migration, and cell invasion. CONCLUSION: BBD suppressed cell migration and invasion by inhibiting TGF-ß-induced EMT and inactivating TGF-ß/Smad signaling in GC cells.