Pentamethylquercetin Protects Against Cognitive Dysfunction In Diabetes Rats

Objective:Diabetes is a metabolic disease characterized by hyperglycemia due to a lack of insulin secretion or insulin resistance, which can cause many complications. Diabetes encephalopathy is one of the most complicated chronic complications of diabetes, which main clinical symptoms presented cognitive dysfunction, the ability of learning and memory of the light symptoms is significantly decreased, spatial orientation is difficult, the person whith weigh can develop dementia. So far the exact pathological damage mechanism is still unclear, which bring great difficulty in getting diabetes encephalopathy in the prevention and control.The cAMP-responsive element-binding protein (CREB) is a member of a large family of structurally related transcription factors, which regulated the expression of memory-related proteines in the healthy brain. A number of signal pathways regulate CREB activity, in which Akt signaling pathways may be important one in the regulation of CREB phosphorylation. In diabetic state, the activity of Akt was inhibited, so it has been natural thought that diabetes-induced cognitive dysfunction may be because the Akt pathway was inhibited in the hippocampus, the expression of phosphorylated CREB was significantly downregulated and ultimately led to behaviour deficits. Thus, improving the Akt/CREB pathway activity may be a useful therapeutic strategy in treating humans with diabetic encephalopathy.Polymethoxylated flavones (PMF) are a group of highly methoxylated phenolic compounds existing widely in the natural world. Numerous reports have demonstrated that a few PMF family members possess the health beneficial effects including improving insulin resistance, anti-inflammatory, antioxidative and radical-scavenging capacities and ameliorating cognitive dysfunction. Pentamethylquercetin (PMQ) is a typical member of the polymethoxylated flavone family in the natural world. Previous studies have demonstrated that PMQ possesses many health beneficial effects including anti-diabetic, anti-carcinogenic and cardioprotective properties. However, there is no report whether PMQ can improve diabetes-induced cognitive dysfunction.Therefore, we have conducted studies using neonatally streptozotocin-induced diabetic rats (n-STZ rats) and genetic diabetic Goto-Kakizaki rats (GK rats) to assess the effects of insulin resistance and hyperglycemia on synaptic plasticity and behaviour, explore the mechanisms of Akt/CREB signaling pathways in diabetes-induced cognitive dysfunction and hippocampal neuron damage. At the same time, we investigated the protective role of PMQ on diabetes-induced cognitive dysfunction, using GK rats.Methods:1、The2-day-old male SD pups were treated with a single intraperitoneal injection of streptozotocin (90mg/kg, i.p.).Age-matched control SD pups were injected with sodium citrate solution. In4weeks, twenty-four rats were were randomly divided into control group (n=12) and STZ group (n=12), continuous fed for29weeks. Twelve-week-old (male) GK rats (n=12) and age-matched healthy Wistar rats (n=9) were continuous fed for20weeks. Tap water was provided ad libitum. Food and water intakes were measured on every2days, and rats were weighed weekly. On32weeks, the learning and memory of the rats were evaluated by water maze. After the evaluation of learning and memory, the fasting blood glucose and serum insulin were determined. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Then the rats were sacrificed under phenobarbital anesthesia and transcardial perfusion with ice-cold normal saline was performed. The hippocampus were rapidly removed and weighed. One part of the hippocampus were selected for further Golgi, Nissl and immunohistochemical staining, the other part were assayed for Akt, CREB, SYP and BDNF protein expression by western blot analysis.2-. The rats were randomized into six groups as follows (n=9-12):GK group; PMQ groups (GK rats given PMQ at2.5,5,10mg kg-1day-1, respectively); Metformin (MET) group (GK rats given MET at300mg kg-1day-1); Wistar group. All rats were acclimatized to their environment for4week prior to the beginning of the study. The Wistar group and GK group were fed on a standard rat chow (60%carbohydrate,20%protein,10%vitamin and mineral mix,5%fat and5%cellulose), while PMQ groups and MET group were given a diet containing2.5,5,10mg kg-1day-1of PMQ and300mg kg-1day-1of MET for16consecutive weeks. Tap water was provided ad libitum. Food and water intakes were measured on every2days, and rats were weighed weekly. On16weeks after PMQ treatment, the learning and memory of the rats were evaluated by water maze. After the evaluation of learning and memory, the fasting blood glucose and serum insulin were determined. Oral glucose tolerance test (OGTT) was performed. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Then the rats were sacrificed under phenobarbital anesthesia and transcardial perfusion with ice-cold normal saline was performed. The hippocampus were rapidly removed and weighed. One part of the hippocampus were selected for further Golgi, Nissl and immunohistochemical staining, the other part were assayed for Akt, CREB, SYP and GluR1protein expression by western blot analysis.3、Primary cultures of rat hippocampal neurons were prepared from the hippocampus of E18-E19Wistar rat embryos. In brief, the rats were decapitated and hippocampus was carefully isolated. After being washed twice using D-Hank’s buffer, hippocampus was digested with equal volumes of0.125%trypsin/ethylene diamine tetraacetic acid (EDTA) for approximately15min at37℃. The hippocampus was then washed with Hank’s solution containing10%fetal bovine serum (Biochrom, Cambridge, UK) to stop digestion. Then, neurons were plated in six-well plates (8.75×104cells/cm2) or in96well plate plates (0.85×104cells/cm2). The cultured neurons were incubated with DMEM in a humidified5%CO2incubator at37℃for6h, and the medium was then replaced with Neurobasal/B27medium. Neurobasal/B27medium containing25mM glucose (control condition) is intended to give optimal growth and long-term survival to rat embryonic hippocampal neurons. After7days in culture, cells were incubated with50,75,100,125mM of glucose or with75mM mannitol (plus75mM mannitol in normal medium), which was used as an osmotic control, and maintained for further24,48,72and96h. The optimal time point and glucose concentration were selected for further study as a representation of hyperglycemic condition. At day8, hippocampal neurons were incubated with various concentrations of PMQ (0.1,0.3,1,3and10μM, PMQ were dissolved in dimethylsulphoxide.) or vehicle under this optimal time point and glucose concentration. To study the mechanism involved in PMQ effects on high glucose-induced neurotoxicity, hippocampal neurons were incubated under this optimal time point and glucose concentration with PMQ (1μM), either with or without LY294002(a specific inhibitor of PI3kinase;50μM; Beyotime Institute of Biotechnology, China).Results:1. GK and n-STZ rats exhibited severe hyperglycemia, hyperinsulinemia and significant insulin resistance, as well as significantly reduced weight gain and increased food and water uptake compared with Wistar rats and SD rats. At28weeks of age, GK and n-STZ rats displayed significantly increased escape latencies in the hidden platform paradigm, while spending less time in the target quadrant in a probe trial (Fig.1G) compared with Wistar rats and SD rats. The performance of all groups in the trial with the visible platform was not significantly different. Furthermore, velocities were similar. The escape latency time varied in proportion to the insulin resistance. In the CA1sectors of GK and n-STZ rats, cells were sparsely arranged and the cell shapes were fuzzy; cells with eumorphism were significantly reduced; the%cell viability was significantly lower. The dendritic spines density was significantly decreased in the CA1hippocampus of the GK and n-STZ rats compared with Wistar rats and SD rats. The fasted glucose levels and the neuronal cell viability and dendritic spine density were observed a significant positive correlation between these parameters. The GK and n-STZ rats showed a significant decrease in phosphorylation of Akt and CREB, and the levels BDNF and synaptophysin also was a significant decrease.2. PMQ treatment alleviated weight loss, ameliorated the polyphagia and polydipsia symptom, reduced levels of fed blood glucose, fasting insulin levels and ameliorated glucose intolerance. PMQ intervention significantly dose-dependently improved insulin resistance. Treatment with MET in GK rats showed similar results.PMQ treatment significantly decreased escape latency and swimming distance and increased the time spent in target quadrant compared to GK rats group, Treatment with MET in GK rats showed similar results. The escape latency time varied in proportion to the insulin resistance.The administration of PMQ and MET significantly increased the dendritic spines density compared with the GK rats. However, the dendritic length of the pyramidal cells of the CA1hippocampus was not significantly different in all groups. In PMQ (10mg/kg group) and MET group, cell shapes were clear and the cell structure was compact; cells were relative large and had abundant cytoplasm and Nissl bodies. The%cell viability was significantly higher. PMQ significantly activated the Akt/cAMP response element-binding protein pathway and increased the expression of memory-related proteins in the downstream part of the Akt/cAMP response element-binding protein pathway, such as SYP and GluR1.3. When incubating cells for24hours, we did not find any detrimental effect of high glucose (HG) on cell viability at concentrations ranging from50to125mM; when incubated for48,72and96h, cells activities were inhibited. Dealt72h with100mM of glucose, the hippocampus neurons activity dropped to58.8%(P<0.01vs. control group). So this time point and glucose concentration were selected for further study as a representation of high glucose condition. To support the in vivo findings reported above, hippocampal neurons grown in HG media were treated with PMQ. As expected, the cells treated with0.1,0.3,1,3and10μM of PMQ in the presence of100mM glucose for72h exhibited higher viability than HG group. Similarly, we have found that the high glucose condition can lead to shortening dendrite of hippocampal neurons, decreasing the numbers of the branches, influencing the normal growth of neuronal cells, while PMQ (1μM) can ameliorate the pathological changes. Pretreatment with LY294002resulted in a part loss of PMQ-mediated protective effects in hippocampal neurons. Concurrent treatment with1μM PMQ resulted in elevations in p-Akt, p-CREB, SYP, GluRl protein. When hippocampal neurons were pretreated with LY294002for30min, PMQ did not increase the levels of p-Akt, p-CREB, SYP, GluRl protein.Conclusion:1. Insulin resistance could predominantly reduce Akt/cAMP response element-binding protein activation in the brain, which is associated with a higher risk of cognitive dysfunction.2. Pentamethylquercetin ameliorates cognitive deficits through enhancing of Akt/CREB signaling pathways in diabetic goto-kakizaki rats.3. Pentamethylquercetin could provide a new potential option for prevention of the cognitive dysfunction in diabetes.