Inhibitory Effects and Mechanism of the Natural Compound Diaporthein B Extracted from Marine-Derived Fungi on Colon Cancer Cells.

This study aimed to investigate the inhibitory effects and mechanism of diaporthein B (DTB), a natural compound extracted from the fungus Penicillium sclerotiorum GZU-XW03-2, on human colon cancer cells. The inhibitory effect of DTB at different concentrations on the proliferation of colon cancer cells HCT116 and LOVO was detected at 24 and 48 h. The effect of cell migration and clone formation ability were detected by cell scratch and plate cloning experiments. Morphological changes were observed by Hoechst 33342 and Annexin-V/PI staining, and flow cytometry was used to detect the proportion of apoptotic cells. DTB significantly inhibited colon cancer cell proliferation, migration, and apoptosis in a dose-dependent manner without significant effects on normal colonic epithelial cells NCM460. The IC50 inhibition effect can be achieved after treatment with 3 µmol/L DTB for 24 h. Compared with the blank group, the migration and clonal-forming ability of colon cancer cells in the DTB group was significantly decreased (p < 0.01), while the apoptotic cells were significantly increased (p < 0.01) in a concentration-dependent manner. DTB can inhibit the proliferation and migration of human colon cancer cells HCT116 and LOVO and promote the apoptosis of human colon cancer cells.

Molecular Docking and Molecular Dynamics Studies on Selective Synthesis of α-Amyrin and ß-Amyrin by Oxidosqualene Cyclases from Ilex Asprella.

Amyrins are the immediate precursors of many pharmaceutically important pentacyclic triterpenoids. Although various amyrin synthases have been identified, little is known about the relationship between protein structures and the constituent and content of the products. IaAS1 and IaAS2 identified from Ilex asprella in our previous work belong to multifunctional oxidosqualene cyclases and can produce α-amyrin and ß-amyrin at different ratios. More than 80% of total production of IaAS1 is α-amyrin; while IaAS2 mainly produces ß-amyrin with a yield of 95%. Here, we present a molecular modeling approach to explore the underlying mechanism for selective synthesis. The structures of IaAS1 and IaAS2 were constructed by homology modeling, and were evaluated by Ramachandran Plot and Verify 3D program. The enzyme-product conformations generated by molecular docking indicated that ASP484 residue plays an important role in the catalytic process; and TRP611 residue of IaAS2 had interaction with ß-amyrin through π-σ interaction. MM/GBSA binding free energy calculations and free energy decomposition after 50 ns molecular dynamics simulations were performed. The binding affinity between the main product and corresponding enzyme was higher than that of the by-product. Conserved amino acid residues such as TRP257; TYR259; PHE47; TRP534; TRP612; and TYR728 for IaAS1 (TRP257; TYR259; PHE473; TRP533; TRP611; and TYR727 for IaAS2) had strong interactions with both products. GLN450 and LYS372 had negative contribution to binding affinity between α-amyrin or ß-amyrin and IaAS1. LYS372 and ARG261 had strong repulsive effects for the binding of α-amyrin with IaAS2. The importance of Lys372 and TRP612 of IaAS1, and Lys372 and TRP611 of IaAS2, for synthesizing amyrins were confirmed by site-directed mutagenesis. The different patterns of residue-product interactions is the cause for the difference in the yields of two products.

Baicalin Protects against TNF-α-Induced Injury by Down-Regulating miR-191a That Targets the Tight Junction Protein ZO-1 in IEC-6 Cells.

Tumor necrosis factor-alpha (TNF-α) plays an important role in the developing process of inflammatory bowel disease. Tight junction protein zonula occludens-1 (ZO-1), one of epithelial junctional proteins, maintains the permeability of intestinal barrier. The objective of this study was to investigate the mechanism of the protective effect of baicalin on TNF-α-induced injury and ZO-1 expression in intestinal epithelial cells (IECs). We found that baicalin pretreatment significantly improved cell viability and cell migration following TNF-α stimulation. miR-191a inhibitor increased the protective effect of baicalin on cell motility injured by TNF-α. In addition, miR-191a down-regulated the mRNA and protein level of its target gene ZO-1. TNF-α stimulation increased miR-191a expression, leading to the decline of ZO-1 mRNA and protein. Moreover, pretreatment with baicalin reversed TNF-α induced decrease of ZO-1 and increase of miR-191a, miR-191a inhibitor significantly enhanced ZO-1 protein expression restored by baicalin. These results indicate that baicalin exerts a protective effect on IEC-6 (rat small intestinal epithelial cells) cells against TNF-α-induced injury, which is at least partly via inhibiting the expression of miR-191a, thus increasing ZO-1 mRNA and protein levels.

Protective effects of baicalin on LPS-induced injury in intestinal epithelial cells and intercellular tight junctions.

AIMS: To investigate the protective effects and mechanisms of baicalin on lipopolysaccharide (LPS)-induced injury in intestinal epithelial cells and intercellular tight junctions. METHODS: IEC-6 cells were stimulated with LPS (1.0 µg/mL), with or without baicalin, for 24 h. The levels of the inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α were determined using ELISA. Quantitative real-time PCR was used for determining the mRNA expression level of claudin-3, occludin, and ZO-1; Western blot and immunofluorescence analysis were used for analyzing the expression level and the distribution patterns of ZO-1 protein. RESULTS: Pretreatment with baicalin (10.0 µg/mL) improved LPS-stimulated cell viability and repressed IL-6 and TNF-α levels. In addition, pretreatment with baicalin up-regulated mRNA and protein expression levels of ZO-1 and kept the protein intact in IEC-6 cells injured with LPS. CONCLUSION: Baicalin has the capacity to protect IEC-6 cells and the intercellular tight junctions from LPS-induced injury. The mechanisms may be associated with inhibiting the production of inflammatory cytokines, and up-regulating the mRNA and protein expression of ZO-1.

[Community structure and difference of endophtic bacteria in Aquilaria sinensis with and without agarwood].

Aquilaria sinensis can generate agarwood, which is closely related with endophyte. Up to now, studies mainly focused on the effects of endophytic fungi on agarwood formation, but studies about endophytic bacteria are rarely reported. In our research, the T-RFs and Shannon index of endophytic bacteria in samples of agarwood increase. The number of distinctive T-RFs fragments of corresponding samples in the same group accounted for more than 60% the number of total T-RFs fragments. In samples of no-agarwood, the dominant bacterial population are Anoxybacillus, Clostridium, Candidatus endobugula, Lysinibacillus. In samples of agarwood, the dominant bacterial population are Clostridium, Lysinibacillus, Luteimonas, phytoplasma. Besides, there are. specific T-RFs fragment in samples of agarwood and no-agarwood respectively. When we perform cluster analysis, we found samples of agarwood highly gather together and samples of no-agarwood highly gather together. This means community of endophytic bacteria emerge significant and regular changes during agarwood formation, which may be result of agarwood production, or maybe it is important reason of agarwood production. In this paper, we obtain more comprehensive and accurate community of endophytic bacteria in Aquilaria sinensis and it's variation during agarwood formation using T-RFLP, which is first study of effects of endophytic bacteria on agarwood formation, and will help to exploit resource of endophytic bacteria more reasonably.

pH-Responsive Calcium Ions and Crocetin Releasing Hydrogel for Accelerating Skin Wound Healing.

The ideal and highly anticipated dressing for skin wounds should provide a moist environment, possess antibacterial properties, and ensure sustained drug release. In the present work, a hyaluronic acid-based hydrogel was formed by cross-linking crocetin and CaCO3@polyelectrolyte materials (CaCO3@PEM) microspheres with HA hydrogels via hydrogen bond and amido bonding (CaCO3@PEM@Cro@HA hydrogel, CPC@HA hydrogel). Moreover, the CPC@HA hydrogel had the capability of sustained, controlled release of calcium ions and crocetin via pH-sensitive and accelerated skin wound healing. The experiment results showed that the CPC@HA hydrogel exhibited porous network structures, stable physical properties, and had antibacterial properties and biocompatibility in vitro. In addition, the CPC@HA hydrogel covering on the skin wound could reduce inflammation and promote wound healing. The high expression of angiogenic cytokines (CD31) and epidermal terminal differentiation markers (Loricrin) of wound healing tissue suggested the CPC@HA hydrogel also had the function of promoting the remodeling of regenerated skin. Overall, CPC@HA hydrogel has promising potential for clinical applications in accelerating skin wound repair.

The effect of acupuncture on depression and its correlation with metabolic alterations: A randomized controlled trial.

BACKGROUND: Electroacupuncture (EA) treatment has antidepressant effect and when patients were treated with EA and antidepressants, the effect could be maintained for a longer time. However, the effect of EA combined with antidepressants based on metabolism is still in the initial observation stage, which requires further research. METHODS: A total of 60 patients with moderate depression were assigned into 2 groups at a ratio of 1:1, the EA group (receiving EA and antidepressants) and the control group (taking antidepressants only) in this randomized controlled pilot trial. The EA treatment was performed 3 times a week for 8 consecutive weeks and then follow up for 4 weeks. The patients' depressive mood was measured by the Hamilton Depression scale (HAMD) at baseline, week 4, week 8 and week 12. Before and after 8-week treatment, morning urine samples from all patients were analyzed by the gas chromatography-mass spectrometry (GC-MS) to find possible metabolic markers of depression and of EA treatment related changes. RESULTS: Compared with the control group, the EA group showed more significant improvements in depressive symptoms measured by HAMD at week 4 (16.89 ±â€Š5.74 vs 25.58 ±â€Š7.03, P < .001), week 8 (9.59 ±â€Š5.13 vs 25.04 ±â€Š7.49, P < .001) and week 12 (11.07 ±â€Š6.85 vs 27.25 ±â€Š7.14, P < .001). The significant differences in urinary specific metabolites before and after EA treatment were malonic acid (fatty acid biosynthesis), cysteine (glutamate metabolism), glutathione (glutamate metabolism), tryptophan (tryptophan metabolism), proline (glutamate metabolism), and N-acetyl-5-hydroxytryptamine. These metabolites are involved in tryptophan metabolism, glutamate metabolism, and fatty acid biosynthesis. CONCLUSION: EA treatment combined with antidepressants is more effective in improving depressive symptoms than antidepressants alone. EA may treat depression by acting on tryptophan metabolism, glutamate metabolism, and fatty acid biosynthesis. TRIAL REGISTRATION: Chinese Clinical Trial Registry: ChiCTR-2000030786.

AICD: an integrated anti-inflammatory compounds database for drug discovery.

Systemic or local inflammation drives the pathogenesis of various human diseases. Small compounds with anti-inflammatory properties hold great potential for clinical translation. Over recent decades, many compounds have been screened for their action against inflammation-related targets. Databases that integrate the physicochemical properties and bioassay results of these compounds are lacking. We created an "Anti-Inflammatory Compounds Database" (AICD) to deposit compounds with potential anti-inflammation activities. A total of 232 inflammation-related targets were recruited by the AICD. Gene set enrichment analysis showed that these targets were involved in various human diseases. Bioassays of these targets were collected from open-access databases and adopted to extract 79,781 small molecules with information on chemical properties, candidate targets, bioassay models and bioassay results. Principal component analysis demonstrated that these deposited compounds were closely related to US Food and Drug Administration-approved drugs with respect to chemical space and chemical properties. Finally, pathway-based screening for drug combination/multi-target drugs provided a case study for drug discovery using the AICD. The AICD focuses on inflammation-related drug targets and contains substantial candidate compounds with high chemical diversity and good drug-like properties. It could be serviced for the discovery of anti-inflammatory medicines and can be accessed freely at http://956023.ichengyun.net/AICD/index.php .

In silico Identification and Mechanism Exploration of Hepatotoxic Ingredients in Traditional Chinese Medicine.

BACKGROUNDS AND AIMS: Recently, a growing number of hepatotoxicity cases aroused by Traditional Chinese Medicine (TCM) have been reported, causing increasing concern. To date, the reported predictive models for drug induced liver injury show low prediction accuracy and there are still no related reports for hepatotoxicity evaluation of TCM systematically. Additionally, the mechanism of herb induced liver injury (HILI) still remains unknown. The aim of the study was to identify potential hepatotoxic ingredients in TCM and explore the molecular mechanism of TCM against HILI. MATERIALS AND METHODS: In this study, we developed consensus models for HILI prediction by integrating the best single classifiers. The consensus model with best performance was applied to identify the potential hepatotoxic ingredients from the Traditional Chinese Medicine Systems Pharmacology database (TCMSP). Systems pharmacology analyses, including multiple network construction and KEGG pathway enrichment, were performed to further explore the hepatotoxicity mechanism of TCM. RESULTS: 16 single classifiers were built by combining four machine learning methods with four different sets of fingerprints. After systematic evaluation, the best four single classifiers were selected, which achieved a Matthews correlation coefficient (MCC) value of 0.702, 0.691, 0.659, and 0.717, respectively. To improve the predictive capacity of single models, consensus prediction method was used to integrate the best four single classifiers. Results showed that the consensus model C-3 (MCC = 0.78) outperformed the four single classifiers and other consensus models. Subsequently, 5,666 potential hepatotoxic compounds were identified by C-3 model. We integrated the top 10 hepatotoxic herbs and discussed the hepatotoxicity mechanism of TCM via systems pharmacology approach. Finally, Chaihu was selected as the case study for exploring the molecular mechanism of hepatotoxicity. CONCLUSION: Overall, this study provides a high accurate approach to predict HILI and an in silico perspective into understanding the hepatotoxicity mechanism of TCM, which might facilitate the discovery and development of new drugs.

[Thoughts on pathogenesis of Helicobacter pylori related gastrosis with splenogastric hygropyrexia syndrome].

Splenogastric hygropyrexia syndrome, a common syndome in clinical practice, responds well to the therapy of traditional Chinese medicine (TCM). In view of the complexity of TCM syndrome formation, proceeding from different aspects such as Helicobacter pylori (Hp) infection, expression of inflammatory factors, mucosal protection and change in micro-ecosystem, the authors proposed that Hp infection in gastric mucosa, nuclear factor-kappa B activation, over-expression of heat shock protein 70, and "excessive evil, dyssplenism and confliction between healthy energy and evils" due to disequilibrium between Hp and lactobacillus acidophilus in gastric mucosa and coated tongue may be the important links in the formation of Hp related gastrosis with splenogastric hygropyrexia syndrome.

Endophytic bacterial and fungal communities transmitted from cotyledons and germs in peanut (Arachis hypogaea L.) sprouts.

Seed-borne endophytes could be transmitted into sprouts. Whether this happened in peanuts and the difference between microbial taxa in peanut germs and cotyledons remain unknown. In this research, Illumina-based sequencing was employed to investigate the microbial taxa in peanut germs, cotyledons, and sprouts. Sulfur-oxidizing bacteria was isolated and inoculated into peanut sprouts, and then, the growth of peanut seedlings was measured. The results illustrated that diverse bacteria and fungi were detected in peanut germs, cotyledons, and sprouts. The number of bacterial OTUs declined with the germination from germs and cotyledons to sprouts. However, the number of fungal OTUs increased during the seedling procedure. Seed-borne dominant bacterial genera Halothiobacillus and Synechococcus and fungal genera Humicola, Emericella, and Penicillium were detected in sprouts. Based on the endophytic community information, the Halothiobacillus strains were isolated from sprouts. Pot experiments that illustrated the growth of peanut seedlings inoculated with the strain were promoted. These results provide new understanding into plant-microbe interactions in peanut and suggest that the selection for biocontrol agents based on mycobiome and bacteriome analysis is reliable and feasible compared with the present greenhouse selection.

[Preliminary study on intestinal flora in diarrhea type irritable bowel syndrome with pi-wei dampness-heat syndrome].

OBJECTIVE: To observe the changes of intestinal flora in diarrhea type irritable bowel syndrome with Pi-wei dampness-heat syndrome (IBS-PDS). METHODS: The seven kinds of common intestinal bacteria in feces, including enteri bacillus, enterococci, saccharomycete, bifid bacteria, lactobacillus, bacteroides and peptococcus were studied in 21 patients suffered from IBS-PDS, and compared with those in 22 patients with IBS with deficiency of Pi syndrome (DPS) and 25 healthy subjects as control. RESULTS: As compared with the healthy subjects, the levels of enteri bacillus and enterococci were significantly increased (P<0.01), the levels of bifid bacteria, Lactobacillus and Peptococcus were significantly decreased (P < 0.01), and saccharomycete and Bacteroides were insignificantly different in patients with PDS. As compared with patients with DPS, the levels of enteri bacillus, enterococci, bifid bacteria, Lactobacillus, Peptococcus and Bacteroidaceae were significantly increased except the level of saccharomycete. CONCLUSION: There may be alteration of intestinal flora in patients with IBS-PDS.

Identification of UQCRB as an oxymatrine recognizing protein using a T7 phage display screen.

ETHNOPHARMACOLOGICAL RELEVANCE: Sophora flavescens Aiton (Radix Sophorae Flavescentis, Kushen) is used in traditional Chinese medicine to treat chronic hepatitis B (CHB), and has the ability to clear heat and dampness from the body. Oxymatrine is one of the major bioactive compounds extracted from Sophora flavescens Aiton and constitutes more than 90% of the oxymatrine injection commonly used for CHB treatment in clinics in China. AIM OF THE STUDY: We aim to analyze the protein binding target of oxymatrine in treating CHB by screening a T7 phage display cDNA library of human CHB and examine the biochemistry of protein-ligand binding between oxymatrine and its ligands. MATERIALS AND METHODS: A T7 phage cDNA library of human CHB was biopanned by affinity selection using oxymatrine as bait. The interaction of oxymatrine with its candidate binding protein was investigated by affinity assay, molecular docking, Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR). RESULTS: A library of potential oxymatrine binding peptides was generated. Ubiquinol-cytochrome c reductase binding protein (UQCRB) was one of the candidate binding proteins of oxymatrine. UQCRB-displaying T7 phage binding numbers in the oxymatrine group were significantly higher than that in the control group, biotin group, and matrine group (p<0.05 or p<0.01). Three-dimensional structure modeling of the UQCRB with oxymatrine showed that their binding interfaces matched and oxymatrine inserted into a deeper pocket of UQCRB, which mainly involved amino acid residues Tyr21, Arg33, Tyr83, Glu84, Asp86, Pro88, and Glu91. The binding affinity constant (Kb) from SPR was 4.2mM. The Kb from ITC experiment was 3.9mM and stoichiometry was fixed as 1, which fit very well with the result of SPR. The binding of oxymatrine to UQCRB was driven by strong enthalpy forces such as hydrogen bonds and polar interactions as the heat released was about 157kcal/mol and ΔG was less than zero. CONCLUSIONS: In this study, using the T7 phage display system, we have identified UQCRB as a direct binding protein of oxymatrine. Furthermore, the specificity and molecular interaction of oxymatrine with UQCRB were also determined. The binding of UQCRB to oxymatrine suggests that UQCRB is a potential target of oxymatrine in treating CHB. These results provide new understanding into the mechanism of oxymatrine and insights into the strategy on the treatment of CHB.