Intravenous administration of xenogenic adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes markedly reduced brain infarct volume and preserved neurological function in rat after acute ischemic stroke

We tested the hypothesis that combined xenogenic (from mini-pig) adipose-derived mesenchymal stem cell (ADMSC) and ADMSC-derived exosome therapy could reduce brain-infarct zone (BIZ) and enhance neurological recovery in rat after acute ischemic stroke (AIS) induced by 50-min left middle cerebral artery occlusion. Adult-male Sprague-Dawley rats (n = 60) were divided equally into group 1 (sham-control), group 2 (AIS), group 3 [AIS-ADMSC (1.2×106 cells)], group 4 [AIS-exosome (100μg)], and group 5 (AIS-exosome-ADMSC). All therapies were provided intravenously at 3h after AIS procedure. BIZ determined by histopathology (by day-60) and brain MRI (by day-28) were highest in group 2, lowest in group 1, higher in groups 3 and 4 than in group 5, but they showed no difference between groups 3 and 4 (all p < 0.0001). By day-28, sensorimotor functional results exhibited an opposite pattern to BIZ among the five groups (p < 0.005). Protein expressions of inflammatory (inducible nitric oxide synthase/tumor necrosis factor-α/nuclear factor-κB/interleukin-1β/matrix metalloproteinase-9/plasminogen activator inhibitor-1/RANTES), oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/ Poly-ADP-ribose polymerase), and fibrotic (Smad3/transforming growth factor-β) biomarkers, and cellular expressions of brain-damaged (γ-H2AX+/ XRCC1-CD90+/p53BP1-CD90+), inflammatory (CD11+/CD68+/glial fibrillary acid protein+) and brain-edema (aquaporin-4+) markers showed a similar pattern of BIZ among the groups (all n < 0.0001). In conclusion, xenogenic ADMSC/ADMSC-derived exosome therapy was safe and offered the additional benefit of reducing BIZ and improving neurological function in rat AIS.


INTRODUCTION
Acute ischemic stroke (AIS) is the leading cause of long-term disability and the second cause of death worldwide [1][2][3][4]. Currently, a universally accepted effective and safe management strategy for patients with AIS remains undefined [5][6][7][8][9][10][11]. Thrombolysis with tissue plasminogen activator (tPA) and endovascular intracranial treatment are two emerging treatments for AIS with promising results in specific patient groups; however, strict enrollment criteria and contraindications restrict their scope in clinical practice [12][13][14][15][16][17][18]. Furthermore, tPA appears to be associated with a relatively high incidence of intracranial bleeding complications [12][13][14][15][16][17] and poor patency rate in large-vessel occlusion, and yet endovascular intracranial treatment is only suitable for patients with large-vessel occlusion [18]. Accordingly, the majority of AIS patients still lack a treatment that is both effective and safe. An alternative treatment needs to be developed for AIS patients, particularly for those who are not candidates for thrombolysis or endovascular intracranial treatment.
Plentiful data have demonstrated that stem cell therapies were effective and promising for many diseases refractory to traditional management strategies [18][19][20][21]. Of these stem cells, adipose-derived mesenchymal stem cells (ADMSCs) induce angiogenesis, leading to improved ischemia-related organ dysfunction, and have anti-inflammatory and immunomodulatory capacity for attenuating IR-induced organ dysfunction [22][23][24][25]. Autologous stem cells were utilized in most of these studies [18][19][20][21][22][23][24][25]; however, allogenic stem cells may also be suitable. Investigators often emphasize that allogenic stem cells are immune privileged and safe for recipients, but this is not universally accepted. Perhaps, a new innovation is required to resolve this issue. Exosomes contain distinct subsets of microRNAs depending upon the cell type from which they are secreted [26]. Several experimental studies have demonstrated that the MSCderived exosomes have distinctive features of (1) angiogenesis, (2) immunomodulation, and (3) a paracrine effect that improves organ function following injury in preclinical studies [25,[27][28][29][30][31]. Recently, our study [25] showed that ADMSCs and ADMSC-derived exosome therapy significantly protected kidney from ischemiareperfusion injury. Based on the aforementioned issues, this study tested the therapeutic impact of xenogenic (from mini-pig) ADMSCs and ADMSC-derived exosomes on brain anatomical and neurological functional recoveries in rat after AIS. We also clarified whether xenogenic MSCs exhibit immune privilege like allogenic MSCs reportedly do.

RESULTS
H&E staining for examination of the five major organs of brain, heart, lung, liver and kidney by day 21 after ADMSC/ADMSC-derived exosome treatment in healthy animals ( Figure 1) As expected, H&E microscopy revealed no abnormal histopathological findings in these five organs. IF microscopy also did not illustrate any ADMS/ADMSCderived exosomes present in these organs. Our findings highlighted that exogenic AMDMS/ADMSC-derived exosomes administered to rodent animals should maintain immune privilege without immune reaction and without harm to the healthy animals.
By day 3 after AIS, the ratio of left brain infarct volume to total left brain volume (LIV/TLBV) and the ratio of LIV/TBV were lowest in SC and highest in AIS only, and significantly lower in AIS-ADMSC-Ex than in AIS-ADMSC and AIS-Ex, but they showed no difference between AIS-ADMSC and AIS-Ex ( Figure 2). Additionally, by day 28, the ratios of LIV/TLBV and LIV/ TBV exhibited an identical pattern of day 3 among the five groups ( Figure 3).
By day 60, the left brain infarct area (i.e., identified by H.E. staining and calculated by summations of five whole brain cross sections in each animal) showed an identical pattern of LIV by 28 after AIS among the five groups ( Figure 3). Importantly, no tumorigenesis was evident on brain MRI or pathology (i.e., H.E., stain) in ADMS/ADMSC-derived exosome-treated animals (i.e., in AIS-ADMSC, AIS-Ex and AIS-ADMSC-Ex).
Corner test (Figure 3) demonstrated the attainment of a steady state of neurological functional impairment on day 3 following AIS among AIS-ADMSC, AIS-Ex and Oncotarget 74541 www.impactjournals.com/oncotarget AIS-ADMSC-Ex. Additionally, significant improvement in neurological function became apparent in AIS-ADMSC, AIS-Ex and AIS-ADMSC-Ex but not in AIS only compared with SC on day 14. Further substantial improvements in AIS-ADMSC and AIS-Ex and more substantial improvements in AIS-ADMSC-Ex were noted on day 28 after AIS.

Protein expression of inflammatory and oxidative stress biomarkers in brain infarct zone by day 60 after AIS (Figures 4 and 5)
The protein expressions of MMP-9, IL-1β, TNFα, RANTES, PAI-1, NF-κB and iNOS, seven indicators of inflammation, were highest in AIS only, lowest in SC, significantly higher in AIS-ADMSC and AIS-Ex than in AIS-ADMSC-Ex, and significantly higher in AIS-Ex than in AIS-ADMSC. This identical pattern was found among the five groups for protein expression of NOX-1, NOX-2 and oxidized protein, three indicators of oxidative stress.
Protein expressions of apoptotic, fibrotic and antifibrotic biomarkers in brain infarct zone by day 60 after AIS ( Figure 6) The protein expressions of cleaved caspase 3 and cleaved PARP, two indicators of apoptosis, were highest in AIS only, lowest in SC, significantly higher in AIS-ADMSC and AIS-Ex than in AIS-ADMSC-Ex, and significantly higher in AIS-Ex than in AIS-ADMSC. The protein expressions of Smad3 and TGF-β, two indices of fibrosis, exhibited an identical pattern of apoptosis among the five groups. The protein expressions of Smad1/5 and BMP-2, two indicators of anti-fibrosis, displayed an opposite pattern of apoptosis among the five groups.

Protein expressions of DNA-damage, mitochondrial-damage and angiogenesis markers in brain infarct zone by day 60 after AIS (Figure 7)
The protein expressions of γ-H2AX, a DNA-damage marker, and cytosolic cytochrome C, a mitochondrial- Oncotarget 74542 www.impactjournals.com/oncotarget damage marker, was highest in AIS only, lowest in SC, significantly higher in AIS-ADMSC and AIS-Ex than in AIS-ADMSC-Ex, and significantly higher in AIS-Ex than in AIS-ADMSC. On the other hand, protein expression of mitochondrial cytochrome C, an indicator of mitochondrial integrity, showed an opposite pattern to γ-H2AX among the five groups. Additionally, the protein expressions of CD31 and eNOS, two indicators of endothelial function, exhibited an identical pattern to mitochondrial cytochrome C among the five groups. Furthermore, the protein expressions of VEGF and CXCR4, two indices of angiogenesis, exhibited significantly progressive increases from groups SC to AIS-ADMSC-Ex, suggesting an intrinsic response to ischemic stimulation. The IF microscopic findings demonstrated that the cellular expressions (Figure 8) of CD11 and CD68, two indicators of inflammation, were highest in AIS only, lowest in SC, significantly higher in AIS-ADMSC and AIS-Ex than in AIS-ADMSC-Ex, and significantly higher in AIS-Ex than in AIS-ADMSC. IF microscopic findings also showed that the cellular expressions ( Figure 9) of XRCC1/CD90 and p53BP1/CD90, two indicators of double-stranded DNA damage, exhibited an identical pattern to inflammation among the five groups. Furthermore, the cellular expressions ( Figure 10) of GFAP (a specific glial cell marker, indicating inflammation) and AQP4 (an indicator of brain edema) showed an identical pattern to inflammation among the five groups.
Microscopic findings of angiogenesis and endothelial cellular expressions in brain infarct zone by day 60 after AIS (Figures 11 and 12) The cellular expressions of CD31 and vWF ( Figure  11), two indicators of endothelial function integrity, were Oncotarget 74544 www.impactjournals.com/oncotarget highest in SC, lowest in AIS only, significantly higher in AIS-ADMSC-Ex than in AIS-ADMSC and AIS-Ex, and significantly higher in AIS-ADMSC than in AIS-Ex. Additionally, the cellular expressions of CXCR4 and SDF-1α ( Figure 12), two indicators of angiogenesis, showed significant and progressive increases from groups SC to AIS-ADMSC-Ex, implicating an intrinsic response to ischemic stimulation.
Small vessel density and γ-H2AX+ cell expressions in brain infarct zone (BIZ) by day 60 after AIS ( Figure 13) The number of small vessels in BIZ, an indicator of angiogenesis, was highest in SC, lowest in AIS only, significantly higher in AIS-ADMSC-Ex than in AIS-ADMSC and AIS-Ex, and significantly higher in AIS-ADMSC than in AIS-Ex. The γ-H2AX+ cell expression, another DNA-damage biomarker, was highest in AIS only, lowest in SC, significantly higher in AIS-ADMSC and AIS-Ex than in AIS-ADMSC-Ex, and significantly higher in AIS-Ex than in AIS-ADMSC.

DISCUSSION
Our recent study showed that allogenic ADMSC/ ADMSC-derived exosome therapy significantly protected kidney architecture, glomerular ultrastructure and renal function from acute kidney ischemia-reperfusion injury in rodent [25]. In addition, this study proved that allogenic ADMSC/ADMSC-derived exosomes were immune privileged in rodent [25]. An essential finding in the present study was that immune reaction was not identified and major organs (brain, heart, lung, liver and kidney) were not injured by xenogenic ADMSC/ADMSC-derived exosome treatment. Additionally, the most important findings in the present study were that, compared with AIS animals, the BIZ (i.e., by MRI and histopathological findings) was reduced and neurological function (i.e., by sensorimotor functional test) was improved in AIS animals treated by xenogenic ADMSC or ADMSC-derived exosomes. Also, combined therapy with the two regimens offered additional benefit over either one therapy alone for reducing brain infarct volume and enhancing recovery of neurological function. Our findings, in addition to extending the findings of our recent study [25], highlighted Oncotarget 74545 www.impactjournals.com/oncotarget two important issues: (1) xenogenic ADMSC/ADMSCderived exosome has the property of immune privilege; (2) these xenogenic cell-based therapies safely and effectively protect the brain from AIS in rodent.
It has long been known that innate immune reactions are quickly activated following tissue/organ ischemic injury and necrosis, which, in turn, elicit the complement cascade, inflammation and the generation of oxidative Oncotarget 74546 www.impactjournals.com/oncotarget stress and reactive oxygen species (ROS) [19,[32][33][34][35]. These cellular-molecular perturbations, finally, directly participate in further tissue/organ damage after ischemic injury [2-5, 19, 21-25, 32, 35-39]. On the other hand, numerous studies have previously shown that MSCs, especially ADMSCs, have powerful anti-inflammatory and immunomodulatory properties that can attenuate ischemia-reperfusion or sepsis induced organ damage [21-  25,39]. One important finding in the present study was that the protein and cellular levels of inflammation in the BIZ were substantially higher in AIS animals compared to control animals. Additionally, oxidative stress biomarkers were higher in AIS animals than in control animals. Accordingly, our findings corroborate those of previous studies [19,22,[32][33][34][35]39]. Importantly, inflammation, the generation of ROS and oxidative stress were significantly Oncotarget 74548 www.impactjournals.com/oncotarget suppressed by ADMSC-derived exosome therapy, more significantly suppressed by ADMSC therapy, and most significantly suppressed by their combined therapy in AIS animals. Interestingly, our recent study [31] showed that ADMSC-derived exosome therapy markedly suppressed the growth of hepatocellular carcinoma mainly Additionally, our other studies [21][22][23][24]32] have recently shown that allogenic and autologous ADMSCs have immunomodulatory and anti-inflammatory properties, and suppress the production of oxidative stress in the setting of ischemia-reperfusion injury. Furthermore, other studies Oncotarget 74550 www.impactjournals.com/oncotarget [40,41] have exhibited that human-derived endothelial progenitor cell (EPC) therapy effectively improved ischemia-related organ dysfunction in rat (i.e. xenogenic EPC therapy). Accordingly, our findings strengthen those of our recent studies [21-24, 31, 32, 40, 41] and extend the indications for ADMSC/ADMSC-derived exosomes, in that they protect organs from ischemic attack (i.e., from allogenic to xenogenic levels). Oncotarget 74551 www.impactjournals.com/oncotarget A strong correlation between inflammation/ oxidative stress and cellular apoptosis/death, fibrosis of organ and tissues, as well as DNA/mitochondria damage in the setting of ischemic injury has been keenly investigated by previous studies [19, 22-25, 32, 35, 37-39]. These molecular-cellular perturbations, in turn, resulted in organ damage and dysfunction [19, 22-25, 32, 35, 37-39]. A principal finding in the present study was that, when compared with the control group, these aforementioned molecular-cellular perturbations were substantially increased in AIS animals. In this way, our findings could, in addition to being comparable with the findings of several previous studies [19, 22-25, 32, 35, 37-39], explain why the BIZ was notably increased and the neurological function was significantly impaired in animals after AIS. Importantly, both BIZ and neurological function in AIS animals were significantly reversed after receiving ADMSC-derived exosome treatment and more significantly reversed after ADMSC treatment. Of particular importance, combined therapy with ADMSC/ ADMSC-derived exosomes was superior to either therapy alone at inhibiting the BIZ and improving the recovery of neurological function in animals after AIS.
Another important finding in the present study was that the angiogenesis biomarkers (i.e., CD31+ and vWF+ cell, protein expression of eNOS, VEGF, CXCR4 and number of small vessels in BIZ) were significantly higher in AIS animals with xenogenic ADMSC/ADMSC-derived exosome treatment than in those without. Intriguingly, our recent studies [22,39] have also displayed that ADMSC treatment for rat with acute ischemia-reperfusion injury markedly enhanced the generation of angiogenesis biomarkers in the region of ischemia. Accordingly, our findings could, in addition to corroborating the findings of our previous studies [22,39], explain the improvement of neurological function and reduction of brain infarct volume, at least in part, through restoring blood flow in the ischemic region.

Study limitation
This study has limitations. First, this study only considered the management of AIS and therefore did not provide any information regarding the impact of xenogenic ADMSC/ADMSC-derived exosome therapy on chronic IS. Second, this study did not test the optimal dosage of ADMSCs or ADMSC-derived exosomes on improving the neurological outcome. Accordingly, we remain uncertain whether ADMSC therapy or ADMSCderived exosome therapy was superior at improving the outcomes in rat after AIS, when both dosage regimens have been optimized.
In conclusion, the present study provided evidence that xenogenic cell-based therapy was safe and efficacious for improving outcomes in rat after AIS. Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-approved animal facility in our hospital with controlled temperature and light cycles (24°C and 12/12 light cycle).

Animal model of acute ischemic stroke and corner test for assessment of neurological function
The protocol and procedure for the experimental rodent AIS model have previously been described [32,36]. In detail, adult male Sprague-Dawley rats, weighing 350-375 g (Charles River Technology, BioLASCO Taiwan Co., Ltd., Taiwan), were utilized in the current study. Each animal was anesthetized by 2% inhalational isoflurane in a supine position on a warming pad (37 ºC). After exposure of the left common carotid artery (LCCA) through a transverse neck incision, a small arteriotomy was performed on the LCCA through which a 0.28 mm diameter nylon monofilament was carefully advanced into the distal left internal carotid artery for occlusion of the left middle cerebral artery; this caused brain ischemia and infarction of its supplied area. The nylon monofilament was removed 50 min after occlusion, followed by closure of the muscle and skin in layers. The rats were then cared for in a portable animal intensive care unit (ThermoCare ® ) with food and water for 24 hours.
The sensorimotor functional test (Corner test) was conducted for each rat at baseline and on days 1, 3, 14 and 28 after AIS induction as we recently described [32,36]. Briefly, the rat was allowed to walk through a tunnel and then into a corner, the angle of which was 60 degrees. To exit the corner, the rat could turn either left or right. The results were recorded by a technician who was blind to the study design. This test was repeated 10 to 15 times with at least 30 seconds between each trial. We recorded the number of right and left turns from 10 successful trials for each animal and used the results for statistical analysis.

Procedure and protocol for isolating and culturing ADMSCs, exosome protein quantification, characterization, and preparation
The procedure and protocol for ADMSC isolation and culture have been described in details in our recent reports [25,31]. Briefly, abdominal adipose tissue from eight mini-pigs (about 16-18kg) was carefully dissected and excised. ADMSCs were automatically isolated using the KISO™ System (Keai Bioresearch, BC, Canada). For purification, the harvested cells were cultured in a 100 mm diameter dish with MEM-α culture medium containing 10% FBS for 14 days. By this time plentiful ADMSCs were obtained in the culture plate; they were collected to treat AIS animals. Surface markers for ADMSCs were identified by flow cytometric analyses as reported in our previous studies [25,31].
The procedure to isolate exosomes from the culture medium has been detailed in our recent report [25,31]. Briefly, exosomes were isolated from the culture medium of ADMSCs and were pooled for electron microscopic assessment, protein separation and characterization, and western blot analysis. The proteins in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% serum before and after cell culture were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The exosomes in DMEM were purified and the proteins in different exosome fractions (1 μg, 2 μg, 10 μg, and 50 μg) were separated by SDS-PAGE. The gel was stained with Coomassie blue for analysis. The following primary antibodies were used: mouse monoclonal anti-CD63 (Santa Cruz Biotechnology), rabbit polyclonal antitumor susceptibility gene-101 (TSG101) (Abcam) and anti-β-catenin (Abcam).

Histopathological findings for identification of ADMSC/ADMSC-derived exosomes in brain, heart liver and lung tissues
To elucidate whether treatment with xenogenic ADMSC or ADMSC-derived exosomes (stained by CM-Dil dye for tracking the ADMSC/exosome) would be harmful to the normal organs/tissues, an additional eight healthy animals (4 per group) were utilized to receive these two different therapies intravenously. By day 21, all animals were euthanized and specimens were collected. Microscopic investigations (Figure 1) demonstrated that no any abnormal findings were identified in any individual organ/tissue.

Procedure and protocol of brain magnetic resonance imaging (MRI) study
The procedure and protocol for brain magnetic resonance imaging (MRI) study were based on our recent report [36]. MRI was performed at days 3 and 28 after AIS induction. Briefly, during MRI measurements, mice were anesthetized by 3% inhalational isoflurane with room air and placed in an MRI-compatible holder (Biospec 94/20, Bruker, Ettingen, Germany). Rectal temperature and respiration were monitored throughout the procedure to ensure normal physiological conditions were being maintained. MRI data were collected using a Varian 9.4T animal scanner (Biospec 94/20, Bruker, Ettingen, Germany) with a rat surface array. The MRI protocol consisted of 40 T2-weighted images. Forty continuous slice locations were imaged with a field-of-view of 30 mm x 30 mm, an acquisition matrix dimension of 256 x 256 and slice thickness of 0.5 mm. The repetition time (TR) and echo time (TE) for each fast spin-echo volume were 4200 ms and 30 ms, respectively. Custom software, ImageJ (1.43i, NIH, USA), was used to process the region of interest (ROI). Planimetric measurements of images from MRI T2 were performed to calculate the stroke volumes of cortex.

Specimen collection and preparation for individual study
Animals were euthanized by day 60 after the AIS procedure. For examination of protein expression, the brain of each rat (n = 6 for each group) was promptly removed, immersed in cold saline, snap-frozen in liquid nitrogen and then stored at -80 º C for individual study. For immunofluorescent (IF) and immunohistochemical (IHC) staining studies, the brains of 6 other animals in each group were reperfused with normal saline via the carotid artery, removed, fixed with 4% paraformaldehyde in 1x PBS (pH7.4), and soaked in 20% sucrose in 1x PBS (freshly prepared) until the brain took on a completely sunken appearance. The sucrose was then discarded and the brain soaked in 30% sucrose in 1x PBS (freshly prepared) for 48 h. The infarcted and non-infarcted parts were then collected. Finally, the OCT block (Tissue-Tek, Sakura, Netherlands) was prepared for IHC and IF staining.

Immunofluorescent (IF) staining of brain specimens
The procedure and protocol of IF and IHC staining were based on our previous reports [36][37][38]. In detail, frozen sections (4 µm thick) were obtained from the brain infarct zone (BIZ) of each animal, permeated with 0.5% Triton X-100, and incubated with antibodies against glial fibrillary acid protein (GFAP) (1:500, DAKO), aquaporin4 Three brain sections were analyzed for each rat. For quantification, three randomly selected high-power fields (HPFs; 400x for IF study) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.

Vessel density in BIZ
The procedure and protocol for identifying small vessels in the BIZ were based on our previous reports [32,[36][37][38]. In detail, IHC staining of small blood vessels was performed with α-SMA (1:400) as primary antibody at room temperature for 1 hour, followed by washing with PBS thrice. Ten minutes after the addition of anti-mouse-HRP conjugated secondary antibody, the tissue sections were washed with PBS thrice. Then 3,3' diaminobenzidine (DAB) (0.7 gm/tablet) (Sigma) was added, followed by washing with PBS thrice after one minute. Finally, hematoxylin was added as a counter-stain for nuclei, followed by washing twice with PBS after one minute. Three brain sections were analyzed in each rat. For quantification, three randomly selected HPFs (100x) were analyzed in each section. The mean number per HPF for each animal was then determined by summation of all numbers divided by 9.

Western blot analysis
The procedure and protocol for Western blot analysis were based on our recent reports [19, 21-25, 32, 36]. Briefly, equal amounts (50 mg) of protein extracts were loaded and separated by SDS-PAGE using acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride membrane (GE, UK). Nonspecific sites were blocked by incubation of the membrane in blocking buffer Signaling), and Actin (1: 1000, Millipore)] for 1 hour at room temperature. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (1:2000, Cell Signaling, Danvers, MA, USA) was used as a secondary antibody for one-hour incubation at room temperature. The washing procedure was repeated eight times within one hour. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Amersham, UK) and exposed to Biomax L film (Kodak, Rochester, NY, USA). For the purpose of quantification, ECL signals were digitized using Labwork software (UVP, Waltham, MA, USA).

Assessment of oxidative stress
The procedure and protocol for evaluating the protein expression of oxidative stress have been described in detail in our previous reports [19, 21-25, 32, 36]. The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon, Billerica, MA, USA (S7150). DNPH derivatization was carried out on 6 μg of protein for 15 minutes according to the manufacturer's instructions. Onedimensional electrophoresis was carried out on 12% SDS/ polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes that were then incubated in the primary antibody solution (anti-DNP 1: 150) for 2 hours, followed by incubation in secondary antibody solution (1:300) for 1 hour at room temperature. The washing procedure was repeated eight times within