Probiotics Prevent Intestinal Barrier Dysfunction in Acute Pancreatitis in Rats via Induction of Ileal Mucosal Glutathione Biosynthesis

BACKGROUND: During acute pancreatitis (AP), oxidative stress contributes to intestinal barrier failure. We studied actions of multispecies probiotics on barrier dysfunction and oxidative stress in experimental AP. METHODOLOGY/PRINCIPAL FINDINGS: Fifty-three male Spraque-Dawley rats were randomly allocated into five groups: 1) controls, non-operated, 2) sham-operated, 3) AP, 4) AP and probiotics and 5) AP and placebo. AP was induced by intraductal glycodeoxycholate infusion and intravenous cerulein (6 h). Daily probiotics or placebo were administered intragastrically, starting five days prior to AP. After cerulein infusion, ileal mucosa was collected for measurements of E. coli K12 and (51)Cr-EDTA passage in Ussing chambers. Tight junction proteins were investigated by confocal immunofluorescence imaging. Ileal mucosal apoptosis, lipid peroxidation, and glutathione levels were determined and glutamate-cysteine-ligase activity and expression were quantified. AP-induced barrier dysfunction was characterized by epithelial cell apoptosis and alterations of tight junction proteins (i.e. disruption of occludin and claudin-1 and up-regulation of claudin-2) and correlated with lipid peroxidation (r>0.8). Probiotic pre-treatment diminished the AP-induced increase in E. coli passage (probiotics 57.4+/-33.5 vs. placebo 223.7+/-93.7 a.u.; P<0.001), (51)Cr-EDTA flux (16.7+/-10.1 vs. 32.1+/-10.0 cm/s10(-6); P<0.005), apoptosis, lipid peroxidation (0.42+/-0.13 vs. 1.62+/-0.53 pmol MDA/mg protein; P<0.001), and prevented tight junction protein disruption. AP-induced decline in glutathione was not only prevented (14.33+/-1.47 vs. 8.82+/-1.30 nmol/mg protein, P<0.001), but probiotics even increased mucosal glutathione compared with sham rats (14.33+/-1.47 vs. 10.70+/-1.74 nmol/mg protein, P<0.001). Glutamate-cysteine-ligase activity, which is rate-limiting in glutathione biosynthesis, was enhanced in probiotic pre-treated animals (probiotics 2.88+/-1.21 vs. placebo 1.94+/-0.55 nmol/min/mg protein; P<0.05) coinciding with an increase in mRNA expression of glutamate-cysteine-ligase catalytic (GCLc) and modifier (GCLm) subunits. CONCLUSIONS: Probiotic pre-treatment diminished AP-induced intestinal barrier dysfunction and prevented oxidative stress via mechanisms mainly involving mucosal glutathione biosynthesis.Original Publication:Femke Lutgendorff, Rian M Nijmeijer, Per A Sandström, Lena M Trulsson, Karl-Eric Magnusson, Harro M Timmerman, L Paul van Minnen, Ger T Rijkers, Hein G Gooszen, Louis M A Akkermans and Johan D Söderholm, Probiotics prevent intestinal barrier dysfunction in acute pancreatitis in rats via induction of ileal mucosal glutathione biosynthesis., 2009, PLoS ONE, (4), 2, e4512.http://dx.doi.org/10.1371/journal.pone.0004512Licensee: Public Library of Science (PLoS)http://www.plos.org

Probiotics enhance pancreatic glutathione biosynthesis and reduce oxidative stress in experimental acute pancreatitis

Factors determining severity of acute pancreatitis (AP) are poorly understood. Oxidative stress causes acinar cell injury and contributes to the severity, whereas prophylactic probiotics ameliorate experimental pancreatitis. Our objective was to study how probiotics affect oxidative stress, inflammation, and acinar cell injury during the early phase of AP. Fifty-three male Sprague-Dawley rats were randomly allocated into groups: 1) control, 2) sham procedure, 3) AP with no treatment, 4) AP with probiotics, and 5) AP with placebo. AP was induced under general anesthesia by intraductal glycodeoxycholate infusion (15 mM) and intravenous cerulein (5 mu g . kg(-1) . h(-1), for 6 h). Daily probiotics or placebo were administered intragastrically, starting 5 days prior to AP. After cerulein infusion, pancreas samples were collected for analysis including lipid peroxidation, glutathione, glutamate-cysteine-ligase activity, histological grading of pancreatic injury, and NF-kappa B activation. The severity of pancreatic injury correlated to oxidative damage (r = 0.9) and was ameliorated by probiotics (1.5 vs. placebo 5.5; P = 0.014). AP-induced NF-kappa B activation was reduced by probiotics (0.20 vs. placebo 0.53 OD450(nm)/mg nuclear protein; P <0.001). Probiotics attenuated AP-induced lipid peroxidation (0.25 vs. placebo 0.51 pmol malondialdehyde/mg protein; P <0.001). Not only was AP-induced glutathione depletion prevented (8.81 vs. placebo 4.1 mu mol/mg protein, P <0.001), probiotic pretreatment even increased glutathione compared with sham rats (8.81 vs. sham 6.18 mu mol/mg protein, P <0.001). Biosynthesis of glutathione (glutamate-cysteine-ligase activity) was enhanced in probiotic-pretreated animals. Probiotics enhanced the biosynthesis of glutathione, which may have reduced activation of inflammation and acinar cell injury and ameliorated experimental AP, via a reduction in oxidative stress

http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-17082 Probiotics Prevent Intestinal Barrier Dysfunction in Acute Pancreatitis in Rats via Induction of Ileal Mucosal

Probiotics prevent intestinal barrier dysfunction in acute pancreatitis in rats via induction of ileal mucosal glutathione biosynthesis

Probiotics induce systemic increase in GSH levels and GCL activity.

<p>After 5 days of pre-treatment with placebo (pla, n = 12) or probiotics (pro, n = 12), rats were subjected to acute pancreatitis (AP, n = 12), a sham-procedure (n = 12) or not operated (control, n = 5). Whole blood was sampled 1) before treatment, 2) after 5 days of pre-treatment, immediately before induction of AP and 3) six hours after induction of AP or sham-procedure. Time course of plasma GSH levels (A) and GCL activity in red blood cells (B) was monitored. The graphs show average (±SD). All analyses were run in duplicates. Comparisons were performed using ANOVA followed by Tukey's HSD. *<i>P</i><0.001, probiotics <i>vs.</i> placebo. Associations between (C) ileal lipid peroxidation, (D) bacterial passage, (E) ⁵¹Cr-EDTA flux, (F) ileal GSH content six hours after induction of AP and GCL activity in red blood cells immediately before subjection to AP.</p

Probiotics prevented disruption of tight junction proteins.

<p>After 5 days of pre-treatment with placebo (pla) or probiotics (pro), rats were subjected to acute pancreatitis (AP), or a sham-procedure. Ileal sections were stained with occludin, claudin-1 or -2 antibodies (green), counterstained with DAPI (blue) and visualized by confocal laser scanning microscopy. Bar = 500 µm. The higher magnification (100×/1.30) images shown in the insets are typical details of crypts (left) and villi (right). Probiotics prevented the deleterious effects of AP on occludin and caused redistribution of occludin to the apical surface (arrowhead). Acute pancreatitis-induced detachment of epithelial cells seems to be preceded by loss of claudin-1 (arrowhead) and was reduced by probiotic pre-treatment; though probiotics could not prevent the AP-induced formation of aggregates of claudin-1 in the cytosol (arrowhead). Probiotics prevented AP-associated up-regulation of claudin-2 in both crypts and villi (arrowhead). The patterns of staining are typical of that seen in 4 sections of 4 rats per group.</p

Probiotics have no effect on cysteine availability, but induce glutamate-cysteine-ligase activity.

<p>After 5 days of pre-treatment with placebo (pla, n = 12) or probiotics (pro, n = 12), rats were subjected to acute pancreatitis (AP, n = 12), a sham-procedure (n = 12) or not operated (control, n = 5). Six hours after induction of the AP or sham-procedure, tissue cysteine availability (A) and mucosal glutamate-cysteine-ligase (GCL) activity (B) were determined in ileum samples. (C) RT-PCR was conducted on ileal mRNA. PCR products of specific primers for the catalytic (GCLc, 65 bp) and the modulatory (GCLm, 81 bp) subunit of GCL and for 18S rRNA (65 bp) as control were identified on 2.5% agarose gel, using a GeneRuler 50 bp DNA Ladder (Fermentas GMBH, St. Leon-Rot, Germany). mRNA expression of (D) GCLc and (E) GCLm were quantified. Data are normalized to 18S rRNA expression and expressed as ratio to control animals. The graphs show average (±SD). All analyses were run in triplicates. Comparisons were performed using ANOVA followed by Tukey's HSD.</p

Probiotics prevented acute pancreatitis-induced ileal permeability but not ion secretion.

<p>After 5 days of pre-treatment with placebo (pla, n = 12) or probiotics (pro, n = 12), rats were subjected to acute pancreatitis (AP, n = 12), a sham-procedure (n = 12) or not operated (control, n = 5). Ileal segments were mounted in Ussing chambers and (A) horseradish peroxidase (HRP) and (B) ⁵¹Cr-EDTA flux were studied for two hours. (C) Baseline conductance, (D) baseline short circuit current (Isc), (E) passage of <i>Escherichia coli</i> K12 and (F) elevation of conductance during one hour after challenge with <i>E. coli</i> were measured. The graphs show average (±SD). The data were collected from independently acquired sets of 3 tissue segments per rat. Comparisons were performed using ANOVA followed by Tukey's HSD.</p

Probiotics attenuated acute pancreatitis-associated mucosal damage.

<p>After 5 days of pre-treatment with placebo (pla, n = 12) or probiotics (pro, n = 12), rats were subjected to acute pancreatitis (AP, n = 12), or a sham-procedure (n = 12). (A) Sections of ileum were H&E stained and graded according to Chiu <i>et al.</i> (25). The graph shows median (±range). Comparisons were performed using Kruskal-Wallis followed by Mann–Whitney <i>U</i> test. (B) Compared with sham-operated animals (Sham), acute pancreatitis (AP) caused widespread destruction of villi. Placebo treated animals (AP pla) also showed a severe degree of mucosal damage. Probiotic animals (AP pro) showed extensive epithelial lifting, but with intact epithelium. The mucosal damage is typical of that seen in 4 sections from 12 rats per group. Bar = 200 µm. (C) Lipid peroxidation (MDA levels) (control n = 5, sham n = 12, AP n = 12, AP pla n = 12 and AP pro n = 12). The graph shows the average (±SD). Comparisons were performed using ANOVA followed by Tukey's HSD.</p

Probiotics reduced pancreatitis-associated intestinal apoptosis.

<p>After 5 days of pre-treatment with placebo or probiotics, rats were subjected to acute pancreatitis, or a sham-procedure. (A) Sections of ileum were TUNEL stained. The results shown are typical images from 4 sections of 4 rats per group. Bar = 200 µm. (B) DNA-fragmentation (control n = 5, sham n = 12, AP n = 12, AP pla n = 12 and AP pro n = 12). The graph shows the average (±SD). Comparisons were performed using ANOVA followed by Tukey's HSD. (C) Positive correlation between intestinal apoptosis and <i>Escherichia coli</i> K12 passage was computed using Spearman's rank correlation coefficients.</p