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STAT3 pathway
Biomedicine & Pharmacotherapy 95 (2017) 1799–1808
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Original article
Effects of apigenin pretreatment against renal ischemia/reperfusion injury via activation of the JAK2/STAT3 pathway
MARK
Yang Liua, Lei Wanga, Yang Dua, Zhiyuan Chena, Jia Guoa, Xiaodong Wenga, Xiao Wanga, ⁎ Min Wanga, Danyang Chenb, Xiuheng Liua, a b
Department of Urology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China Department of Plastic Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Apigenin JAK2/STAT3 AZD1480 Ischemia/reperfusion si-JAK2
The aim of our study is to investigate the protective effect of apigenin and the role of the JAK2/STAT3 signaling pathway in renal ischemia/reperfusion injury (IRI) in rats. For in vivo experiments, rat kidneys were subjected to 45 min of ischemia, followed by 24 h of reperfusion. The kidneys were pretreated for 24 h with apigenin (4 mg/ kg) intraperitoneally in the absence or presence of the JAK2 kinase-specific inhibitor AZD1480 (30 mg/kg). The serum creatinine and urea nitrogen levels were analyzed. Histologic examinations were evaluated. Expression of p-JAK2, p-STAT3, Bcl-2, Bax and Caspase-3 was detected by immunohistochemistry or western blot. For In vitro experiments, NRK-52E cells were exposed to I/R in the absence or presence of apigenin and JAK2 siRNA was used to explore JAK2/STAT3 activity. Cell viability, cell apoptosis and expression of p-JAK2, p-STAT3, Bcl-2, Bax and Caspase-3 were examined in NRK-52E culture after I/R. Consequently, apigenin conferred a renoprotective effect on the kidneys against IRI, as evidenced by decreased serum creatinine and urea nitrogen, mitigated renal histologic damage, improved NRK-52E cell viability and a decreased apoptotic index, including up-regulation of the anti-apoptotic protein Bcl-2 and down-regulation of the pro-apoptotic proteins Bax and Caspase3. However, AZD1480 and JAK2 siRNA blocked the apigenin-mediated renoprotective effects by attenuating the JAK2/ STAT3 signaling pathway as well as abolished the effect of anti-oxidative stress and anti-apoptosis of apigenin. Our study demonstrates that apigenin pretreatment can protect against renal IRI via the activation of the JAK2/ STAT3 signaling pathway.
1. Introduction
and renal functions [5]. Therefore, it is essential to explore novel therapies for effective management of renal IRI. Apigenin is a plant flavone that is present in a variety of fruits and vegetables, such as celery, parsley and wheat sprouts [6,7]. Apigenin has been proven to possess a series of biological properties, such as anti-inflammatory, anti-oxidant, and anti-tumor effects [8]. Apigenin is a type of antioxidant that has been reported to have protective effects on several organs. Apigenin can protect cells against apoptosis and necrosis by inhibiting oxidative stress. Recent studies have shown that apigenin has a potent therapeutic effects on liver in rats [9]. However, the effects of apigenin on renal ischemia/reperfusion injury remain unknown. Evidence indicates that the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway plays a pivotal role in mediating protection from IRI. Additionally, the JAK2/ STAT3 signaling pathway is associated with a mass of growth factors, cytokines and hormones. Accumulated evidence shows that the JAK2/ STAT3 signaling pathway is involved in preventing renal IRI.
Renal ischemia/reperfusion (I/R) is a primary cause of acute kidney injury (AKI) and an inevitable consequence of a series of operations and clinical entities, such as renal transplantation, partial nephrectomy, surgical revascularization of the renal artery, iatrogenic trauma and shock [1,2]. Renal ischemia/reperfusion injury (IRI) is an unavoidable incident in the setting of renal transplantation and entire blockage of renal blood flow; however, the pathologic mechanisms of renal IRI has not been fully researched. The pathogenesis underlying renal IRI is complicated and involves necrosis, apoptosis, oxidative stress, calcium ion overloading, and inflammation among others [3,4]. Despite impressive advances in medicine, the therapies available to clinicians for curing renal IRI are very limited. Presently, the available therapeutic options merely target symptoms, such as the drugs, vasodilators and diuretics that improve renal blood flow, including dopamine and fenoldopam as well as natriuretic peptide to protect cardiac
⁎
Corresponding author at: Department of Urology, Renmin Hospital of Wuhan University, Jiefang Road 238, Wuhan 430060, Hubei Province, China. E-mail addresses: [email protected] (Y. Liu), [email protected] (X. Liu).
http://dx.doi.org/10.1016/j.biopha.2017.09.091 Received 15 June 2017; Received in revised form 6 September 2017; Accepted 18 September 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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spectrophotometer (UV-1700; Shimadzu Corporation, Tokyo, Japan), and the absorbance was detected at 520 nm. The concentrations of BUN and Cr were then calculated.
Furthermore, previous studies have reported that apigenin inhibits hepatocyte necrosis and reduces the infarct size and apoptotic rate of cardiomyocytes after IR injury [10,11]. However, whether the protective effects of apigenin on renal IRI are induced via the JAK2/STAT3 signaling pathway requires further investigation. The purpose of our study was to investigate whether apigenin shows any protective effects on the renal IRI in vivo and anoxia/reoxygenation (A/R) in vitro and to evaluate whether the protective mechanisms of apigenin involve the JAK2/STAT3 signaling pathway.
2.3.3. Measurements of oxidative stress markers The renal tissues were homogenized and centrifuged at 3,000 × g for 10 min at 4 °C, and the supernatants were then collected for analysis of MDA, SOD and GSH-Px activity using commercial SOD, MDA and GSH-Px assay kits (A001-1-1; A003-1; A005. Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocols.
2. Materials and methods 2.3.4. Histological examination Half of each kidney was removed and fixed in 4% paraformaldehyde, followed by routine paraffin embedding. According to standard procedures, the tissue sections were cut at a thickness of 4 μm and stained with HE for histological grading. These morphological sections were assessed by an experienced renal pathologist. A grading scale described by Paller's standard [12] was used for the histopathological assessment of renal ischemia-reperfusion injury.
2.1. Drugs and reagents Apigenin (Api, purity > 98%, A106676) was purchased from Aladdin Biochemical Company (Shanghai, China). AZD1480 (purity > 98.81%; HY-10193) was purchased from MedChemExpress (MCE, USA). Anti-Bax (sc-493) and anti-Caspase-3 (sc-271759) antibodies were purchased from Santa Cruz Biotechnology (USA); anti-Bcl2 (A2212) and anti-GAPDH (A10868) antibodies were purchased from ABclonal Technology (USA); anti-cleaved caspase3 (9664), anti-pSTAT3 (9145) and anti-STAT3 (9139) antibodies were purchased from Cell Signaling Technology (CST, USA); anti-p-JAK2 (Bs-3206r) antibody were purchased from BIOSS (peking, China); anti-JAK2 (17670-1-AP) antibody were purchased from Proteintech Group (Wuhan, China).
2.3.5. Immunohistochemistry assay The expression of Bax, Caspase-3, Bcl-2, p-JAK2, p-STAT3 were assessed by immunohistochemical staining. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide at 37 °C for 10 min. The sections were then treated with 1:50 normal horse serum in Trisbuffered saline (TBS) for 30 min at 37 °C. Rabbit primary antibody was then added and incubated overnight at 4 °C. The sections were washed three times with PBS. After incubating with the secondary antibody for 30 min at 20 °C, these sections were treated with the color reagent DAB. The average optical density (AOD) was calculated from 5 random fields per slide using Image-Pro Plus software, version 5.0 (MediaCybernetics, Shanghai, China), and AOD is presented as the mean (SD) based on 3 detection times.
2.2. Animal preparation This study was approved by the local ethical committee, and the experimental procedures were carried out in accordance with the principles of the Helsinki Declaration. Fifty adult male Sprague-Dawley rats (weighing 220 ± 20 g) were provided by Hubei Provincial Academy of Preventive Medicine. 2.3. In vivo experiments
2.3.6. Western blotting assay The kidney tissues were dissociated by using the total protein extraction kit (purchased from Wuhan Goodbio technology company) according to the specification supplied with the kit, and total proteins extracted were examined via western blotting, in which 40 μg of protein from each sample was separated on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. Then, the membranes were then blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 (TBST) buffer and incubated with then primary polyclonal antibodies (1:500) anti-Bax (sc-493; SANTA CRUZ), anti-Bcl-2 (A2212; ABclonal), antiCaspase-3 (sc-271759; SANTA CRUZ), anti-cleaved Caspase3 (9664; CST), anti-p-JAK2 (Bs-3206r; BIOSS, China), anti-p-STAT3 (9145; CST), anti-JAK2 (17670-1-AP; PROTEINTECH, China), anti-STAT3 (9139; CST) and anti-GAPDH (A10868; ABclonal) at 4 °C overnight. After being washed three times with TBST for approximately 15 min, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:1000 dilution; BA1054; Boster Biological Technology co.ltd) or goat anti-mouse secondary antibody (1:1000 dilution; BA1051; Boster Biological Technology co. ltd). The membranes were then washed 3 times with TBS-T. Specific bands were visualized using an enhanced chemiluminescence detection kit. The immune complexes were visualized by an enhanced chemiluminescence detection kit. Ultimately, the band intensity were detected using Quantity One software.
2.3.1. Experimental protocol Fifty rats were randomly separated into five different groups (10 rats in each group): (1) sham-operation group: rats were pretreated with normal saline; (2) I/R group: the rats were pretreated with normal saline; (3) I/R + apigenin group: the rats were intraperitoneally injected with apigenin (4 mg/kg) 24 h before the experiment; (4) I/R + apigenin + AZD1480 group: the rats were intraperitoneally injected with both apigenin (4 mg/kg) and AZD1480 (30 mg/kg) 24 h before the experiment; (5) I/R + AZD1480 group: The rats were intraperitoneally injected with AZD1480 (30 mg/kg) alone. All rats were intraperitoneally anesthetized with chloral hydrate (350 mg/kg). After i.v. Injection of heparin (1000 UI/kg), body temperature was maintained at 37 °C, and a midline laparotomy was performed. Renal ischemia was induced by clamping both renal pedicles for 45 min using the artery clips, and the clamp was removed to restore kidney blood flow. A change in color of the kidneys to a paler shade and reperfusion resulting in a blush verified occlusion. sham operation group was subjected to the same surgical procedures as the I/R group without renal clamping. At 24 h after I/R injury, all rats were sacrificed. Blood samples (1 ml) were collected from the heart to measure urea creatinine (Cr) and nitrogen (BUN). The left kidney was removed and fixed in 4% paraformaldehyde or immediately frozen and stored at −80 °C for routine paraffin embedding and different determinations.
2.4. In vitro experiments 2.3.2. 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, C011-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance was measured using a
2.4.1. Experimental protocol The NRK-52E cells were randomly divided into 5 groups: (1) Control group: cells were pretreated with PBS; (2) I/R group: cells were pretreated with PBS; (3) I/R +apigenin group: cells were pretreated for 24 h with apigenin (1 μM); (4) I/R +apigenin + si-JAK2 group: cells 1800
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Fig. 1. Effects of apigenin and AZD1480 on renal function. (A) Effects of apigenin and AZD1480 on serum BUN concentrations after 45 min of ischemia; (B) effects of apigenin and AZD1480 on serum Cr concentrations after 45 min of ischemia. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
for 5 min at room temperature in the dark, followed by flow cytometry analysis.
were treated with JAK2 siRNA and pretreated for 24 h with apigenin (1 μM); (5) I/R +si-JAK2 group: cells were pretreated with JAK2 siRNA; (6) I/R +si-NC group: cells were pretreated with a control siRNA. Before the process, all the groups were cultured for 24 h simultaneously. After that, the control group was incubated as a routine culture by adding a control medium (NaHCO3 24.0 mM, Na2HPO4 0.8 mM, NaH2PO4 0.2 mM, NaCl 86.5 mM, KCl 5.4 mM, CaCl2 1.2 mM, MgCl2 0.8 mM, HEPES 20 mM and 5 mM glucose; pH 7.4). The I/R group was cultured with anoxia medium (NaHCO3 4.5 mM, Na2HPO4 0.8 mM, NaH2PO4 0.2 mM, NaCl 106.0 mM, KCl 5.4 mM, CaCl2 1.2 mM, MgCl2 0.8 mM and morpholinoethanesulfonic acid 20 mM; pH 6.6) and then exposed to hypoxia (37 °C, 0.5% O2, 5% C02, 94.5% N2) for 3 h and to reoxygenation (37 °C, 21% O2, 5% CO2, and 74% N2) for 24 h.
2.4.5. Western blotting assay The cells were washed 3 times with PBS and then trypsinized and suspended in DMEM. Cell suspensions were centrifuged at 1000 rpm for 5 min to remove the supernatant. Total proteins were examined by western blotting, in which 40 μg of protein from each sample was separated on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. The membranes were then blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 (TBST) buffer and incubated with primary polyclonal antibodies (1:500) of anti-Bax (sc-493; SANTA CRUZ), anti-Bcl-2 (A2212; ABclonal), anti-Caspase-3 (sc-271759; SANTA CRUZ), anti-cleaved caspase3 (9664; CST), anti-p-JAK2 (Bs3206r; BIOSS, China), anti-p-STAT3 (9145; CST), anti-JAK2 (17670-1AP; PROTEINTECH, China), anti-STAT3 (9139; CST) and anti-GAPDH (A10868; ABclonal) at 4 °C overnight. After being washing three times with TBST for approximately 15 min, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:1000 dilution; BA1054; Boster Biological Technology co. ltd) or goat anti-mouse secondary antibody (1:1000 dilution; BA1051; Boster Biological Technology co. ltd). The membranes were then washed 3 times with TBS-T. Specific bands were visualized using an enhanced chemiluminescence detection kit. The immune complexes were visualized using an enhanced chemiluminescence detection kit. Ultimately, the band intensity were detected using Quantity One software.
2.4.2. Small interference RNA (siRNA) treatment The plasmid including the small interfering RNA (siRNA) targeting JAK2 (si-JAK2: 5′-GCAGTTCAGTCAGTGCAAACCTAAGGACTTCAA CAAATGCAGTATACATCCCAGATA-3′) and a control siRNA (si-NC: 5′TTCTCCGAACGTGTCACGT-3′) were provided by Biossci Company (Wuhan, China). The NRK-52E cells were transfected using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocol. The expression of JAK2 was confirmed by western blot analysis. Eventually, the cells were collected for further analyses. 2.4.3. Analysis of cell viability The NRK-52E cells were inoculated in a 96-well plate. After 24 h of culture, the cells were treated with CCK-8 solution, and the plate was incubated in an incubator for 4 h. The absorbance (OD) of cells at a wavelength of 450 nm was measured using a microplate reader. The calculation formula for cell survival rate was as follows: [treatment group (OD) − blank group (OD)]/[control group (OD) − blank group (OD)] × 100%.
2.4.6. Statistical analysis All data are expressed as the mean ± SD. Significant differences between groups were tested by analysis of variance, and all statistical analyses were processed using SPSS 11.0 statistic software. P < 0.05 was regarded as statistically significant. 3. Results
2.4.4. Annexin V-FITC/PI detection of apoptosis The Annexin V-FITC/PI assay was carried out using the Annexin VFITC Apoptosis Detection Kit according the instructions provided for flow cytometry. NRK-52E cells were collected and washed twice with cold PBS and then resuspended in binding buffer and incubated with 10 μl Annexin V-fluorescein isothiocyanate and 5 μl propidium iodide
3.1. The effects of apigenin and the JAK2 inhibitor AZD1480 on the renal function in vivo Initially, to investigate the effects of apigenin and the JAK2 1801
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Fig. 2. Histologic features were evaluated by HE staining. (A) HE staining, original magnification × 400; the yellow arrow indicates the glomerulus, and the black arrow indicates the kidney tubules. (B) Paller scores for the histological appearance of acute tubular necrosis. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇ P < 0.01 versus IR + apigenin.
antioxidant effect of apigenin (p < 0.01, Fig. 3). Our results showed that apigenin could protect the kidney against damage from oxidative stress and that AZD1480 could attenuate the antioxidant capacity of apigenin.
inhibitor AZD1480, we evaluated renal function by measuring serum urea nitrogen (BUN) and creatinine (Cr). Compared with the shamoperated group, the I/R group showed significant increases in BUN and Cr (p < 0.01). However, apigenin treatment decreased the levels of BUN and Cr significantly compared with the I/R group (p < 0.01). In addition, the BUN and Cr levels in the IR + apigenin + AZD1480 groups and IR + AZD1480 group were significantly higher than those in the IR + apigenin group(p < 0.01), which suggest that renal protection by apigenin was significantly weakened by treatment with AZD1480 (Fig. 1).
3.4. The effects of apigenin and AZD1480 on JAK2 and STAT3 phosphorylation in IRI kidneys in vivo The expression levels of the phosphorylation of JAK2 (p-JAK2) and STAT3 (p-STAT3) were measured by immunohistochemical staining and western blot analysis. Western blot analysis demonstrated that pJAK2 and p-STAT3 expression was significantly increased in response to renal IR injury (p < 0.01). Apigenin dramatically enhanced the phosphorylation of JAK2 and STAT3 in IR-exposed kidneys (p < 0.01), whereas cotreatment with AZD1480 and apigenin decreased the expression of p-JAK2 and p-STAT3 (p < 0.01, Fig. 4A and B). In addition, p-JAK2 and p-STAT3 were localized by immunohistochemical staining, consistent with western blot analysis. The blue color indicated the cell nuclei of renal tubular cells and glomerular cells, and the brown color indicated positive expression of p-JAK2 and p-STAT3 in kidneys. Stained sections showed that renal tissues were highly positive for p-JAK2 and p-STAT3 expression in the I/R group (p < 0.01), and this tendency was strengthened by apigenin treatment (p < 0.01). However, the expression of p-JAK2 and p-STAT3 was inhibited by AZD1480 (p < 0.01, Fig. 4C–E). These results indicated that apigenin could activate the JAK2/STAT3 signaling pathway, and inhibition of JAK2/STAT3 signaling pathway could weaken the renoprotective effect of apigenin.
3.2. The effects of apigenin and AZD1480 on the morphological features of injury in vivo In the I/R group, morphologic abnormalities including tubular cell necrosis, cytoplasmic vacuolization and tubular lumen obstruction and impairments, were observed by in HE. Apigenin treatment relieved this severe renal damage. However, compared with the IR + apigenin group, this damage was greater in the IR + apigenin + AZD1480 group (Fig. 2A). Furthermore, the renal histological evaluation was quantified according to the Paller scale histological grading scores. Quantitative analysis showed a lower histologic score in the IR + apigenin group compared with IR group (p < 0.01). Additionally, the histologic scores in the IR + apigenin + AZD1480 group were clearly higher than those in I/R group (p < 0.01, Fig. 2B). These results demonstrated that apigenin could relieve renal damage and AZD1480 could reverse the renoprotective effect of apigenin. 3.3. The effects of apigenin and AZD1480 on the levels of SOD, MDA and GSH-Px in vivo
3.5. The effects of apigenin and AZD1480 on the expression of apoptosisrelated proteins in vivo
The I/R group showed a significant decrease in SOD and GSH-Px levels and a significant increase in MDA levels compared with the shamoperated after I/R (p < 0.01). Nevertheless, apigenin inhibited the decrease in SOD and GSH-Px levels and reduced the level of MDA (p < 0.01). However, treatment with AZD1480 abrogated the
Expression of the pro-apoptotic proteins Bax and Caspase-3 is often associated with increased apoptosis, and the anti-apoptotic protein Bcl2 has been associated with inhibition of apoptosis in target cells [13]. To survey the differences in expression of apoptosis-related proteins, 1802
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Fig. 3. Effects of apigenin and AZD1480 on oxidative stress in rats at 24 h after reperfusion. (A) The level of SOD in the renal tissue; (B) the level of MDA in the renal tissue; (C) the level of GSH-Px in the renal tissue. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
Fig. 4. The effects of apigenin and AZD1480 on JAK2 and STAT3 phosphorylation following the IR procedure. (A)Representative western blots showing the effects of apigenin and AZD1480 on p-JAK2, JAK2, p-STAT3 and STAT3 expression levels; (B) quantitative analyses of the expression of p-JAK2/JAK2 and p-STAT3/STAT3 by western blotting; (C) immunohistochemistry was performed to measure the expression of p-JAK2 and p-STAT3; (D) average optical density (AOD) of p-JAK2 and p-STAT3 were determined as described. Bars represent the means ± SE (n = 10). *P[Please apply a consistent format for the formatting of the ‘p’ to denote p-values.] < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
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Fig. 5. The expression of Bcl-2, Bax, pro-Caspase-3 and cleaved-Caspase-3 by western blotting. (A) Representative western blots showing the effects of apigenin and AZD1480 on Bax, Bcl2, pro-Caspase-3 and cleaved-Caspase-3 expression levels; (B) quantitative analyses of the expression of Bcl-2, Bax, pro-Caspase-3 and cleaved-Caspase-3 in NRK-52E cells by western blotting. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
apigenin inhibited the expression of pro-apoptotic proteins and increased the expression of anti-apoptotic proteins; however, AZD1480 abated the anti-apoptosis effect of apigenin.
the levels of Bax, Bcl-2, pro-Caspase-3 and cleaved-Caspase-3 were measured by western blot analysis. The results showed that the expression of Bcl-2 and pro-Caspase-3 in kidney tissues at the protein level decreased significantly in the I/R group compared with the sham-operated group (p < 0.01), and apigenin improved the expression of Bcl2 and pro-Caspase-3 in the I/R + apigenin group (p < 0.01). However, the increases were drastically abolished by AZD1480 treatment (p < 0.01, Fig. 5A). In contrast, Bax and cleaved-Caspase-3 expression showed an opposite result, such that the IR procedure clearly increased Bax and cleaved-Caspase-3 expression (p < 0.01). Additionally, the expression levels of Bax and Caspase-3 were reduced when the IR kidneys were subjected to apigenin treatment (p < 0.01). However, the decreases in Bax and cleaved-Caspase-3 expression induced by apigenin treatment were reversed by AZD1480 (p < 0.01, Fig. 5B and C). As shown,
3.6. The effects of apigenin and JAK2 siRNA on the levels of SOD, MDA and GSH-Px in vitro The levels of SOD, MDA and GSH-Px were evaluated in NRK-52E cells in vitro. As shown in Fig. 6, compared with the I/R group, apigenin pretreatment could decrease the MDA concentrations (p < 0.01). However, JAK2 siRNA treatment dramatically increased the MDA level in I/R-injured NRK-52E cells (p < 0.01). In contrast, I/R alone significantly decreased the levels of SOD and GSH-Px compared with the control group. Apigenin pretreatment increased the levels of SOD and GSH-Px compared with the I/R group (p < 0.01). However, a decrease
Fig. 6. Effect of apigenin and JAK2 siRNA on oxidative stress in NRK-52E cells subjected to I/R injury. (A) The level of SOD in NRK-52E cells; (B) the level of MDA in NRK-52E cells; (C) the level of GSH-Px in NRK-52E cells. Bars represent the means ± SE (n = 10). *P < 0.01 versus control; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
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3.9. The effects of apigenin and JAK2 siRNA on JAK2 and STAT3 phosphorylation in I/R-injured NRK-52E cells We examined the effect of apigenin and JAK2 siRNA on the JAK2/ STAT3 signaling pathway in I/R-injured NRK-52E cells by western blotting. These results indicated that p-JAK2 and p-STAT3 expression was elevated after IRI compared with the control group (p < 0.01). Apigenin improved the levels of p-JAK2 and p-STAT3 (p < 0.01). However, treatment with apigenin and si-JAK2 produced a significant decrease in the expression of p-JAK2 and p-STAT3 (p < 0.01, Fig. 9). These results suggested that the JAK2/STAT3 pathway was involved in the process of renal IRI, and apigenin activated the JAK2/STAT3 signaling pathway to exert a renoprotective effect. 4. Discussion Renal IRI, which often arises from kidney transplantation and nephrectomy, is an unresolved problem in clinical practice. Renal IRI is one of the main causes of acute renal failure (ARF), which leads to disease with high mortality and morbidity [14]. During the I/R procedure, after the aorta or renal pedicle is blocked, restoration of blood flow to ischemic tissue exacerbates the damage to the kidney [15]. The main mechanism of renal IRI is associated with multiple mediators including inflammation, oxidative stress and activation of adhesion molecules, ultimately resulting in renal tubular damage, inflammation, endothelial disordering and cell apoptosis [16,17]. At present, a therapeutic strategy using anti-apoptotic and anti-oxidative stress drugs may prevent the decline in renal function and alleviate tubular injury. In our study, renal protection of apigenin was evaluated as a potential therapeutic agent for renal IRI. Apigenin is an edible plant flavonoid that exists widely in a variety of fruits and vegetables and has gained extensive attention for its potential use in IRI and cancers [18]. The detailed mechanisms underlying the protective effect of apigenin vary extensively among studies, involving, e.g., reactive oxygen species (ROS), the Fas/FasL signaling pathway, nuclear factor-kappa B (NF-κB), endothelial nitric oxide synthase (eNOS) and cell autophagy [19–21]. Chuanjun et al. [10] reported the cardioprotective effects of apigenin pretreatment in vitro and in vivo. Moreover, we further verified that apigenin pretreatment exerted significant renoprotection against renal IRI in vitro and in vivo. The safe and effective dosage range of apigenin has been explored in multiple studies [10,22]. Therefore, we used a concentration of 4 mg/kg apigenin in a rat kidney model and 1 μM apigenin in a cultured renal tubular epithelial cell model. Our results demonstrated that apigenin suppressed the functional and histological alterations of I/R-injured kidneys associated with oxidative stress and apoptosis. Lipid peroxidation plays a key role in the I/R procedure. We found that activation of SOD and GSH-Px reduced and the increased level of MDA after I/R. When we administered apigenin to rats subjected to I/R, SOD and GSH-Px levels were increased, and the level of MDA decreased. The results supported the antioxidant activity of apigenin. Furthermore, the effect of apigenin on renal tubular epithelial cell apoptosis was also studied. The expression levels of the pro-apoptotic protein Bax and Caspase-3 and the anti-apoptotic protein Bcl-2 were detected by immunohistochemistry. The cell viability assay and flow cytometry were performed to assess cell viability and cell apoptosis. We found that apigenin treatment observably alleviated cell apoptosis and increased cell viability, as evidenced by increased Bcl-2 expression and decreased Bax and caspase-3 expression. In summary, the renal protective effect of apigenin may be related to anti-apoptosis and anti-oxidative stress. A previous study showed that myocardial IRI was inhibited by activation of the JAK2/STAT3 signaling pathway [23]. The JAK2/STAT3 signaling pathways has been reported to be a pivotal signaling pathway for a series of stress responses such as oxidative stress, ischemia and hypoxia [24–26]. The JAK2/STAT3 signaling pathway, a core component of organ protection, is involved in various organs, including brain,
Fig. 7. Effects of apigenin and JAK2 siRNA on cell viability in NRK-52E cells subjected to I/R injury. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; # P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
in SOD and GSH-Px levels was observed in the IR + apigenin + siJAK2 group (p < 0.01), which indicated that JAK2 siRNA treatment significantly decreased the SOD level and GSH-Px level in I/R-injured NRK-52E cells (Fig. 6).
3.7. The effects of apigenin and JAK2 siRNA on the viability of I/R-injured NRK-52E cells To survey the effects of apigenin and AZD1480 on the viability of I/ R-injured NRK-52E cells, the cells were subjected to the CCK-8 assay. As depicted in Fig. 7, IR exposure significantly decreased the rate of cell survival compared with the control group (p < 0.01), and apigenin resulted in a distinct increase in viability (p < 0.01). However, JAK2 siRNA treatment significantly decreased the viability of I/R-injured NRK-52E cells (p < 0.01, Fig. 7).
3.8. The effects of apigenin and JAK2 siRNA on the apoptotic index of I/Rinjured NRK-52E cells To verify and quantify the apoptotic cells induced by apigenin, we used the Annexin V-FITC/PI assay to analyze the percentage of apoptotic cells. As shown in Fig. 8, the Annexin V-FITC/PI assay showed that cell apoptosis increased significantly in the I/R group compared with the control group (p < 0.01). Additionally, cell apoptosis was clearly attenuated by treatment with apigenin (p < 0.01). Treatment with JAK2 siRNA produced a conspicuous increase in cell apoptosis compared with the I/R+ apigenin group (p < 0.01, Fig. 8A and B). Furthermore, the expression of apoptosis-related proteins was detected. Western blot analysis showed that the expression of Bcl-2 and pro-Caspase-3 was reduced and the expression of Bax and cleavedCaspase-3 was increased after I/R (p < 0.01). Compared with the I/R group, a significant increase in expression of Bcl-2 and pro-Caspase-3 and a significant decrease in expression of Bax and cleaved-Caspase-3 were observed in the I/R+ apigenin group (p < 0.01). Moreover, when the NRK-52E cells were treated with JAK2 siRNA and I/R, there was a significant decrease in Bcl-2 and pro-Caspase-3 expression and an increase in Bax and cleaved-Caspase-3 expression compared with the I/ R+ apigenin group (p < 0.01, Fig. 8C and D). The results indicated that apigenin inhibited the apoptosis of renal tubular epithelial cells, while AZD1480 had stronger apoptosis-inducing effects.
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Fig. 8. Effects of apigenin and JAK2 siRNA on the apoptosis of NRK-52E cells subjected to I/R injury. (A) Flow cytometry assays showing apoptosis of the NRK-52E cells treated with apigenin and JAK2 siRNA; (B) quantification of the apoptosis rate in NRK-52e cells; (C) representative western blots showing the effects of apigenin and JAK2 siRNA on Bax, Bcl-2, proCaspase-3 and cleaved–Caspase-3 expression levels; (D) quantitative analyses of the expression of Bcl-2, Bax, pro-Caspase-3 and cleaved–Caspase-3 in NRK-52E cells by western blotting. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
pathway in the protection of apigenin against renal IRI. Our results demonstrated that AZD1480 and JAK2 siRNA abolished the renoprotective effect of apigenin, as evidenced by a decrease in cell viability and an increase in the apoptotic index. AZD1480 and JAK2 siRNA strongly suppressed p-JAK2 and p-STAT3 expression and attenuated the apigenin-induced decrease in Bax and Caspase-3 expression and increase in Bcl-2 expression. Furthermore, AZD1480 and JAK2 siRNA caused a significant decrease in SOD and GSH-Px levels and an increase in MDA in vivo, which indicated the JAK2/STAT3 signaling pathway may be involved in anti-apoptotic pathways and antioxidant effects. In summary, the current study demonstrated that apigenin provided protection against renal ischemia/reperfusion injury via activation of the JAK2/STAT3 pathway and that inhibition of the JAK2/STAT3 signaling
liver, kidney and heart [27–30]. Cumulative evidences has suggested that the JAK2/STAT3 signaling pathway plays a crucial role in IRI, which is associated with the upregulation of protective and anti-apoptosis proteins [31]. However, the role of the JAK2/STAT3 signaling pathway in the renal protective effects of apigenin has not been investigated. Our experiment was performed to determine whether apigenin could protect against renal IRI via the JAK2-STAT3 signaling pathway. These results indicated that the renoprotective effect of apigenin treatment was dependent on activation of the JAK2/STAT3 signaling pathway. AZD1480 is an ATP-competitive small molecule inhibitor of JAK2 that has been applied to attenuate the JAK2/STAT3 signaling pathway in various studies [32–34]. We used 1 μM AZD1480 and JAK2 siRNA to explore the role of the JAK2/STAT3 signaling 1806
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Fig. 9. The expression of p-JAK2 and p-STAT3 in NRK-52E cells by western blotting. (A) Representative western blots showing the effects of apigenin and JAK2 siRNA on p-JAK2, JAK2, p-STAT3 and STAT3 expression levels; (B) quantitative analyses of the expression of p-JAK2 and p-STAT3 in NRK-52E cells by western blotting. Bars represent the means ± SE (n = 10). * P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
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pathway could reverse the renoprotective effect of apigenin. There are some limitations to our study. Our studies were performed to verify the renoprotective effect of apigenin and the role of the JAK2/ STAT3 signaling pathway in renal IRI. However, the role of other signaling pathways should be explored in future studies. Moreover, our study did not use other JAK2 inhibitors such as AG490 and LY2784544. Therefore, these factors should be considered in further studies. Disclosure Authors have no conflict of interests, and the work was not supported or funded by any drug company. References [1] C. Shingu, H. Koga, S. Hagiwara, S. Matsumoto, K. Goto, I. Yokoi, T. Noguchi, Hydrogen-rich saline solution attenuates renal ischemia-reperfusion injury, J. Anesth. 24 (2010) 569. [2] Y. Wang, S.W. Seto, J. Golledge, Therapeutic effects of renal denervation on renal failure, Curr. Neurovasc. Res. 10 (2013) 172. [3] J. Zhang, Y.R. Zou, X. Zhong, H.D. Deng, L. Pu, K. Peng, L. Wang, Erythropoietin pretreatment ameliorates renal ischaemia-reperfusion injury by activating PI3K/ Akt signalling, Nephrology (Carlton) 20 (2015) 266. [4] H. Ozturk, A. Cetinkaya, F. Yilmaz, H. Ozturk, Protective effect of oxymatrine against renal ischemia/reperfusion injury in rats, Bratisl. Lek. Listy 118 (2017) 217. [5] A.P. Singh, N. Singh, P. Bedi, Estradiol mitigates ischemia reperfusion-induced acute renal failure through NMDA receptor antagonism in rats, Mol. Cell. Biochem. 434 (2017) 33. [6] D.F. Birt, S. Hendrich, W. Wang, Dietary agents in cancer prevention: flavonoids and isoflavonoids, Pharmacol. Ther. 90 (2001) 157. [7] D. Patel, S. Shukla, S. Gupta, Apigenin and cancer chemoprevention: progress, potential and promise (review), Int. J. Oncol. 30 (2007) 233. [8] C. Nicholas, S. Batra, M.A. Vargo, O.H. Voss, M.A. Gavrilin, M.D. Wewers, D.C. Guttridge, E. Grotewold, A.I. Doseff, Apigenin blocks lipopolysaccharide-induced lethality in vivo and proinflammatory cytokines expression by inactivating NF-kappaB through the suppression of p65 phosphorylation, J. Immunol. 179 (2007) 7121. [9] A.K. Tsaroucha, A. Tsiaousidou, N. Ouzounidis, E. Tsalkidou, M. Lambropoulou, D. Giakoustidis, E. Chatzaki, C. Simopoulos, Intraperitoneal administration of apigenin in liver ischemia/reperfusion injury protective effects, Saudi J. Gastroenterol. 22 (2016) 415. [10] C. Chen, H. He, Y. Luo, M. Zhou, D. Yin, M. He, Involvement of Bcl-2 signal pathway in the protective effects of apigenin on anoxia/reoxygenation-induced myocardium injury, J. Cardiovasc. Pharmacol. 67 (2016) 152. [11] A.K. Tsaroucha, A. Tsiaousidou, N. Ouzounidis, E. Tsalkidou, M. Lambropoulou, D. Giakoustidis, E. Chatzaki, C. Simopoulos, Intraperitoneal administration of apigenin in liver ischemia/reperfusion injury protective effects, Saudi J. Gastroenterol. 22 (2016) 415. [12] M.S. Paller, J.R. Hoidal, T.F. Ferris, Oxygen free radicals in ischemic acute renal failure in the rat, J. Clin. Invest. 74 (1984) 1156. [13] S. Khazaei, V. Ramachandran, H.R. Abdul, E.N. Mohd, A. Etemad, S. Moradipoor, P. Ismail, Flower extract of Allium atroviolaceum triggered apoptosis, activated caspase-3 and down-regulated antiapoptotic Bcl-2 gene in HeLa cancer cell line, Biomed. Pharmacother. 89 (2017) 1216. [14] A.E. El-Sisi, S.S. Sokar, S.E. Abu-Risha, H.A. Ibrahim, Combination of tadalafil and diltiazem attenuates renal ischemia reperfusion-induced acute renal failure in rats,
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Original article
Effects of apigenin pretreatment against renal ischemia/reperfusion injury via activation of the JAK2/STAT3 pathway
MARK
Yang Liua, Lei Wanga, Yang Dua, Zhiyuan Chena, Jia Guoa, Xiaodong Wenga, Xiao Wanga, ⁎ Min Wanga, Danyang Chenb, Xiuheng Liua, a b
Department of Urology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China Department of Plastic Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Apigenin JAK2/STAT3 AZD1480 Ischemia/reperfusion si-JAK2
The aim of our study is to investigate the protective effect of apigenin and the role of the JAK2/STAT3 signaling pathway in renal ischemia/reperfusion injury (IRI) in rats. For in vivo experiments, rat kidneys were subjected to 45 min of ischemia, followed by 24 h of reperfusion. The kidneys were pretreated for 24 h with apigenin (4 mg/ kg) intraperitoneally in the absence or presence of the JAK2 kinase-specific inhibitor AZD1480 (30 mg/kg). The serum creatinine and urea nitrogen levels were analyzed. Histologic examinations were evaluated. Expression of p-JAK2, p-STAT3, Bcl-2, Bax and Caspase-3 was detected by immunohistochemistry or western blot. For In vitro experiments, NRK-52E cells were exposed to I/R in the absence or presence of apigenin and JAK2 siRNA was used to explore JAK2/STAT3 activity. Cell viability, cell apoptosis and expression of p-JAK2, p-STAT3, Bcl-2, Bax and Caspase-3 were examined in NRK-52E culture after I/R. Consequently, apigenin conferred a renoprotective effect on the kidneys against IRI, as evidenced by decreased serum creatinine and urea nitrogen, mitigated renal histologic damage, improved NRK-52E cell viability and a decreased apoptotic index, including up-regulation of the anti-apoptotic protein Bcl-2 and down-regulation of the pro-apoptotic proteins Bax and Caspase3. However, AZD1480 and JAK2 siRNA blocked the apigenin-mediated renoprotective effects by attenuating the JAK2/ STAT3 signaling pathway as well as abolished the effect of anti-oxidative stress and anti-apoptosis of apigenin. Our study demonstrates that apigenin pretreatment can protect against renal IRI via the activation of the JAK2/ STAT3 signaling pathway.
1. Introduction
and renal functions [5]. Therefore, it is essential to explore novel therapies for effective management of renal IRI. Apigenin is a plant flavone that is present in a variety of fruits and vegetables, such as celery, parsley and wheat sprouts [6,7]. Apigenin has been proven to possess a series of biological properties, such as anti-inflammatory, anti-oxidant, and anti-tumor effects [8]. Apigenin is a type of antioxidant that has been reported to have protective effects on several organs. Apigenin can protect cells against apoptosis and necrosis by inhibiting oxidative stress. Recent studies have shown that apigenin has a potent therapeutic effects on liver in rats [9]. However, the effects of apigenin on renal ischemia/reperfusion injury remain unknown. Evidence indicates that the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway plays a pivotal role in mediating protection from IRI. Additionally, the JAK2/ STAT3 signaling pathway is associated with a mass of growth factors, cytokines and hormones. Accumulated evidence shows that the JAK2/ STAT3 signaling pathway is involved in preventing renal IRI.
Renal ischemia/reperfusion (I/R) is a primary cause of acute kidney injury (AKI) and an inevitable consequence of a series of operations and clinical entities, such as renal transplantation, partial nephrectomy, surgical revascularization of the renal artery, iatrogenic trauma and shock [1,2]. Renal ischemia/reperfusion injury (IRI) is an unavoidable incident in the setting of renal transplantation and entire blockage of renal blood flow; however, the pathologic mechanisms of renal IRI has not been fully researched. The pathogenesis underlying renal IRI is complicated and involves necrosis, apoptosis, oxidative stress, calcium ion overloading, and inflammation among others [3,4]. Despite impressive advances in medicine, the therapies available to clinicians for curing renal IRI are very limited. Presently, the available therapeutic options merely target symptoms, such as the drugs, vasodilators and diuretics that improve renal blood flow, including dopamine and fenoldopam as well as natriuretic peptide to protect cardiac
⁎
Corresponding author at: Department of Urology, Renmin Hospital of Wuhan University, Jiefang Road 238, Wuhan 430060, Hubei Province, China. E-mail addresses: [email protected] (Y. Liu), [email protected] (X. Liu).
http://dx.doi.org/10.1016/j.biopha.2017.09.091 Received 15 June 2017; Received in revised form 6 September 2017; Accepted 18 September 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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spectrophotometer (UV-1700; Shimadzu Corporation, Tokyo, Japan), and the absorbance was detected at 520 nm. The concentrations of BUN and Cr were then calculated.
Furthermore, previous studies have reported that apigenin inhibits hepatocyte necrosis and reduces the infarct size and apoptotic rate of cardiomyocytes after IR injury [10,11]. However, whether the protective effects of apigenin on renal IRI are induced via the JAK2/STAT3 signaling pathway requires further investigation. The purpose of our study was to investigate whether apigenin shows any protective effects on the renal IRI in vivo and anoxia/reoxygenation (A/R) in vitro and to evaluate whether the protective mechanisms of apigenin involve the JAK2/STAT3 signaling pathway.
2.3.3. Measurements of oxidative stress markers The renal tissues were homogenized and centrifuged at 3,000 × g for 10 min at 4 °C, and the supernatants were then collected for analysis of MDA, SOD and GSH-Px activity using commercial SOD, MDA and GSH-Px assay kits (A001-1-1; A003-1; A005. Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocols.
2. Materials and methods 2.3.4. Histological examination Half of each kidney was removed and fixed in 4% paraformaldehyde, followed by routine paraffin embedding. According to standard procedures, the tissue sections were cut at a thickness of 4 μm and stained with HE for histological grading. These morphological sections were assessed by an experienced renal pathologist. A grading scale described by Paller's standard [12] was used for the histopathological assessment of renal ischemia-reperfusion injury.
2.1. Drugs and reagents Apigenin (Api, purity > 98%, A106676) was purchased from Aladdin Biochemical Company (Shanghai, China). AZD1480 (purity > 98.81%; HY-10193) was purchased from MedChemExpress (MCE, USA). Anti-Bax (sc-493) and anti-Caspase-3 (sc-271759) antibodies were purchased from Santa Cruz Biotechnology (USA); anti-Bcl2 (A2212) and anti-GAPDH (A10868) antibodies were purchased from ABclonal Technology (USA); anti-cleaved caspase3 (9664), anti-pSTAT3 (9145) and anti-STAT3 (9139) antibodies were purchased from Cell Signaling Technology (CST, USA); anti-p-JAK2 (Bs-3206r) antibody were purchased from BIOSS (peking, China); anti-JAK2 (17670-1-AP) antibody were purchased from Proteintech Group (Wuhan, China).
2.3.5. Immunohistochemistry assay The expression of Bax, Caspase-3, Bcl-2, p-JAK2, p-STAT3 were assessed by immunohistochemical staining. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide at 37 °C for 10 min. The sections were then treated with 1:50 normal horse serum in Trisbuffered saline (TBS) for 30 min at 37 °C. Rabbit primary antibody was then added and incubated overnight at 4 °C. The sections were washed three times with PBS. After incubating with the secondary antibody for 30 min at 20 °C, these sections were treated with the color reagent DAB. The average optical density (AOD) was calculated from 5 random fields per slide using Image-Pro Plus software, version 5.0 (MediaCybernetics, Shanghai, China), and AOD is presented as the mean (SD) based on 3 detection times.
2.2. Animal preparation This study was approved by the local ethical committee, and the experimental procedures were carried out in accordance with the principles of the Helsinki Declaration. Fifty adult male Sprague-Dawley rats (weighing 220 ± 20 g) were provided by Hubei Provincial Academy of Preventive Medicine. 2.3. In vivo experiments
2.3.6. Western blotting assay The kidney tissues were dissociated by using the total protein extraction kit (purchased from Wuhan Goodbio technology company) according to the specification supplied with the kit, and total proteins extracted were examined via western blotting, in which 40 μg of protein from each sample was separated on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. Then, the membranes were then blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 (TBST) buffer and incubated with then primary polyclonal antibodies (1:500) anti-Bax (sc-493; SANTA CRUZ), anti-Bcl-2 (A2212; ABclonal), antiCaspase-3 (sc-271759; SANTA CRUZ), anti-cleaved Caspase3 (9664; CST), anti-p-JAK2 (Bs-3206r; BIOSS, China), anti-p-STAT3 (9145; CST), anti-JAK2 (17670-1-AP; PROTEINTECH, China), anti-STAT3 (9139; CST) and anti-GAPDH (A10868; ABclonal) at 4 °C overnight. After being washed three times with TBST for approximately 15 min, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:1000 dilution; BA1054; Boster Biological Technology co.ltd) or goat anti-mouse secondary antibody (1:1000 dilution; BA1051; Boster Biological Technology co. ltd). The membranes were then washed 3 times with TBS-T. Specific bands were visualized using an enhanced chemiluminescence detection kit. The immune complexes were visualized by an enhanced chemiluminescence detection kit. Ultimately, the band intensity were detected using Quantity One software.
2.3.1. Experimental protocol Fifty rats were randomly separated into five different groups (10 rats in each group): (1) sham-operation group: rats were pretreated with normal saline; (2) I/R group: the rats were pretreated with normal saline; (3) I/R + apigenin group: the rats were intraperitoneally injected with apigenin (4 mg/kg) 24 h before the experiment; (4) I/R + apigenin + AZD1480 group: the rats were intraperitoneally injected with both apigenin (4 mg/kg) and AZD1480 (30 mg/kg) 24 h before the experiment; (5) I/R + AZD1480 group: The rats were intraperitoneally injected with AZD1480 (30 mg/kg) alone. All rats were intraperitoneally anesthetized with chloral hydrate (350 mg/kg). After i.v. Injection of heparin (1000 UI/kg), body temperature was maintained at 37 °C, and a midline laparotomy was performed. Renal ischemia was induced by clamping both renal pedicles for 45 min using the artery clips, and the clamp was removed to restore kidney blood flow. A change in color of the kidneys to a paler shade and reperfusion resulting in a blush verified occlusion. sham operation group was subjected to the same surgical procedures as the I/R group without renal clamping. At 24 h after I/R injury, all rats were sacrificed. Blood samples (1 ml) were collected from the heart to measure urea creatinine (Cr) and nitrogen (BUN). The left kidney was removed and fixed in 4% paraformaldehyde or immediately frozen and stored at −80 °C for routine paraffin embedding and different determinations.
2.4. In vitro experiments 2.3.2. 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, C011-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance was measured using a
2.4.1. Experimental protocol The NRK-52E cells were randomly divided into 5 groups: (1) Control group: cells were pretreated with PBS; (2) I/R group: cells were pretreated with PBS; (3) I/R +apigenin group: cells were pretreated for 24 h with apigenin (1 μM); (4) I/R +apigenin + si-JAK2 group: cells 1800
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Fig. 1. Effects of apigenin and AZD1480 on renal function. (A) Effects of apigenin and AZD1480 on serum BUN concentrations after 45 min of ischemia; (B) effects of apigenin and AZD1480 on serum Cr concentrations after 45 min of ischemia. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
for 5 min at room temperature in the dark, followed by flow cytometry analysis.
were treated with JAK2 siRNA and pretreated for 24 h with apigenin (1 μM); (5) I/R +si-JAK2 group: cells were pretreated with JAK2 siRNA; (6) I/R +si-NC group: cells were pretreated with a control siRNA. Before the process, all the groups were cultured for 24 h simultaneously. After that, the control group was incubated as a routine culture by adding a control medium (NaHCO3 24.0 mM, Na2HPO4 0.8 mM, NaH2PO4 0.2 mM, NaCl 86.5 mM, KCl 5.4 mM, CaCl2 1.2 mM, MgCl2 0.8 mM, HEPES 20 mM and 5 mM glucose; pH 7.4). The I/R group was cultured with anoxia medium (NaHCO3 4.5 mM, Na2HPO4 0.8 mM, NaH2PO4 0.2 mM, NaCl 106.0 mM, KCl 5.4 mM, CaCl2 1.2 mM, MgCl2 0.8 mM and morpholinoethanesulfonic acid 20 mM; pH 6.6) and then exposed to hypoxia (37 °C, 0.5% O2, 5% C02, 94.5% N2) for 3 h and to reoxygenation (37 °C, 21% O2, 5% CO2, and 74% N2) for 24 h.
2.4.5. Western blotting assay The cells were washed 3 times with PBS and then trypsinized and suspended in DMEM. Cell suspensions were centrifuged at 1000 rpm for 5 min to remove the supernatant. Total proteins were examined by western blotting, in which 40 μg of protein from each sample was separated on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. The membranes were then blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 (TBST) buffer and incubated with primary polyclonal antibodies (1:500) of anti-Bax (sc-493; SANTA CRUZ), anti-Bcl-2 (A2212; ABclonal), anti-Caspase-3 (sc-271759; SANTA CRUZ), anti-cleaved caspase3 (9664; CST), anti-p-JAK2 (Bs3206r; BIOSS, China), anti-p-STAT3 (9145; CST), anti-JAK2 (17670-1AP; PROTEINTECH, China), anti-STAT3 (9139; CST) and anti-GAPDH (A10868; ABclonal) at 4 °C overnight. After being washing three times with TBST for approximately 15 min, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:1000 dilution; BA1054; Boster Biological Technology co. ltd) or goat anti-mouse secondary antibody (1:1000 dilution; BA1051; Boster Biological Technology co. ltd). The membranes were then washed 3 times with TBS-T. Specific bands were visualized using an enhanced chemiluminescence detection kit. The immune complexes were visualized using an enhanced chemiluminescence detection kit. Ultimately, the band intensity were detected using Quantity One software.
2.4.2. Small interference RNA (siRNA) treatment The plasmid including the small interfering RNA (siRNA) targeting JAK2 (si-JAK2: 5′-GCAGTTCAGTCAGTGCAAACCTAAGGACTTCAA CAAATGCAGTATACATCCCAGATA-3′) and a control siRNA (si-NC: 5′TTCTCCGAACGTGTCACGT-3′) were provided by Biossci Company (Wuhan, China). The NRK-52E cells were transfected using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocol. The expression of JAK2 was confirmed by western blot analysis. Eventually, the cells were collected for further analyses. 2.4.3. Analysis of cell viability The NRK-52E cells were inoculated in a 96-well plate. After 24 h of culture, the cells were treated with CCK-8 solution, and the plate was incubated in an incubator for 4 h. The absorbance (OD) of cells at a wavelength of 450 nm was measured using a microplate reader. The calculation formula for cell survival rate was as follows: [treatment group (OD) − blank group (OD)]/[control group (OD) − blank group (OD)] × 100%.
2.4.6. Statistical analysis All data are expressed as the mean ± SD. Significant differences between groups were tested by analysis of variance, and all statistical analyses were processed using SPSS 11.0 statistic software. P < 0.05 was regarded as statistically significant. 3. Results
2.4.4. Annexin V-FITC/PI detection of apoptosis The Annexin V-FITC/PI assay was carried out using the Annexin VFITC Apoptosis Detection Kit according the instructions provided for flow cytometry. NRK-52E cells were collected and washed twice with cold PBS and then resuspended in binding buffer and incubated with 10 μl Annexin V-fluorescein isothiocyanate and 5 μl propidium iodide
3.1. The effects of apigenin and the JAK2 inhibitor AZD1480 on the renal function in vivo Initially, to investigate the effects of apigenin and the JAK2 1801
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Fig. 2. Histologic features were evaluated by HE staining. (A) HE staining, original magnification × 400; the yellow arrow indicates the glomerulus, and the black arrow indicates the kidney tubules. (B) Paller scores for the histological appearance of acute tubular necrosis. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇ P < 0.01 versus IR + apigenin.
antioxidant effect of apigenin (p < 0.01, Fig. 3). Our results showed that apigenin could protect the kidney against damage from oxidative stress and that AZD1480 could attenuate the antioxidant capacity of apigenin.
inhibitor AZD1480, we evaluated renal function by measuring serum urea nitrogen (BUN) and creatinine (Cr). Compared with the shamoperated group, the I/R group showed significant increases in BUN and Cr (p < 0.01). However, apigenin treatment decreased the levels of BUN and Cr significantly compared with the I/R group (p < 0.01). In addition, the BUN and Cr levels in the IR + apigenin + AZD1480 groups and IR + AZD1480 group were significantly higher than those in the IR + apigenin group(p < 0.01), which suggest that renal protection by apigenin was significantly weakened by treatment with AZD1480 (Fig. 1).
3.4. The effects of apigenin and AZD1480 on JAK2 and STAT3 phosphorylation in IRI kidneys in vivo The expression levels of the phosphorylation of JAK2 (p-JAK2) and STAT3 (p-STAT3) were measured by immunohistochemical staining and western blot analysis. Western blot analysis demonstrated that pJAK2 and p-STAT3 expression was significantly increased in response to renal IR injury (p < 0.01). Apigenin dramatically enhanced the phosphorylation of JAK2 and STAT3 in IR-exposed kidneys (p < 0.01), whereas cotreatment with AZD1480 and apigenin decreased the expression of p-JAK2 and p-STAT3 (p < 0.01, Fig. 4A and B). In addition, p-JAK2 and p-STAT3 were localized by immunohistochemical staining, consistent with western blot analysis. The blue color indicated the cell nuclei of renal tubular cells and glomerular cells, and the brown color indicated positive expression of p-JAK2 and p-STAT3 in kidneys. Stained sections showed that renal tissues were highly positive for p-JAK2 and p-STAT3 expression in the I/R group (p < 0.01), and this tendency was strengthened by apigenin treatment (p < 0.01). However, the expression of p-JAK2 and p-STAT3 was inhibited by AZD1480 (p < 0.01, Fig. 4C–E). These results indicated that apigenin could activate the JAK2/STAT3 signaling pathway, and inhibition of JAK2/STAT3 signaling pathway could weaken the renoprotective effect of apigenin.
3.2. The effects of apigenin and AZD1480 on the morphological features of injury in vivo In the I/R group, morphologic abnormalities including tubular cell necrosis, cytoplasmic vacuolization and tubular lumen obstruction and impairments, were observed by in HE. Apigenin treatment relieved this severe renal damage. However, compared with the IR + apigenin group, this damage was greater in the IR + apigenin + AZD1480 group (Fig. 2A). Furthermore, the renal histological evaluation was quantified according to the Paller scale histological grading scores. Quantitative analysis showed a lower histologic score in the IR + apigenin group compared with IR group (p < 0.01). Additionally, the histologic scores in the IR + apigenin + AZD1480 group were clearly higher than those in I/R group (p < 0.01, Fig. 2B). These results demonstrated that apigenin could relieve renal damage and AZD1480 could reverse the renoprotective effect of apigenin. 3.3. The effects of apigenin and AZD1480 on the levels of SOD, MDA and GSH-Px in vivo
3.5. The effects of apigenin and AZD1480 on the expression of apoptosisrelated proteins in vivo
The I/R group showed a significant decrease in SOD and GSH-Px levels and a significant increase in MDA levels compared with the shamoperated after I/R (p < 0.01). Nevertheless, apigenin inhibited the decrease in SOD and GSH-Px levels and reduced the level of MDA (p < 0.01). However, treatment with AZD1480 abrogated the
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Fig. 3. Effects of apigenin and AZD1480 on oxidative stress in rats at 24 h after reperfusion. (A) The level of SOD in the renal tissue; (B) the level of MDA in the renal tissue; (C) the level of GSH-Px in the renal tissue. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
Fig. 4. The effects of apigenin and AZD1480 on JAK2 and STAT3 phosphorylation following the IR procedure. (A)Representative western blots showing the effects of apigenin and AZD1480 on p-JAK2, JAK2, p-STAT3 and STAT3 expression levels; (B) quantitative analyses of the expression of p-JAK2/JAK2 and p-STAT3/STAT3 by western blotting; (C) immunohistochemistry was performed to measure the expression of p-JAK2 and p-STAT3; (D) average optical density (AOD) of p-JAK2 and p-STAT3 were determined as described. Bars represent the means ± SE (n = 10). *P[Please apply a consistent format for the formatting of the ‘p’ to denote p-values.] < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
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Fig. 5. The expression of Bcl-2, Bax, pro-Caspase-3 and cleaved-Caspase-3 by western blotting. (A) Representative western blots showing the effects of apigenin and AZD1480 on Bax, Bcl2, pro-Caspase-3 and cleaved-Caspase-3 expression levels; (B) quantitative analyses of the expression of Bcl-2, Bax, pro-Caspase-3 and cleaved-Caspase-3 in NRK-52E cells by western blotting. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
apigenin inhibited the expression of pro-apoptotic proteins and increased the expression of anti-apoptotic proteins; however, AZD1480 abated the anti-apoptosis effect of apigenin.
the levels of Bax, Bcl-2, pro-Caspase-3 and cleaved-Caspase-3 were measured by western blot analysis. The results showed that the expression of Bcl-2 and pro-Caspase-3 in kidney tissues at the protein level decreased significantly in the I/R group compared with the sham-operated group (p < 0.01), and apigenin improved the expression of Bcl2 and pro-Caspase-3 in the I/R + apigenin group (p < 0.01). However, the increases were drastically abolished by AZD1480 treatment (p < 0.01, Fig. 5A). In contrast, Bax and cleaved-Caspase-3 expression showed an opposite result, such that the IR procedure clearly increased Bax and cleaved-Caspase-3 expression (p < 0.01). Additionally, the expression levels of Bax and Caspase-3 were reduced when the IR kidneys were subjected to apigenin treatment (p < 0.01). However, the decreases in Bax and cleaved-Caspase-3 expression induced by apigenin treatment were reversed by AZD1480 (p < 0.01, Fig. 5B and C). As shown,
3.6. The effects of apigenin and JAK2 siRNA on the levels of SOD, MDA and GSH-Px in vitro The levels of SOD, MDA and GSH-Px were evaluated in NRK-52E cells in vitro. As shown in Fig. 6, compared with the I/R group, apigenin pretreatment could decrease the MDA concentrations (p < 0.01). However, JAK2 siRNA treatment dramatically increased the MDA level in I/R-injured NRK-52E cells (p < 0.01). In contrast, I/R alone significantly decreased the levels of SOD and GSH-Px compared with the control group. Apigenin pretreatment increased the levels of SOD and GSH-Px compared with the I/R group (p < 0.01). However, a decrease
Fig. 6. Effect of apigenin and JAK2 siRNA on oxidative stress in NRK-52E cells subjected to I/R injury. (A) The level of SOD in NRK-52E cells; (B) the level of MDA in NRK-52E cells; (C) the level of GSH-Px in NRK-52E cells. Bars represent the means ± SE (n = 10). *P < 0.01 versus control; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
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3.9. The effects of apigenin and JAK2 siRNA on JAK2 and STAT3 phosphorylation in I/R-injured NRK-52E cells We examined the effect of apigenin and JAK2 siRNA on the JAK2/ STAT3 signaling pathway in I/R-injured NRK-52E cells by western blotting. These results indicated that p-JAK2 and p-STAT3 expression was elevated after IRI compared with the control group (p < 0.01). Apigenin improved the levels of p-JAK2 and p-STAT3 (p < 0.01). However, treatment with apigenin and si-JAK2 produced a significant decrease in the expression of p-JAK2 and p-STAT3 (p < 0.01, Fig. 9). These results suggested that the JAK2/STAT3 pathway was involved in the process of renal IRI, and apigenin activated the JAK2/STAT3 signaling pathway to exert a renoprotective effect. 4. Discussion Renal IRI, which often arises from kidney transplantation and nephrectomy, is an unresolved problem in clinical practice. Renal IRI is one of the main causes of acute renal failure (ARF), which leads to disease with high mortality and morbidity [14]. During the I/R procedure, after the aorta or renal pedicle is blocked, restoration of blood flow to ischemic tissue exacerbates the damage to the kidney [15]. The main mechanism of renal IRI is associated with multiple mediators including inflammation, oxidative stress and activation of adhesion molecules, ultimately resulting in renal tubular damage, inflammation, endothelial disordering and cell apoptosis [16,17]. At present, a therapeutic strategy using anti-apoptotic and anti-oxidative stress drugs may prevent the decline in renal function and alleviate tubular injury. In our study, renal protection of apigenin was evaluated as a potential therapeutic agent for renal IRI. Apigenin is an edible plant flavonoid that exists widely in a variety of fruits and vegetables and has gained extensive attention for its potential use in IRI and cancers [18]. The detailed mechanisms underlying the protective effect of apigenin vary extensively among studies, involving, e.g., reactive oxygen species (ROS), the Fas/FasL signaling pathway, nuclear factor-kappa B (NF-κB), endothelial nitric oxide synthase (eNOS) and cell autophagy [19–21]. Chuanjun et al. [10] reported the cardioprotective effects of apigenin pretreatment in vitro and in vivo. Moreover, we further verified that apigenin pretreatment exerted significant renoprotection against renal IRI in vitro and in vivo. The safe and effective dosage range of apigenin has been explored in multiple studies [10,22]. Therefore, we used a concentration of 4 mg/kg apigenin in a rat kidney model and 1 μM apigenin in a cultured renal tubular epithelial cell model. Our results demonstrated that apigenin suppressed the functional and histological alterations of I/R-injured kidneys associated with oxidative stress and apoptosis. Lipid peroxidation plays a key role in the I/R procedure. We found that activation of SOD and GSH-Px reduced and the increased level of MDA after I/R. When we administered apigenin to rats subjected to I/R, SOD and GSH-Px levels were increased, and the level of MDA decreased. The results supported the antioxidant activity of apigenin. Furthermore, the effect of apigenin on renal tubular epithelial cell apoptosis was also studied. The expression levels of the pro-apoptotic protein Bax and Caspase-3 and the anti-apoptotic protein Bcl-2 were detected by immunohistochemistry. The cell viability assay and flow cytometry were performed to assess cell viability and cell apoptosis. We found that apigenin treatment observably alleviated cell apoptosis and increased cell viability, as evidenced by increased Bcl-2 expression and decreased Bax and caspase-3 expression. In summary, the renal protective effect of apigenin may be related to anti-apoptosis and anti-oxidative stress. A previous study showed that myocardial IRI was inhibited by activation of the JAK2/STAT3 signaling pathway [23]. The JAK2/STAT3 signaling pathways has been reported to be a pivotal signaling pathway for a series of stress responses such as oxidative stress, ischemia and hypoxia [24–26]. The JAK2/STAT3 signaling pathway, a core component of organ protection, is involved in various organs, including brain,
Fig. 7. Effects of apigenin and JAK2 siRNA on cell viability in NRK-52E cells subjected to I/R injury. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; # P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
in SOD and GSH-Px levels was observed in the IR + apigenin + siJAK2 group (p < 0.01), which indicated that JAK2 siRNA treatment significantly decreased the SOD level and GSH-Px level in I/R-injured NRK-52E cells (Fig. 6).
3.7. The effects of apigenin and JAK2 siRNA on the viability of I/R-injured NRK-52E cells To survey the effects of apigenin and AZD1480 on the viability of I/ R-injured NRK-52E cells, the cells were subjected to the CCK-8 assay. As depicted in Fig. 7, IR exposure significantly decreased the rate of cell survival compared with the control group (p < 0.01), and apigenin resulted in a distinct increase in viability (p < 0.01). However, JAK2 siRNA treatment significantly decreased the viability of I/R-injured NRK-52E cells (p < 0.01, Fig. 7).
3.8. The effects of apigenin and JAK2 siRNA on the apoptotic index of I/Rinjured NRK-52E cells To verify and quantify the apoptotic cells induced by apigenin, we used the Annexin V-FITC/PI assay to analyze the percentage of apoptotic cells. As shown in Fig. 8, the Annexin V-FITC/PI assay showed that cell apoptosis increased significantly in the I/R group compared with the control group (p < 0.01). Additionally, cell apoptosis was clearly attenuated by treatment with apigenin (p < 0.01). Treatment with JAK2 siRNA produced a conspicuous increase in cell apoptosis compared with the I/R+ apigenin group (p < 0.01, Fig. 8A and B). Furthermore, the expression of apoptosis-related proteins was detected. Western blot analysis showed that the expression of Bcl-2 and pro-Caspase-3 was reduced and the expression of Bax and cleavedCaspase-3 was increased after I/R (p < 0.01). Compared with the I/R group, a significant increase in expression of Bcl-2 and pro-Caspase-3 and a significant decrease in expression of Bax and cleaved-Caspase-3 were observed in the I/R+ apigenin group (p < 0.01). Moreover, when the NRK-52E cells were treated with JAK2 siRNA and I/R, there was a significant decrease in Bcl-2 and pro-Caspase-3 expression and an increase in Bax and cleaved-Caspase-3 expression compared with the I/ R+ apigenin group (p < 0.01, Fig. 8C and D). The results indicated that apigenin inhibited the apoptosis of renal tubular epithelial cells, while AZD1480 had stronger apoptosis-inducing effects.
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Fig. 8. Effects of apigenin and JAK2 siRNA on the apoptosis of NRK-52E cells subjected to I/R injury. (A) Flow cytometry assays showing apoptosis of the NRK-52E cells treated with apigenin and JAK2 siRNA; (B) quantification of the apoptosis rate in NRK-52e cells; (C) representative western blots showing the effects of apigenin and JAK2 siRNA on Bax, Bcl-2, proCaspase-3 and cleaved–Caspase-3 expression levels; (D) quantitative analyses of the expression of Bcl-2, Bax, pro-Caspase-3 and cleaved–Caspase-3 in NRK-52E cells by western blotting. Bars represent the means ± SE (n = 10). *P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
pathway in the protection of apigenin against renal IRI. Our results demonstrated that AZD1480 and JAK2 siRNA abolished the renoprotective effect of apigenin, as evidenced by a decrease in cell viability and an increase in the apoptotic index. AZD1480 and JAK2 siRNA strongly suppressed p-JAK2 and p-STAT3 expression and attenuated the apigenin-induced decrease in Bax and Caspase-3 expression and increase in Bcl-2 expression. Furthermore, AZD1480 and JAK2 siRNA caused a significant decrease in SOD and GSH-Px levels and an increase in MDA in vivo, which indicated the JAK2/STAT3 signaling pathway may be involved in anti-apoptotic pathways and antioxidant effects. In summary, the current study demonstrated that apigenin provided protection against renal ischemia/reperfusion injury via activation of the JAK2/STAT3 pathway and that inhibition of the JAK2/STAT3 signaling
liver, kidney and heart [27–30]. Cumulative evidences has suggested that the JAK2/STAT3 signaling pathway plays a crucial role in IRI, which is associated with the upregulation of protective and anti-apoptosis proteins [31]. However, the role of the JAK2/STAT3 signaling pathway in the renal protective effects of apigenin has not been investigated. Our experiment was performed to determine whether apigenin could protect against renal IRI via the JAK2-STAT3 signaling pathway. These results indicated that the renoprotective effect of apigenin treatment was dependent on activation of the JAK2/STAT3 signaling pathway. AZD1480 is an ATP-competitive small molecule inhibitor of JAK2 that has been applied to attenuate the JAK2/STAT3 signaling pathway in various studies [32–34]. We used 1 μM AZD1480 and JAK2 siRNA to explore the role of the JAK2/STAT3 signaling 1806
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Fig. 9. The expression of p-JAK2 and p-STAT3 in NRK-52E cells by western blotting. (A) Representative western blots showing the effects of apigenin and JAK2 siRNA on p-JAK2, JAK2, p-STAT3 and STAT3 expression levels; (B) quantitative analyses of the expression of p-JAK2 and p-STAT3 in NRK-52E cells by western blotting. Bars represent the means ± SE (n = 10). * P < 0.01 versus sham; #P < 0.01 versus I/R; ⋇P < 0.01 versus IR + apigenin.
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