Korean J Gastroenterol.  2012 Aug;60(2):86-93. 10.4166/kjg.2012.60.2.86.

Anti-inflammatory Mechanism of Lactobacillus rhamnosus GG in Lipopolysaccharide-stimulated HT-29 Cell

• Lee SK
• Yang KM
• Cheon JH
• Kim TI
• Kim WH

• KMID: 1387336
• DOI: http://doi.org/10.4166/kjg.2012.60.2.86

Abstract

BACKGROUND/AIMS
Probiotics are live non-pathogenic organisms that belong to the resident microflora, and confer health benefits by multiple mechanisms. Lactobacillus rhamnosus GG (LGG) is one of the probiotic bacteria that ameliorates intestinal injury and inflammation caused by various stimuli. We aimed to evaluate the anti-inflammatory effect and mechanism of LGG in lipopolysaccharide (LPS)-stimulated HT-29 cells.
METHODS
HT-29 cells were stimulated with interleukin (IL)-1beta (2 ng/mL), tumor necrosis factor (TNF)-alpha (20 ng/mL), and LPS (20 microg/mL) in the presence or absence of LGG (107-109 colony forming units/mL). Production of the pro-inflammatory chemokine IL-8 was measured by ELISA and semi-quantitative PCR. Transcriptional activity of NF-kappaB-responsive gene was evaluated by luciferase assay with reporter gene. Toll-like receptor 4 (TLR4) mRNA expression was assessed by semi-quantitative PCR. The IkappaBalpha degradation was evaluated by western blot and intranuclear translocation of NF-kappaB was determined by western blot and immunofluorescence.
RESULTS
LGG did not affect the viability of HT-29 cells. Pretreatment of HT-29 cells with LGG significantly blocked TNF-alpha, and LPS induced IL-8 activation at both mRNA and protein level (p<0.05). Pretreatment of HT-29 cells with LGG attenuated LPS-induced NF-kappaB nuclear translocation and also blocked LPS-induced IkappaBalpha degradation. LGG also down-regulated TLR4 mRNA activated by LPS.
CONCLUSIONS
LGG attenuates LPS induced inflammation, and this may be associated with TLR4/NF-kappaB down-regulation.

Keyword

Lactobacillus rhamnosus; Lipopolysaccharides; NF-kappaB

MeSH Terms

Cell Survival/drug effects
Down-Regulation/drug effects
HT29 Cells
Humans
I-kappa B Proteins/metabolism
Interleukin-1beta/metabolism
Interleukin-8/genetics/metabolism
Lactobacillus rhamnosus/*metabolism
Lipopolysaccharides/toxicity
NF-kappa B/metabolism
Probiotics/pharmacology
Toll-Like Receptor 4/genetics/metabolism
Tumor Necrosis Factor-alpha/genetics/metabolism

Figure

• 

  Fig. 1 Lactobacillus rhamnosus GG (LGG) did not affect the viability of HT-29 cell. HT-29 cells were seeded at the density of 1×104 cells/well in 6-well plates and maintained in the medium with 10% FBS for 24 hours. (A) LGG were added to the HT-29 cell culture wells at the appropriated dilution to reach a final concentration of 105, 106, 107, 108, 109, and 1010 colony forming units (CFU) per mL of the incubation medium without antibiotics. After 4 hours incubation, cell viability was determined by MTT assay. (B) The 109 CFU/mL concentration of LGG were added to HT-29 cell culture well and incubated with various time intervals (6, 12, 18, and 24 hours). Data are the mean of triplicate assays and represent the relative viability compared to untreated controls.

• 

  Fig. 2 Attenuation of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, or lipopolysaccharide (LPS)-mediated suppression of IL-8 expression by Lactobacillus rhamnosus GG (LGG) in HT-29 cells. (A) HT-29 cells were pre-incubated with LGG (1×109 colony forming units [CFU]/well) for 1 hour before treatment with 20 ng/mL of TNF-α, 2 ng/mL of IL-1β, and 20 µg/mL of LPS. Supernatant were harvested for IL-8 ELISA. (B) HT-29 cells were pre-incubated with different concentration of LGG (1×107, 1×108, and 1×109 CFU/mL) for 1 hour before treatment with 20 µg/mL of LPS. Supernatant were harvested for IL-8 ELISA. (C) Cells were harvested for RT-PCR analysis of IL-8 mRNA expression. GAPDH expression was used as control. The increases in the percentages of IL-8 mRNA expression were quantified by densitometric analysis. *p<0.05, **p<0.01 in Mann-Whitney U test.

• 

  Fig. 3 Attenuation of lipopolysaccharide (LPS)-mediated induction of TLR-4 expression by Lactobacillus rhamnosus GG (LGG) in HT-29 cells. HT-29 cells were pre-incubated with different concentration of LGG (1×107, 1×108, and 1×109 CFU/well) for 1 hour before treatment with 20 ng/mL of tumor necrosis factor (TNF)-α, 2 ng/mL of interleukin (IL)-1β, and 20 µg/mL of LPS. Cells were harvested for RT-PCR analysis of toll-like receptor 4 (TLR4) mRNA expression. GAPDH expression was used as control. The increases in the percentages of TLR4 mRNA expression were quantified by densitometric analysis.

• 

  Fig. 4 Effect of Lactobacillus rhamnosus GG (LGG) on NF-κB transcriptional activation induced by lipopolysaccharide (LPS). (A) SW480 cells were cotransfected with an HIV-1 long-terminal repeat luciferase construct containing NF-κB binding sites and pCMV β-gal plasmid. pCMV β-gal served as a marker of transfection efficiency. Cotransfected cells were stimulated with LPS (20 µg/mL) with or without pretreatment of LGG (1×109 colony forming units [CFU]/mL), and NF-κB-dependent luciferase activity was measured 4 hours after stimulation. Data represent the mean with SEM and are expressed as fold increase over the media control cells. Results are expressed as means of triplicate determinations and are representative of three independent experiments. *p<0.05 compared with LPS-stimulated cells without LGG. (B) HT-29 cells were lysed at different times (15, 30, 60 and 120 minutes) after LPS (20 µg/mL) stimulation with or without pretreatment of LGG (1×109 CFU/mL). Nuclear and cytoplasmic fractions were separately prepared for Western blot. Samples were resolved by SDS-PAGE and analyzed by Western blotting with anti-NFκB/p65, anti-IκBα antibody and an anti-β-actin antibody for control. (C) HT-29 cell were plated in 4-chamber slides, grown to 70% confluence, and pre-treated with or without LGG (1×109 CFU/mL). LPS (20 µg/mL) was added to the medium and incubation continued for 4 hours. Fluorescein isothiocyanate (FITC) conjugated secondary antibody and 4',6-diamidino-2-phenylindole (DAPI) was used for immunofluorescence. DAPI staining served to visualize the nucleus (×500). These results are representative of three independent experiments.

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