Open Access

Effects of apigenin on the expression levels of B‑cell lymphoma‑2, Fas and Fas ligand in renal ischemia‑reperfusion injury in rats

  • Authors:
    • Yang Liu
    • Xiuheng Liu
    • Lei Wang
    • Yang Du
    • Zhiyuan Chen
    • Hui Chen
    • Jia Guo
    • Xiaodong Weng
    • Xiao Wang
    • Ming Wang
    • Zhishun Wang
  • View Affiliations

  • Published online on: October 2, 2017     https://doi.org/10.3892/etm.2017.5241
  • Pages: 5345-5354
  • Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of the present study was to investigate the effect and possible mechanism of apigenin on renal ischemia-reperfusion (I/R) injury in rats, as well as in in vitro experiments. In total, 36 rats were subjected to 45 min of renal ischemia, with or without treatment prior to ischemia with different concentrations of apigenin (2, 10 and 50 mg/kg) administered intravenously. All rats were sacrificed at 24 h after I/R injury. The serum creatinine (Cr) and blood urea nitrogen (BUN) levels were analyzed, and histological examination was conducted. In addition, the expression levels of B‑cell lymphoma 2 (Bcl‑2) and Fas/Fas ligand (FasL) were detected by immunohistochemistry, reverse transcription‑quantitative polymerase chain reaction and western blot analysis. For in vitro experiments, the NRK‑52E cell line was employed. The viability, apoptosis and expression levels of Fas, FasL and Bcl‑2 were examined in the culture of NRK‑52E cells following the I/R. The results indicated that apigenin significantly decreased the levels of serum Cr and BUN induced by renal I/R, demonstrating an improvement in renal function. The histological evidence of renal damage associated with I/R was also mitigated by apigenin in vivo. Furthermore, apigenin increased the cell viability and decreased cell apoptosis in the culture of NRK52E after I/R in vitro. Compared with the I/R group, the expression of Bcl‑2 was upregulated and the expression levels of Fas and FasL were downregulated by apigenin at the mRNA and protein levels in vivo and in vitro. In conclusion, apigenin appeared to increase the expression of Bcl‑2 and reduce Fas/FasL expression in renal I/R injury, providing evident protection against renal I/R injury in rats.

Introduction

Renal ischemia/reperfusion (I/R) injury is a primary cause of acute renal failure, which is commonly observed in a number of clinical settings, including shock and renal transplantation (1,2). In addition, renal I/R injury is a leading contributor to the morbidity and mortality of the aging population, and results from the process of recovering the blood or oxygen supply following ischemia in the kidney. This process may then lead to cellular damage in renal tissues (35). Under these conditions, the tissue structure and renal function deteriorate, and tubular necrosis, medullary hemorrhage and congestion are observed. Patients with I/R injury have a poor prognosis and there is currently no available effective therapy to mitigate this injury (6).

Apigenin is a plant flavone that exist in a variety of fruits and vegetables, such as celery, parsley and wheat sprouts (7,8). This compound has been proven to possess a number of biological properties, including anti-inflammatory, antioxidant and antitumor effects (9), as well as a protective effect on several organs. Apigenin can protect cells against apoptosis and necrosis by inhibiting oxidative stress. Furthermore, it has been demonstrated that apigenin has a potent therapeutic effect on liver in rats (10). However, the effects of apigenin on renal I/R injury remain unclear.

In the kidney, the cellular death receptors Fas and Fas ligand (FasL) are major mediators of the apoptotic pathway. Fas belongs to the tumor necrosis factor (TNF) receptor superfamily of cell surface death receptors, and FasL is a member of the TNF family that induces apoptosis by cross-linking its Fas receptor (11,12). Several studies revealed that Fas/FasL are involved in multifarious forms of renal injury, including I/R injury and tubular injury in glomerulonephritis (13,14). Furthermore, the role of B-cell lymphoma 2 (Bcl-2) in the development of apoptotic cell death has been widely investigated (15,16). The Bcl-2 family of proteins regulates cell apoptosis and cell necrosis (17). The Fas/FasL and Bcl-2 genes are two of the key factors influencing apoptosis and regulating the intrinsic apoptosis pathway in renal tubular epithelial cells (18,19). In addition, the intrinsic apoptosis pathway is activated by oxidative stress, which is mediated by increased mitochondrial membrane permeability (20). Previous studies have suggested that oxidative stress is involved in the apoptotic mechanism mediated by the Fas/FasL signaling pathway (2123). Therefore, it is important to verify whether the renoprotective effect of apigenin are associated with the modulation of the Fas/FasL and Bcl-2 pathway.

The aim of the present study was to investigate the protective role of apigenin against renal I/R injury in rats and the underlying mechanism of its action. The study examined whether the protective effect of apigenin on renal function was through the modulation of the Fas/FasL pathway and improvement of the expression of Bcl-2.

Materials and methods

Ethical approval

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.

Animals and in vivo experimental protocol

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.

Serum assays

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.

Measurement of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in renal tissues

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.

Histological examination of renal tissues

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.

Immunohistochemical analysis of renal tissues

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.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

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.

Western blot assay

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).

Cell culture for in vitro experiments

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.

In vitro experimental groups and treatments

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).

Cell counting kit-8 (CCK8) assay

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%.

Annexin V-FITC/propidium iodide (PI) detection of apoptosis in NRK-52E cells

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.

Immunohistochemical analysis in NRK-52E cells

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.

Western blot assay in NRK-52E cells

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.

Statistical analyses

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.

Results

Serum BUN and Cr Levels

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).

Measurement of MDA, SOD and GSH-Px

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.

Morphological features of kidneys and immunohistochemical staining

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).

Bcl-2, Fas and FasL mRNA expression levels in the kidneys

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.

Effects of apigenin on NRK-52E cell viability

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.

Effects of apigenin on apoptosis in NRK-52E cells

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).

Bcl-2, Fas and FasL protein expression levels in NRK-52E cells

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).

Discussion

Renal I/R injury associated with kidney transplantation and nephrectomy are unresolved problems in clinical practice (25). Acute renal failure can be induced by the renal I/R injury, and it has been suggested that cell apoptosis and necrosis are the main features of this disease (26). Cell apoptosis serves an important role in the process of cell development and the internal environment homeostasis (27). Dysfunction of the apoptosis regulatory mechanism results in a variety of clinical diseases, including neurodegenerative diseases, autoimmune diseases, hematopoietic dysfunction, infertility and cancer (2832). A large number of animal experiments have demonstrated that renal tubular cell apoptosis can be observed subsequent to kidney damage caused by ureteral obstructive diseases, renal artery stenosis, biological toxin exposure and trauma (3336). Furthermore, renal I/R injury can induce renal tubular epithelial cell apoptosis and necrosis (37). These two modes exist in kidney pathology as interdependent phenomena resulting from the activation of shared pathways and signals (38).

The present study demonstrated that apigenin, a common edible plant flavonoid and a well-characterized antioxidant (39), had a renoprotective effect by inhibiting apoptosis in renal I/R injury. However, the mechanisms underlying cell apoptosis are complicated. To clarify the possible mechanisms mediating the anti-apoptotic effect of apigenin, the expression levels of Bcl-2 and Fas/FasL, as well as the levels of MDA, SOD and GSH-Px, were investigated in the current study. The results revealed that the expression of Bcl-2 was decreased by I/R injury and improved by apigenin treatment, while the opposite expression alterations were observed for Fas/FasL. Furthermore, apigenin exhibited protective effects by reducing the oxidative stress through reduced production of free radical derivatives and increased production of antioxidants, as evidenced by the decreased MDA level and the increased levels of SOD and GSH-Px. These results suggest that apigenin inhibits renal cell apoptosis and protects against the oxidation of renal cellular membrane damage by enhancing the activation of the Bcl-2 pathway and blocking the activation of the Fas/FasL pathway in renal I/R injury. Furthermore, the current data indicated that the 10 and 50 mg/kg treatment subgroups exhibited a marked renoprotective effect as compared with the 2 mg/kg treatment.

In order to examine the toxicity of apigenin, the renal function and protein levels of Bcl-2, Fas and FasL in the sham surgery and apigenin + sham groups were first compared. No significant differences were observed between these two groups in terms of the BUN, serum Cr, Bcl-2, Fas and FasL protein levels (P>0.1), indicating that apigenin was non-toxic for normal renal tissues.

The Bcl-2 family is one of the primary regulatory factors of cell apoptosis (40), and has received the most attention among a variety of relevant proteins functioning in apoptosis regulation (41). Bcl-2 is one of the most important anti-apoptotic genes, and is expressed at low levels in renal tissues following I/R (42). When I/R injury was induced in the present study, the amount of apoptotic cells in distal convoluted tubules increased when compared with the sham surgery group. However, compared with the I/R group, Bcl-2 was highly expressed at the distal convoluted tubules of the I/R + apigenin groups. High expression of Bcl-2 serves an important role in the anti-apoptosis mechanism of renal tissues treated with I/R. Thus, the present study suggested that apigenin regulated the expression of the apoptosis-inhibitory Bcl-2 gene in I/R injury. The anti-apoptosis mechanism of Bcl-2 may be associated with several factors: i) Bcl-2 functions as an anti-apoptotic protein causing endoplasmic reticulum Ca2+ depletion and helping to keep the luminal Ca2+ concentration at physiological levels, decreasing the cellular Ca2+ concentration by inhibiting Ca2+ release (43); ii) cell apoptosis gene signaling and apoptosis-associated gene products are blocked by Bcl-2; iii) oxidative stress triggers multiple signaling pathways, including the pathways involving the Bcl-2 proteins. A previous study suggested that the upregulated expression of Bcl-2 can lead to the decrease of oxidative stress (44). Bcl-2 gene is a terminal part of apoptotic regulation, and high expression of Bcl-2 protein may reduce the formation of lipid peroxide and oxygen free radicals. Thus, Bcl-2 is an important survival factor, which is sensitive to oxidative stress and an increase in Bcl-2 suppress cell apoptosis.

Fas, a transmembrane protein that belongs to the TNF superfamily, is one of the death receptors in the cell membrane (45). Death receptors and the apoptotic cascade are activated upon engagement with the corresponding ligand, which is FasL in the case of Fas (46). Fas/FasL are considered as cell apoptosis genes, and the combination of Fas and its ligand result in Fas-associated cell apoptosis (47). In addition, FasL, expressed in renal tubular epithelial cells of normal rat kidney, may accelerate the apoptosis of lymphocytes (48). The apoptosis mechanism of Fas/FasL may be associated with the overexpression of the immediate early gene, immunological dysfunction and the effect of certain inflammatory cytokines. In the present experimental study, the expression of the Fas protein was significantly increased following I/R injury, in comparison with the sham surgery group. Similar results were observed for the expression of FasL protein. Thus, Fas/FasL induced cell apoptosis in the kidney following the induction of I/R injury. Furthermore, the data demonstrated that there was a significant difference between the I/R and I/R + apigenin groups. It was observed that apigenin was able to block the interaction between Fas and FasL, strongly inhibiting renal injury after I/R. The Fas system may be involved in the triggering of renal tissue cell apoptosis by oxidative stress. Thus, the present study also validated the effect of oxidative stress on membrane Fas/FasL expression in renal tubular epithelial cells. Nevertheless, the mechanism of Fas/FasL expression induced by oxidative stress remains unclear. A possible mechanism of oxidative stress-induced Fas/FasL expression is the tyrosine phosphorylation of signaling molecules, including p38 mitogen-activated protein kinase and Jun-N-terminal kinase, which have been implicated in Fas/FasL expression (49).

In conclusion, the results of the present study revealed that apigenin increased the expression of Bcl-2 and reduced Fas/FasL expression in renal I/R injury, providing marked protection against this injury in rats. The present study also suggested that the upregulation of Bcl-2 and downregulation of Fas/FasL protein expression levels are involved in anti-apoptosis and antioxidation, and may be one of the mechanisms underlying the protective effect of apigenin on renal I/R injury.

Acknowledgements

The current study was supported by grants from the Application and Basic Research Project Of Wuhan City (no. 2015060101010049), Hubei Province Health and Family Planning Scientific Research Project (nos. WJ2017M025 and WJ2017Z005) and the Bureau of Public Health of Hubei Province (no. JX6B62).

References

1 

Jang HR, Ko GJ, Wasowska BA and Rabb H: The interaction between ischemia-reperfusion and immune responses in the kidney. J Mol Med (Berl). 87:859–864. 2009. View Article : Google Scholar : PubMed/NCBI

2 

Sementilli A and Franco M: Renal acute cellular rejection: Correlation between the immunophenotype and cytokine expression of the inflammatory cells in acute glomerulitis, arterial intimitis, and tubulointerstitial nephritis. Transplant Proc. 42:1671–1676. 2010. View Article : Google Scholar : PubMed/NCBI

3 

Di Nardo M, Ficarella A, Ricci Z, Luciano R, Stoppa F, Picardo S, Picca S, Muraca M and Cogo P: Impact of severe sepsis on serum and urinary biomarkers of acute kidney injury in critically ill children: An observational study. Blood Purif. 35:172–176. 2013. View Article : Google Scholar : PubMed/NCBI

4 

Levy EM, Viscoli CM and Horwitz RI: The effect of acute renal failure on mortality. A cohort analysis. JAMA. 275:1489–1494. 1996. View Article : Google Scholar : PubMed/NCBI

5 

Santos WJ, Zanetta DM, Pires AC, Lobo SM, Lima EQ and Burdmann EA: Patients with ischaemic, mixed and nephrotoxic acute tubular necrosis in the intensive care unit-a homogeneous population? Crit Care. 10:R682006. View Article : Google Scholar : PubMed/NCBI

6 

Jia Y, Zhao J, Liu M, Li B, Song Y, Li Y, Wen A and Shi L: Brazilin exerts protective effects against renal ischemia-reperfusion injury by inhibiting the NF-κB signaling pathway. Int J Mol Med. 38:210–216. 2016. View Article : Google Scholar : PubMed/NCBI

7 

Birt DF, Hendrich S and Wang W: Dietary agents in cancer prevention: Flavonoids and isoflavonoids. Pharmacol Ther. 90:157–177. 2001. View Article : Google Scholar : PubMed/NCBI

8 

Patel D, Shukla S and Gupta S: Apigenin and cancer chemoprevention: Progress, potential and promise (review). Int J Oncol. 30:233–245. 2007.PubMed/NCBI

9 

Nicholas C, Batra S, Vargo MA, Voss OH, Gavrilin MA, Wewers MD, Guttridge DC, Grotewold E and Doseff AI: Apigenin blocks lipopolysaccharide-induced lethality in vivo and proinflammatory cytokines expression by inactivating NF-kappaB through the suppression of p65 phosphorylation. J Immunol. 179:7121–7127. 2007. View Article : Google Scholar : PubMed/NCBI

10 

Tsalkidou EG, Tsaroucha AK, Chatzaki E, Lambropoulou M, Papachristou F, Trypsianis G, Pitiakoudis M, Vaos G and Simopoulos C: The effects of apigenin on the expression of Fas/FasL apoptotic pathway in warm liver ischemia-reperfusion injury in rats. Biomed Res Int. 2014:1572162014. View Article : Google Scholar : PubMed/NCBI

11 

Brunner T, Mogil RJ, LaFace D, Yoo NJ, Mahboubi A, Echeverri F, Martin SJ, Force WR, Lynch DH, Ware CF, et al: Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas. Nature. 373:441–444. 1995. View Article : Google Scholar : PubMed/NCBI

12 

Zhao J, Duan S, Zhou J, Sun R, Zhang L and Wang D: Mild hypothermia reduces expression of Fas/FasL and MMP-3 after cerebral ischemia-reperfusion in rats. Iran J Basic Med Sci. 17:454–459. 2014.PubMed/NCBI

13 

Del RM, Imam A, DeLeon M, Gomez G, Mishra J, Ma Q, Parikh S and Devarajan P: The death domain of kidney ankyrin interacts with Fas and promotes Fas-mediated cell death in renal epithelia. J Am Soc Nephrol. 15:41–51. 2004. View Article : Google Scholar : PubMed/NCBI

14 

Erkan E, Garcia CD, Patterson LT, Mishra J, Mitsnefes MM, Kaskel FJ and Devarajan P: Induction of renal tubular cell apoptosis in focal segmental glomerulosclerosis: Roles of proteinuria and Fas-dependent pathways. J Am Soc Nephrol. 16:398–407. 2005. View Article : Google Scholar : PubMed/NCBI

15 

Gahl RF, Dwivedi P and Tjandra N: Bcl-2 proteins bid and bax form a network to permeabilize the mitochondria at the onset of apoptosis. Cell Death Dis. 7:e24242016. View Article : Google Scholar : PubMed/NCBI

16 

Wang Y, Wang W and Qiu E: Protection of oxidative stress induced apoptosis in osteosarcoma cells by dihydromyricetin through down-regulation of caspase activation and up-regulation of BcL-2. Saudi J Biol Sci. 24:837–842. 2017. View Article : Google Scholar : PubMed/NCBI

17 

Tsujimoto Y and Shimizu S: Bcl-2 family: Life-or-death switch. FEBS Lett. 466:6–10. 2000. View Article : Google Scholar : PubMed/NCBI

18 

Furuichi K, Kokubo S, Hara A, Imamura R, Wang Q, Kitajima S, Toyama T, Okumura T, Matsushima K, Suda T, et al: Fas ligand has a greater impact than TNF-α on apoptosis and inflammation in ischemic acute kidney injury. Nephron Extra. 2:27–38. 2012. View Article : Google Scholar : PubMed/NCBI

19 

Zhu MX, Ran B, Feng ZQ and Pan QW: Effects of Rb1 and Rg1 on the expression of Bcl-2, Bax in apoptosis of HK-2 cells induced by the serum of kidney ischemia/reperfusion. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 25:496–499. 2009.(In Chinese). PubMed/NCBI

20 

Wang J, Su B, Zhu H, Chen C and Zhao G: Protective effect of geraniol inhibits inflammatory response, oxidative stress and apoptosis in traumatic injury of the spinal cord through modulation of NF-κB and p38 MAPK. Exp Ther Med. 12:3607–3613. 2016. View Article : Google Scholar : PubMed/NCBI

21 

Vogt M, Bauer MK, Ferrari D and Schulze-Osthoff K: Oxidative stress and hypoxia/reoxygenation trigger CD95 (APO-1/Fas) ligand expression in microglial cells. FEBS Lett. 429:67–72. 1998. View Article : Google Scholar : PubMed/NCBI

22 

Chang WT, Hsieh BS, Cheng HL, Lee KT and Chang KL: Progesterone augments epirubicin-induced apoptosis in HA22T/VGH cells by increasing oxidative stress and upregulating Fas/FasL. J Surg Res. 188:432–441. 2014. View Article : Google Scholar : PubMed/NCBI

23 

Bussuan LA, Fagundes DJ, Marks G, Bussuan PM and Teruya R: The role of Fas ligand protein in the oxidative stress induced by azoxymethane on crypt colon of rats. Acta Cir Bras. 25:501–506. 2010. View Article : Google Scholar : PubMed/NCBI

24 

Jablonski P, Howden BO, Rae DA, Birrell CS, Marshall VC and Tange J: An experimental model for assessment of renal recovery from warm ischemia. Transplantation. 35:198–204. 1983. View Article : Google Scholar : PubMed/NCBI

25 

Gianello P, Squifflet JP, Carlier M, Lambotte L, Ketelslegers JM and Alexandre GP: Atrial natriuretic factor: A protective role after acute renal ischemia? Is there room for it in kidney transplantation? Transpl Int. 3:41–46. 1990. View Article : Google Scholar : PubMed/NCBI

26 

Gobé G, Willgoss D, Hogg N, Schoch E and Endre Z: Cell survival or death in renal tubular epithelium after ischemia-reperfusion injury. Kidney Int. 56:1299–1304. 1999. View Article : Google Scholar : PubMed/NCBI

27 

Ameisen JC: On the origin, evolution, and nature of programmed cell death: A timeline of four billion years. Cell Death Differ. 9:367–393. 2002. View Article : Google Scholar : PubMed/NCBI

28 

Hanlon MG, Gacis ML, Kakakios AM and Kilham H: Investigation of suspected deficient Fas-mediated apoptosis in a father and son. Cytometry. 43:195–198. 2001. View Article : Google Scholar : PubMed/NCBI

29 

Kiechle FL and Zhang X: The postgenomic era: Implications for the clinical laboratory. Arch Pathol Lab Med. 126:255–262. 2002.PubMed/NCBI

30 

Markström E, Svensson ECh, Shao R, Svanberg B and Billig H: Survival factors regulating ovarian apoptosis-dependence on follicle differentiation. Reproduction. 123:23–30. 2002. View Article : Google Scholar : PubMed/NCBI

31 

Müllauer L, Gruber P, Sebinger D, Buch J, Wohlfart S and Chott A: Mutations in apoptosis genes: A pathogenetic factor for human disease. Mutat Res. 488:211–231. 2001. View Article : Google Scholar : PubMed/NCBI

32 

Yuan J and Yankner BA: Apoptosis in the nervous system. Nature. 407:802–809. 2000. View Article : Google Scholar : PubMed/NCBI

33 

Hashemi M: The study of pentoxifylline drug effects on renal apoptosis and BCL-2 gene expression changes following ischemic reperfusion injury in rat. Iran J Pharm Res. 13:181–189. 2014.PubMed/NCBI

34 

Nilsson L, Madsen K, Krag S, Frøkiær J, Jensen BL and Nørregaard R: Disruption of cyclooxygenase type 2 exacerbates apoptosis and renal damage during obstructive nephropathy. Am J Physiol Renal Physiol. 309:F1035–F1048. 2015. View Article : Google Scholar : PubMed/NCBI

35 

Li H, Sun JJ, Chen GY, Wang WW, Xie ZT, Tang GF and Wei SD: Carnosic acid nanoparticles suppress liver ischemia/reperfusion injury by inhibition of ROS, Caspases and NF-κB signaling pathway in mice. Biomed Pharmacother. 82:237–246. 2016. View Article : Google Scholar : PubMed/NCBI

36 

Liu A, Huang L, Fan H, Fang H, Yang Y, Liu S, Hu J, Hu Q, Dirsch O and Dahmen U: Baicalein pretreatment protects against liver ischemia/reperfusion injury via inhibition of NF-κB pathway in mice. Int Immunopharmacol. 24:72–79. 2015. View Article : Google Scholar : PubMed/NCBI

37 

Zweier JL and Talukder MA: The role of oxidants and free radicals in reperfusion injury. Cardiovasc Res. 70:181–190. 2006. View Article : Google Scholar : PubMed/NCBI

38 

Lemasters JJ: V. Necrapoptosis and the mitochondrial permeability transition: Shared pathways to necrosis and apoptosis. Am J Physiol. 276:G1–G6. 1999.PubMed/NCBI

39 

Bruno A, Siena L, Gerbino S, Ferraro M, Chanez P, Giammanco M, Gjomarkaj M and Pace E: Apigenin affects leptin/leptin receptor pathway and induces cell apoptosis in lung adenocarcinoma cell line. Eur J Cancer. 47:2042–2051. 2011. View Article : Google Scholar : PubMed/NCBI

40 

Chen C, He H, Luo Y, Zhou M, Yin D and He M: Involvement of Bcl-2 signal pathway in the protective effects of apigenin on Anoxia/Reoxygenation-induced myocardium injury. J Cardiovasc Pharmacol. 67:152–163. 2016. View Article : Google Scholar : PubMed/NCBI

41 

Walls KC, Ghosh AP, Ballestas ME, Klocke BJ and Roth KA: bcl-2/Adenovirus E1B 19-kd interacting protein 3 (BNIP3) regulates hypoxia-induced neural precursor cell death. J Neuropathol Exp Neurol. 68:1326–1338. 2009. View Article : Google Scholar : PubMed/NCBI

42 

Meier P, Finch A and Evan G: Apoptosis in development. Nature. 407:796–801. 2000. View Article : Google Scholar : PubMed/NCBI

43 

Lam M, Dubyak G, Chen L, Nuñez G, Miesfeld RL and Distelhorst CW: Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes. Proc Natl Acad Sci USA. 91:pp. 6569–6573. 1994, View Article : Google Scholar : PubMed/NCBI

44 

Jin GF, Hurst JS and Godley BF: Hydrogen peroxide stimulates apoptosis in cultured human retinal pigment epithelial cells. Curr Eye Res. 22:165–173. 2001. View Article : Google Scholar : PubMed/NCBI

45 

Tsalkidou EG, Tsaroucha AK, Chatzaki E, Lambropoulou M, Papachristou F, Trypsianis G, Pitiakoudis M, Vaos G and Simopoulos C: The effects of apigenin on the expression of Fas/FasL apoptotic pathway in warm liver ischemia-reperfusion injury in rats. Biomed Res Int. 2014:1572162014. View Article : Google Scholar : PubMed/NCBI

46 

Malhi H, Gores GJ and Lemasters JJ: Apoptosis and necrosis in the liver: A tale of two deaths? Hepatology. 43 2 Suppl 1:S31–S44. 2006. View Article : Google Scholar : PubMed/NCBI

47 

Mor G, Straszewski S and Kamsteeg M: Role of the Fas/Fas ligand system in female reproductive organs: Survival and apoptosis. Biochem Pharmacol. 64:1305–1315. 2002. View Article : Google Scholar : PubMed/NCBI

48 

Ortiz A, Lorz C and Egido J: The Fas ligand/Fas system in renal injury. Nephrol Dial Transplant. 14:1831–1834. 1999. View Article : Google Scholar : PubMed/NCBI

49 

Hsu SC, Gavrilin MA, Tsai MH, Han J and Lai MZ: p38 mitogen-activated protein kinase is involved in Fas ligand expression. J Biol Chem. 274:25769–25776. 1999. View Article : Google Scholar : PubMed/NCBI

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December 2017
Volume 14 Issue 6

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Copy and paste a formatted citation
APA
Liu, Y., Liu, X., Wang, L., Du, Y., Chen, Z., Chen, H. ... Wang, Z. (2017). Effects of apigenin on the expression levels of B‑cell lymphoma‑2, Fas and Fas ligand in renal ischemia‑reperfusion injury in rats. Experimental and Therapeutic Medicine, 14, 5345-5354. https://doi.org/10.3892/etm.2017.5241
MLA
Liu, Y., Liu, X., Wang, L., Du, Y., Chen, Z., Chen, H., Guo, J., Weng, X., Wang, X., Wang, M., Wang, Z."Effects of apigenin on the expression levels of B‑cell lymphoma‑2, Fas and Fas ligand in renal ischemia‑reperfusion injury in rats". Experimental and Therapeutic Medicine 14.6 (2017): 5345-5354.
Chicago
Liu, Y., Liu, X., Wang, L., Du, Y., Chen, Z., Chen, H., Guo, J., Weng, X., Wang, X., Wang, M., Wang, Z."Effects of apigenin on the expression levels of B‑cell lymphoma‑2, Fas and Fas ligand in renal ischemia‑reperfusion injury in rats". Experimental and Therapeutic Medicine 14, no. 6 (2017): 5345-5354. https://doi.org/10.3892/etm.2017.5241