Ginsenoside Rd attenuates early oxidative damage and sequential inflammatory response after transient focal ischemia in rats

Neurochemistry International 58 (2011) 391–398

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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint

Ginsenoside Rd attenuates early oxidative damage and sequential inflammatory response after transient focal ischemia in rats Ruidong Ye a,1, Qianzi Yang b,1, Xiangwei Kong c,1, Junliang Han a,1, Xiao Zhang a, Yunxia Zhang a, Ping Li d, Juanfang Liu a, Ming Shi a, Lize Xiong c, Gang Zhao a,* a

Department of Neurology, Xijing Hospital, the Fourth Military Medical University, Xi’an, China Department of Anaesthesiology, Xijing Hospital, the Fourth Military Medical University, Xi’an, China College of Stomatology, the Fourth Military Medical University, Xi’an, China d Department of Orthopedics, Xijing Hospital, the Fourth Military Medical University, Xi’an, China b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 September 2010 Received in revised form 15 December 2010 Accepted 17 December 2010 Available online 24 December 2010

We previously found that ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, attenuates neuronal oxidative damage in vitro induced by hydrogen peroxide and oxygen–glucose deprivation. In this study, we sought to investigate the potential protective effects and associated mechanisms of Rd in a rat model of focal cerebral ischemia. Rats administered with Rd (0.1–200 mg/kg) or vehicle was subjected to transient middle cerebral artery occlusion. Rd at the dose of 10–50 mg/kg significantly reduced the infarct volume and improved the long-term neurological outcome up to 6 weeks after ischemia. To evaluate the underlying mechanisms, in vivo free radical generation was monitored using microdialysis, oxidative DNA damage was identified by 8-hydroxy-deoxyguanosine immunostaining, oxidative protein damage was identified by the assessment of protein carbonyl and advanced glycosylation end products, and lipid peroxidation was estimated by determining the malondialdehyde and 4-hydroxynonenal formations. Microdialysis results displayed a prominent inhibitory effect of Rd on the hydroxy radical formation trapped as 2,3- and 2,5-DHBA. Early accumulations of DNA, protein and lipid peroxidation products were also suppressed by Rd treatment. Although Rd partly preserved endogenous antioxidant activities in the ischemic penumbra, in sham rats without stroke, endogenous antioxidant activities were not affected by Rd. Furthermore, we assayed sequential inflammatory response in a later phase after ischemia. Rd significantly eliminated inflammatory injury as indicated by the suppression of microglial activation, inducible nitric oxide synthase and cyclooxygenase-2 expression. Collectively, these findings demonstrated that Rd exerts neuroprotection in transient focal ischemia, which may involve early free radicals scavenging pathway and a late anti-inflammatory effect. ß 2010 Elsevier Ltd. All rights reserved.

Keywords: Ginsenoside Rd Neuroprotection Oxidative stress Stroke

1. Introduction Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), has been used as a tonic to treat a wide variety of disorders in China for millennia. Extensive studies have proved that the molecular components responsible for the pharmacologic effects of ginseng are ginseng saponins, namely ginsenosides. Currently, over 30 ginsenosides have been identified and isolated from ginseng. Dammar-24(25)-ene-3b,12b,20(S)-triol-(20-O-b-D-glucopyranosyl)-3-O-b-D-glucopyranosyl-(1 ! 2)-b-D-glucopyranoside (ginsenoside Rd, Rd) is one of the major active components of

* Corresponding author. Tel.: +86 29 84775361. E-mail addresses: [email protected] (R. Ye), [email protected] (G. Zhao). 1 These authors contributed equally to this work. 0197-0186/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2010.12.015

ginsenosides. It has been demonstrated to have a number of pharmacologic actions such as inhibiting Ca2+ influx through receptor-and store-operated Ca2+ channels (Guan et al., 2006), preventing glutamate-induced apoptosis in rat cortical neurons (Li et al., 2010), and significantly reducing the 3-nitropropionic acidinduced motor impairment and cell loss in the striatum (Lian et al., 2005). Ginsenoside Rd is also one of the major ingredients in the total saponins from Panax notoginseng and its content in the total notoginseng saponins is 4.07%, making it inexpensive for practical use. Overwhelming evidence indicated that free radicals play a pivotal role in the ischemic cascade in a consecutive 2-phase pattern (Lipton, 1999). In addition to the direct cytotoxic effects of oxidative DNA, lipid and protein damage occurring immediately after ischemia/reperfusion, the burst of free radicals also induces the formation of inflammatory mediators through redox-mediated

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signaling pathways, leading to post-ischemia/reperfusion inflammatory injury (Zhang et al., 2005). Therefore, the control of these 2 impacts of oxidative damage is important to achieve neuroprotection. There is evidence indicating that Rd presents antioxidant effects in kidney injury model (Yokozawa et al., 1998; Yokozawa and Owada, 1999) and in senescence-accelerated mice (Yokozawa et al., 2004). In the CNS, Rd was shown to be effective in decreasing reactive oxygen species (ROS) formation in cultured astrocytes (Lopez et al., 2007). Consistently, we previously reported that Rd protects PC12 cells from hydrogen peroxide-induced oxidative damage (Ye et al., 2008). Rd has also been noted to mitigate neuroinflammation (Lin et al., 2007) and nitric oxide overproduction (Choi et al., 2003). More recently, we found that Rd attenuates neuronal oxidative damage induced by oxygen–glucose deprivation (OGD) (Ye et al., 2009), an in vitro model of cerebral ischemia. However, the efficacy of Rd has not been established in animal models of stroke. Thus, in this study, we examined the potential protective effects of Rd in a rat model of transient middle cerebral artery occlusion (MCAO). Effects of Rd on the oxidative damage at an early period and sequential inflammatory response were also assessed. 2. Materials and methods 2.1. Focal cerebral ischemia A total of 161 male Sprague–Dawley rats weighing 270–320 g were used in this study. Animal protocols were approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University. The focal cerebral ischemia was induced by 2-h MCAO as described earlier (Wang et al., 2008, 2009). Briefly, animals were anesthetized with a mixture of isoflurane (1.5–2%), oxygen, and nitrogen. A 4-0 nylon monofilament coated with poly-Llysine was introduced through the internal carotid artery to occlude the origin of the MCA. We maintained temperature (37.0  0.5 8C) using a thermostatically controlled heating blanket connected to a thermometer probe in the rectum, and, at the same time, monitored physiological parameters including blood gases (pCO2 and pO2), pH, glucose level and blood pressure. The induction of focal cerebral ischemia was verified with laser Doppler flowmetry (PeriFlux 5000; Perimed AB, Sweden). A drop in regional cerebral blood flow (CBF) below 30% from baseline after the insertion of the filament was considered to be sufficient for induction of focal cerebral ischemia. Control animals were subjected to the same surgical procedures except that the suture was not advanced into the MCA. Rd with a purity of 98% was obtained from Tai-He Biopharmaceutical Co. Ltd. (Guangzhou, China). Rd stock solutions were prepared in saline containing 10% 1,3propanediol (v/v). Rd at concentrations ranging from 0.1 to 200 mg/kg or vehicle was applied intraperitoneally 30 min before MCAO. 2.2. Infarct size assessment On post-operative day (POD) 1, animals were decapitated and 2-mm thick coronal sections from throughout the brain were stained with 2% 2,3,5triphenyltetrazolium chloride (TTC, Amresco Inc., USA) to evaluate the infarct volume (Isayama et al., 1991). To compensate for the effect of brain edema, the corrected infarct volume was calculated as previously described: corrected infarct area = left hemisphere area  (right hemisphere area  infarct area) (Lin et al., 1993; Schabitz et al., 2003). Infarct volume was manually quantified using ImageJ software and expressed as a percentage of the contralateral structure.

microdialysis samples were continuously collected into capped microvials (containing 10 ml of 0.4 mM perchloric acid for stabilization) at 20 min intervals and measured immediately by high-performance liquid chromatographic-electrochemical (HPLC-EC) procedure. Three samples were collected as baseline values at the end of 2 h equilibration period. The HPLC-ECD system included a pump (PM-92, BAS, West Lafayette, IN, USA), a ODS analytical column (3 mm, 100 mm  3 mm), and ECD (Epsilon, BAS, West Lafayette, IN, USA). 1 l mobile phase consist of 5.3 g of chloroacetic acid, 0.612 g of EDTA, 1.72 g of camphorsulfonic acid, 4.6 g of sodium acetate and 100 ml methylalcohol (pH 3.5). The mobile phase was delivered at 300 ml/min. DHBA were detected with a carbon electrode held at +600 mV relative to a Ag/AgCl reference electrode. Level changes for all measured chemicals are expressed as percent variations from the mean baseline value. 2.5. Western blot analysis Total cell lysates from target brain tissue were extracted and prepared by using the RIPA lysis buffer (Beyotime, China) containing 0.5 mM PMSF on ice. Western blot was performed as described previously (Wang et al., 2008, 2009). Briefly, after addition of sample loading buffer, protein samples were electrophoresed on a 10% or 8% SDS-PAGE and subsequently transferred to nitrocellulose membrane (Millipore, USA). The membrane was incubated in fresh blocking buffer (0.1% Tween 20 in Tris-buffered saline, pH 7.4, containing 5% nonfat dried milk) at room temperature for 30 min and then probed with the primary antibody in blocking buffer at 4 8C overnight. We used primary antibodies to 4-hydroxynonenal (4-HNE; goat, Chemicon, USA, 1:3000), advanced glycosylation end products (AGEs; rabbit, Biosynthesis Biotechnology, China, 1:400), iNOS (rabbit, Chemicon, USA, 1:1000), cyclooxygenase-2 (COX-2; rabbit, Abcam, UK, 1:500) and b-actin (rabbit, Biosynthesis Biotechnology, China, 1:400). The membrane was washed three times for 5 min each using PBST (PBS and 0.1% Tween 20). After that it was incubated in the appropriate HRP-conjugated secondary antibody at room temperature for another 1 h and washed again three times in PBST buffer. Specific signals of proteins were visualized by chemiluminescence using the ECL western blotting detection system (GE Healthcare, UK). For quantitative analysis, the ratio for specific signals of protein (relative density of the signal) and the constitutively expressed b-actin protein was calculated to normalize for loading and transfer artifacts introduced in western blotting. 2.6. Immunohistochemistry Immunohistochemistry was performed on 20 mm-thick free-floating coronal sections, which were prepared as described previously (Yang et al., 2010a). After incubation in 0.5% H2O2 followed by normal goat serum to avoid nonspecific immunoreactions, the sections were stained overnight at 4 8C using primary antibodies against 8-hydroxy-deoxyguanosine (8-OHdG; mouse, JaICA, Japan, 1:50) and ionized calcium-binding adapter molecule 1 (Iba-1; rabbit, Wako Pure Chemicals, Japan, 1:1000). After incubation, they were washed with PBS and incubated in biotinylated goat anti-mouse or goat anti-rabbit secondary antibodies (1:500; Sigma–Aldrich, USA) for 2 h at room temperature, followed by rinsing in PBS and incubation in avidin–biotin–peroxidase complex (1:500; Sigma–Aldrich, USA) for 2 h. After a final wash, sections were reacted for peroxidase enzyme activity using 3,3diaminobenzidine (0.5 mg/ml, Sigma–Aldrich, USA). The specificity of immunolabeling was verified by controls in which the primary antibody was omitted. 2.7. Malondialdehyde assay The ischemic penumbra in the ipsilateral hemisphere to the occlusion was dissected on POD 1 as described (Ashwal et al., 1998; Li et al., 2008). The tissues were sonicated with 10 ml of RIPA buffer with 0.1 mg/ml PMSF and 5 mM betahydroxytoluene per gram of tissue for 15 s at 40 V over ice. Then tissues were centrifuged at 1600  g for 10 min at 4 8C. The supernatant was used to measure malondialdehyde (MDA) according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China).

2.3. Behavioral tests 2.8. Protein carbonyl assay Modified neurological severity scores (mNSS), a composite of motor, sensory, reflex, and balance tests (Chen et al., 2001), were performed before MCAO and at POD 1, 3, 7, 14, 21, 28 and 42 by two investigators (Q.Y. and X.K.) who were blinded to the experimental groups. All tests were conducted in the light phase. 2.4. Microdialysis Free radical formation in the brain in vivo was measured by the method previously described in detail (Teismann and Ferger, 2000; Yang et al., 2010b). The method relies on the fact that hydroxyl free radicals react with salicylic acid to generate 2,3- and 2,5-dihydroxybenzoic acids (2,3- and 2,5-DHBA) and this reaction is utilized by measuring the formation of 2,3- and 2,5-DHBA in the dialysate of a microdialysis probe perfused with salicylic acid. Briefly, a microdialysis probe (BAS MD-2204, 4 mm membrane) was inserted through the cannula guide into the right striatum. Artificial cerebrospinal fluid incorporated with salicylate (5 mM) was perfused at 2 ml/min by means of a microinjection pump (BeeHive BAS, USA). The

The ischemic penumbra in the ipsilateral hemisphere to the occlusion was dissected on POD 1 as described (Ashwal et al., 1998; Li et al., 2008). Brain tissues were sonicated with 10 ml of 0.1 M PBS containing 10 mM EDTA per gram of tissue over ice. Then tissues were centrifuged at 10,000  g for 15 min at 4 8C. The supernatant was removed and stored on ice. To avoid undesired integration of the nucleic acid, the samples were incubated with streptomycin sulfate at a final concentration of 1% for 15 min and then centrifuged at 6000  g for 10 min at 4 8C. The supernatant was used to determine protein carbonyl content with an ELISA kit (Cell Biolab, USA). Total protein concentration was determined using Enhanced BCA Protein Assay Kit (Beijing Biosynthesis Biotechnology, China). 2.9. Determination of antioxidant enzyme activities and glutathione level The ischemic core and penumbra in the ipsilateral hemisphere to the occlusion, and contralateral brain tissue were dissected on POD 1 as described (Ashwal et al.,

R. Ye et al. / Neurochemistry International 58 (2011) 391–398

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Table 1 Physiological parameters.

Rd 0 mg/kg Preischemia During ischemia Rd 0.1 mg/kg Preischemia During ischemia Rd 1 mg/kg Preischemia During ischemia Rd 10 mg/kg Preischemia During ischemia Rd 50 mg/kg Preischemia During ischemia Rd 200 mg/kg Preischemia During ischemia

Rectal temperature (8C)

pH

PaO2 (mmHg)

PaCO2 (mmHg)

Glucose (mg/dL)

MAP (mmHg)

37.2  0.11 37.3  0.07

7.37  0.03 7.39  0.05

125  7.3 114  5.2

38  1.2 38  1.3

136  19 143  18

102  4 97  3

37.0  0.05 37.3  0.07

7.40  0.05 7.38  0.06

129  7.9 122  5.3

38  0.4 41  1.3

132  17 124  10

94  3 96  7

36.9  0.04 37.1  0.06

7.40  0.04 7.35  0.06

119  6.7 126  3.2

39  0.5 40  1.3

136  12 128  15

102  3 101  6

36.8  0.08 37.1  0.06

7.38  0.06 7.42  0.02

128  6.9 118  4.9

39  1.0 39  0.8

149  13 132  17

101  7 107  4

37.1  0.09 37.3  0.10

7.37  0.02 7.40  0.04

126  6.0 128  6.2

40  1.2 38  1.1

151  15 137  15

99  5 106  7

36.7  0.07 37.3  0.11

7.43  0.04 7.35  0.07

130  8.0 122  6.5

38  1.5 41  1.0

146  11 132  19

105  7 96  4

MAP: mean arterial pressure.

1998; Li et al., 2008). The samples were then homogenized in cold saline with a weight-to-volume ratio of 1:9. The homogenate was centrifuged at 3000 rpm for 10 min at 4 8C and the supernatant was assayed for protein concentration by Enhanced BCA Protein Assay Kit (Beijing Biosynthesis Biotechnology, China). The enzyme activities of superoxide dismutase (SOD 1 and 2), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione content (reduced glutathione (GSH) and glutathione disulfide (GSSG)) in tissue homogenate were measured according to the technical manual of the detection kits (Cayman Chemical, USA). 2.10. Statistical analysis All results are presented as average with SEM. For histological quantification, micrographs were made using a 40 objective lens on an Olympus BX51 microscope equipped with a digital imaging system. Sections from at least 3 animals were analyzed for each data point. Behavioral test and microdialysis results were analyzed with repeated-measures analysis of variance, followed by Tukey HSD post hoc test. Antioxidant enzyme activities were analyzed using unpaired ttest. Other results were analyzed with one-way analysis of variance followed by Tukey HSD post test for multiple comparisons. We used SPSS software for statistical tests. Statistical significance was established at P < 0.05.

3.3. Rd reduces 2,3- and 2,5-DHBA in the in the dialysate during ischemia and reperfusion We have previously noted that Rd can neutralize ROS in neuronal cells exposed to hydrogen peroxide (Ye et al., 2008) and OGD (Ye et al., 2009). In the present study, we predicted that Rd would suppress oxidative damage induced by MCAO. To this end, we monitored the in vivo hydroxy radical formation by microdialysis during ischemia and recirculation. Quantitative HPLC measurements indicated that MCAO induced a gradual increase of 2,3-DHBA (Fig. 3A) and 2,5-DHBA (Fig. 3B) levels, which reached approximately 3-fold of baseline during 80 and 120 min of ischemia, and remained elevated during the entire reperfusion period. Rd did decrease the levels of 2,3- and 2,5-DHBA derived from hydroxy radical. In the Rd-treated animals, 2,3- and 2,5-DHBA increased more slowly, reached approximately 2-fold of baseline at 80 min of ischemia, and decreased to approximately 1.5-fold at 60 min of reperfusion.

3. Results

3.4. Rd suppresses oxidative DNA, lipid and protein damage

3.1. Physiologic parameters are normal and equivalent in all study groups

We identified oxidative DNA damage by anti-8-OHdG antibody (Fig. 4). Rare positive staining was detected in the sham operation group. After 24 h of ischemia, strong 8-OHdG immunoreactivity

All animals of this study showed similar values for rectal temperature, mean arterial blood pressure, arterial blood gases, and plasma glucose (Table 1). The laser Doppler flowmetry signal showed that regional CBF was reduced equivalently in all groups during ischemia (Fig. 1). Rats whose CBF remained >30% of baseline were excluded from further experiment.

[()TD$FIG]

3.2. Rd is neuroprotective in transient MCAO injury First, we attempted to investigate the efficacy of Rd for histological and functional improvement after MCAO. The reduction in infarct size was observed at 10 mg/kg and was greatest at 50 mg/kg, while the 0.1, 1 and 200 mg/kg doses were ineffective (Fig. 2A and B). Infarct volume corrected for brain swelling was reduced by 59% on POD 1 after the treatment with 50 mg/kg Rd. Accordingly, this infarct reduction ass associated with remarkable behavioral improvements (Fig. 2C). Repeated measures ANOVA revealed ischemic rats treated with Rd 10 or 50 mg/kg performed significantly better in mNSS than vehicle-treated animals (P = 0.018, 10 mg/kg; P = 0.001, 50 mg/kg).

Fig. 1. Laser Doppler flowmetry showed that cerebral blood flow (CBF) was not altered by Rd during ischemia. CBF was normalized by comparison to the mean CBF before administration of MCAO in each animal. There was no difference in the decrease of CBF during MCAO between animals assigned to different treatment groups.

[()TD$FIG]

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Fig. 2. Neuroprotection of Rd in transient middle cerebral artery occlusion (MCAO). (A) Representative ischemic lesions in coronal slices on post-operative day (POD) 1. Pale areas represent infarction. Quantitative analysis of total infarct volume (corrected for edema) is displayed in (B). The infarct size was significantly reduced in rats pretreated with 10 and 50 mg/kg Rd, but not in animals treated with 0.1, 1, or 200 mg/kg, as compared with vehicle-treated counterparts. (C) Rd treatment also improves long-term neurological recovery in stroke rats as measured by modified Neurological Severity Scores (mNSS). Data are expressed as mean  SEM (n = 9–12 rats for each group). *P < 0.05; **P < 0.01 vs vehicle value by one way analysis of variance with Tukey post test.

was evident in the nuclei of neurons located in the ischemic penumbra. In comparison, nuclei of neurons in the Rd group showed a weakly positive immunoreactivity for 8-OHdG. Quantitative analysis showed that the number of positive cells for 8OHdG were significantly smaller in the Rd groups than in the vehicle group. Lipid peroxidation was estimated by determining the 4-HNE and MDA formation. Fig. 5A illustrates different 4-HNE-protein adducts with apparent masses ranging from 70 to 25 kDa in the ipsilateral hemisphere to MCAO. Quantitative analysis showed that total levels of 4-HNE-protein adducts were significantly decreased in stroke rats treated with Rd, as compared with vehicletreated animals. Consistently, Rd administration also showed a marked decrease in MDA level in ischemic penumbra (Fig. 5B). Oxidative protein damage was identified by the assessment of protein carbonyl and AGEs. Western blot analysis showed lower intensity of AGEs-positive band in stroke rats treated with Rd, as

[()TD$FIG]

compared with vehicle-treated animals (Fig. 6A). Carbonyl levels in rats subjected to MCAO also showed a 3.7-fold increase when compared with sham rats (Fig. 6B). Administration of Rd significantly alleviated the levels of protein carbonyls in stroke rats (2.4-fold). 3.5. Rd preserves endogenous antioxidant activities after MCAO We next probed whether Rd could affect the endogenous antioxidant system activities. The enzyme activities of CAT (Fig. 7A), SOD1 (Fig. 7B), SOD2 (Fig. 7C), GPX (Fig. 7D), GR (Fig. 7E), and non-enzymic antioxidants GSH (Fig. 7F) were measured from sham rats, contralateral hemisphere to the lesion, ischemic penumbra and core, respectively. Administration of Rd to the stroke rats resulted in a remarkable augmentation of the endogenous antioxidant status. Despite minimal effects of Rd on the GPX; CAT, SOD1, SOD2, GR, and

Fig. 3. Rd reduces 2,3- and 2,5-DHBA in the in the dialysate during ischemia and reperfusion. The temporal changes of hydroxyl radicals formation was monitored by the continuous administration of salicylate and collection of its hydroxylation products 2,3-DHBA (A) and 2,5-DHBA (B) via microdialysis probe. The increase of 2,3-DHBA and 2,5-DHBA levels was significantly attenuated in Rd-treated rats during both ischemia and reperfusion periods. There were no significant changes in 2,3-DHBA or 2,5-DHBA levels before MCAO (baseline). Data are expressed as mean  SEM (n = 7–8 rats for each group). *P < 0.05; **P < 0.01 vs MCAO control value by one way analysis of variance with Tukey post test.

[()TD$FIG]

[()TD$FIG]

R. Ye et al. / Neurochemistry International 58 (2011) 391–398

Fig. 4. Rd attenuates oxidative DNA damage following MCAO. On POD 1, coronal sections from the ischemic penumbra area in the temporal cortex were stained with antibody to 8-hydroxy-deoxyguanosine (8-OHdG; a marker to oxidative DNA damage). Scale bar, 100 mm. The right panels showed the quantitative analysis demonstrating that the number of positive cells for 8-OHdG per field of view (FOV) was significantly smaller in stroke animals receiving Rd treatment as compared with those receiving vehicle. Histograms represent mean  SEM (n = 3). *P < 0.05; **P < 0.01 vs ischemic control value by one way analysis of variance with Tukey post test.

GSH/GSSG showed significant increment in their activities in ischemic penumbra on POD 1. SOD2 was even increased in ischemic core area. However, there was no significant alteration of these antioxidant activities in sham rats without stroke after Rd supplementation. 3.6. Rd ameliorates subsequent inflammatory injury In addition to these direct cytotoxic effects, the burst of free radicals also induces the formation of inflammatory mediators through redox-mediated signaling pathways, leading to post[()TD$FIG]ischemic inflammatory injury (Chamorro, 2004). Therefore, we

395

Fig. 6. Rd attenuates oxidative protein damage following MCAO. Protein peroxidation was assessed by measuring advanced glycosylation end products (AGEs) (A) and protein carbonyl (B) concentrations. Data are expressed as mean  SEM (n = 3). Mean values in sham-treated groups were scaled to 100%. *P < 0.05; **P < 0.01 vs ischemic control value by one way analysis of variance with Tukey post test.

further evaluated the subsequent inflammatory responses by immunostaining for Iba-1, which is specific to activated microglia; and Western blot assays for iNOS and COX-2 expression on POD 1 and 7. In the ischemic core, where tissues became fully necrotic and cavity formation was noted, the intensity of the staining was much stronger than in penumbra. Immunoreactive cells were present throughout the entire ischemic core. There is no significant difference between Rdtreated and vehicle-treated groups. In the ischemic penumbra, we found a distinct Rd-dependent decrease in the accumulation of microglia, indicative of inflammation and remodeling (Fig. 8A and B). Postischemic syntheses of two damaging enzymes, COX-2 and iNOS, were also significantly inhibited by Rd treatment (Fig. 8C–E). 4. Discussion

Fig. 5. Rd attenuates lipid peroxidation following MCAO. Lipid peroxidation was assessed by measuring 4-hydroxynonenal (4-HNE) (A) and malondialdehyde (MDA) (B) concentrations. Data are expressed as mean  SEM (n = 3). Mean values in sham-treated groups were scaled to 100%. *P < 0.05; **P < 0.01 vs ischemic control value by one way analysis of variance with Tukey post test.

Here we demonstrated the neuroprotective effects of Rd in a rat model of focal cerebral ischemia injury. The major findings of the present study were that Rd reduced free radical generation during ischemia and recirculation, suppressed early oxidative damage, and inhibited the sequential inflammatory injury. In this study, first we found that the neuroprotection of Rd presented at the dose of 10–50 mg/kg was abrogated at higher dose (200 mg/kg) or lower doses (0.1 and 1 mg/kg). Various neuroprotective antioxidants with diverse structures and activities also display a biphasic dose–response relationship (Ley et al., 2007). Deficiencies in antioxidants, especially in the case of elevated oxidative stress, are deleterious, though excessive doses of antioxidant supplementation confer no benefit and indeed may be potentially detrimental (Bjelakovic et al., 2007). ROS act as cellular messengers necessary for normal function, and a brief increase may activate protective mechanisms as seen in preconditioning and post-conditioning. Thus, the efficacy of freeradical-scavenging antioxidants may be limited to their ability to restore levels of free radicals and oxidative stress to within narrow homeostatic limits (Ley et al., 2007). Overwhelming evidence has accumulated implying that free radicals play a pivotal role in the ischemic cascade in a consecutive 2-phase pattern, an immediately occurring direct cytotoxic damage and a post-ischemia/reperfusion inflammatory injury

[()TD$FIG]

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Fig. 7. Rd attenuates the consumption of the endogenous antioxidant system activities. The enzyme activities of catalase (CAT) (A), superoxide dismutase (SOD) 1 (B) and 2 (C), glutathione peroxidase (GPX) (D), glutathione reductase (GR) (E); and glutathione content (reduced glutathione (GSH) and glutathione disulfide (GSSG)) (F) were measured on POD 1. Rd induced a noticeable elevation in the activities of CAT, SOD1, SOD2, GR, and GSH/GSSG in the infarct penumbra. However, no significant difference of enzyme activities was observed in other regions except for SOD2 in the infarct core. Data are expressed as mean  SEM of percentage value in sham-treated group (n = 5). *P < 0.05, **P < 0.01 vs respective vehicle value using unpaired t-test.

(Zhang et al., 2005). Here we measured both direct (using the salicylate trap) and indirect (lipid, protein and DNA peroxidation products; and loss of scavengers) free radical accumulation. Rd was found to be effective in scavenging early accumulation of free radicals. Specifically, Rd reduced hydroxyl radical as indicated by the inhibition of hydroxylated salicylate formation. These findings are in agreement with the in vitro results observed in living astrocytes (Lopez et al., 2007) and in neuronal cells exposed to hydrogen peroxide (Ye et al., 2008) and OGD (Ye et al., 2009). It is worthwhile to note that Rd did not affect cerebral antioxidant activities under normal conditions, which indicates that the antioxidant ability of Rd may be due to its direct scavenging of ROS rather than the recruitment of the endogenous antioxidative system. Ginsenosides are steroid-like molecules that have a gonane steroid nucleus with different sugar moieties attached. It has been suggested that ginsenosides are capable of accessing intracellular locations thanks to their steroid-like structures, justifying their ability to attenuate the oxidative stress caused by diverse stimuli (Liu et al., 2003; Tang and Eisenbrand, 1992). The chemical structure of Rd (sugar moiety attached to the 20position of the triterpene dammarane) may contribute to its direct antioxidant property (Liu et al., 2003). The other interrelated mechanism that may account for the neuroprotective effect of Rd is its anti-inflammatory effect mediated by the inhibition of microglial activity, iNOS and COX2 expression. As a cellular response to cerebral ischemia, microglial

activation is observed in regions of neuronal death after the onset of cerebral ischemia. Activated microglia support tissue repair processes by rapid removal of debris, but they also induce cytotoxic mediators such as NO and inflammatory cytokines, which may contribute to the infarct progression in the postischemic period. Accordingly, pharmacological inhibition of microglial activation after ischemia has attenuated the extent of tissue damage and neurologic impairment (Hayakawa et al., 2008; Yenari et al., 2006). Cerebral ischemia up-regulates COX-2 and iNOS expression. Following cerebral ischemia and reperfusion, COX-2 induction is closely associated with postischemic inflammation and NO production. Also, postischemic COX-2 reaction product prostaglandin E2 accumulation was reduced significantly in iNOS null mice (Nogawa et al., 1998). Upregulation of these enzymes will lead to superoxide and peroxynitrite synthesis during the later phases of reperfusion (Lipton, 1999). Based on our results that Rd decreased Iba-1-positive microglia and lowered iNOS and COX-2 enzyme concentrations, the possible scenario behind Rd-induced reduction of the infarct volume may include suppression of inflammatory injury during the later phase, which might be important for extension of the therapeutic time window in acute cerebral ischemia. Although many compounds with neuroprotective action are in various stages of the pharmaceutical pipeline, Rd represents unique advantages. P. ginseng and its related species have already had thousands of years of human exposure with little reported

[()TD$FIG]

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Fig. 8. Rd attenuates sequential inflammatory responses after MCAO. (A) On POD 1 and 7, coronal sections from the ischemic penumbra area in the temporal cortex were stained with antibody to ionized calcium-binding adapter molecule 1 (Iba1; a marker to activated microglia). Scale bar, 200 mm. (B) Cells positive for Iba1 per field of view (FOV) were counted as indicated in (A). (C) Western blot analyses of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were performed on POD 1 and 7. The right panels show the densitometric analyses of bands of iNOS (D) and COX-2 (E). Histograms represent mean  SEM (n = 3). **P < 0.01 vs respective vehicle value using unpaired t-test.

toxicity. Rd is highly lipophilic and can easily diffuse across biological membranes and blood–brain barrier in energy deficient environment. Currently Rd is being developed to treat patients with acute ischemic stroke. Several studies have been reported demonstrating the pharmacokinetics, safety and preliminary efficacy of Rd in human subjects (Liu et al., 2009; Yang et al., 2007; Zeng et al., 2010). More recently, a phase III multi-center clinical trial of Rd showed a benefit in terms of the prespecified primary end point, the distribution of disability as measured by the modified Rankin score at 90 days after stroke onset (unpublished data). Together, both laboratory and clinical evidence indicate that Rd may be a promising neuroprotectant and provide support for future studies to confirm whether Rd is beneficial in ischemic stroke. In summary, this study demonstrated the neuroprotection of Rd in transient focal ischemia, which may involve an integrated process of early free radicals scavenging pathway and a late antiinflammatory effect. Acknowledgements This study was supported in part by the National Natural Science Foundation of China (grant no. 81073094). The authors are

grateful to Dongyun Feng and Shiquan Wang for their excellent technical assistance with Western blot and MCAO model experiments.

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