Glycyrrhizin ameliorating sterile inflammation induced by low-dose radiation exposure

Abstract

Glycyrrhizin (GL) is a direct inhibitor of HMGB1 which acts as an alarmin when excreted into the extracellular space. High-dose radiation in radiotherapy induces collateral damage to the normal tissue, which can be mitigated by GL inhibiting HMGB1. The purpose of this study was to assess changes in HMGB1 and pro-inflammatory cytokines and to evaluate the protective effect of GL after low-dose radiation exposure. BALB/c mice were irradiated with 0.1 Gy (n = 10) and 1 Gy (n = 10) with GL being administered to half of the mice (n = 5, respectively) before irradiation. Blood and spleen samples were harvested and assessed for oxidative stress, HMGB1, pro-inflammatory cytokines, and cell viability. HMGB1 and pro-inflammatory cytokines increased and cell viability decreased after irradiation in a dose-dependent manner. Oxidative stress also increased after irradiation, but did not differ between 0.1 Gy and 1 Gy. With the pretreatment of GL, oxidative stress, HMGB1, and all of the pro-inflammatory cytokines decreased while cell viability was preserved. Our findings indicate that even low-dose radiation can induce sterile inflammation by increasing serum HMGB1 and pro-inflammatory cytokines and that GL can ameliorate the sterile inflammatory process by inhibiting HMGB1 to preserve cell viability.

Figure 1

Oxidative stress measured by the MDA assay according to radiation dose. (A) The graph shows the normalized levels of MDA for the IR with/without GL groups compared to the control group. Oxidative stress was significantly higher in the IR only groups compared to the controls, whereas it was attenuated in the IR + GL groups. (B) ROS fluorescence was measured using flow cytometry at 24 h. It significantly higher in the IR only groups compared to the positive controls, whereas it was lower in the IR + GL groups. Measeured values normalized to the positive controls (= H₂DCFDA stained cells). Results are expressed as means ± SEM from duplicate experiments (n = 5) performed in each group. The asterisk (*) indicates statistically significant differences between the control and IR groups (both IR only and IR + GL). The hash sign (#) indicates significant differences between the IR only groups and IR + GL groups. *p < 0.05, ***p < 0.001 #P < 0.05 and ###p < 0.001. Abbreviation: IR irradiation, GL glycyrrhizin, MDA malonaldehyde acid, ROS reactive oxidative species, NC negative control, PC positive control.

Figure 2

mRNA expression (A) and serum protein levels (B) of HMGB1 according to irradiation dose. Both the mRNA expression (A) and serum protein levels (B) of HMGB1 were significantly higher in the IR only groups (both in 0.1 Gy and 1 Gy) compared to the control group, whereas, they were significantly lower in the IR + GL groups compared to the IR only groups. Results are expressed as means ± SEM from duplicate experiments (n = 5) performed for each group. The asterisk (*) indicates statistically significant differences between the control and IR groups (both IR only and IR + GL). The hash sign (#) indicates significant differences between the IR only groups and IR + GL groups. ***p < 0.001, ###p < 0.001. IR irradiation, GL glycyrrhizin.

Figure 3

The mRNA expression of (A) TNF-α, (B) IL-6, (C) IL-1α, and (D) IL-1β according to radiation exposure. The mRNA expressions of pro-inflammatory cytokines were higher in the IR only groups compared to the controls with/without statistical significance, whereas it was significantly lower in the IR + GL groups than the IR only groups. Results are expressed as means ± SEM from duplicate experiments (n = 5) performed in each group. The asterisk (*) indicates significant differences between the control and IR groups (both IR only and IR + GL). The hash sign (#) indicates significant differences between the IR only groups and IR + GL groups. ***p < 0.001, ##p < 0.01, ###p < 0.001. IR irradiation, GL glycyrrhizin.

Figure 4

Cell viability of the isolated splenocytes according to radiation dose. Cell viabilities were significantly lower in the IR only groups (both in 0.1 Gy and 1 Gy) compared to the control group, whereas they were significantly higher in the IR + GL groups compared to the IR only groups. Results are expressed as means ± SEM from duplicate experiments for each group. The asterisk (*) indicates significant differences between the control and IR groups (both of the untreated and GL treated groups). The hash sign (#) indicates significant differences between the IR groups and IR + GL groups. **p < 0.01, ##p < 0.01, #p < 0.05. IR irradiation, GL glycyrrhizin.

Figure 5

The mRNA expression of HMGB1 and pro-inflammatory cytokines in isolated splenocytes according to radiation dose. The mRNA expression levels of HMGB1 and pro-inflammatory cytokines were higher in the IR only group except for IL-1β (both in 0.1 Gy and 1 Gy) compared to the control group. With glycyrrhizin pretreatment, the mRNA expression levels of HMGB1 and pro-inflammatory cytokines were lower than IR only groups with and without statistical significance. Results are expressed as means ± SEM from duplicate experiments performed for each group. The asterisk (*) indicates statistically significant differences between the control and IR groups (both IR only and IR + GL). The hash sign (#) indicates significant differences between the IR only and IR + GL groups. *p < 0.05, #p < 0.05.

Figure 6

Scheme of the animal experiments. (A) Thirty mice were divided into 5 groups which were the control (n = 10), 0.1 Gy IR only (n = 5), 0.1 Gy IR + GL (n = 5), 1 Gy IR only (n = 5) and 1 Gy IR + GL (n = 5) group. Except for the control group, all mice received whole body irradiation with a total single dose of 0.1 Gy or 1 Gy (300 kV, 12.5 mA). GL was administrated intraperitoneally before 2 h of irradiation. All mice were sacrificed after 24 h of radiation exposure and blood samples were collected and spleen tissue was harvested. (B) To assess cell viability and mRNA expression , the radiation-exposed splenocytes were incubated for 24 h after being isolated from the mice (no-irradiation). These samples were divided into the five groups (Control, 0.1 Gy IR only, 0.1 Gy IR + GL, 1 Gy IR only, and 1 Gy IR + GL) as well. GL was applied 2 h before radiation exposure for the 0.1 Gy IR + GL and 1 Gy IR + GL groups. IR irradiation, GL glycyrrhizin, N/S normal saline.