The present study was approved by the Local Ethical Committee of the Renmin Hospital of Wuhan University (Wuhan, China), and the experimental procedures were performed in accordance with the principles of the Declaration of Helsinki.

A total of 36 Sprague-Dawley rats (male; weight, 220±20 g; Hubei Provincial Academy of Preventive Medicine, Wuhan, China) were randomly separated into six groups (n=6 per group) based on the randomized block design method, as follows: i) Sham surgery; ii) I/R injury only; iii) apigenin (50 mg/kg) + sham; iv) I/R injury + 2 mg/kg apigenin treatment; v) I/R injury + 10 mg/kg apigenin treatment; and vi) I/R injury + 10 mg/kg apigenin treatment. In the three I/R + apigenin groups, apigenin was intravenously injected at 10 min prior to the induction of ischemia. Apigenin (purity, >98%) was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China).

All the rats were anesthetized with chloral hydrate intraperitoneally (350 mg/kg). After intravenous injection of heparin (1,000 UI/kg) and maintaining the body temperature at 37°C, a midline laparotomy was performed. In the I/R groups, a right nephrectomy was performed, followed by isolation of the left renal pedicles (artery, vein and nerve). The left kidney was then subjected to 45 min of ischemia followed by reperfusion subsequent to right nephrectomy. During ischemia, the color of the kidneys changed to a purple shade, which was altered to a blush color during reperfusion. In the sham and apigenin + sham groups, rats were subjected to the same surgical procedures as the I/R groups without left renal clamping. At 24 h after I/R injury, all rats were sacrificed. Blood samples (1 ml) were collected from the heart for the measurement of serum creatinine (Cr) and blood urea nitrogen (BUN) levels. The left kidney was removed and fixed in 4% paraformaldehyde or immediately frozen, and stored at −80°C for routine paraffin embedding and further examinations.

Blood samples were centrifuged at 15,000 × g for 10 min at 20°C and the serum was stored at −20°C until further analyses. Serum was analyzed according to the protocols of the Creatinine and Urea Assay kits (C013-1 and C011-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance was measured using a spectrophotometer (UV-1700; Shimadzu Corporation, Tokyo, Japan) at 520 nm. Then, the concentrations of BUN and Cr were calculated.

The renal tissues were homogenized and centrifuged at 3,000 × g for 10 min at 4°C. Then, the supernatants were collected for analysis of the MDA, SOD and GSH-Px activity using commercial SOD, MDA and GSH-Px assay kits (A001-1-1, A003-1 and A005; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocols.

Half of each kidney was removed from the rats and fixed in 4% paraformaldehyde, followed by routine paraffin embedding. According to standard procedures, the tissues were cut into 4-µm sections and stained with hematoxylin and eosin (H&E) for histological grading. The sections were assessed by an experienced renal pathologist to determine the grading scores by Jablonski's standard (24) for the histopathological assessment of renal I/R injury.

The expression levels of Fas/FasL and Bcl-2 in renal tissues were examined by immunohistochemical staining. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide at 37°C for 10 min. Next, the tissue sections were treated with 1:50 normal horse serum in Tris-buffered saline (TBS) for 30 min at 37°C. Subsequently, rabbit anti-Bcl-2 (1:1,000 dilution; A2212; ABclonal, Woburn, MA, USA) or rabbit anti-Fas/FasL (1:500 dilution; A2639/A0234; ABclonal) antibodies were added to the tissues and incubated overnight at 4°C. Phosphate-buffered saline (PBS) was then used for washing these sections three times. Subsequent to incubating with the anti-rabbit (1:100; sc-3753; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) secondary antibody for 30 min at 20°C, the sections were treated with the DAB chromogen for visualization. The average optical density (AOD) was calculated from five random fields-of-view per slide using Image-Pro Plus software, version 5.0 (Media Cybernetics, Inc., Shanghai, China), and the AOD was presented as the mean value of three detections for each sample.

The extraction of total RNA from rat kidney tissues was performed using TRIzol RNA Reagent kit (Takara Bio, Inc., Otsu, Japan). RNA concentration was obtained by spectrophotometry. Single-stranded cDNA was synthesized using the cDNA synthesis kit (Takara Bio, Inc.) according to the manufacturer's protocol. Subsequently, qPCR was conducted with the Applied Biosystems SYBR-Green Mix kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR reaction mixture contained: 2 µl cDNA, 12.5 µl 2X SYBR-Green mix, 1 µl forward primer, 1 µl reverse primer and 8.5 µl ddH 2 O, in a final volume of 25 µl. The primers used were as follows: Bcl-2 forward, 5′-TTTGATTTCTCCTGGCTGTCT-3′ and reverse, 5′-CTGATTTGACCATTTGCCTG-3′; Fas forward, 5′-CAAGGGACTGATAGCATCTTTGAGG-3′ and reverse, 5′-GTCCTTAACTTTTCGTTCACCCAGG-3′; FasL forward, 5′-TCCACCACCACCTCCATCAC-3′ and reverse, 5′-CCAACCTTACCCCAATCCTT-3′; GAPDH forward, 5-GGTCATCAACGGGAAACCC-3′ and reverse, 5′-TCTGAGTGGCAGTGATGGCA-3′. Analysis of the relative gene expression levels was performed using GAPDH as an endogenous reference gene. Bcl-2, Fas and FasL transcript levels were normalized to GAPDH transcript levels, with the mean values reported for each group.

The kidney tissues were dissociated using a Total Protein Extraction kit (Wuhan Goodbio Technology Co., Ltd., Wuhan, China) according to the specifications of the kit. Total proteins extracted were then examined via western blot analysis. Briefly, 40 µg protein from each sample was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were then blocked by 5% non-fat milk in TBS/Tween-20 (TBST) buffer and incubated with polyclonal primary antibodies of anti-Bcl-2 (1:500 dilution; A2212; ABclonal), anti-Fas/FasL (1:500 dilution; A2639/A0234; ABclonal) and anti-GAPDH (1:500 dilution; A10868; ABclonal) at 4°C overnight. Subsequent to washing three times with TBST for ~15 min, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (1:200 dilution; sc-3753; Santa Cruz Biotechnology, Inc.). Membranes were then washed three times with TBST, and specific bands were visualized using an enhanced chemiluminescence detection kit (Immobilon Western Chemiluminescence HRP Substrate; Merck KGaA, Darmstadt, Germany). The band intensity was detected using the Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Rat renal tubular epithelial NRK-52E cells were purchased from the Shanghai Institutes for Biological Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), 0.1 mg/ml streptomycin and 100 U/ml penicillin. The medium was replaced every 24 h.

The NRK-52E cells were randomly divided into six groups, as follows: i) Normal; ii) I/R only; iii) normal + 1,000 nM apigenin; iv) I/R + 10 nM apigenin; v) I/R + 100 nM apigenin; and vi) I/R + 1,000 nM apigenin. Prior to the experiment, all the groups were cultured for 24 h simultaneously. The normal group was incubated with a control medium (24.0 mM NaHCO₃, 0.8 mM Na₂HPO₄, 0.2 mM NaH₂PO₄, 86.5 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl₂, 0.8 mM MgCl₂, 20 mM HEPES and 5 mM glucose; pH 7.4). The I/R group was cultured with an anoxia medium (4.5 mM NaHCO₃, 0.8 mM Na₂HPO₄, 0.2 mM NaH₂PO₄, 106.0 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl₂, 0.8 mM MgCl₂ and 20 mM morpholinoethanesulfonic acid; pH 6.6), and then exposed to hypoxia (0.5% O₂, 5% CO₂ and 94.5% N₂) at 37°C for 3 h, followed by reoxygenation (21% O₂, 5% CO₂ and 74% N₂) at 37°C for 24 h. Similarly, the I/R + apigenin (10, 100 and 1,000 nM) groups were subjected to the same procedure as the I/R group, but cells were pretreated for 24 h with apigenin prior to the procedure. In the normal + apigenin group, the same procedures were performed as for the normal group, along with pretreatment for 24 h with apigenin (1,000 nM).

The NRK-52E cells were inoculated in a 96-well plate at a cell density of 10⁵ cells/well. After 24 h of culture at 37°C, the cells were treated with CCK8 solution, and the plate was incubated in an incubator for 4 h. The absorbance of cells at wavelength of 450 nm in terms of the OD was measured using a microplate reader. The cell survival rate was calculated according to the following formula: Survival (%) = (treatment group OD-blank control OD)/(normal group OD-blank control OD) ×100%.

An Annexin V-FITC/PI assay was conducted using the Annexin V-FITC Apoptosis Detection kit (70-AP101-100; Liankebio, Hangzhou, China) according to the manufacturer's protocol, followed by flow cytometric analysis. Briefly, NRK-52E cells were collected, washed twice with cold PBS and then resuspended in binding buffer. The cells were subsequently incubated with 10 µl Annexin V-FITC and 5 µl PI for 5 min at room temperature in the dark, followed by analysis by flow cytometry.

The NRK-52E cells were inoculated in a 96-well plate at a cell density of 10⁶ cells/well. The NRK-52E cells were fixed with 4% paraformaldehyde and incubated with rabbit anti-Bcl-2 (1:1,000 dilution; A2212; ABclonal) or rabbit anti-Fas (1:500 dilution; A2639; ABclonal) antibodies overnight at 4°C. Next, cells were washed three times with PBS, incubated with the anti-rabbit secondary antibody (1:100; sc-3753; Santa Cruz Biotechnology, Inc.) for 30 min at 20°C and then visualized by addition of DAB. The AOD was calculated in five random fields per slide using Image-Pro Plus software (version 5.0) and presented as the mean of three independent measurements.

The cells were washed three times with PBS, trypsinized, suspended in DMEM and then centrifuged at 3,000 × g for 5 min at 4°C to remove the supernatant. The total proteins extracted were examined via western blot analysis. Briefly, 40 µg protein from each sample was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Next, the membrane was blocked with 5% non-fat milk in TBST buffer and incubated at 4°C overnight with the following primary polyclonal antibodies: Anti-Bcl-2, anti-Fas/FasL and anti-GAPDH (dilution, all 1:500). Following three washed with TBST for ~15 min, the membrane was incubated with a secondary antibody conjugated with horseradish peroxidase (dilution, 1:2,000), followed by further washing with TBST for three times. Specific bands were visualized using an enhanced chemiluminescence detection kit, and the band intensity was detected using the Quantity One software.

All data are expressed as the mean ± standard deviation. Statistically significant differences between groups were tested by analysis of variance, and all statistical analyses were processed by SPSS version 11.0 statistical software (SPSS, Inc., Chicago, IL, USA). P<0.05 was regarded as an indicator of statistically significant differences.

The levels of the renal function parameters BUN and serum Cr in rats of the various groups are shown in Fig. 1A and B. Compared with the sham surgery rats, the I/R group demonstrated a significant increase in BUN and Cr levels (P<0.01). However, the renal function of rats subjected to I/R was improved by treatment with apigenin as observed by the significantly reduced levels of BUN and Cr in the I/R + apigenin groups compared with I/R alone groups (P<0.05). In addition, the difference in the BUN and Cr levels between the apigenin + sham and the sham-operated group was not statistically significant. Furthermore, the BUN and Cr levels of the I/R + api (2 mg/kg) group was higher than in other apigenin treatment groups (10 and 50 mg/kg), which indicated that the concentration of 10 and 50 mg/kg apigenin were the most effective at reducing I/R injury (Fig. 1).

As shown in Fig. 1C-E, the I/R group exhibited a significant increase in MDA (P<0.01), a marker of lipid peroxidation, as well as significant decreases in the SOD and GSH-Px levels compared with the sham group (P<0.01). By contrast, apigenin pretreatment in I/R rats significantly inhibited the decrease in the SOD and GSH-Px levels compared with the I/R only group (P<0.05). Furthermore, no significant difference was identified between the sham and apigenin + sham groups (P>0.05; Fig. 1C-E). Similarly, high doses of apigenin exerted a marked increase in renoprotection against renal I/R injury compared with lower doses of apigenin.

In the I/R group, various morphological abnormalities were identified by H&E staining, including tubular cell necrosis, cytoplasmic vacuolization and tubular lumen obstruction and impairments. However, apigenin pretreatment relieved the severe renal damage caused by I/R injury. Quantitative analysis indicated that 45 min of renal ischemia followed by 24 h of reperfusion resulted in severe acute tubular necrosis, but the Jablonski histological scores in the I/R + apigenin groups were significantly lower than those in the I/R group, which indicated that this renal damage was attenuated by apigenin (P<0.05; Fig. 2A and B).

Immunohistological staining for Bcl-2, Fas and FasL was also conducted in the kidney tissues (Fig. 2A, C and D). The results indicated that Fas/FasL expression significantly increased in the I/R group compared with the sham group (P<0.01), and this tendency was suppressed by apigenin treatment (P<0.05). The immunohistochemical results of Fas gene were almost identical with those for FasL, thus only the immunohistochemical results for FasL are illustrated in Fig. 2. In contrast to the Fas/FasL results, Bcl-2 expression significantly decreased in the I/R group compared with the sham group (P<0.01); however, apigenin pretreatment significantly improved this expression (P<0.05).

RT-qPCR was used to investigate the differences in the mRNA expression levels of Bcl-2, Fas and FasL. As shown in Fig. 3A, the expression of Bcl-2 in kidney tissues at the mRNA level demonstrated a significant decrease in the I/R group compared with that in the sham group (P<0.01). However, apigenin evidently improved the mRNA expression of Bcl-2 in the I/R + apigenin groups, since an increased level was observed compared with the I/R group (P<0.05). On the contrary, the mRNA levels of Fas and FasL were significantly higher in I/R group in comparison with the sham group (P<0.01), while apigenin pretreatment inhibited the mRNA expression of Fas and FasL in the I/R + apigenin group compared with the I/R only group (P<0.05; Fig. 3B and C).

Similarly, western blot analysis revealed a significant increase in Fas and FasL protein expression levels in the I/R group compared with the sham group (P<0.01), and these levels were decreased by apigenin pretreatment in the I/R + apigenin groups (P<0.05). Furthermore, as compared with the sham-operated group, the I/R group induced a significant decrease in Bcl-2 protein expression (P<0.01), whereas these effects were antagonized in the I/R + apigenin groups (P<0.05; Fig. 4). The western blot analysis results were consistent with the immunohistochemical findings on Bcl-2 and Fas/FasL expression levels. Furthermore, apigenin was indicated to increase Bcl-2 expression and decrease Fas/FasL expression significantly in a dose-dependent manner. The Bcl-2 expression was increased to the highest at a final concentration of 10 and 50 mg/kg, which indicated that apigenin protected kidneys against I/R injury.

In order to further investigate the effects of apigenin, in vitro experiments were also conducted in an I/R cell model established by anoxia/reoxygenation. The effects of apigenin on the viability of NRK-52E cells induced by I/R were determined quantitatively with a CCK8 assay. I/R in cells significantly decreased the cell survival rate as compared with the normal group. However, apigenin pretreatment prior to I/R significantly increased the viability of NRK-52E cells (Fig. 5). In addition, the effect of apigenin was dose dependent, since the viability of NRK-52E cells was the highest at the final concentration of 1,000 nM apigenin, although the difference among the 1,000 and 100 nM doses was not statistically significant (Fig. 5). Thus, the concentration of 1 µM apigenin was used in subsequent experiments. In addition, there was no evident difference in cell viability between the normal and normal + apigenin groups, therefore, only the normal group was used in subsequent experiments.

Flow cytometry was conducted to determine whether apigenin was able to inhibit the apoptosis of NRK-52E cells. Compared with the normal group, the I/R group demonstrated significantly increased cell apoptosis. However, the cell apoptosis induced by I/R was reduced upon pretreatment with apigenin (Fig. 6).

To determine the effect of Bcl-2 and Fas/FasL in NRK-52E cells, immunohistochemical analysis was performed. As shown in Fig. 7, the I/R group displayed low protein expression of Bcl-2 and strong protein expression of Fas compared with the normal group. By contrast, pretreatment with apigenin at a concentration of 1,000 nM significantly increased the Bcl-2 protein levels and decreased the Fas protein levels in the I/R + apigenin (1,000 nM) group when compared with the I/R group (Fig. 7). Furthermore, western blot analysis was conducted to analyze the expression levels of Bcl-2 and Fas/FasL. The results indicated that the protein expression levels of Fas and FasL were significantly increased, while the Bcl-2 level was significantly decreased, as compared with the normal group. In addition, pretreatment with apigenin at a concentration of 1,000 nM attenuated the increase in Fas/FasL and the decrease in Bcl-2 expression levels (Fig. 8).