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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. [ABSTRACT FROM AUTHOR]

The microbial community compositions of a chemostat enriched in a thermophilic (55 °C) mixed culture fermentation (MCF) for hydrogen production under different operational conditions were revealed in this work by integrating denaturing gradient gel electrophoresis (DGGE), Illumina Miseq high-throughput sequencing, and 16S rRNA clone library sequencing. The results showed that the community structure of the enriched cultures was relatively simple. Clones close to the genera of Thermoanaerobacter and/or Bacillus mainly dominated the bacteria. And homoacetogens and archaea were washed out and not detected even by Illumina Miseq high-throughput sequencing which supported the benefit for hydrogen production. On the other hand, the results revealed that the metabolic shift was clearly associated with the change of dominated bacterial groups. The effects of hydrogen partial pressure (P) and pH from 4.0 to 5.5 on the microbial compositions were not notable and Thermoanaerobacter was dominant, thus, the metabolites were also not changed. While Bacillus, Thermoanaerobacter and Propionispora hippei dominated the bacteria communities at neutral pH, or Bacillus and Thermoanaerobacter dominated at high influent glucose concentrations, consequently the main metabolites shifted to acetate, ethanol, propionate, or lactate. Thereby, the effect of microbial composition on the metabolite distribution and shift shall be considered when modeling thermophilic MCF in the future. [ABSTRACT FROM AUTHOR]

Immune dysfunction is a major cause of mortality in septic patients. Current evidence indicates an important role for dendritic cells (DCs) in the pathophysiology of immune dysfunction, and these cells are potential targets of immunomodulation therapies. In the present study, our aim was to enhance the resistance of endotoxemic mice to bacterial translocation and secondary infection and to improve the outcome of these infections using a combination therapy consisting of thymosin alpha1 and dexamethasone in a timely manner according to the changes of DCs' number. The effect of treatment with dexamethasone (DXM) and thymosin alpha1 (Tα1) on DCs was investigated by examining their number, MHCII and CD86 expression and their capacity to induce T cell activation. Endotoxemic mice were randomly divided into five treatment groups. The survival rates, the levels of TNF-α and IL-10, the occurrence of bacterial translocation, and the ability to clear secondary infections were determined. Additionally, the behavior of DCs over time was also evaluated. Tα1 induced significant increases in DC numbers in vivo, whereas DXM reduced cell numbers both in vitro and in vivo. However, neither drug induced significant changes in the capacity of DCs to induce T cell activation or their expression of MHCII or CD86. Among the five treatment groups, the mice treated with a combination of DXM and Tα1 had the highest survival rate; this increased survival was associated with a decrease in bacterial translocation to extra-intestinal organs and an enhanced ability to eradicate secondary infections by reversing the change in DC numbers during endotoxemia. Immunomodulatory therapy that combines Tα1 and DXM in a timely manner and was based on changes in DCs enhanced the resistance of endotoxemic mice to bacterial translocation and secondary infections, improving the outcome of the infection. [ABSTRACT FROM AUTHOR]

Background: Gut barrier failure caused by endotoxemia is a life-threatening problem. The present study aimed to determine whether any specific intestinal site is highly correlated with gut barrier failure, and whether recombinant human growth hormone (rhGH) can ameliorate gut barrier failure in a rat model of endotoxemia. Methods: Enterostomy tubes were surgically placed in adult male Sprague-Dawley rats three days before induction of endotoxemia by lipopolysaccharide (LPS) injection. Controls received no LPS. Rats were then randomly assigned to receive subcutaneous injections of rhGH (experimental, n = 30) or 0.9 % saline (control, n = 15) at 24, 48, or 72 h after LPS injection. Escherichia coli labeled with green fluorescent protein (GFP) were injected into the intestinal segment of all rats through the enterostomy tubes. The number of GFP-labeled E. coli detected in mesenteric lymph nodes was examined after 96 h. Apoptosis and proliferation rates of intestinal epithelial cells, and intestinal permeability were measured. Results: Endotoxemia led to high mortality, compared with the control group, and rhGH treatment did not improve survival. Intestinal permeability, reflected by translocation rates of GFP-labeled E. coli, and apoptosis rates in the LPS-induced endotoxemia group were higher than those in the non-endotoxemia control group, and the endotoxemia ileum group had the highest rates of both bacterial translocation and apoptosis. The LPS+GH group had less bacterial translocation and apoptosis than the LPS-induced endotoxemia group. In contrast, the proliferation rates were lower in the LPS group compared to the LPS+GH group. Conclusions: Endotoxemia can induce gut barrier failure in rats, and the ileum is the site of greatest risk. The GH can reduce the incidence of endotoxemia-induced gut barrier failure, but not the associated mortality. [ABSTRACT FROM AUTHOR]