Sepsis Encephalopathy Is Partly Mediated by miR370-3p-Induced Mitochondrial Injury but Attenuated by BAM15 in Cecal Ligation and Puncture Sepsis Male Mice.

BAM15 (a mitochondrial uncoupling agent) was tested on cecal ligation and puncture (CLP) sepsis mice with in vitro experiments. BAM15 attenuated sepsis as indicated by survival, organ histology (kidneys and livers), spleen apoptosis (activated caspase 3), brain injury (SHIRPA score, serum s100ß, serum miR370-3p, brain miR370-3p, brain TNF-α, and apoptosis), systemic inflammation (cytokines, cell-free DNA, endotoxemia, and bacteremia), and blood-brain barrier (BBB) damage (Evan's blue dye and the presence of green fluorescent E. coli in brain after an oral administration). In parallel, brain miR arrays demonstrated miR370-3p at 24 h but not 120 h post-CLP, which was correlated with metabolic pathways. Either lipopolysaccharide (LPS) or TNF-α upregulated miR370-3p in PC12 (neuron cells). An activation by sepsis factors (LPS, TNF-α, or miR370-3p transfection) damaged mitochondria (fluorescent color staining) and reduced cell ATP, possibly through profound mitochondrial activity (extracellular flux analysis) that was attenuated by BAM15. In bone-marrow-derived macrophages, LPS caused mitochondrial injury, decreased cell ATP, enhanced glycolysis activity (extracellular flux analysis), and induced pro-inflammatory macrophages (iNOS and IL-1ß) which were neutralized by BAM15. In conclusion, BAM15 attenuated sepsis through decreased mitochondrial damage, reduced neuronal miR370-3p upregulation, and induced anti-inflammatory macrophages. BAM15 is proposed to be used as an adjuvant therapy against sepsis hyperinflammation.

Lactobacillus rhamnosus attenuates Thai chili extracts induced gut inflammation and dysbiosis despite capsaicin bactericidal effect against the probiotics, a possible toxicity of high dose capsaicin.

Because of a possible impact of capsaicin in the high concentrations on enterocyte injury (cytotoxicity) and bactericidal activity on probiotics, Lactobacillus rhamnosus L34 (L34) and Lactobacillus rhamnosus GG (LGG), the probiotics derived from Thai and Caucasian population, respectively, were tested in the chili-extract administered C57BL/6 mice and in vitro experiments. In comparison with placebo, 2 weeks administration of the extract from Thai chili in mice caused loose feces and induced intestinal permeability defect as indicated by FITC-dextran assay and the reduction in tight junction molecules (occludin and zona occludens-1) using fluorescent staining and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the chili extracts also induced the translocation of gut pathogen molecules; lipopolysaccharide (LPS) and (1→3)-ß-d-glucan (BG) and fecal dysbiosis (microbiome analysis), including reduced Firmicutes, increased Bacteroides, and enhanced total Gram-negative bacteria in feces. Both L34 and LGG attenuated gut barrier defect (FITC-dextran, the fluorescent staining and gene expression of tight junction molecules) but not improved fecal consistency. Additionally, high concentrations of capsaicin (0.02-2 mM) damage enterocytes (Caco-2 and HT-29) as indicated by cell viability test, supernatant cytokine (IL-8), transepithelial electrical resistance (TEER) and transepithelial FITC-dextran (4.4 kDa) but were attenuated by Lactobacillus condition media (LCM) from both probiotic-strains. The 24 h incubation with 2 mM capsaicin (but not the lower concentrations) reduced the abundance of LGG (but not L34) implying a higher capsaicin tolerance of L34. However, Lactobacillus rhamnosus fecal abundance, using qRT-PCR, of L34 or LGG after 3, 7, and 20 days of the administration in the Thai healthy volunteers demonstrated the similarity between both strains. In conclusion, high dose chili extracts impaired gut permeability and induced gut dysbiosis but were attenuated by probiotics. Despite a better capsaicin tolerance of L34 compared with LGG in vitro, L34 abundance in feces was not different to LGG in the healthy volunteers. More studies on probiotics with a higher intake of chili in human are interesting.

Pathogen-Associated Molecules from Gut Translocation Enhance Severity of Cecal Ligation and Puncture Sepsis in Iron-Overload ß-Thalassemia Mice.

INTRODUCTION: Systemic inflammation induced by gut translocation of lipopolysaccharide (LPS), a major component of Gram-negative bacteria, in thalassemia with iron-overload worsens sepsis. However, the impact of (1→3)-ß-D-glucan (BG), a major fungal molecule, in iron-overload thalassemia is still unclear. Hence, the influence of BG was explored in 1) iron-overload mice with sepsis induced by cecal ligation and puncture (CLP) surgery; and 2) in bone marrow-derived macrophages (BMMs). METHODS: The heterozygous ß-globin-deficient mice, Hbbth3/+ mice, were used as representative thalassemia (TH) mice. Iron overload was generated by 6 months of oral iron administration before CLP surgery- induced sepsis in TH mice and wild-type (WT) mice. Additionally, BMMs from both mouse strains were used to explore the impact of BG. RESULTS: Without sepsis, iron-overload TH mice demonstrated more severe intestinal mucosal injury (gut leakage) with higher LPS and BG in serum, from gut translocation, when compared with WT mice. With CLP in iron-overload mice, sepsis severity in TH mice was more severe than WT as determined by survival analysis, organ injury (kidney and liver), bacteremia, endotoxemia, gut leakage (FITC-dextran) and serum BG. Activation by LPS plus BG (LPS+BG) in BMMs and in peripheral blood-derived neutrophils (both WT and TH cells) demonstrated more prominent cytokine production when compared with LPS activation alone. In parallel, LPS+BG also prominently induced genes expression of M1 macrophage polarization (iNOS, TNF-α and IL-1ß) in both WT and TH cells in comparison with LPS activation alone. In addition, LPS+BG activated macrophage cytokine production was enhanced by a high dose of ferric ion (800 mM), more predominantly in TH macrophages compared with WT cells. Moreover, LPS+BG induced higher glycolysis activity with similar respiratory capacity in RAW264.7 (a macrophage cell line) compared with LPS activation alone. These data support an additive pro-inflammatory effect of BG upon LPS. CONCLUSION: The enhanced-severity of sepsis in iron-overload TH mice was due to 1) increased LPS and BG in serum from iron-induced gut-mucosal injury; and 2) the pro-inflammatory amplification by ferric ion on LPS+BG activation.

Dibromopinocembrin and Dibromopinostrobin Are Potential Anti-Dengue Leads with Mild Animal Toxicity.

Dengue infection is one of the most deleterious public health concerns for two-billion world population being at risk. Plasma leakage, hemorrhage, and shock in severe cases were caused by immunological derangement from secondary heterotypic infection. Flavanone, commonly found in medicinal plants, previously showed potential as anti-dengue inhibitors for its direct antiviral effects and suppressing the pro-inflammatory cytokine from dengue immunopathogenesis. Here, we chemically modified flavanones, pinocembrin and pinostrobin, by halogenation and characterized them as potential dengue 2 inhibitors and performed toxicity tests in human-derived cells and in vivo animal model. Dibromopinocembrin and dibromopinostrobin inhibited dengue serotype 2 at the EC50s of 2.0640 ± 0.7537 and 5.8567 ± 0.5074 µM with at the CC50s of 67.2082 ± 0.9731 and >100 µM, respectively. Both of the compounds also showed minimal toxicity against adult C57BL/6 mice assessed by ALT and Cr levels in day one, three, and eight post-intravenous administration. Computational studies suggested the potential target be likely the NS5 methyltransferase at SAM-binding pocket. Taken together, these two brominated flavanones are potential leads for further drug discovery investigation.

The cooperation of pharmacologic-dose ascorbate with ceftriaxone against Staphylococcus aureus through bactericidal synergy and enhanced macrophage killing activity.

BACKGROUND: Ascorbate is a low-cost compound with a known bactericidal-synergy to antibitics. However, the synergy depends on concentrations and organisms. Thus, the synergy test by time-kill assay might be appropriate for the screening of the synergy. OBJECTIVE: We aimed to test the adjuvant property of ascorbate with ceftriaxone, a frequently prescribed ß-lactam antibiotic. METHOD: Ascorbate was tested with several bacteria from the American Type Culture Collection (ATCC) including Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli for i) bactericidal property of ascorbate, alone or with ceftriaxone-combination, by time-kill assay, ii) an influence on the killing-activity of bone -marrow-derived macrophage and iii) the attenuation of myositis mouse model. RESULT: The bactericidal synergy (determined with time-kill assay at 24 h) against S. aureus, but not other selected bacteria, was demonstrated in ascorbate (10 and 40 mM) plus ceftriaxone at the minimal inhibitory concentration (1x MIC). Ascorbate alone, without antibiotic, enhanced macrophage killing-activity and directly eliminated bacteria at the concentration 10-40mM and 250mM, respectively (both properties presented against S. aureus and P. aeruginosa, but not other bacteria). Ascorbate with ceftriaxone also reduced bacterial burdens in muscle and serum cytokines of S. aureus -myositis mouse model. Moreover, the synergy against the clinical isolated methicillin resistant S. aureus (MRSA) by time-kill assay and myositis model also presented. CONCLUSION: Ascorbate-ceftriaxone synergy against S. aureus was demonstrated by time-kill assay and myositis model. Time-kill assy might be valuable as a screening test to select the patients that potentially benefit from ascorbate- ceftriaxone adjuvant therapy.