reperfusion

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Research Report

12/15-Lipoxygenase inhibitor baicalein suppresses PPARγ expression and nuclear translocation induced by cerebral ischemia/reperfusion Yan-Wei Xu 1 , Li Sun⁎,1 , Hao Liang, Guo-Min Sun, Yan Cheng Tianjin Neurology Institute, Tianjin Medical University General Hospital, 154 Anshan Road Heping District, Tianjin 300052, China

A R T I C LE I N FO

AB S T R A C T

Article history:

Accumulating evidences have demonstrated the beneficial actions of peroxisome

Accepted 15 October 2009

proliferator-activated receptor γ (PPARγ) in a variety of animal stroke models. Following

Available online 22 October 2009

middle cerebral artery occlusion (60 min) and 2–24 hr reperfusion in rats, we observed cerebral ischemia/reperfusion (I/R) induced up-regulation of PPARγ protein expression and

Keywords:

translocation from the cytoplasm into the nucleus in a time-dependent manner. We also

PPARγ (peroxisome

found that PPARγ agonist rosiglitazone enhanced whereas PPARγ antagonist GW9662

proliferator-activated receptor γ)

inhibited the alteration of PPARγ stimulated by I/R, suggesting that the changes of PPARγ

Ischemia/reperfusion

may result from the activation by endogenous ligands. Moreover, the link between the 12/15-

Baicalein

lipoxygenase and the production of activating ligands for PPARγ has been proved in various

Translocation

tissues. However, the relation of them in brain tissue has not been identified. We

Rosiglitazone

demonstrated that the I/R-induced PPARγ alteration was reversed by baicalein, the specific

GW9662

inhibitor of 12/15-lipoxygenase. Baicalein treatment significantly inhibited the up-regulation of PPARγ expression and, furthermore, suppressed PPARγ nuclear accumulation as well as maintained PPARγ cytoplasmic retention. Together, these results suggest that I/R induces both PPARγ expression and translocation, probably through the activation by endogenous ligands in a 12/15-lipoxygenase inhibitor-sensitive way. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor that regulates lipid metabolism and glucose homoeostasis (Boyle, 2007). Ligands for PPARγ exist as natural and synthetic classes. Examples are modified fatty acids (e.g., oxidized metabolites of linoleic and

arachidonic acids, 13-hydroxyoctadecadienoic acid (13-HODE), and 15-hydroxyeicosatetraenoic acid (15-HETE), which are derived from oxidized low-density lipoprotein) (Huang et al., 1999), 15-deoxy-delta prostaglandin J2 (15d-PGJ2), and the synthetic thiazolidinedione (TZD) class of insulin-sensitizing agents or “glitazones” (troglitazone, pioglitazone, ciglitazone, and rosiglitazone) which are used to treat type II diabetes.

⁎ Corresponding author. Fax: +86 22 6036 2766. E-mail address: [email protected] (L. Sun). Abbreviations: PPAR, peroxisome proliferator-activated receptor; I/R, ischemia/reperfusion; 13-HODE, 13-hydroxyoctadecadienoic acid; 15-HETE, 15-hydroxyeicosatetraenoic acid; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; Ros, rosiglitazone; DMSO, dimethylsulfoxide; PBS, phosphate-buffered saline; TRITC, tetraethyl rhodamine isothiocyanate; DAPI, 4',6-diamidino-2phenylindole; NF-Κκ, nuclear factor-κB; TZDs, thiazolidinediones; 15d-PGJ2, 15-deoxy-delta prostaglandin J2 1 Yan-Wei Xu and Li Sun contributed equally to this study. 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.10.038

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Upon activation by its ligands, PPARγ translocates from cytoplasm into nucleus to regulate gene transcription (Bogazzi et al., 2005). Nitration of PPARγ causes dysfunction of the transcription factor by inhibiting its agonist-stimulated nuclear translocation (Shibuya et al., 2002). In addition to the multiple physiological roles, PPARγ is also involved in the control of inflammation, in particular in modulating the production of inflammatory mediators (Han et al., 2008; Hunter et al., 2008; Kielian et al., 2008; Ko et al., 2008; Randy and Guoying, 2007). The endogenous PPARγ pathway has been reported to act against inflammatory responses in the gastrointestinal tract in animal model of ischemia-induced colitis (Nakajima et al., 2001). Consistently, using neuronal PPARγ deficient mice and PPARγ agonists, Zhao et al. (2009) showed that PPARγ in neurons plays an intrinsic protective role in the brain against ischemia/reperfusion (I/R) injury. Activation of PPARγ by its agonist significantly reduced infarction size and improved neurologic function in a variety of animal models of stroke (Luo et al., 2006; Sundararajan et al., 2005; Zhao et al., 2005). Improvement was associated with the inhibition of nuclear factor-κB (NF-κB) from inducing transcription of genes involved in the inflammatory and oxidative responses (Collino et al., 2006; Nakajima et al., 2001; Remels et al., 2009). Therefore, the endogenous PPARγ pathway is considered a self-defense system to protect brain and a potential novel therapeutic target against ischemic stroke. 12/15-Lipoxygenase is a lipid-peroxidating enzyme that can oxygenate free polyunsaturated fatty acids and phospholipids

in biomembranes (Yamamoto, 1992). In mammalian cells, linoleic acid and arachidonic acid are the major polyunsaturated fatty acid substrates for 12/15-lipoxygenase (Kuhn, 1996). Linoleic acid is oxygenated by 12/15-lipoxygenase to 13-HODE (Kuhn, 1996), whereas 12-HETE and 15-HETE are the major metabolites of arachidonic acid by the 12/15-LO pathway (Heydeck et al., 1998). Cell-based assays have proven that 12HETE, 15-HETE, and 13-HODE function as activating ligands of PPARγ and induce PPARγ and its target gene CD36 expression in multiple cells (Berry et al., 2007; Limor et al., 2008; Li et al., 2004). The present study thus aimed to evaluate the alteration in protein expression and nuclear translocation of endogenous PPARγ after focal cerebral I/R in rats. We also investigated whether the changes of PPARγ may associate with the activation by endogenous ligands during cerebral I/R.

2.

Results

2.1. I/R induced PPARγ protein up-regulation and nuclear translocation Brain tissue is known to express PPARγ though the protein level is rather low (Sun et al., 2008). We explored the alteration of PPARγ expression in a rat model of 60-min middle cerebral artery occlusion (MCAO) and 2–24 hr reperfusion by Western blot (Fig. 1A). Clearly, an analysis of whole-cell extracts revealed a time-dependent up-regulation of PPARγ expression

Fig. 1 – PPARγ protein expression in the rat ischemic cortex after I/R. (A) Western blot analysis of PPARγ and β-actin protein products was carried out in rats of sham operation (Sham) and 60 min-ischemia and reperfusion for 2, 4, 8, and 24 hr. The bar graph illustrates the densitometrical analysis of the related bands. Data are expressed as mean ± SEM (n = 6). *P < 0.05 versus Sham. (B) Immunohistochemical demonstration of PPARγ protein expression in frontoparietal cortex of Sham and I/R (60 min/24 hr).

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following 4 hr of reperfusion when compared with the shamoperated control. To confirm the elevation, we detected PPARγ by immunocytochemistry (Fig. 1B). In sham-operated rats, the intensity of PPARγ immunostaining was faint to moderate throughout the hemisphere. After 60 min of ischemia and 24 hr of reperfusion, however, robust increases in numbers of PPARγ-positive cells were demonstrated and the intensity of staining became moderate to strong in the ischemia-affected region of the ipsilateral middle cerebral artery (MCA) territory (ipsilateral cortex). As PPARγ is a nuclear receptor, we then carried out cell fractionation analysis of PPARγ affected by I/R. As shown in Fig. 2A, in sham-operated animals, PPARγ expressed in both cytosol and nuclear fractions. An increase of PPARγ in the nucleus could be noted with a simultaneous reduction in the cytosol following I/R, indicating the induction of nuclear translocation by I/R. A time-dependent enhancement in PPARγ translocation was demonstrated by the analysis of cytosol and nuclear level of PPARγ at 2, 4, 8, and 24 hr of reperfusion. It was worthy of note that this nuclear translocation of PPARγ occurred as early as 2 hr of reperfusion, at which time point no detectable increase in total PPARγ expression was observed, implying that nuclear translocation of PPARγ was a quicker and earlier

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response to I/R relative to the up-regulation of protein level. Immunofluorescence staining was also carried out to evaluate the nuclear translocation (Fig. 2B). In accordance with the result of Western blot, the localization of PPARγ was visualized in both cytoplasm and nucleus in sham-operated rats. After the exposure to ischemia and 2 hr of reperfusion, the majority of PPARγ accumulated in the nucleus and became slightly detectable in the cytoplasm. Thus, an induction of nuclear import of PPARγ by I/R was occurred and this relocalization suggested the activation of PPARγ.

2.2. PPARγ agonist enhanced whereas PPARγ antagonist inhibited the I/R-increased expression and nuclear translocation of PPARγ The influence of PPARγ agonist (rosiglitazone) and antagonist (GW9662) on I/R-induced changes of PPARγ was also examined in our study. Rats were injected with rosiglitazone, GW9662, or vehicle alone, 30 min before exposure to ischemia plus 24 hr of reperfusion. Western blot (Fig. 3A, upper panel) and immunoflorescence staining (Fig. 3B) analysis revealed that rosiglitazone dramatically increased I/R-induced total protein level and cytoplasm-to-nuclear enrichment of PPARγ, indicating

Fig. 2 – PPARγ translocation from the cytosol to the nucleus in the rat ischemic cortex after I/R. (A) PPARγ translocation was evaluated with cytosol and nuclear protein fractions by anti-PPARγ antibody in rats of sham operation (Sham) and 60 min of ischemia and reperfusion for 2, 4, 8, and 24 hr. The bar graph illustrates the densitometrical analysis of the related bands. Data are expressed as mean ± SEM (n = 6). *P < 0.05 versus Sham. (B) Immunofluorescence visualization of PPARγ translocation in frontoparietal cortex of Sham and I/R (60 min/2 hr). Color in red and blue represent PPARγ and nucleus, respectively. Pink fluorescence indicates co-localization of PPARγ/nuclei. Arrows indicate PPARγ-positive cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3 – The effects of PPARγ agonists rosiglitazone and antagonist GW9662 on PPARγ expression and nuclear translocation induced by I/R. For rosiglitazone-treated I/R rats (Ros) or GW9662-treated I/R rats (GW9662), rosiglitazone (6 mg/kg) or GW9662 (4 mg/kg) was injected intraperitoneally 30 min before 60 min of ischemia plus 24 hr of reperfusion. Sham-operated (Sham) and I/R rats received equal volumes of vehicle. (A) Western blot analysis of the actions of rosiglitazone and GW9662. Total PPARγ levels were measured with the whole protein lysate. PPARγ translocation was evaluated with cytosol and nuclear protein fractions. The bar graphs illustrate the densitometrical analysis of the related bands. Data are expressed as mean ± SEM (n = 6). *P < 0.05 versus Sham, **P < 0.05 versus I/R. (B) Visualization of PPARγ expression and nuclear translocation in frontoparietal cortex by fluorescence microscopy. A higher-power photomicrograph is included as an insert.

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that rosiglitazone promoted the actions of I/R on PPARγ. GW9662 treatment, on the other hand, inhibited I/R-stimulated changes of PPARγ by analyzing the whole cell lysate and subcellular fraction, respectively (Fig. 3A, lower panel). These results indicate that the I/R-induced PPARγ expression and translocation probably results from the activation by endogenous ligands.

2.3. 12/15-Lipoxygenase inhibitor, baicalein, reversed I/R-induced PPARγ alteration 12/15-Lipoxygenase, which belongs to the lipoxygenase family, can effectively catalyse the conversion of arachidonic acid and linoleic acid to endogenous ligands for PPARγ (Huang et al., 1999). Furthermore, the up-regulated 12/15-lipoxygenase expression has been reported in the ischemic regions following cerebral I/R in mice (van Leyen et al., 2006). Combined with our study, the coordinate induction of both 12/15-lipoxygenase and PPARγ indicates a possible link between 12/15-lipoxygenase and the alteration of PPARγ in the context of cerebral I/R. To investigate this possibility, we inhibited 12/15-lipoxygenase by intraperitoneal injection of its specific inhibitor, baicalein, 30 min before the exposure to I/R. First, different concentrations of baicalein (150 and 300 mg/kg) were delivered to rats suffering 60 min of ischemia and 24 hr of reperfusion (Fig. 4A). Second, baicalein (300 mg/kg)-treated rats were subjected to 60 min of ischemia and reperfusion of different duration (4 and 24 hr) (Fig. 4B). As seen in Fig. 4A, the majority of PPARγ localized in the nucleus under the I/R condition (lane 1). However, baicalein treatment significantly suppressed I/R-induced PPARγ nuclear

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accumulation and maintained PPARγ cytoplasmic retention, implying that baicalein could hinder PPARγ nuclear translocation. Furthermore, baicalein also inhibited the up-regulation of PPARγ expression. Consistently, Fig 4B showed that in the presence of baicalein, I/R-induced increase in PPARγ expression and nuclear translocation became less pronounced compared with vehicle-treated I/R rats at both 4 hr and 24 hr after ischemia. These data suggest the I/R-induced changes of PPARγ probably result from the activation by endogenous ligands generated through 12/15-lipoxygenase pathway.

3.

Discussion

The present data demonstrated that cerebral I/R markedly induced PPARγ nuclear translocation and the up-regulation of protein level. It also showed that I/R-stimulated alteration of PPARγ was strengthened or attenuated by PPARγ agonist rosiglitazone or antagonist GW9662, respectively, implying the changes of PPARγ resulting from the activation by endogenous ligands. 12/15-Lipoxygenase-derived metabolites have been identified as activating ligands of PPARγ (Huang et al., 1999). Our findings revealed that 12/15-lipoxygenase inhibition was associated with suppression of I/R-induced PPARγ alteration, indicating the possibility that I/R induced PPARγ expression and translocation through the 12/15-lipoxygenase pathway. As a transcription factor, PPARγ functions primarily in the nucleus and the binding of specific ligand activates PPARγ and enables its translocation from the cytosol into the nucleus. In our study, the combined and final effect of simultaneous

Fig. 4 – The effects of 12/15-lipoxygenase inhibitor baicalein on PPAR γ expression and nuclear translocation induced by I/R. PPARγ expression in whole cell extract and subcellular fractions of the ischemic cortices was examined by Western blotting. (A) Baicalein of indicated concentrations was injected intraperitoneally 30 min before ischemia started, and then animals were subjected to I/R (60 min/24 hr). (B) Baicalein (300 mg/kg) treatment was adopted to rats suffering 60 min of ischemia and 4 hr or 24 hr of reperfusion. The bar graphs illustrate the densitometrical analysis of the related bands. Data are expressed as mean ± SEM (n = 6). *P < 0.05 versus I/R. B indicates baicalein.

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induction of protein expression and translocation of PPARγ by I/R is to bring about an elevation of nuclear PPARγ, which probably associates with the enhancement of its nuclear activity, especially its well-established NF-κB-inhibiting effect. However, the onset of translocation was earlier than that of the up-regulation of protein expression (Fig. 2A), indicating that nuclear translocation of PPARγ was a quick response to I/R. In sham-operated rats, we observed that PPARγ expression was not only nuclear but also cytoplasmic. Thus, relative to increasing protein level, the control of cytoplasm-to-nuclear shuttling utilizing the existing cytoplasmic PPARγ is probably a more direct and convenient means to regulate the transcription factor. In the present observation, we detected that the supplementation of rosiglitazone, the exogenous ligand, promoted the alteration of PPARγ, whereas the treatment with PPARγ antagonist GW9662 inhibited the changes of PPARγ. These findings indicate I/R-induced changes of PPARγ resulting from the activation by endogenous ligands. Consistently, a previous study by Victor et al. (2006) showed that PPARγ antagonist T0070907 not only reversed the rosiglitazone-mediated protective effects after stroke but also augmented the degree of brain injury in the absence of PPARγ agonist. Their results also suggested the release of endogenous PPARγ ligands during cerebral I/R. Given that PPARγ is activated by a range of naturally occurring substances, including polyunsaturated fatty acids, 15d-PGJ2 and components of oxidized low-density lipoprotein, such as 13-HODE and 15-HETE. 13-HODE and 15HETE can be generated from linoleic and arachidonic acids by 12/15-lipoxygenase (Huang et al., 1999). Moreover, the 12/15lipoxygenase pathway has also been proven to be able to activate PPARγ and to induce PPARγ and its target gene CD36 expression in multiple cells and tissues (Berry et al., 2007; Limor et al., 2008; Li et al., 2004). However, the link between 12/ 15-lipoxygenase pathway and PPARγ has not been identified in the brain tissue. Furthermore, 12/15-lipoxygenase was previously reported to be up-regulated after brain I/R in mice (van Leyen et al., 2006). Immunohistochemical detection demonstrated that the increased presence of 12/15-lipoxygenase was predominantly in neurons of the peri-infarct area, which is similar to the localization of I/R-induced PPARγ expression (van Leyen et al., 2006). These findings indicate the possible link between 12/15-lipoxygenase and PPARγ. This possibility is further supported by our present study. We observed that the 12/15-lipoxygenase inhibitor baicalein reduced I/R-induced PPARγ expression and translocation. It therefore seems that I/R induces PPARγ expression and translocation through the activation of the 12/15-lipoxygenase pathway. 12/15-Lipoxygenase has been reported to generate peroxides from arachidonic acids and cause cell death in cultured neurons (Li et al., 1997). However, controversial results that 12/ 15-lipoxygenase and their metabolites exhibit anti-inflammatory properties have also been documented. For instance, 15HETE and 13-HODE were shown to inhibit the production of leukotriene-B4 and reactive oxygen species by polymorphonuclear neutrophils (Smith et al., 1993) and the generation of IL-8 by colonic epithelial cells (Altmann et al., 2007). In addition, 12/15-lipoxygenase was shown to mediate the suppressive effect of the anti-inflammatory cytokine IL-4 on

NF-κB trans-activation in glial cells and to protect oligodendrocyte progenitors under neuroinflammatory disease conditions (Paintlia et al., 2006). Reduced inflammation and tissue damage was also observed in transgenic animals overexpressing 12/15-lipoxygenase (Munger et al., 1999; Serhan et al., 2003; Shen et al., 1996). The mechanism by which 12/15-lipoxygenase and their metabolites exert anti-inflammatory actions has been evidenced to be through activation of PPARγ (Chabane et al., 2009). Thus, the reduced PPARγ expression and nuclear translocation in response to inhibition of 12/15-lipoxygenase by baicalein (Fig. 4) may compromise the beneficial actions of PPARγ. In addition to 12/15-lipoxygenase inhibition, baicalein is also a polyphenolic antioxidant by scavenging free radical and by inhibiting xanthine oxidase. It attenuated free radical production, lipid peroxidation, and cell death in hippocampal HT22 cells (Lapchak et al., 2007). Baicalein could also inhibit microglial activation and free radicals' production induced by LPS exposure in primary midbrain neuron–glia cultures (Li et al., 2005). Thus, in our study, baicalein could also decrease oxidative stress, which would prevent activation of the 12/15lipoxygenase (Kühn et al., 2002; Li et al., 1997) and subsequent generation of endogenous ligands for PPARγ. Further studies are warranted to confirm the relation between 12/15-lipoxygenase pathway and PPARγ in the context of I/R, including the measurement of 12/15-lipoxygenase metabolites (13-HODE and 15-HETE) and their direct ligand-activating effect on PPARγ in ischemic brains. Taken together, we showed that despite causing deleterious injury on the brain, cerebral I/R also triggers PPARγ protective response, including the induction of nuclear translocation and the increase in the total protein level of PPARγ. The mechanism underlying I/R-induced changes of PPARγ may be related to the activation by endogenous ligands in a 12/15-lipoxygenase inhibitor-sensitive way.

4.

Experimental procedures

4.1.

Rat model of transient focal ischemia

All animal experiments were carried out according to an institutionally approved protocol, in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University. Sprague-Dawley male rats (280–330 g) were obtained from the Academy of Military Medical Sciences (Beijing, China). Focal cerebral ischemia was induced by the right MCAO with a nylon monofilament suture. Briefly, the external and internal carotid arteries were dissected from the surrounding connective tissue. The MCA was occluded by advancing a suture with an expanded (heated) tip from the external carotid artery into the lumen of the internal carotid artery to block the origin of the MCA. After 60 min of occlusion, the suture was withdrawn to allow reperfusion for 2, 4, 8, or 24 hr. The body temperature was maintained at 37.0 ± 0.5 °C with a heated blanket. Animals were allowed free access to food and water after recovering from anesthesia. Sham-operated rats underwent identical surgical procedures without occlusion of MCA.

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4.2.

Drug treatment

Drug treatment was given to animals 30 min before the MCAO. PPARγ agonist rosiglitazone and antagonist GW9662 were from Cayman Chemical (Ann Arbor, MI, USA). 12/15-Lipoxygenase inhibitor baicalein was purchased from the Sigma-Aldrich (St. Louis, MO, USA). All drugs were dissolved in dimethylsulfoxide (DMSO, 0.1 ml/250 g animal weight) (Sundararajan et al., 2005; Victor et al., 2006) and injected intraperitoneally 30 min before the MCAO plus 24 hr of reperfusion. The final concentrations for PPARγ agonist and antagonist were 6 mg/kg (15 mg/ml DMSO) and 4 mg/kg (10 mg/ml DMSO), respectively. This dosing regimen was chosen to resemble that previously shown to confer neuroprotection by rosiglitazone and prevention PPARγmediated actions by GW9662 (Luo et al., 2006; Yi et al., 2008). For 12/15-lipoxygenase inhibitor studies, the lipoxygenase inhibitor, baicalein, was delivered in doses of 150 and 300 mg/kg (van Leyen et al., 2006). Control animals were injected with the equal volume DMSO. Six animals were observed in each treatment.

4.3.

Tissue preparation and nuclear extracts isolation

Tissues representing the ipsilateral cortices were harvested at 2, 4, 8, and 24 hr after the onset of reperfusion. The tissues were homogenized at 4 °C in phosphate-buffered saline (PBS) containing protease inhibitors (10 μg/ml soybean trypsin inhibitor, 10 μg/ml benzamadine, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 5 μg/ml antipain, 0.2 mM PMSF, and 0.1 mM ethylene diamine tetra-acetic acid). Each sample was homogenized and sonicated on ice. The homogenates were centrifuged at 3000 × g for 15 min at 4 °C, and the supernatant was obtained and used for total protein analysis. Nuclear and cytoplasmic fractions were separated with the Active Motif nuclear extract kit (Carlsbad, CA, USA). Isolated brain tissue were finely diced and homogenized in ice-cold buffer, and the manufacturer's instructions were followed. Both fractions were stored at −80 °C before analysis of the subcellular fractions of PPARγ.

4.4.

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secondary biotinylated antibody at a dilution of 1:200 (Vector Laboratories, Burlingame, CA, USA) for 1 hr at room temperature. After washing, sections were treated with avidin and biotinylated horseradish peroxidase complex (ABC Elite kit, Vector Laboratories). Immunostaining was visualized using 3, 3′-diaminobenzidine as a chromogen.

4.6.

Immunoflorescence staining

Frozen sections were stained for subcellular localization of PPARγ. Sections were permeabilized with 0.3% (vol./vol.) Triton X-100 in PBS for 30 min at room temperature and blocked with 1% (wt./vol.) BSA in PBS for 1 hr at 37 °C. After washing three times in PBS, sections were incubated in 1% BSA in PBS containing mouse antibody for PPARγ (1:50, Santa Cruz Biotechnology) overnight at 4 °C. Sections were washed six times in PBS and incubated in 1% BSA in PBS containing tetraethyl rhodamine isothiocyanate (TRITC)-labeled goat anti-mouse secondary antibody for 1 hr at 37 °C in the dark. Washing again for six times in PBS, sections were exposed to 4′,6-diamidino-2-phenylindole (DAPI) in the dark at room temperature for 10 min. The fluorescent images were observed under a fluorescent microscope.

4.7.

Statistical analyses

The experimental data were expressed as mean ± SEM and the SPSS 11.0 software package was used for data processing. Oneway analysis of variance (ANOVA) was used to compare the means of different groups. Comparisons between the two groups were conducted by t test. A P value < 0.05 was considered as statistically significant.

Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (No. 30500169 to L.S.).

Western blot analysis REFERENCES

Proteins were separated on 10% SDS–PAGE, then transferred onto nitrocellulose membrane. After blocking for 1 hr in 0.1% Tween 20/PBS (PBS-T) containing 5% fat-free milk, the blot was incubated with the primary antibody for PPARγ at a dilution of 1:200 (sc-7273, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight followed by re-incubation with horseradish peroxidase-conjugated secondary antibody for 1 hr at room temperature. After washing 3 × 10 min with PBS-T, the blot was visualized by chemiluminescence. The density of the bands was evaluated densitometrically using the program Quantity One 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA).

4.5.

Immunohistochemical detection

Paraffin-embedded sections of the brain were stained for PPARγ. Standard methodology of immunohistochemistry was used. Briefly, the brain samples were incubated overnight with a primary antibody for PPARγ at a dilution of 1:100 (Santa Cruz Biotechnology) at 4 °C, followed by incubation with the

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