M1 cholinergic signaling in the brain modulates cytokine levels and splenic cell sub-phenotypes following cecal ligation and puncture.

BACKGROUND: The contribution of the central nervous system to sepsis pathobiology is incompletely understood. In previous studies, administration of endotoxin to mice decreased activity of the vagus anti-inflammatory reflex. Treatment with the centrally-acting M1 muscarinic acetylcholine (ACh) receptor (M1AChR) attenuated this endotoxin-mediated change. We hypothesize that decreased M1AChR-mediated activity contributes to inflammation following cecal ligation and puncture (CLP), a mouse model of sepsis. METHODS: In male C57Bl/6 mice, we quantified basal forebrain cholinergic activity (immunostaining), hippocampal neuronal activity, serum cytokine/chemokine levels (ELISA) and splenic cell subtypes (flow cytometry) at baseline, following CLP and following CLP in mice also treated with the M1AChR agonist xanomeline. RESULTS: At 48 h. post-CLP, activity in basal forebrain cells expressing choline acetyltransferase (ChAT) was half of that observed at baseline. Lower activity was also noted in the hippocampus, which contains projections from ChAT-expressing basal forebrain neurons. Serum levels of TNFα, IL-1ß, MIP-1α, IL-6, KC and G-CSF were higher post-CLP than at baseline. Post-CLP numbers of splenic macrophages and inflammatory monocytes, TNFα+ and ILß+ neutrophils and ILß+ monocytes were higher than baseline while numbers of central Dendritic Cells (cDCs), CD4+ and CD8+ T cells were lower. When, following CLP, mice were treated with xanomeline activity in basal forebrain ChAT-expressing neurons and in the hippocampus was significantly higher than in untreated animals. Post-CLP serum concentrations of TNFα, IL-1ß, and MIP-1α, but not of IL-6, KC and G-CSF, were significantly lower in xanomeline-treated mice than in untreated mice. Post-CLP numbers of splenic neutrophils, macrophages, inflammatory monocytes and TNFα+ neutrophils also were lower in xanomeline-treated mice than in untreated animals. Percentages of IL-1ß+ neutrophils, IL-1ß+ monocytes, cDCs, CD4+ T cells and CD8+ T cells were similar in xanomeline-treated and untreated post-CLP mice. CONCLUSION: Our findings indicate that M1AChR-mediated responses modulate CLP-induced alterations in serum levels of some, but not all, cytokines/chemokines and affected splenic immune response phenotypes.

Evaluating the effects of tannic acid on rabbit growth performance, digestibility, antioxidant status, intestinal morphology and caecal fermentation and microbiota.

The objective of this study was to assess the effects of dietary supplementation with tannic acid (TA) on the growth performance, digestibility, antioxidant status, intestinal morphology and the caecal fermentation and microbiota in rabbits. A total number of 120 Ira rabbits (30 days of age) were randomly allotted to four dietary treatment groups: TA 0 (control), TA 0.75, TA 1.5 and TA 3, administered basal diets with 0, 0.75, 1.5 and 3 g TA/kg of feed for 28 days. Compared to the control group, dietary 3 g TA/kg inclusion decreased the average daily feed intake (p < 0.05). No significant differences were found in the digestibility among the groups (p > 0.05). Serum total antioxidant capacity was significantly higher in the 3 g/kg TA group than in the other groups (p < 0.05). There was a significant increase in the concentration of propionic acid and butyric acid in the 3 g/kg TA group. The addition of TA had no effect on villus height and crypt depth of small intestine (p > 0.05). The 16S rRNA high-throughput sequencing results showed that at the phylum level, dietary 3 g/kg TA increased the abundance of Bacteroidetes in the caecum of rabbits (p < 0.05). Based on the results, dietary TA is effective in antioxidant capacity of rabbits, improving caecal fermentation and optimizing the caecal microflora. However, the appropriate dosage supplementation of TA in rabbits needs further research.

Single-cell profiling of the pig cecum at various developmental stages.

The gastrointestinal tract is essential for food digestion, nutrient absorption, waste elimination, and microbial defense. Single-cell transcriptome profiling of the intestinal tract has greatly enriched our understanding of cellular diversity, functional heterogeneity, and their importance in intestinal tract development and disease. Although such profiling has been extensively conducted in humans and mice, the single-cell gene expression landscape of the pig cecum remains unexplored. Here, single-cell RNA sequencing was performed on 45 572 cells obtained from seven cecal samples in pigs at four different developmental stages (days (D) 30, 42, 150, and 730). Analysis revealed 12 major cell types and 38 subtypes, as well as their distinctive genes, transcription factors, and regulons, many of which were conserved in humans. An increase in the relative proportions of CD8 + T and Granzyme A (low expression) natural killer T cells (GZMA low NKT) cells and a decrease in the relative proportions of epithelial stem cells, Tregs, RHEX + T cells, and plasmacytoid dendritic cells (pDCs) were noted across the developmental stages. Moreover, the post-weaning period exhibited an up-regulation in mitochondrial genes, COX2 and ND2, as well as genes involved in immune activation in multiple cell types. Cell-cell crosstalk analysis indicated that IBP6 + fibroblasts were the main signal senders at D30, whereas IBP6 - fibroblasts assumed this role at the other stages. NKT cells established interactions with epithelial cells and IBP6 + fibroblasts in the D730 cecum through mediation of GZMA-F2RL1/F2RL2 pairs. This study provides valuable insights into cellular heterogeneity and function in the pig cecum at different development stages.

An in vitro model for caecal proteolytic fermentation potential of ingredients in broilers.

Fermentation of protein in the caeca of chickens may lead to the production of potentially detrimental metabolites, which can reduce gut health. A poor precaecal digestion is expected to increase protein fermentation (PF), as more proteins are likely to enter the caeca. It is unknown if the undigested protein that enters the caeca differs in fermentability depending on their ingredient source. In order to predict which feed ingredients increase the risk of PF, an in vitro procedure was developed, which simulates the gastric and enteric digestion, subsequent caecal fermentation. After digestion, amino acids and peptides smaller than 3.5 kD in the soluble fraction were removed by means of dialysis. These amino acids and peptides are assumed to be hydrolysed and absorbed in the small intestine of poultry and therefore not used in the fermentation assay. The remaining soluble and fine digesta fractions were inoculated with caecal microbes. In chicken, the soluble and fine fractions enter the caeca, to be fermented, while insoluble and coarse fractions bypass them. The inoculum was made N-free to ensure bacteria would require the N from the digesta fractions for their growth and activity. The gas production (GP) from the inoculum, therefore, reflected the ability of bacteria to use N from substrates and was an indirect measure for PF. The Maximum GP rate of ingredients averaged 21.3 ± 0.9 ml/h (mean ± SEM) and was in some cases more rapid than the positive control (urea, maximum GP rate = 16.5 ml/h). Only small differences in GP kinetics were found between protein ingredients. Branched-chain fatty acids and ammonia concentrations in the fermentation fluid after 24 hours showed no differences between ingredients. Results indicate that solubilised undigested proteins larger than 3.5 kD are rapidly fermented independent of its source when an equal amount of N is present.

Downregulation of the gene expression of Cyp2c29 and Cyp3a11 by cecal ligation and puncture-induced sepsis is associated with interleukin-6.

Sepsis is a pathological condition that affects the metabolism of administered drugs, leading to changes in the duration and intensity of their intended efficacies. Proinflammatory cytokines downregulate the expression of cytochrome P450s (P450s). The effects of P450 expression under inflammatory conditions have been studied using prophlogistic substances such as lipopolysaccharide; however, few studies have focused on clinical models of sepsis. Here, we show that cecal ligation and puncture (CLP), an approach for the study of human polymicrobial sepsis, leads to the expression of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNFα) at 24 h after the CLP operation. Following CLP, IL-6-/- mice exhibited markedly lower survival than WT mice. In addition, CLP led to the significant downregulation of Cyp2c29 and Cyp3a11 gene expression in IL-1α-/-/ß-/- (IL-1-/-) and TNFα-/- mice as well as in WT mice. In contrast, CLP elicited no significant effect on Cyp3a11 expression in IL-6-/- mice. Although CLP reduced the Cyp2c29 expression level in IL-6-/- mice, the expression of Cyp2c29 was lower in CLP-operated WT mice than in CLP-operated IL-6-/- mice. The reduction in the respective P450 protein levels and activities due to CLP-induced sepsis, reflected in the mRNA expression levels, was abolished by IL-6 depletion. Thus, CLP-induced sepsis downregulates P450 gene expression, particularly Cyp2c expression, and this effect is associated with IL-6 without affecting resistance to CLP-induced sepsis. These findings demonstrate the usefulness of CLP for studying the regulation of P450s and highlight IL-6 as a potential indicator of drug-metabolizing capacity under septic conditions.

Th17-Related Cytokines Involved in Fluoride-Induced Cecal and Rectal Barrier Damage of Ovariectomized Rats.

To investigate fluoride (F)-induced intestine barrier damage and the role of estrogen deficiency in this progress, a rat model of estrogen deficiency was established through bilateral surgical removal of ovaries. The F exposure model was then continued by adding sodium fluoride (0, 25, 50, and 100 mg/L, calculated on a fluorine ion basis) to drinking water for 90 days. Afterward, intestinal mucosal structure, barrier function, and inflammatory cytokines were evaluated. The results showed that excessive F decreased the developmental parameters (crypt depth) of the cecum and rectum and inhibited the proliferation capacity of the intestinal epithelia, which are more obvious in the state of estrogen deficiency. The distribution of goblet cells and glycoproteins in the intestinal mucosa decreased with the increase in F concentration, and estrogen deficiency led to a further decline, especially in the rectum. Using the immunofluorescence method, the study showed that excessive F caused interleukin-17A (IL-17A) significantly decrease in the cecum and increase in the rectum. Meanwhile, F treatment remarkably upregulated the expression of intestinal IL-1ß, IL-23, and IL-22, while the level of IL-6 was downregulated. In addition, estrogen deficiency increased IL-1ß, IL-6, IL-23, and IL-22, but decreased IL-17A expression in the cecum and rectum. Collectively, F exposure damaged intestinal morphological structure, inhibited epithelial cell proliferation and mucus barrier function, and resulted in the disturbance of T helper (Th) 17 cell-related cytokines expression. Estrogen deficiency may further aggravate F-induced damage to the cecum and rectum.

Oxalate secretion is stimulated by a cAMP-dependent pathway in the mouse cecum.

Elevated levels of the intracellular second messenger cAMP can stimulate intestinal oxalate secretion however the membrane transporters responsible are unclear. Oxalate transport by the chloride/bicarbonate (Cl-/HCO3-) exchanger Slc26a6 or PAT-1 (Putative Anion Transporter 1), is regulated via cAMP when expressed in Xenopus oocytes and cultured cells but whether this translates to the native epithelia is unknown. This study investigated the regulation of oxalate transport by the mouse intestine focusing on transport at the apical membrane hypothesizing PAT-1 is the target of a cAMP-dependent signaling pathway. Adopting the Ussing chamber technique we measured unidirectional 14C-oxalate and 36Cl- flux ([Formula: see text] and [Formula: see text]) across distal ileum, cecum and distal colon, employing forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) to trigger cAMP production. FSK/IBMX initiated a robust secretory response by all segments but the stimulation of net oxalate secretion was confined to the cecum only involving activation of [Formula: see text] and distinct from net Cl- secretion produced by inhibiting [Formula: see text]. Using the PAT-1 knockout (KO) mouse we determined cAMP-stimulated [Formula: see text] was not directly dependent on PAT-1, but it was sensitive to mucosal DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid), although unlikely to be another Cl-/HCO3- exchanger given the lack of trans-stimulation or cis-inhibition by luminal Cl- or HCO3-. The cAMP-activated oxalate efflux was reliant on CFTR (Cystic Fibrosis Transmembrane conductance Regulator) activity, but only in the presence of PAT-1, leading to speculation on the involvement of a multi-transporter regulatory complex. Further investigations at the cellular and molecular level are necessary to define the mechanism and transporter(s) responsible.

A rare case of poorly differentiated mixed neuroendocrine-nonneuroendocrine tumor of the caecum with long term survival: A case report.

A 59-year-old woman presented with flushing attacks accompanied by tachycardia and hypotension, which lasted approximately 30 to 60 minutes, underwent 18 years ago a gastrointestinal tumor resection. The histologic examination revealed a poorly differentiated mixed neuroendocrine/adenocarcinoma located in the caecum with regional metastases. Postoperatively, the patient received combined chemotherapy of 5-fluorouracil with interferon for six months and since has remained asymptomatic. Her examination revealed positivity for chromogranin A (CgA) and a-Fetoprotein (aFP) (580 ng/24 h, normal range 27-94, and 10 IU/mL, normal range 0-6, respectively). Urinary 5-hydroxy indole acetic acid excretion was remarkably high (41.8 mg/24 h, normal range 2-10 mg/24 h). An abdominal Magnetic Resonance Imaging scan revealed multiple focal loci in the liver whose histological examination revealed a carcinoid tumor confirmed by an Octreoscan. Additional uptake was noted on the right shoulder and the right sternum-clavicle joint confirmed by Tc-99m MDP scan. The patient received somatostatin analogue therapy followed by long-acting release octreotide analogue therapy (30 mg/month) showing a partial improvement of relevant biomarkers. Two years later, carcinoid syndrome symptoms reappeared and due to the tumors expression of somatostatin receptors the patient received peptide receptor radionuclide therapy with 177Lu-DOTATATE that resulted in both clinical and biochemical improvements.

Grape seed alleviates lindane-induced oxidative stress and improves growth performance, caecal fermentation and antioxidant capacity in growing rabbits.

This study evaluated the protective effect of grape seed on performance, caecal characteristics, blood metabolites and liver antioxidant status in lindane-treated rabbits. Four-week-old New Zealand White rabbits (n = 96) with an initial body weight of 0.750 ± 0.23 g were randomly divided into four groups (n = 24). One group was the control received only corn oil orally, while group L were received lindane daily via gavage in corn oil (4 mg/kg BW; 1/50 LD50 for oral dose), group GS was treated with 50 g grape seed /kg diet, and group LGS treated with a combination of both L and GS for 98 days. Results revealed that final body weight (FBW), average daily gain (ADG), dry matter intake and feed efficiency (FE) were similar between GS and control groups, and achieved the highest FBW and ADG, and the best FE. Caecum pH of the L group increased, while the caecum pH of the GS group decreased sharply. There was a significant increase in the concentration of total VFA, acetic acid, propionic acid and NH3 -N in the GS group, but butyric acid level decreased. GS treatment resulted in a significant increase in the concentrations of total protein, albumin and AChE. GPx, GST, CAT and SOD activity decreased, but TBARS activity significantly increased in the group L, while GS caused a significant elevation of antioxidant activity in the liver. These results confirm that the antioxidant compounds present in grape seed can alleviate the oxidative stress caused by lindane-induced hepatotoxicity and could be a useful supplement to maintain health and improve performance in rabbits.

Contributory Role of BLT2 in the Production of Proinflammatory Cytokines in Cecal Ligation and Puncture-Induced Sepsis.

BLT2 is a low-affinity receptor for leukotriene B4, a potent lipid mediator of inflammation generated from arachidonic acid via the 5-lipoxygenase pathway. The aim of this study was to investigate whether BLT2 plays any role in sepsis, a systemic inflammatory response syndrome caused by infection. A murine model of cecal ligation and puncture (CLP)-induced sepsis was used to evaluate the role of BLT2 in septic inflammation. In the present study, we observed that the levels of ligands for BLT2 (LTB4 [leukotriene B4] and 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid]) were significantly increased in the peritoneal lavage fluid and serum from mice with CLP-induced sepsis. We also observed that the levels of BLT2 as well as 5-LO and 12-LO, which are synthesizing enzymes for LTB4 and 12(S)-HETE, were significantly increased in lung and liver tissues in the CLP mouse model. Blockade of BLT2 markedly suppressed the production of sepsis-associated cytokines (IL-6 [interleukin-6], TNF-α [tumor necrosis factor alpha], and IL-1ß [interleukin-1ß] as well as IL-17 [interleukin-17]) and alleviated lung inflammation in the CLP group. Taken together, our results suggest that BLT2 cascade contributes to lung inflammation in CLP-induced sepsis by mediating the production of inflammatory cytokines. These findings suggest that BLT2 may be a potential therapeutic target for sepsis patients.

Pharmacological Effects of Marine-Derived Enterococcus faecium EA9 against Acute Lung Injury and Inflammation in Cecal Ligated and Punctured Septic Rats.

Microorganisms obtained from the marine environment may represent a potential therapeutic value for multiple diseases. This study explored the possible protective role of marine-derived potential probiotic Enterococcus faecium EA9 (E. faecium) against pulmonary inflammation and oxidative stress using the cecal ligation and puncture (CLP) model of sepsis in Wistar rats. Animals were pretreated with E. faecium for 10 days before either sham or CLP surgeries. Animals were sacrificed 72 hours following the surgical intervention. The histological architecture of lung tissues was evaluated as indicated by the lung injury score. In addition, the extend of pulmonary edema was determined as wet/dry weight ratio. The inflammatory cytokines were estimated in lung tissues, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1ß) using the enzyme-linked-immunosorbent-assay (ELISA) technique. Moreover, markers for lipid peroxidation such as thiobarbituric acid reaction substances (TBARs), and endogenous antioxidants, including reduced glutathione (GSH) were determined in lung tissues. Finally, the enzymatic activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were assayed in the lungs. Pretreatment with E. faecium markedly attenuated CLP-induced lung injury and pulmonary edema. Markers for inflammation, including TNF-α, IL-6, and IL-1ß were augmented in the lung tissues of CLP animals, while E. faecium ameliorated their augmented levels. E. faecium pretreatment also restored the elevated TBARS levels and the prohibited CAT, SOD, and GPx enzymatic activities in CLP animals. GSH levels were corrected by E. faecium in CLP animals. The inflammatory and lipid peroxidation mediators were positively correlated, while antioxidant enzymatic activities were negatively correlated with CLP-induced lung injury and pulmonary edema. Collectively, marine-derived Enterococcus faecium EA9 might be considered as a prospective therapeutic tool for the management of pulmonary dysfunction associated with sepsis.

Interference on Cytosolic DNA Activation Attenuates Sepsis Severity: Experiments on Cyclic GMP-AMP Synthase (cGAS) Deficient Mice.

Although the enhanced responses against serum cell-free DNA (cfDNA) in cases of sepsis-a life-threatening organ dysfunction due to systemic infection-are understood, the influence of the cytosolic DNA receptor cGAS (cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase) on sepsis is still unclear. Here, experiments on cGAS deficient (cGAS-/-) mice were conducted using cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) injection sepsis models and macrophages. Severity of CLP in cGAS-/- mice was less severe than in wildtype (WT) mice, as indicated by mortality, serum LPS, cfDNA, leukopenia, cytokines (TNF-α, IL-6 and IL-10), organ histology (lung, liver and kidney) and spleen apoptosis. With the LPS injection model, serum cytokines in cGAS-/- mice were lower than in WT mice, despite the similar serum cfDNA level. Likewise, in LPS-activated WT macrophages, the expression of several mitochondria-associated genes (as revealed by RNA sequencing analysis) and a profound reduction in mitochondrial parameters, including maximal respiration (determined by extracellular flux analysis), DNA (mtDNA) and mitochondrial abundance (revealed by fluorescent staining), were demonstrated. These data implied the impact of cfDNA resulting from LPS-induced cell injury. In parallel, an additive effect of bacterial DNA on LPS, seen in comparison with LPS alone, was demonstrated in WT macrophages, but not in cGAS-/- cells, as indicated by supernatant cytokines (TNF-α and IL-6), M1 proinflammatory polarization (iNOS and IL-1ß), cGAS, IFN-γ and supernatant cyclic GMP-AMP (cGAMP). In conclusion, cGAS activation by cfDNA from hosts (especially mtDNA) and bacteria was found to induce an additive proinflammatory effect on LPS-activated macrophages which was perhaps responsible for the more pronounced sepsis hyperinflammation observed in WT mice compared with the cGAS-/- group.

LncRNA-Cox2 regulates macrophage polarization and inflammatory response through the CREB-C/EBPß signaling pathway in septic mice.

LncRNA-Cox2 has been reported to regulate macrophage polarization, and the activation of macrophages is a major participant in the pathogenesis of sepsis. Therefore, we explored whether lncRNA-Cox2 was involved in the progression of sepsis. In this study, we established a cecal ligation and puncture (CLP) mouse model and found that silencing lncRNA-Cox2 in CLP mice improved the 7-day survival rate, and alleviated the increase of blood bacterial burdens, systemic inflammatory response, and pulmonary dysfunction induced by CLP. Besides, interference with lncRNA-Cox2 declined the percentage of M1 macrophages and increased the percentage of M2 macrophages in the spleens of CLP mice. In vitro, the knockdown of lncRNA-Cox2 suppressed LPS-induced inflammation and M1 macrophage marker expression, and promoted M2 macrophage marker expression in primary peritoneal macrophages and RAW264.7 cells. Moreover, lncRNA-Cox2 induced CREB phosphorylation by binding to CREB, and increased phosphorylated-CREB enrichment in the C/EBPß promoter region, so as to promote C/EBPß transcription, thereby activating the CREB-C/EBPß cascade. In addition, overexpressing lncRNA-Cox2 enhanced the effect of LPS on inflammation and macrophage polarization, which was reversed by treatment with 666-15 (an inhibitor of CREB). In conclusion, silencing lncRNA-Cox2 restrained the progression of sepsis in mice by modulating macrophage polarization and inflammatory response through suppressing CREB-C/EBPß pathway.

Effects of Acute 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Exposure on the Circulating and Cecal Metabolome Profile.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a polyhalogenated planar hydrocarbon belonging to a group of highly toxic and persistent environmental contaminants known as "dioxins". TCDD is an animal teratogen and carcinogen that is well characterized for causing immunosuppression through activation of aryl hydrocarbon receptor (AHR). In this study, we investigated the effect of exposure of mice to an acute dose of TCDD on the metabolic profile within the serum and cecal contents to better define the effects of TCDD on host physiology. Our findings demonstrated that within the circulating metabolome following acute TCDD exposure, there was significant dysregulation in the metabolism of bioactive lipids, amino acids, and carbohydrates when compared with the vehicle (VEH)-treated mice. These widespread changes in metabolite abundance were identified to regulate host immunity via modulating nuclear factor-kappa B (NF-κB) and extracellular signal-regulated protein kinase (ERK1/2) activity and work as biomarkers for a variety of organ injuries and dysfunctions that follow TCDD exposure. Within the cecal content of mice exposed to TCDD, we were able to detect changes in inflammatory markers that regulate NF-κB, markers of injury-related inflammation, and changes in lysine degradation, nicotinamide metabolism, and butanoate metabolism, which collectively suggested an immediate suppression of broad-scale metabolic processes in the gastrointestinal tract. Collectively, these results demonstrate that acute TCDD exposure results in immediate irregularities in the circulating and intestinal metabolome, which likely contribute to TCDD toxicity and can be used as biomarkers for the early detection of individual exposure.

Tong­fu­li­fei decoction attenuates immunosuppression to protect the intestinal­mucosal barrier in sepsis by inhibiting the PD­1/PD­L1 signaling pathway.

The aim of the present study was to investigate the therapeutic effects of Tong­fu­li­fei (TFL) decoction on sepsis­induced injury to the intestinal mucosal barrier and the underlying mechanism. Cecal ligation and puncture (CLP) was used to establish a sepsis model in rats. The post­surgery death of the rats was recorded to calculate the survival rate. A 4­kD fluorescein isothiocyanate (FITC)­dextran assay was used to evaluate the intestinal permeability of the rats. The pathological state of the intestine tissues was detected by hematoxylin and eosin staining and the ultrastructural changes in the endometrium were evaluated by transmission electron microscopy. Enzyme­linked immunosorbent assay was used to determine the concentrations of interleukin (IL)­6 and tumor necrosis factor (TNF)­α in the intestinal tissues and cells. The expression levels of SHP­2 and PI3K were detected by reverse transcription­quantitative PCR and western blotting. Sorting by flow cytometry was used to obtain pure dendritic cells (DC), CD8+ T cells and natural killer cells. Western blotting was used to evaluate the expression levels of phosphorylated (p)­AKT and AKT. The results demonstrated that the significantly decreased survival rate caused by CLP surgery was elevated by glutamine (Gln) and TFL treatment. Intestinal permeability was increased by CLP, and greatly suppressed by Gln or TFL treatment. Histopathological changes in the intestinal tissues, such as thinner barrier and atrophied mucosa, and ultrastructure changes such as sharply decreased microvilli and mitochondria dropsy, were observed on sepsis animals; these effects were ameliorated by the introduction of Gln or TFL. The upregulation of SHP­2, PI3K and p­AKT induced by CLP was reversed by TFL. The release of IL­6 and TNF­α was elevated and the expression of SHP­2, PI3K and p­AKT was suppressed in the co­cultural system of DC cells and CD8+ T cells by TFL. Overall, TFL decoction may attenuate immunosuppression to protect intestinal mucosal barrier in sepsis via inhibiting the programmed death1/programmed cell death ligand 1 signal pathway.

Ulinastatin protects against sepsis­induced myocardial injury by inhibiting NLRP3 inflammasome activation.

Myocardial injury is the primary manifestation of multiple organ dysfunction during sepsis, however, the mechanisms underlying sepsis­induced myocardial injury remain unclear. Similarly, no effective therapeutics have yet been developed for myocardial injury. In the present study, the role of the NOD­like receptor 3 (NLRP3) inflammasome on cardiac function were characterized and the effects of different ulinastatin (UTI) doses in protecting a septic rat model from myocardial injury were elucidated. To evaluate UTI efficacy on cardiac function, its effects on anti­inflammatory mediators were analyzed and its cardioprotective effects were investigated. It was demonstrated that circulatory levels of tumor necrosis factor­α and interleukin­1ß were elevated during sepsis. It was also observed that NLRP3 and caspase­1 expression enhanced post­cecal ligation and puncture (CLP), and that high UTI levels protected against myocardial injury induced by sepsis. To the best of our knowledge, this is the first study to demonstrate that the mechanisms underpinning UTI­mediated myocardial protection were due to the downregulation of the NLRP3/caspase­1/IL­1ß signaling pathway. Based on these findings, it is proposed that UTI exerts beneficial effects during sepsis­induced myocardial injury.

Discriminatory and cooperative effects within the mouse gut microbiota in response to flaxseed and its oil and lignan components.

Gut microbial processing of dietary flaxseed (FS) contributes to its health benefits, but the relative effects of its bioactive components (lignans, omega-3 fatty acids, fiber) on the microbiota are unclear. We investigated the gut microbial compositional and functional responses to whole FS and its isolated components, FS oil (FSO) and secoisolariciresinol diglucoside (SDG) (precursor to microbial-derived enterolignans) to help understand their contribution to whole FS benefits. Cecum content and fecal samples were collected from C57BL/6 female mice fed a basal diet (AIN93G) or isocaloric diets containing 10% FS or 10% FS-equivalent amounts of FSO or SDG for 21 days. Cecal and fecal microbiota composition and predicted genomic functions, and their relationship with serum enterolignans were evaluated. Only FS modified the community structure. Shared- and diet-specific enriched taxa and functions were identified. Carbohydrate and protein processing functions were enriched in FS mice, and there was a positive correlation between select enriched taxa, encompassing fiber degraders and SDG metabolizers, and serum enterolignans. This was not observed in mice receiving isolated FSO and SDG, suggesting that FS fiber supports SDG microbial metabolism. In conclusion, the cooperative activities of a diverse microbiota are necessary to process FS components and, when administered at the amount present in FS, these components may act together to affect SDG-derived enterolignans production. This has implications for the use of FS, FSO and SDG in clinical practice.

Antithrombin Together with NETs Inhibitor Protected Against Postoperative Adhesion Formation in Mice.

BACKGROUND/AIMS: Postoperative adhesions may induce adverse outcomes in patients. Adhesion formation is initiated by fibrin accumulation at the surgical site which is followed by local neutrophilia and the establishment of neutrophil extracellular traps (NET). Previous reports have suggested that the preventive efficacy of reagents designed to reduce postoperative adhesion is inversely correlated with neutrophilia and NET production. Antithrombin (AT) is a natural inhibitor of thrombin, a key factor in coagulation. Here, we evaluate whether treatment with AT and/or NET inhibitors prevent or reduce postoperative adhesion formation in mice. METHODS: Mice were treated with AT and/or NET inhibitors before and/or after cecum cauterization and their adhesion scores were evaluated on day 7 post-operation. Immunochemistry/ immunofluorescence analyses were also performed and we used GSK484, an inhibitor of peptidyl arginine deiminase 4 (PAD4), as the NET inhibitor. RESULTS: AT or GSK484 partially rescued postoperative adhesion formation in mice. AT prevented thrombin-induced plasminogen activator inhibitor 1 and interleukin-6 expression in mesothelial cells in vitro. However, AT could not prevent neutrophilia or NETs formation around the injured serosa. Finally, we investigated a combination of AT and a PAD4 inhibitor and found that this could inhibit almost all adhesion formation in these animals. Since AT-inactivating proteases are liberated following NET release, they might dampen the biological action of the AT treatment. This suggests that NET inhibitors might allow AT to exert its full action in the surgically injured serosa. CONCLUSION: Combined treatment with AT and GSK484 may effectively attenuate postoperative adhesion production in mice.

Depletion of the gut microbiota differentially affects the impact of whey protein on high-fat diet-induced obesity and intestinal permeability.

Whey protein isolate (WPI) is considered a dietary solution to obesity. However, the exact mechanism of WPI action is still poorly understood but is probably connected to its beneficial effect on energy balance, adiposity, and metabolism. More recently its ability to modulate the gut microbiota has received increasing attention. Here, we used a microbiota depletion, by antibiotic cocktail (ABX) administration, to investigate if the gut microbiota mediates the physiological and metabolic changes observed during high-fat diet (HFD)-WPI consumption. C57BL/6J mice received a HFD containing WPI (HFD-WPI) or the control non-whey milk protein casein (HFD-CAS) for 5 or 10 weeks. HFD-fed mice supplemented with WPI showed reduced body weight gain, adiposity, Ob gene expression level in the epidydimal adipose tissue (eWAT) and plasma leptin relative to HFD-CAS-fed mice, after 5- or 10-weeks intervention both with or without ABX treatment. Following 10-weeks intervention, ABX and WPI had an additive effect in lowering adiposity and leptin availability. HFD-WPI-fed mice showed a decrease in the expression of genes encoding pro-inflammatory markers (MCP-1, TNFα and CD68) within the ileum and eWAT, compared to HFD-CAS-fed mice, without showing alterations following microbiota depletion. Additionally, WPI supplementation decreased HFD-induced intestinal permeability disruption in the distal ileum; an effect that was reversed by chronic ABX treatment. In summary, WPI reverses the effects of HFD on metabolic and physiological functions through mainly microbiota-independent mechanisms. Moreover, we demonstrate a protective effect of WPI on HFD-induced inflammation and ileal permeability disruption, with the latter being reversed by gut microbiota depletion.

Pressurized intra-peritoneal aerosol chemotherapy (PIPAC): increased intraperitoneal pressure does not affect distribution patterns but leads to deeper penetration depth of doxorubicin in a sheep model.

BACKGROUND: Pressurized Intra-Peritoneal Aerosol Chemotherapy (PIPAC) is an innovative treatment against peritoneal carcinomatosis. Doxorubicin is a common intra-venous chemotherapy used for peritoneal carcinomatosis and for PIPAC. This study evaluated the impact of increased PIPAC intraperitoneal pressure on the distribution and cell penetration of doxorubicin in a sheep model. METHODS: Doxorubicin was aerosolized using PIPAC into the peritoneal cavity of 6 ewes (pre-alpes breed): N = 3 with 12 mmHg intraperitoneal pressure ("group 12") and N = 3 with 20 mmHg ("group 20"). Samples from peritoneum (N = 6), ovarian (N = 1), omentum (N = 1) and caecum (N = 1) were collected for each ewe. The number of doxorubicin positive cells was determined using the ratio between doxorubicine fluorescence-positive cell nuclei (DOXO+) over total number of DAPI positive cell nuclei (DAPI+). Penetration depth (µm) was defined as the distance between the luminal surface and the location of the deepest DOXO+ nuclei over the total number of cell nuclei that were stained with DAPI. Penetration depth (µm) was defined as the distance between the luminal surface and the location of the deepest DOXO+ nuclei. RESULTS: DOXO+ nuclei were identified in 87% of samples. All omental samples, directly localized in front of the nebulizer head, had 100% DOXO+ nuclei whereas very few nuclei were DOXO+ for caecum. Distribution patterns were not different between the two groups but penetration depth in ovary and caecum samples was significantly deeper in group 20. CONCLUSIONS: This study showed that applying a higher intra-peritoneal pressure during PIPAC treatment leads to a deeper penetration of doxorubicin in ovarian and caecum but does not affect distribution patterns.