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Dipotassium Glycyrrhizate Inhibits HMGB1-Dependent Inflammation and Ameliorates Colitis in Mice.

Vitali R, Palone F, Cucchiara S, Negroni A, Cavone L, Costanzo M, Aloi M, Dilillo A, Stronati L - PLoS ONE (2013)

Bottom Line: DPG significantly reduces in vitro the release of HMGB1 in the extracellular matrix as well as expression levels of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, by inhibiting HMGB1.Murine colonic samples show decreased mRNA levels of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6, as well as HMGB1 receptors, RAGE and TLR4.DPG exerts inhibitory effects against HMGB1 activity, significantly reducing intestinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiobiology and Human Health, ENEA, Rome, Italy.

ABSTRACT

Background: High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. Advances in targeting HMGB1 represent a major challenge to improve the treatment of acute/chronic inflammation.

Aim: This study is aimed at verifying whether the inhibition of HMGB1 through dipotassium glycyrrhizate (DPG) is a good strategy to reduce intestinal inflammation.

Methods: Human colon adenocarcinoma cell line, HT29, human epithelial colorectal adenocarcinoma, Caco2, and murine macrophage cell line, RAW 264.7, were cultured to investigate the effect of DPG on the secretion of HMGB1. Acute colitis was induced in C57BL/6 mice through administration of 3% dextran sodium sulphate (DSS); a combined treatment with DSS and 3 or 8 mg/kg/day DPG was used to investigate the effects of DPG on intestinal inflammation. Animals were euthanized at seventh day and colonic samples underwent molecular and histological analyses.

Results: DPG significantly reduces in vitro the release of HMGB1 in the extracellular matrix as well as expression levels of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, by inhibiting HMGB1. Moreover, DPG significantly decreases the severity of DSS-induced colitis in mice. Murine colonic samples show decreased mRNA levels of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6, as well as HMGB1 receptors, RAGE and TLR4. Finally, HMGB1, abundantly present in the feces of mice with DSS-induced colitis, is strongly reduced by DPG.

Conclusions: HMGB1 is an early pro-inflammatory cytokine and an active protagonist of mucosal gut inflammation. DPG exerts inhibitory effects against HMGB1 activity, significantly reducing intestinal inflammation. Thus, we reason that DPG could represent an innovative tool for the management of human intestinal inflammation.

No MeSH data available.


Related in: MedlinePlus

In vitro effects of DPG on gene/protein expression and extracellular level of HMGB1.RAW267.4 cell line was treated for 4 hours with LPS, DPG and B-box (a) Quantitative real-time PCR analysis. Data represent the target gene expression normalized to the reference gene; (b) western blot assay; (c) RAW267.4, HT29 and Caco2 cell lines were treated for 24–48 hours with LPS, DPG; extracellular HMGB1 was then analysed in the culture medium by western blot. Densitometric analysis is showed. Values (optical density units, O.D.) are the mean ± sd, of three independent experiments, referred to the optical density values of untreated samples. UN, untreated samples; LPS, lipopolysaccharide; DPG, dipotassium glycyrrhizate; B-box, recombinant truncated form of HMGB1 consisting of the B box domain. *p<0.01.
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pone-0066527-g001: In vitro effects of DPG on gene/protein expression and extracellular level of HMGB1.RAW267.4 cell line was treated for 4 hours with LPS, DPG and B-box (a) Quantitative real-time PCR analysis. Data represent the target gene expression normalized to the reference gene; (b) western blot assay; (c) RAW267.4, HT29 and Caco2 cell lines were treated for 24–48 hours with LPS, DPG; extracellular HMGB1 was then analysed in the culture medium by western blot. Densitometric analysis is showed. Values (optical density units, O.D.) are the mean ± sd, of three independent experiments, referred to the optical density values of untreated samples. UN, untreated samples; LPS, lipopolysaccharide; DPG, dipotassium glycyrrhizate; B-box, recombinant truncated form of HMGB1 consisting of the B box domain. *p<0.01.

Mentions: HMGB1 carries out primary functions in the nucleus, where it is abundantly expressed in almost all eukaryotic cells. It is known that, when the inflammation is triggered, HMGB1 leaves the nucleus and, through the cytoplasm, is secreted in the extracellular matrix. We performed a pilot in vitro experiment, by using the murine macrophage cell line, RAW 264.7, to investigate the effect of DPG on the expression of HMGB1. First we evaluated the effect of DPG on HMGB1 gene/protein expression. To trigger inflammation, cells were exposed for 4 hours to LPS or to HMGB1 B box (B-box), which is the recombinant truncated form of the full protein consisting of the pro-inflammatory component, the B box domain, (1 and 10 µg/ml), in presence or absence of DPG. As expected, gene/protein expression levels of cellular HMGB1 did not change in response to LPS nor to DPG, due to the physiological large amount of the protein inside the cell (Fig. 1a, 1b). Then, to evaluate the effect of DPG on HMGB1 secretion, we exposed cells for 24 and 48 hours to LPS, in presence or absence of DPG. As a main source of HMGB1 in vivo are epithelial cells, we performed these experiments on two other cell lines, the human intestinal colorectal adenocarcinoma cell line, Caco2, and the human colon carcinoma cell line HT29, in addition to the macrophage RAW 264.7. Results obtained were the same for all the three cell lines used. We observed a significant increase of the secreted HMGB1 in the culture medium at both exposure times (p<0.01); however, this amount was significantly reduced in presence of DPG (p<0.01) at 48 hours in RAW264.7 and at both exposure times in Caco2 and HT29, demonstrating the ability of DPG to influence the release of HMGB1 (Fig. 1c).


Dipotassium Glycyrrhizate Inhibits HMGB1-Dependent Inflammation and Ameliorates Colitis in Mice.

Vitali R, Palone F, Cucchiara S, Negroni A, Cavone L, Costanzo M, Aloi M, Dilillo A, Stronati L - PLoS ONE (2013)

In vitro effects of DPG on gene/protein expression and extracellular level of HMGB1.RAW267.4 cell line was treated for 4 hours with LPS, DPG and B-box (a) Quantitative real-time PCR analysis. Data represent the target gene expression normalized to the reference gene; (b) western blot assay; (c) RAW267.4, HT29 and Caco2 cell lines were treated for 24–48 hours with LPS, DPG; extracellular HMGB1 was then analysed in the culture medium by western blot. Densitometric analysis is showed. Values (optical density units, O.D.) are the mean ± sd, of three independent experiments, referred to the optical density values of untreated samples. UN, untreated samples; LPS, lipopolysaccharide; DPG, dipotassium glycyrrhizate; B-box, recombinant truncated form of HMGB1 consisting of the B box domain. *p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3686690&req=5

pone-0066527-g001: In vitro effects of DPG on gene/protein expression and extracellular level of HMGB1.RAW267.4 cell line was treated for 4 hours with LPS, DPG and B-box (a) Quantitative real-time PCR analysis. Data represent the target gene expression normalized to the reference gene; (b) western blot assay; (c) RAW267.4, HT29 and Caco2 cell lines were treated for 24–48 hours with LPS, DPG; extracellular HMGB1 was then analysed in the culture medium by western blot. Densitometric analysis is showed. Values (optical density units, O.D.) are the mean ± sd, of three independent experiments, referred to the optical density values of untreated samples. UN, untreated samples; LPS, lipopolysaccharide; DPG, dipotassium glycyrrhizate; B-box, recombinant truncated form of HMGB1 consisting of the B box domain. *p<0.01.
Mentions: HMGB1 carries out primary functions in the nucleus, where it is abundantly expressed in almost all eukaryotic cells. It is known that, when the inflammation is triggered, HMGB1 leaves the nucleus and, through the cytoplasm, is secreted in the extracellular matrix. We performed a pilot in vitro experiment, by using the murine macrophage cell line, RAW 264.7, to investigate the effect of DPG on the expression of HMGB1. First we evaluated the effect of DPG on HMGB1 gene/protein expression. To trigger inflammation, cells were exposed for 4 hours to LPS or to HMGB1 B box (B-box), which is the recombinant truncated form of the full protein consisting of the pro-inflammatory component, the B box domain, (1 and 10 µg/ml), in presence or absence of DPG. As expected, gene/protein expression levels of cellular HMGB1 did not change in response to LPS nor to DPG, due to the physiological large amount of the protein inside the cell (Fig. 1a, 1b). Then, to evaluate the effect of DPG on HMGB1 secretion, we exposed cells for 24 and 48 hours to LPS, in presence or absence of DPG. As a main source of HMGB1 in vivo are epithelial cells, we performed these experiments on two other cell lines, the human intestinal colorectal adenocarcinoma cell line, Caco2, and the human colon carcinoma cell line HT29, in addition to the macrophage RAW 264.7. Results obtained were the same for all the three cell lines used. We observed a significant increase of the secreted HMGB1 in the culture medium at both exposure times (p<0.01); however, this amount was significantly reduced in presence of DPG (p<0.01) at 48 hours in RAW264.7 and at both exposure times in Caco2 and HT29, demonstrating the ability of DPG to influence the release of HMGB1 (Fig. 1c).

Bottom Line: DPG significantly reduces in vitro the release of HMGB1 in the extracellular matrix as well as expression levels of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, by inhibiting HMGB1.Murine colonic samples show decreased mRNA levels of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6, as well as HMGB1 receptors, RAGE and TLR4.DPG exerts inhibitory effects against HMGB1 activity, significantly reducing intestinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiobiology and Human Health, ENEA, Rome, Italy.

ABSTRACT

Background: High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. Advances in targeting HMGB1 represent a major challenge to improve the treatment of acute/chronic inflammation.

Aim: This study is aimed at verifying whether the inhibition of HMGB1 through dipotassium glycyrrhizate (DPG) is a good strategy to reduce intestinal inflammation.

Methods: Human colon adenocarcinoma cell line, HT29, human epithelial colorectal adenocarcinoma, Caco2, and murine macrophage cell line, RAW 264.7, were cultured to investigate the effect of DPG on the secretion of HMGB1. Acute colitis was induced in C57BL/6 mice through administration of 3% dextran sodium sulphate (DSS); a combined treatment with DSS and 3 or 8 mg/kg/day DPG was used to investigate the effects of DPG on intestinal inflammation. Animals were euthanized at seventh day and colonic samples underwent molecular and histological analyses.

Results: DPG significantly reduces in vitro the release of HMGB1 in the extracellular matrix as well as expression levels of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, by inhibiting HMGB1. Moreover, DPG significantly decreases the severity of DSS-induced colitis in mice. Murine colonic samples show decreased mRNA levels of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6, as well as HMGB1 receptors, RAGE and TLR4. Finally, HMGB1, abundantly present in the feces of mice with DSS-induced colitis, is strongly reduced by DPG.

Conclusions: HMGB1 is an early pro-inflammatory cytokine and an active protagonist of mucosal gut inflammation. DPG exerts inhibitory effects against HMGB1 activity, significantly reducing intestinal inflammation. Thus, we reason that DPG could represent an innovative tool for the management of human intestinal inflammation.

No MeSH data available.


Related in: MedlinePlus