1:Effect Of INOS On Learning Amnesia Induced By High-fat Diet2:Piceid Attenuates Behavioral Impairment In A Rotenone-induced Rat Model Of Parkinson’s Disease

Objective:The incidence of cognitive dysfunction increases with age, it become a very common clinical symptoms in today’s aging society. Severe cases of cognitive impairment were called dementia. According to statistics, the current global dementia patients is about24million, of which more than half are Alzheimer’s disease (AD). AD is the most usual disease of old age cognitive dysfunction. AD is a progressive cognitive impairment of central neurodegenerative diseases, its pathology is characterized by the formation of senile plaques (SP), neurofibrillary tangles (NFT) and a large number of neurons lost. Ideal AD animal model is very important to study the pathogenesis of AD and the treatment. There are many ways to establish the AD animal models. They mainly can be grouped into the following three categories: simulate the characteristics of AD symptoms model, simulate histopathological features of AD model and multi-factor composite animal model. After decades of hard work AD animal model studies has made a lot of progress. Each animal model simulates a certain degree, or some aspects of the symptoms and pathological changes of AD. But as yet no one can truly reflect the various cases of stage of development characterized and widely popular animal model. Colton CA. in Duke University School of Medicine first proposed that genetic removal of iNOS in mice expressing mutated amyloid precursor protein results in pathological hyperphosphorylation of mouse tau, its redistribution to the somatodendritic compartment in cortical and hippocampal neurons, and aggregate formation. Lack of iNOS in the amyloid precursor protein Swedish mutant mouse increased insoluble β-amyloid peptide levels, neuronal degeneration, caspase-3activation, and tau cleavage, suggesting that NO acts at a junction point between β-amyloid peptides, caspase activation, and tau aggregation. Lot of people questioned this theory when Colton CA first proposed. iNOS gene is located on the17pairs of autosomes. It only can be expressed when the cells were stimulated and activated. NO, the production of iNOS can last a long time about several hours to days. NO is an important intracellular and intercellular signaling regulating molecular. It has a dual nature in human physiological and pathological processes. NO, a cytotoxic effector molecule is a tissue damage enhancement factor and predisposing factors of variety of lesions. This is the pathology bad side. In physiological good side, NO is also can be a signaling molecules in many organisms, innate immune response regulatory effector molecules and as an information material to maintain normal physiological function. In rodents experiments suggesting that iNOS activity play a harmful role in experimental autoimmune or chronic inflammatory processes, and several other diseases. But the detial of iNOS specific roles in many diseases are still unknown; it needs to be further in-depth study. The purpose of this project is to study the relationship of iNOS with learning and memory; study the effect and mechanism of iNOS on learning amnesia induced by high-fat diet.Methods:3-month-old iNOS gene knockout mice (iNOS-/-) and the same background (C57BL/6) with littermate wild-type mice (WT) were rearing to adapt to the environment one week. PCR was carried out to genotype the transgenic mice before the experiment. iNOS knockout mice and WT mice were fed with chow diet (Chow) and/or high-fat diet (HFC). After20weeks feeding mice in each group were did anxiety behavior test, including open field test, the elevated plus maze, and light dark box test. Memory behavior test were conducted after anxiety behavior, including the Morris water maze and fear condition test. At the same time for each group of mice did intraperitoneally tolerance test (IPGTT). All mice were fasted for ahout16hours and weighed, injected2g/kg dose of glucose. After0,15,30,60,90and120min, using micro-blood glucose meter and supporting blood glucose test strips to detect the blood glucose levels. After the end of the in vivo behavior and IPGTT test, some of mice were i.p. injected50mg/kg dose of Evans blue dye (EBD), three hours later, sacrificed mice and separated left and right brain hemispheres, cerebellum, as well as the heart, liver, kidney, spleen, and mouse thigh muscle tissue and plasma. Measure the EBD concentration in different tissues and plasma. For some other mice:(1) after heart perfusion remove the brain to do the GFAP and Aβ40immunofluorescence staining;(2) sacrificed mice, separated frontal cortex and hippocampus tissue; homogenated and measured the protein concentration. Using Western blotting method to analysis the Akt signaling molecule and Tau protein level. In addition, another frontal cortex and hippocampus tissue samples were added trisol to extracted RNA. Using real-time PCR to detected inflammatory cytokines, leukocyte interleukin-1β (IL-1β), tumor necrosis factor a (TNF-a) changes in gene expression levels, and GFAP expression in astrocytes.Results:(1) The results from open field test, elevated plus maze test, and light-dark box test showed that the mice in each group including iNOS knockout and up to20weeks of high-fat diet feeding, mice does not have anxiety behavior, nor does it affect the spontaneous motor activity.(2) Memory behavior of each group of mice were detected in the state of non-anxiety behavior. Morris water maze:1)5days acquisition training experiments results showed that mice in each group have no significant difference, which further illustrates iNOS gene knock-out and up to20weeks of high-fat diet feeding did not affect the visual acuity and swimming ability.2)10days exploratory experiment: with the increasing of test days, the time of animals in each group to find the platform have different degrees of shortening. In the first1-5days, the differences between the groups is not very obvious, but iNOS knockout mice fed with high-fat diet the latency of finding the platform is slightly slower than the other groups. After6days, the escape latency of iNOS-/-HFC group was significantly higher than the WT-Chow normal group. It showed that iNOS gene knockout mice fed HFC had a certain degree of damage in spatial learning and memory. In the6th days, the escape latency of iNOS-/-HFC group was higher than the iNOS-/-Chow group, which indicated that high-fat diet will further aggravate the iNOS-/-mice learning and memory damage. WT mice fed a high-fat diet compared with the WT-Chow normal group have no significant difference in the statistics, but the escape latency of WT mice fed a high-fat diet is higher than in normal mice.Fear conditioning experiment:The first day of each group of mice conditioned fear training by electrical stimulation. The fear freezing time has increased after time moving. It suggested that each group of mice to fear almost have the same degree of respond. At the2th days contextual fear test result showed that chronic fed high-fat diet will damage the spatial memory in mice, especially the iNOS gene knockout mice fed HFC. This result suggests that the iNOS knockout exacerbated the damage of the high-fat diet on learning and memory. The tone test results showed that there was no significant difference among the groups.(3) The IPGTT experimental results show that the mice in each group after the intraperitoneal injection2g/kg glucose, the blood glucose concentration increased rapidly. WT-Chow and iNOS-/-Chow group mice reached the peaked blood glucose at15min. While the WT-HFC and iNOS-/-HFC group mice reached peaked in30min, and were significantly higher than the WT-Chow and iNOS-/-Chow group. After2hours, WT-Chow and iNOS-/-Chow mice blood glucose tends to normal levels; the WT-HFC and iNOS-/-HFC mice still showed a high level (>150mg/dl glucose), and the peaks after the extension, which indicates that fed with high fat diet of WT and iNOS-/-mice have abnormal glucose tolerance. INOS-/-mice blood glucose values overall were lower than with WT mice fed high-fat diet, especially in the first30min and90min; after120min the glucose values tends to normal levels. It inferred that iNOS gene knockout can help to recovery the abnormal glucose tolerance under the pathological conditions like diabetes.Based on the IPGTT results, we further examined the phosphorylated Akt and total Akt level in frontal cortex and hippocampus to analysis its possible mechanism. The results show that the WT-Chow and WT-HFC mice are relatively low baseline levels of p-Akt in the frontal cortex. Interestingly, p-Akt level in WT-HFC mice was low expression in the hippocampus than WT mice fed a normal diet, which perhaps can be explained why the IPGTT in WT-HFC mice of abnormal glucose tolerance, and the hippocampus of mice selectively reduce the level of p-Akt. iNOS knockout mice, both fed a normal diet or a high fat diet, their frontal cortex have high levels of p-Akt than WT mice, indicating that iNOS knockout contribute to the activation of Akt signaling molecules. More interesting the iNOS-/-mice fed the high fat diet, p-Akt levels in the frontal cortex and hippocampus was significantly increased compared with WT-HFC group and iNOS-/-Chow group of mice. Therefore, we concluded that iNOS knockout can help improve abnormal glucose tolerance by activating the Akt signaling molecules and decreased glucose level, this hypothesis is consistent with the above IPGTT results.(4) Intraperitoneal injection of EBD experimental results:the left and right brain hemispheres of WT mice fed a high-fat diet EBD concentration was significantly increased than WT-Chow group, indicating that high-fat diet can cause damage to the blood-brain barrier. Cerebellum EBD concentration is relatively high than left and right brain hemispheres. Fed high-fat diet iNOS gene knockout mouse brain EBD concentration significantly reduced compared with WT-HFC mice. This shows that iNOS knockout contribute to the restoration and reconstruction of the BBB induced by high-fat diet. At the same time, we also observed the similar situation in the kidneys. But iNOS gene knockout does not affect other tissues or organs such as the spleen, liver, heart and muscle vascular permeability. In order to ensure that each group of mice can absorb the equivalent concentration of EBD into the blood circulation, and intraperitoneal injection technology, we examined plasma EBD concentration. The results showed the EBD’s concentration in each group of mice plasma were no significant differences.(5) Immune and inflammatory experiment:test results showed that long-term high fat diet induced inflammation. The frontal cortex and hippocampus IL-1β and TNF-a gene level were higher in WT-Chow group than WT-Chow group, especially in the prefrontal cortex tissue. iNOS knockout does not affect the immune system, as the inflammatory cytokines IL-1β and TNF-a gene levels did not change significantly in iNOS-/-Chow group compared with WT-Chow mice. iNOS knockout mice fed a high fat diet, the frontal cortex and hippocampus inflammatory cytokines IL-1β and TNF-a gene level were higher than other groups, than surprisingly compared with WT-HFC mice, despite the significant difference is not very obvious, only TNF-a in the frontal cortex has the significant. Thus, we speculate that under the pathological conditions (high fat diet), iNOS knockout induced increase in the levels of other inflammatory cytokines, which can also be interpreted as a compensatory response.(6) Astrocyte GFAP expression:The GFAP gene levels in the frontal cortex and hippocampus significantly higher in WT-HFC group than WT-Chow group. GFAP gene level did not change significantly in iNOS-/-Chow mice than WT-Chow mice. This suggests that iNOS gene knockout does not affect the numbers of astrocyte in the frontal cortex and hippocampus. iNOS knockout mice fed high fat diet, the level of GFAP gene in the frontal cortex are higher than other groups, even higher than the WT-HFC mice, despite the significant difference is not very obvious. Thus, we speculate that the pathological conditions (high-fat diet), iNOS knockout further induced upregulation of GFAP expression, increasing the AD-like memory injury. These results were consistent with the memory behavior test results. Based on these, we tested the GFAP protein expression in brain by immunofluorescence staining method. The result was consistent with the results of GFAP mRNA expression in frontal cortex and hippocampus. iNOS gene knockout mice fed a high fat diet the GFAP fluorescence intensity is higher than the other groups, increasing the density of astrocytes.(7) Detection of AD-like phenotype (Tau, Aβ40):detection the phosphorylation of Tau protein (p-Tau (AT8)) and total Tau protein (Tau46) expression levels in frontal cortex and hippocampus of each group of mice. The results showed that the ratio of phosphorylation tau with total Tau protein the relative level were significantly higher in the frontal cortex and hippocampus in WT-HFC mice than WT-Chow group. p-Tau and Tau ratio did not change significantly in iNOS-/-Chow group than WT-Chow group. This suggested that under normal physiological state iNOS knockout does not induce abnormal phosphorylation of Tau protein expression. iNOS knockout mice fed high fat diet, the relative level of p-Tau proteins is higher in the frontal cortex than other groups, eventualy higher than the WT-HFC mice. Thus, we speculate that in pathological conditions (high-fat diet) iNOS knockout further induced abnormally phosphorylated Tau protein upregulation in the frontal cortex, increasing the AD-like phenotype and memory injury. Detection the expression of Aβ40in frontal cortex and hippocampus of each group mice by immunofluorescence assay. The results show that the high-fat diet and iNOS gene knock can induce the Aβ40expression increasing trend.Conclusion:iNOS knock-out addition to the high-fat diet for20weeks does not produce anxiety behavior, affect its own activities, visual acuity and swimming ability. Excluding the anxiety behavior interference, the high-fat diet iNOS knockout mice of spatial learning and memory ability still have a certain degree of injury. iNOS knockout exacerbated the damage of the high-fat diet on learning and memory, and is dependent on the hippocampus, has the tissue selectivity. High-fat diet is one of the risk factors to impaired glucose tolerance. IPGTT results showed that WT and iNOS-/-mice fed with high-fat diet existed abnormal glucose tolerance. In pathological conditions like diabetes, iNOS gene knockout help the recovery of abnormal glucose tolerance. Based on this phenomenon, we explore the possible mechanism of its occurrence. iNOS knockout contribute to the activation of Akt signaling molecules. It inferred that iNOS knockout on the improvement of abnormal glucose tolerance may through the activation of Akt signaling molecules and decline blood glucose level. This hypothesis is consistent with the results of the IPGTT. EBD experimental results confirmed that the high-fat diet can cause damage of the blood-brain barrier, and iNOS knockout contributed to the restoration and reconstruction of the blood-brain barrier disorder induced by high-fat diet. iNOS gene has the same similar role in the kidney, but does not affect other tissues and organs such as the spleen, liver, heart and muscle vascular permeability and blood absorption and distribution functions. The long-term high-fat diet can induce inflammation, IL-1β and TNF-α genes have higher levels in the frontal cortex and hippocampus, especially in the frontal cortex. iNOS knockout does not affect the immune system; but in pathological conditions (high-fat diet), iNOS knockout induced increase the levels of other inflammatory cytokines, which may be a compensatory response. iNOS knockout does not affect the number of astrocyte cells in the frontal cortex and hippocampus. While in pathological conditions (high-fat diet) iNOS knockout further induced upregulation of GFAP expression, increasing the AD-like phenotype, damage memory behavior of the mice. Normal physiological conditions, iNOS knockout does not induce abnormal phosphorylation of Tau protein expression; pathological conditions (high-fat diet), iNOS knockout will further induce abnormal phosphorylation of Tau protein upregulation in the frontal cortex, increasing the AD-like phenotype, damage memory behavior of the mice. These test results are consistent with previous memory behavior results. Detection the expression of A04O in frontal cortex and hippocampus of each group mice by immunofluorescence assay. The results showed that the high-fat diet and iNOS gene knockout can induced the Aβ40expression increasing trend. This project detail discussed the role of iNOS in learning amnesia induced by high-fat diet and the possible mechanisms, which may provide a possible to establish a new ideal AD animal model. Objective:Oxidative stress has been implicated in the etiology of Parkinson’s disease (PD) and may be an effective target of intervention. Peripherally and locally administered rotenone has been proposed as a well acknowledged preclinical model of PD as it induces selective nigrostriatal DA-ergic neuronal degeneration and produces motor dysfunction. Neuroprotective effects of piceid have been shown to be associated with its antioxidant properties, but the mechanisms remain unknown. Considering that enzymatic defense systems and the thioredoxin (Trx) systems, are the important cellular redox modulation systems, change under oxidative stress and could exert protective effects, the relationship between the antioxidant effects of piceid and these systems regulation were explored.Methods and Results:Male Sprague Dawley (SD) rats were treated with rotenone2.0mg/kg/day i.p. for5weeks. Behavioral data showed a strong increase in catalepsy score, a decrease in motor coordination activity from4-5weeks, and biochemical data showed profound deficient in the contents of reduced glutathione (GSH), the activities of superoxide dismutase (SOD) and the contents of adenosine triphosphate (ATP), and a significant increase the contents of malondialdehyde (MDA), and down-regulated the antioxidative enzyme thioredoxin (Trx) expression in the striatum in rotenone treated animals compared to vehicle. Chronic administration of rotenone to SD rats resulted in marked oxidative damage in the striatum region compared to other regions (cortex and hippocampus) of the brain. Oral administration of piceid (20,40,80mg/kg) or L-DOPA (10mg/kg) co-treatment is able to successfully attenuate these motor defects and most of biochemical changes in dose dependent manners. The administration with dose (40,80mg/kg) of piceid and L-DOPA (10mg/kg) caused the most marked symptomatic improvement in catalepsy score and motor coordination activity when compared to its administration with low dose of piceid (20mg/kg) or when compared to rotenone treated animals, respectively. Piceid in a dose40mg/kg especially in a high dose80mg/kg significantly attenuated rotenone-induced depletion in GSH and decreased the contents of MDA. In addition, the drug prevented the decrease in ATP level and also enhanced the activity of SOD. Western blot result showed piceid (80mg/kg) co-treatment could up-regulate Trx expression in the striatum region compared to cortex and hippocampus.Conclusion:Taken together, these results strongly indicate the possible therapeutic potential of piceid as an antioxidant in PD and other movement disorders based on its up-regulating effects on enzymatic defense systems and neuroprotective protein thioredoxin, a substrate in the Trx redox system.