The neuroprotection of deproteinized calf blood extractives injection against Alzheimer's disease via regulation of Nrf-2 signaling

The detection of DCBEI compositions

Seventeen amino acids were identified in DCBEI, the most abundant of which were phenylalanine (15.37 g/kg), histidine (10.21 g/kg) and glutamic acid (10.16 g/kg) (Table 1). Of the five types of nucleotide that were detected, the levels of hypoxanthine nucleotide (9.19 mg/100 g) and uridylic acid (9.04 mg/100 g) were highest (Table 1). The ribose content of DBCEI was 140.07 mg/kg, and the molecular weight distribution of DBCEI was predominantly between 0 and 500 kDa (93.92%) (Table 1).

DCBEI protects HT22 cells against mitochondrial apoptosis

L-Glu reduced the viability of HT22 cells by 30.1%, whereas DCBEI improved >20% cell viability, especially at concentrations of 6 mg/mL to 8 mg/mL (p. < 0.01; Figure 1A). Compared to cells treated only with L-Glu, those co-treated with DCBEI showed suppressed cell apoptosis rate by >9.5% (p. < 0.01, Figure 1B) and reduced caspase-3/7 activity by 37.5% (p. < 0.01) (Figure 1C), caspase-8 activity by 78.3% (p. < 0.001) (Figure 1D) and caspase-9 activity by 64.2% (p. < 0.001) (Figure 1E). DCBEI at concentrations of 4 mg/mL and 8 mg/mL strongly suppressed the accumulation of ROS, as indicated by the reduced red fluorescence intensity (Figure 2A) and green fluorescence intensity (Figure 2B), and prevented the dissipation of MMP, as indicated by the reduced green/red fluorescence ratio (Figure 2C). DCBEI also inhibited Ca²⁺ influx in L-Glu-treated HT22 cells, as indicated by the reduced green fluorescence intensity of the reporter (Figure 2D). Additionally, DCBEI suppressed the induction of pro-apoptotic proteins including Bax, Bid, Bad, cleaved PARP-1, Calpain-1 and phospho-Drp1, and increased the levels of anti-apoptotic proteins including Bcl-2, Bcl-XL and phospho-RSK1 p90 in L-Glu-treated HT22 cells (Figure 2E).

Figure 1. DCBEI protects HT22 cells against L-Glu-induced damage. HT22 cells were pre-incubated with DCBEI (4 mg/mL or 8 mg/mL) for 3 h, and co-treated with L-Glu for a further 24 h. (A) DCBEI increased cell viability. (B) DCBEI inhibited apoptosis. DCBEI attenuated the activation of caspase-3/7 (C), caspase-8 (D) and caspase-9 (E). Data are expressed as a percentage of corresponding control cells and means ± S.D. (n = 6). ## P < 0.01 and ### P < 0.001 vs. CTRL, * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. L-Glu-treated cells.

Figure 2. HT22 cells were pre-incubated with DCBEI (4 mg/mL or 8 mg/mL) for 3 h, and co-treated with L-Glu for a further 24 h. (A) DCBEI reduced the intracellular ROS activity in L-Glu-treated HT22 cells (n = 3). (B) DCBEI reduced L-Glu-induced ROS production (200×) (Scale bar: 50 μm) (n = 3). (C) DCBEI attenuated L-Glu-induced MMP dissipation (200×) (Scale bar: 50 μm) (n = 3). (D) Fluo 4-AM staining indicated that DCBEI suppressed L-Glu-induced Ca²⁺ increases (200×) (Scale bar: 50 μm) (n = 3). (E) DCBEI regulated the expression of proteins related to mitochondrial function. DCBEI increased the levels of anti-apoptotic proteins (Bcl-2, Bcl-xL and P-RSK1 p90) and reduced the levels of pro-apoptotic proteins (Bax, Bid, Bad, cleaved PARP-1, Calpain-1 and p-Drp1) in L-Glu-treated HT22 cells. Quantification data were normalized to GAPDH and the corresponding total proteins, and reported as fold change relative to CTRL (n = 3). Data are expressed as a percentage of corresponding control cells and means ± S.D. # P < 0.05, ## P < 0.01 and ### P < 0.001 vs. CTRL, * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. L-Glu-treated cells.

Aβ1-42 treatment of U251 significantly reduced survival due to the induction of apoptosis; this effect was reversed by treatment with 4 mg/mL to mg/mL DCBEI (P < 0.05). Compared to cells treated only with Aβ1-42, those co-treated with DCBEI showed suppressed cell apoptosis rate (P < 0.01, Supplementary Figure 1).

DCBEI alleviated the AD-like symptoms in APP/PS1 mice

In the behavioral tests, administration of DCBEI for 28-days reduced the escape latency that APP/PS1 mice in the Morris water maze test (30.50 ± 7.84 s [DCBEI at 160 mg/kg, p < 0.05] and 19.33 ± 5.64 s [DCBEI at 320 mg/kg, p < 0.01] versus 51.67 ± 10.54 s [APP/PS1mice]; Figure 3A). It also reduced the time of mice took to enter the central area in the open field test (66.53 ± 12.79 s [DCBEI at 160 mg/kg, p < 0.05] and 35.58 ± 13.94 s [DCBEI at 320 mg/kg, p < 0.01] versus 112.07 ± 9.94 s [APP/PS1mice]; Figure 3B).

Figure 3. DCBEI improves the behavioral performance of APP/PS1 mice. Compared with non-treated APP/PS1 mice, a 28-day course of DCBEI (A) reduced the escape latency time in the Morris water maze test, (B) decreased the time taken for mice to enter the central area in an open field test. Data are expressed as means ± S.D. (n = 10). # P < 0.05 and ## P < 0.01 vs. WT, * P < 0.05 and ** P < 0.01 vs. APP/PS1 mice.

As Aβ1-42 forms the core of amyloid plaques, it is heavily implicated in the pathology associated with plaque deposition. Compared with non-treated APP/PS1 mice, treatment with 160 mg/kg and 320 mg/kg DCBEI for 28 days reduced hippocampal Aβ1-42 concentrations by 17.1% and 14.8%, respectively (p < 0.01) (Figure 4A). There were concomitant increases of 11.7% and 7.4% in the serum concentration of Aβ1-42 with 160 mg/kg and 320 mg/kg DCBEI, respectively (p < 0.05) (Figure 4B).

Figure 4. DCBEI attenuates pathology in the brains of APP/PS1 mice. DCBEI (A) reduced the expression of Aβ1-42 in the hippocampus (n = 10), and (B) increased the levels of Aβ1-42 in the serum (n = 10). Data are expressed as means ± S.D. (n = 10). # P < 0.05 and ## P < 0.01 vs. WT, * P < 0.05 and ** P < 0.01 vs. APP/PS1 mice. (C) DCBEI reduced the level of Aβ in the hippocampus (n = 3). (D) DCBEI attenuated the increase in p-Tau levels in the hippocampus (n = 3), (E) but did not affect the levels of total Tau (n = 3). (F) DCBEI significantly reduced the levels of 4-HNE in the brains of APP/PS1 mice (n = 3) (40×) (Scale bar: 200 μm) (200×) (Scale bar: 50 μm). (G) TUNEL staining shows that a 28-day course of DCBEI significantly reduces apoptosis (n = 3) (200×) (Scale bar: 50 μm).

The DCBEI-dependent reduction of Aβ1-42 deposition—especially in the hippocampus—was further confirmed by pathologic analysis (Figure 4C). Compared with non-treated APP/PS1 mice, DCBEI suppressed the induction of phospho-Tau (p-Tau) in the hippocampus (Figure 4D), but had no effect on the levels of total Tau (Figure 4E). DCBEI strongly reduced the levels of 4 Hydroxynonenal (4-HNE), a key mediator of oxidative stress-induced damage, in the hippocampus (Figure 4F). DCBEI also significantly reduced the amount of TUNEL-positive apoptotic neurons in the brains of APP/PS1 mice (Figure 4G). DCBEI did not induce significant gross pathology in the brain, spleen or kidney (Supplementary Figure 2), suggesting that it could be used safely.

The regulation of DCBE on neurotransmitters of APP/PS1 mice

The serum content of nine neurotransmitters including r-Amino-butyric acid (GABA), Norepinephrine (NA), 5-hydroxyindoleacetic acid (5-HIAA), serotonin (5-HT), Histamine (HIS), glutamine (Gln) and isoprenaline hydrochloride (iso-Hyd) was quantified using HPLC-MS/MS (Figure 5C and Supplementary Figure 3). Based on this initial quantification, Enzyme-linked immunosorbent assay (ELISA) was used to quantify the levels of seven of these neurotransmitters in whole brain tissues. Compared with non-treated APP/PS1 mice, DCBEI-treated mice (160 mg/kg and 320 mg/kg) showed enhanced levels of GABA (p < 0.01), iso-Hyd (p < 0.001), 5-HT (p < 0.01), NA (p < 0.001), HIS (p < 0.001) and 5-HIAA (p < 0.01) that were >71.4%, >73.9%, >63.9%, >59.3%, >7 9.0%, and >96.6%, respectively (Table 2).

Figure 5. Proteomic and metabonomic profiling of the effects of DCBEI in APP/PS1 mice. (A) Heat map of differentially expressed factors (n = 3). (B) The relationship between proteins processed through STRINGdb. (C) Serum neurotransmitter levels were quantified using HPLC-MS/MS. Significant differences in the levels of nine neurotransmitters (R-amino-butyric acid, norepinephrine, 5-HIAA, serotonin, methoxytyramine, histamine, tyramine, glutamine and normetanephrine) was observed (n = 3).

The regulation of DCBEI on Nuclear factor-erythroid 2 related factor 2 (Nrf-2) signaling

Proteomics revealed that 362 proteins were differentially expressed between WT and APP/PS1 mice, and 309 proteins were differentially expressed between DCBEI-treated and untreated APP/PS1 mice (Supplementary Figure 4). After applying cut-off values of A/B > 1.5 or A/B < 0.66, we observed that 64 proteins were differentially expressed following DCBEI treatment (32 were upregulated and 32 were downregulated; Figure 5A). Gene Ontology analysis indicated an enrichment of mitochondrial proteins, suggesting that DCBEI-mediated neuroprotection is linked to the regulation of apoptosis and oxidative stress (Supplementary Figure 5). We next used STRINGdb to analyze interactions between proteins that were differentially expressed between experimental groups; this revealed 333 enriched interactions and 165 expected interactions (Figure 5B).

Liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) analysis revealed changes in the levels of anti- and pro-oxidative factors in the serum and hippocampus of APP/PS1 mice. Compared with vehicle-treated APP/PS1 mice, DCBEI-treated mice showed increased concentration of catalase (CAT, p < 0.05) and reduced levels of ROS (p < 0.05) in the serum and hippocampus, enhanced levels of glutathione peroxidase (GSH-Px, p < 0.05) and superoxide dismutase (SOD, p < 0.05) in the hippocampus, and reduced levels of malondialdehyde (MDA, p < 0.05) in the serum (Table 3). The effect was particularly striking at the highest DCBEI dose (320 mg/kg).

Following L-Glu exposure, Nrf-2 and its downstream targets (including HO-1, SOD-1, SOD-2, GCLC, GCLM and NQO1) were downregulated, whereas C-Maf and PKC were upregulated. These effects were reversed by a 3-h preincubation with DCBEI (Figure 6A). Compared with cells treated with only L-Glu, those pre-treated with DCBEI showed enhanced phosphorylation of AKT and mTOR and reduced phosphorylation of JNK, PTEN and P38 (Figure 6B). Additionally, DCBEI increased the levels of nuclear CytoC and the nuclear translocation of Nrf-2 and phospho-ERK in L-Glu-treated HT22 cells (Figure 6C).

Figure 6. DCBEI ameliorates AD symptoms by reducing apoptosis, attenuating oxidative stress, and regulating neurotransmitter levels via Nrf2 signaling. (A) In L-Glu-exposed HT22 cells, DCBEI increased the expression of Nrf-2 and its downstream targets including HO-1, SOD-1, SOD-2, GCLC, GCLM and NQO1, and reduced the expression levels of C-Maf. (B) Analysis of MAPKs and Akt in L-Glu-treated HT22 cells. DCBEI increased the expression of p-AKT and p-mTOR, and reduced the expression of p-JNK, p-PTEN and p-P38 in L-Glu-treated HT22 cells. (C) DCBEI increases p-ERK levels and causes a redistribution of p-ERK, Nrf-2, and Cyto C from the cytoplasm to the nucleus. Quantification data were normalized to GAPDH and the corresponding total proteins, and are reported as fold change relative to CTRL (n = 3).

Analysis of DCBEI composition

As described in previous studies [47–50], we determined the molecular weight distribution and the nucleotide and ribose contents of Deproteinized Calf Blood Extractive Injection (DCBEI)(CAS: 20160803; obtained from Jilin Connell Pharmaceutical Co. LTD., Jilin, China) samples using high-performance liquid chromatography (HPLC) (E2695, Waters Co., Ltd., USA) systems and gel permeation chromatography (1515, Waters Co., Ltd., USA).

DCBEI was hydrolyzed by the addition of 6 mol/L of hydrochloric acid at 110° C for 24 h, and then dried under vacuum. Deionized water was added to prepare DCBEI test samples and amino acid standards. Seventeen distinct free amino acids were detected using the HPLC system as described in a previous study [51].

Cell culture

HT22, a mouse hippocampal neuronal cell line (BNCC-337709) (Chinese Academy of Sciences Cell Bank, Shanghai, China), and U251, a human astroglioma cell line (No. BNCC337874), were cultured in Dulbecco’s Modified Eagle’s Media (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen, USA) under a humidified atmosphere containing 5%/95% CO₂/air at 37° C.

Cell viability and caspase activity analyses

HT22 cells (6 × 10³ cells/100 μL) were seeded into 96-well plates, pre-treated with DCBEI at doses of 1 mg/mL to 8 mg/mL for 3 h, and then treated with L-Glu (25 mM) for another 24 h.

U251 cells (6 × 10³ cells/100 μL) were seeded into 96-well plates, pre-treated with DCBEI at doses of 1 mg/mL to 8 mg/mL for 3 h, and then treated with Aβ1-42 (10 μM) (purity ≥ 95%, Gill Biochemical Co., Ltd., Shanghai, China) for another 24 h [52].

A cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed as described in our previous study [53].

HT22 cells (3 × 10⁵ cells/mL) were seeded into 6-well plates, pre-treated with 4 mL or 8 mL of DCBEI for 3 h, and then treated with L-Glu (25 mM) for another 24 h. The activities of Caspase-3/7 (APT423), -8 (APT408) and -9 (APT409) were analyzed using commercial kits (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.

Quantification of apoptosis and ROS activities via flow cytometry

HT22 cells (3 × 10⁵ cells/mL) were seeded into 6-well plates, pre-treated with DCBEI at doses of 4 mL and 8 mL for 3 h, and then treated with L-Glu (25 mM) for another 24 h.

U251 cells (3 × 10⁵ cells/mL) were seeded into 6-well plates, pre-treated with 4 mL or 8 mL of DCBEI for 3 h, and then treated with Aβ1-42 (10 μM) for another 24 h.

Cells were collected and stained with reagents of the Dead Cell Kit (MCH100105, Millipore, Billerica, MA, USA) and Oxidative Stress Kit (MCH100111, Millipore, Billerica, MA, USA) at 37° C in the dark for 15 min. Fluorescence intensity, as an indicator of apoptosis and ROS activity, was measured using a Muse™ Cell Analyzer (Millipore, Billerica, MA, USA).

Assessment of the dissipation of MMP, and intracellular levels of ROS and Ca²⁺

HT22 cells (1 × 10⁵ cells/mL) were seeded into 6-well plates, pre-treated with 4 mL or 8 mL of DCBEI for 3 h, and then treated with L-Glu (25 mM) for another 24 h.

Treated cells were stained with 10 μM of 2,7-dichlorofluorescein diacetate (DCFH-DA; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), 5 μM of Fluo-4-AM (Molecular Probes, USA), or 2 μmol/L of 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, Millipore, Billerica, MA, USA) for 20 min at room temperature in the dark. Intracellular ROS and Ca²⁺ levels, which were indirectly reported by the green fluorescence, and the degree of MMP l, which was reported by the ratio of red/green fluorescence, were quantified using a Nikon TE2000 fluorescence microscope with a CCD camera (Japan) (200×).

Western blot

HT22 cells were seeded into 6-well plates (3 × 10⁵ cells/well), pre-treated with 4 mL or 8 mL of DCBEI for 3 h, and then treated with L-Glu (25 mM) for another 24 h. The concentration of protein in cell lysates was measured using BCA protein assay kit (Millipore, Billerica, MA, USA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (10–12%) was used to separate the protein lysates (40 μg), which were transferred to polyvinylidene difluoride membranes (0.45 μm, Merck Millipore, Billerica, MA, USA). The membranes were blotted with 5% bovine serum albumin (Sigma, USA) at 4° C for 4 h, and then incubated with primary antibodies (Supplementary Table 1) at 4° C for 12 h. After washing with Tris-buffered saline-Tween-20 buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody [anti-mouse (IH-0031) and anti-rabbit (IH-0011)] (Dingguo, Beijing, China) at 4° C for 4 h. An ECL kit (WBKLS0500, Millipore, Billerica, MA, USA) was used to visualize the protein bands, and the intensity of the bands was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Animal husbandry and drug treatment

The experiment was conducted with the approval of the Institution Animal Ethics Committee of Jilin University (License No. SY0604). Forty-five B6C3-Tg (APPswePSEN1dE9)/Nju double transgenic male mice [Genotype: (Appswe)T, (Psen1) T] (APP/PS1) (8 months old, 45–50 g) and 15 wild-type male mice [Genotype: (Appswe)W, (Psen1) W] (WT) (8 months old, 45–50 g) were purchased from Nanjing Biomedical Research Institute of Nanjing University, Jiangsu, China [SCXK (SU) 2015-0001]. All mice were raised in a room held at 23 ± 2° C with a humidity of 40–60% under a 12-h:12-h light/dark cycle; the animals were given access to water and food ad libitum.

APP/PS1 mice were randomly assigned to three groups (n = 15/group), and received an intraperitoneal injection of DCBEI (160 mg/kg and 320 mg/kg), or an equivalent volume of saline for 28 days. After the last behavioral test, the mice were euthanized by injection with 150 mg/kg of 1.5% pentobarbital solution. The serum together with tissues of the brain, spleen and kidney were collected for biochemical and pathological analyses.

Behavioral tests

Morris water maze experiment test

The long-term spatial memory and learning capabilities of mice were evaluated using the Morris water maze [54]. After 5 days of training, mice were put into the MT-200 Water Labyrinth Video Tracking Analysis System (S7200) with a circular pool (122 cm diameter, 62.5 cm deep) filled with water to a depth of 40 cm (24 ± 2° C) containing titanium dioxide; this pool was higher than the platform. The amount of time spent searching the platform over a 60-s period and the movement trajectory were recorded by a video camera.

Open field experiment test

An open field test was used to evaluate the autonomic activities, exploration behaviors, and anxiety behaviors of experimental animals when placed in new environments [54]. The open field test apparatus (50 cm long × 50 cm wide × 50 cm deep) was protected with an isolator, and divided into a central area (25 cm long × 25 cm wide) and a surrounding area. The movements of mice were recorded for 5 min, and their trajectory was analyzed by software (Any-maze™, Stoelting Co., Chicago, IL, USA). The apparatus was cleaned between successive mice to ensure that no information from the preceding animal was present.

Immunohistochemistry examination

Paraffin sections of tissues were deparaffinized in xylene, rehydrated in different concentrations of ethanol, and washed twice in phosphate-buffered saline solution (PBS). The tissue slides were heated with citrate buffer for 15 min to retrieve antigens, incubated with hydrogen peroxide for 12 min at room temperature to block endogenous peroxidase, and then blocked with 5% normal horse serum and 1% normal goat serum dissolved in PBS. The slides were then incubated with anti-amyloid precursor (Aβ) antibody (1:500 dilution; ab32136), anti-Tau antibody (1:750 dilution; ab32057), anti-phospho-Tau (ser404) (1:200 dilution; ab 92676) and anti-4-HNE antibody (1:200 dilution; ab46545) (Abcam, Cambridge, MA, USA) at 25° C for 1 h. The slides were stained with 3,3′-diaminobenzidine and Mayer’s hematoxylin and visualized and photographed using a microscope (40× and 200×; IX73, Olympus, Tokyo, Japan).

TUNEL assay

Brain tissue sections were deparaffinized at 37° C, and then exposed to permeabilization reagent (Proteinase K solution) for 15 min. Sections of mouse hippocampus were labelled with the Click-it™ Plus TUNEL assay kit (C10617, Invitrogen, USA) according to the manufacturer’s instructions. The slides were stained with 3,3′-diaminobenzidine and apoptosis was then quantified using a microscope (200×; IX73, Olympus, Tokyo, Japan).

LC-MS/MS analysis of serum neurotransmitters

Based on previous studies [55, 56], 100 μL of serum from each mouse was homogenized in 400 μL ice cold methanol (Sigma Aldrich Fluka, USA)/acetonitrile (ACN; Merck, GER) (V:V = 1:1) with 1% formic acid (Merck, GER), and the mixture was incubated for 60 min at 37° C until a liquid supernatant was obtained. After vacuum drying, the precipitate of liquid supernatant was dissolved in ACN/water (1:1, v/v) with 1% FA.

The concentrations of NA, 5-HT, and 5-HIAA, Glu, GABA, Gln, HIS, normetanephrine, tyramine and methoxytyramine were quantified using an integrated HPLC-MS/MS system. All standards were purchased from Sigma-Aldrich (USA).

Label-free quantification (LFQ) analysis of proteins in the brains

Preparation of protein samples

The brain tissue samples were homogenized in RIPA buffer containing protease inhibitor cocktail (Kangchen Bio-tech, Shanghai, China) and 1 mM Phenylmethylsulfonyl fluoride (PMSF), followed by 1 min of sonication. Samples were then centrifuged and the supernatant containing the whole tissue extract was retained. Protein concentration was determined using a BCA assay. Sample preparation was based on previous studies [57], and all samples were processed simultaneously. Samples were analyzed using LC-MS/MS.

LC-MS/MS analysis

For each sample, 2 μg of peptide was separated and analyzed with a Nano-HPLC (EASY-nLC1200) coupled to a Q-Exactive mass spectrometer (Thermo Finnigan). The separation conditions used were the same as those reported in a previous study.

Data-dependent acquisition (DDA) was performed in the profile and positive modes with an Orbitrap analyzer under the same conditions as those used in a previous study [57].

MaxQuant database search and protein quantification

Raw MS files were processed with MaxQuant (Version 1.5.6.0). LFQ was performed using a label-free, intensity-based absolute quantification approach.

Significant differences in protein levels were defined for proteins with different expression multiples (ratio A/B > 1.5 or ratio A/B < 0.66). Subsequently, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, as well as protein-protein interaction analysis.

ELISA

The levels of Ach (E20535), acetylcholine esterase (AchE, E2148), choline acetyltransferase (ChAT, E21422), Gln (E92310M), GABA (E20434M), iso-Hyd (E20542M), 5-HT (E20435M), NA (E91467M), HIS (E20452M) and 5-HIAA (E91494M) in the hippocampus were measured using the relevant ELISA kits according to the corresponding manufacturer’s instructions. The levels of Aβ1-42 (E20118), MDA (E20347M), CAT (E21414), SOD (E20348M), ROS (E20634) and GSH-Px (E20584) in the hippocampus and serum were measured using the relevant ELISA kits according to the corresponding manufacturer’s instructions (Shanghai Yuanye Biological Technology Co. Ltd., Shanghai, China).

Statistical analysis

Data are expressed as the mean ± standard deviation (S.D.). Differences were determined by one-way analysis of variance followed by post-hoc multiple comparisons (Dunn’s test) using SPSS 16.0 software (IBM Corporation, Armonk, USA). Statistical significance was declared for p-values less than 0.05.