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Nutrition 30 (2014) 210–217

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Nutrition

journal homepage: www.nutr i t ionjrnl .com

Basic nutritional investigation

Cinnamon polyphenols regulate S100b, sirtuins, and neuroactive proteinsin rat C6 glioma cells

Bolin Qin Ph.D., M.D. a,b,*, Kiran S. Panickar Ph.D. a,c, Richard A. Anderson Ph.D. a

aDiet, Genomics and Immunology Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD, USAb Integrity Nutraceuticals International, Spring Hill, TN, USAcDepartment of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, USA

a r t i c l e i n f o

Article history:Received 12 February 2013Accepted 2 July 2013

Keywords:PolyphenolsS100bSirtuinsPhospho-NF-kB and Bcl-xlSpice

* Corresponding author. Tel.: þ1 301 504 5253; faxE-mail address: Bolin.Qin@ars.usda.gov (B. Qin).

0899-9007/$ - see front matter � 2014 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.nut.2013.07.001

a b s t r a c t

Objective: Increasing evidence suggests that cinnamon has many health benefits when used inherbal medicine and as a dietary ingredient. The aim of this study was to investigate the effects ofan aqueous extract of cinnamon, high in type A polyphenols, on molecular targets in rat C6 gliomacells that underlie their protective effects.Methods: C6 rat glioma cells were seeded in 35-mm culture dishes or six-well plates, then wereincubated with cinnamon polyphenols at doses of 10 and 20 mg/mL for 24 h. The targeting proteinexpression, secretion, and phosphorylation were evaluated by immunoprecitation/immunoblottingand immunofluorescence imaging.Results: Cinnamon polyphenols significantly enhanced secretion of S100b, a Ca2þ-binding protein,and increased intracellular S100b expression after 24 h of incubation, in rat C6 glioma cells. Cin-namon polyphenols also enhanced protein levels of sirtuin 1, 2, and 3, deacetylases important incell survival, and the tumor suppressor protein, p53, and inhibited the inflammatory factors, tumornecrosis factor alpha, and phospho-p65, a subunit of nuclear factor-kb. Cinnamon polyphenols alsoup-regulated levels of phospho-p38, extracellular signal-regulated protein and mitogen-activatedprotein and kinase-activated protein kinases that may be important for prosurvival functions.Conclusion: Our results indicate that the effects of cinnamon polyphenols on upregulating pro-survival proteins, activating mitogen-activated protein kinase pathways, and decreasing proin-flammatory cytokines may contribute to their neuroprotective effects.

� 2014 Elsevier Inc. All rights reserved.

Introduction

Plant-derived natural polyphenols represent a wide variety ofcompounds that are enriched in fruits, vegetables, wine, tea,extra virgin olive oil, chocolate, and other plant products. Accu-mulating evidence suggests that cinnamon has many healthbenefits when used both in herbal medicine and as a spice [1].We have isolated and characterized a polyphenol-rich water-soluble extract from cinnamon polyphenols (CP), that is high indoubly-linked polyphenol type-A polymers [2,3] that hasinsulin-potentiating [2], antioxidant [4], and antiinflammatoryproprieties [5,6]. In human studies, water-soluble CP have beenshown to reduce fasting blood sugar levels in prediabetic pa-tients with metabolic syndrome [7], improve antioxidant status,decrease risk factors associated with diabetes and cardiovascular

: þ1 301 504 9062.

ll rights reserved.

diseases [4], and improve insulin sensitivity in women withpolycystic ovary syndrome [8]. Metabolic syndrome is associatedwith insulin resistance, elevated glucose and lipids, inflamma-tion, decreased antioxidant activity, increased weight gain, andincreased glycation of proteins. CP have been shown to improveall of these variables in in vitro, animal, and/or human studies[1].

CP also have been reported to exert neuroprotective effects,such as inhibiting tau aggregation associated with Alzheimer’sdisease [9], reducing cell swelling associated with ischemicinjury [10], attenuating mitochondrial dysfunction in ischemicinjury [10,11], and improving glutamate uptake followingischemia-like injury in vitro [11]. Moreover, an aqueous extract ofcinnamon has been shown to inhibit Ab oligomer formation andameliorate Alzheimer’s disease symptoms [12]. Although poly-phenols have been reported to exert neuroprotective actionsagainst injury induced by neurotoxins, and display the ability tosuppress neuroinflammation [13], it is not clear whether such

B. Qin et al. / Nutrition 30 (2014) 210–217 211

polyphenols exert protective effects by modulating the functionsof other prosurvival proteins, including S100b, sirtuins, Bcl-xl,as well as intracellular signaling pathways mediated bymitogen-activated protein kinases (MAPKs), and nuclear fac-tor-kB (NF-kB).

S100b, is a calcium-binding protein primarily expressed in thecentral nervous system (CNS), and synthesized and secreted byastrocytes [14]. Increasing evidence suggests that S100b plays adouble function as an intracellular regulator and an extracellularsignal of the CNS. S100b is found in the cytoplasm in a solubleform and also is associated with intracellular membranes, cen-trosomes, microtubules, and type III intermediate filaments [15].Within cells, it is involved in the regulation of cell proliferation,differentiation, shape, Ca2þ homeostasis, protein phosphoryla-tion, transcription, enzyme activity, and metabolism [14]. Whensecreted into the extracellular medium, S100b exerts regulatoryeffects on neighboring cells including astrocytes, neurons, andmicroglia. At nanomolar levels, S100b has neurotrophic activityin the extracellular medium, but at micromolar levels it stimu-lates apoptosis [16,17]. The mechanisms of regulating S100bsecretion are not completely understood and appear to berelated to many factors [14], such as the proinflammatory cyto-kines, tumor necrosis factor alpha (TNF-a) [18], interleukin(IL)-1b [19], and metabolic stress [20]. Moreover, a previousstudy suggested that S100b secretion involves the MAPKpathway and apparently could involve NF-kB signaling [19].

A Sirts activator, resveratrol, has been shown to increaseglutamate uptake and S100b secretion in cortical astrocyte cul-tures [21]. It also has a protective effect against acute H2O2-induced oxidative stress in astrocyte cultures, enhancing theimpaired S100b secretion [22]. Sirtuins (Sirt), a family of nico-tinamide adenine nucleotide (NAD)-dependent deacetylases, areimplicated in energy metabolism and lifespan. Sirtuins are noveltargets for Alzheimer’s disease and other neurodegenerativedisorders. Sirt1 has been found to protect cell DNA damage andreduce apoptotic death in vitro [23]. Sirt2 activity plays a key roleinmaintaining the survival of glioma cells [24], and Sirt3 acts as asurvival factor playing an essential role to protect neurons underexcitotoxic injury [25].

The C6 glioma cell line exhibits many astrocytic features, andhas been used widely to characterize the mechanisms of anastrocytic model system, such as the study of neurotrophic ac-tions [26], nerve growth factors [27], and insulin-like growthfactors [28]. Here, we investigated the in vitro effects of CP on theintracellular expression and the extracellular secretion of S100bin rat C6 glioma cultures. We also evaluated whether CP regu-lates Sirtuin 1, 2, and 3 expression. Additionally, the anti-apoptotic protein, Bcl-xl, inflammatory factor, TNF-a,transcription factor, phospho-NF-kB (p-NF-kB) p65, MAPKphospho-p38, extracellular signal-regulated protein (ERK) andmitogen-activated protein and kinase-activated protein(MAPKAPK2) kinases also were investigated.

Materials and methods

Cinnamon polyphenols

The aqueous extract of cinnamon contained 0.74% of a type A tetramer,cassiatannin A, with a molecular weight of 1152 and 2.30% of cinnamtannin D1,molecular weight 864, 0.72% of cinnamtannin B1, and 0.22 and 0.12% of two typeA trimer isomers, whose exact structure is not known,molecular weight 864. Thetotal percent of the type A polymers that were quantitated was 4.10%. Type Apolymers were identified using electrospray ionization mass spectrometry ana-lyses using an LCQ ion trap instrument (ThermoElectron Co., San Jose, CA) andhigh-performance liquid chromatography. The dried CP, Cinnulin PF�, was

provided by Integrity Nutraceuticals International (Spring Hill, TN, USA) and alsocan be prepared as described [2].

Cell culture

C6 glioma cells (CCL-107) were purchased from American Type CultureCollection (ATCC; Manassas, VA, USA). Cell cultures were grown in F-12 K me-dium (Gibco/Invitrogen) supplemented with 10% horse serum and 2% fetalbovine serum and maintained at 37�C with 5% CO2/95% air. Cultures were grownto 85% confluence in 75-mm flasks and, after trypsinization, were seeded in35-mm culture dishes or six-well plates (0.25 � 106); and allowed to confluenceduring the experimental period. All cultures used in the experiment were be-tween passages 22 and 32.

Extracellular S100b secretion of rat C6 glioma cells and Western blotting analysis

C6 glioma cells were incubated with CP (10 and 20 mg/mL) in the fresh me-dium, at 37�C. The selection of the doses of CP was based on our previous studiesshowing significant effects of CP in primary enterocytes [5], macrophages [6], andC6 cells [10]. After 24 h, the cultures were harvested for immunoprecipitationusing rabbit anti-S100b immunoglobulin G and Dynabeads� protein A (Invi-trogen, Carlsbad, CA, USA), according to the manufacturer’s directions. The elutedsamples were separated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis and analyzed by Western blotting with anti-S100b antibody, thensubjected to fluorography. Immunoblotting protocols were performed asdescribed in previous studies [29,30].

Immunofluorescence cell staining

Cells were grown on 35-mm culture dishes, rinsed with ice-cold phosphatebuffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min at roomtemperature, followed by permeabilization with 0.3% Triton x-100 for 10 min.After being washed with PBS three times, cells were incubated for 1 h in PBScontaining 10% normal goat serum-blocking solution. The cells were subjected toimmunofluorescence staining with the specific antibodies overnight at 4�C. Thecells were then washed with cold PBS three times for 3 min each, and incubatedwith Alexa-labeled secondary antibodies (Invitrogen) at room temperature for 1h. Nuclei were stained with NucBlue� fixed cell stain kit (Invitrogen, Carlsbad,CA, USA) for 5min. The cells were examined by fluorescencemicroscopy (a NikonTE2000-S microscope, Nikon, Tokyo, Japan). Fluorescence intensities from im-ages of five randomly selected microscopic fields of cells were semiquantitativelyanalyzed by densitometry (ImageJ software, NIH Image).

Extraction of cell nuclear protein and analysis of phospho-p65, subunit of NF-kB

Following 24-h treatment with CP, the cells were harvested by scraping andwashed with cold PBS. The nuclear fractions were isolated using NE-PER� nu-clear and cytoplasm extraction reagents (NER and CER) according to the manu-facturer’s instructions (Pierce, Rockford, IL). Briefly, the cell suspensions werecentrifuged, CER I solutionwas added to cell pellets and vortexed, and then CER IIsolution was added and vortexed. After centrifugation, the supernatant cyto-plasmic extracts were removed. Nuclear pellets were suspended in 50 ml of NERsolution, vortexed every 10 min, incubated in an ice bath for 40 min, and thesupernatant containing nuclear extracts collected. The nuclear extracts wereanalyzed by Western blotting using antibody against p-NF-kB p65.

Statistical analyses

Data were analyzed by one-way analysis of variance followed by post hocanalysis of between group mean differences by Fisher’s least significant differ-ence (LSD) test. P-values < 0.05 were significant for all analyses.

Results

CP enhanced the intracellular expression and the extracellularsecretion of S100b in C6 cells

To investigate whether CP increases S100b secretion in C6cells, extracellular S100b content was measured after exposureto CP for 24 h by immunoprecitation and subsequent immuno-blotting (Fig. 1). After CP incubation, a significant increase inS100b secretion was observed with both concentrations of CP(147% and 243% of controls; P < 0.05, respectively). Increasedsecretion of S100b also was observed after 4 h of CP treatment(data not shown). Furthermore, intracellular S100b expression

Fig. 1. CP enhanced extracellular S100b secretion in C6 glioma cells. Cells weretreated with PBS or CP (10 or 20 mg/mL) in the medium for 24 h at 37�C. Thecollected medium samples were subjected to immunoprecipitation and thenanalyzed by SDS-PAGE and fluorography. Data are the means � SE (n ¼ 6). *Significantly different from the respective control; P < 0.05.

B. Qin et al. / Nutrition 30 (2014) 210–217212

was significantly increased by 4 h (Fig. 2A, B) and 24 h (Fig. 2C, D).Both concentrations of CP (10 and 20 mg/mL) increased thenumber of S100b-immunopositive cells compared with non-treated control cultures (4-h incubation: 136% and 147% of con-trols; 24-h incubation: 223% and 200% of controls; P < 0.05,respectively).

CP increased sirtuin 1, 2, and 3 expression in C6 cells

Because sirtuins 1 to 3 have been linked with neuroprotectiveeffects [2,24], Sirt1 to 3 protein levels were measured by immu-nofluorescense in C6 glial cells. Following 24-h treatment, bothCP dosages (10 and 20 mg/mL) significantly increased Sirt1(145%and 297% of controls), Sirt2 (155% and 165% of controls), and Sirt3(194% and 180% of controls) immunoreactive cells comparedwithcontrol cultures (Fig. 3; P < 0.05, respectively).

CP inhibited TNF-a protein levels and phospho-p65 in C6 cells

To assess the antiinflammatory effects of CP, we measuredproteins that are important in inflammatory processes, includingTNF-a and NF-kB. When compared with controls, there was asignificant decrease in TNF-a protein levels in cultures treatedwith CP (10 and 20 mg/mL) for 24 h (Fig. 4A). The transcriptionfactor, NF-kB, is a key regulator of inflammation and cell survival.NF-kB activation increases following cerebral ischemia. As shownin Figure 4B, there was a significant decrease in p-NF-kB p65levels in the nuclear fractions upon treatment with CP for 24 h.

CP inhibited Bcl-xl and induced p53 expressions in C6 cells

We then examined the effects of CP on Bcl-xl, an anti-apoptotic protein of the Bcl-2 family, and p53, a tumor sup-pressor protein. Cultures were treated with CP (10 and 20 mg/mL)for 24 h. CP treatment significantly decreased Bcl-xl protein

levels measured by immunofluorescense (Fig. 5A, B: 50% and 73%of controls; P < 0.05, respectively) and immunoblot (Fig. 5C). CPincreased p53-immunoreactive cells (Fig. 5D: 152% and 174% ofcontrols; P < 0.05, respectively).

CP increased phosphorylation of p38, ERK and MAPKAPK2 in C6cells

The activation of MAPK has been linked to neuroprotection invarious cellular model systems [31,32]. Cultures were treatedwith CP (10 and 20 mg/mL) for 24 h. As shown in Figure 6, CPenhanced phosphor-p38 and phosphor-ERK levels by Westernblot analysis; both doses of CP also stimulatedphospho-MAPKAPK2 levels by immunofluorescence (157% and155% of controls; P < 0.05, respectively).

Discussion

CP have been reported previously to exert neuroprotectiveeffects [9–11,33]. This study adds to mounting evidence that CPincreases intracellular S100b expression and extracellular S100bsecretion in C6 glial cultures. Additionally, CP also increased thelevels of sirtuins, phosphorylation of MAPK, and decreased levelsof proinflammatory proteins.

S100b is involved in the regulation of cell proliferation, dif-ferentiation, shape, Ca2þ homeostasis, protein phosphorylation,transcription, enzyme activity, and metabolism [14]. S100b hasbeen also implicated in glial–neuronal communication [34]. Atnanomolar levels, extracellular S100b may protect hippocampalneurons against glutamate-induced damage [35]. Antibody anti-S100b addition decreased glutamate uptake without affectingcell integrity or viability. Low levels of S100b stimulate glutamateuptake [36]. In previous studies, CP were shown to attenuate cellswelling and mitochondrial dysfunction following ischemia-likeinjury and to increase glutamate uptake in glial cultures [10,11].It is possible that CP-induced S100b secretion, may be associatedwith increased glutamate uptake [10,11,36,37]. Consistent withthis, resveratrol, a plant-derived polyphenol with multiple bio-logical effects including antioxidant and antiinflammatoryproperties, has been shown to be neuroprotective by increasingglutamate uptake, and it also stimulates S100b secretion. All ofthis contributes to functional recovery after brain injury [38,39].

Sirtuins are neuroprotective in various disease models andhave multiple beneficial activities on metabolic syndrome-related diseases, such as protecting cell DNA damage andreducing apoptotic death [23], maintaining the survival of gli-oma cells [24], and protecting neurons under excitotoxic injury[25]. In this study, our data indicate that CP treatment enhancesSirt1, 2, and 3 protein expressions in C6 glioma cells (Fig. 3).Resveratrol, which has neuroprotective effects, also has beenidentified as a potent pharmacologic agonist of sirtuin activity[40]. An increase in sirtuins in the present study supports thehypothesis that one mechanism by which CP exert their neu-roprotective effects is by increasing sirtuin levels. It is alsopossible that sirtuins are involved in other beneficial effects ofCP, including antioxidant [4,41], insulin-potentiating [2,29,42],and antiinflammatory effects [6], as well as improving lipidmetabolism [5,43].

At micromolar levels, S100b can induce neuronal apoptosis,mediated by the induction of proinflammatory cytokines, such asIL-1b, TNF-a, and inflammatory stress-related enzymes, such asinducible nitric oxide synthase [44]. There is a crosstalk betweenproinflammatory cytokines and S100b. Previous studies havereported an increase in cellular S100b in C6 glioma cells after

Fig. 2. CP increased intracellular S100b protein in C6 glioma cells. C6 cells were seeded in 35-mm culture dishes and treated with PBS or CP (10 or 20 mg/mL) for 4 h or 24 h,then cells were assessed by immunofluorescense staining with an anti-S100b antibody as described in the Materials and methods section. In the left panels of A and C, cellswere stained with anti-S100b primary antibody (Abcam Inc, Cambridge, MA) and AlexaFluor� 594-conjugated normal rabbit IgG (red) secondary antibody; the middle panelsof A and C are stained with DAPI (blue), identifies nuclei; the right panels are merged images which correspond to the overlay of the anti-S100b and DAPI staining. A (4 h) andC (24 h): Representative immunofluorescence images; B (4 h) and D (24 h): Fluorescence intensities from images were measured and values are mean � SE of two separateexperiments performed in duplicate. * versus control, P < 0.05.

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24 h of exposure to IL-1b [45], but no change in astrocyte culturesafter 48 h [46]. S100b secretion in brain tissue is stimulatedrapidly and persistently (at least for 24 h) by intra-cerebroventricular administration of lipopolysaccharide (LPS)[47]. TNF-a, a potent proinflammatory cytokinewith a major rolein initiating a cascade of activation of other cytokines and growthfactors in inflammatory responses, which is synthesized bymicroglia, astrocytes, and some populations of neurons, hasseveral important functions in the CNS, including astrocyteactivation, glutamatergic transmission, and synaptic plasticity[48]. It has been proposed that TNF-a is able to mediate S100bsecretion in astrocytes and decrease S100b secretion in culturedastrocytes after 3 d of exposure to TNF-a [18]. Our data indicatethat CP inhibit cellular TNF-a protein expression (Fig. 4), thatmay be associated with S100b expression and secretion. Ourprevious studies have shown that CP attenuated TNF-a–induced

intestinal lipoprotein apoB48 overproduction by regulating in-flammatory, insulin, and lipoprotein pathways [5], and increasedantiinflammatory protein expression [6].

NF-kb, a transcription factor, is a downstream signal of theTNF-a pathway and a ubiquitous transcription factor in virtuallyall cell types; it plays a pivotal role in inflammatory and immuneresponses [49]. Moreover, the activation of NF-kB is required forthe neuroprotective and potentially neuroregenerative effects ofS100b. A previous study suggested that very low concentrationsof S100b significantly protect primary rat hippocampal neuronsagainst N-methyl-D-aspartate (NMDA) toxicity by activation oftranscription factors of the Rel/NF-kB family [50]. NF-kB alsopartially regulates the expression of Bcl-xl [51], which is anantiapoptotic member of the Bcl-2 family. In the current study,our data suggest that CP decreased Bcl-xl protein levels (Fig. 5)and the p-NF-kB p65 expression (Fig. 4). Cinnamon extract has

Fig. 3. CP stimulated Sirt1, 2, and 3 protein expressions in C6 glioma cells. Cultures were treated for 24 h with PBS or CP (10 or 20 mg/mL). The cells were then fixed,permeabilized, and stained as described in Materials and methods. In the left panels of A, C, and E, the cells were stained with anti-Sirt1 primary antibody (Abcam Inc,Cambridge MA), or anti-Sirt2 antibody or anti-Sirt3 antibody and AlexaFluor� 488-conjugated normal mouse IgG (green) or AlexaFluor� 594-conjugated normal rabbit IgG(red) secondary antibodies; in the middle panels of A, C, and E, DAPI staining (blue) identifies nuclei; the right: merged images correspond to the overlay of the anti-Sirt1–3and DAPI staining. A, C, and E: Representative immunofluorescence images for Sirt1–3; B, D, and F: fluorescence intensities from images were measured and values are mean� SE of two separate experiments performed in duplicate. *versus control, P < 0.05.

Fig. 4. CP inhibited TNF-a and phospho-p65 expressions in C6 glioma cells. The immunoblots show decreased TNF-a (A) and phospho-NF-kB p65 (B) expression in 24-hCP-treated C6 cells; the blots were reprobed with anti-GAPDH or anti-Lamin a/c as the loading control; the immunoblots shown are representative of three independentexperiments.

B. Qin et al. / Nutrition 30 (2014) 210–217214

Fig. 5. CP inhibited Bcl-xl and p53 expressions in C6 glioma cells. The experimental conditions described in Figure 2 and in Materials and methods. In the left panels of A, cellswere stained with anti-Bcl-xl antibody (Abcam Inc, Cambridge MA), and AlexaFluor� 594-conjugated Normal rabbit IgG (red); In the middle panels of A, DAPI staining (blue)identifies nuclei; the right: merged images correspond to the overlay of the anti-Bcl-xl and DAPI staining. A and D: Representative immunofluorescence images for Bcl-xl andp53; B and E: Fluorescence intensities from images were measured and values are mean � SE of two separate experiments performed in duplicate; * versus control, P < 0.05.C: Representative immunoblots show decreased Bcl-xl expression in CP treated C6 cells; the blots were reprobed with anti-GAPDH as a loading control; the immunoblotsshown are representative of three independent experiments.

B. Qin et al. / Nutrition 30 (2014) 210–217 215

been shown to strongly inhibit tumor cell proliferation anddecrease NF-kB activity and a target gene, Bcl-xl, in vitro and alsoin vivo in a mouse melanoma model [52]. Epigallocatechin-3-gallate also has been shown to decrease the expressions of Bcl-2a and Bcl-xl by inhibition of NF-kB in human hepatocellularcarcinoma [53]. The antiapoptotic protein Bcl-xl has beenimplicated in both cancer cell and normal cell survival [54].Recently, S100b has been shown to bind directly to the p53protein [55], and enhance [56] or downregulate [55] p53expression. The p53 protein, a non-histone substrate of Sirt1, alsoactivates the transcription of proapoptotic factors, as well assuppresses the transcription of antiapoptotic genes such as Bcl-xl[57]. There are protein–protein interactions between S100b andp53, which are crucial for the proper function of cells. TheS100b–p53 interactions are regulated by post-translationalmodifications [58]. Moreover, the effects of S100 proteins onp53 may be different in various cell types particularly, becauseS100 proteins themselves are cell-specifically distributed [59]. In

this study, our data indicate that CP also increased p53 proteinlevels (Fig. 5), which are associated with improved S100bsecretion and expression.

MAPK pathways constitute a large modular network thatregulates a variety of physiological processes, such as cellgrowth, differentiation, and apoptotic cell death. The activationof MAPK cascades has been linked to neuroprotection in variouscellular model systems [31,32], and the phosphatase-mediatedcrosstalk between MAPK signaling pathways in the regulationof cell survival [60]. In response to cytokines, stress, andchemotactic factors, MAPKAPK2 is rapidly phosphorylated andactivated. MAPKAPK2 also is activated in vitro by thep42/p44MAPK [61]. It has been shown thatMAPKAPK2 is a directtarget of p38 MAPK [61]. A previous study has shown that verylow concentrations of S100b protect neurons against NMDAtoxicity, and that S100b-mediated neuroprotection mightinvolve the MAPK pathway [50]. We have reported previouslythat CP induces phosphor-p38, ERK, and Jun N-terminal kinase

Fig. 6. CP induced phospho-p38, anti-Phospho-ERK1/2 and phospho-MAPKAPK2 expressions in C6 glioma cells. Cultures were treated for 24 h with PBS or CP (10 or 20 mg/mL). The cells were subjected to immunoblot or immunofluorescence as described in Materials and methods. A and B: Representative immunoblots show increased phospho-p38, -ERK expressions in CP-treated C6 cells; the blots were reprobed with anti-GAPDH as a loading control; the immunoblots shown are representative of three independentexperiments; C: Representative immunofluorescence images for phosphor-MAPKAPK2; D: fluorescence intensities from images were measured and values are mean � SE oftwo separate experiments performed in duplicate. *versus control, P < 0.05.

B. Qin et al. / Nutrition 30 (2014) 210–217216

levels in primary enterocytes [62]. In the current study, CPenhanced phospho-p38, ERK, and MAPKAPK2 levels, and theseeffects were associated with increased S100b secretion and areconsistent with the neuroprotective effects of CP.

In summary, in agreement with the protective roles previ-ously demonstrated for CP [10,11], our data show that CP areassociated with significant improvements in the intracellularexpression and extracellular secretion of S100b in C6 gliomacells. CP also enhanced sirtuins, which have been shown to havetherapeutic potential in neurodegenerative diseases such asstroke, ischemic brain injury, Alzheimer’s disease, and Parkin-son’s disease [63]. CP also up-regulated multiple prosurvivalproteins and increased phosphorylation of MAPK pathways. Aninteresting new study has suggested that polyphenols from olivetreatment may increase the levels of nerve growth factor andbrain-derived neurotrophic factor in vivo, which are both neu-rotrophins and play key roles in brain cell development, growth,and survival [64].

Conclusions

The results of the current investigation, in conjunction withthe findings from our previous studies, indicate that CP mayexert neuroprotective effects in astroglial cells by upregulatingprosurvival proteins and decreasing proinflammatory proteins.

Acknowledgments

This work was supported in part by a USDA CooperativeResearch and Development Agreement (CRADA No.58-3K95-7-1184) with Integrity Nutraceuticals International, Spring Hill, TN.BQ has a joint appointment with the U.S. Department of Agri-culture and Integrity Nutraceutical International (Spring Hill,TN). Mention of trade names or commercial products is solely forthe purpose of providing specific information and does not implyrecommendation or endorsement by the U.S. Department ofAgriculture. The U.S. Department of Agriculture is an equal op-portunity provider and employer.

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