Xanthohumol protects against renal ischaemia reperfusion (I/R) injury by scavenging ROS and inhibition of JAK-2/STAT-3 inflammatory pathway

Peer review under responsibility of Taibah University.

Abstract

Recent evidence has shown that reactive oxygen species (ROS), inflammation and the activation of JAK/STAT signalling are major pathways in the induction and progression of renal ischaemia/reperfusion (I/R) injury. The protective effect of Xanthohumol (XN) against I/R induced renal injury has not been investigated. Hence, this study aimed to investigate the in vivo effect of XN against renal I/R injury. The rats used in this study were divided into 2 main groups of either 1) sham-operated or 2) subjected to renal I/R, in which each group was further divided into 2 subgroups: treated with oral administration of normal saline or XN (1 mg/kg) for 28 days. Renal function, histology, markers and expression levels of oxidative stress, inflammation and apoptosis were examined. The expression levels of the activated JAK-2/Stat-3 signalling pathway were also assessed. XN treatment in the sham group resulted in a normal response, as seen in the sham operated as control group. However, XN significantly improved renal function and attenuated histological changes by reducing the levels of oxidative stress and lipid peroxidation, inflammatory markers, adhesive molecules and the activity of myeloperoxidase (MPO). Concomitantly, reduced expression levels of activated caspase with a parallel decrease in JAK-1/Stat-3 phosphorylation were also noticed. In conclusion, these findings show, for the first time, a protective effect of XN against renal I/R injury, and the mechanisms of protection involve ROS scavenging and an anti-inflammatory effect mediated by the inhibition of the JAK-2/STAT-3 pathway.

Keywords:

• Renal ischaemia reperfusion (I/R)
• Xanthohumol
• JAK-2/Stat-3 signalling pathway
• Reactive oxygen species (ROS)

1 Introduction

Renal injury as a result of ischaemia reperfusion (I/R), which usually occurs after renal transplantation, shock, sepsis, and renal artery stenosis, is considered a major cause of acute renal failure (ARF) and is responsible for more than 50% of global mortality [1]. A high generation rate of reactive oxygen species (ROS), endothelial cell injury, and apoptosis have been shown to be implicated in the pathogenesis of the early phases of renal ischaemia/reperfusion (I/R) injury [2]. In addition, there is accumulating evidence that I/R injury results in inflammatory disease, as manifested by the infiltration of leucocytes, up-regulation of chemotactic factors by endothelial cells and generation of pro-inflammatory mediators by renal tubular epithelial cells [3].

During early I/R events, a massive influx of neutrophils after reperfusion occurs which result in the release of cytotoxic proteases and exacerbates the generation of ROS that worsen the oxidative damage [3,4], making apoptosis a common form of death of renal cells [5]. This process is enhanced due to the overexpression of endothelial cell adhesion molecules such as intracellular cell adhesion molecules (ICAMs), complement components such as C5a, and a number of chemokines including monocyte chemoattractant protein-1 (MCP-1) (of which the murine homologue is keratinocyte-derived chemokine (KC)) and macrophage inflammatory protein-2 (MIP-2) [3].

On the other hand, the JAK/STAT pathway is composed of a family of receptor-associated cytosolic tyrosine kinases (JAKs) that phosphorylate a tyrosine residue on bound transcription factors (STATs) [6]. Once phosphorylated, STAT family members are translocated to the nucleus where they augment gene transcription [7]. The activation of the JAK/STAT pathway has been confirmed as a major pathway involved in the development of renal I/R injury, during which many pro-inflammatory cytokines and adhesive molecules are up-regulated [6]. Activation of the JAK/STAT pathway mediates the up-regulation of MCP-1 and ICAM [8]. It has been found that the expression of MCP-1 and ICAMs is blocked by JAK/STAT inhibitors, resulting in decreased levels of ROS and inflammation under various conditions [8–10].

Xanthohumol (XN, 3-[3,3-dimethylallyl]-2,4,4-trihydroxy-6-methoxychalcone) is the principal prenylated chalcone of the female inflorescences (hop cones, hops) of the hop plant Humulus lupulus L. Hops (Humulus lupulus L.) flowers (Fig. 1A,B) are widely used throughout the world as a raw material in the brewing industry, to preserve beer and to give beer its characteristic aroma and flavour. In addition to their application in the brewing industry, hops have for a long time been used for various medical purposes [11]. Although XN has been traditionally used since ancient times to treat pain and improve appetite and digestion, recent studies have confirmed the ability of this flavonoid to treat various medical disorders including cancer [11]. Very recently, numerous studies have shown a protective effect of XN against ischaemia-related disorders due to its antioxidant and anti-inflammatory actions. In cerebral ischaemic rats, XN resulted in a reduction of the infarct volume and the improvement of neuro-behaviours due to its antioxidant and anti-inflammatory roles (i.e., decrease of hypoxia-inducible factor-1α (HIF-1α), iNOS expression, and free radical formation), apoptosis (i.e., TNF-α, active caspase-3) and platelet activation [12]. Additionally, XN reduced liver damage in a mouse model of warm I/R injury in a mechanism related to the inhibition of proinflammatory genes including IL-6, MCP-1 and ICAM-1 [13].

Fig. 1 Kidney function parameters in the serum and urine of all groups. Values are expressed as the means ± SD for 10 rats/group. Values were considered significantly different at P < 0.05. (a) Significantly different when compared to Sham group. (b) Significantly different when compared to Sham + XN group. (c) Significantly different when compared to I/R group.

To our knowledge, the protective effect of XN against I/R induced renal injury has not been investigated. Moreover, the anti-inflammatory role of XN being mediated by inhibition of the JAK/STAT pathway has not been studied. Therefore, given the important role of ROS and the JAK/STAT pathway in the development and progression of renal I/R injury and the antioxidant and anti-inflammatory effects of XN, this study aimed to investigate the protective role of XN against renal I/R injury and to investigate if such an effect is mediated through inhibition of the JAK/STAT pathway.

2 Materials and methods

2.1 Chemicals

Xanthohumol (XN, Cat. No. X0379) was obtained from Sigma Aldrich (St Louis, MO, USA), with a purity ≥96% determined by HPLC and was freshly prepared in normal saline to the final concentration used in the experimental procedure.

2.2 Animals

This study was performed between March 2015 and Decembers 2015. Forty male Wistar rats aged 12 weeks and weighing 250 ± 10 g from the same lineage and breeding stock were obtained from the animal house of the College of Science at King Khalid University (Abha, Saudi Arabia) and housed (5 rats/cage, 2 cages/group) under controlled conditions (temperature of 23 ± 1 °C, and a 12 h light:12 h dark cycle). The animals were allowed free access to normal chow and tap water. The experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). All experimental procedures in this study were approved by the ethical committee of the College of Science at King Khalid University.

2.3 Experimental design

The rats were divided into four groups each of 10 rats and classified as follows: (1) Sham group: Pre-treated with normal saline for 28 consecutive days and then exposed to the surgical procedure only as that in I/R exposed rats but without clamping the renal arteries; (2) Sham group + XN: Pre-treated with XN (1 mg/kg b.w) for 28 consecutive 28 and then exposed to surgical procedure only as in group 1; (3) I/R group: received normal saline for 28 days and then exposed to renal I/R as discussed below; (4): Xanthohumol (XN) pre-treated group (XH + I/R): pre-treated with XN (1 mg/kg b.w) for 28 days and then exposed to renal I/R on day 28.

However, selection of this dose was based on Dorn's study [13] that showed an antioxidant and anti-inflammatory protective role of XN against hepatic I/R injury at a similar dose. However, it is important to mention here that XN has been well tolerated and is considered safe, even at very high doses (up to 1000 mg/kg), as this dose did not affect the major organ functions, homoeostasis, or the protein, lipid, and carbohydrate metabolism in rats [14].

2.4 Induction of renal I/R injury

By the end of the treatment period, on day 28, I/R injury was performed under sodium pentobarbital anaesthesia (60–70 mg/kg, intraperitoneally) (Sigma Life Science, St Louis, MO, USA) where an abdominal incision was made and the renal vessels on both sides were identified. The renal arteries were bilaterally occluded with a micro-vascular clamp for 60 min. Then, the clamps were removed, and blood flow to the kidneys was re-established. The surgical incision was sutured, and rats were allowed to recover for 48 h with free access to food and water only. Groups 1 and 2 underwent the same surgical procedure without IR.

2.5 Blood, urine and tissue collection

At the end of the experimental period (end of the post-ischaemia 48 h period), blood samples were withdrawn, centrifuged (5000 rpm, 10 min) to collect serum. Then, the rats were housed in individual metabolic cages to collect a 24-h urine specimen to measure urine volume and urinary creatinine. After that, the rats were sacrificed by cervical dislocation after being anaesthetized with sodium pentobarbital (6–70 mg/kg, intraperitoneally), after which both kidneys were removed. Parts of the first kidney were used for histological evaluation and the other parts were immediately frozen and used later for western blot studies. Parts of the other kidney were frozen and homogenized in appropriate buffers as per kit instructions to prepare homogenates, which were stored at −80 °C and used for biochemical analysis.

2.6 Parameters of urine and serum and creatinine (Cr) clearance

Serum Cr concentration was determined by the commercial colorimetric assay kit purchased from the Cayman Company (Ann Arbor, MI, USA; Cat., no. 700460). The concentration of Cr in urine samples was determined by the commercial colorimetric assay kit (Cayman Company, Ann Arbor, MI, USA; Cat., no. 500701). Blood urea nitrogen (BUN) was determined by analysis with a kinetic reagent (Diagnostic Chemicals Limited, Cat., no. 283-30). All analyses were performed in accordance with the manuals provided by the manufacturers. Cr clearance (Ccr) was calculated using the following equation (Liu et al., 2012):$\text{Ccr}(\text{ml/min/kg})=\left[\frac{\text{urinary}\text{Cr}(\text{mg/dl})\times \text{urinary}\text{volume}(\text{ml/min})}{\text{serum}\text{Cr}(\text{mg/dl})}\right]\times [1000/\text{body}\text{weight}(\text{g})]\times [1/1440(\min )]\text{.}$

2.7 Biochemical measurements in renal homogenates

The levels of malondialdehyde (MDA), a lipid peroxidation marker, were measured as levels of thiobarbituric acid reactive substances (TBARS) using a commercial assay kit (Cat No. NWK-MDA01, NWLSS, USA). Reduced glutathione (GSH) and glutathione disulphide (GSSG) concentrations were measured using an assay kit (Cat. No. 703002, Cayman Chemical, Ann Arbor, MI, USA). Myeloperoxidase (MPO) activity was measured spectrophotometrically by a commercially available colorimetric kit purchased from Hycult Biotec. Inc. (Netherland, Cat. no. HK105-02) using a UV-visible spectrophotometer (UV-160A, Shimadzu, Japan). Special ELISA kits to measure levels IL-6 (Cat No. ELR-IL6-001, RayBio, MO, USA), TNF-α (ab100785, Abcam), MIP-2 (Cat. No. KRC1021, Life Technology, USA), KC (Cat. No. KMC1061, Invitrogen, USA), and IL-1β (Cat. No. ab100768, Abcam) were used according to the manufacturer's instructions.

2.8 Western blot

Total proteins of the frozen renal tissue were extracted with I ml RIPA buffer (150 mM sodium chloride, 1.0% NP-40 or Triton X-100 0.5%, sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate), 50 mM Tris, pH 8.0) to which protease and phosphatase inhibitors were added. The protein concentration in this whole cell lysate was measured by Bradford assay. The sample, diluted in loading buffer and heated at 95 °C for 5 min, was then subjected to electrophoresis on 10% SDS-PAGE gel at 100 V for 2 h (60 μg protein/well). After electrophoresis of the gel and transfer of the proteins to nitrocellulose membrane, the membranes were rinsed briefly in Tris-buffered saline containing 0.05% Tween (TBST) buffer, blocked in blocking buffer (5% milk and 0.5% BSA) for 1 h, and washed three times with TBST buffer. The membranes were incubated with different primary monoclonal and polyclonal antibodies: JAK-2 (SC344479, 128 kDa), p-JAK-2 (CS21780, 128 kDa), STAT-3, p-STAT-3, STAT1 (SC482, 84/91 kDa) and p-STAT1 (SC-7993, 84/91 kDa)-, β actin (Sc-1616, 43 kDa) 8 hydroxyguanine (8-OHdG, sc-66036) and 4-Hydroxynonenal (4HNE, sc-130083), all of which were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA and Caspase 3 (32 kDa) and its resultant cleaved large fragment (17 kDa) (9662) were purchased from Cell signalling, Technology, USA) (ASP 175, Cell signalling, Technology, USA) at a dilution of 1:1000 for 2 h and then washed and reacted with corresponding secondary horseradish peroxidase-conjugated antibodies (Cell Signalling Technology) for other 2 h. Antigen-antibody complexes were then visualized using a Pierce ECL kit (Thermofisher, USA, Piscataway, NJ) photographed and bands intensities were quantified using Image J software (1.46R, NIH, USA). All experiments were performed in triplicate to represent the mean plus or minus SD of all data.

2.9 Histopathological studies

Parts of the kidney were rapidly fixed in 10% neutral buffered formalin, dehydrated in ascending concentrations of ethyl alcohol (70 to 100%) and then prepared using standard procedures for haematoxylin and eosin staining. Light microscopic examination of multiple tissue sections from each sample was performed and image representatives of the typical histological profile were examined.

2.10 Statistical analysis

Statistical analyses were performed by using Graphpad Prism statistical software package (version 6). The data are represented as the means ± SD. All comparisons were analyzed by one-way ANOVA followed by post hoc Tukey's test and were accepted as significant at P < 0.05.

3 Results

3.1 Markers of kidney function

Markers of kidney functions are depicted in Fig. 1. There were no significant changes in the levels of serum urea and creatinine (Cr) or urinary levels of Cr, urine volume or Cr clearance between the sham groups that received normal saline or XN. However, the levels of serum urea and Cr were significantly (P < 0.0001) increased and the urine volume, urinary Cr levels and Cr clearance were significantly (P < 0.0001) decreased in the I/R induced rats compared to sham rats. On the other hand, the levels of all these parameters showed significant improvement towards their normal levels seen in the sham rats in I/R rats pre-administered with XN. However, the levels of all these parameters remained slightly higher than those levels seen in the sham group.

3.2 Levels of inflammatory IL-6 and TNF-α

There were no significant changes in the renal levels of TNF-α and IL-6 between sham rats and sham rats pre-treated with XN. However, significant elevations (P < 0.001) in the levels of TNF-α and IL-6 were noticed in renal homogenates of I/R-induced rats compared to sham rats. However, pre-administration of XN prior to the induction of I/R injury resulted in significant decreases (P < 0.01) in the levels of both cytokines, when compared to I/R rats. In spite of this reduction, ANOVA analysis revealed significantly higher levels (P < 0.05) of both IL-6 and TNF-α in I/R + NX compared to sham (Fig. 2).

Fig. 2 Levels of tumour necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in the renal homogenates of all groups. Values are expressed as the means ± SD for 10 rats/group. Values were considered significantly different at P < 0.05. (a) Significantly different when compared to Sham operated group. (b) Significantly different when compared to Sham + XN group. (c) Significantly different when compared to I/R group.

3.3 Levels of chemokines and adhesion molecules

As depicted in Fig. 3, XN administration to sham rats did not significantly affect the renal levels of IL-1β, ICAM-1, MIP-1 and KC compared to sham rats that received normal saline. On the other hand, I/R resulted in significant elevations of all these parameters in the renal homogenates of ischaemic rats. However, significant decreases in the renal levels of these parameters were seen in the renal homogenates of I/R rats pre-administered XN compared to the model I/R group. ANOVA analysis showed that XN did not completely normalize the levels of these parameters and the levels remained significantly higher (P < 0.05) than their corresponding levels measured in the renal homogenates of the sham group.

Fig. 3 Levels of inflammatory chemokines in renal homogenates of the all experimental groups of rats. Values are expressed as the means ± SD for 10 rats/group. Values were considered significantly different at P < 0.05. (a) Significantly different when compared to Sham operated group. (b) Significantly different when compared to Sham + XN group. (c) Significantly different when compared to I/R group.

3.4 Activity of myeloperoxidase (MPO) and levels of oxidative stress

There were no significant alterations in the activities of renal MPO (Fig. 4) and renal levels of MDA, GSH, GSSG, the ratio of GSH/GSSG (Fig. 5), or the expression levels of 4HNE and 8-HG (Fig. 6) in sham groups pre-administered XN compared to the sham group that received normal saline. However, significant elevations (P < 0.05) in the activities of renal MPO and renal levels of MDA and GSSG, as well as the expression levels of 4HNE and 8-HG, with concomitant significant decreases (P < 0.05) in the renal levels of GSH and GSH/GSSG ratio were noticed in the renal homogenates of I/R rats (Figs. 4–6).

Fig. 4 Activity of myeloperoxidase activity (MPO) in renal homogenates of all experimental groups of rats. Values are expressed as the means ± SD for 10 rats/group. Values were considered significantly different at P < 0.05. (a) Significantly different when compared to Sham group. (b) Significantly different when compared to Sham + XN group. (c) Significantly different when compared to I/R group.

Fig. 5 Levels of malondialdehyde (MDA), reduced glutathione (GSH), glutathione disulfide (GSSG) and the ratio of GSH/GSSG in renal homogenates of all experimental groups of rats. Values are expressed as the means ± SD for 10 rats/group. Values were considered significantly different at P < 0.05. (a) Significantly different when compared to Sham group. (b) Significantly different when compared to Sham + XN group. (c) Significantly different when compared to I/R group.

Fig. 6 Levels of 4-hydroxyguanine (4-HG) and 4-hydroxynonenal (4HNE) in the renal tissue of all experimental groups of rats. Values are expressed as the means ± SD for 10 rats/group. Values were considered significantly different at P < 0.05. (a) Significantly different when compared to Sham group. (b) Significantly different when compared to Sham + XN group. (c) Significantly different when compared to I/R group.

Significant improvement characterized by a significant reduction in the activities of renal MPO and renal levels of MDA and GSSG, as well as expression levels of 4HNE and 8-HG, with parallel increases in the renal levels of GSG and GSH/GSSG ratio, were seen in I/R rats pre-administered XN compared to sham rats. Among all, only the renal levels of GSSG and GSH/GSSG ratio and expression levels of 4HNE and 8-HG were completely normalized, and their levels were not significantly different with those measured in the sham group.

3.5 Expression levels of Caspase 3 and cleaved Caspase 3

The expression levels of Caspase 3 remained constant and not significantly different between all experimental groups of rats. However, the levels of cleaved Caspase 3 were barely detected in the sham groups that received normal saline or XN. I/R significantly raised (P < 0.001) the expression of cleaved Caspase 3 compared to sham group. A significant reduction in the expression levels of cleaved Caspase 3 was seen in I/R rats pre-treated with XN (Fig. 7).

Fig. 7 Expression levels of Caspase 3 and Cleaved Caspase fragment in the renal tissue of all groups of rats. Values are expressed as the means ± SD for 6 rats/group. Values were considered significantly different at P < 0.05. (a) Significantly different when compared to Sham group. (b) Significantly different when compared to Sham + XN group. (c) Significantly different when compared to I/R group.

3.6 Activation of JAK-2/STAT-3

While there were no significant changes in the levels of JAK-2 and STAT-3 between the sham and sham + XN experimental groups, I/R significantly enhanced (P < 0.001) the expression levels of both p-JAK-2 and p-STAT-3, which resulted in their activation (Fig. 8). On the other hand, pre-administration of XN prior to I/R injury resulted in significant decreases (P < 0.001) in the phosphorylation levels of both JAK-2 and STAT-3 compared to I/R rats, but they remained significantly higher (P < 0.001) than their expression levels in sham rats (Fig. 8).

Fig. 8 Expression levels and activation ratios of JAK-2 and STAT-3 in the renal tissue of all groups of rats. Activation ratio was calculated as expressed levels of phosphorylated to unphosphorylated forms after being expressed normalized to β actin. Values are expressed as the means ± SD for 6 rats/group. Values were considered significantly different at P < 0.05. (a) Significantly different when compared to Sham group. (b) Significantly different when compared to Sham + XN group. (c) Significantly different when compared to I/R group.

3.7 Histopathological findings

Kidney sections obtained from all groups of rats are presented in Fig. 9. The sections of the sham groups that received normal saline or XN (A and B, respectively) showed normal histological structure of the renal corpuscles and renal tubules. The renal corpuscle consisted of a tuft of blood capillaries surrounded by Bowmann's capsule. The latter has a parietal layer lined by squamous cells and a visceral layer lined by podocytes. Sections of I/R rats (C) showed a high degree of loss of the brush border in renal tubules, tubular dilatation, severe tubular necrosis and apoptosis, dilatation of Bowman's capsule and increased urinary space. In addition, degeneration and shrinkage of the glomerular capillaries was evident. On the other hand, XN pre-treatment for 28 days prior the I/R injury significantly ameliorated the pathological findings seen in the renal tissues of the I/R injured group, and resulted in almost normal architectures as seen in the control group. However, some cortical tubules were still dilated and mild glomerular capillary degeneration and tubular necrosis was still seen.

Fig. 9 Photomicrographs of kidneys obtained from all groups of rats. (A) and (B) were taken from Sham and Sham + XN, respectively. These groups show normal architecture of kidney with prominent Bowman's capsule, epithelial cells and normal tubules. (C) was taken from I/R rat. The kidney sections of this rat shows mild thickening of the basement membrane along with changes in the density of mesenchyme, atrophy and degeneration of glomerular capillaries with increased Bowman's space (urinary space) and tubular necrosis and apoptosis. Glomerular capillaries showed severe shrinkage and damage. (D) was taken from treated rat and shows normal architecture of glomerular capillaries, intact epithelial cells with the presence of some degeneration in the tubules and slight shrinkage of the glomerular capillaries.

4 Discussion

Ischaemic acute renal failure (IARF), which is mainly caused by the reduction of blood supply to the kidney, is a common clinical event. Despite current medications and supportive preventive strategies, this disease continues to be associated with significant morbidity and mortality [15]. Indeed, complications of renal failure associated with ischaemia/reperfusion (I/R) during or after surgical and anaesthesia procedures are still major problems [16]. Thus, improving the abilities of the kidneys to tolerate I/R injury associated with surgery would have important implications [17]. Hence, this study aimed to investigate the protective effect of Xanthohumol (XN) against I/R induced renal damage and to investigate its molecular mechanisms of protection. Interestingly, the results of this study showed that in addition to its antioxidant potential, XN ameliorates renal I/R damage through the inhibition of JAK/STAT phosphorylation, which could explain its potent anti-inflammatory action.

To validate our model and safety of XN, the I/R model group showed severe renal damage associated with a decline in kidney function, increase of serum urea and creatinine, and decrease of urinary creatinine and decreased creatinine clearance. These changes correlated to extensive histopathological damages such as cellular vacoulation, apoptosis, tubular necrosis, glomerular damage and tubulointerstitial injury. Such effects were completely absent in the sham rats that received normal saline or XN, indicating success of our model, safety of our drug on renal function and confirming that the damage occurred after induction of I/R injury.

However, endothelial cell injury, generation of reactive oxygen species (ROS), and apoptosis have been implicated in the pathogenesis of the early phases of renal I/R injury [2]. During renal IR, hypoxanthine and xanthine accumulate within the ischaemic cells [18,19] and reperfusion stimulates the transformation of these metabolites to uric acid and excessive production of ROS [18,19]. Another source of ROS could originate from the massive early influx of neutrophils, which results in the release of ROS through the action of NADPH oxidase or MPO system [3,4]. It has been shown that excessive amount of ROS can cause cellular oxidative damage through inhibition of mitochondrial oxidative phosphorylation, ATP depletion, increasing intracellular calcium and activation of membrane phospholipid proteases [2]. Moreover, these ROS damage the cells by the peroxidation of membrane lipids and causing oxidative damage to the cellular proteins and DNA, contributing to apoptosis and cell death [20] to worsen the condition, and reduced levels of intracellular enzymatic and non-enzymatic antioxidants have been reported during and after early phases of renal I/R injury [21]. Therefore, inhibiting this pathway by preventing free radical production, or stimulating antioxidants and ROS scavenger levels are interesting strategies to protect the renal tissue during I/R. Indeed, numerous studies have shown beneficial effects of natural free radical scavengers and antioxidants on IRI [18,22]. In the current study, XN protected the animals from ROS oxidative damage by enhancing the levels of reduced glutathione (GSH) and reducing levels of lipid peroxidation as measured by MDA. In addition, XN reduced subcellular damage as indicated by decreased levels of 4-HNE and 8-HG, common markers of oxidative lipid peroxidation and DNA damage as a result of ROS, as depicted by western blot data. Thus, these results suggest that the protective effect of XN against renal I/R involves an antioxidant scavenger capacity. However, further investigation on the effect of XN on GSH synthesis pathway should be considered, and were not performed in this study. These results are supported by previous studies that have shown the antioxidant potential of XN in various conditions where XN could directly scavenge ROS [23]. Similar to our findings, Hartkorn et al. [24] and Dorn et al. [13] reported an antioxidant preventative effect of XN against liver induced I/R injury. Furthermore, XN has cellular defence mechanisms against oxidative damages induced by chemicals [25]. Moreover, XN protects human LDL from oxidation [26].

On the other hand, accumulated evidence shows that IARF is also an inflammatory disease, as manifested by the infiltration of leucocytes, up-regulation of chemotactic factors by endothelial cells and generation of pro-inflammatory mediators by renal tubular epithelial cells [27]. Leucocytes are usually recruited to the sites of injury by pro-inflammatory cytokines interleukin-1, (IL-1), g-interferon (IFN-g) and TNF-a [2] During renal I/R injury, renal tubular epithelial cells also facilitate the process by generating proinflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1), TNF-a, IL-1b, IL-6, IL-18 [3]. In addition, enhanced expression of adhesion molecules such as intracellular cell adhesive molecule (ICAM-1) (murine homologue is keratinocyte-derived chemokine (KC)) and macrophage inflammatory protein-2 (MIP-2) exacerbates the infiltration of neutrophils to the ischaemic tissue [3]. Pro-inflammatory cytokines such as interleukin 6 (IL6) [28] and activation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway have been implicated to play an essential role in the pathogenesis of I/R injury [17]. Moreover, it has been reported that the activation of JAK/STAT pathway causes the release of many pro-inflammatory cytokines that are involved in the progression of renal I/R, including MCP-1, ICAM and KC [17,29,30]. Among all, recent evidence has shown that the JAK-2/STAT-3 pathway is activated during the early phases of renal I/R and is a determinant of the progression of I/R inflammation [17,29,30], and investigating the role of this pathway in the anti-inflammatory action of XN was one of our targets.

In the current study, in accordance with previously mentioned findings, I/R injury was associated with enhanced levels of major cytokines and adhesion molecules with a concomitant increase in MPO activity and activation of the JAK-2/STAT-3 pathway, as indicated by their enhanced phosphorylated forms detected by western blot. These results further confirmed the previous findings that activation of JAK-2/STAT-3 pathway plays an essential role during renal I/R injury and is involved in neutrophilic infiltration damage [17,29,30]. On the other hand, beside its antioxidant potential, XN ameliorated I/R cell damage and resulted in decreased levels of all inflammatory cytokines and adhesion molecules. Numerous mechanisms of action have been suggested for the anti-inflammatory effect of XN in various disease conditions, such as inhibition of inducible NO synthase (iNOS) [31], suppression of NFκB activation [32,33], inhibiting the binding activity of STAT-1α and interferon regulatory factor-1 [32], modulating the T-cell mediated response [34], inhibition of cyclooxygenase 2 (COX-2) [35] and decreasing prostaglandin-E2 (PGE2) expression [36]. For the first time, this study shows a novel mechanism by which XN may ameliorate renal I/R induced inflammation and renal damage through inhibition of the JAK-2/STAT-3 pathway.

On the other hand, a mounting body of evidence now indicates that apoptosis is the major mechanism of early tubular cell death in both clinical acute renal failure and experimental I/R injury [21]. A multitude of pathways, including the intrinsic (Bcl-2 family, cytochrome c and caspase 9), and regulatory (p53 and NF-kβ) factors, seem to be activated by I/R [21]. ROS species are believed to activate both of these pathways. On the other hand, the acute inflammatory response initiated by I/R contributes to the death of renal cells as a combination of both necrosis and apoptosis [2]. In this study, apoptosis and necrosis were seen in the histopathological slides obtained from I/R rats. Furthermore, enhanced expression of cleaved caspase 3 is clearly demonstrates activation of apoptotic cascade. XN completely ameliorated the expression levels of activated Caspase 3 and resulted in improvement of glomerulus and epithelium tissue of renal tubules, suggesting decreased levels of necrosis and apoptosis. Recent evidence has shown that inhibition of JAK-2 or STAT-3 by AG490 or dominant-negative STAT-3 adenovirus, respectively, leads to increased ERK activation and survival of mouse proximal tubular epithelial (TKPTS) cells during severe oxidative stress [37]. Based on these findings and its antioxidant effect, XN could enhance the survival of tubular epithelial cells by decreasing the activation of JAK-2/STAT-2 pathway. However, such findings should be confirmed in further studies since XN has both antioxidant and anti-inflammatory effects and it is not clear whether the mechanism responsible for such decreases in phosphorylated JAK and STAT-1 are due to a direct effect of XN on JAK/STAT tyrosine activities or due to decreased ROS and/or inflammatory mediators found in this study.

5 Conclusion

Based on our results, there was a protective effect of XN against renal I/R injury and the mechanisms of protection involved ROS scavenging and an anti-inflammatory effect mediated by inhibition of the JAK-2/STAT-3 pathway. Further experiments and clinical trials remain necessary to validate these findings.

Conflicts of interest

The author has no conflicts of interest, and the work was not supported or funded by any company.

Acknowledgements

The author would like to thank Mr. Mahmoud Yousef for his technical assistance in the laboratory.

Notes

Peer review under responsibility of Taibah University.