Effects of lactoferrin on collagen gel contractile activity and myosin light chain phosphorylation in human fibroblasts

AbstractWhen fibroblasts are plated on a type I collagen gel they reduce the size of the gel and the extent of collagen gel contraction reflects the motile activity of the fibroblasts. We found that both bovine and human lactoferrin (Lf) enhanced the collagen gel contractile activity of WI-38 human fibroblasts. Rho inhibitor (exoenzyme C3), Rho kinase inhibitor (Y-27632), myosin light chain kinase inhibitor (ML-7), MEK inhibitor (PD98059) and Src family tyrosine kinase inhibitor inhibited the Lf-enhanced collagen gel contraction. Treatment of fibroblasts with Lf induced the phosphorylation of myosin light chain (MLC) within 30 min. Lf-enhanced MLC phosphorylation was inhibited by Y-27632 and ML-7. These results suggest that Lf promotes the motility of fibroblasts by regulating MLC phosphorylation

The effect of GG administration on the number of CXCR5-positive ILFs in the murine small intestine.

<p>After 2 weeks of GG administration (1 × 10⁹ CFU/day) or PBS (n = 5 in each group), the small intestines were divided into four equal segments, from proximal to distal (A–D), labeled with anti-CXCR5 antibody and HRP-conjugated anti-rabbit antibody, and visualized with DAB. The CXCR5-positive ILFs in each intestinal fragment were counted under a stereomicroscope.</p

The distinct effects of orally administered <i>Lactobacillus rhamnosus</i> GG and <i>Lactococcus lactis</i> subsp. <i>lactis</i> C59 on gene expression in the murine small intestine

<div><p>The molecular mechanisms of strain-specific probiotic effects and the impact of the oral administration of probiotic strains on the host’s gene expression are not yet well understood. The aim of this study was to investigate the strain-specific effects of probiotic strain intake on gene expression in the murine small intestine. Two distinct strains of lactic acid bacteria, <i>Lactobacillus rhamnosu</i>s GG (GG) and <i>Lactococcus lactis</i> subsp. <i>lactis</i> C59 (C59), were orally administered to BALB/c mice, daily for 2 weeks. The total RNA was isolated from the upper (including the duodenum) and lower (the terminal ileum) small intestine, and gene expression was assessed by microarray analysis. The data revealed (1) oral administration of C59 and GG markedly down-regulated the expression of genes encoding fibrinogen subunits and plasminogen in the upper small intestine; (2) administration of more than 1 × 10⁷ CFU/day of GG changed the gene expression of the host ileum. (3) strain- and dose-related effects on various GO biological processes; and (4) enrichment for B cell-related Gene Ontology terms among up-regulated genes in the terminal ileum of mice administered the 1 × 10⁹ CFU/day of GG. The distinct effects of GG and C59 on gene expression in the intact small intestine provide clues to understand how the health beneficial effects of specific strains of probiotic bacteria are mediated by interactions with intestinal cells.</p></div

Principal component analysis of gene expression of the terminal ileum after the administration of various doses of C59 and GG.

<p>A. PCA plot using PC1 and PC3 gene expression data quantified by RMA. Each dot indicates control (Δ), C59-10^7 (○), C59-10^8 (⊗), C59-10^9 (●), GG-10^7 (□), GG-10^8 (⊠), and GG-10^9 (■) (n = 5 in each group). B, Plot of component loadings of PC1 and PC3 for all genes. Gene with high PC values were numbered: 1, <i>Hist1h2ao/Hist1h2ap</i>; 2, <i>C430003N24Rik</i>; 3, <i>Ms4a1</i>; 4, <i>Glycam</i>; 5, <i>Faim3</i>; 6, <i>Ighv14-2</i>; 7, <i>Cr2</i>,; 8, <i>Serpina1a</i>; 9, <i>P2ry10</i>; 10. <i>Mndal/Ifi205/Ifi204/Ifi211</i>; 11, <i>Ighg/Ighg2b</i>; 12, <i>Hmgb1l/Hmgb1</i>; 13, <i>Arfgef3</i>; 14, <i>Igkv6-20</i>; 15, <i>2810043O03Rik</i>; 16, <i>B830007D08Rik</i>; 17, <i>Slc6a14</i>; 18, <i>Retnlb</i>; 19. <i>Saa1</i>; and 20, <i>Car1</i>.</p

Outline of the animal experiments.

<p>(A) Experiment 1. The mice were divided into three groups (control, C59, and GG groups; <i>n</i> = 6 per group) and administered 0.2 ml of PBS (control) or LAB suspensions (C59 and GG groups) for 2 weeks. (B) Experiment 2. The mice were divided into seven groups (control, GG-10^7, GG-10^8, GG-10^9, C59-10^7, C59-10^8, and C59-10^9 groups; <i>n</i> = 6 per group per experiment) and was performed in duplicate (round-1 and round-2). Round 2 (n = 3 per group) started a week after the start of the round 1 (n = 3 per group).</p

Number of differentially expressed genes (DEGs, FDR < 0.05) in the terminal ileum of mice administered different doses of C59 and GG.

<p>Number of differentially expressed genes (DEGs, FDR < 0.05) in the terminal ileum of mice administered different doses of C59 and GG.</p

The effect of GG administration on the number of CXCR5-positive ILFs in the murine small intestine.

<p>After 2 weeks of GG administration (1 × 10⁹ CFU/day) or PBS (n = 5 in each group), the small intestines were divided into four equal segments, from proximal to distal (A–D), labeled with anti-CXCR5 antibody and HRP-conjugated anti-rabbit antibody, and visualized with DAB. The CXCR5-positive ILFs in each intestinal fragment were counted under a stereomicroscope.</p

PANTHER Overrepresentation Test of gene expression in upper small intestine and terminal ileum of mice administered C59 and GG.

<p>PANTHER Overrepresentation Test of gene expression in upper small intestine and terminal ileum of mice administered C59 and GG.</p

PANTHER Overrepresentation Test of gene expression in terminal ileum of mice administered GG and C59.

<p>PANTHER Overrepresentation Test of gene expression in terminal ileum of mice administered GG and C59.</p