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Apigenin attenuates hippocampal oxidative events, inflammation and pathological alterations in rats fed high fat, fructose diet
Biomedicine & Pharmacotherapy 89 (2017) 323–331
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Apigenin attenuates hippocampal oxidative events, inflammation and pathological alterations in rats fed high fat, fructose diet Kalivarathan Jagan, Chandrasekaran Sathiya Priya, Kalaivanan Kalpana, Ramachandran Vidhya, Carani Venkatraman Anuradha* Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India
A R T I C L E I N F O
Article history: Received 23 November 2016 Received in revised form 23 January 2017 Accepted 29 January 2017 Keywords: High calorie diet Hippocampus Oxidative stress Inflammation Apigenin Sitagliptin
A B S T R A C T
High calorie diet promotes oxidative stress and chronic low grade inflammation that predispose to brain dysfunction and neurodegeneration. Hippocampus region of the brain has been shown to be particularly sensitive to high calorie diet. We hypothesize that apigenin (API), a flavonoid could attenuate hippocampal derangements induced by high fat-high fructose diet (HFFD). In this study, we investigated the effects of API on oxidative stress and inflammation in the hippocampus, and compared with those of sitagliptin (STG), a standard drug with neuroprotective properties. The markers of oxidative stress and inflammation were examined using biochemical assays, western blotting and immunohistochemistry techniques. HFFD-fed rats showed severe pathological alterations and API treatment rescued the hippocampus from the derangements. API significantly improved the antioxidant machinery, reduced ROS levels and prevented the activation of the stress kinases, inhibitor of kappa B kinase beta (IKKb) and c-Jun NH2 terminal kinase (JNK), and the nuclear translocation and activation of nuclear factor kappa B (NF-kB). The plasma levels of inflammatory cytokines were also reduced. Our findings suggest that hippocampal derangements triggered by HFFD feeding were effectively curtailed by API. Suppression of oxidative stress, NF-kB activation and JNK phosphorylation in the hippocampus are the mechanisms by which API offers neuroprotection in this model. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Intake of calorie-abundant foods is steadily on the rise in parallel with an escalating incidence of metabolic diseases such as obesity and type 2 diabetes (T2D). Calorie rich diet provides excess fats and sugars creating a stress on the metabolic machinery. Recent research provides evidence for the adverse effects of high calorie diet on brain functions which can increase the susceptibility to neurodegenerative disorders [1]. Hippocampal region of the brain is important for learning and memory and is the most
Abbreviations: HFFD, high fat-high fructose diet; API, apigenin; STG, sitagliptin; IKKb, inhibitor of kappa B kinase beta; NF-kB, nuclear factor kappa B; JNK, c-Jun NH2 terminal kinase; T2D, type 2 diabetes; TNF-a, tumour necrosis factor-a; IL-6, interleukin-6; 3-NT, 3-nitrotyrosine; 4-HNE, 4-hydroxynonenol; TBARS, thiobarbituric acid reactive substances; LHP, lipid hydroperoxides; PCO, protein carbonyl; AOPP, advanced oxidation protein products; FRAP, ferric reducing antioxidant power; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidise; GSH, reduced glutathione. * Corresponding author. E-mail address: [email protected] (A. Carani Venkatraman). http://dx.doi.org/10.1016/j.biopha.2017.01.162 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
sensitive to metabolic changes associated with dietary habits. High-calorie diet impairs the structure and function of the hippocampus causing a decline in neurogenesis, synaptic plasticity and cognition [2]. Cells suffer from oxidative stress when there is an imbalance between free radical production and cellular antioxidant defense mechanisms. The brain tissue is highly susceptible to oxidative insults because of its high oxygen consumption rate, abundant lipid content and relative paucity of antioxidant enzymes. Many studies show that reactive oxygen species (ROS) production in the neuronal cells plays an important role in neurodegenerative disorders [3]. Inflammation is recognized to be yet another causative mechanism that facilitates neuronal dysfunction and neurodegeneration. Nuclear factor-kB (NF-kB) signaling and c-Jun NH2 terminal kinase (JNK) activation are suggested to be involved in the pathology of brain inflammation leading to neuronal apoptosis, neuronal loss and impaired cognition [4]. ROS can promote inflammation by activating cellular JNK and another kinase called inhibitor of kappa B kinase beta (IKKb). Activation of
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JNK modulates a transcription factor called activator protein-1 (AP-1), the activation of which provokes an inflammatory response while IKKb activation facilitates the nuclear transcription of NF-kB which triggers the gene transcription of pro-inflammatory cytokines like tumor necrosis factor-a (TNF-a) and interleukin6 (IL-6). Sitagliptin (STG), is a selective dipeptidyl peptidase-IV (DPP-IV) inhibitor approved for use in patients with T2D. This drug is known to improve glucose and lipid metabolism, and insulin sensitivity [5]. Besides this, STG recovers the antioxidant defense system in the brain and ameliorates cognitive impairment in insulin resistant rats [6]. STG administration for 21 days is further shown to prevent brain mitochondrial dysfunction and improve learning and memory in high fat diet-fed rats [7]. However, treatment with STG has several side effects like gastrointestinal disturbances, upper respiratory infection, joint pain and urinary tract infection [8]. The flavonoid apigenin (API) is found abundant in fruits, vegetables, nuts and in medicinal herbs. API scavenges superoxide, singlet oxygen and hydroxyl radicals in vitro [9] and boosts up the cellular antioxidant defense system. We previously showed that API can bind and interact with DPP-IV and also inhibit the enzyme DPP-IV in the hippocampus of high fat, high fructose diet (HFFD)fed insulin resistant rats [10]. API improves blood–brain barrier integrity, learning and memory and lowers the levels of amyloid b peptide (Ab)25–35 in an Alzheimer's disease (AD) model [11]. API has been proven to be safe, non-toxic and non-mutagenic when administered to rats [12]. We hypothesized that API can protect hippocampus of rats challenged with a high calorie diet. We tested this hypothesis by investigating the effects of API on oxidant– antioxidant balance, NF-kB activation and JNK phosphorylation in the hippocampus of HFFD-fed rats. 2. Materials and methods 2.1. Chemicals, antibodies and kits API with 98% HPLC grade purity and STG (Januvia) were purchased from the Cayman Chemical Company, USA and Merck Pvt. Ltd., USA respectively. Assay kits for glucose, insulin, TNF-a and IL-6 were purchased from Agappe diagnostics Pvt. Ltd., Kerala, India; Accubind, Monobind Inc., CA, USA; BD Biosciences, San Jose, CA, USA and Koma Biotech, Seoul, South Korea respectively. Anti-3nitrotyrosine (3-NT) and anti-4-hydroxynonenol (4-HNE) antibodies were purchased from Invitrogen, USA, and Merck (Calbiochem), Darmstadt, Germany, respectively. Anti-IKKb, anti-pIKKb, anti-pJNK and b-actin were purchased from Cell Signaling Technologies, USA. Anti-NF-kB and anti-JNK were purchased from Santacruz Biotechnology, CA, USA. Chemiluminescence assay kit was purchased from Thermo Scientific, Rockford, IL, USA. Antirabbit and anti-mouse secondary antibodies were purchased from GeNei, Bangalore, India. Fine chemicals and reagents were acquired from Himedia Laboratories Pvt. Ltd., Mumbai, India or Sisco Research Laboratories Pvt. Ltd., Mumbai, India.
2.3. Animals Male albino Wistar rats of body weight 100–120 g were obtained and maintained in the Central Animal House, Raja Muthiah Medical College and Hospital, Annamalai Nagar, India. The animals were kept in polypropylene cages in a clean room and had free access to standard pellet and water, standard light–dark (12 h light/12 h dark) cycle and temperature (22–24 C). This study was approved by the Institutional Animal Ethics Committee of Animal Care (IAEC), Annamalai University (no. 160/1999/CPCSEA/ 1101). 2.4. Experimental design and treatment Age and weight-matched animals were used in the study. A total number of 45 rats were grouped into five, each consisting of nine animals (n = 9): Group I—Control (CON), Group II—HFFD, Group III—HFFD + API, Group IV—HFFD + STG, Group V—CON + API. The animals were maintained for a period of 60 days. API (1.5 mg/ kg bw) dissolved in 0.1% dimethyl sulfoxide (DMSO) was injected intraperitoneally (i.p.) every alternate day for the last 30 days of the experimental period [12]. STG (30 mg/kg bw) dissolved in 0.9% saline was administered by intragastric intubation for the last 30 days of the experimental period [7]. Body weight and food intake of animals were noted every day. The dosage of API used for this study is based on the report by Chowdhury et al. [12] and our previous study [10]. 2.5. Blood and tissue sampling At the end of experimental period, the animals were fasted for 12 h and then sacrificed by cervical dislocation. Blood was collected by sino-ocular puncture. Hippocampus was dissected out and washed immediately in ice-cold saline (0.89% sodium chloride). Hippocampus were either frozen immediately in liquid nitrogen or fixed in 10% formalin for histological and immunohistochemical studies. Hippocampal homogenate (10%) was prepared in ice-cold 0.1 M Tris–HCl buffer, pH 7.4. Biochemical estimations were done in plasma and hippocampal homogenate obtained from six animals in each group (n = 6). Portions of tissue were collected from three animals in each group and used for western blotting and immunohistological studies (n = 3). 2.6. Biochemical, molecular and pathological studies 2.6.1. Plasma glucose and insulin The levels of glucose, insulin, TNF-a and IL-6 were measured in plasma according to the manufactures’ protocol.
2.2. Diet
2.6.2. Lipid peroxidation and protein damage Lipid damage was assessed by monitoring the levels of thiobarbituric acid reactive substances (TBARS) [13] and lipid hydroperoxides (LHP) [14] in plasma and hippocampus and protein damage by measuring the levels of protein carbonyl (PCO) in plasma and tissue [15]. Advanced oxidation protein products (AOPP) formation in plasma was studied by the method of WitkoSarsat et al. [16].
HFFD had the following ingredients (g/100 g): fructose 45.0, groundnut oil 10.0, beef tallow 10.0, casein 22.5, DL-methionine 0.3, vitamin mixture 1.2, mineral mixture 5.5 and wheat bran 5.5. The standard laboratory chow (Amrut animal feed, Pranav Agro Industries Ltd., Bangalore, India) consisted of 60% (w/w) starch, 22.08% (w/w) protein, and 4.38% (w/w) fat. The normal chow provided 382.61 cal/100 g while HFFD provided 471.25 cal/100 g. HFFD was freshly prepared every day.
2.6.3. Antioxidant status The total antioxidant potential in plasma was measured by ferric reducing antioxidant power (FRAP) assay [17]. The activities of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidise (GPx) and the levels of non-enzymatic anti-oxidants such as ascorbic acid (vitamin-C), a-tocopherol (vitamin-E) and reduced glutathione (GSH) were assayed by the methods outlined elsewhere [18].
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Table 1 Initial and final body weights of experimental animals. Parameters
CON
HFFD
HFFD + API
HFFD + STG
CON + API
Body weight Initial (g) Final (g)
128.24 6.8 165.42 10.3a
125.31 5.1 242.78 13.8b
135.21 6.2 202.12 5.3c
132.16 7.9 184.26 9.3d
137.65 4.7 172.81 8.4a,d
Values are means SD of six rats from each group. CON, control rats; HFFD, high fat-high fructose diet-fed rats; HFFD + API, high fat-high fructose diet-fed rats treated with API; HFFD + STG, high fat-high fructose diet-fed rats treated with STG; CON + API, control rats treated with API. Values that bear different superscripts are significantly different from one another [One way ANOVA followed by Tukey's test (P < 0.05)].
2.6.4. SDS-PAGE and Western blotting Hippocampal homogenates were prepared using ice-cold homogenization buffer (50 mM Tris, 0.25% SDS, 150 mM NaCl, 1% NP-40 and 1 mM EDTA, pH 7.4) and centrifuged at 10,000 g, 15 min at 4 C. For NF-kB analysis alone, nuclear fraction was extracted using kit from Cayman Chemical Company, MI, USA. After quantifying the protein content (50 mg), equal amount of protein from each sample were resolved by SDS-PAGE. The separated proteins were then electrotransferred onto the polyvinylidene difluoride (PVDF) membrane using transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) at 8 mA or 40 V for 1.5 h. Transfer of proteins from gel to membrane was checked by adding ponceau-S dye (0.5% dye in 5% glacial acetic acid) to membrane. After washing, the PVDF membranes were blocked with blocking buffer and were incubated overnight at 4 C with antibodies specific to pIKKb, NF-kB and pJNK. Next day, the membranes were washed with TBST and incubated with respective secondary antibody for 2 h at room temperature. The membranes were stripped and re-probed with anti-IKKb (for pIKKb), anti-JNK (for pJNK) or anti-b-actin (for NF-kB) antibodies for normalization. The protein bands were visualized by enhanced chemiluminescence detection using Immobilon HRP Western Substrate (Millipore, Bangalore, India). Finally, the densitometry analyses of the bands were performed using Image J software (National Institute of Health, Bethesda, MD, USA). 2.6.5. Histopathological studies Hippocampal tissue excised from the experimental animals were fixed in 10% formalin and embedded in paraffin. Sections of 4-5 mm thickness were mounted on glass slides, deparaffinized and dehydrated in graded alcohol. Then, the sections were stained with haematoxylin and eosin and observed under phase contrast microscope (40). 2.6.6. Immunohistochemistry For immunohistochemistry, 4–5 mm paraffin embedded hippocampal sections were deparaffinized with xylene and dehydrated with gradually increasing concentrations of isopropyl alcohol. Slides were incubated overnight with anti-4 HNE or 3-NT antibody (1: 200 dilution). The slides were rinsed well with phosphate buffer and incubated with super enhancer reagent for 30 min. After rinsing with phosphate buffer, incubation was done with supersensitive polymer–HRP immunohistochemistry detection system. Sections were washed with buffer and incubated with a
diaminobenzidene (DAB) solution for 5 min. Sections were observed under phase contrast microscope (40). 2.7. Statistical analysis The data obtained were tested for statistical significance by one-way analysis of variance (ANOVA) followed by Tukey's Multiple Range Test for multiple comparisons. SPSS statistical software (version 20.0; SPSS, Chicago, IL, USA) was used. P value <0.05 was considered significant. 3. Results 3.1. API decreased body weight gain in HFFD fed rats The initial and final body weights of experimental rats are given in (Table 1). HFFD feeding for 60 days resulted in an increase in body weight compared to normal pellet fed rats. HFFD-fed rats treated with API showed a significant reduction in body weight compared to rats fed HFFD alone. No significant differences in final body weight were observed between CON, HFFD + STG and CON + API groups. Food intake (g/rat/day) was CON, 14.73 0.73; HFFD, 14.31 0.71; HFFD + API, 14.56 0.72; HFFD + STG, 14.62 0.73; CON + STG, 14.79 0.74. No significant differences in food intake were observed between the experimental groups. 3.2. Levels of plasma glucose, insulin, TNF-a and IL-6 in HFFD-fed rats Glucose and insulin levels in plasma of experimental rats are shown in (Table 2). Rats fed HFFD alone showed significant increase in the levels of glucose and insulin as compared to control rats. API- and STG-administration to HFFD-fed rats lowered glucose and insulin levels as compared to HFFD group. Between the two, API addition to HFFD rats brought a better reduction than STG treatment. However, the levels in these two groups were still significantly different from CON group. No significant differences were observed between the CON and CON + API treated rats with respect to glucose and insulin levels. The levels of TNF-a and IL-6 in plasma are tabulated in (Table 2). HFFD feeding caused significant increase in the levels of TNF-a and IL-6 as compared to control rats. The levels were close to normal in API- and STG-treated HFFD-fed rats. No significant differences were observed between the CON and CON + API treated rats.
Table 2 Plasma glucose, insulin and proinflammatory cytokines in experimental rats. Parameters
CON
HFFD
HFFD + API
HFFD + STG
CON + API
Glucose (mg/dl) Insulin (mIU/ml) TNF-a (pg/ml) IL-6 (pg/ml)
100.90 10.1a 16.8 0.5a 14.67 1.01a 81.37 3.5a
158.44 4.3b 31.0 2.7b 42.68 2.6b 198.66 9.5b
116.04 12.1c 23.3 1.5c 29.74 2.3c 129.42 6.9c
134.19 6.2d 26.7 0.7d 22.36 1.3d 155.88 4.1d
94.38 7.8a 16.5 0.1a 15.05 0.9a 84.09 6.3a
Values are means SD of six rats from each group. CON, control rats; HFFD, high fat-high fructose diet-fed rats; HFFD + API, high fat-high fructose diet-fed rats treated with API; HFFD + STG, high fat-high fructose diet-fed rats treated with STG; CON + API, control rats treated with API. Values that bear different superscripts are significantly different from one another [One way ANOVA followed by Tukey's test (P < 0.05)].
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Table 3 Levels of oxidative stress in experimental rats. Parameters Plasma TBARS (A) LHP (B) PC (B) AOPP (C) Hippocampus TBARS (D) LHP (D) PC (D)
CON
HFFD
HFFD + API
HFFD + STG
CON + API
1.12 0.08a 2.03 0.06a 3.98 0.3a 85.17 7.4a
6.12 0.6b 5.21 0.3b 9.15 0.5b 143.12 9.6b
4.36 0.1c 4.56 0.15c 7.06 0.1c 128.05 4.3c
3.19 0.3d 3.13 0.08d 5.56 0.4d 107.47 6.2d
1.05 0.07a 1.98 0.04a 3.86 0.3a 83.45 4.2a
2.31 0.1a 4.16 0.3a 4.02 0.4a
7.86 0.7b 7.38 0.6b 9.36 0.3b
5.32 0.3c 6.16 0.2c 7.78 0.5c
4.11 0.2d 5.07 0.5d 5.87 0.5d
2.17 0.08a 4.38 0.3a,d 3.91 0.2a
Values are means SD of six rats from each group. CON, control rats; HFFD, high fat-high fructose diet-fed rats; HFFD + API, high fat-high fructose diet-fed rats treated with API; HFFD + STG, high fat-high fructose diet-fed rats treated with STG; CON + API, control rats treated with API. Values that bear different superscripts are significantly different from one another [One way ANOVA followed by Tukey's test (P < 0.05)]. A = mmol/dl; B = nmol/dl; C = mmol/l; D = nmol/mg protein.
Fig. 1. Immunohistochemical localization of 3-NT (A) and 4-HNE (B) adducts in hippocampus of experimental animals (40). Brown color indicates DAB staining and the occurrence of antigen–antibody reaction. Blue color indicates hemotoxylin staining and the absence of antigen–antibody reaction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.3. API alleviates oxidative changes in HFFD fed rats The levels of plasma AOPP and TBARS, LHP and PCO in plasma and hippocampus are shown in (Table 3). The levels of lipid peroxidation and protein damage markers in HFFD rats were significantly higher than those seen in CON. On the other hand, administration of API- and STG-lowered the levels of these oxidative stress markers. No significant differences in the levels of AOPP, TBARS, LHP and PCO were observed between the CON and CON + API groups. API- and STG-administered HFFD-fed animals had reduced AOPP, TBARS, LHP and PCO levels significantly as compared to HFFD group. The extent of lipid and protein damage was also assessed by examining the localization of 3-NT and 4-HNE adducts respectively in hippocampal sections by immunohistochemistry (Fig. 1A and B). Positive immunoreactivity for these adducts was observed in HFFD group. HFFD + API and HFFD + STG groups had minimum levels of adducts as compared to HFFD group. There was negligible accumulation of 3-NT and 4-HNE adducts in CON and CON + API groups. The activities of enzymatic antioxidants of SOD, CAT and GPx in the hippocampus are presented in (Table 4). HFFD-fed rats had poor antioxidant status as compared to CON group. API or STG treatment improved the levels of antioxidant enzymes. No significant differences were found between the CON, CON + API and HFFD + STG treated groups in the hemolysate of CAT and GPx. Levels of SOD in hippocampus were not significantly different in HFFD + STG as compared to CON animals and in HFFD + API as compared to HFFD + STG treated group. The levels of GSH and vitamins C and E in circulation and hippocampus and the results of FRAP assay in plasma of animals in each group are listed in (Table 5). The antioxidant status was significantly decreased in HFFD-fed animals as compared to CON animals. Levels of vitamins C and E were not significantly different
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between CON and CON + API treated animals. API and STG treatment prevented the detrimental effects of HFFD by uplifting the levels of antioxidants. No significant differences in GSH were observed between HFFD + STG and CON groups. 3.4. API modulates IKKb/NF-kB associated neuroinflammation IKKb, the stress kinase activated by phosphorylation and required for the release of cytosolic NF-kB to nucleus from bound IkB was increased in HFFD-fed rats compared to control rats. The phospho-form of IKKb was reduced in API- and STG-treated animals (Fig. 2A and B). The results obtained for immunohistochemical staining of pIKKb and NF-kB-p65 are concordant with the western blotting analysis (Fig. 2C and D). Fig. 2C and D represents the photomicrographs on the expression of pIKKb and NF-kB-p65 in the hippocampus of experimental animals. The intensity of immunostaining for pIKKb and NF-kB-p65 were more pronounced in HFFD treated group. It is apparent that there is a marked reduction in pIKKb and NF-kB-p65 immunoreactivity in HFFD + API and HFFD + STG treated group. There is no marked difference in the immunoreactivity for these antibodies between CON and CON + API groups. HFFD-fed rats displayed increased expression of NF-kB in the nuclear fraction of the hippocampus as compared to control rats (Fig. 2A and B) suggesting translocation and activation in HFFD group. The translocation of NF-kB to nucleus was reduced upon treatment with API- and STG- as compared to HFFD-fed rats. This is revealed by reduced nuclear expression. 3.5. API prevents JNK activation Activation of JNK by phosphorylation at Thr183/Y185 was observed in the HFFD-fed rats as compared to normal diet-fed rats. The rise in JNK phosphorylation promoted by HFFD feeding was
Table 4 Levels of enzymatic antioxidants in experimental animals. Parameters Hemolysate SOD (A) CAT (B) GPx (C) Hippocampus SOD (A) CAT (B) GPx (C)
CON
HFFD
HFFD + API
HFFD + STG
CON + API
3.78 0.02a 43.21 4.2a,d 9.2 0.8a,d
1.09 0.07b 22.14 2.0b 5.75 0.3b
2.58 0.2c 34.68 2.3c 7.18 0.6c
3.05 0.3d 40.08 2.0d 8.24 0.7d
3.92 0.1a 45.16 2.1a 9.5 0.5a
4.21 0.4a,d 49.16 2.8a 4.68 0.3a
2.04 0.2b 24.18 1.5b 2.24 0.2b
3.16 0.3c 33.32 2.1c 3.18 0.2c
3.69 0.2c,d 41.16 2.5d 3.97 0.2d
4.24 0.4a 51.44 4.2a 4.97 0.4a
Values are means SD of six rats from each group. CON, control rats; HFFD, high fat-high fructose diet-fed rats; HFFD + API, high fat-high fructose diet-fed rats treated with API; HFFD + STG, high fat-high fructose diet-fed rats treated with STG; CON + API, control rats treated with API. Values that bear different superscripts are significantly different from one another [One way ANOVA followed by Tukey's test (P < 0.05)]. A = U/mg Hb for hemolysate; U/mg protein for hippocampus; B = mmol of H2O2 consumed/ min/mg Hb for hemolysate; mmol of H2O2 consumed/min/mg protein for hippocampus; C = mmol GSH consumed/min/mg Hb hemolysate; mmol GSH consumed/min/mg protein for hippocampus; U = enzyme concentration required to produce 50% inhibition of chromogen formation in 1 min under standard conditions.
Table 5 Levels of FRAP and non-enzymatic antioxidants in experimental animals. Parameters Plasma FRAP (A) Vitamin C (B) Vitamin E (B) GSH (B) Hippocampus Vitamin C (C) Vitamin E (C) GSH (C)
CON
HFFD
HFFD + API
HFFD + STG
CON + API
1032.4 6.4a 2.61 0.08a 1.92 0.07a 19.46 1.6a,d
798.2 7.4b 0.91 0.05b 0.46 0.2b 11.22 0.1b
843.7 3.8c 1.67 0.1c 0.85 0.04c 14.76 0.7c
968.2 4.1d 2.03 0.1d 1.46 0.1d 18.12 0.4d
1056.3 5.4a 2.75 0.1a 1.98 0.06a 20.18 1.0a
46.36 3.5a 2.12 0.1a 42.18 0.4a,d
21.09 2.0b 0.82 0.06b 28.12 2.0b
34.58 1.5c 1.35 0.1c 34.69 2.3c
40.69 3.9d 1.86 0.1d 39.14 1.6d
48.12 3.0a 2.24 0.2a 44.36 4.3a
Values are means SD of six rats from each group. CON, control rats; HFFD, high fat-high fructose diet-fed rats; HFFD + API, high fat-high fructose diet-fed rats treated with API; HFFD + STG, high fat-high fructose diet-fed rats treated with STG; CON + API, control rats treated with API. Values that bear different superscripts are significantly different from one another [One way ANOVA followed by Tukey's test (P <.05)]. A = mmol/l; B = mg/dl; C = mg/mg protein.
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Fig. 2. Phoshphorylation status of IKKb and protein expression of NF-kB p65 subunit. (A) Representative western blot of IKKb, pIKKb and NF-kB-p65 subunit in hippocampus. Lane 1, CON; lane 2, HFFD; lane 3, HFFD + API; lane 4, HFFD + STG; lane 5, CON + API. (B) Densitometry data obtained after normalization of pIKKb with total IKKb and NF-kB-p65 with b-actin. Fold change for a group was calculated with respect to control. Results are expressed as means S.D (n = 3). (C and D) Immunohistochemical localization of pIKKb (C) and NF-kB (D) in the hippocampus (40). Brown color indicates DAB staining and the occurrence of antigen–antibody reaction. Blue color indicates hemotoxylin staining and the absence of antigen–antibody reaction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
reversed with API and STG treatment (Fig. 3A and B). Results obtained from the immunohistochemical investigation in the hippocampus of control and experimental animals are in agreement with the Western blotting results (Fig. 3C). Fig. 3C represents the photomicrographs on the expression of JNK in the hippocampus of experimental animals. The intensity of immunostaining for JNK was more pronounced in HFFD treated group. It is apparent that there is a marked reduction in JNK immunoreactivity in HFFD + API and HFFD + STG treated group. There is no marked difference in the immunoreactivity for this antibody between CON and CON + API groups. 3.6. API prevents histopathological alterations in HFFD fed rats Fig. 4 depicts the photographs of hippocampal sections stained using hematoxylin and eosin. Cells appear normal in sections from CON and CON + API groups. Sections from HFFD group show shrinkage in the size of the pyramidal cells, disorganization of layer, dark nuclei and many apoptotic bodies and large vacuolated cells. Sections from HFFD + API and HFFD + STG groups show restoration of pyramidal cell size and normal architecture of CA1 region and dentate gyrus with few degenerative neurons. 4. Discussion The present study investigated the neuroprotective actions of API in high calorie-diet fed rats by determining its effects on oxidative stress, inflammation and pathological changes in the hippocampus. Free radical-induced damage to macromolecules is a central event in the progression of neurodegeneration. Accumulation of toxic products of lipid peroxidation such as TBARS, LHP and 4-HNE and protein damage such as 3-NT, AOPP and PCO were observed in the hippocampus of HFFD-fed rats suggesting oxidative damage to hippocampus. Excessive nutrients, particularly fats and sugars
when transported into cells, disturb metabolic homeostasis and directly increase mitochondrial oxidative workload, which causes increased production of ROS. A study by Liu et al. [19] shows that hippocampal neurons exposed to high glucose develop oxidative stress through high ROS generation. Also, palmitic acid can induce lipotoxicity in neuronal progenitor cells [20]. Thus, administration of excess nutrients may directly induce lipo- or glucose toxicity in brain cells. In addition, the depletion of antioxidant enzymes and molecules that can scavenge ROS or prevent oxidative damage in HFFD-fed rats could also be responsible for the rise in levels of oxidative damage markers. The levels of ROS, TBARS and PCO were reported to be significantly increased in the brains of fructosedrinking insulin resistant rats [21]. Progressive damage due to oxidative stress causes histological alterations. Histopathological examination is therefore important for evaluating the extent of tissue damage and reversal. The hippocampus consists of the hippocampus proprius, gyrus dentatus and the subiculum. The hippocampus proper is subdivided into four regions according to density, size and branching of axons and dendrites of pyramidal cells (Cornu Ammonis (CA) 1–4). Each of these regions consists of three layers: stratum moleculare, stratum pyramidale and stratum multiforme. The stratum pyramidale contains bodies of the pyramidal cells [22]. The CA1 region is characterized by densely packed medium-sized pyramidal cells and is more sensitive to various insults [23]. Examination of CA1 area in the hippocampus of HFFD-fed rats revealed loss of cell integrity, significant decrease in pyramidal cells number and shrinkage in the size of pyramidal cells. A similar observation has been observed and reported by El-Khair et al. [24] in high cholesterol diet-fed adult rats. Significant reduction in pyramidal cells, attenuation and thickening of small blood vessels, apparent increase of astrocytes and decrease of Nissl's granules in the hippocampus of high cholesterol diet-fed rats have been reported [24]. Reduced hippocampal dendritic spine density, and reduced long-term potentiation at Schaffer collateral-CA1
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Fig. 3. Phoshphorylation status of JNK. (A) Representative western blots of JNK and pJNK in the hippocampus. Lane 1, CON; lane 2, HFFD; lane 3, HFFD + API; lane 4, HFFD + STG; lane 5, CON + API. (B) Densitometry data was obtained after normalization of pJNK with total JNK. Fold change for each group was calculated with respect to control. Results are expressed as means S.D (n = 3). (C) Immunohistochemical localization of pJNK in the hippocampus (40). Brown color indicates DAB staining and the occurrence of antigen–antibody reaction. Blue color indicates hemotoxylin staining and the absence of antigen–antibody reaction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
synapses are found in rats fed high-fat, high-glucose diet supplemented with high-fructose corn syrup for 8 months [25]. Over nutrition induces neuroinflammation and dysfunction of the central nervous system. Oxidative stress is the pathogenic link that bridges over-nutrition with inflammation via NF-kB activation. Upon stimulation by ROS, the enzyme IKKb phosphorylates an inhibitor of kappa B (IkB) attached to p50/p65 heterodimer of NF-kB resulting in its degradation by the ubiquitin proteasome pathway. Dissociation of IkB from the dimer facilitates the entry of NF-kB in to the nucleus. Activated NF-kB can trigger the generation of inflammatory molecules like TNF-a and IL-6. The levels of these molecules are also increased in HFFD-fed rats as a consequence of NF-kB activation. Insulin regulates enormous brain functions and fructose induced perturbation of insulin signaling/insulin resistance may
be linked to the hippocampal derangements. Insulin resistance in the hippocampus is associated with learning and memory decline [26]. JNK and IKKb are shown to be critically involved in promotion of diet-induced insulin resistance by negative modulation of insulin signaling by serine phosphorylation of insulin receptor substrate at Ser307 residue [27]. Thus insulin resistance observed in HFFD-fed rats might contribute damage to the hippocampus. API administration significantly reduced oxidative stress, histopathological changes, IKKb/NF-kB signaling and JNK phosphorylation in the hippocampus of HFFD-fed rats. API is reported to regulate redox imbalance and inhibit JNK signaling during Abmediated toxicity [28]. API restored enzymatic (SOD, CAT and GPx) and non-enzymatic (GSH, vitamins C and E) antioxidants. A study by Guo et al. [29] reported that API enhanced the mRNA expression of antioxidant
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Fig. 4. Representative photographs of CA1 region of hippocampus stained with haematoxylin and eosin: (A) CON—layer of compact granular cells with normal nuclei in dentate gyrus (G), small pyramidal cells (P) and dispersed small vacuoles (V). (B) HFFD—shrinkage and darkening of many large pyramidal cells, disorganization of pyramidal cells. (C) HFFD + API—preservation of small pyramidal cells of CA1 region. (D) HFFD + STG—decreased thickness of layer of small pyramidal cells of CA1 region. (E) CON + API— normal architecture of hippocampus (40). Insets show the enlarged view of pyramidal cells.
enzymes in OGD/R-treated PC12 cells. The improved enzymatic activities of SOD, CAT and GPx by API may be a direct effect of API in the brain at the protein level or at the mRNA level or an indirect result of improved insulin sensitivity by API. Vitamin E-dependent protection of the hippocampus has been described in some studies suggesting the neuroprotective role of antioxidants [30,31]. API is a proven antioxidant. The presence of C2–C3 double bond on the C ring confers ROS-scavenging activity [32]. The API protective action may be attributed to a combination of improved insulin sensitivity, antioxidant and anti-inflammatory effects. Further, rats fed HFFD for 60 days were found to be obese which may have an influence on the investigated parameters. API administration reduced body weight gain in HFFD-fed rats, even when the food intake was not significantly different. Hiranya et al. [7] showed that STG prevents hippocampal mitochondrial dysfunction, improves the learning behavior and
decreases brain oxidative stress levels in high fat diet-fed rats and that STG can be useful in the treatment of neurodegenerative events associated with insulin resistance and metabolic syndrome. Administration of STG reduces brain oxidative stress induced by high fat diet consumption [7]. STG increases glucagon like peptide1 (GLP-1) levels by inhibiting DPP-IV enzyme and acts as an antidiabetic agent. We previously reported that API has binding efficacy at Glu206 in the active site of DPP-IV enzyme [10]. Possibly API could act in a similar manner to increase brain active GLP-1 levels which remains to be studied. 5. Conclusions Unhealthy dietary habits could lead to changes in hippocampal integrity and loss of hippocampal function. Intake of nutritious dietary components enriched diets can be productive strategy not
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only to counteract metabolic dysfunction but also to protect brain health. The present findings indicate that API could offer significant neuroprotection. Since API appears to be a promising compound that protects hippocampus, it can be taken to the next level of investigation. Our future studies are to evaluate the effects of API on the expression of neurotrophic peptides and on insulin signaling events in the hippocampus. Conflict of interest The authors declare no conflicts of interest. References [1] R. Agrawal, F. Gomez-Pinilla, Metabolic syndrome in the brain: deficiency in omega-3 fatty acid exacerbates dysfunctions in insulin receptor signalling and cognition, J. Physiol. 590 (2012) 2485–2499. [2] C.E. Greenwood, G. Winocur, Learning and memory impairment in rats fed a high saturated fat diet, Behav. Neural Biol. 53 (1990) 74–87. [3] J. Yuan, B.A. Yankner, Apoptosis in the nervous system, Nature 407 (2000) 802– 809. [4] D. Cai, T. Liu, Inflammatory cause of metabolic syndrome via brain stress and NF-kB, Aging 4 (2012) 98–115. [5] H. Yanai, H. Adachi, H. Hamasaki, Y. Masui, R. Yoshikawa, et al., Effects of 6month sitagliptin treatment on glucose and lipid metabolism, blood pressure, body weight and renal function in type 2 diabetic patients: a chart-based analysis, J. Clin. Med. Res. 4 (2012) 251–258. [6] V.A. Gault, R. Lennox, P.R. Flatt, Sitagliptin, a dipeptidyl peptidase-4 inhibitor, improves recognition memory, oxidative stress and hippocampal neurogenesis and upregulates key genes involved in cognitive decline, Diabetes Obes. Metab. 17 (2015) 403–413. [7] P. Hiranya, A. Nattayaporn, C. Nipon, C.C. Siriporn, DPP4 inhibitors improve cognition and brain mitochondrial functions of insulin-resistant rats, J. Endocrinol. 218 (2013) 1–11. [8] A. Barnett, DPP-4 inhibitors and their potential role in the management of type 2 diabetes, Int. J. Clin. Pract. 60 (2006) 1454–1470. [9] J.Y. Han, S.Y. Ahn, C.S. Kim, S.K. Yoo, S.K. Kim, et al., Protection of apigenin against kainate-induced excitotoxicity by anti-oxidative effects, Biol. Pharm. Bull. 35 (2012) 1440–1446. [10] K. Jagan, M.K. Radika, E. Priyadarshini, C.V. Anuradha, A study on the inhibitory potential of DPP-IV enzyme by apigenin through in silico and in vivo approaches, Res. J. Rec. Sci. 4 (2015) 22–29. [11] R. Liu, T. Zhang, H. Yang, X. Lan, J. Ying, G. Du, The flavonoid apigenin protects brain neurovascular coupling against amyloid-beta25-35-induced toxicity in mice, J. Alzheimers Dis. 24 (2011) 85–100. [12] M.H. Chowdhury, K.G. Miltu, S.S. Bhabani, S.D. Niladri, M. Biswajit, Apigenin causes biochemical modulation, GLUT4 and CD38 alterations to improve diabetes and to protect damages of some vital organs in experimental diabetes, Am. J. Pharmacol. Toxicol. 9 (2014) 39–52. [13] B. Vanithadevi, C.V. Anuradha, Effect of rosmarinic acid on insulin sensitivity, glyoxalase system and oxidative events in liver of fructose-fed mice, Int. J. Diabetes Metab. 16 (2008) 35–44.
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Apigenin attenuates hippocampal oxidative events, inflammation and pathological alterations in rats fed high fat, fructose diet Kalivarathan Jagan, Chandrasekaran Sathiya Priya, Kalaivanan Kalpana, Ramachandran Vidhya, Carani Venkatraman Anuradha* Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India
A R T I C L E I N F O
Article history: Received 23 November 2016 Received in revised form 23 January 2017 Accepted 29 January 2017 Keywords: High calorie diet Hippocampus Oxidative stress Inflammation Apigenin Sitagliptin
A B S T R A C T
High calorie diet promotes oxidative stress and chronic low grade inflammation that predispose to brain dysfunction and neurodegeneration. Hippocampus region of the brain has been shown to be particularly sensitive to high calorie diet. We hypothesize that apigenin (API), a flavonoid could attenuate hippocampal derangements induced by high fat-high fructose diet (HFFD). In this study, we investigated the effects of API on oxidative stress and inflammation in the hippocampus, and compared with those of sitagliptin (STG), a standard drug with neuroprotective properties. The markers of oxidative stress and inflammation were examined using biochemical assays, western blotting and immunohistochemistry techniques. HFFD-fed rats showed severe pathological alterations and API treatment rescued the hippocampus from the derangements. API significantly improved the antioxidant machinery, reduced ROS levels and prevented the activation of the stress kinases, inhibitor of kappa B kinase beta (IKKb) and c-Jun NH2 terminal kinase (JNK), and the nuclear translocation and activation of nuclear factor kappa B (NF-kB). The plasma levels of inflammatory cytokines were also reduced. Our findings suggest that hippocampal derangements triggered by HFFD feeding were effectively curtailed by API. Suppression of oxidative stress, NF-kB activation and JNK phosphorylation in the hippocampus are the mechanisms by which API offers neuroprotection in this model. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Intake of calorie-abundant foods is steadily on the rise in parallel with an escalating incidence of metabolic diseases such as obesity and type 2 diabetes (T2D). Calorie rich diet provides excess fats and sugars creating a stress on the metabolic machinery. Recent research provides evidence for the adverse effects of high calorie diet on brain functions which can increase the susceptibility to neurodegenerative disorders [1]. Hippocampal region of the brain is important for learning and memory and is the most
Abbreviations: HFFD, high fat-high fructose diet; API, apigenin; STG, sitagliptin; IKKb, inhibitor of kappa B kinase beta; NF-kB, nuclear factor kappa B; JNK, c-Jun NH2 terminal kinase; T2D, type 2 diabetes; TNF-a, tumour necrosis factor-a; IL-6, interleukin-6; 3-NT, 3-nitrotyrosine; 4-HNE, 4-hydroxynonenol; TBARS, thiobarbituric acid reactive substances; LHP, lipid hydroperoxides; PCO, protein carbonyl; AOPP, advanced oxidation protein products; FRAP, ferric reducing antioxidant power; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidise; GSH, reduced glutathione. * Corresponding author. E-mail address: [email protected] (A. Carani Venkatraman). http://dx.doi.org/10.1016/j.biopha.2017.01.162 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
sensitive to metabolic changes associated with dietary habits. High-calorie diet impairs the structure and function of the hippocampus causing a decline in neurogenesis, synaptic plasticity and cognition [2]. Cells suffer from oxidative stress when there is an imbalance between free radical production and cellular antioxidant defense mechanisms. The brain tissue is highly susceptible to oxidative insults because of its high oxygen consumption rate, abundant lipid content and relative paucity of antioxidant enzymes. Many studies show that reactive oxygen species (ROS) production in the neuronal cells plays an important role in neurodegenerative disorders [3]. Inflammation is recognized to be yet another causative mechanism that facilitates neuronal dysfunction and neurodegeneration. Nuclear factor-kB (NF-kB) signaling and c-Jun NH2 terminal kinase (JNK) activation are suggested to be involved in the pathology of brain inflammation leading to neuronal apoptosis, neuronal loss and impaired cognition [4]. ROS can promote inflammation by activating cellular JNK and another kinase called inhibitor of kappa B kinase beta (IKKb). Activation of
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JNK modulates a transcription factor called activator protein-1 (AP-1), the activation of which provokes an inflammatory response while IKKb activation facilitates the nuclear transcription of NF-kB which triggers the gene transcription of pro-inflammatory cytokines like tumor necrosis factor-a (TNF-a) and interleukin6 (IL-6). Sitagliptin (STG), is a selective dipeptidyl peptidase-IV (DPP-IV) inhibitor approved for use in patients with T2D. This drug is known to improve glucose and lipid metabolism, and insulin sensitivity [5]. Besides this, STG recovers the antioxidant defense system in the brain and ameliorates cognitive impairment in insulin resistant rats [6]. STG administration for 21 days is further shown to prevent brain mitochondrial dysfunction and improve learning and memory in high fat diet-fed rats [7]. However, treatment with STG has several side effects like gastrointestinal disturbances, upper respiratory infection, joint pain and urinary tract infection [8]. The flavonoid apigenin (API) is found abundant in fruits, vegetables, nuts and in medicinal herbs. API scavenges superoxide, singlet oxygen and hydroxyl radicals in vitro [9] and boosts up the cellular antioxidant defense system. We previously showed that API can bind and interact with DPP-IV and also inhibit the enzyme DPP-IV in the hippocampus of high fat, high fructose diet (HFFD)fed insulin resistant rats [10]. API improves blood–brain barrier integrity, learning and memory and lowers the levels of amyloid b peptide (Ab)25–35 in an Alzheimer's disease (AD) model [11]. API has been proven to be safe, non-toxic and non-mutagenic when administered to rats [12]. We hypothesized that API can protect hippocampus of rats challenged with a high calorie diet. We tested this hypothesis by investigating the effects of API on oxidant– antioxidant balance, NF-kB activation and JNK phosphorylation in the hippocampus of HFFD-fed rats. 2. Materials and methods 2.1. Chemicals, antibodies and kits API with 98% HPLC grade purity and STG (Januvia) were purchased from the Cayman Chemical Company, USA and Merck Pvt. Ltd., USA respectively. Assay kits for glucose, insulin, TNF-a and IL-6 were purchased from Agappe diagnostics Pvt. Ltd., Kerala, India; Accubind, Monobind Inc., CA, USA; BD Biosciences, San Jose, CA, USA and Koma Biotech, Seoul, South Korea respectively. Anti-3nitrotyrosine (3-NT) and anti-4-hydroxynonenol (4-HNE) antibodies were purchased from Invitrogen, USA, and Merck (Calbiochem), Darmstadt, Germany, respectively. Anti-IKKb, anti-pIKKb, anti-pJNK and b-actin were purchased from Cell Signaling Technologies, USA. Anti-NF-kB and anti-JNK were purchased from Santacruz Biotechnology, CA, USA. Chemiluminescence assay kit was purchased from Thermo Scientific, Rockford, IL, USA. Antirabbit and anti-mouse secondary antibodies were purchased from GeNei, Bangalore, India. Fine chemicals and reagents were acquired from Himedia Laboratories Pvt. Ltd., Mumbai, India or Sisco Research Laboratories Pvt. Ltd., Mumbai, India.
2.3. Animals Male albino Wistar rats of body weight 100–120 g were obtained and maintained in the Central Animal House, Raja Muthiah Medical College and Hospital, Annamalai Nagar, India. The animals were kept in polypropylene cages in a clean room and had free access to standard pellet and water, standard light–dark (12 h light/12 h dark) cycle and temperature (22–24 C). This study was approved by the Institutional Animal Ethics Committee of Animal Care (IAEC), Annamalai University (no. 160/1999/CPCSEA/ 1101). 2.4. Experimental design and treatment Age and weight-matched animals were used in the study. A total number of 45 rats were grouped into five, each consisting of nine animals (n = 9): Group I—Control (CON), Group II—HFFD, Group III—HFFD + API, Group IV—HFFD + STG, Group V—CON + API. The animals were maintained for a period of 60 days. API (1.5 mg/ kg bw) dissolved in 0.1% dimethyl sulfoxide (DMSO) was injected intraperitoneally (i.p.) every alternate day for the last 30 days of the experimental period [12]. STG (30 mg/kg bw) dissolved in 0.9% saline was administered by intragastric intubation for the last 30 days of the experimental period [7]. Body weight and food intake of animals were noted every day. The dosage of API used for this study is based on the report by Chowdhury et al. [12] and our previous study [10]. 2.5. Blood and tissue sampling At the end of experimental period, the animals were fasted for 12 h and then sacrificed by cervical dislocation. Blood was collected by sino-ocular puncture. Hippocampus was dissected out and washed immediately in ice-cold saline (0.89% sodium chloride). Hippocampus were either frozen immediately in liquid nitrogen or fixed in 10% formalin for histological and immunohistochemical studies. Hippocampal homogenate (10%) was prepared in ice-cold 0.1 M Tris–HCl buffer, pH 7.4. Biochemical estimations were done in plasma and hippocampal homogenate obtained from six animals in each group (n = 6). Portions of tissue were collected from three animals in each group and used for western blotting and immunohistological studies (n = 3). 2.6. Biochemical, molecular and pathological studies 2.6.1. Plasma glucose and insulin The levels of glucose, insulin, TNF-a and IL-6 were measured in plasma according to the manufactures’ protocol.
2.2. Diet
2.6.2. Lipid peroxidation and protein damage Lipid damage was assessed by monitoring the levels of thiobarbituric acid reactive substances (TBARS) [13] and lipid hydroperoxides (LHP) [14] in plasma and hippocampus and protein damage by measuring the levels of protein carbonyl (PCO) in plasma and tissue [15]. Advanced oxidation protein products (AOPP) formation in plasma was studied by the method of WitkoSarsat et al. [16].
HFFD had the following ingredients (g/100 g): fructose 45.0, groundnut oil 10.0, beef tallow 10.0, casein 22.5, DL-methionine 0.3, vitamin mixture 1.2, mineral mixture 5.5 and wheat bran 5.5. The standard laboratory chow (Amrut animal feed, Pranav Agro Industries Ltd., Bangalore, India) consisted of 60% (w/w) starch, 22.08% (w/w) protein, and 4.38% (w/w) fat. The normal chow provided 382.61 cal/100 g while HFFD provided 471.25 cal/100 g. HFFD was freshly prepared every day.
2.6.3. Antioxidant status The total antioxidant potential in plasma was measured by ferric reducing antioxidant power (FRAP) assay [17]. The activities of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidise (GPx) and the levels of non-enzymatic anti-oxidants such as ascorbic acid (vitamin-C), a-tocopherol (vitamin-E) and reduced glutathione (GSH) were assayed by the methods outlined elsewhere [18].
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Table 1 Initial and final body weights of experimental animals. Parameters
CON
HFFD
HFFD + API
HFFD + STG
CON + API
Body weight Initial (g) Final (g)
128.24 6.8 165.42 10.3a
125.31 5.1 242.78 13.8b
135.21 6.2 202.12 5.3c
132.16 7.9 184.26 9.3d
137.65 4.7 172.81 8.4a,d
Values are means SD of six rats from each group. CON, control rats; HFFD, high fat-high fructose diet-fed rats; HFFD + API, high fat-high fructose diet-fed rats treated with API; HFFD + STG, high fat-high fructose diet-fed rats treated with STG; CON + API, control rats treated with API. Values that bear different superscripts are significantly different from one another [One way ANOVA followed by Tukey's test (P < 0.05)].
2.6.4. SDS-PAGE and Western blotting Hippocampal homogenates were prepared using ice-cold homogenization buffer (50 mM Tris, 0.25% SDS, 150 mM NaCl, 1% NP-40 and 1 mM EDTA, pH 7.4) and centrifuged at 10,000 g, 15 min at 4 C. For NF-kB analysis alone, nuclear fraction was extracted using kit from Cayman Chemical Company, MI, USA. After quantifying the protein content (50 mg), equal amount of protein from each sample were resolved by SDS-PAGE. The separated proteins were then electrotransferred onto the polyvinylidene difluoride (PVDF) membrane using transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) at 8 mA or 40 V for 1.5 h. Transfer of proteins from gel to membrane was checked by adding ponceau-S dye (0.5% dye in 5% glacial acetic acid) to membrane. After washing, the PVDF membranes were blocked with blocking buffer and were incubated overnight at 4 C with antibodies specific to pIKKb, NF-kB and pJNK. Next day, the membranes were washed with TBST and incubated with respective secondary antibody for 2 h at room temperature. The membranes were stripped and re-probed with anti-IKKb (for pIKKb), anti-JNK (for pJNK) or anti-b-actin (for NF-kB) antibodies for normalization. The protein bands were visualized by enhanced chemiluminescence detection using Immobilon HRP Western Substrate (Millipore, Bangalore, India). Finally, the densitometry analyses of the bands were performed using Image J software (National Institute of Health, Bethesda, MD, USA). 2.6.5. Histopathological studies Hippocampal tissue excised from the experimental animals were fixed in 10% formalin and embedded in paraffin. Sections of 4-5 mm thickness were mounted on glass slides, deparaffinized and dehydrated in graded alcohol. Then, the sections were stained with haematoxylin and eosin and observed under phase contrast microscope (40). 2.6.6. Immunohistochemistry For immunohistochemistry, 4–5 mm paraffin embedded hippocampal sections were deparaffinized with xylene and dehydrated with gradually increasing concentrations of isopropyl alcohol. Slides were incubated overnight with anti-4 HNE or 3-NT antibody (1: 200 dilution). The slides were rinsed well with phosphate buffer and incubated with super enhancer reagent for 30 min. After rinsing with phosphate buffer, incubation was done with supersensitive polymer–HRP immunohistochemistry detection system. Sections were washed with buffer and incubated with a
diaminobenzidene (DAB) solution for 5 min. Sections were observed under phase contrast microscope (40). 2.7. Statistical analysis The data obtained were tested for statistical significance by one-way analysis of variance (ANOVA) followed by Tukey's Multiple Range Test for multiple comparisons. SPSS statistical software (version 20.0; SPSS, Chicago, IL, USA) was used. P value <0.05 was considered significant. 3. Results 3.1. API decreased body weight gain in HFFD fed rats The initial and final body weights of experimental rats are given in (Table 1). HFFD feeding for 60 days resulted in an increase in body weight compared to normal pellet fed rats. HFFD-fed rats treated with API showed a significant reduction in body weight compared to rats fed HFFD alone. No significant differences in final body weight were observed between CON, HFFD + STG and CON + API groups. Food intake (g/rat/day) was CON, 14.73 0.73; HFFD, 14.31 0.71; HFFD + API, 14.56 0.72; HFFD + STG, 14.62 0.73; CON + STG, 14.79 0.74. No significant differences in food intake were observed between the experimental groups. 3.2. Levels of plasma glucose, insulin, TNF-a and IL-6 in HFFD-fed rats Glucose and insulin levels in plasma of experimental rats are shown in (Table 2). Rats fed HFFD alone showed significant increase in the levels of glucose and insulin as compared to control rats. API- and STG-administration to HFFD-fed rats lowered glucose and insulin levels as compared to HFFD group. Between the two, API addition to HFFD rats brought a better reduction than STG treatment. However, the levels in these two groups were still significantly different from CON group. No significant differences were observed between the CON and CON + API treated rats with respect to glucose and insulin levels. The levels of TNF-a and IL-6 in plasma are tabulated in (Table 2). HFFD feeding caused significant increase in the levels of TNF-a and IL-6 as compared to control rats. The levels were close to normal in API- and STG-treated HFFD-fed rats. No significant differences were observed between the CON and CON + API treated rats.
Table 2 Plasma glucose, insulin and proinflammatory cytokines in experimental rats. Parameters
CON
HFFD
HFFD + API
HFFD + STG
CON + API
Glucose (mg/dl) Insulin (mIU/ml) TNF-a (pg/ml) IL-6 (pg/ml)
100.90 10.1a 16.8 0.5a 14.67 1.01a 81.37 3.5a
158.44 4.3b 31.0 2.7b 42.68 2.6b 198.66 9.5b
116.04 12.1c 23.3 1.5c 29.74 2.3c 129.42 6.9c
134.19 6.2d 26.7 0.7d 22.36 1.3d 155.88 4.1d
94.38 7.8a 16.5 0.1a 15.05 0.9a 84.09 6.3a
Values are means SD of six rats from each group. CON, control rats; HFFD, high fat-high fructose diet-fed rats; HFFD + API, high fat-high fructose diet-fed rats treated with API; HFFD + STG, high fat-high fructose diet-fed rats treated with STG; CON + API, control rats treated with API. Values that bear different superscripts are significantly different from one another [One way ANOVA followed by Tukey's test (P < 0.05)].
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Table 3 Levels of oxidative stress in experimental rats. Parameters Plasma TBARS (A) LHP (B) PC (B) AOPP (C) Hippocampus TBARS (D) LHP (D) PC (D)
CON
HFFD
HFFD + API
HFFD + STG
CON + API
1.12 0.08a 2.03 0.06a 3.98 0.3a 85.17 7.4a
6.12 0.6b 5.21 0.3b 9.15 0.5b 143.12 9.6b
4.36 0.1c 4.56 0.15c 7.06 0.1c 128.05 4.3c
3.19 0.3d 3.13 0.08d 5.56 0.4d 107.47 6.2d
1.05 0.07a 1.98 0.04a 3.86 0.3a 83.45 4.2a
2.31 0.1a 4.16 0.3a 4.02 0.4a
7.86 0.7b 7.38 0.6b 9.36 0.3b
5.32 0.3c 6.16 0.2c 7.78 0.5c
4.11 0.2d 5.07 0.5d 5.87 0.5d
2.17 0.08a 4.38 0.3a,d 3.91 0.2a
Values are means SD of six rats from each group. CON, control rats; HFFD, high fat-high fructose diet-fed rats; HFFD + API, high fat-high fructose diet-fed rats treated with API; HFFD + STG, high fat-high fructose diet-fed rats treated with STG; CON + API, control rats treated with API. Values that bear different superscripts are significantly different from one another [One way ANOVA followed by Tukey's test (P < 0.05)]. A = mmol/dl; B = nmol/dl; C = mmol/l; D = nmol/mg protein.
Fig. 1. Immunohistochemical localization of 3-NT (A) and 4-HNE (B) adducts in hippocampus of experimental animals (40). Brown color indicates DAB staining and the occurrence of antigen–antibody reaction. Blue color indicates hemotoxylin staining and the absence of antigen–antibody reaction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.3. API alleviates oxidative changes in HFFD fed rats The levels of plasma AOPP and TBARS, LHP and PCO in plasma and hippocampus are shown in (Table 3). The levels of lipid peroxidation and protein damage markers in HFFD rats were significantly higher than those seen in CON. On the other hand, administration of API- and STG-lowered the levels of these oxidative stress markers. No significant differences in the levels of AOPP, TBARS, LHP and PCO were observed between the CON and CON + API groups. API- and STG-administered HFFD-fed animals had reduced AOPP, TBARS, LHP and PCO levels significantly as compared to HFFD group. The extent of lipid and protein damage was also assessed by examining the localization of 3-NT and 4-HNE adducts respectively in hippocampal sections by immunohistochemistry (Fig. 1A and B). Positive immunoreactivity for these adducts was observed in HFFD group. HFFD + API and HFFD + STG groups had minimum levels of adducts as compared to HFFD group. There was negligible accumulation of 3-NT and 4-HNE adducts in CON and CON + API groups. The activities of enzymatic antioxidants of SOD, CAT and GPx in the hippocampus are presented in (Table 4). HFFD-fed rats had poor antioxidant status as compared to CON group. API or STG treatment improved the levels of antioxidant enzymes. No significant differences were found between the CON, CON + API and HFFD + STG treated groups in the hemolysate of CAT and GPx. Levels of SOD in hippocampus were not significantly different in HFFD + STG as compared to CON animals and in HFFD + API as compared to HFFD + STG treated group. The levels of GSH and vitamins C and E in circulation and hippocampus and the results of FRAP assay in plasma of animals in each group are listed in (Table 5). The antioxidant status was significantly decreased in HFFD-fed animals as compared to CON animals. Levels of vitamins C and E were not significantly different
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between CON and CON + API treated animals. API and STG treatment prevented the detrimental effects of HFFD by uplifting the levels of antioxidants. No significant differences in GSH were observed between HFFD + STG and CON groups. 3.4. API modulates IKKb/NF-kB associated neuroinflammation IKKb, the stress kinase activated by phosphorylation and required for the release of cytosolic NF-kB to nucleus from bound IkB was increased in HFFD-fed rats compared to control rats. The phospho-form of IKKb was reduced in API- and STG-treated animals (Fig. 2A and B). The results obtained for immunohistochemical staining of pIKKb and NF-kB-p65 are concordant with the western blotting analysis (Fig. 2C and D). Fig. 2C and D represents the photomicrographs on the expression of pIKKb and NF-kB-p65 in the hippocampus of experimental animals. The intensity of immunostaining for pIKKb and NF-kB-p65 were more pronounced in HFFD treated group. It is apparent that there is a marked reduction in pIKKb and NF-kB-p65 immunoreactivity in HFFD + API and HFFD + STG treated group. There is no marked difference in the immunoreactivity for these antibodies between CON and CON + API groups. HFFD-fed rats displayed increased expression of NF-kB in the nuclear fraction of the hippocampus as compared to control rats (Fig. 2A and B) suggesting translocation and activation in HFFD group. The translocation of NF-kB to nucleus was reduced upon treatment with API- and STG- as compared to HFFD-fed rats. This is revealed by reduced nuclear expression. 3.5. API prevents JNK activation Activation of JNK by phosphorylation at Thr183/Y185 was observed in the HFFD-fed rats as compared to normal diet-fed rats. The rise in JNK phosphorylation promoted by HFFD feeding was
Table 4 Levels of enzymatic antioxidants in experimental animals. Parameters Hemolysate SOD (A) CAT (B) GPx (C) Hippocampus SOD (A) CAT (B) GPx (C)
CON
HFFD
HFFD + API
HFFD + STG
CON + API
3.78 0.02a 43.21 4.2a,d 9.2 0.8a,d
1.09 0.07b 22.14 2.0b 5.75 0.3b
2.58 0.2c 34.68 2.3c 7.18 0.6c
3.05 0.3d 40.08 2.0d 8.24 0.7d
3.92 0.1a 45.16 2.1a 9.5 0.5a
4.21 0.4a,d 49.16 2.8a 4.68 0.3a
2.04 0.2b 24.18 1.5b 2.24 0.2b
3.16 0.3c 33.32 2.1c 3.18 0.2c
3.69 0.2c,d 41.16 2.5d 3.97 0.2d
4.24 0.4a 51.44 4.2a 4.97 0.4a
Values are means SD of six rats from each group. CON, control rats; HFFD, high fat-high fructose diet-fed rats; HFFD + API, high fat-high fructose diet-fed rats treated with API; HFFD + STG, high fat-high fructose diet-fed rats treated with STG; CON + API, control rats treated with API. Values that bear different superscripts are significantly different from one another [One way ANOVA followed by Tukey's test (P < 0.05)]. A = U/mg Hb for hemolysate; U/mg protein for hippocampus; B = mmol of H2O2 consumed/ min/mg Hb for hemolysate; mmol of H2O2 consumed/min/mg protein for hippocampus; C = mmol GSH consumed/min/mg Hb hemolysate; mmol GSH consumed/min/mg protein for hippocampus; U = enzyme concentration required to produce 50% inhibition of chromogen formation in 1 min under standard conditions.
Table 5 Levels of FRAP and non-enzymatic antioxidants in experimental animals. Parameters Plasma FRAP (A) Vitamin C (B) Vitamin E (B) GSH (B) Hippocampus Vitamin C (C) Vitamin E (C) GSH (C)
CON
HFFD
HFFD + API
HFFD + STG
CON + API
1032.4 6.4a 2.61 0.08a 1.92 0.07a 19.46 1.6a,d
798.2 7.4b 0.91 0.05b 0.46 0.2b 11.22 0.1b
843.7 3.8c 1.67 0.1c 0.85 0.04c 14.76 0.7c
968.2 4.1d 2.03 0.1d 1.46 0.1d 18.12 0.4d
1056.3 5.4a 2.75 0.1a 1.98 0.06a 20.18 1.0a
46.36 3.5a 2.12 0.1a 42.18 0.4a,d
21.09 2.0b 0.82 0.06b 28.12 2.0b
34.58 1.5c 1.35 0.1c 34.69 2.3c
40.69 3.9d 1.86 0.1d 39.14 1.6d
48.12 3.0a 2.24 0.2a 44.36 4.3a
Values are means SD of six rats from each group. CON, control rats; HFFD, high fat-high fructose diet-fed rats; HFFD + API, high fat-high fructose diet-fed rats treated with API; HFFD + STG, high fat-high fructose diet-fed rats treated with STG; CON + API, control rats treated with API. Values that bear different superscripts are significantly different from one another [One way ANOVA followed by Tukey's test (P <.05)]. A = mmol/l; B = mg/dl; C = mg/mg protein.
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Fig. 2. Phoshphorylation status of IKKb and protein expression of NF-kB p65 subunit. (A) Representative western blot of IKKb, pIKKb and NF-kB-p65 subunit in hippocampus. Lane 1, CON; lane 2, HFFD; lane 3, HFFD + API; lane 4, HFFD + STG; lane 5, CON + API. (B) Densitometry data obtained after normalization of pIKKb with total IKKb and NF-kB-p65 with b-actin. Fold change for a group was calculated with respect to control. Results are expressed as means S.D (n = 3). (C and D) Immunohistochemical localization of pIKKb (C) and NF-kB (D) in the hippocampus (40). Brown color indicates DAB staining and the occurrence of antigen–antibody reaction. Blue color indicates hemotoxylin staining and the absence of antigen–antibody reaction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
reversed with API and STG treatment (Fig. 3A and B). Results obtained from the immunohistochemical investigation in the hippocampus of control and experimental animals are in agreement with the Western blotting results (Fig. 3C). Fig. 3C represents the photomicrographs on the expression of JNK in the hippocampus of experimental animals. The intensity of immunostaining for JNK was more pronounced in HFFD treated group. It is apparent that there is a marked reduction in JNK immunoreactivity in HFFD + API and HFFD + STG treated group. There is no marked difference in the immunoreactivity for this antibody between CON and CON + API groups. 3.6. API prevents histopathological alterations in HFFD fed rats Fig. 4 depicts the photographs of hippocampal sections stained using hematoxylin and eosin. Cells appear normal in sections from CON and CON + API groups. Sections from HFFD group show shrinkage in the size of the pyramidal cells, disorganization of layer, dark nuclei and many apoptotic bodies and large vacuolated cells. Sections from HFFD + API and HFFD + STG groups show restoration of pyramidal cell size and normal architecture of CA1 region and dentate gyrus with few degenerative neurons. 4. Discussion The present study investigated the neuroprotective actions of API in high calorie-diet fed rats by determining its effects on oxidative stress, inflammation and pathological changes in the hippocampus. Free radical-induced damage to macromolecules is a central event in the progression of neurodegeneration. Accumulation of toxic products of lipid peroxidation such as TBARS, LHP and 4-HNE and protein damage such as 3-NT, AOPP and PCO were observed in the hippocampus of HFFD-fed rats suggesting oxidative damage to hippocampus. Excessive nutrients, particularly fats and sugars
when transported into cells, disturb metabolic homeostasis and directly increase mitochondrial oxidative workload, which causes increased production of ROS. A study by Liu et al. [19] shows that hippocampal neurons exposed to high glucose develop oxidative stress through high ROS generation. Also, palmitic acid can induce lipotoxicity in neuronal progenitor cells [20]. Thus, administration of excess nutrients may directly induce lipo- or glucose toxicity in brain cells. In addition, the depletion of antioxidant enzymes and molecules that can scavenge ROS or prevent oxidative damage in HFFD-fed rats could also be responsible for the rise in levels of oxidative damage markers. The levels of ROS, TBARS and PCO were reported to be significantly increased in the brains of fructosedrinking insulin resistant rats [21]. Progressive damage due to oxidative stress causes histological alterations. Histopathological examination is therefore important for evaluating the extent of tissue damage and reversal. The hippocampus consists of the hippocampus proprius, gyrus dentatus and the subiculum. The hippocampus proper is subdivided into four regions according to density, size and branching of axons and dendrites of pyramidal cells (Cornu Ammonis (CA) 1–4). Each of these regions consists of three layers: stratum moleculare, stratum pyramidale and stratum multiforme. The stratum pyramidale contains bodies of the pyramidal cells [22]. The CA1 region is characterized by densely packed medium-sized pyramidal cells and is more sensitive to various insults [23]. Examination of CA1 area in the hippocampus of HFFD-fed rats revealed loss of cell integrity, significant decrease in pyramidal cells number and shrinkage in the size of pyramidal cells. A similar observation has been observed and reported by El-Khair et al. [24] in high cholesterol diet-fed adult rats. Significant reduction in pyramidal cells, attenuation and thickening of small blood vessels, apparent increase of astrocytes and decrease of Nissl's granules in the hippocampus of high cholesterol diet-fed rats have been reported [24]. Reduced hippocampal dendritic spine density, and reduced long-term potentiation at Schaffer collateral-CA1
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Fig. 3. Phoshphorylation status of JNK. (A) Representative western blots of JNK and pJNK in the hippocampus. Lane 1, CON; lane 2, HFFD; lane 3, HFFD + API; lane 4, HFFD + STG; lane 5, CON + API. (B) Densitometry data was obtained after normalization of pJNK with total JNK. Fold change for each group was calculated with respect to control. Results are expressed as means S.D (n = 3). (C) Immunohistochemical localization of pJNK in the hippocampus (40). Brown color indicates DAB staining and the occurrence of antigen–antibody reaction. Blue color indicates hemotoxylin staining and the absence of antigen–antibody reaction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
synapses are found in rats fed high-fat, high-glucose diet supplemented with high-fructose corn syrup for 8 months [25]. Over nutrition induces neuroinflammation and dysfunction of the central nervous system. Oxidative stress is the pathogenic link that bridges over-nutrition with inflammation via NF-kB activation. Upon stimulation by ROS, the enzyme IKKb phosphorylates an inhibitor of kappa B (IkB) attached to p50/p65 heterodimer of NF-kB resulting in its degradation by the ubiquitin proteasome pathway. Dissociation of IkB from the dimer facilitates the entry of NF-kB in to the nucleus. Activated NF-kB can trigger the generation of inflammatory molecules like TNF-a and IL-6. The levels of these molecules are also increased in HFFD-fed rats as a consequence of NF-kB activation. Insulin regulates enormous brain functions and fructose induced perturbation of insulin signaling/insulin resistance may
be linked to the hippocampal derangements. Insulin resistance in the hippocampus is associated with learning and memory decline [26]. JNK and IKKb are shown to be critically involved in promotion of diet-induced insulin resistance by negative modulation of insulin signaling by serine phosphorylation of insulin receptor substrate at Ser307 residue [27]. Thus insulin resistance observed in HFFD-fed rats might contribute damage to the hippocampus. API administration significantly reduced oxidative stress, histopathological changes, IKKb/NF-kB signaling and JNK phosphorylation in the hippocampus of HFFD-fed rats. API is reported to regulate redox imbalance and inhibit JNK signaling during Abmediated toxicity [28]. API restored enzymatic (SOD, CAT and GPx) and non-enzymatic (GSH, vitamins C and E) antioxidants. A study by Guo et al. [29] reported that API enhanced the mRNA expression of antioxidant
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Fig. 4. Representative photographs of CA1 region of hippocampus stained with haematoxylin and eosin: (A) CON—layer of compact granular cells with normal nuclei in dentate gyrus (G), small pyramidal cells (P) and dispersed small vacuoles (V). (B) HFFD—shrinkage and darkening of many large pyramidal cells, disorganization of pyramidal cells. (C) HFFD + API—preservation of small pyramidal cells of CA1 region. (D) HFFD + STG—decreased thickness of layer of small pyramidal cells of CA1 region. (E) CON + API— normal architecture of hippocampus (40). Insets show the enlarged view of pyramidal cells.
enzymes in OGD/R-treated PC12 cells. The improved enzymatic activities of SOD, CAT and GPx by API may be a direct effect of API in the brain at the protein level or at the mRNA level or an indirect result of improved insulin sensitivity by API. Vitamin E-dependent protection of the hippocampus has been described in some studies suggesting the neuroprotective role of antioxidants [30,31]. API is a proven antioxidant. The presence of C2–C3 double bond on the C ring confers ROS-scavenging activity [32]. The API protective action may be attributed to a combination of improved insulin sensitivity, antioxidant and anti-inflammatory effects. Further, rats fed HFFD for 60 days were found to be obese which may have an influence on the investigated parameters. API administration reduced body weight gain in HFFD-fed rats, even when the food intake was not significantly different. Hiranya et al. [7] showed that STG prevents hippocampal mitochondrial dysfunction, improves the learning behavior and
decreases brain oxidative stress levels in high fat diet-fed rats and that STG can be useful in the treatment of neurodegenerative events associated with insulin resistance and metabolic syndrome. Administration of STG reduces brain oxidative stress induced by high fat diet consumption [7]. STG increases glucagon like peptide1 (GLP-1) levels by inhibiting DPP-IV enzyme and acts as an antidiabetic agent. We previously reported that API has binding efficacy at Glu206 in the active site of DPP-IV enzyme [10]. Possibly API could act in a similar manner to increase brain active GLP-1 levels which remains to be studied. 5. Conclusions Unhealthy dietary habits could lead to changes in hippocampal integrity and loss of hippocampal function. Intake of nutritious dietary components enriched diets can be productive strategy not
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