Food and Chemical Toxicology 72 (2014) 295–302

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Proanthocyanidins improves lead-induced cognitive impairments by blocking endoplasmic reticulum stress and nuclear factor-jB-mediated inflammatory pathways in rats Chan-Min Liu a,⇑, Jie-Qiong Ma b, Si-Si Liu a, Gui-Hong Zheng a, Zhao-Jun Feng a, Jian-Mei Sun a a b

School of Life Science, Jiangsu Normal University, No. 101, Shanghai Road, Tangshan New Area, 221116 Xuzhou City, Jiangsu Province, PR China School of Chemistry and Pharmaceutical Engineering, Sichuan University of Science and Engineering, Xuyuan Road, 643000 Zigong City, Sichuan Province, PR China

a r t i c l e

i n f o

Article history: Received 7 May 2014 Accepted 22 July 2014 Available online 31 July 2014 Keywords: Proanthocyanidins Lead Oxidative stress Beta amyloid Endoplasmic reticulum stress Brain inflammation

a b s t r a c t Proanthocyanidins (PCs), a class of naturally occurring flavonoids, had been reported to possess a variety of biological activities, including anti-oxidant, anti-tumor and anti-inflammatory. In this study, we examined the protective effect of PCs against lead-induced inflammatory response in the rat brain and explored the potential mechanism of its action. The results showed that PCs administration significantly improved behavioral performance of lead-exposed rats. One of the potential mechanisms was that PCs decreased reactive oxygen species production and increased the total antioxidant capacity in the brains of lead-exposed rats. Furthermore, the results also showed that PCs significantly decreased the levels of tumor necrosis factor-a, interleukin 1b and cyclooxygenase-2 in the brains of lead-exposed rats. Moreover, PCs significantly decreased the levels of beta amyloid and phosphorylated tau in the brains of lead-treated rats, which in turn inhibited endoplasmic reticulum (ER) stress. PCs also decreased the phosphorylation of protein kinase RNA-like ER kinase, eukaryotic translation initiation factor-2, inositolrequiring protein-1, c-Jun N-terminal kinase, p38 and inhibited nuclear factor-jB nuclear translocation in the brains of lead-exposed rats. In conclusion, these results suggested that PCs could improve cognitive impairments by inhibiting brain oxidative stress and inflammatory response. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Proanthocyanidins (PCs), the widely distributed flavonoids in fruits and vegetables, possess many biological activities, including antioxidant, anti-allergic, anti-inflammatory, vasodilatory, antibacterial, cardioprotective, immune-stimulating, anti-viral and antitumoral properties (Gong et al., 2008; Singh et al., 2011; Lee et al., 2012). PCs mostly contain dimers, trimers and other oligomers of catechin and epicatechin and their gallic acid esters. A previous study confirmed that 6 months of 250 or 500 mg/kg body weight (BW) PCs treatment was safe and did not cause any detrimental effects in vivo (Ray et al., 2001; Ding et al., 2014). Several studies Abbreviations: Ab, beta amyloid; COX-2, cyclooxygenase-2; eIF2a, eukaryotic translation initiation factor-2; ER, endoplasmic reticulum; IL-1b, interleukin-1beta; IRE1, inositol-requiring protein-1; JNK, the c-Jun N-terminal kinases; NF-jB, nuclear factor-jB; Pb, lead; PCs, proanthocyanidins; PERK, protein kinase RNAlike ER kinase; ROS, reactive oxygen species; TAC, the total antioxidant capacity; TBARS, thiobarbituric acid reactive substances; TNF-a, tumor necrosis factor-alpha. ⇑ Corresponding author. Tel.: +86 516 83403170; fax: +86 516 83500171. E-mail address: [email protected] (C.-M. Liu). http://dx.doi.org/10.1016/j.fct.2014.07.033 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.

had demonstrated that PCs could ameliorate age-related depression and cognitive decline by inhibiting oxidative stress and regulating immune function (Asha Devi et al., 2011; Ogle et al., 2013). PCs conferred neuroprotection in the models of Parkinson’s disease and Alzheimer’s disease (Moreira et al., 2010; Wang et al., 2012; Strathearn et al., 2014). PCs exerted the protective effects on the brains of diabetic rats through modulating AGEs/RAGE/NF-kappaB pathway (Xu et al., 2008; Lu et al., 2010). Increasing evidence showed that PCs could protect brains from injury induced by neurotoxin (Gong et al., 2008; Asha Devi et al., 2011; Wang et al., 2012). Lead (Pb) is a well known hazardous material. Pb is also a nonthreshold multi-targeted toxicant that causes alterations in different organs of the body (Seddik et al., 2011; Liu et al., 2012). Pb is a nonessential heavy metal, which is taken up from the environment into the body through pulmonary and enteral pathways (Liu et al., 2011). Pb is widely recognized as a potent central neurotoxin known to produce detrimental effects in the nervous system that interferes with neuronal functions (Liu et al., 2013a). Because neuronal injury mediated by Pb involves a complex cascade of events,

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Pb induced memory loss is not fully understood. Substantial evidence had demonstrated that Pb exposure is associated with both peripheral and central neurological effects including memory deficits and Alzheimer’s disease-like pathology (Gu et al., 2011; Bakulski et al., 2012; Chander et al., 2014). Many researches also suggested that increase of immunoactivity for interleukin-1beta (IL-1b), tumor necrosis factor-alpha (TNF-a), beta-amyloid (Ab) and tau proteins may contribute to the progression of memory decline in animals with maternal Pb exposure (Bakulski et al., 2012; Zhang et al., 2012; Li et al., 2014). The endoplasmic reticulum (ER) is an organelle present in all eukaryotic cells, which is also a multifunctional signaling organelle that controls a wide range of cellular processes (Hu et al., 2006; Liu et al., 2013b). The ER plays a critical role in the synthesis, folding, modification, and trafficking of proteins. Whereas, a number of cellular stress conditions can disturb the ER functions, which causes accumulation of misfolded (ER stress) and activates the unfolded protein response (UPR) (Endres and Reinhardt, 2013; Liu et al., 2013b). The ER stress can induce several pathways which activate nuclear factor-jB (NF-jB) and cause inflammation (Salminen et al., 2009). Previous reports have demonstrated that Pb could induce the ER stress in rat liver and inflammation in rat kidney, and these effects were shown to be associated with reactive oxygen species (ROS) formation (Liu et al., 2012, 2013b). However, the molecular mechanisms of lead-induced nerve injury and neuroprotective effects of PCs are not yet completely understood. Therefore, we hypothesized that Pb-induced memory loss could also involve ER stress and inflammatory response. In the present study, we aimed to determine whether PCs could protect rat brains from Pb-induced cognitive impairments and inflammation by modulating the Ab, tau and ER stress pathway. 2. Materials and methods 2.1. Chemicals and reagents Lead acetate (Pb(CH3COO)2) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). USA); Proanthocyanidins were purchased from Tianjin Jianfeng Natural Products Co. (proanthocyanidins contents > 95%). The possible contaminants of endotoxin contained in proanthocyanidins were removed using AffiPrep Polymyxin Matrix (Bio-Rad). The phospho-PERK (Thr980), phospho-IRE1 and phospho-eIF2a (Ser51) antibody were purchased Cell Signaling Technology (Beverly, MA, USA); IL-1b antibody, TNF-a antibody, COX-2 antibody, phospho-JNK, phospho-JNK, phospho-p38, p38, NF-jB p65, IRE1, PERK, eIF2a and GRP78 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The protein concentration was measured using the bicinchonininc acid (BCA) assay kit from Pierce Biotechnology, Inc. (Rockford, IL, USA). All other reagents unless indicated were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

2.2. Animals and treatment Male Wistar rats (4-week-old) were purchased from the Branch of National Breeder Center of Rodents (Shanghai). Fifty rats were maintained under constant conditions (23 ± 1 °C and 60% humidity) with free access to rodent food and tap water under 12 h light/dark schedule (lights on from 08:30 to 20:30 h) (Liu et al., 2012). After acclimatization to the laboratory conditions, the animals were randomly divided into five groups (ten rats in each). Five rats were put into one cage. (1) Control group, the rats received lead-free redistilled water and daily given physiological saline (0.9% NaCl) by oral gavage during the whole course of the experiment; (2) lead treated group, animals received an aqueous solution of lead acetate (Pb(CH3COO)2) (Sigma–Aldrich, MO, USA) at a concentration of 250 mg Pb/L of drinking water and daily given physiological saline (0.9% NaCl) by oral gavage; (3) lead + PCs (100 mg/kg) treated group, animals received an aqueous solution of lead acetate (250 mg Pb/L in the drinking water) and received a daily oral gavage administration of PCs at dose of 100 mg/kg/body weight dissolved in normal saline solution; (4) lead + PCs (200 mg/kg) treated group, animals received an aqueous solution of lead acetate (250 mg Pb/L in the drinking water) and received a daily oral gavage administration of PCs at dose of 200 mg/kg/body weight dissolved in normal saline solution; (5) PCs treated group, the rats received lead-free redistilled water and received a daily oral gavage administration of PCs at dose of 200 mg/kg/body weight dissolved in normal saline solution. Rats received

an aqueous solution of lead acetate at a concentration of 250 mg Pb/L of drinking water as reported previously by Seddik et al., 2011. The choice of PCs dose is based on previous findings (El-Alfy et al., 2008; Lu et al., 2010; Singh et al., 2011). The experiment lasted for 75 days. There was not any anaesthesia or confinement used for application via oral gavage. At the end of treatment, all the rats (ten rats in each group) were used for behavioral test. Then, the rats were sacrificed by decapitation. Brain tissues were quickly collected, placed in ice-cold 0.9% NaCl solution, perfused with the physiological saline solution to remove blood cells, blotted on filter paper, and stored at 70 °C for later use. All the brains of the rats were used for further analysis. This research was conducted in accordance with Chinese laws and NIH publications on the use and care of laboratory animals. Relevant university committees for animal experiments approved these experiments. Fifty rats were approved in this animal testings. 2.3. Behavioral testing This task evaluates motor performance in the training section and non-associative memory in the retention test session (Comim et al., 2012; Liu et al., 2013c). Habituation to an open field was carried out in a 40 cm  60 cm open field surrounded by 50 cm high walls made of brown plywood with a frontal glass wall. The floor of the open field was divided into 9 equal rectangles by black lines. The animals were gently placed on the left rear quadrant and left to explore the area for 5 min (training session). The number of crossing the black lines and rearing performed in this training session was counted. Immediately following this, the animals were taken back to their home cage and submitted again to a similar openfield session 24 h later (test session). The number of crossing the black lines and rearing performed in this test session was also counted. The decrease in the number of crossings and rearing between the two sessions was taken as a measure of the retention of habituation. 2.4. Reactive oxygen species (ROS) assay ROS was measured as described previously, based on the oxidation of 20 70 dichlorodihydrofluorescein diacetate to 20 70 -dichloro-fluorescein (Shinomol and Muralidhara, 2007; Liu et al., 2012). Briefly, the homogenate was diluted 1:20 times with ice-cold Locke’s buffer to obtain a concentration of 5 mg tissue/ml. The reaction mixture (1 ml) containing Locke’s buffer (154 Mm NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 5 mM glucose, 5 mM HEPES), 0.2 ml homogenate (0.5 mg protein) and 10 ll of DCFH-DA (5 lM) was incubated for 15 min at room temperature to allow the DCFH-DA to be incorporated into any membranebound vesicles and the diacetate group cleaved by esterases. After 30 min of further incubation, the conversion of DCFH-DA to the fluorescent product DCF was measured using a spectrofluorimeter with excitation at 484 nm and emission at 530 nm. Background fluorescence (conversion of DCFH-DA in the absence of homogenate) was corrected by the inclusion of parallel blanks. The DCF standard curve was established in the presence of homogenate. ROS formation was quantified from a DCF-standard curve and data are expressed as pmol DCF formed/min/ mg protein. 2.5. Lipid peroxidation assay Estimation of lipid peroxidation (TBARS) was performed by our previous method (Liu et al., 2012). A standard calibration curve was prepared by using 1– 10 nM of 1,1,3,3-tetra methoxy propane. Protein levels were determined using the bicinchoninic acid (BCA) assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). The concentration was expressed in terms of nanomoles of TBARS per mg of protein. 2.6. The total antioxidant capacity (TAC) assay The total antioxidant capacity in brain was assayed with a commercially available assay kit (Jiancheng Biochemical, Inc., Nanjing, China) (Liu et al., 2012, 2013a). This method is based on the reduction of iron (III) in acidic medium by intracellular antioxidants. Liberated iron (II) reacts with 1,10-phenanthroline to form a colored complex, which is measured at 520 nm. One unit of TAC is defined as 0.01 optical densities (OD520) units per mg protein per min at 37 °C. 2.7. Western blot analyses Western blot analyses were performed as described previously (Liu et al., 2011, 2013a). The hippocampus was homogenized in 1/3 (w/v) ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (tris-buffered saline (TBS), 1% Nonidet P-40 (NP40), 0.5% sodium deoxycholate, 0.1% SDS and 0.004% sodium azide) containing 30 ll of 10 mg/ml phenylmethylsulphonyl fluoride (PMSF), 30 ll of Na3VO4, 30 ll of NaF and 30 ll of protease inhibitor cocktail per gram of tissue. The homogenates were sonicated four times for 30 s with 20 s intervals using a sonicator and centrifuged at 15,000g for 10 min at 4 °C. Protein levels were determined using the bicinchoninic acid (BCA) assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA).

C.-M. Liu et al. / Food and Chemical Toxicology 72 (2014) 295–302 2.8. Statistic analysis All statistical analyses were performed using the SPSS software, version 11.5. A one-way analysis of variance (ANOVA; P < 0.05) was used to determine significant differences between groups and the individual comparisons were obtained by Tukey’s HSD post hoc test. Statistical significance was set at P < 0.05.

3. Results 3.1. PCs attenuated Pb-induced locomotor damage The effects of lead exposure plus PCs on motor activity in the open-field test are shown in Fig. 1. Lead exposure caused a marked decrease in crossing and rearing number in relation to the control group, respectively (P < 0.05; the Pb group vs. the control group). Interestingly, treatment with low and high dose of PCs in Pb-treated rats significantly increased crossing number and rearing number (P < 0.05, vs. the Pb group). There was no significant difference between the control group and the PCs group (Fig. 1).

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difference in the levels of ROS, TBARS and TAC was found between the control group and the PCs group. 3.3. PCs blocked ER stress and inhibits Ab and tau activity in the brains of rats exposed to Pb Previous studies have shown that ER stress and Ab and tau activation play an important role in the development of Alzheimer’s disease by triggering metabolic inflammation. As shown in Fig. 3, level of GRP78 (ER stress marker) was significantly upregulated in the Pb exposure rats compared to the control group, indicating that ER stress is increased under Pb exposure (P < 0.01; the Pb group vs. the control group). Moreover, Pb exposure significantly increased the Ab production and the phosphorylation of tau protein (P < 0.01; the Pb group vs. the control group) in the rat hippocampus. Oral administration of PCs to rats of Pb exposure for 75 days significantly reversed these changes (P < 0.01 vs. the Pb

3.2. PCs inhibited Pb-induced oxidative stress in brain PCs decreased Pb-induced ROS and TBARS (Fig. 2). On the other hand, Pb treatment markedly increased brain ROS and TBARS levels by 64% and 71% as compared with those of the controls, respectively (P < 0.01; the Pb group vs. the control group). However, treatment with low and high dose of PCs in Pb-treated rats significantly increased ROS (by 12% and 21%) and TBARS (by 13% and 23%) (P < 0.01, vs. the Pb group) (Fig. 2). Many studies suggested that the TAC level might be an indicator of oxidative stress. As shown in Fig. 2D, the TAC level in the Pbtreated rats was markedly decreased by 27% as compared with that of the control (P < 0.01). Interestingly, treatment with low and high dose of PCs in the Pb-treated rats significantly increased TAC level (by 15% and 20%) (P < 0.01, vs. the Pb group). No significant

Fig. 1. Effects of proanthocyanidins (PCs) treatments on the locomotor behavior of Pb-treated rats. (A) Number of crossing; (B) number of rearing. All values are expressed as mean ± S.E.M. (n = 10). #P < 0.05, compared with the control group; * P < 0.05, vs. Pb-treated group.

Fig. 2. Effect of proanthocyanidins (PCs) on oxidative stress in the brains of Pbtreated rats. (A) Level of ROS; (B) level of TBARS; (C) level of TAC. Each value is expressed as mean ± S.E.M. (n = 10). ##P < 0.01, compared with the control group; ** P < 0.01, vs. Pb-treated group.

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Fig. 3. Proanthocyanidins (PCs) reduced the levels of Ab, p-tau and blocks ER stress in the hippocampus of rats exposed to Pb. (A) Western blot analysis of the Ab, p-tau and GRP78 proteins in the hippocampus; (B) relative density analysis of the Ab protein bands; (C) relative density analysis of the p-tau protein bands; (D) relative density analysis of the GRP78 protein bands; b-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Values are averages from three independent experiments. Each value is expressed as mean ± S.E.M. ##P < 0.01, compared with the control group; **P < 0.01, vs. the Pb-treated group.

Fig. 4. Proanthocyanidins (PCs) reduced the levels of inflammatory cytokines in the hippocampus of rats exposed to Pb. (A) Relative density analysis of the NF-jB p65 protein bands in cytosol and nucleus; (B) relative density analysis of the TNF-a protein bands; (C) relative density analysis of the IL-1b protein bands; (D) relative density analysis of the COX-2 protein bands; b-actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Values are averages from three independent experiments. Each value is expressed as mean ± S.E.M. **P < 0.01, compared with the control group; ##P < 0.01, vs. the Pb-treated group.

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group). No significant difference in the protein levels of Ab, tau and GRP78 was found between the control group and the PCs group (Fig. 3). 3.4. PCs reduced the NF-jB activity and the levels of inflammatory cytokines in the hippocampus of rats exposed to Pb NF-jB is a transcription factor that has crucial roles in inflammation. We further examined the protein levels of inflammatory cytokines, including TNF-a, IL-1b and COX-2, in the hippocampus of each group by western blot analysis. The Pb exposure increased the inflammatory response through ER stress in the mouse hippocampus (P < 0.01 vs. the control group). In addition, the Pb significantly increased nuclear translocation of NF-jB p65 in the rat hippocampus (P < 0.01, vs. the control group). However, PCs administration markedly inhibited the inflammatory response in the hippocampus of rats exposed to Pb (P < 0.01, vs. the Pb-treated group). There was no significant difference between the control group and the PCs group. 3.5. PCs inhibited IRE1/JNK-mediated inflammatory signaling in the hippocampus of rats exposed to Pb ER stress can activate NF-jB signaling by IRE1-ASK1-JNK/p38 pathway. Recent work from our group also demonstrated that ROS induced ER stress plays a key role in the Pb-induced injury. In this study, we found that Pb exposure induced ER stress and inflammation by IRE1-ASK1-JNK/p38 pathway. We used western blot analysis to assess the level of p-IRE1 and its interactions with p-JNK and p-p38 (Fig. 5). Our results showed that ER stress significantly enhanced p-IRE1 level and increased the phosphorylation of JNK and p38 in the hippocampus of rats exposed to Pb (P < 0.01 vs. the control group). Interestingly, oral administration of PCs to Pb-treated rats markedly blocked the ER stress pathway and inhibited these changes in protein level (P < 0.01, vs. the Pbtreated group). No significant difference in the levels of p-IRE1,

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p-JNK and p-p38 was found between the control group and the PCs group (Fig. 5). 3.6. PCs inhibited PREK/eIF2a-mediated inflammatory signaling in the hippocampus of rats exposed to Pb PERK is one of the stress kinases that can phosphorylate eIF2a protein and thus induce inflammatory responses through activation of the transcription factor NF-jB. In this study, we further explored the effect of PCs on the level of phosphorylated PERK and eIF2a in the brains of rats exposed to Pb (Fig. 6). Results showed that Pb significantly increased the levels of phosphorylated PERK and eIF2a, which are associated with ER stress and NF-jB activation and lead to inflammation (P < 0.01, vs. the control group). Interestingly, after oral administration of PCs, these protein level changes were restored to near-normal levels in rats exposed to Pb. There was no significant difference between the control group and the PCs group (Fig. 6). 4. Discussion Procyanidins (PCs) are the most abundant polyphenols found in foods such as grapes and red wine, which had been reported to possess a variety of potent properties (Cedó et al., 2013; Gong et al., 2008; Singh et al., 2011; Lee et al., 2012). This study showed that PCs possessed the protective effects on Pb-induced nerve damage rats. These results indicated that PCs improved cognitive impairments by blocking ER stress and NF-jB-mediated inflammatory pathways in rats. Lead is an environmentally persistent toxin that causes neurological immunological pathologies. In this study, PCs administration restored the Pb-induced cognitive deficits and behavioral dysfunctions to near-normal levels (Fig. 1). Although the mechanisms underlying Pb-induced neuronal degeneration are not well understood, accumulating evidence suggests that oxidative

Fig. 5. Proanthocyanidins (PCs) inhibited inflammatory responses by IRE1/JNK pathway in the hippocampus of rats exposed to Pb. (A) Western blot analysis of the IRE1, JNK and p38 proteins; (B) relative density analysis of the Phospho-IRE1 protein bands; (C) relative density analysis of the phospho-JNK1/2 protein bands; (D) relative density analysis of the Phospho-p38 protein bands. b-Actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Values are averages from three independent experiments. Each value is expressed as mean ± S.E.M. ##P < 0.01, compared with the control group; **P < 0.01, vs. the Pb-treated group.

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Fig. 6. Proanthocyanidins (PCs) inhibited inflammatory responses by PERK/eIF2a pathway in the hippocampus of rats exposed to Pb. (A) Relative density analysis of the p-PERK protein bands; (B) relative density analysis of the p-eIF2a protein bands. b-Actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Values are averages from three independent experiments. Each value is expressed as mean ± S.E.M. **P < 0.01, compared with the control group; ## P < 0.01, vs. the Pb-treated group.

stress-mediated nerve damage may play a central role in Pbinduced neurotoxicity (Liu et al., 2013a,c). Several evidences showed that Pb affects mammalian systems by directly lowering antioxidant reserves and generating ROS, specifically hydroperoxides and lipoperoxides (Nava-Ruiz et al., 2012; Liu et al., 2013a). These ROS alter cellular membranes and tissue, resulting in vascular, neurological and genetic damage (Patrick, 2006; Nava-Ruiz et al., 2012). In this study, levels of ROS and TBARS were markedly increased, and TAC significantly decreased in Pb-treated group as compared with those of the control, implying that Pb exposure induced oxidative stress. However, PCs significantly decreased ROS and TBARS levels in the brains of rats exposed to Pb (Fig. 2), which may be due to the powerful antioxidant and free radical scavenging activities (Asha Devi et al., 2011; Cui et al., 2008; Tekiner et al., 2009). This suggests that PCs could at least partly attenuate oxidative stress by decreasing ROS level and increasing TAC in the brains of rats exposed to Pb. Many studies showed that the toxic amyloid-b peptides are processed from the APP (amyloid-b precursor protein) via cleavage by BACE1 (b-secretase) and c-secretase complexes (Tanzi and Bertram, 2005; LaFerla et al., 2007). APP is a transmembrane protein which is folded and modified in ER and transported through the Golgi complex to the outer membrane (LaFerla et al., 2007; Salminen et al., 2009). Animal studies indicate that Pb exposure led to Ab accumulation by disturbing the balance between Ab production and elimination (Gu et al., 2011; Bakulski et al., 2012). Accumulating evidence has also shown that Pb can cause abnormally hyperphosphorylation of tau (Zhu et al., 2011; Zhang et al., 2012; Bihaqi and Zawia, 2013). In the brains of rats exposed to

Pb, increased the Ab levels promoted the phosphorylation of tau, resulting in memory impairment and behavioral abnormalities (Zhang et al., 2012; Bihaqi and Zawia, 2013). ER is a sensitive organelle which can recognize disturbances in cellular homeostasis. Previous report had demonstrated that Pb can induce the ER stress in rat liver and these effects were shown to be associated with ROS formation (Liu et al., 2013b). Recent study showed that Pb induced the ER stress in rat brain by excess accumulation of hyperphosphorylation of tau (Zhang et al., 2012). Consistent with these viewpoints, excessive ROS and Ab in the hippocampus of the Pbtreated rats significantly promoted the phosphorylation of tau and increased the levels of GRP78, indicating ER stress. Whereas, PCs remarkably decreased the levels of GRP78, Ab and phosphorylation of tau in the brains of rats exposed to Pb (Fig. 3). These results suggested that PCs could alleviate Pb-induced neurotoxicity by decreasing the toxic proteins and inhibiting ER stress. A large body of evidence indicates that Pb can stimulate circulating monocytes and tissue macrophages, which lead to the synthesis and release of a variety of proinflammatory cytokines (Liu et al., 2012; Li et al., 2014). Many studies had demonstrated that the accumulation of Ab, phosphorylated tau and ER stress could also induced inflammation (Salminen et al., 2009; Endres and Reinhardt, 2013; Li et al., 2014). TNF-a is an adipokine involved in systemic inflammation and is a member of a group of cytokines that stimulate the acute phase reaction. IL-1b is a cytokine protein that in humans is encoded by the IL1b gene. Several evidences also revealed that IL-1b and TNF-a play an important role in the regulation of immune and inflammatory responses and those cytokines elevation is associated with many neurological diseases (Gu et al., 2011; Li et al., 2014). COX-2 is an inducible enzyme often found at sites of inflammation. COX-2 is the enzyme largely responsible for causing inflammation (Liu et al., 2012). The present study showed that Pb treatment markedly up-regulated the IL-1b, TNF-a and COX-2 levels in the rat brains. However, PCs significantly attenuated this up-regulation (Fig. 4). These results suggested that PCs could alleviate brain injury caused by Pb through suppressing inflammatory response. NF-jB is a major transcription factor that modulates the production of pro-inflammatory cytokines (Liu et al., 2012). ER stress induces several pathways which can activate NF-jB signaling. The activation of IRE1 in ER stress stimulates IKK kinases which subsequently phosphorylate IjB proteins and trigger NF-jB signaling (Hu et al., 2006). ER stress activates IRE1 and ASK1, which subsequently can trigger JNK and p38 MAPK signaling (Salminen et al., 2009). ASK1-mediated JNK and p38 activation can (i) regulate APP processing and induce accumulation of intracellular amyloid-b (Colombo et al., 2009), (ii) phosphorylate tau protein and trigger aggregation of neurofibrillary tangles (Vogel et al., 2009), and (iii) potentiate inflammatory responses via AP-1 activation (Manning and Davis, 2003). PERK is one of the stress kinases that can phosphorylate eIF2a protein and thus inhibit protein synthesis and activate NF-jB signaling (Salminen et al., 2009; Laszlo and Wu, 2009). This study demonstrated that Pb exposure significantly induced oxidative stress and ER stress in the rat hippocampus, which promoted the phosphorylation of PERK, eIF2a, IRE1, JNK, p38 and induced NF-kB activation (Figs. 5 and 6). Subsequently, the activated NF-kB up-regulated inflammatory cytokines IL-1b, TNF-a and COX-2 (Fig. 4). These results indicate that the ER stress pathway plays a key role in Pb-induced inflammation. Whereas, PCs blocked Pb-induced ER stress and inhibited NF-jB-mediated inflammatory responses in the hippocampus of rats, which inactivated the PERK/eIF2a and IRE1/ASK1/JNK pathway. These results explained the potential mechanism for the neuroprotective effect of PCs in the hippocampus of rats exposed to Pb. In conclusion, this study demonstrates for the first time that PCs reversed Pb-induced cognitive deficits through inhibiting ER stress

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Fig. 7. Schematic diagram shows the protective signaling of proanthocyanidins in brain inflammation of rats exposed to Pb. The ? indicates activation or induction, and a indicates inhibition or blockade.

and NF-jB-mediated inflammatory responses. Here we demonstrated that PCs attenuated Pb-induced oxidative damage by inhibiting ROS generation in brain. PCs decreased levels of Ab and tau phosphorylation in the hippocampus of rats exposed to Pb. PCs inhibited inflammation through the PERK/eIF2a and IRE1/ASK1/ JNK pathway. PCs might be possible candidates for the prevention or treatment of cognitive deficits in excitotoxic and other brain disorders. PCs may be useful for diseases associated with leadinduced inflammation, oxidative stress and ER stress. Dietary PCs supplementation may also mitigate misfolded tau-mediated neuropathologic and clinical phenotypes in human. PCs would be more probable candidates for the development of proanthocyanidinsbased functional foods, pharmaceuticals, and cosmetic products. A schematic diagram of the neuroprotective effects of PCs against Pb-induced cognitive deficits is shown in Fig. 7. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

Acknowledgments This work grants from the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). This work is supported from Natural Science Project Foundation of Jiangsu Province (BK20141147) and Startup Project of Doctor scientific research by Sichuan University of Science and Engineering (2013RC14). References Asha Devi, S., Sagar Chandrasekar, B.K., Manjula, K.R., Ishii, N., 2011. Grape seed proanthocyanidin lowers brain oxidative stress in adult and middle-aged rats. Exp. Gerontol. 46, 958–964. Bakulski, K.M., Rozek, L.S., Dolinoy, D.C., Paulson, H.L., Hu, H., 2012. Alzheimer’s disease and environmental exposure to lead: the epidemiologic evidence and potential role of epigenetics. Curr. Alzheimer Res. 9, 563–573. Bihaqi, S.W., Zawia, N.H., 2013. Enhanced tauopathy and AD-like pathology in aged primate brains decades after infantile exposure to lead (Pb). NeuroToxicology 39, 95–101.

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