Uncaria rhynchophylla and rhynchophylline improved kainic acid-induced epileptic seizures via IL-1[beta] and brain-derived neurotrophic factor.
Uncaria rhynchophylla (UR) has been used for the treatment of convulsions and epilepsy in traditional Chinese medicine. This study reported the major anti-convulsive signaling pathways and effective targets of UR and rhynchophylline (RP) using genomic and immunohistochemical studies. Epileptic seizure model was established by intraperitoneal injection of kainic acid (KA) in rats. Electroencephalogram and electromyogram recordings indicated that UR and RP improved KA-induced epileptic seizures. Toll-like receptor (TLR) and neurotrophin signaling pathways were regulated by UR in both cortex and hippocampus of KA-treated rats. KA upregulated the expression levels of interleukin-1[beta] (IL-1[beta]) and brain-derived neurotrophin factor (BDNF), which were involved in TLR and neurotrophin signaling pathways, respectively. However, UR and RP downregulated the KA-induced IL-lfl and BDNF gene expressions. Our findings suggested that UR and RP exhibited anti-convulsive effects in KA-induced rats via the regulation of TLR and neurotrophin signaling pathways, and the subsequent inhibition of IL-1[beta] and BDNF gene expressions.
Brain-derived neurotrophic factor
Uncaria rhynchophylla (Miq.) Jacks (UR), the dried stems of Uncaria, is an important traditional Chinese medicinal herb commonly used for the treatment of neural associated disorders for years in China (Shi et al., 2003). Its aqueous extract has been used to treat neuropsychiatrie symptoms, such as headache, dizziness, tremors, hypertension-induced convulsion, and epilepsy (Hsieh et al., 1999a,b; Heitzman et al., 2005; Lin and Hsieh, 2011). The extracts of UR contain various components, such as corynoxine, corynoxeine, isocorynoxeine, rhynchophylline (RP), isorhynchophylline, and hirsutine (Xian et al., 2012). Among these compounds, RP and isorhynchophylline have been known as neuroprotective compounds (Kang et al., 2004; Yuan et al., 2009).
UR exhibits anti-epileptic effects in kainic acid (KA)-induced epileptic seizures and its inhibition is associated with a scavenging activity of oxygen free radicals (Hsieh et al., 1999a,b). In addition, UR displays neuroprotective effects via the inhibition of cyciooxygenase-2 (COX-2) and the subsequent reduction of inflammation in rats with transient global ischemia (Suk et al., 2002). UR inhibits microglia activation, neuronal nitric oxide synthase (nNOS) activity, and inducible NOS (iNOS) activity (Tang et al., 2010), and attenuates glial cell proliferation and S100B expression in KA-treated rats (Lin and Hsieh, 2011). Moreover, UR inhibits N-methyl-D-aspartate (NMDA) receptor activation and suppresses NMDA-induced apoptosis (Lee et al., 2003a,b). RP, one of the major components of UR, displays neuroprotection and anti-convulsive actions (Kang et al., 2002, 2004). Several studies indicated that RP inhibits [Ca.sup.2+] influx and plays a neuroprotection in glutamate-induced neuronal death (Shimada et al, 1999; Xu et al., 2012). Although the anti-convulsive effects of UR and RP has been studied, the molecular mechanisms of UR and RP remains to be elucidated.
The anti-epileptic effects of UR in KA-induced epileptic seizures might be associated with various signaling pathways and therapeutic targets. Microarray analysis which provides the whole genomic expression profiling can be applied to identify signaling pathways and therapeutic targets of drugs. By the comparison of expression profiles, we have identified and classified the differentially expressed genes to find the EGb761-affected signaling pathways (Su et al., 2009). In addition, based on the similarity of gene expression signatures, we have predicted the therapeutic effects and safeties of Chinese herbal formulae (Cheng et al., 2010). Moreover, the novel therapeutic targets of San-Huang-Xie-Xin-Tang, silymarin, genipin, and Momordica charanda have been recognized by microarray analysis (Cheng et al., 2008; Li et al., 2012a,b,c; Lo et al., 2013). Therefore, the purpose of the present study was to investigate the anti-convulsive signaling pathways and targets of UR and RP using genomic and immunohistochemistry (1HC) studies.
Materials and methods
Preparation and high-performance liquid chromatography (HPLC) analysis of UR extract
UR purchased from China, was authenticated and extracted by Koda Pharmaceutical Company (Taoyuan, Taiwan), a good manufacturing pharmaceutical factory in Taiwan. Extraction of UR was performed as described previously (Hsieh et al., 2009). Briefly, UR (80 kg) was extracted by boiling in 64 kg of 70% alcohol for 35 min. The extracts were filtered with 100-mesh filters, freeze-dried, and then stored in a dried box. The total yield was 566.63 g (7.08%). The HPLC fingerprint of UR extract was analyzed as described previously (Wagner et al., 2011). HPLC was performed on a Hitachi HPLC system (Japan) equipped with Hitachi Chromaster 5110 pump, 5210 auto sampler, 5310 column oven, and 5410 UV detector. The column used was LiChrospher[R] 60 RP-select B (125 mm x 4 mm, 5 [micro]m) with LiChroCART[R] 4-4 (Merck, Germany). The mobile phase was composed of solvent A (10 mM phosphate buffer, pH 6.6) and solvent B (acetonitrile-methanol at 1:1). The solvent gradient was as follows: 20% solvent B to 75% solvent B in 25 min; 75% solvent B for 13 min. The HPLC fingerprint is shown in Fig. 1.The amount of RP in freeze-dried extracts of UR were also quantitated by a HPLC system (Hitachi D-7000 interface, L-7100 pump, L-7200 Autosampler, L-7455DAD, Japan) using RP (Matsuura Yakugyo Co. Ltd., Japan) as a reference standard (Supplementary Fig. S1). The column used was COSMOSIL 5C18-MS (250 mm x 4.6 mm, 5 pm) (Nacalai USA, San Diego, CA, USA). The mobile phase was acetonitrile-acetic buffer at 1:4. Each gram of freeze-dried extract of UR contained 1.81 mg of pure alkaloid RP component.
Male Sprague-Dawley (SD) rats, weighing 200-300g, were purchased from BioLasco Taiwan Co., Ltd (Taipei, Taiwan). Rats were housed in standard iron cages on a 12-h light-dark cycle at 22 [+ or -] 2[degrees]C and 55 [+ or -] 5% humidity, and had free access to food and water. All animal experiments were performed according to the Guidelines of the Chinese Society for Laboratory.
A total of 45 SD rats were examined in this study. At least four days prior to electroencephalogram (EEG) and electromyogram (EMG) recordings, the head of rat was fixed in a stereotactic apparatus under chloral hydrate (400 mg/kg, intraperitoneal) anesthesia. The stainless steel screws were implanted over epidural region located on the bilateral sensory-motor cortices to serve as the recording electrodes, while another electrode was placed on the frontal sinus to serve as a reference for EEG recordings. Two electrode wires were implanted around the neck muscle for EMG recording. Finally, these electrodes were plugged into a relay and then connected to an EEG and EMG-recorder(MP100WSW, BIOPAC System Inc., Goleta, CA, USA).
Rats were randomly divided into five groups of 9 rats: (1) phosphate buffered saline (PBS) group, intraperitoneal injection of PBS (1ml/kg) only; (2) KA group, intraperitoneal injection of KA (12 mg/kg; Sigma, St. Louis, MO, USA) only; (3) UR group, oral administration of UR (1 g/kg/day) 2 days and 15 min prior to KA injection; (4) RP group, intraperitoneal injection of RP (0.25 mg/kg/day) 2 days and 15 min prior to KA injection; (5) valproic acid (VA) group, intraperitoneal injection of VA (250 mg/kg/day; Sigma, St. Louis, MO, USA) 2 days and 15 min prior to KA injection. The behavioral observation, and EEG and EMG recordings were performed from 15 min before and 3h after KA injection. Six rats in groups 1-4 were sacrificed 3h after KA injection and rat brains were divided into right and left brains. Right brains were applied for microarray analysis and left brains were applied for IHC staining. The other 3 rats in groups 1-4 were sacrificed 24 h after KA injection for IHC study.
Total RNAs were extracted from cortex and hippocampus tissues of rats in PBS, KA, and UR groups using RNeasy Mini kit (Qiagen, Valencia, CA, USA). RNAs were evaluated by Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and the RNA samples with RNA integrity numbers greater than 8.0 were accepted for microarray analysis. Microarray analysis was performed as described previously (Chang et al., 2011; Li et al., 2012a,b,c). Briefly, fluorescent RNA targets were prepared using Message AMP[TM] aRNA kit (Ambion, Austin, TX, USA) and Cy5 dye (Amersham Pharmacia, Piscataway, NJ, USA), and then hybridized to Rat Ref-12 Expression BeadChip (Illumina, San Diego, CA, USA). The Cy5 fluorescent intensity of each spot was scanned by an Axon 4000 scanner (Molecular Devices, Sunnyvale, CA, USA) and analyzed by genepix 4.1 software (Molecular Devices). The signal intensity of each spot was corrected by subtracting background signals in the surroundings. Spots were normalized by the R program in limma package using quantiles normalization (Smyth and Speed, 2003) and normalized data were tested for differential expression using Gene Expression Pattern Analysis Suite (GEPAS) (Montaner et al., 2006). The p-value of each gene was calculated by t-statistics using Differential Expression tool on GEPAS web site (http://gepas3.bioinfo.cipf.es/). These differentially expressed genes (p < 0.01) were further analyzed for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway by geneSetTest function in the R program. This function computes a p-value to test the hypothesis that the selected genes in a pathway tend to be differentially expressed. The p-values were adjusted for multiple testing by calculating the false discovery rate (FDR) for each pathway. Pathways with FDR <0.5 were considered significantly.
Sections of 15 [micro]m were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase was quenched with 0.9% hydrogen peroxide in methanol for 15 min and the non-specific binding was blocked with 1% bovine serum albumin at room temperature for 1 h. Sections were incubated with interleukin-1[beta] (IL-[beta]) antibody (Abbiotec[TM], San Diego, CA, USA) at 1:200 dilution or brain-derived neurotrophic factor (BDNF) antibody (Millipore, Billerica, MA, USA) at 1:1000 dilution at 4[degrees]C overnight and then incubated with biotinylated secondary antibody at room temperature for 20 min. Finally, slides were incubated with avidin-biotin complex reagent and stained with 3,3-diaminobenzidine tetrahydrochloride (Histostain[R]-Plus kit, Zymed Laboratories, San Francisco, CA, USA).
Data were presented as mean [+ or -] standard error. Student's t test was used for comparisons between two experiments. A value of p < 0.05 was considered statistically significant.
Effects of UR and RP on KA-induced epileptic seizures in SD rats
KA, an excitotoxic analog of glutamate, induces epileptic seizures in rats that resemble to temporal lobe epilepsy in human (Tanaka et al., 1992). After KA administration, all rats developed epileptic seizures, which were judged by wet dog shakes, paw tremors, and facial myoclonia. Moreover, each type of seizure behaviors had its own characteristic EEG activity (Fig. 2). Total counts of wet dog shakes, paw tremors, and facial myoclonia were significantly reduced by the pretreatment of UR (1g/kg) and RP (0.25mg/kg) (Fig. 3). As expected, the antiepileptic drug VA also significantly the total counts of wet dog shakes, paw tremors, and facial myoclonia in KA-induced rats. These data indicated that UR and RP ameliorated KA-induced epileptic seizures in rats.
Pathway analysis of UR-regulated gene expression profiles in rats with KA-induced epileptic seizures
To investigate which biological pathways were involved in anticonvulsive effect of UR, we harvested cortex and hippocampus tissues from KA-induced rats given without or with 1 g/kg of UR. The gene expression profiles were analyzed by oligonucleotide microarray and microarray data were then analyzed by CEPAS to identify the differentially expressed genes. We further selected UR-regulated genes in cortex and hippocampus regions and tested for the biological pathways by geneSetTest function. Table 1 shows that a total of 16 pathways in both cortex and hippocampus regions was regulated (FDR<0.5) by KA and UR. Among 16 pathways, 12 pathways, such as Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling pathways, were related to immune responses, and two pathways, including neurotrophin signaling pathway and prion diseases, were associated with nervous system. These findings suggested that UR might exhibit anti-convulsive effects via the regulation of immune response and neurotrophin signaling pathway in KA-induced epileptic seizures.
Expression levels of UR-regulated genes in neurotrophin and TLR signaling pathways
To explore the pharmacological and molecular mechanisms of UR, we examined the expression levels of genes in neurotrophin and TLR signaling pathways. Table 2 shows that KA upregulated the expressions of genes in both neurotrophin and TLR signaling pathways in cortex and hippocampus regions, while UR suppressed the expression of KA-upregulated genes. KA upregulated and UR down-regulated the expressions of nuclear factor of kappa light chain (Nfkbia) gene and IL-1[beta] gene, which are associated with inflammation. The expressions of Fos gene, Jun oncogene, and Map2k3 gene, which are related to MAPK pathways, were also regulated by KA and UR. Moreover, Bdnf gene, which is involved in neuron survival, was affected by KA and UR. These findings suggested that UR might ameliorate KA-induced seizures via regulating the expression of genes involved in neuron survival and inflammation.
Verification of the expression of IL-1[beta] and BDNF genes in KA-induced rats given with UR and RP by IHC staining
Rat brains were removed, sectioned, and stained with anti-IL-1[beta] and anti-BDNF antibodies. As shown in Fig. 4, no prominent IL-1[beta]-immunoreactive cells and BDNF-immunoreactive cells were observed in both cortex and hippocampus at 3 h after KA administration. However, IL-1[beta]- and BDNF-immunoreactive cells were prominently noted in the cortex and hippocampus at 24 h after KA administration. Administration of UR or RP decreased the number of IL-1 (3- and BDNF-immunoreactive cells in rat brains. These findings suggested that KA stimulated the production of IL-1[beta] and BDNF, while UR and RP suppressed KA-induced expressions of IL-1[beta] and BDNF genes in brains.
Present study indicated that rats developed epileptic seizures after KA administration, and the behaviors of epileptic seizure included wet shakes, paw tremor, and facial myoclonia. Administration of UR (1 g/kg) and RP (0.25 mg/kg) improved the behaviors of wet shakes, paw tremor, and facial myoclonia. These results suggested that UR and RP had anti-convulsive effects. Our previous study has shown that UR and RP reduce superoxide anion production, c-Jun-N-terminal kinase phosphorylation, and nuclear factor-[kappa]B (NF-[kappa]B) activity, and in turn, decrease the epileptic seizures induced by KA (Hsieh et al., 2009). In this study, we found that the expressions of IL-1[beta] and BDNF genes were upregulated at 3 h after KA administration, and these upregulations were suppressed by the pretreatment of UR and RP. Although IL-1[beta]- and BDNF-immunoreactive cells in cortex and hippocampus could not be found clearly at 3 h after KA administration, these cells were noted prominently at 24 h after KA administration and the number of immunoreactive cells was decreased by UR and RP pretreatment. Taken together, we suggested that both UR and RP exhibited anticonvulsive effects and their anti-convulsive potentials might be related to the inhibition of IL-1[beta] and BDNF gene expression.
Seizures induce the inflammatory response of central nervous system, and this neuro-inflammation may influence the development and the severity of seizures (Vezzani et al., 2011). Inflammatory response plays a critical role in neuronal excitability, seizure frequency, and duration (Silveira et al., 2012). The pro-inflammatory pathways, such as IL-1 receptor (IL-1R)/TLR, transforming growth factor and COX-2 signaling pathways, are involved in epileptogenesis because seizures may activate microglias, astrocytes, and neurons, and followed by the release of IL-1[beta]. Moreover, neuronal hyperexcitability induced by IL-1R/TLR-mediated NF-[kappa]B activation also contributes to the development of epileptogenesis and inflammation (Vezzani et al., 2013). Seizure possibly mediates the activation of IL-1R/TLR signaling pathway to induce brain inflammation because anakinra, a human recombinant IL-1[beta] receptor antagonist, can terminate seizures and prevent seizures recurrent (Librizzi et al., 2012). The productions of pro-inflammatory cytokines, such as IL-1[beta], IL-6 and tumor necrosis factor-[alpha] (TNF-[alpha]), are increased in the hippocampus after seizure in limbic epileptic animal model (Li et al., 2011). IL-1[beta] increases at 2 h after limbic status epilepticus in rats, the maximal increase is achieved at 6 h, and IL-1[beta] mRNA maintains 60 days after seizures (De Simoni et al., 2000). Moreover, seizures induce IL-1 (3 release in cortex and hippocampus in bicuculline rat model and further cause blood-brain barrier damage with protein extravasation (Li et al., 2011; Librizzi et al., 2012). The seizure activities are increased by intrahippocampal injection of IL-1 (3, but the seizure is delayed by IL-Ira, a powerful anticonvulsant (Vezzani et al., 2002). The decreased serum levels of IL-1[beta], TNF-[alpha], and macrophage inflammatory protein-la after resection of epileptic focus abolish epilepsy in human with temporal lobe epilepsy (Quirico-Santos et al., 2013). IL-1[beta], like a proconvulsant, triggers neuronal excitability and contributes to seizure and neuronal death. IL-1[beta] mediates NF-[kappa]B and MAPK-dependent genes to change the structure and function of glia and neuronal network, and results in epileptogenesis (Vezzani et al., 2008). These findings suggested that IL-1[beta] play a crucial role in the pathogenesis of seizures.
BDNF is a growth factor belonging to neurotrophin family. It distributes widespread in the rat brain, including cortex and hippocampus (Kawamoto et al., 1996). The levels of BDNF mRNA in hippocampus increase 4.1-fold in temporal lobe epilepsy (TLP) patients with hippocampal sclerosis (HS) than TLP patients without HS, and BDNF mediates NMDA current to induce the excitability of dentate granule cells (Wang et al., 2011). Dysregulation of BDNF function induces neurodegenerative diseases, like seizure and Huntington's disease (Zuccato and Cattaneo, 2009). BDNF increases after KA administration and reaches a peak at 24 h in the hippocampus, suggesting that BDNF is associated with the severity of seizures (Rudge et al., 1998). Furthermore, seizure induces the upregulation of BDNF, and BDNF-induced neuronal excitability contributes to epileptogenesis (Binder et al., 2001). BDNF can stimulate neurotrophin receptor tyrosine kinase B (trkB) located on the hilar segment of mossy fiber sprouting and evoke axonal branching, which may contribute to the formation of hyperexcitable dentate circuits (Koyama et al., 2004). The hyperexcitability develops after BDNF administration in combined entorhinal/hippocampal slices of rat brains, and this effect may be due to a presynaptic action on trkB receptor (Scharfman, 1997). BDNF can mediate trkB to cause mossy fiber sprouting and to establish hyperexcitablity reentrant circuits of dentate gyrus (Koyama et al., 2004). Because neuro-inflammation and mossy fiber sprouting play critical roles in epileptogenesis, and IL-1[beta] and BDNF seemly play trigger roles in these events, we suggested that UR and RP may inhibit KA-induced IL-1[beta] and BDNF gene expressions, resulting in the amelioration of epileptic seizure in rats.
In conclusion, KA induced epileptic seizures, and UR and RP ameliorated the seizure behaviors induced by KA in rats. By genomic and IHC studies, we found that UR affected TLR and neurotrophin signaling pathways, and UR and RP suppressed KA-induced IL-1[beta] and BDNF gene expressions. These findings suggested that UR and RP exhibited anti-convulsive effects in KA-induced rats via the regulation of TLR and neurotrophin signaling pathways, and the subsequent inhibition of IL-1[beta] and BDNF gene expressions (Fig. 5).
Conflict of interest
The authors have declared that no competing interests exist.
This work was supported by grants from Taiwanese Department of Health, Clinical Trials and Research Center of Excellence (DOH102-TD-B-111-004), National Research Program for Biopharmaceuticals (NSC101-2325-B-039-007 and NSC102-2325-B-039-007), National Science Council (NSC101-2320-B-039-034-MY3, NSC101 -3114-Y-466-002, and NSC 102-2632-B-039-001-MY3), and China Medical University (CMU101-S-21, CMU101-AWARD-09, and CMU102-NSC-04).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.Org/10.1016/j.phymed.2014. 01.011.
Received 23 July 2013
Received in revised form 8 November 2013
Accepted 31 January 2014
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Tin-Yun Ho (a), Nou-Ying Tang (b), Chien-Yun Hsiang (c), Ching-Liang Hsieh (d,e,f), *
(a) Graduate Institute of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
(b) School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
(c) Department of Microbiology, College of Medicine, China Medical University, Taichung 40402, Taiwan
(d) Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
(e) Acupuncture Research Center, China Medical University, Taichung 40402, Taiwan
(f) Department of Chinese Medicine, China Medical University Hospital, Taichung 40402, Taiwan
Abbreviations: BDNF, brain-derived neurotrophic factor; COX-2, cyclooxygenase-2; EEG, electroencephalogram; EMG, electromyogram; FDR, false discovery rate; GEPAS, gene expression pattern analysis suite; HS, hippocampal sclerosis; IHC, immunohistochemistry; IL-1R, IL-1 receptor; IL-1[beta], interleukin-1[beta]; iNOS, inducible NOS; KA, kainic acid; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; Nfkbia, nuclear factor of kappa light chain; NF-[kappa]B, nuclear factor-[kappa]B; NMDA, N-methyl-D-aspartate; nNOS, neuronal nitric oxide synthase; PBS, phosphate buffered saline; RP, rhynchophylline; SD, Sprague-Dawley; TLP, temporal lobe epilepsy; TLR, Toll-like receptor; TNF-[alpha], tumor necrosis factor-[alpha]; trkB, receptor tyrosine kinase B; UR, Uncaria rhynchophylla (Miq.) Jacks; VA, valproic acid.
* Corresponding author at: Graduate Institute of Integrated Medicine, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan.
Tel.: +886 4 22053366x3500; fax: +886 4 22037690.
E-mail address: clhsieh<5>mail.cmu.org.tw (C.-L. Hsieh).
Table 1 Pathway analysis of genes in cortex and hippocampus of rats treated with KA and/or UR. KEGG pathway Cortex (FDR value) KA UR Nervous system Neurotrophin signaling pathway 0.1137 0.3342 Prion diseases 0.0292 0.0025 Immune response TLR signaling pathway 0.0469 0.0024 Chemokine signaling pathway 0.0221 0.0049 Transforming growth factor-p 0.4293 0.3653 signaling pathway NOD-like receptor signaling 0.0210 0.2275 pathway MAPK signaling pathway 0.0051 0.0012 Leukocyte transendothelial 0.0353 0.0087 migration Vascular endothelial growthy 0.1065 0.1057 factor signaling pathwa p53 signaling pathway 0.0184 0.0033 Wnt signaling pathway 0.0975 0.3806 B cell receptor signaling pathway 0.1145 0.0327 Complement and coagulation 0.0204 9.75 x cascades [10.sup.-6] Antigen processing and 0.0153 0.0021 presentation Other Type I diabetes mellitus 0.1502 0.0035 Arachidonic acid metabolism 0.0382 0.0223 KEGG pathway Hippocampus (FDR value) KA UR Nervous system Neurotrophin signaling pathway 0.0479 0.4828 Prion diseases 0.1035 0.0148 Immune response TLR signaling pathway 0.0014 0.0096 Chemokine signaling pathway 0.0007 0.0101 Transforming growth factor-p 0.0391 0.3374 signaling pathway NOD-like receptor signaling 0.0018 0.2522 pathway MAPK signaling pathway 2.95 x 0.1555 [10.sup.-7] Leukocyte transendothelial 0.2673 0.1107 migration Vascular endothelial growthy 0.3883 0.0519 factor signaling pathwa p53 signaling pathway 0.3149 0.2765 Wnt signaling pathway 0.4386 0.1587 B cell receptor signaling pathway 0.4243 0.3226 Complement and coagulation 0.1524 1.11 x cascades [10.sup.-6] Antigen processing and 0.2632 0.3482 presentation Other Type I diabetes mellitus 0.14 0.2804 Arachidonic acid metabolism 0.0184 0.005 Table 2 Fold changes of KA-and UR-regulated genes in neurotrophin and TLR signaling pathways. Accession No. Gene symbol Definition Neurotrophin signaling pathway NM-012513.2 Bdnf Brain derived neurotrophic factor XM-343065.3 Nfkbia Nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha NM-021835.3 Jun Jun oncogene Toll-like receptor signaling pathway NM_022197.1 Fos FBJ murine osteosarcoma viral oncogene homolog XM_343065.3 Nfkbia Nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha XM_239239.3 Map2k3 Mitogen activated protein kinase kinase 3 NM_031512.1 nib Interleukin 1 beta NM_021835.3 Jun Jun oncogene Accession No. Cortex Hippocampus KA (a) UR (b) KA (a) UR (b) Neurotrophin signaling pathway NM-012513.2 13.8 -1.6 17.0 -1.0 XM-343065.3 3.9 -1.2 3.6 1.0 NM-021835.3 2.8 -1.2 2.1 -1.0 Toll-like receptor signaling pathway NM_022197.1 29.5 -1.7 62.1 -1.1 XM_343065.3 3.9 -1.2 3.6 1.0 XM_239239.3 2.8 -1.4 2.8 -1.1 NM_031512.1 2.4 -1.6 1.8 -1.3 NM_021835.3 2.8 -1.2 2.1 -1.0 (a) Fold changes of genes were calculated by dividing the normalized signal intensities of genes in KA-treated rats by those in PBS-treated rats. (b) Fold changes of genes were calculated by dividing the normalized signal intensities of genes in UR-treated rats by those in KA-treated rats.