Anti-glycative and anti-inflammatory effects of protocatechuic acid in brain of
mice treated by d-galactose
Shih-jei Tsaia,b, Mei-chin Yinc,d,*
aSchool of Medicine, Chung Shan Medical University, Taichung, Taiwan
bDepartment of Neurology, Chung Shan Medical University Hospital, Taichung, Taiwan cDepartment of Health and Nutrition Biotechnology, Asia University, Taichung City, Taiwan
dDepartment of Nutrition, China Medical University, Taichung City, Taiwan
Running title: neuro-protection of protocatechuic acid
*To whom correspondence should be addressed: Dr. Mei-chin Yin, Professor, Department of Nutrition, China Medical University, 91, Hsueh-shih Rd., Taichung City, Taiwan
Abstract
Protocatechuic acid (PCA) at 0.5, 1 or 2% was supplied to d-galactose (DG) treated mice for 8 wks. PCA intake at 2% increased its deposit in brain. DG treatment increased brain level of reactive oxygen species, protein carbonyl, carboxymethyllysine, pentosidine, sorbitol, fructose and methylglyoxal (P<0.05). PCA intake, at 1 and 2%, lowered brain level of these parameters (P<0.05). DG treatments enhanced activity and protein expression of aldose reductase (AR) and sorbitol dehydrogenase, as well as declined glyoxalase I (GLI) activity and protein expression (P<0.05). PCA intake at 1 and 2% reduced activity and protein expression of AR (P<0.05), and at 2% restored GLI activity and expression (P<0.05). DG injection also elevated cyclooxygenase (COX)-2 activity and expression, and increased the release of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha and prostaglandin E2 in brain (P<0.05). PCA intake decreased these cytokines (P<0.05), and at 1 and 2% suppressed COX-2 activity and expression (P<0.05). PCA intake at 1 and 2% also lowered DG-induced elevation in activity, mRNA expression and protein production of nuclear factor kappa B p65 (P<0.05). These findings suggest that the supplement of protocatechuic acid might be helpful for the prevention or alleviation of aging.
Abbreviation:
AGE, advanced glycation end product; AR, aldose reductase; COX-2, cyclooxygenase-2; CML, carboxymethyllysine; DG, D-galactose; GLI, glyoxalase I; GSH, glutathione; IL, interleukin; MDA, malonyldialdehyde; NF-κB, nuclear factor kappa B; PCA, protocatechuic acid; PGE2, prostaglandin E2; ROS, reactive oxygen species; SDH, sorbitol dehydrogenase; TNF, tumor necrosis factor.
1. Introduction
The brain accumulation of advanced glycation end products (AGEs) such as
carboxymethyllysine (CML) and pentosidine in patients with aging related neurodegenerative diseases such as Alzheimer's disease has been observed (Gironès et al., 2004). Aldose reductase (AR) and sorbitol dehydrogenase (SDH) are major enzymes in polyol pathway and responsible for AGEs formation (Takeuchi and Yamagishi, 2004). Contrarily, glyoxalase I (GLI) could metabolize physiological reactive -carbonyl compounds such as methylglyoxal, and decrease the available precursors for AGEs production (Auburger and Kurz, 2011). Age-dependent changes of GLI in human brain have been notified (Kuhla et al., 2007). Thus, any agent with the potential to reduce AGE generation, and regulate activity or expression of AR or GLI may attenuate glycative stress and delay the progression of neurodegenerative diseases. In addition, AGEs could up-regulate membrane-anchored receptor for AGEs (RAGE) in the brain. Engagement of RAGE by AGEs could activate transcription factor nuclear factor kappa B (NF-κB), which further evokes oxidative stress via increasing reactive oxygen species (ROS) formation and stimulates the transcription of genes encoding for cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines (Saha et al., 2008; Bansal et al., 2012), and leads to the excessive production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1. These events finally cause neuronal perturbation. Thus, any agent with the ability to suppress RAGE, NF-κB and COX-2, or decline the production of ROS and TNF-alpha may alleviate oxidative and inflammatory stress.
Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a phenolic compound found in many plant foods such as olives, Hibiscus sabdariffa (roselle) and Eucommia ulmoides (du-zhong) (Lin et al., 2003; Pacheco-Palencia et al., 2008). Our previous study reported that PCA
exhibited anti-oxidative and anti-inflammatory protection against diabetic deterioration in mice (Lin et al., 2009). Our another study further found that this compound attenuated renal glycative stress in diabetic mice via decreasing AGEs production (Lin et al., 2011). The results of Guan et al. (2011) revealed that PCA promoted neuronal differentiation and survival via mediating endogenous antioxidant enzymes. Zhang et al. (2010) indicated that PCA protected mice brain against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced dopamine loss. These previous studies implied that PCA was a potent neuro-protective agent; however, it remains unknown that this compound could provide anti-glycative and anti-inflammatory effects in brain.
D-galactose (DG)-induced neuro-pathological alteration has been used as an aging model because DG over-supply results in galactitol accumulation, excessive production of ROS and AGEs (Tsai et al., 2011). In our present study, DG injected mice were used to examine the neuro-protection of PCA. The effects of this compound at various doses upon brain level of ROS, AGEs and cytokines were measured. The impact of this agent upon activity, mRNA expression and/or protein production of AR, GLI, COX-2 and NF-κB were also determined in order to elucidate its possible action modes.
2. Materials and Methods
2.1. Materials
Protocatechuic acid (PCA, 99.5%) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The purity was further checked according to the method of Caccetta et al. (2000), and it was 99.3%. All chemicals used in these measurements were of the highest purity commercially available.
2.2. Animals and diet
(National Science Council, Taipei City, Taiwan). Mice were housed on a 12-h light:dark schedule; water and mouse standard diet were consumed ad libitum. The use of mice was reviewed and approved by China Medical University animal care committee (CMU-98-23-N). PCA at 0.5, 1 or 2 g was mixed with 99.5, 99 or 98 g powder diet (PMI Nutrition International LLC, Brentwood, MO, USA) to prepare 0.5, 1 or 2% PCA diet.
2.3. Experimental design
Mice at 19-20 wk old were used for experiments. Mice were divided into two groups, in which one group was treated with DG (100 mg/kg body weight) via i.p. daily injection. DG treated mice were further divided into four sub-groups, in which normal diet, 0.5, 1 or 2% PCA was supplied. All mice had free access to food and water at all times. Non-DG treated mice were divided into two sub-groups, and normal diet or 2% PCA was supplied. Consumed water volume and body weight were recorded weekly. After 8-wk DG treatment and PCA supplementation, mice were sacrificed by decapitation. Brain was quickly removed and at 0.1 g was homogenized on ice in 2 ml of phosphate buffer saline (PBS, pH 7.2). Protein concentration of filtrate was determined by a commercial assay kit (Pierce Biotechnology Inc., Rockford, IL, USA) with bovine serum albumin as standard. In all experiments, the sample was diluted to a final concentration of 1 mg protein/ml.
2.4. Content of PCA
PCA content in brain was measured according to a GC-MS method described in Caccetta et al. (2000). Briefly, lyophilized sample was mixed with 1-hydroxy-2-naphthoic acid, as internal standard, and followed by acidification with 6 N HCl and extraction with ethyl acetate and 5% NaHCO3. The ethyl acetate extract was dried and derivatized with equal amount of
5890 gas chromatograph (Hewlett-Packard, Palo Alto, CA, USA) equipped with a HP 5970 series mass-selective detector and a HP-1 cross-linked methyl silicone column (12 m x 0.20 mm, 0.33 µm film thickness). The electron-impact mode was used for mass spectrometer, and the mass-to-charge ratio of PCA was 370. The limit of detection and quantification was 0.2 and 0.8 ng/ml, respectively.
2.5. Measurement of CML and pentosidine
For CML determination, 50 mg brain was homogenized and digested with proteinase K (1 mg/ml) for 3 h at 37 °C, and reaction was stopped by 2 mmol/l phenylmethylsulfonyl fluoride. CML was immunochemically determined with a competitive ELISA kit (Roche Diagnostics, Penzberg, Germany) using the CML-specific monoclonal antibody 4G9,and calibration with 6-(N-carboxymethylamino)caproic acid. Absorbance was read in a microtiter ELISA plate reader (Bio-Rad, Hercules, CA, USA) at 405 nm (reference 603 nm). Intra- and interassay variability of this assay were 5.2 and 6.1%, respectively. Pentosidine level was analyzed by a HPLC equipped with a C18 reverse-phase column and a fluorescence detector according to the method described in Miyata et al. (1996). Briefly, sample was lyophilized and acid hydrolyzed in 500 µl 6 N HCl for 16 hat 110 °C in screw-cap tubes purged with nitrogen. After neutralization with NaOH and diluted with PBS, sample was usedfor HPLC measurement.
2.6. Determination of sorbitol and fructose
Brain sample was homogenized with PBS (pH 7.4) containing U-[13C]-sorbitol as an internal standard. After precipitating protein by ethanol, the supernatant was lyophilised. The content of sorbitol and fructose in lyophilized sample was determined by liquid chromatography with tandem mass spectrometry, according to the method of Guerrant and Moss (1984).
2.7. Determination of malonyldialdehyde (MDA), ROS, protein carbonyl and glutathione (GSH)
Intracellular ROS level was determined using a oxidation sensitive dye, 2',7'-dichlorofluorescein diacetate (DCFH-DA) according to the method of Olalekan et al. (2011). Briefly, 500 l homogenate was mixed with 500 l of 2 mg/ml DCFH-DA for 30 min at 37 °C. Fluorescence was measured at 488 nm excitation and 525 nm emission using a fluorescence plate reader. Results are expressed as relative fluorescence unit (RFU) per mg protein. Protein carbonyls were determined with the Zentech PC kit (BioCell, Auckland, New Zealand). Briefly, 50 µl sample was mixed with a 200 l dinitrophenylhydrazine (DNP) solution. The adsorbed DNP–protein was reacted with an anti-DNP-biotin antibody, and followed by reacting with streptavidin-linked horseradish peroxidase probe and chromatin reagent. The absorbance at 450 nm was measured. The concentration of reduced GSH was determined by a commercial colorimetric GSH assay kit (OxisResearch, Portland, OR, USA).
2.8. Activity of AR, SDH and GLI
The method of Nishinaka and Yabe-Nishimura (2001) was used to measure AR activity by monitoring the decrease in absorbance at 340 nm due to NADPH oxidation. SDH activity was assayed according to the method of Ulrich (1974) by mixing 100 l homogenate, 200 l NADH (12 mM) and 1.6 ml triethanolamine buffer (0.2 M, pH 7.4), and monitoring the absorbance change at 365 nm. The method of McLellan and Thornalley (1989) was used to assay GLI activity. Brain tissue at 50 mg was homogenized in 1 ml NaPB containing 0.02% Triton X-100. After centrifugation at 20,000 xg for 20 min at 4 °C, supernatant was collected. GLI activity was assayed by monitoring the increase in absorbance at 240 nm due to the formation of S-(D)-lactoylglutathione.
2.9. Cytokines and prostaglandin E (PGE)2 determination
M NaCl, 1 mM ethylenediaminetetraacetic acid, 0.01% Tween 80, 1 mM phenylmethylsulfonyl fluoride, and centrifuged at 9000 xg for 30 min at 4 °C. The resultant supernatant was used for cytokine determination. The levels of IL-1beta, IL-6 and TNF-alpha were measured by ELISA using cytoscreen immunoassay kits (BioSource International, Camarillo, CA, USA). The level of PGE2 was determined using a PGE2 EIA kit (Cayman Chemical Co., Ann Arbor, MI, USA).
2.10. NF-κB and COX-2 activity assay
Nuclear protein extracts were prepared from fresh brain tissues, and 10 g nuclear protein extract was used for determining NF-κB activity using antibody against NF-κB p50 and NF-κB p65 according to the manufacturer's guidelines of TransFactor kit (Clontech Laboratories Inc, Mountain View, CA, USA). After color development, absorbance was measured at 655 nm with a microtiter plate reader. Values are expressed as relative optical density (OD) per mg protein. COX-2 activity was assayed by a commercial assay kit (Cayman Chemical Co., Ann Arbor, MI, USA), and colorimetrically monitoring oxidized N,N,N',N'-tetramethyl-p-phenylenediamine at 590 nm.
2.11. Real time polymerase chain reaction for mRNA expression
Total RNA was isolated using Trizol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA). One g RNA was used to generate cDNA, which was amplified using Taq DNA polymerase. PCR was carried out in 50 l of reaction mixture containing Taq DNA polymerase buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl, 200 mM dNTP, 2.5 mM MgCl2, 0.5 mM of each primer) and 2.5 U Taq DNA polymerase. The specific oligonucleotide primers of targets are shown in Table 1. The cDNA was amplified under the following reaction conditions: 95 ºC for 1 min, 55 ºC for 1 min, and 72 ºC for 1 min. 28 cycles were performed for glyceraldehyde-3-phosphatedehydrogenase (GAPDH, the housekeeping gene) and 35 cycles were performed for
others. Generated fluorescence from each cycle was quantitatively analyzed by using the Taqman system based on real-time sequence detection system (ABI Prism 7700, Perkin-Elmer Inc., Foster City, CA, USA). In this study, mRNA level was calculated as percentage of the control group.
2.12. Western blot analysis
Brain tissue was homogenized in buffer containing 0.5% Triton X-100 and protease-inhibitor cocktail (1:1000, Sigma-Aldrich Chemical Co., St. Louis, MO, USA). This homogenate was further mixed with buffer (60 mM Tris-HCl, 2% SDS, and 2% β-mercaptoethanol, pH 7.2), and boiled for 5 min. Sample at 40 μg protein was applied to 10% SDS-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA) for 1 h. After blocking with a solution containing 5% nonfat milk for 1 h to prevent non-specific binding of antibody, membrane was incubated with mouse anti-RAGE (1:2000), anti-AR (1:1000), anti-SDH (1:1000), anti-GLI (1:2000), anti-NF-κB p50 (1:2000), anti-NF-κB p65 (1:2000), anti-COX-2 (1:1000) monoclonal antibody (Boehringer-Mannheim, Indianapolis, IN, USA) at 4 ºC overnight, and followed by reacting with horseradish peroxidase-conjugated antibody 3.5 h at room temperature. The blot was imaged by autoradiography, and quantified by densitometric analysis. Results were normalized to GAPDH, and given as arbitrary units (AU).
2.13. Statistical analysis
The effect of each measurement was analyzed from 10 mice (n = 10/group). All data were expressed as mean ± standard deviation (SD). Statistical analysis was done using one-way analysis of variance, and post-hoc comparisons were carried out using Dunnett's t-test. Statistical significance is defined as P< 0.05.
DG injection and PCA intake did not significantly affect body weight and brain weight (data not shown, P>0.05). As shown in Table 2, PCA intake at 2% increased its deposit in brain of mice with or without DG treatment. DG treatment decreased brain GSH content and increased brain level of ROS and protein carbonyl (P<0.05). PCA intake reduced ROS and protein carbonyl levels, and at 1 and 2% retained GSH content (P<0.05). DG treatment increased the production of CML, pentosidine, sorbitol, fructose and methylglyoxal (Table 3,
P<0.05). PCA intake, at 1 and 2%, lowered brain level of these parameters (P<0.05). As shown
in Table 4 and Figure 1, DG treatments enhanced activity, mRNA expression and protein production of AR and SDH, as well as declined GLI activity and protein expression (P<0.05). PCA intake at 1 and 2% reduced activity, mRNA expression and protein production of AR (P<0.05), and at 2% restored GLI activity and expression (P<0.05). PCA treatments did not affect SDH (P>0.05).
DG injection also elevated COX-2 activity, and increased the release of IL-1beta, IL-6, TNF-alpha and PGE2 in brain (Table 5, P<0.05). PCA intake lowered COX-2 activity and these cytokines (P<0.05), in which dose-dependent effect was in decreasing TNF-alpha (P<0.05). As shown in Figure 2, PCA treatments failed to affect RAGE, but at 1 and 2% declined COX-2 mRNA and protein expression (P<0.05). PCA intake at 1 and 2% reduced DG-induced elevation in activity, mRNA expression and protein level of NF-κB p65, not NF-κB p50 (Figure 3,
P<0.05).
4. Discussion
In our present study, PCA intake at 2% increased its deposit in brain of mice with or without DG treatments. Zhang et al. (2011) reported that PCA could be detected in rat brain tissues after oral administration of danshen extract. Those studies indicated that PCA could penetrate blood
brain barrier and exerted neuro-protective activities. Furthermore, we found that PCA intake lowered production of ROS, inflammatory cytokines and AGEs, declined activity of COX-2 and AR, as well as suppressed NF-κB activation in brain of DG-treated mice. These findings suggest that PCA, via its anti-oxidative, anti-inflammatory and anti-glycative actions, was an effective agent to delay aging. The anti-oxidative activities of PCA in rat brain have been reported (Shi et al., 2006). Our present study also revealed PCA effectively reduced ROS and protein carbonyl formation, as well as retained GSH content in brain of DG-treated mice. These results indicated that this compound could protect brain against DG-induced oxidative injury. ROS is a stimulator for generation of cytokines and AGEs, and even NF-κB activation (Mukherjee et al., 2005; Wu et al., 2009). Thus, the decreased ROS in brain of PCA-treated mice benefited the alleviation of inflammatory and glycative stress.
CML and pentosidine, two AGEs, have been implied in aging associated pathological development (Jono et al., 2002; Gironès et al., 2004). AR and SDH are two key enzymes in polyol pathway. Enhanced activity and expression of these enzymes facilitate the production of sorbitol and fructose, which in turn promote AGEs formation and glycative stress. Our present study found that PCA declined activity and protein expression of AR, which subsequently lowered the production of sorbitol and fructose. Thus, the decreased CML and pentosidine could be partially explained. These findings support that this compound could inhibit brain AGEs formation via suppressing polyol pathway. On the other hand, AGEs level could be diminished via elevating activity or expression of GLI to catalyze the detoxification of alpha-oxoaldehydes to corresponding aldonic acids (Vander, 2008). Methylglyoxal, the most reactive AGE precursor, is a physiological substrate of GLI (Kilhovd et al., 2003). We notified that PCA at 2% was able to restore the activity and protein expression of GLI, which in turn favored methylglyoxal metabolism. Since the available methylglyoxal was reduced, it seems reasonable
to detect lower AGEs. Obviously, the anti-glycative activity of PCA also involved its up-regulation upon GLI.
Suppressing TNF-alpha response has been considered as a promising target for anti-inflammatory treatment in neuro-degenerative diseases (Saha et al., 2008). Our present study found that PCA intake effectively diminished over-expression of TNF-alpha and IL-6, which consequently mitigated inflammatory stress in brain. COX-2 is the rate-limiting enzyme for synthesis of PGE2, a pro-inflammatory mediator. Enhanced COX-2 expression facilitated microglial activation of substantia nigra pars compacta, which accelerated the loss of neuro-transmitters such as dopamine and promoted the release of inflammatory cytokines including IL-1beta and TNF-alpha (Liu and Hong, 2003; Teismann et al., 2003). In our present study, elevated expression and activity of COX-2 in brain from DG-treated mice indicated that COX-2 was responsible for increased production of PGE2 and inflammatory cytokines in DG-induced aging. However, PCA intake suppressed COX-2 expression and abated COX-2 activity, which in turn diminished brain production of PGE2 and inflammatory cytokines. These findings suggest that the anti-inflammatory effects of PCA in brain were partially due to this compound repress COX-2 and PGE2.
The interaction of RAGE with AGEs activates NF-κB and other signaling pathways, which favor glycative and inflammatory reactions in aging progression (Gao et al., 2008; Miller et al., 2008). In our present study, the raised mRNA expression and protein production of RAGE and NF-κB in brain of DG-treated mice implied that the interaction of RAGE and its ligands, as well as NF-κB activation were involved in DG-induced aging. We notified PCA treatments failed to affect RAGE expression, but substantially suppressed NF-κB activation and expression based on our RT-PCR and western blot data. Obviously, the observed anti-aging effects of PCA were RAGE independent. It is highly possible that the less available AGEs due to PCA’s anti-glycative
activities led to less ligands to interact with RAGE, which declined the subsequent NF-κB activation. Activated NF-κB regulates the gene expression of many mediators participated in inflammatory reactions, such as TNF-alpha and COX-2 (Munhoz et al., 2008; Kaushik et al., 2010). Therefore, NF-κB has been considered as a target for novel anti-inflammatory therapy. Our present study found that PCA supplementation suppressed the activity, mRNA expression and protein production of NF-κB p65, which subsequently reduced activity and expression of COX-2. These findings supported that the anti-inflammatory effects of PCA were NF-κB-dependent. PCA failed to counteract DG enhanced RAGE expression. This result implied that other pathways such as the interaction of RAGE and amyloid-beta peptide might be involved in DG induced neurotoxicity. PCA is a natural phenolic compound, and could be absorbed in human large intestine after microbial metabolization (Vitaglione et al., 2007). Therefore, the application of this compound against aging seems feasible. However, further study is necessary to ensure the safety of this compound at these doses before it is applied for human.
In summary, the intake of protocatechuic acid increased its deposit in brain of senescent mice. Protocatechuic acid treatments at 2% protected brain against glycative and inflammatory stresses via decreasing AGEs, PGE2, inflammatory cytokines and ROS levels. This compound also repressed brain activity and/or expression of AR, COX-2 and NF-κB. Therefore, the supplement of protocatechuic acid or foods rich in this compound might be helpful for the prevention or alleviation of aging.
Conflict of interest statement None
Acknowledgement
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Table 1.
Forward and reverse primers for real time PCR analysis.
Target forward reverse
AR 5’-CCC AGG TGT ACC AGA ATG AGA-3’ 5’-TGG CTG CAA TTG CTT TGA TCC-3’
SDH 5’-TGG GAG CTG CTC AAG TTG TG-3′ 5′-GGT CTC TTT GCC AAC CTG GAT-3′
GLI 5’-CGT GAG ACA GCA AGC AGC TAG A-3’ 5’-ACC ATG AGG CAT AGG CAT ACC C-3’
RAGE 5’-CCA TCC TAC CTT CTC CTG-3’ 5’-AGC GAC TAT TCC ACC TTC-3’
NF-κB p65 5’-GCG TAC ACA TTC TGG GGA GT-3’ 5’-CCG AAG CAG GAG CTA TCA AC-3’
NF-κB p50 5’-GGA GGC ATG TTC GGT AGT GG-3’ 5’-CCC TGC GTT GGA TTT CGT G-3’
COX-2 5’-CCA GCA GGC TCA TAC TGA TAG GA-3’ 5’-GCA GGT CTG GGT CGA ACT TG-3’
Table 2.
Level of PCA, GSH, ROS and protein carbonyl in brain from mice with or without DG treatment and consumed PCA. Data are meanSD (n = 10). PCA nmol/mg protein GSH ng/mg protin ROS RFU/mg protein protein carbonyl pmol/mg protein Control -* 894d 0.200.06a 13.70.9a PCA 2% 0.240.09b 916d 0.190.04a 12.91.0a DG+PCA 0% - 493a 1.930.24d 148.17.5d DG+PCA 0.5% - 536a 1.480.16c 116.75.4c DG+PCA 1% 0.080.05a 664b 1.020.19b 109.46.3c DG+PCA 2% 0.130.07a 755c 0.890.13b 73.04.8b
*Means too low to be detected.
Table 3.
Level of CML, pentosidine, sorbitol, fructose and methylglyoxal in brain from mice with or without DG treatment and consumed PCA. Data are meanSD (n = 10).
CML pmol/mg protein Pentosidine pmol/mg protein Sorbitol nmol/mg protein Fructose nmol/mg protein Methylglyoxal nmol/mg protein Control 83a 0.330.09a 3.040.23a 10.10.5a 1.020.16a PCA 2% 94a 0.210.05a 3.100.16a 8.60.7a 0.970.11a DG+PCA 0% 1029d 1.620.12c 8.150.27c 70.24.3c 9.060.41c DG+PCA 0.5% 976d 1.530.16c 7.900.19c 67.53.0c 8.550.38c DG+PCA 1% 7310c 1.050.10b 6.410.26b 54.61.9b 7.160.34b DG+PCA 2% 508b 0.840.13b 6.160.14b 49.72.1b 6.600.31b
Table 4.
Activity of AR, SDH and GLI in brain from mice with or without DG treatment and consumed PCA. Data are meanSD (n = 10). AR nmol/min/mg protein SDH U/g protein GLI nmol/min/mg protein Control 1.540.21a 3.340.40a 29318c PCA 2% 1.460.19a 3.180.36a 31815c DG+PCA 0% 4.160.31d 7.870.57b 9710a DG+PCA 0.5% 3.950.24d 7.720.42b 1058a DG+PCA 1% 3.060.17c 7.600.37b 11312a DG+PCA 2% 2.250.20b 7.540.61b 14914b
Fig. 1. mRNA expression (A) and protein production (B) of brain AR, SDH and GLI in mice with or without DG treatment and consumed PCA. Data are meanSD (n = 10). a-dMeans among bars without a common letter differ, P<0.05.
B AR SDH GLI GAPDH DG - - + + + + PCA - 2 - 0.5 1 2
Table 5.
Brain IL-1beta, IL-6, TNF-alpha and PGE2 levels, and COX-2 activity in mice with or without DG treatment and consumed PCA. Data are meanSD (n = 10).
IL-1beta pg/mg protein IL-6 pg/mg protein TNF-alpha pg/mg protein PGE2 pg/g protein COX-2 U/mg protein Control 0.920.14a 1.050.14a 1.240.3a 98583a 0.230.06a PCA 2% 0.880.15a 0.940.10a 1.300.4a 934107a 0.190.08a DG+PCA 0% 3.190.33d 3.290.25d 4.950.62e 2297151d 1.870.12d DG+PCA 0.5% 2.630.24c 2.550.31c 4.070.45d 210696d 1.700.07d DG+PCA 1% 2.470.17c 2.310.17c 3.460.61c 1602115c 1.260.10c DG+PCA 2% 1.900.12b 1.870.14b 2.230.30b 124569b 0.810.09b
Fig. 2. mRNA expression (A) and protein level (B) of COX-2 and RAGE in brain of mice with or without DG treatment and consumed PCA. Data are meanSD (n = 10). a-dMeans among bars without a common letter differ, P<0.05.
B COX-2 RAGE GAPDN DG - - + + + + PCA - 2 - 0.5 1 2
Fig. 3. Activity (A), mRNA expression (B) and protein level (C) of NF-B p65 and NF-B p50
in brain from mice with or without DG treatment and consumed PCA. Data are meanSD (n = 10). a-dMeans among bars without a common letter differ, P<0.05.
C NF-κB p65 F-B p50 GAPDH DG - - + + + + PCA - 2 - 0.5 1 2
