Protective effect of Phellodendri Cortex against lipopolysaccharide-induced memory impairment in rats
Protective effect of Phellodendri Cortex against lipopolysaccharide-induced memory impairment in...
Lee, Bombi; Sur, Bongjun; Cho, Sehyung; Yeom, Mijung; Shim, Insop; Lee, Hyejung; Hahm, Dae-Hyun
2012-08-01 00:00:00
Animal Cells and Systems Vol. 16, No. 4, August 2012, 302312 Protective effect of Phellodendri Cortex against lipopolysaccharide-induced memory impairment in rats a b c a a,b a,b a,b Bombi Lee , Bongjun Sur , Sehyung Cho , Mijung Yeom , Insop Shim , Hyejung Lee and Dae-Hyun Hahm * Acupuncture and Meridian Science Research Center, College of Oriental Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea; The Graduate School of Basic Science of Oriental Medicine, College of Oriental Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea; Department of Physiology, Neurodegeneration Control Research Center, Kyung Hee University School of Medicine, Seoul, 130-701, Republic of Korea (Received 16 January 2012; received in revised form 20 May 2012; accepted 29 May 2012) The purpose of this study was to examine whether Phellodendri Cortex extract (PCE) could improve learning and memory impairments caused by lipopolysaccharide (LPS)-induced inflammation in the rat brain. The effect of PCE on modulating pro-inflammatory mediators in the hippocampus and its underlying mechanism were investigated. Injection of LPS into the lateral ventricle caused acute regional inflammation and subsequent deficits in spatial learning ability in the rats. Daily administration of PCE (50, 100, and 200 mg/kg, i.p.) for 21 days markedly improved the LPS-induced learning and memory disabilities in the Morris water maze and passive avoidance test. PCE administration significantly decreased the expression of pro-inflammatory mediators such as tumor necrosis factor-a, interleukin-1b, and cyclooxygenase-2 mRNA in the hippocampus, as assessed by RT-PCR analysis and immunohistochemistry. Together, these findings suggest that PCE significantly attenuated LPS-induced spatial cognitive impairment through inhibiting the expression of pro-inflammatory mediators in the rat brain. These results suggested that PCE may be effective in preventing or slowing the development of neurological disorders, including Alzheimer’s disease, by improving cognitive and memory function because of its anti-inflammation activity in the brain. Keywords: lipopolysaccharide; memory; inflammation; Phellodendri Cortex; Morris water maze Introduction deficits in learning and memory in a variety of behavioral tasks (Frank-Cannon et al. 2009). The Inflammation has been implicated as a common cause hippocampal neurons that are known to play an in various neurodegenerative diseases, such as Alzhei- important role in learning and memory functions are mer’s disease (AD), Parkinson’s disease, and ischemic particularly vulnerable to a neuronal damage such as stroke (Mrak 2009). Much evidence suggests that that from LPS-induced inflammation, which results in neuroinflammation and neurodegenerative disorders serious deficits in spatial memory tasks (Hwang et al. with sustained increase in various pro-inflammatory 2011) and synaptic plasticity (Min et al. 2009). cytokines, such as tumor necrosis factor-a (TNF-a) Thus, many studies have suggested that chronic use and interleukin-1b (IL-1b), are closely correlated with of non-steroidal anti-inflammatory drugs (NSAIDs) the cognitive dysfunction primarily associated with the can prevent cognitive decline in individuals diagnosed progression of AD pathogenesis (Schwab and McGeer with developing AD dementia or in elderly people 2008; Mrak 2009). (Szekely et al. 2008). However, the long-term treat- Lipopolysaccharide (LPS), a non-infectious com- ments using NSAIDs can cause gastrointestinal side ponent of the outer membranes of Gram-negative effects and even occasional liver and kidney toxicities bacteria, when administered by intraventricular micro- (Knight et al. 1996; Warner et al. 1999; Graupera et al. injection or chronic infusion, induces neuroinflamma- 2003). These side effects have encouraged the develop- tion and impairment of memory function (Kim et al. ment of new NSAIDs that are safer for long-term 2000; Kitazawa et al. 2005). The memory dysfunction caused by LPS-induced inflammation has been hy- treatments (Kelloff et al. 2000). Recent studies have pothesized to play an important role in the pathogen- also suggested the use of herbal medicines or natural esis of the degenerative changes and cognitive products for treating Alzheimer’s-type, dementia- impairments (Cunningham et al. 2009). The pathogen- related disorders exhibiting cognitive memory impair- ment and neuroinflammation (Ho et al. 2011). esis of various neurodegenerative changes, and cogni- In Korean traditional medicine, Phellodendri tive and memory impairments, closely associated with Cortex extract (PCE) has been widely used to prevent AD or senile CNS dysfunction (Lukiw and Bazan bacterial infection of the respiratory system and to treat 2000). In fact, it is well established that LPS-induced inflammation in the hippocampus produces severe various chronic inflammatory diseases (Lu et al. 2011). *Corresponding author. Email: dhhahm@khu.ac.kr; dhahm@paran.com ISSN 1976-8354 print/ISSN 2151-2485 online # 2012 Korean Society for Integrative Biology http://dx.doi.org/10.1080/19768354.2012.699004 http://www.tandfonline.com NEUROBIOLOGY & PHYSIOLOGY Animal Cells and Systems 303 PCE has multiple pharmacological activities and 1.2 mM Na2HPO , and maintained at pH 7.4. The produces a variety of biological effects in the central lateral ventricle in the stereotaxic coordinate was nervous system (Zhang et al. 2009). Some studies have designated according to the Paxinos and Watson brain demonstrated that PCE alleviated LPS-induced acute atlas (AP: 0.2, L:90.3, DV: 6.2 referenced to the airway inflammation through decreasing the infiltra- bregma; Paxinos and Watson 1986). Artificial CSF or tion of inflammatory cells and releasing inflammatory LPS solution was injected for 5 min at a flow rate of mediators (Mao et al. 2010). It also attenuated LPS- 2 ml/min using 22-gauge Hamilton syringe and micro- induced inflammatory responses and inhibited the injection pump (Pump 22; Harvard Apparatus Inc., expression of TNF-a, IL-1b, and nitric oxide mRNAs Holliston, MA, USA). LPS (Escherichia coli; 055:B5) in BV2 mouse microglia cells (Park et al. 2007). These and ibuprofen were purchased from Sigma-Aldrich Co. studies suggest that PCE could be useful for suppres- (St. Louis, MO, USA). Administration of PCE was sing inflammation in neurodegenerative diseases. How- started 24 h after the lesion generation. ever, the underlying mechanism of pharmacological effect of PCE on improving LPS-induced cognitive Experimental groups deficits and spatial learning disability remains poorly understood. Rats were randomly divided into seven groups of seven The aim of the present study was to evaluate the individuals as follows: CSF-injected sham group being anti-inflammatory effects of PCE in improving learning regarded as normal (CON group, n7), CSF-injected and memory function in rats exposed to LPS-induced plus 200 mg/kg PCE-treated group (PCE group, n7), inflammation, as measured by performance on the LPS-injected plus saline-treated group (LPS group as passive avoidance test (PAT) and the Morris water a negative control, n7), LPS-injected plus 50 mg/kg maze (MWM) tests. We also examined how these PCE-treated group (PCE50 LPS group, n7), LPS- effects were related to the molecular modulation of injected plus 100 mg/kg PCE-treated group inflammation in terms of the neural mechanism under- (PCE100 LPS group, n7), LPS-injected plus lying the memory-enhancing activity of PCE. 200 mg/kg PCE-treated group (PCE200 LPS group, n7), and LPS-injected plus 40 mg/kg ibuprofen- treated (IBU LPS group as a positive control, n7). PCE extract was manufactured in the oriental Materials and methods pharmacy in Kyung Hee Hospital of Oriental Medi- Animals cine, Kyung Hee University (Seoul, Korea), according Adult male SpragueDawley (SD) rats weighing 260 to the protocol of Lee et al. (2004). IBU, a centrally 280 g were obtained from Samtako Animal Co. (Seoul, acting cholinesterase inhibitor, was used as a positive Korea). The rats were housed up to five per poly- control. The rats were intraperitoneally administrated carbonate cage in a limited-access rodent facility. The with PCE and IBU for 21 days, and PCE and IBU were room controls were set to maintain the temperature at dissolved in 0.9% physiological saline before use. The 22928C and the relative humidity at 55915%. Cages experimental schedule of all drug administration and were lit by artificial light for 12 h each day. Sterilized behavioral tests is shown in Figure 1. drinking water and standard chow diet were supplied ad libitum to each cage during the experiments. All animal experiments began at least seven days after the Open field test animals arrived. Prior to water maze testing, the rats were individually housed in a rectangular container that was made of black polyethylene (454535 cm) to provide best Lesion generation and LPS administration contrast to the white rats in a dimly lit room equipped Learning and memory deficits in the rats were induced with a video camera above the center of the room, and by bilateral intracranial injection (right and left side) of their locomotor activities (animal’s movements) were LPS, according to the procedures described previously then measured. The locomotor activity indicated by the (Guo et al. 2010; Lim et al. 2008). Fifty micrograms of speed and the distance of movements was monitored by LPS dissolved in 10-ml cerebrospinal fluid (CSF) a computerized video-tracking system using S-MART (Sigma-Aldrich Co., St. Louis, MO, USA) was micro- program (PanLab Co., Barcelona, Spain). After 5 min injected into the lateral ventricle of the rat brains in all of adaptation, the distance they traveled in the contain- lesion groups. Sham animals as a vehicle control er was recorded for another 5 min. The locomotor received microinjection of artificial CSF instead of activity was expressed in meters. The number of rearing LPS (Hwang et al. 2011). Artificial CSF consists of events of the rats was also recorded to analyze 140 mM NaCl, 3.0 Mm KCl, 2.5 mM CaCl locomotor activity in the open field test. , and 2 304 B. Lee et al. Figure 1. Experimental schedule of lesion generation, PCE administration, and behavioral tests in rats. PCE, Phellodendri Cortex; LPS, lipopolysaccharide. Passive avoidance test polypropylene and internally painted white. The pool was half-filled with water to 30 cm in depth. The water The test was performed according to the step-through in the pool was made opaque by adding 1 kg skim milk method described previously (Lee et al. 2011). The powder and continuously maintained at 22928C. The Gemini Avoidance System (SD Instruments., San Diego, pool was divided into four quadrants of equal area. CA, USA) was used for this test. The step-through During the MWM test, an escape platform (15 cm in passive avoidance apparatus consists of a tilting floor diameter) was located in one of four sections of the acrylic box divided into two compartments, a lightened pool being hidden 1.5 cm below the water surface and compartment connected to darkened one, by an auto- apart approximately 50 cm from the sidewalls. Several matic guillotine door and a control unit generating visual cues were placed around the pool in plain sight electric shock (Behbood Pardaz Co., Ghaem, Iran). of the animals. A digital camera was mounted to Electric shock can be delivered to the grid floors, made of stainless steel rods (3 mm diameter) spaced 1 cm the ceiling straight above the center of the pool and apart, in both compartments. First, the rats were taken was connected to a computerized recording system trials to acquisition test in the apparatus. In the training equipped with a tracking program (S-MART: PanLab session, a rat was placed in a lightened compartment of Co., Barcelona, Spain), which permitted on- and off- the passive avoidance apparatus facing away from the line automated tracking of the paths taken by the rats. entrance to the dark compartment, and then the guillotine door was opened. Because of intrinsic pre- ference to the dark environment, the rat immediately Hidden platform trial for acquisition test entered the dark compartment and the door was closed. The MWM test was initiated on the 14th day after During acquisition test, the latency time before entry injections of PCE extract and LPS. The MWM test was into the dark compartment was recorded for each rat. basically performed according to the MWM apparatus After 30 min, the rat was placed in the lightened and the method described previously (Lee et al. 2011). compartment once again. After entering the dark The animals received three trials per day. The rats were compartment, the guillotine door was closed and subse- trained to find the hidden platform, which remained in quently a mild electrical shock (0.5 mA) was applied a fixed location throughout the test. The trials lasted for 3 s. The retention test was started 24 h after the for a maximum of 180 s, and the escape latency was acquisition trial for training. The rat was again placed in expressed by the swimming time (or swim distance) to the lightened compartment and the guillotine door was find the submerged platform in the pool. The animals opened. In the retention test, the rat was placed in the were tested in three trials per day for five days, and they passive avoidance apparatus as previously described and received a 60-s probe trial on the sixth day. Finding the the time required for the rat to enter the dark compart- platform was defined as staying on it for at least 4 s ment was measured for a maximum period of 3 min in before the acquisition time of 180 s ended. When the the same method with the acquisition test. The rat did rat failed to find the platform in the limited time in the not enter the dark compartment within this period received a latency time of 180 s (Mohamed et al. 2000). first trial of hidden platform test, the rat should be placed on the platform for 20 s and assigned a latency of 180 s. Between one trial and the next, the water in MWM test the pool was stirred to remove olfactory traces of MWM apparatus previous swim patterns. The entire schedule proceeded for six days and each animal had three trials for The MWM test was performed in a small circular pool (2.0 m in diameter and 0.35 m deep) made of training per day with 3040 min inter-trial interval. Animal Cells and Systems 305 Probe trial for retention test the numbers of cells within 100100-um grids were counted by observers blinded to the experimental For the probe trial, a rat was placed in the quadrant groups. Counting immunopositive cells was performed located diagonally from the target quadrant and in at least three different hippocampal sections per rat allowed to swim to the quadrant from which the escape brain. Stained sections were randomly chosen from platform had been removed for a maximum of 60 s. equal levels of serial sections along the rostralcaudal The probe trial was expressed by the ratio of the time axis. The TNF-a-, IL-1b-, and COX-2-immunopositive spent (or the distance) for searching the platform in the cells were only counted when their densities reached a target quadrant to total duration spent for swimming defined darkness above the background level (Guo in the pool. et al. 2010). Distinct brown spots for TNF-a, IL-1b, and COX-2 were observed in the cytoplasms and in the membranes of the cone-shaped cells of the hippocam- Immunohistochemistry for proinflammatory markers pus. The cells were anatomically localized according to For immunohistochemical studies, the animals were the stereotactic rat brain atlas of Paxinos and Watson deeply anesthetized with sodium pentobarbital (80 mg/ (1986). The brightness and contrast between images kg, by intraperitoneal injection) and perfused through were not adjusted to exclude any possibility of sub- the ascending aorta with normal saline (0.9%) followed jective selection of immunoreactive cells. by 300 ml (per rat) of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The brains were Total RNA preparation and RT-PCR analysis removed in a randomized order, post-fixed over-night, and cryoprotected with 20% sucrose in 0.1 M PBS at The expression levels of TNF-a, IL-1b, and COX-2 48C. Coronal sections with 30 mm thickness were cut mRNAs were determined by the reverse transcription- through the hippocampus using a cryostat (Leica polymerase chain reaction (RT-PCR). The brain hip- CM1850; Leica Microsystems Ltd., Nussloch, Ger- pocampus was isolated from three rats per group. The many). The sections were obtained according to the rat total RNAs were prepared from the brain tissues atlas of Paxinos and Watson (hippocampus; between using a TRIzol reagent (Invitrogen Co., Carlsbad, bregma 2.6 and 3.6) (Paxinos and Watson 1986). CA, USA) according to the supplier’s instruction. The sections were immunostained for TNF-a, IL-1b, Complementary DNA was first synthesized from total and cyclooxygenase-2 (COX-2) expression using the RNA using a reverse transcriptase (Takara Co., Shiga, avidinbiotinperoxidase complex (ABC) method. The Japan). PCR was performed using a PTC-100 pro- sections were incubated with primary rabbit anti-TNF- grammable thermal controller (MJ Research, Inc., a antibody (1:200 dilution; Novus Biochemicals LLC., Watertown, MA, USA). All primers were designed Littleton, CO, USA), rabbit anti-IL-1b antibody (1:200 using published mRNA sequences of those cytokines dilution; Santa Cruz Biotechnology Inc., Santa Cruz, and a primer designing software, Primer 3, offered by CA, USA), and goat anti-COX-2 antibody (1:200 the Whitehead Institute for Biomedical Research dilution; Cambridge Research Biochemicals Co., (Cambridge, MA, USA; www.genome.wi.mit.edu) on Bellingham, UK) in PBST (PBS plus 0.3% Triton the website. The PCR products were separated on 1.2% X-100) for 72 h at 48C, respectively. The sections were agarose gels and stained with ethidium bromide. The incubated for 120 min at room temperature with density of each band was quantified using an image- TM biotinylated anti-rabbit goat IgG (for the anti-TNF-a analyzing system (i-Max , CoreBio System Co., antibody and anti-IL-1b antibody) and biotinylated Seoul, Korea). The expression levels were compared anti-goat rabbit IgG (for the anti-COX-2 antibody). each other by calculating the relative density of target The secondary antibodies were obtained from Vector band, such as TNF-a, IL-1b, and COX-2, to that of Laboratories Co. (Burlingame, CA, USA) and diluted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). 1:200 in PBST containing 2% normal serum. To visualize immunoreactivity, the sections were incubated Statistical analysis for 90 min in ABC reagent (Vectastain Elite ABC kit; Vector Labs. Co., Burlingame, CA, USA), and incu- All measurements were performed by an independent bated in a solution containing 3,3?-diaminobenzidine investigator blinded to the experimental conditions. (DAB; Sigma-Aldrich Co., St. Louis, MO, USA) and Results in figures are expressed as mean9standard 0.01% H O for 1 min. Images were captured using an error of means. Differences within or between normally 2 2 AxioVision 3.0 imaging system (Carl Zeiss, Inc., distributed data were analyzed by analysis of variance Oberkochen, Germany) and processed using Adobe using SPSS (Version 13.0; SPSS, Inc., Chicago, IL, Photoshop (Adobe Systems, Inc., San Jose, CA, USA). USA) followed by Tukey’s post hoc test. Statistical The sections were viewed at 200 magnification, and significance was set at pB0.05. 306 B. Lee et al. Results behavior in the open field test. However, the PCE- treated rats (200 mg/kg) significantly increased their Effect of PCE on LPS-induced body weight loss locomotor activity and rearing, as compared to those Rats exposed to LPS infusion begin to lose their body in the LPS group (pB0.05). Therefore, the PCE-treated weights on the first day of LPS infusion, and this LPS- rats showed remarkable enhancements of the active induced initial reduction of body weight is sustained responses via stimulating motivation required for for three days without restoring to normal level or even exploring for 21 days, ultimately indicating that the exacerbated in some cases (Gaab et al. 2005). The LPS- treatment of PCE significantly restored locomotor injected rats gradually gained less body weights over activity and exploratory behavior and accordingly the treatment period than did the sham control rats in alleviated LPS-induced impairment of motor function the CON group (Figure 2a). Analysis of body weight behavior in rats. values revealed no significant differences among the seven groups on the first day prior to starting LPS infusion. However, there was a significant reduction in Effect of PCE on LPS-induced step-through latency body weight gain during 21 days of the treatment in the deficit in the PAT LPS group, as compared to the CON group (pB0.001). Interestingly, body weights of the 100 mg/kg PCE- To determine whether PCE promotes the recovery of treated rats and 200 mg/kg PCE-treated rats showed memory dysfunction, PCE was administrated in the significant inhibition of reductions of body weight gain rats with LPS-induced impairment of memory, and during 21 days of the treatment, as compared to the their memory and cognitive functions were examined LPS group (pB0.05 and pB0.01, respectively). It also in the PAT (Figure 3). It was verified that the rats in indicated that the recovery of body weights in the all groups had no physiological defects (i.e. motor PCE100 LPS and PCE200 LPS groups was closely function) or intrinsic cognitive impairments through associated with those in the IBU LPS group. acquisition trials without electric challenge. In the time for acquisition trials, indicated by the latencies for entering the dark compartment, there were no signifi- Effect of PCE on LPS-induced locomotor disability in cant differences among all groups. After acquisition the open field test trials, the effect of PCE on the retention latency was examined 24 hours after applying electric shock in the Open field activity was used to evaluate locomotor dark box in the PAT. In the retention, it was shown that activity and exploratory behavior in the rats that the rats in the PCE200 LPS group had significantly received an LPS injection into the lateral ventricle increased latencies to enter the dark compartment for (Figure 2b). These results indicated that the rats with retention as compared to those in the LPS group LPS infusion significantly reduced their locomotor (pB0.05). This study indicated that the LPS activities and total numbers of rearing, as compared infusion severely impaired long-term memory, and with those in the CON group (pB0.05). It suggested that the rats, treated with LPS, subsequently produced the treatment of PCE significantly attenuated LPS- impairments of motor and exploration activities which induced memory deficit in the PAT. It also indicated have been closely associated with motor function that the restoration of memory function in the Figure 2. Effects of PCE on body weight gain on the first day prior to LPS injection and on the 21th day after LPS injection, and the activity counts of locomotor activity and the total numbers of rearings in the open field test on the 21th day. PCE, * *** # Phellodendri Cortex extract; LPS, lipopolysaccharide; IBU, ibuprofen. pB0.01, pB0.001 vs. CON group; pB0.05 and ## ### pB0.01, pB0.001 vs. LPS group. Animal Cells and Systems 307 Figure 3. Effects of PCE on the latencies of entering the dark compartment on the acquisition trial and on the retention test in ** # the passive avoidance test. pB0.01 vs. CON group; pB0.05 vs. LPS group. PCE200 LPS group was almost close to that in the The rats with LPS infusion showed severe impairment IBU LPS group. of spatial performance in the MWM (pB0.001). The rats in the 200 mg/kg PCE-treated group spent more time (pB0.05) and distance (pB0.001) around the platform area than did those in the LPS group. Effect of PCE on LPS-induced spatial memory The mean swimming speeds, as calculated by dividing impairment in the water maze test the total swim distance by latency (Figure 4e), of the The effect of the PCE treatment on swimming to reach rats in the LPS group were not significantly different the submerged platform in the MWM test is shown in from those in other groups. On the basis of these Figure 4. In the present study, a spatial learning and results, it was suggested that 200 mg/kg PCE-treated memory deficit rat model was established by infusion rats exhibited better improvement of acquisition in the of LPS into the lateral ventricle, by which the escape hidden platform trial, and this memory improvement latency and the searching distance were significantly resulted in more rapid escape from the water than did increased as compared with those in the CON group. the LPS-treated rats. Administration of PCE signifi- Analysis of escape latency revealed that the rats in the cantly attenuated LPS-induced deficits of learning and PCE200 LPS group significantly reduced swimming memory demonstrated in the MWM test. Thus, PCE- latency as compared with those in the LPS group treated rats showed a significant amelioration in the (PCE200 LPS group: pB0.05 on day 3, and 5, memory retention test because they spent more time pB0.01 on day 4; Figure 4a). Swimming distance and distance in the quadrant where the platform was traveled in each group was closely associated with formerly located and swam over the location of plat- escape latency in this task. Analysis of searching form more frequently. It also indicated that swimming distance values revealed that the rats in the latency of the LPS-injected rats receiving 200 mg/kg PCE200 LPS group significantly reduced swimming PCE was closely associated with those of rats receiving distance as compared with those in the LPS group 40 mg/kg IBU. (PCE200 LPS group: pB0.05 on day 4, pB0.01 on day 5; Figure 4b). The PCE treatment of 200 mg/kg improved spatial learning ability and memory function Effects of PCE on LPS-induced immunohistochemical in the rats with chronic inflammation in their brain by changes of inflammatory mediators LPS infusion, as evidenced by decreases in escape latency and searching distance throughout the training Following the behavioral tasks, brain tissue samples period. To investigate the effect on the spatial memory, from the subjects were analyzed using immunohisto- the performance in the probe trial on day 6 was chemistry to investigate the effect of PCE administra- examined by analyzing the percentages of time and tion on the expression of pro-inflammatory markers distance required for swimming to the expected posi- formerly activated by LPS-induced inflammation in the tion of the platform, respectively (Figure 4c,d). The rat brains (Figure 5A). The brain immunohistochem- swimming times and distances are reduced in the rat istry in the LPS group showed a significant increase that directly swims to the target quadrant where the in TNF-a, IL-1b, and COX-2 expression in the escape platform had been removed without confusion. hippocampus, as compared to those in the CON 308 B. Lee et al. Figure 4. Effects of PCE on latency of escaping from water (a) and swimming distance (b) during acquisition trials using a submerged platform, the percentages of time (c) and distance (d) in a probe trial without a platform, and swimming speed (e) in * *** # ## ### the Morris water maze test. pB0.05 and pB0.001 vs. CON group; pB0.05, pB0.01 and pB0.001 vs. LPS group. group (pB0.01). TNF-a immunoreactive cells signifi- IBU LPS group. These results clearly indicated that cantly decreased in the hippocampal regions in the the LPS-induced increase in the expression of TNF-a PCE100 LPS (pB0.01) and PCE200 LPS groups and COX-2 in the rat hippocampus was significantly (pB0.05), as compared to those in the LPS group attenuated by the treatment of PCE. (Figure 5B). Despite appearance of IL-1b-immunor- eactive cells in the hippocampal regions in the Effect of PCE on LPS-induced expression of TNF-a, PCE200 LPS group, there was no statistical signifi- IL-1 b, and COX-2 mRNAs in the hippocampus cance as compared to the LPS group (Figure 5B). Similarly, the COX-2-immunoreactive cells were sig- The effect of PCE administration on LPS-induced nificantly increased in the hippocampal region in the expression of TNF-a, IL-1b, and COX-2 mRNAs in PCE200 LPS group (pB0.05), as compared to the rat hippocampus was investigated using RT-PCR those in the LPS group (Figure 5B). The increases in analysis (Figure 6). Hippocampal expression of TNF-a, the pro-inflammatory mediators such as TNF-a, IL-1b, IL-1b, and COX-2 mRNA in the LPS group was and COX-2 by LPS-induced inflammation were significantly increased as compared to that in the CON significantly inhibited by PCE administration, and group (pB0.01). In case of PCE administration of their restorations were almost close to those in the 200 mg/kg, the increased expression of TNF-a, IL-1b, Animal Cells and Systems 309 Figure 5. Effects of PCE on the mean number of tumor necrosis factor-a (TNF-a)-, interleukin-1b (IL-1b)-, and cyclooxygenase- 2 (COX-2)-stained hippocampal areas after the Morris water maze test. Representative photographs and the relative percentage values are indicated in (A) and (B), respectively. (a) TNF-a expression in the CON group, (b) TNF-a expression in the LPS group, (c) TNF-a expression in the PCE200 LPS group, (d) IL-1b expression in the CON group, (e) IL-1b expression in the LPS group, (f) IL-1b expression in the PCE200 LPS group, (g) COX-2 expression in the CON group, (h) COX-2 expression in the LPS group, (i) COX-2 expression in the PCE200 LPS group. Sections were cut coronally at 30 mm. Scale bar indicates 50 mm. ** # ## pB0.01 vs. CON group; pB0.05 and pB0.01 vs. LPS group. and COX-2 mRNA in the LPS group was significantly also alleviated LPS-stimulated inflammatory responses, restored (pB0.05). The restored levels of these inflam- including LPS-induced expression of TNF-a, IL-1b, matory mediators were almost close to those in the and iNOS through the negative modulation of nuclear IBU LPS group. factor-kappaB (NF-kB) signaling in the microglial cells (Park et al. 2007). Thus, these studies suggest a new medicinal role of PCE on suppressing inflammatory Discussion response in the central nervous system, which can lead to developing a novel therapeutics using PCE for In the Korean traditional medicine, PCE has been treating neurodegenerative diseases in humans. widely used to treat chronic inflammatory diseases and Our results clearly demonstrated that the admin- prevent bacterial infection of the respiratory system istration of PCE significantly improved spatial learn- and gastrointestinal tract (Yang et al. 2010; Lu et al. ing and memory impairments induced by LPS 2011). Recently, several studies have shown that PCE infusions into the bilateral ventricles, as evidenced had a protective role against ischemia-reperfusion- by the behavior in the PAT and MWM tests. It induced brain injury through the reduction of neutro- phil infiltration into the brain (Hwang et al. 2002b). It also attenuated the increase in pro-inflammatory Figure 6. Effects of PCE on the expression of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and cyclooxygenase-2 (COX-2) mRNAs in rats with LPS-induced hippocampal impairment. PCR bands on agarose gel and their relative intensities are indicated in (a) and (b), respectively. The expression levels of TNF-a, IL-1b, and COX-2 mRNAs were normalized to that of ** # glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. pB0.01 vs. CON group; pB0.05 vs. LPS group. 310 B. Lee et al. markers such as TNF-a, IL-1b, and COX-2 in the brain dysfunction (Ringheim and Conant 2004). hippocampus, as assessed by RT-PCR analysis and According to the inflammation hypothesis, the memory immunohistochemistry. impairment in patients with senile dementia is due We found that the administration of PCE after LPS to selective and irreversible dysfunction and chronic infusion into the lateral ventricle markedly restored inflammation in the brain (Hoozemans et al. 2001). body weight gain and motor activity, suggesting that Thus, we propose that the anti-inflammatory effects PCE could inhibit the physiological changes and the of PCE significantly reversed the impairment in sickness behavioral symptoms caused by LPS-induced memory retention and the increased expression of pro- neuroinflammation. It can be suggested that PCE inflammatory cytokines such as TNF-a and IL-1b. enhanced resistance against stress and various psycho- Also, much experimental evidence has suggested somatic disorders and exerted various benefits, that the inducible gene encoding COX-2 is a key strengthening the immune system. element that modulates the generation of proinflam- To identify the effects of PCE on two different types matory mediators including various prostagladins of memory, cognitive memory and spatial learning, we (Fehe ´r et al. 2010). It is known that the expression of used the PAT and MWM, respectively. Our results COX-2 and the synthesis of prostaglandin E2, one demonstrated that the administration of PCE signifi- of its products, are increased in the hippocampus of cantly increased the step-through latency in the mem- patients with AD (Fujimi et al. 2007), which may be ory retention trial, which had been shortened by LPS related to the pathogenesis of the degenerative changes infusion, in the PAT. During the trial sessions in the and cognitive impairments (Hwang et al. 2002a). These MWM, administration of PCE resulted in a significant results suggest that inflammatory responses to LPS reduction in the escape latency, enhanced cognitive infusion significantly stimulated the expression of performance, and ameliorated of memory deficits, COX-2 protein or mRNA in the hippocampus through associated with LPS injection. These results showed modulating the NF-kB pathway (Gong et al. 2011). that LPS infusion into the lateral ventricle of rat brains The increase in COX-2 expression by NF-kB activation retarded escape latency in the MWM test, indicating can accelerate inflammatory responses and subse- deficits in spatial learning ability and reference mem- quently contribute to learning and memory deficits. ory. The scoring of the escape latency from water in Thus, treatment with long-lasting COX-2 inhibitors the spatial probe tests in the MWM test is primarily during the initial stages of AD, before clinical symp- considered to reflect long-term spatial memory ability toms of dementia appear, may suppress inflammatory (Lee et al. 2011). In this study, administration of PCE responses and the synthesis of pro-inflammatory med- shortened the escape latency without affecting swim- iators in the brain (Kumar et al. 2006). In the present ming velocity and extended the time spent swimming in study, PCE significantly decreased LPS-stimulated the place where the platform was previously located. behavioral changes and memory disturbances through This indicated that the administration of PCE signifi- inhibition of COX-2 mRNA expression. cantly improved the long-term memory deficit in rats In summary, we demonstrated that PCE signifi- that had suffered LPS-induced spatial memory impair- cantly improved spatial learning and memory abilities ment. From the results of the PAT and MWM tests, we in the rats with LPS-induced brain dysfunction, as concluded that a chronic inflammatory response played evidenced by performance in the PAT and the MWM an important role in learning acquisition and synaptic tests. PCE also suppressed LPS-simulated mRNA plasticity (Collister and Albensi 2005). expression of pro-inflammatory mediators such as Our results also showed that LPS infusion into the TNF-a, IL-1b, and COX-2 in the hippocampus. lateral ventricle significantly increased the expression Thus, PCE may be useful as an alternative medicine of TNF-a and IL-1b in the hippocampus, ultimately for treating or retarding the development of neurode- leading to a chronic neuroinflammatory response in the generative diseases including AD. brain. Thus, LPS-stimulated sustained increases in the expression of pro-inflammatory cytokines have been directly linked to neurodegenerative disorders asso- Acknowledgements ciated with decreased working memory (Frank-Cannon This work was supported by a grant from the Kyung Hee et al. 2009). 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http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.pngAnimal Cells and SystemsTaylor & Francishttp://www.deepdyve.com/lp/taylor-francis/protective-effect-of-phellodendri-cortex-against-lipopolysaccharide-erEQfdhMN6
Protective effect of Phellodendri Cortex against lipopolysaccharide-induced memory impairment in rats
Protective effect of Phellodendri Cortex against lipopolysaccharide-induced memory impairment in rats
Abstract
Abstract The purpose of this study was to examine whether Phellodendri Cortex extract (PCE) could improve learning and memory impairments caused by lipopolysaccharide (LPS)-induced inflammation in the rat brain. The effect of PCE on modulating pro-inflammatory mediators in the hippocampus and its underlying mechanism were investigated. Injection of LPS into the lateral ventricle caused acute regional inflammation and subsequent deficits in spatial learning ability in the rats. Daily...
Animal Cells and Systems Vol. 16, No. 4, August 2012, 302312 Protective effect of Phellodendri Cortex against lipopolysaccharide-induced memory impairment in rats a b c a a,b a,b a,b Bombi Lee , Bongjun Sur , Sehyung Cho , Mijung Yeom , Insop Shim , Hyejung Lee and Dae-Hyun Hahm * Acupuncture and Meridian Science Research Center, College of Oriental Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea; The Graduate School of Basic Science of Oriental Medicine, College of Oriental Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea; Department of Physiology, Neurodegeneration Control Research Center, Kyung Hee University School of Medicine, Seoul, 130-701, Republic of Korea (Received 16 January 2012; received in revised form 20 May 2012; accepted 29 May 2012) The purpose of this study was to examine whether Phellodendri Cortex extract (PCE) could improve learning and memory impairments caused by lipopolysaccharide (LPS)-induced inflammation in the rat brain. The effect of PCE on modulating pro-inflammatory mediators in the hippocampus and its underlying mechanism were investigated. Injection of LPS into the lateral ventricle caused acute regional inflammation and subsequent deficits in spatial learning ability in the rats. Daily administration of PCE (50, 100, and 200 mg/kg, i.p.) for 21 days markedly improved the LPS-induced learning and memory disabilities in the Morris water maze and passive avoidance test. PCE administration significantly decreased the expression of pro-inflammatory mediators such as tumor necrosis factor-a, interleukin-1b, and cyclooxygenase-2 mRNA in the hippocampus, as assessed by RT-PCR analysis and immunohistochemistry. Together, these findings suggest that PCE significantly attenuated LPS-induced spatial cognitive impairment through inhibiting the expression of pro-inflammatory mediators in the rat brain. These results suggested that PCE may be effective in preventing or slowing the development of neurological disorders, including Alzheimer’s disease, by improving cognitive and memory function because of its anti-inflammation activity in the brain. Keywords: lipopolysaccharide; memory; inflammation; Phellodendri Cortex; Morris water maze Introduction deficits in learning and memory in a variety of behavioral tasks (Frank-Cannon et al. 2009). The Inflammation has been implicated as a common cause hippocampal neurons that are known to play an in various neurodegenerative diseases, such as Alzhei- important role in learning and memory functions are mer’s disease (AD), Parkinson’s disease, and ischemic particularly vulnerable to a neuronal damage such as stroke (Mrak 2009). Much evidence suggests that that from LPS-induced inflammation, which results in neuroinflammation and neurodegenerative disorders serious deficits in spatial memory tasks (Hwang et al. with sustained increase in various pro-inflammatory 2011) and synaptic plasticity (Min et al. 2009). cytokines, such as tumor necrosis factor-a (TNF-a) Thus, many studies have suggested that chronic use and interleukin-1b (IL-1b), are closely correlated with of non-steroidal anti-inflammatory drugs (NSAIDs) the cognitive dysfunction primarily associated with the can prevent cognitive decline in individuals diagnosed progression of AD pathogenesis (Schwab and McGeer with developing AD dementia or in elderly people 2008; Mrak 2009). (Szekely et al. 2008). However, the long-term treat- Lipopolysaccharide (LPS), a non-infectious com- ments using NSAIDs can cause gastrointestinal side ponent of the outer membranes of Gram-negative effects and even occasional liver and kidney toxicities bacteria, when administered by intraventricular micro- (Knight et al. 1996; Warner et al. 1999; Graupera et al. injection or chronic infusion, induces neuroinflamma- 2003). These side effects have encouraged the develop- tion and impairment of memory function (Kim et al. ment of new NSAIDs that are safer for long-term 2000; Kitazawa et al. 2005). The memory dysfunction caused by LPS-induced inflammation has been hy- treatments (Kelloff et al. 2000). Recent studies have pothesized to play an important role in the pathogen- also suggested the use of herbal medicines or natural esis of the degenerative changes and cognitive products for treating Alzheimer’s-type, dementia- impairments (Cunningham et al. 2009). The pathogen- related disorders exhibiting cognitive memory impair- ment and neuroinflammation (Ho et al. 2011). esis of various neurodegenerative changes, and cogni- In Korean traditional medicine, Phellodendri tive and memory impairments, closely associated with Cortex extract (PCE) has been widely used to prevent AD or senile CNS dysfunction (Lukiw and Bazan bacterial infection of the respiratory system and to treat 2000). In fact, it is well established that LPS-induced inflammation in the hippocampus produces severe various chronic inflammatory diseases (Lu et al. 2011). *Corresponding author. Email: dhhahm@khu.ac.kr; dhahm@paran.com ISSN 1976-8354 print/ISSN 2151-2485 online # 2012 Korean Society for Integrative Biology http://dx.doi.org/10.1080/19768354.2012.699004 http://www.tandfonline.com NEUROBIOLOGY & PHYSIOLOGY Animal Cells and Systems 303 PCE has multiple pharmacological activities and 1.2 mM Na2HPO , and maintained at pH 7.4. The produces a variety of biological effects in the central lateral ventricle in the stereotaxic coordinate was nervous system (Zhang et al. 2009). Some studies have designated according to the Paxinos and Watson brain demonstrated that PCE alleviated LPS-induced acute atlas (AP: 0.2, L:90.3, DV: 6.2 referenced to the airway inflammation through decreasing the infiltra- bregma; Paxinos and Watson 1986). Artificial CSF or tion of inflammatory cells and releasing inflammatory LPS solution was injected for 5 min at a flow rate of mediators (Mao et al. 2010). It also attenuated LPS- 2 ml/min using 22-gauge Hamilton syringe and micro- induced inflammatory responses and inhibited the injection pump (Pump 22; Harvard Apparatus Inc., expression of TNF-a, IL-1b, and nitric oxide mRNAs Holliston, MA, USA). LPS (Escherichia coli; 055:B5) in BV2 mouse microglia cells (Park et al. 2007). These and ibuprofen were purchased from Sigma-Aldrich Co. studies suggest that PCE could be useful for suppres- (St. Louis, MO, USA). Administration of PCE was sing inflammation in neurodegenerative diseases. How- started 24 h after the lesion generation. ever, the underlying mechanism of pharmacological effect of PCE on improving LPS-induced cognitive Experimental groups deficits and spatial learning disability remains poorly understood. Rats were randomly divided into seven groups of seven The aim of the present study was to evaluate the individuals as follows: CSF-injected sham group being anti-inflammatory effects of PCE in improving learning regarded as normal (CON group, n7), CSF-injected and memory function in rats exposed to LPS-induced plus 200 mg/kg PCE-treated group (PCE group, n7), inflammation, as measured by performance on the LPS-injected plus saline-treated group (LPS group as passive avoidance test (PAT) and the Morris water a negative control, n7), LPS-injected plus 50 mg/kg maze (MWM) tests. We also examined how these PCE-treated group (PCE50 LPS group, n7), LPS- effects were related to the molecular modulation of injected plus 100 mg/kg PCE-treated group inflammation in terms of the neural mechanism under- (PCE100 LPS group, n7), LPS-injected plus lying the memory-enhancing activity of PCE. 200 mg/kg PCE-treated group (PCE200 LPS group, n7), and LPS-injected plus 40 mg/kg ibuprofen- treated (IBU LPS group as a positive control, n7). PCE extract was manufactured in the oriental Materials and methods pharmacy in Kyung Hee Hospital of Oriental Medi- Animals cine, Kyung Hee University (Seoul, Korea), according Adult male SpragueDawley (SD) rats weighing 260 to the protocol of Lee et al. (2004). IBU, a centrally 280 g were obtained from Samtako Animal Co. (Seoul, acting cholinesterase inhibitor, was used as a positive Korea). The rats were housed up to five per poly- control. The rats were intraperitoneally administrated carbonate cage in a limited-access rodent facility. The with PCE and IBU for 21 days, and PCE and IBU were room controls were set to maintain the temperature at dissolved in 0.9% physiological saline before use. The 22928C and the relative humidity at 55915%. Cages experimental schedule of all drug administration and were lit by artificial light for 12 h each day. Sterilized behavioral tests is shown in Figure 1. drinking water and standard chow diet were supplied ad libitum to each cage during the experiments. All animal experiments began at least seven days after the Open field test animals arrived. Prior to water maze testing, the rats were individually housed in a rectangular container that was made of black polyethylene (454535 cm) to provide best Lesion generation and LPS administration contrast to the white rats in a dimly lit room equipped Learning and memory deficits in the rats were induced with a video camera above the center of the room, and by bilateral intracranial injection (right and left side) of their locomotor activities (animal’s movements) were LPS, according to the procedures described previously then measured. The locomotor activity indicated by the (Guo et al. 2010; Lim et al. 2008). Fifty micrograms of speed and the distance of movements was monitored by LPS dissolved in 10-ml cerebrospinal fluid (CSF) a computerized video-tracking system using S-MART (Sigma-Aldrich Co., St. Louis, MO, USA) was micro- program (PanLab Co., Barcelona, Spain). After 5 min injected into the lateral ventricle of the rat brains in all of adaptation, the distance they traveled in the contain- lesion groups. Sham animals as a vehicle control er was recorded for another 5 min. The locomotor received microinjection of artificial CSF instead of activity was expressed in meters. The number of rearing LPS (Hwang et al. 2011). Artificial CSF consists of events of the rats was also recorded to analyze 140 mM NaCl, 3.0 Mm KCl, 2.5 mM CaCl locomotor activity in the open field test. , and 2 304 B. Lee et al. Figure 1. Experimental schedule of lesion generation, PCE administration, and behavioral tests in rats. PCE, Phellodendri Cortex; LPS, lipopolysaccharide. Passive avoidance test polypropylene and internally painted white. The pool was half-filled with water to 30 cm in depth. The water The test was performed according to the step-through in the pool was made opaque by adding 1 kg skim milk method described previously (Lee et al. 2011). The powder and continuously maintained at 22928C. The Gemini Avoidance System (SD Instruments., San Diego, pool was divided into four quadrants of equal area. CA, USA) was used for this test. The step-through During the MWM test, an escape platform (15 cm in passive avoidance apparatus consists of a tilting floor diameter) was located in one of four sections of the acrylic box divided into two compartments, a lightened pool being hidden 1.5 cm below the water surface and compartment connected to darkened one, by an auto- apart approximately 50 cm from the sidewalls. Several matic guillotine door and a control unit generating visual cues were placed around the pool in plain sight electric shock (Behbood Pardaz Co., Ghaem, Iran). of the animals. A digital camera was mounted to Electric shock can be delivered to the grid floors, made of stainless steel rods (3 mm diameter) spaced 1 cm the ceiling straight above the center of the pool and apart, in both compartments. First, the rats were taken was connected to a computerized recording system trials to acquisition test in the apparatus. In the training equipped with a tracking program (S-MART: PanLab session, a rat was placed in a lightened compartment of Co., Barcelona, Spain), which permitted on- and off- the passive avoidance apparatus facing away from the line automated tracking of the paths taken by the rats. entrance to the dark compartment, and then the guillotine door was opened. Because of intrinsic pre- ference to the dark environment, the rat immediately Hidden platform trial for acquisition test entered the dark compartment and the door was closed. The MWM test was initiated on the 14th day after During acquisition test, the latency time before entry injections of PCE extract and LPS. The MWM test was into the dark compartment was recorded for each rat. basically performed according to the MWM apparatus After 30 min, the rat was placed in the lightened and the method described previously (Lee et al. 2011). compartment once again. After entering the dark The animals received three trials per day. The rats were compartment, the guillotine door was closed and subse- trained to find the hidden platform, which remained in quently a mild electrical shock (0.5 mA) was applied a fixed location throughout the test. The trials lasted for 3 s. The retention test was started 24 h after the for a maximum of 180 s, and the escape latency was acquisition trial for training. The rat was again placed in expressed by the swimming time (or swim distance) to the lightened compartment and the guillotine door was find the submerged platform in the pool. The animals opened. In the retention test, the rat was placed in the were tested in three trials per day for five days, and they passive avoidance apparatus as previously described and received a 60-s probe trial on the sixth day. Finding the the time required for the rat to enter the dark compart- platform was defined as staying on it for at least 4 s ment was measured for a maximum period of 3 min in before the acquisition time of 180 s ended. When the the same method with the acquisition test. The rat did rat failed to find the platform in the limited time in the not enter the dark compartment within this period received a latency time of 180 s (Mohamed et al. 2000). first trial of hidden platform test, the rat should be placed on the platform for 20 s and assigned a latency of 180 s. Between one trial and the next, the water in MWM test the pool was stirred to remove olfactory traces of MWM apparatus previous swim patterns. The entire schedule proceeded for six days and each animal had three trials for The MWM test was performed in a small circular pool (2.0 m in diameter and 0.35 m deep) made of training per day with 3040 min inter-trial interval. Animal Cells and Systems 305 Probe trial for retention test the numbers of cells within 100100-um grids were counted by observers blinded to the experimental For the probe trial, a rat was placed in the quadrant groups. Counting immunopositive cells was performed located diagonally from the target quadrant and in at least three different hippocampal sections per rat allowed to swim to the quadrant from which the escape brain. Stained sections were randomly chosen from platform had been removed for a maximum of 60 s. equal levels of serial sections along the rostralcaudal The probe trial was expressed by the ratio of the time axis. The TNF-a-, IL-1b-, and COX-2-immunopositive spent (or the distance) for searching the platform in the cells were only counted when their densities reached a target quadrant to total duration spent for swimming defined darkness above the background level (Guo in the pool. et al. 2010). Distinct brown spots for TNF-a, IL-1b, and COX-2 were observed in the cytoplasms and in the membranes of the cone-shaped cells of the hippocam- Immunohistochemistry for proinflammatory markers pus. The cells were anatomically localized according to For immunohistochemical studies, the animals were the stereotactic rat brain atlas of Paxinos and Watson deeply anesthetized with sodium pentobarbital (80 mg/ (1986). The brightness and contrast between images kg, by intraperitoneal injection) and perfused through were not adjusted to exclude any possibility of sub- the ascending aorta with normal saline (0.9%) followed jective selection of immunoreactive cells. by 300 ml (per rat) of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The brains were Total RNA preparation and RT-PCR analysis removed in a randomized order, post-fixed over-night, and cryoprotected with 20% sucrose in 0.1 M PBS at The expression levels of TNF-a, IL-1b, and COX-2 48C. Coronal sections with 30 mm thickness were cut mRNAs were determined by the reverse transcription- through the hippocampus using a cryostat (Leica polymerase chain reaction (RT-PCR). The brain hip- CM1850; Leica Microsystems Ltd., Nussloch, Ger- pocampus was isolated from three rats per group. The many). The sections were obtained according to the rat total RNAs were prepared from the brain tissues atlas of Paxinos and Watson (hippocampus; between using a TRIzol reagent (Invitrogen Co., Carlsbad, bregma 2.6 and 3.6) (Paxinos and Watson 1986). CA, USA) according to the supplier’s instruction. The sections were immunostained for TNF-a, IL-1b, Complementary DNA was first synthesized from total and cyclooxygenase-2 (COX-2) expression using the RNA using a reverse transcriptase (Takara Co., Shiga, avidinbiotinperoxidase complex (ABC) method. The Japan). PCR was performed using a PTC-100 pro- sections were incubated with primary rabbit anti-TNF- grammable thermal controller (MJ Research, Inc., a antibody (1:200 dilution; Novus Biochemicals LLC., Watertown, MA, USA). All primers were designed Littleton, CO, USA), rabbit anti-IL-1b antibody (1:200 using published mRNA sequences of those cytokines dilution; Santa Cruz Biotechnology Inc., Santa Cruz, and a primer designing software, Primer 3, offered by CA, USA), and goat anti-COX-2 antibody (1:200 the Whitehead Institute for Biomedical Research dilution; Cambridge Research Biochemicals Co., (Cambridge, MA, USA; www.genome.wi.mit.edu) on Bellingham, UK) in PBST (PBS plus 0.3% Triton the website. The PCR products were separated on 1.2% X-100) for 72 h at 48C, respectively. The sections were agarose gels and stained with ethidium bromide. The incubated for 120 min at room temperature with density of each band was quantified using an image- TM biotinylated anti-rabbit goat IgG (for the anti-TNF-a analyzing system (i-Max , CoreBio System Co., antibody and anti-IL-1b antibody) and biotinylated Seoul, Korea). The expression levels were compared anti-goat rabbit IgG (for the anti-COX-2 antibody). each other by calculating the relative density of target The secondary antibodies were obtained from Vector band, such as TNF-a, IL-1b, and COX-2, to that of Laboratories Co. (Burlingame, CA, USA) and diluted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). 1:200 in PBST containing 2% normal serum. To visualize immunoreactivity, the sections were incubated Statistical analysis for 90 min in ABC reagent (Vectastain Elite ABC kit; Vector Labs. Co., Burlingame, CA, USA), and incu- All measurements were performed by an independent bated in a solution containing 3,3?-diaminobenzidine investigator blinded to the experimental conditions. (DAB; Sigma-Aldrich Co., St. Louis, MO, USA) and Results in figures are expressed as mean9standard 0.01% H O for 1 min. Images were captured using an error of means. Differences within or between normally 2 2 AxioVision 3.0 imaging system (Carl Zeiss, Inc., distributed data were analyzed by analysis of variance Oberkochen, Germany) and processed using Adobe using SPSS (Version 13.0; SPSS, Inc., Chicago, IL, Photoshop (Adobe Systems, Inc., San Jose, CA, USA). USA) followed by Tukey’s post hoc test. Statistical The sections were viewed at 200 magnification, and significance was set at pB0.05. 306 B. Lee et al. Results behavior in the open field test. However, the PCE- treated rats (200 mg/kg) significantly increased their Effect of PCE on LPS-induced body weight loss locomotor activity and rearing, as compared to those Rats exposed to LPS infusion begin to lose their body in the LPS group (pB0.05). Therefore, the PCE-treated weights on the first day of LPS infusion, and this LPS- rats showed remarkable enhancements of the active induced initial reduction of body weight is sustained responses via stimulating motivation required for for three days without restoring to normal level or even exploring for 21 days, ultimately indicating that the exacerbated in some cases (Gaab et al. 2005). The LPS- treatment of PCE significantly restored locomotor injected rats gradually gained less body weights over activity and exploratory behavior and accordingly the treatment period than did the sham control rats in alleviated LPS-induced impairment of motor function the CON group (Figure 2a). Analysis of body weight behavior in rats. values revealed no significant differences among the seven groups on the first day prior to starting LPS infusion. However, there was a significant reduction in Effect of PCE on LPS-induced step-through latency body weight gain during 21 days of the treatment in the deficit in the PAT LPS group, as compared to the CON group (pB0.001). Interestingly, body weights of the 100 mg/kg PCE- To determine whether PCE promotes the recovery of treated rats and 200 mg/kg PCE-treated rats showed memory dysfunction, PCE was administrated in the significant inhibition of reductions of body weight gain rats with LPS-induced impairment of memory, and during 21 days of the treatment, as compared to the their memory and cognitive functions were examined LPS group (pB0.05 and pB0.01, respectively). It also in the PAT (Figure 3). It was verified that the rats in indicated that the recovery of body weights in the all groups had no physiological defects (i.e. motor PCE100 LPS and PCE200 LPS groups was closely function) or intrinsic cognitive impairments through associated with those in the IBU LPS group. acquisition trials without electric challenge. In the time for acquisition trials, indicated by the latencies for entering the dark compartment, there were no signifi- Effect of PCE on LPS-induced locomotor disability in cant differences among all groups. After acquisition the open field test trials, the effect of PCE on the retention latency was examined 24 hours after applying electric shock in the Open field activity was used to evaluate locomotor dark box in the PAT. In the retention, it was shown that activity and exploratory behavior in the rats that the rats in the PCE200 LPS group had significantly received an LPS injection into the lateral ventricle increased latencies to enter the dark compartment for (Figure 2b). These results indicated that the rats with retention as compared to those in the LPS group LPS infusion significantly reduced their locomotor (pB0.05). This study indicated that the LPS activities and total numbers of rearing, as compared infusion severely impaired long-term memory, and with those in the CON group (pB0.05). It suggested that the rats, treated with LPS, subsequently produced the treatment of PCE significantly attenuated LPS- impairments of motor and exploration activities which induced memory deficit in the PAT. It also indicated have been closely associated with motor function that the restoration of memory function in the Figure 2. Effects of PCE on body weight gain on the first day prior to LPS injection and on the 21th day after LPS injection, and the activity counts of locomotor activity and the total numbers of rearings in the open field test on the 21th day. PCE, * *** # Phellodendri Cortex extract; LPS, lipopolysaccharide; IBU, ibuprofen. pB0.01, pB0.001 vs. CON group; pB0.05 and ## ### pB0.01, pB0.001 vs. LPS group. Animal Cells and Systems 307 Figure 3. Effects of PCE on the latencies of entering the dark compartment on the acquisition trial and on the retention test in ** # the passive avoidance test. pB0.01 vs. CON group; pB0.05 vs. LPS group. PCE200 LPS group was almost close to that in the The rats with LPS infusion showed severe impairment IBU LPS group. of spatial performance in the MWM (pB0.001). The rats in the 200 mg/kg PCE-treated group spent more time (pB0.05) and distance (pB0.001) around the platform area than did those in the LPS group. Effect of PCE on LPS-induced spatial memory The mean swimming speeds, as calculated by dividing impairment in the water maze test the total swim distance by latency (Figure 4e), of the The effect of the PCE treatment on swimming to reach rats in the LPS group were not significantly different the submerged platform in the MWM test is shown in from those in other groups. On the basis of these Figure 4. In the present study, a spatial learning and results, it was suggested that 200 mg/kg PCE-treated memory deficit rat model was established by infusion rats exhibited better improvement of acquisition in the of LPS into the lateral ventricle, by which the escape hidden platform trial, and this memory improvement latency and the searching distance were significantly resulted in more rapid escape from the water than did increased as compared with those in the CON group. the LPS-treated rats. Administration of PCE signifi- Analysis of escape latency revealed that the rats in the cantly attenuated LPS-induced deficits of learning and PCE200 LPS group significantly reduced swimming memory demonstrated in the MWM test. Thus, PCE- latency as compared with those in the LPS group treated rats showed a significant amelioration in the (PCE200 LPS group: pB0.05 on day 3, and 5, memory retention test because they spent more time pB0.01 on day 4; Figure 4a). Swimming distance and distance in the quadrant where the platform was traveled in each group was closely associated with formerly located and swam over the location of plat- escape latency in this task. Analysis of searching form more frequently. It also indicated that swimming distance values revealed that the rats in the latency of the LPS-injected rats receiving 200 mg/kg PCE200 LPS group significantly reduced swimming PCE was closely associated with those of rats receiving distance as compared with those in the LPS group 40 mg/kg IBU. (PCE200 LPS group: pB0.05 on day 4, pB0.01 on day 5; Figure 4b). The PCE treatment of 200 mg/kg improved spatial learning ability and memory function Effects of PCE on LPS-induced immunohistochemical in the rats with chronic inflammation in their brain by changes of inflammatory mediators LPS infusion, as evidenced by decreases in escape latency and searching distance throughout the training Following the behavioral tasks, brain tissue samples period. To investigate the effect on the spatial memory, from the subjects were analyzed using immunohisto- the performance in the probe trial on day 6 was chemistry to investigate the effect of PCE administra- examined by analyzing the percentages of time and tion on the expression of pro-inflammatory markers distance required for swimming to the expected posi- formerly activated by LPS-induced inflammation in the tion of the platform, respectively (Figure 4c,d). The rat brains (Figure 5A). The brain immunohistochem- swimming times and distances are reduced in the rat istry in the LPS group showed a significant increase that directly swims to the target quadrant where the in TNF-a, IL-1b, and COX-2 expression in the escape platform had been removed without confusion. hippocampus, as compared to those in the CON 308 B. Lee et al. Figure 4. Effects of PCE on latency of escaping from water (a) and swimming distance (b) during acquisition trials using a submerged platform, the percentages of time (c) and distance (d) in a probe trial without a platform, and swimming speed (e) in * *** # ## ### the Morris water maze test. pB0.05 and pB0.001 vs. CON group; pB0.05, pB0.01 and pB0.001 vs. LPS group. group (pB0.01). TNF-a immunoreactive cells signifi- IBU LPS group. These results clearly indicated that cantly decreased in the hippocampal regions in the the LPS-induced increase in the expression of TNF-a PCE100 LPS (pB0.01) and PCE200 LPS groups and COX-2 in the rat hippocampus was significantly (pB0.05), as compared to those in the LPS group attenuated by the treatment of PCE. (Figure 5B). Despite appearance of IL-1b-immunor- eactive cells in the hippocampal regions in the Effect of PCE on LPS-induced expression of TNF-a, PCE200 LPS group, there was no statistical signifi- IL-1 b, and COX-2 mRNAs in the hippocampus cance as compared to the LPS group (Figure 5B). Similarly, the COX-2-immunoreactive cells were sig- The effect of PCE administration on LPS-induced nificantly increased in the hippocampal region in the expression of TNF-a, IL-1b, and COX-2 mRNAs in PCE200 LPS group (pB0.05), as compared to the rat hippocampus was investigated using RT-PCR those in the LPS group (Figure 5B). The increases in analysis (Figure 6). Hippocampal expression of TNF-a, the pro-inflammatory mediators such as TNF-a, IL-1b, IL-1b, and COX-2 mRNA in the LPS group was and COX-2 by LPS-induced inflammation were significantly increased as compared to that in the CON significantly inhibited by PCE administration, and group (pB0.01). In case of PCE administration of their restorations were almost close to those in the 200 mg/kg, the increased expression of TNF-a, IL-1b, Animal Cells and Systems 309 Figure 5. Effects of PCE on the mean number of tumor necrosis factor-a (TNF-a)-, interleukin-1b (IL-1b)-, and cyclooxygenase- 2 (COX-2)-stained hippocampal areas after the Morris water maze test. Representative photographs and the relative percentage values are indicated in (A) and (B), respectively. (a) TNF-a expression in the CON group, (b) TNF-a expression in the LPS group, (c) TNF-a expression in the PCE200 LPS group, (d) IL-1b expression in the CON group, (e) IL-1b expression in the LPS group, (f) IL-1b expression in the PCE200 LPS group, (g) COX-2 expression in the CON group, (h) COX-2 expression in the LPS group, (i) COX-2 expression in the PCE200 LPS group. Sections were cut coronally at 30 mm. Scale bar indicates 50 mm. ** # ## pB0.01 vs. CON group; pB0.05 and pB0.01 vs. LPS group. and COX-2 mRNA in the LPS group was significantly also alleviated LPS-stimulated inflammatory responses, restored (pB0.05). The restored levels of these inflam- including LPS-induced expression of TNF-a, IL-1b, matory mediators were almost close to those in the and iNOS through the negative modulation of nuclear IBU LPS group. factor-kappaB (NF-kB) signaling in the microglial cells (Park et al. 2007). Thus, these studies suggest a new medicinal role of PCE on suppressing inflammatory Discussion response in the central nervous system, which can lead to developing a novel therapeutics using PCE for In the Korean traditional medicine, PCE has been treating neurodegenerative diseases in humans. widely used to treat chronic inflammatory diseases and Our results clearly demonstrated that the admin- prevent bacterial infection of the respiratory system istration of PCE significantly improved spatial learn- and gastrointestinal tract (Yang et al. 2010; Lu et al. ing and memory impairments induced by LPS 2011). Recently, several studies have shown that PCE infusions into the bilateral ventricles, as evidenced had a protective role against ischemia-reperfusion- by the behavior in the PAT and MWM tests. It induced brain injury through the reduction of neutro- phil infiltration into the brain (Hwang et al. 2002b). It also attenuated the increase in pro-inflammatory Figure 6. Effects of PCE on the expression of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and cyclooxygenase-2 (COX-2) mRNAs in rats with LPS-induced hippocampal impairment. PCR bands on agarose gel and their relative intensities are indicated in (a) and (b), respectively. The expression levels of TNF-a, IL-1b, and COX-2 mRNAs were normalized to that of ** # glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. pB0.01 vs. CON group; pB0.05 vs. LPS group. 310 B. Lee et al. markers such as TNF-a, IL-1b, and COX-2 in the brain dysfunction (Ringheim and Conant 2004). hippocampus, as assessed by RT-PCR analysis and According to the inflammation hypothesis, the memory immunohistochemistry. impairment in patients with senile dementia is due We found that the administration of PCE after LPS to selective and irreversible dysfunction and chronic infusion into the lateral ventricle markedly restored inflammation in the brain (Hoozemans et al. 2001). body weight gain and motor activity, suggesting that Thus, we propose that the anti-inflammatory effects PCE could inhibit the physiological changes and the of PCE significantly reversed the impairment in sickness behavioral symptoms caused by LPS-induced memory retention and the increased expression of pro- neuroinflammation. It can be suggested that PCE inflammatory cytokines such as TNF-a and IL-1b. enhanced resistance against stress and various psycho- Also, much experimental evidence has suggested somatic disorders and exerted various benefits, that the inducible gene encoding COX-2 is a key strengthening the immune system. element that modulates the generation of proinflam- To identify the effects of PCE on two different types matory mediators including various prostagladins of memory, cognitive memory and spatial learning, we (Fehe ´r et al. 2010). It is known that the expression of used the PAT and MWM, respectively. Our results COX-2 and the synthesis of prostaglandin E2, one demonstrated that the administration of PCE signifi- of its products, are increased in the hippocampus of cantly increased the step-through latency in the mem- patients with AD (Fujimi et al. 2007), which may be ory retention trial, which had been shortened by LPS related to the pathogenesis of the degenerative changes infusion, in the PAT. During the trial sessions in the and cognitive impairments (Hwang et al. 2002a). These MWM, administration of PCE resulted in a significant results suggest that inflammatory responses to LPS reduction in the escape latency, enhanced cognitive infusion significantly stimulated the expression of performance, and ameliorated of memory deficits, COX-2 protein or mRNA in the hippocampus through associated with LPS injection. These results showed modulating the NF-kB pathway (Gong et al. 2011). that LPS infusion into the lateral ventricle of rat brains The increase in COX-2 expression by NF-kB activation retarded escape latency in the MWM test, indicating can accelerate inflammatory responses and subse- deficits in spatial learning ability and reference mem- quently contribute to learning and memory deficits. ory. The scoring of the escape latency from water in Thus, treatment with long-lasting COX-2 inhibitors the spatial probe tests in the MWM test is primarily during the initial stages of AD, before clinical symp- considered to reflect long-term spatial memory ability toms of dementia appear, may suppress inflammatory (Lee et al. 2011). In this study, administration of PCE responses and the synthesis of pro-inflammatory med- shortened the escape latency without affecting swim- iators in the brain (Kumar et al. 2006). In the present ming velocity and extended the time spent swimming in study, PCE significantly decreased LPS-stimulated the place where the platform was previously located. behavioral changes and memory disturbances through This indicated that the administration of PCE signifi- inhibition of COX-2 mRNA expression. cantly improved the long-term memory deficit in rats In summary, we demonstrated that PCE signifi- that had suffered LPS-induced spatial memory impair- cantly improved spatial learning and memory abilities ment. From the results of the PAT and MWM tests, we in the rats with LPS-induced brain dysfunction, as concluded that a chronic inflammatory response played evidenced by performance in the PAT and the MWM an important role in learning acquisition and synaptic tests. PCE also suppressed LPS-simulated mRNA plasticity (Collister and Albensi 2005). expression of pro-inflammatory mediators such as Our results also showed that LPS infusion into the TNF-a, IL-1b, and COX-2 in the hippocampus. lateral ventricle significantly increased the expression Thus, PCE may be useful as an alternative medicine of TNF-a and IL-1b in the hippocampus, ultimately for treating or retarding the development of neurode- leading to a chronic neuroinflammatory response in the generative diseases including AD. brain. Thus, LPS-stimulated sustained increases in the expression of pro-inflammatory cytokines have been directly linked to neurodegenerative disorders asso- Acknowledgements ciated with decreased working memory (Frank-Cannon This work was supported by a grant from the Kyung Hee et al. 2009). 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Journal
Animal Cells and Systems
– Taylor & Francis
Published: Aug 1, 2012
Keywords: lipopolysaccharide; memory; inflammation; Phellodendri Cortex; Morris water maze
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References
Immunological aspects of alzheimer's disease: therapeutic implications
Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease
Neuropathology and the neuroinflammation idea
Neurodegenerative disease and the neuroimmune axis (Alzheimer's and Parkinson's disease, and viral infections
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