Studies Of Effects Of Gastrodin On 3,3’-iminodipropionitrile-induced Memory Deficit And Lipopolysaccharide-induced Cognitive Dysfunction

BackgroundNitriles are extensively used as organic intermediates in the manufacture of synthetic fibers (nylon), plastics, dyestuffs and pharmaceuticals. Nitriles are easily absorbed via dermal absorption. Their occupational and environmental exposure are considered to be potentially relevant to human health. 3,3’-Iminodipropionitrile (IDPN) is one of the most commonly used reagents for synthetic nitrile compounds. Exposure to IDPN can induce neurotoxicity and lead to neurobehavioral abnormalities in rodents, characterized by repetitive head movements, circling, retropulsion and hyperactivity. Dyskinesia has been well documented to have marked biochemical alterations in the dopaminergic and serotoninergic system of various brain regions, as well as oxidative stress. Symptoms of dyskinesia in animals resemble those of human movement disorders with cognitive decline in Tourette’s syndrome and Huntington’s disease. Furthermore, emerging evidence suggests that IDPN produces adverse effects on brain structure and cognitive function. IDPN has been shown to cause morphometric changes and glisis, decrement in interneuron and inhibition of axonal plasticity in the hippocampus. Noticeably, it was demonstrated that IDPN could cause learning deficit in the T-maze test, impairment of spatial memory as well as disruption of passive avoidance performance in rodents. Accordingly, animal model of IDPN is critical to study the biochemical mechanisms of cognitive decline in neurological disorders and to develop future therapeutic interventions.Gastrodin is a main bioactive constituent of a Chinese herbal medicine (Gastrodia elata Blume) widely used for treating various neurological disorders. Accumulating evidence has revealed that gastrodin exerts a variety of pharmacological properties, including anti-inflammatory, anti-anxiety, anti-depressant and neuroprotection. Notably, gastrodin possesses cognition-enhancing activity. Specifically, treatment of gastrodin can significantly alleviate memory deline in the Morris water maze test in a mouse model of Alzheimer’s disease. Gastrodin can also effectively rescue lead-impaired synaptic plasticity in rat hippocampus. Moreover, gastrodin functions as antioxidant property by decrease in level of lipid hydroperoxides and elevation of antioxidant protein expression. As well, exposure to gastrodin can inhibit glutamate-induced oxidative stress via inhibition of reactive oxygen species (ROS) generation and level of malondialdehyde (MDA), enhancement of mitochondrial membrane potential superoxide dismutase (SOD). However, it has not yet been reported that whether gastrodin could attenuate IDPN-induced memory decline and by what underlying mechanisms.ObjectiveThe objective of this study was to determine whether treatment with gastrodin can alleviate IDPN-induced memory decline and explore the possible neural mechanisms, including cognitive behavior test, dopamine, serotoninergic system and oxidative stress.MethodsWistar rats were randomly divided into four groups as follows:Group Ⅰ (vehicle group):The rats were injected with saline for 7 successive days followed by saline administration for 8 weeks. Group Ⅱ (GAS plus saline): The rats were injected with saline for 1 week followed by gastrodin administration for 8 weeks. Group Ⅲ (IDPN plus saline):The rats were injected with IDPN for 7 days followed by saline administration for 8 weeks. Group Ⅳ (GAS plus IDPN):The rats were injected with IDPN for 1 week followed by gastrodin administration for 8 weeks.In this work, we first examine whether prolonged gastrodin treatment is capable of rescuing IDPN-induced memory deficit using Y-maze and novel object recognition test 60 min after the last drug administration. Furthermore, we investigate alternations in dopamine (DA), serotonin (5-HT) levels and their metabolites by high-performance liquid chromatography, correlated changes in expression of D2 receptor and 5-HT1A receptor, serotonin and dopamine transporter (SERT and DAT) protein are detected by western blot analysis. Additionally, we determine effects of gastrodin on activity of SOD and glutathione peroxidase (GSH-Px), MDA content in IDPN-treated rats using chemistry colorimetry.Results1. Compared with the control group, rats exposed to IDPN produced a significant decrease in spontaneous alternation percentage in Y-maze test. However, there is a significant increase of spontaneous alternation percentage in the gastrodin-treated group compared with the IDPN group. Additionally, rats subjected to IDPN showed increased total arm entries when compared to saline-exposed rats, nevertheless these increases were reversed by gastrodin treatment.During the training session in the novel object recognition test, there were no significant differences between groups regarding exploration time directed to the two identical objects. However, during the retention session, IDPN-treated rats spent less time exploring the novel object compared with saline-treated group. Furthermore, gastrodin treatment significantly elevated the amount of time spent exploring novel object in the retention session, in IDPN-exposed rats. In addition, rats subjected to IDPN showed a decreased discrimination ratio compared with saline treatment, whereas gastrodin was able to increase the discrimination ratio.2. In the hippocampus IDPN induced significantly decreases in DA and its metabolite homovanillic acid (HVA) levels compared to the control group, while these decreases were ameliorated by treatment with gastrodin in DA and HVA levels. Additionally, IDPN induced significant decrement in 5-HT levels. Whereas the reduction of 5-HT levels was significantly mitigated by gastrodin. No significant differences in 5-hydroxyindoleacetic acid (5-HTIAA) levels were observed between groups.3. In the hippocampus, D2 receptor protein expression of IDPN-exposed rats was dramatically increased as compared to control. Whereas administration of gastrodin significantly prevented the increase of D2 receptor expression, when compared to IDPN alone. IDPN significantly decreased the DAT protein levels compared with the corresponding controls. Nevertheless, repeated treatment with gastrodin effectively reversed IDPN-induced the reduction of DAT expression.IDPN exposure significantly increased SERT expression as compared to control in the hippocampus. However, treatment with gastrodin significantly ameliorated the increase of SERT protein compared with the IDPN-treated rats.5-HT1A receptor expression of IDPN-exposed rats was markedly decreased as compared to control rats. However, gastrodin effectively prevented IDPN-induced reduction of 5-HT1A receptor expression in the hippocampus.Statistical analysis showed that SOD activity in the IDPN group was lower than those in the saline-treated group. However, administration of gastrodin effectively prevented IDPN-induced reduction of SOD activity. IDPN exposure significantly increased the MDA levels as compared to controls. Treatment with gastrodin effectively prevented IDPN-induced the increase of MDA levels. No significant differences in GSH-Px levels were detected between groups.Conclusions1. Administration of IDPN produced impairment in spatial memory and object recognition memory in rats.2. Prolonged gastrodin treatment prevented IDPN-induced reductions of DA and HVA levels. Gastrodin also prevented alterations in D2 receptor and DAT expression in the hippocampus. The results suggest that gastrodin can mitigate IDPN-induced memory decline, in part, by normalizing the dopaminergic system.3. The memory-facilitatory effects of gastrodin may be attributive to modulation of the serotonergic system, in part, through alterations in 5-HT, SERT, and 5-HT1A receptor levels.4. Administration of gastrodin effectively prevented IDPN-induced reduction of SOD activity and increase of MDA levels. Gastrodin has a beneficial protection against the memory deficit induced by IDPN, partly through promoting activity of anti-oxidant enzyme, decreasing production of oxidative product.BackgroundIt is now accepted that inflammation is a major underlying pathogenesis of diseases, related to dementia, cancer, diabetes, obesity, arthritis, acute lung injury and inflammatory bowel disease. An increasing number of research efforts have focused on the precise impact of inflammation on cognitive function. Lipopolysaccharide (LPS), a cell wall constituent of Gram-negative bacteria, is one of the critical pathogens of infectious inflammatory diseases. Intraperitoneal injection of LPS causes systemic inflammation. LPS-mediated systemic inflammation triggers a cascade of cellular and molecular events, involving expression of pro-inflammatory cytokines and apoptosis through Toll-like receptor 4, hence can disrupt hippocampus-dependent learning and memory processes. Specifically, previous studies have shown that acquisition of the Morris water maze and consolidation of contextual fear conditioning are disrupted during inflammation. However, there have been mixed results across studies regarding the LPS treatment on the water maze, as well as observations that cytokines can facilitate learning and memory. Thus, whether inflammation can in fact cause cognitive decline, and the underlying mechanisms, remain elusive.The emerging role of epigenetics can contribute greatly to investgate the effect of LPS on cognitive function. DNA methylation is regarded as a major epigenetic modification. Abnormal DNA methylation can alter chromatin conformation and repress gene expression. Multiple reports have demonstrated that LPS-induced inflammation is susceptible to DNA methylation. In particular, epigenetic suppression, mediated by DNA-methylation, of Toll-like receptor 4 gene expression has been linked to a lowered response to LPS in intestinal epithelial cells. Moreover, it has been demonstrated that altered DNA methylation participates in LPS-induced IgM response in splenocytes. LPS-evoked endotoxemia displays reduced DNA methylation levels in the liver of rats. Notably, accumulating evidence suggests that higher level cognitive behaviors, learning and memory are subject to methylate control. Indeed, DNA methylation mediated control of gene expression is crucial for the consolidation and re-consolidation of cued fear conditioning. Contextual fear conditioning could increased DNA methylation at the promoter of the memory-suppressor gene protein phosphatase I, whereas concurrently demethylating at the promoter of the plasticity-associated gene reelin. Therefore, these data suggest that rapid and dynamic methylation and demethylation of specific genes in the brain may play a fundamental role in learning, memory formation, and behavioral plasticity.The inner matrix protein Matrin-3 is an interesting candidate molecule in this context. Matrin-3 contains two RNA recognition motifs (RRM) that bind RNA, which are predicted to bind DNA. Emerging evidence indicates that Matrin-3 has been involved in a variety of functions, such as bounding to the inner nuclear membrane with roles in chromatin organization, DNA replication, transcription, repair, and RNA transport. Matrin-3 mediates neuronal cell death through its phosphorylation by protein kinase A (PKA) following N-methyl-D-aspartic acid (NMDA) receptor activation. More importanly, NMDA receptor-induced, PKA-mediated phosphorylation of Matrin-3 and modulation of the nuclear matrix function could be involved in long-term potentiation. Matrin-3 also interacts with TDP-43, a disease protein linked to amyotrophic lateral sclerosis and frontotemporal dementia. Additonally, the elevation in extracellular matrix expression can lead to impairment in hippocampal long-term potentiation and contextual fear memory in amyloid precursor protein and presenilin 1 (APP/PS1) transgenic mice. Notably, in this study, proteome analysis showed that LPS exhibited a significant increase in Matrin-3 expression. However, it has not yet been reported that whether Matrin-3 could be involved in LPS-induced cognitive decline. Therefore, we provide the benchmark evidence for treatment of diseases, including anti-inflammatory drugs, and protection of the central nervous system.ObjectiveThe aim of the present study was to determine whether alterations in global DNA methylation, Matrin-3 expression, which was the selected differentiated protein by proteome analysis, and related to methylation pattern in Matrin-3 promoters were involved in cogntive decline by LPS-evoked inflammation.MethodsKM mice were randomly allocated into one of the two groups:LPS group and saline group. For LPS-induced inflammation, mice were injected intraperitoneally with LPS for 4 consecutive days. Mice in the control group were administered an equivalent volume of saline intraperitoneally. Specifically, novel object recognition and Morris water maze test were used to evaluate learning and memory ability 2 h after the last drug administration; Levels of 5-methylcytosine in the hippocampus were measured using high-performance liquid chromatography-tandem mass spectrometry; Hippocampus proteomics was further conducted to investigate effects of LPS, and cognition-related potential biomarkers and signal pathways were untangled. Additionally, we validated Matrin-3 expression in the hippocampus by Western blot analysis; Real-time PCR analysis was utilized to detect Matrin-3 mRNA expression; Bisulfite sequencing was used to assess the methylation status of CpG islands in Matrin-3 promoters following LPS exposure.Results1. The results revealed that administration of LPS exhibited a significant decrease in novel object recognition index as compared to controls. Additionally, in the Morris water navigation task, there was a striking increase of the escape latency on day 4 in LPS-treated mice, when compared with controls. Furthermore, in a subsequent probe test, with the platform removed, the number of platform crossings remarkably decreased in LPS-exposed mice, compared with the corresponding controls. There was no significant group difference in percentage path length in the target quadrant.2. Following LPS treatment in mice, we found that a subtle and not statistically significant decrease in global levels of DNA methylation, compared to controls.3. Proteomic analysis of treatment with LPS showed that a total of 463 overlapping proteins confidently identified in the 2 groups. Of these proteins,16 proteins had significant effects in mice injected with LPS compared to controls.7 proteins were found up-regulated, and 9 were down-regulated. Importantly, systemic administration of LPS exhibited a significant increase in Matrin-3 expression compared with controls.The Gene Ontology enrichment of cellular compentents and biological processes was done by searching database using Protein Annotation Through Evolutionary Relationships (PANTHER) system. The resluts showed differential proteins are mostly localized at the cell part, macromolecular complex, organelle and membrane. Expourse to LPS caused changes in a series of proteins mainly involved metabolic process, cellular process, biological regulation and localization. Then, the selected differential proteins were submitted to PANTHER database for molecular function and pathway detail analysis. These proteins mainly function as binding, catalytic activiy, and enzyme regulaor activity as well. PANTHER pathway visual analysis identified these proteins in the epidermal growth factor (EGF) receptor signaling pathway, fibroblast growth factor (FGF) signaling pathway, the inflammation mediated by chemkine and cytokine signaling pathway and Parkinson’s disease.4. Administration of LPS remarkably increased the Matrin-3 protein and mRNA levels in the hippocampus, as compaired with the control group.5. Bisulfite sequencing analysis confirmed the relatively significant decrease in average methylation rate of Matrin-3 promoter following LPS treatment, compared to controls. Further analysis revealed that, in the LPS-treated mice, DNA methylation levels of CpG sites 28 in the Matrin-3 promoter region were lower, compaired with the saline group.Conclusions1. Administration of LPS triggered impairment of novel object recognition and spatial memory in mice.2. No gross DNA methylation changes were detected in mice after LPS treatment.3. LPS exposure altered proteome in the hippocampus. As well, we validated enhanced Matrin-3 protein and mRNA expression following LPS induced inflammation.4. DNA methylation levels and specific CpG sites in Matrin-3 promoters were decreased following exposure to LPS.To conclude, the work presented here shows that enhanced Matrin-3 expression and promoter hypomethylation of Matrin-3 may be responsible for impaired cognition induced by systemic inflammation.