Figure 1.
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.
Figure 2.
In vitro effects of DPG on gene/protein expression of pro-inflammatory cytokines, IL-6, TNF-alpha and IL-1beta.
RAW267.4 cell line was treated for: (a) 4 hours with LPS, DPG and B-box. Cytokine mRNA expression was analysed by Quantitative real-time PCR. Data represent the target gene expression normalized to the reference gene. The error bars represent sd. (b) 48 hours with LPS and DPG. Cytokine mRNA expression was analysed by Quantitative real-time PCR. (c) 48 hours with LPS and DPG. Cytokine presence in the culture medium was analysed 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 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<0.05; p<*<0.01;**p<0.001.
Figure 3.
DPG reduces DSS-induced colitis in mice.
DSS-treated C57BL/6 mice were administered with two different doses (3 and 8 mg/Kg) of DPG for 7 days. Following parameters were analysed: (a) weight loss; (b) total clinical score (stool consistency, presence of blood in stool and general appearance) (c, d) colon length (e) colon weight (f) morphology of the colon evaluated through histological analysis (40xmagnification in the image) (g) histological score. Values are mean ± sd of three independent experiments. UN, untreated animals; DSS, dextran sulfate sodium; DPG, dipotassium glycyrrhizate. *p<0.01; **p<0.001.
Figure 4.
DPG reduces HMGB1 translocation from the nucleus to the cytoplasm in murine inflamed colonic tissues.
Nuclear and cytoplasmic protein fractionation was performed on a subset of murine colonic samples; (a) HMGB1 protein expression assessed by Western blot. Anti-β actin was used as cytoplasmic protein control. Densitometric analysis is showed. Values are the mean ± sd of three independent experiments. (b) HMGB1 intracellular localization was evaluated through histochemical staining. Staining control is showed in the box. Arrows indicate secreted HMGB1. UN, untreated animals; DSS, dextran sulfate sodium; DPG, dipotassium glycyrrhizate. *p<0.01; **p<0.001.
Figure 5.
DPG reduces mRNA expression of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, as well as HMGB1 receptors, TLR4 and RAGE, in inflamed tissues of DSS-treated mice.
Quantitative real-time PCR. Data represent the target gene expression normalized to the reference gene. Values are mean ± sd of three independent experiments. UN, untreated animals; DSS, dextran sulfate sodium; DPG, dipotassium glycyrrhizate. **p<0.001.
Figure 6.
Fecal HMGB1 is strongly reduced by DPG in mice with DSS-induced colitis.
Western blot of HMGB1 in stool specimens of mice. Comassie Blue staining has been used as loading control. Densitometric analysis is showed. Values are the mean ± sd of three independent experiments. UN, untreated animals; DSS, dextran sulfate sodium; DPG, dipotassium glycyrrhizate. **p<0.001.