The animal experiments in the present study were approved by the Ethics Committee of the Third Xiangya Hospital, Central South University (Changsha, China). Male C57BL/6 mice (age, 12 weeks; weight, 20–25 g) were housed at Xiangya Medical Experimental Animal Center, Central South University in a laminar flow, temperature-controlled, pathogen-free environment under a 12 h light/dark cycle. The mice were fasted for 24 h prior to the experiments and provided with tap water ad libitum. During surgery, an intraperitoneal injection of 50 mg/kg pentobarbital was administered to anesthetize the mice. Bilateral flank incisions were made, the right kidney was removed and the left kidney was subjected to ischemia using a microvascular clamp, which was removed after 30 min and the wound was closed. To induce ischemia of the left kidney, a microvascular clip was used to clamp the renal artery after a transverse incision was made to the abdomen. Half an hour later, the clamp was removed, and the wound was closed. The mice were divided into three groups of six; in the sham-operated group, the mice underwent anesthesia, bilateral flank incisions and a right nephrectomy. In the glycyrrhizin-treated group, the mice were injected with 60 mg/kg glycyrrhizin using an infusion pump (Chizhou Kangyuan Medical Equipment Co., Ltd. Chizhou, China) 1 h prior to ischemia. In the saline-treated group, the mice were administered with 60 mg/kg saline. The mice were sacrificed by cervical dislocation under anesthesia 6 h after reperfusion and the blood and kidney samples were immediately collected.

Renal function was assessed via observation of serum creatinine (Cr) and blood urea nitrogen (BUN) levels at the Clinical Laboratory of the Third Xiangya Hospital, Central South University (Changsha, China).

An SOA assay kit (Sigma, St. Louis, MO, USA) was used to determine the level of SOA in the soluble fraction of renal tissue in accordance with the manufacturer’s instructions and the reaction was analyzed using a spectrophotometer (Shimadzu, Kyoto, Japan) at a wavelength of 550 nm.

The SOD activity was measured using the SOD activity assay kit (Biovision, Milpitas, CA, USA) based on the inhibition of adenochrome production by SOD during epinephrine auto-oxidation. Changes in fluorescence were read at a wavelength of 480 nm (Microplate Reader; Bio-Rad, Hercules, CA, USA).

Caspase-3 colorimetric assay kit (Biovision, San Francisco Bay Area, CA, USA) was used to determine the activity of caspase-3, according to the manufacturer’s instructions. Briefly, 20 μg kidney cytosolic protein was extracted and incubated in a solution buffer at room temperature for 30 min. The reaction was initiated by the addition of 200 μM N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin and incubated at 37°C for 2 h. The change in absorbance was measured spectrophotometrically at a wavelength of 400 nm.

MPO is a neutrophil-specific enzyme, the presence of which is considered to be an indicator of neutrophil infiltration in the kidney. Renal tissues were homogenized on ice in phosphate-buffered saline (PBS) with 0.5% hexadecyltrimethyl ammonium bromide (Sigma) and 0.146% EDTA (pH 6.0). The homogenates were centrifuged at 13,400 × g for 30 min at 4°C and the supernatant was incubated at 60°C for 2 h. Hydrogen peroxide (0.005%) and 0.167 mg/ml O-dianisidine dihydrochloride (Sigma) in PBS (pH 6.0) was added and the change in absorbance was measured spectrophotometrically at a wavelength of 460 nm.

Enzyme-linked immunosorbent assay kits (mouse TNF-α ELISA kit, Mouse IFN-γ ELISA kit, mouse IL-1β ELISA kit, mouse IL-6 ELISA kit; Sigma) were used to determine the serum levels of the key pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-1β and IL-6, in accordance with the manufacturer’s instructions.

Cytoplasmic extraction reagents (Pierce Biotechnology, Inc., Rockford, IL, USA) were used to extract the cytosolic proteins from the mice kidney tissues, in accordance with the manufacturer’s instructions. After determining the concentration of protein using the Enhanced BCA Protein Assay kit (Beyotime, Shanghai, China), 20 μg protein was separated using 10% SDS-PAGE, which was transferred to nitrocellulose membranes and maintained at room temperature for 1 h in a buffer solution containing 5% dried skimmed milk. The membrane was incubated at room temperature for 3 h with mouse anti-total-p38 MAPK, mouse anti-phospho-p38 MAPK, mouse anti-cleaved caspase-3 or mouse anti-GAPDH monoclonal antibodies (Abcam, Cambridge, UK), and subsequently with goat anti-mouse secondary antibody (Abcam) for 1 h. The signals on the membranes were detected using an enhanced chemiluminescence reagent (PerkinElmer, Waltham, MA, USA) and the densitometry was analyzed by Image-Pro plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

All of the data were expressed as means ± standard deviation and analyzed by one-way analysis of variance, followed by Student’s t-test to assess the statistical significance. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of glycyrrhizin on I/R-induced renal injury in mice, the serum levels of Cr and BUN were examined 6 h following reperfusion. The mice in the saline-treated group showed significantly higher serum levels of Cr and BUN compared with the mice in the sham-operated group, which indicated marked I/R-induced renal damage. However, the mice that were administered with glycyrrhizin exhibited notably lower serum levels of Cr and BUN, when compared with those in the saline-treated group. These findings indicate that pretreatment with glycyrrhizin provides effective protection against I/R-induced renal injury in mice (Fig. 1A and B).

The SOA level that was observed in the soluble fraction of the renal tissue 6 h after reperfusion was significantly higher in the saline-treated control group, when compared with that observed in the sham-operated and glycyrrhizin-treated groups (Fig. 1C). In addition, the SOD activity in the kidney tissue following I/R was examined and it was demonstrated that the SOD activity was greater in the glycyrrhizin-treated group than in the saline-treated group (Fig. 1D).

To further investigate the effect of renal cell apoptosis in I/R-induced renal injury, western blot analysis was performed to determine the expression of cleaved caspase-3, 6 h after reperfusion. The expression level was notably reduced in the mice that were pretreated with glycyrrhizin when compared with that of the mice pretreated with saline. For further confirmation, the activity of caspase-3 in the renal tissues was determined in each group (Fig. 2A). Consistently, the activity of caspase-3 was downregulated in the mice from the glycyrrhizin-treated group compared with the saline-treated group. Therefore, it was hypothesized that glycyrrhizin protects against I/R-induced renal damage in mice, partially via the inhibition of renal cell apoptosis.

As neutrophil and macrophage infiltration is key in I/R-induced tissue inflammation, and MPO activity is a key indicator of neutrophil and macrophage infiltration, the MPO activity in the renal tissue of each group was assessed. The activity of MPO was identified to be significantly upregulated in the saline-treated group compared with the sham-operated group. However, the activity of MPO in the glycyrrhizin-treated group was significantly reduced compared with the saline-treated group with a value that was comparable to the sham-operated group (Fig. 3A).

To identify the mechanism involved, the effect of glycyrrhizin on the production of inflammatory cytokines, including TNF-α, IFN-γ, IL-1β and IL-6 was examined; these inflammatory cytokines have been demonstrated to be significant in the induction of renal I/R injury (15,16). The production of pro-inflammatory TNF-α, IFN-γ, IL-1β and IL-6 was significantly reduced in the glycyrrhizin-treated mice, when compared with that observed in the saline-treated mice 6 h after reperfusion (Fig. 3B). Accordingly, these findings indicate that pretreatment with glycyrrhizin ameliorates I/R-induced renal injury via the mediation of inflammatory cell infiltration in addition to the production of inflammatory cytokines.

As glycyrrhizin has been reported to exhibit a suppressive effect on the activation of the p38 MAPK pathway in I/R-induced myocardial injury in rabbits, it was hypothesized that it may be similarly involved in the regulation of the p38 MAPK pathway in renal I/R damage in mice. Thus, the activity of the p38 MAPK pathway in the renal tissues was determined in each group via western blot assay. The phosphorylated-p38 MAPK protein level in the saline-treated group was significantly increased compared with the sham-operated group; however, the phosphorylated-p38 protein level in the glycyrrhizin-treated group was notably reduced compared with that in the saline-treated group (Fig. 4). These findings confirmed the hypothesis that administration of glycyrrhizin suppressed the activation of p38 MAPK in I/R-induced renal damage in mice.