A total of 30 male C57BL/6 mice (age, 12 weeks; weight, 20–25 g) were obtained from the Animal Center of Central South University (Changsha, China). The mice were housed at the Xiangya Medical Experimental Animal Center of the Central South University in a laminar flow, temperature-controlled, pathogen-free environment with a 12 h light/dark cycle and with ad libitum access to food and water. Mice were fasted for 24 h prior to the renal I/R procedure, then an intraperitoneal injection of pentobarbital (50 mg/kg) was administered to anesthetize the animals. The animal experiment was approved by the Ethics Committee of The Third Xiangya Hospital of Central South University.

GP was purchased from the China National Pharmaceutical Group Corporation (Beijing, China) and dissolved in saline in accordance with the manufacturer's instructions. Mice were divided into three groups, with each group containing 5 mice. In the sham (control) group, the mice underwent anesthesia with 4% chloral hydrate (8 µl/g; Dalian Meilun Biology Technology Co., Ltd., Dalian, China) and the renal I/R procedure, which included bilateral flank incisions and a right nephrectomy. In the GP-treated I/R (I/R+GP) group, mice were intravenously injected with 50 mg/kg GP using an infusion pump 1 h prior to the renal I/R procedure. In the saline-treated I/R group (I/R), mice were administered 50 mg/kg saline using an infusion pump 1 h prior to the renal I/R procedure.

Renal function was assessed by measuring serum creatinine (Cr) and blood urea nitrogen (BUN) levels. These tests were performed by technicians at the Clinical Laboratory at The Third Xiangya Hospital, Central South University.

Homogenization buffer (Cayman Chemical Company, Ann Arbor, MI, USA), which contained 0.32 mmol/l sucrose, 20 mmol/l N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid), 0.5 mmol/l ethylenediaminetetraacetic acid, 1 mmol/l 1,4-dithio-DL-threitol and 1 mmol/l phenylmethanesulfonyl fluoride, was added to renal tissue (1 ml/0.1 g). Following homogenization, renal tissue was centrifuged at 1,500 × g for 10 min. According to Esterbauer and Cheeseman (17), MDA in tissue reacts with thiobarbituric acid in the sample, and thus, the changes in fluorescence as a result of this were detected at a wavelength of 535 nm in the present study. Recorded concentrations of MDA were divided by 1,000, and the results were expressed in µmol/g wet tissue.

SOD activity was measured using a Superoxide Dismutase Activity Assay kit (BioVision, Inc., Milpitas, CA, USA), based on the inhibition of adenochrome production by SOD by adenochrome production during epinephrine auto-oxidation. Changes in fluorescence were detected at a wavelength of 480 nm using the 721S Spectrophotometer (Shanghai Lengguang Industrial Co., Ltd., Shanghai, China).

Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA). The RevertAid Reverse Transcription kit (Thermo Fisher Scientific, Inc.,) was used to convert RNA (1 µg) into cDNA, according to the manufacturer's instructions. qPCR was then conducted using an Applied Biosystems 7500 thermocycler (Thermo Fisher Scientific, Inc.) and 0.33 µl cDNA solution, 10 µl SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.), 2 µl primers (Sangon Biotech Co., Ltd., Shanghai, China) and 7.67 µl H₂O, to obtain a final reaction volume of 20 µl. The primers used for the qPCR were as follows: TNF-α forward, 5′-CAGGCGGTGCCTATGTCTC-3′ and reverse, 5′-CGATCACCCCGAAGTTCAGTAG-3′; IFN-γ forward, 5′-GCCACGGCACAGTCATTGA-3′ and reverse, 5′-TGCTGATGGCCTGATTGTCTT-3′; IL-1β forward, 5′-GAAATGCCACCTTTTGACAGTG-3′ and reverse, 5′-TGGATGCTCTCATCAGGACAG-3′; IL-6 forward, 5′-CTGCAAGAGACTTCCATCCAG-3′ and reverse, 5′-AGTGGTATAGACAGGTCTGTTGG-3′; and GAPDH forward, 5′-TGACCTCAACTACATGGTCTACA-3′ and reverse, 5′-CTTCCCATTCTCGGCCTTG-3′. The cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 60 sec. GAPDH was used as an internal reference. The relative expression levels were analyzed by the Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Inc.), using the 2^−ΔΔCq method (18).

Enzyme-linked immunosorbent assay (ELISA) kits (Sigma-Aldrich, St. Louis, MO, USA) were used to determine the serum concentration levels of various key proinflammatory cytokines, including TNF-α (RAB0477), IFN-γ (RAB0224), IL-1β (RAB0274) and IL-6 (RAB0308), in accordance with the manufacturer's instructions.

The NE-PER® Nuclear and Cytoplasmic Extraction reagents (Pierce Biotechnology, Inc., Rockford, IL, USA) were used to extract cytosolic proteins from mice renal tissue, in accordance with the manufacturer's protocol. Protein assay reagents (Beyotime Institute of Biotechnology, Haimen, China) were used to determine protein concentration, after which 20 µg protein was separated by 10% SDS-PAGE (initially 80 V and then 150 V; Beyotime Institute of Biotechnology), transferred to nitrocellulose membranes (Thermo Fisher Scientific, Inc.) and maintained at room temperature for 1 h in a buffer solution containing 5% dried skim milk. The membrane was then incubated at room temperature for 3 h with the following antibodies from Abcam (Cambridge, MA, USA): Polyclonal rabbit anti-human p-ERK (1:100; ab65142), ERK (1:200; ab32537), heme oxygenase-1 (HO-1; 1:200; ab13248), Bcl-2 (1:100; ab117115), Bax (1:50; ab79459) and GAPDH (1:50; ab181602). Subsequently, the samples were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20,000; ab6721). The signals on the membranes were detected using enhanced chemiluminescence reagent (Pierce Biotechnology, Inc.) and densitometry was conducted using Image-Pro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Cell apoptosis was analyzed using the Annexin V-FITC Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. Briefly, at 24 h post-transfection, the cells were harvested and washed twice with cold PBS. Subsequently, 10⁶ cells were resuspended in 200 µl binding buffer added to 10 µl Annexin-V-FITC and 5 µl PI-PE, followed by incubation in the dark for 30 min. Finally, 300 µl binding buffer was added, followed by the flow cytometry assay.

All data are presented as the mean ± standard error, and analyzed using one-way analysis of variance. Statistical analyses were conducted using SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Serum concentrations of Cr and BUN were measured following I/R-induced renal injury, and it was observed that mice in the I/R group presented significantly higher serum levels of Cr and BUN compared with the control group (P<0.01; Fig. 1A and B), indicating the occurrence of I/R-induced renal injury. In the I/R+GP group, serum concentration levels of Cr and BUN were significantly reduced compared with the I/R group (P<0.01; Fig. 1A and B), suggesting that pretreatment with GP has a protective effect against I/R-induced renal injury. However, the MDA concentration in the renal tissue at 6 h after reperfusion was significantly increased in the I/R group compared with the control and I/R+GP groups (Fig. 1C). In addition, SOD activity was higher in the I/R+GP group compared with the I/R group (Fig. 1D).

The inflammatory responses resulting from I/R in kidney tissue were examined in mice. It was observed that the mRNA expression levels of the inflammatory cytokines TNF-α, IFN-γ, IL-1β and IL-6 were higher in renal tissue following I/R compared with the control and I/R+GP groups (Fig. 2). To further confirm these results, an ELISA was performed, which demonstrated that the secretion levels of TNF-α, IFN-γ, IL-1β and IL-6 were significantly increased in renal tissue following I/R, but reduced in the GP treatment group (P<0.01; Fig. 3).

Heme oxygenase-1 (HO-1) is crucial in the defense against oxidative damage, and the expression of HO-1 can be induced by I/R (19). In the present study, HO-1 protein expression was detected using western blot analysis. The results indicated that HO-1 expression was significantly increased in the I/R group compared with the control group (P<0.01; Fig. 4), suggesting that oxidative damage had occurred. However, this I/R-induced upregulation of HO-1 was attenuated by pretreatment with GP; the IR+GP group exhibited significantly reduced levels of HO-1 protein, compared with the IR group (P<0.01; Fig. 4). It may therefore be suggested that pretreatment with GP attenuates I/R-induced oxidative damage in mice kidney tissue.

The effect of GP on I/R-induced renal cell injury was investigated using an apoptosis assay. As presented in Fig. 5, the level of apoptosis in kidney tissue following I/R was significantly increased compared with the control group (P<0.01); however, pretreatment with GP significantly attenuated I/R-induced renal cell apoptosis (P<0.01 vs. I/R group).

The expression levels of the apoptosis-associated Bcl-2 and Bax proteins were examined. As demonstrated in Fig. 6A, the protein expression levels of anti-apoptotic Bcl-2 were significantly reduced in the I/R group compared with the control group (P<0.01). However, the expression level was higher in the I/R+GP group compared with the I/R group (P<0.01). In addition, it was observed that the protein level of pro-apoptotic Bax was increased in the I/R group compared with the control group, and that Bax protein expression was attenuated by pretreatment with GP (P<0.01; Fig. 6B). This suggests that the protective effect of GP against I/R-induced renal injury may be attributed to the inhibition of cell apoptosis in the kidney.

As presented in Fig. 6C, the expression level of phosphorylated ERK (p-ERK) in the I/R group was significantly reduced compared with the control group, and p-ERK was significantly attenuated following pretreatment with GP in the I/R+GP group (P<0.01). This suggests that the administration of GP suppressed I/R-induced the downregulation of ERK signaling in renal tissue, which may be associated with the protective effect of GP against I/R-induced renal cell apoptosis.