Temporal evolution of <i>Clostridium</i> groups and <i>Enterobacteriaceae</i> counts and their ratio in control and CCl₄-treated mice.

<p>(A, B, C) Bacterial counts from samples of cecum content were measured by quantitative real-time PCR and represented as <i>Log</i> cell/g intestinal content. Values shown as mean ± standard deviation are represented for control (n = 4/week) and treated mice (n = 6/week) along the 16 weeks of study. (D) Differences in the <i>Enterobacteriaceae/Clostridia</i> ratio are represented as mean ± standard deviation for control (n = 4/week) and treated mice (n = 6/week) along the 16 weeks of study.</p

Fibrosis grade at laparotomy weeks along the study protocol in CCl4-treated mice.

<p>(A) Example of histological changes in CCl4-treated mice along the 16 weeks of study. Hepatic sections stained with Masson Trichrome (original magnification ×10), showing light to strong blue staining, as a reflection of the collagen deposition at different stages of the liver disease: no histopathological changes (week 0); focal mild portal fibrosis with short fibrous septa (week 6); moderate collagen fibber deposition in portal areas with occasional portal bridging (week 10); marked fibrosis in the majority of portal spaces with frequent portal-portal and portal-central bridging and architectural distortion (regenerative nodules) (weeks 13 and 16). (B) Fibrosis grade according to Ishak Scale in treated animals (n = 6/week) at different study weeks are represented as mean ± standard deviation. (C) Relative gene expression levels of different profibrogenic genes at different study weeks. MMP-2: Matrix metalloproteinase 2; TGF-β. Tumour growth factor beta; ProCol-1: Procollagen alpha-1(1); TIMP-1: Tissue inhibitor of metalloproteinase 1. Mean values of CCl4-treated mice (n = 6/week) are represented.</p

Gut microbiota changes during the study protocol in control and CCl₄-treated mice.

<p> <i>Bacterial counts: Log cells/g intestinal content:</i></p><p>**<i> p<0.05 compared with weekly control mice</i>.</p

Characteristics of animals at laparotomies.

<p>*<i>p<0,05 compared with the weekly control group.</i></p

List of primer pairs used in the study.

<p>List of primer pairs used in the study.</p

Bacterial DNA translocation at laparotomies in control and treated mice.

<p>*<i>p<0,05 compared with the weekly control group; MLNs: mesenteric lymph nodes</i>.</p

Temporal pattern of key events related with development of bacterial DNA translocation in CCl₄-treated mice.

<p>The lowest levels of cytokine production, PMN butyrate specific-receptor GPR43 and <i>Clostridium</i> clusters counts from samples of intestinal cecum content along with the highest degrees of liver fibrosis, gut dysbiosis and bacterial translocation observed in treated animals are chronologically represented during the 16 weeks of study protocol.</p

Beta-Adrenergic Receptor 1 Selective Antagonism Inhibits Norepinephrine-Mediated TNF-Alpha Downregulation in Experimental Liver Cirrhosis

<div><h3>Background</h3><p>Bacterial translocation is a frequent event in cirrhosis leading to an increased inflammatory response. Splanchnic adrenergic system hyperactivation has been related with increased bacterial translocation. We aim at evaluating the interacting mechanism between hepatic norepinephrine and inflammation during liver damage in the presence of bacterial-DNA.</p> <h3>Animals and Methods</h3><p>Forty-six mice were included in a 16-week protocol of CCl₄-induced cirrhosis. Laparotomies were performed at weeks 6, 10, 13 and 16. A second set of forty mice injected with a single intraperitoneal dose of CCl₄ was treated with saline, 6-hydroxidopamine, Nebivolol or Butoxamine. After 5 days, mice received <em>E. coli</em>-DNA intraperitoneally. Laparotomies were performed 24 hours later. Liver bacterial-DNA, norepinephrine, TNF-alpha, IL-6 and beta-adrenergic receptor levels were measured.</p> <h3>Results</h3><p>Bacterial-DNA translocation was more frequent in CCl₄-treated animals compared with controls, and increased as fibrosis progressed. Liver norepinephrine and pro-inflammatory cytokines were significantly higher in mice with <em>vs</em> without bacterial-DNA (319.7±120.6 vs 120.7±68.6 pg/g for norepinephrine, 38.4±6.1 vs 29.7±4.2 pg/g for TNF-alpha, 41.8±7.4 vs 28.7±4.3 pg/g for IL-6). Only beta-adrenergic receptor-1 was significantly increased in treated <em>vs</em> control animals (34.6±7.3 <em>vs</em> 12.5±5.3, p = 0.01) and correlated with TNF-alpha, IL-6 and norepinephrine hepatic levels in animals with bacterial-DNA. In the second set of mice, cytokine levels were increased in 6-hydroxidopamine and Nebivolol (beta-adrenergic receptor-1 antagonist) treated mice compared with saline. Butoxamine (beta-adrenergic receptor-2 antagonist) didn’t inhibit liver norepinephrine modulation of pro-inflammatory cytokines.</p> <h3>Conclusions</h3><p>Beta-adrenergic receptor-1 mediates liver norepinephrine modulation of the pro-inflammatory response in CCl₄-treated mice with bacterial-DNA.</p> </div

Interaction between inflammatory and sympathetic activities in the liver.

<p>Correlations between ADRB1 levels and NE, TNF-alpha and IL-6 levels in the liver of CCl₄-treated mice according to bactDNA translocation. r: Spearman’s rank correlation coefficient in bactDNA+ mice; NE: norepinephrine; ADRB1: beta-1 adrenergic receptor; bactDNA: bacterial DNA.</p

Liver NE, cytokine and ADRB levels in control and CCl4-treated mice in basal and protocol Stages.

*<p> <i>p<0,05 compared with basal and the stage control group;</i></p>$<p> <i>p<0,05 compared with CCl4-mice in Stage 1</i></p><p> <i>Stage 1: liver bactDNA≤25%; Stage 2: liver bactDNA>65%; ADRB: beta-adrenergic receptor</i></p