Glycyrrhizin protects against porcine endotoxemia through modulation of systemic inflammatory response

Abstract

Introduction: Glycyrrhizin (GL) was recently found to suppress high-mobility group box 1 (HMGB1)-induced injury by binding directly to it. However, the effect of GL on HMGB1 expression in endotoxemia as well as its underlying molecular mechanism remained unclear.

Methods: Twenty-one pigs were divided into four groups: sham group (n=3), control group (n=6), ethyl pyruvate group (n=6) and glycyrrhizin group (n=6). Pigs were anesthetized, mechanically ventilated, monitored and given a continuous intravenous infusion of lipopolysaccharide (LPS). Twelve hours after the start of the LPS infusion, ethyl pyruvate (30 mg/kg/hr) or glycyrrhizin (1 mg/kg/hr) was administered for 12 hours. Systemic and pulmonary hemodynamics, oxygen exchange, and metabolic status were measured. The concentrations of cytokines in serum and the corresponding gene and protein expressions in tissue samples from liver, lungs, kidneys, small intestine and lymph nodes were measured.

Results: GL maintained the stability of systemic hemodynamics and improved pulmonary oxygen exchange and metabolic status. GL also attenuated organ injury and decreased the serum levels of HMGB1 and other pro-inflammatory cytokines by inhibiting their gene and protein expression.

Conclusions: GL improved systemic hemodynamics and protected vital organs against porcine endotoxemia through modulation of the systemic inflammatory response. By reducing the serum level and gene expression of HMGB1 and other pro-inflammatory cytokines, GL may become a potential agent for the treatment of sepsis.

Figure 1

Experimental design. After stabilization, LPS was infused to pigs for 24 hours and after 12 hours, the EP and GL groups were treated with the corresponding drug. Systemic and pulmonary hemodynamics, gas exchange and metabolic status were measured and blood samples collected at 6, 12, 18 and 24 hours. EP, ethyl pyruvate; GL, glycyrrhizin; LPS, lipopolysaccharide.

Figure 2

Organ injury in each group [hematoxylin and eosin (H&E), yellow bar = 100 μm, white bar = 50 μm]. Lung: A marked infiltration of inflammatory cells and hemorrhage were seen in the control group. The EP and GL groups had slighter leukocyte infiltration and almost no hemorrhage. Liver: The control group was marked by interstitial edema. The EP group had a similar change but to a lesser extent. Kidney: The control group had significant interstitial edema and capillary congestion. The EP and GL groups had slighter edema and almost no capillary congestion. Intestine: Severe edema was observed in the small intestine of the control group. The EP and GL groups had similar change but to a lesser extent. EP, ethyl pyruvate; GL, glycyrrhizin.

Figure 3

mRNA expression of ICAM-1, VCAM-1 and PBEF and protein expression of ICAM-1 and VCAM-1 in lung. A) Real-time PCR showed that the expression levels of ICAM-1, VCAM-1 and PBEF mRNA decreased significantly in the EP and GL groups compared to the control group. B) Western-blot showed that the expression levels of ICAM-1 and VCAM-1 protein decreased significantly in the EP and GL groups compared to the control group. C) Quantitative assessment of protein relative to β-actin showed that the expression levels of ICAM-1 and VCAM-1 protein decreased significantly in the EP and GL groups compared to the control group. * P < 0.05 versus the control group. EP, ethyl pyruvate; GL, glycyrrhizin; ICAM-1, intercellular adhesion molecule-1; PBEF, pre-B-cell colony-enhancing factor; VCAM-1, vascular cell adhesion molecule 1.

Figure 4

Concentration of diamine oxidase and D-lactate in plasma. Diamine oxidase and D-lactate in plasma increased in the control group and decreased after EP or GL was used. *P < 0.05 versus sham group by repeated measures ANOVA; # P < 0.05 versus con group by repeated measures ANOVA. ANOVA, analysis of variance; con, control; EP, ethyl pyruvate; GL, glycyrrhizin.

Figure 5

Concentration of plasma cytokine. *P < 0.05 versus sham group by repeated measures ANOVA; # P < 0.05 versus con group by repeated measures ANOVA. ANOVA, analysis of variance; con, control.

Figure 6

mRNA expression of HMGB1, NF-κB p65, IL-6 and IL-10. Realtime PCR showed the expression levels of HMGB1, NF-κB p65 (NF-κB), IL-6 and IL-10 mRNA in lung, liver, kidney, intestine and lymph node. * P < 0.05 versus control group. HMBG1, high-mobility group box 1; NK-κB, nuclear factor-kappaB.

Figure 7

Protein expression of HMGB1, NF-κB p65, IL-6 and IL-10. A) Western-blot showed the expression levels of HMGB1, NF-κB p65 (NF-κB), IL-6 and IL-10 in lung, liver, kidney, intestine and lymph node. The expression of NF-κB p65 was detected using nuclear protein while others were detected using total protein. B) Quantitative assessment of protein relative to β-actin. * P < 0.05 versus control group. HMBG1, high-mobility group box 1; NK-κB, nuclear factor-kappaB.