| Objective:Diabetes mulllitus (DM) is an endocrine disorder of carbohydrate metabolism resulting from inadequate insulin release (type1diabetes, T1D) or insulin insensitivity (type2diabetes, T2D). It currently affects250million people worldwide, with6million new cases reported each year. The prevalence rises with age from12%in people aged65to15%in people over age80. DM is a systemic disease that can damage many organs in the body. Complications include pathologic changes involving both small and large vessels, cranial and peripheral nerves, skin, and eyes. These organic lesions may lead to hypertension, renal failure, vision loss, automomic and peripheral neuropathy, peripheral vascular disease, myocardial infarction and cerebrovascular disease, including stroke. In recent years, there is evidence illustrating that both T1D and T2D can also cause complications within the central nervous system (CNS). Manifestations of diabetes-induced CNS complications may include structural alterations or brain atrophy, as well as changes in electrophysiological properties that ultimately result in deficits in cognitive performance. The cognitive dysfunction induced by DM represents another serious problem and is rising in prevalence worldwide, especially among the elderly. The diabetes-induced cognitive dysfunction may be associated with the dysfunctional glucose regulation, the insufficient insulin availability, the accumulation of advanced glycation end products, oxidative stress, etc. However, the mechanisms of the deficits in cognitive performance in diabetic patients are still unclear.Aspirin, an anti-inflammatory medication, is often used to relieve minor aches and pains, to reduce fever. Aspirin also has an antiplatelet effect by inhibiting the production of thromboxane, which under normal circumstances binds platelet molecules together to create a patch over damaged walls of blood vessels. Because the platelet patch can become too large and also block blood flow, locally and downstream, aspirin is also used long-term, at low doses, to help prevent heart attacks, strokes, and blood clot formation in people at high risk of developing blood clots. It is reported that low-dose aspirin might be protective for decline in memory in individuals of75years and over. Danshen, a traditional Chinese herbal medicine, is the dried root of the plant Salvia miltiorrhiza Bunge belonging to the Labiata family. It has been widely used in China, Korea, Japan and other Asian countries in the treatment of cerebrovascular diseases. Danshensu is the major water-soluble component of Danshen. Many studies have evaluated the effect of Danshen extracts on cerebral ischemia.Up to date, it remains unclear whether aspirin and Danshensu can ameliorate the cognitive dysfunction induced by hyperglycemia. Therefore, the present study was designed to investigate the effects of aspirin and Danshensu on cognitive decline in diabetes.Methods:There are two parts in the present study.1. The effect of aspirin on hyperglycemia-induced learning and memory decline in miceC57BL/6mice were treated with a single intraperitoneal injection of streptozotocin (150mg/kg body weight, dissolved in sodium citrate solution,0.1mol/L, pH4.5). Age-matched control mice were injected with sodium citrate solution. Seventy two hours after streptozotocin treatment, fasting blood glucose was determined using a glucose analyzer. Animals with blood glucose levels exceeding20mmol/L were selected as the diabetic mice. Twenty-eight diabetic mice were randomly divided into four groups with seven animals in each group:diabetes group, administration with aspirin for1week group, administration with aspirin for4weeks group, administration with aspirin for8weeks group. On the first, fifth and seventh week, the mice in aspirin (8W), aspirin (4W) or aspirin (1W) were continually administered with aspirin at a dose of30mg/kg by intragastric administration once a day for8weeks,4weeks and1week respectively. While the mice in control group and diabetes group were administered with vehicle (distilled water). Eight weeks after aspirin or vehicle treatment, the learning and memory of the mice were evaluated by water maze. The swim path of each subject and calculated mean escape latency (the time between being placed in the water and finding the hidden platform), total path lengths, average swim speed, and the percentage of time spent in target quadrant were analyzed by an automated tracking system. After the evaluation of learning and memory, the body weight, the fasting blood glucose and serum insulin were determined. Then the mice were sacrificed under phenobarbital anesthesia and transcardial perfusion with ice-cold normal saline (10mL/10g body weight) was performed. The hippocampus were rapidly removed and weighed. Then the hippocampus was homogenized. TNF-a and IL-1(3contents in the supernatant were assayed by enzyme-linked immunosorbent assay. The assay for acetylcholinesterase activity in the supernatant was performed with the colorimetric method utilizing acetylthiocholine iodide as a substrate.2. The effect of Danshensu on cognitive decline in streptozotocin-induced diabetic micePrimary hippocampus neuron-glia cultures were prepared from7-day-old C57BL/6mouse pups. Then the cells were exposed to0,25,50,100,200, or400μg/mL of advanced glycation end products (AGEs) for48h. Survival of cells was assessed utilizing the MTT method. LDH in the supernatant was evaluated using LDH cytotoxicity assay kit. The cells were exposed to AGEs (100μg/mL) and0.1,1, or10μM of Sodium Danshensu for48h. Survival of cells was assessed utilizing the MTT method. LDH, NO, PGE2, or TNF-a in the supernatant were assayed. The cultures were rinsed carefully with PBS and fixed in paraformaldehyde. Cells were sequentially incubated with anti-NeuN antibody. The numbers of NeuN-positive neurons were counted at100x magnification with a Nikon inverted microscope.Sprague Dawley rats were randomly divided into two groups with24animals in each group:the control group and the verapamil group. The rats in the verapamil group were administered intraperitoneally with verapamil at a dose of20mg/kg. The rats in the control group were treated with the same volume of normal saline. Ninety minutes later, all rats were treated intravenously with Sodium Danshensu (15mg/kg) by tail vein. At15min,30min and60min after Sodium Danshensu treatment, the animals were anesthetized with chloral hydrate and then5mL heparinized blood was collected. The rats were perfused with100mL of ice-cold normal saline each. The brain was rapidly removed from the cranium and weighed and homogenized in4volumes of ice phosphate buffer. The content of Danshensu in plasma and homogenate was detected by high performance liquid chromatography-electrospray ionization tandem mass spectrometry.Sprague Dawley rats were randomly assigned to three groups (n=27):the sham operation group (sham), the cerebral ischemia/reperfusion group (I/R) and the cerebral ischemia/reperfusion plus Sodium Danshensu group (I/R+Sodium Danshensu). Cerebral ischemia was induced by an intraluminal middle cerebral artery occlusion (MCAO) technique as described by Longa. After2h of MCAO, reperfusion was induced by withdrawing the filament. The sham operation group underwent the same procedure but without vascular occlusion. Immediately after reperfusion, the sham and I/R+Sodium Danshensu group were treated intravenously with Sodium Danshensu by tail vein, the rats in I/R group were treated with the same volume of normal saline. At15min,30min, and60min after Sodium Danshensu treatment, the animals were anesthetized with chloral hydrate and then5mL heparinized blood was collected. Then the rats were perfused with100mL of ice-cold normal saline each. The brain was rapidly removed from the cranium and weighed and homogenized in4volumes of ice phosphate buffer. The content of Danshensu in plasma and homogenate was detected by high performance liquid chromatography-electrospray ionization tandem mass spectrometry. The P-gp expression in brain was assayed by Western blot analysis.C57BL/6mice were treated with a single intraperitoneal injection of streptozotocin (200mg/kg body weight, dissolved in sodium citrate solution,0.1mol/L, pH4.5). Age-matched control mice were injected with sodium citrate solution. Seventy two hours after streptozotocin or vehicle treatment, fasting blood glucose was determined using a glucose analyzer. Animals with blood glucose levels exceeding20mmol/L were selected as the diabetic mice. The diabetic mice were randomly divided into four groups:diabetes group and three Sodium Danshensu groups. The mice in Sodium Danshensu groups were administered with Sodium Danshensu at a dose of15,30, or60mg/kg by gavage once a day for12weeks, while the mice in the control and diabetes groups were dosed with vehicle (distilled water). Twelve weeks after Sodium Danshensu or vehicle treatment, the learning and memory of the mice were evaluated by water maze. The swim path of each subject and calculated mean escape latency (the time between being placed in the water and finding the hidden platform), total path lengths, average swim speed, and the percentage of time spent in the target quadrant were analyzed by an automated tracking system. Then the body weight and the fasting blood glucose were determined. Blood samples were taken from the venae angularis and serum glycosylated hemoglobin (GHb) and insulin were determined using an ELISA kits. The hippocampus was rapidly removed and weighed and then homogenized in4volumes of0.1mol/L ice-cold phosphate buffer. The AGEs, TNF-a, IL-6, and PGE2levels in the supernatant were assayed by ELISA kits according to the manufacturer's instructions. The levels of the receptor of AGEs (RAGE), p38, phospho-p38(p-p38), p65, and COX-2in hippocampus were detected with Western blot method.Results:1. The mean escape latency for the trained mice decreased during the course of the five-day trials. The mean escape latency did not have any difference between any two groups on the first and second day. But on the third day, the mean escape latency of diabetes group was longer than that of control group. Compared with diabetes group, the escape latency of mice treated with aspirin for4or8weeks decreased. There was no difference in average swim speed in the five-day trials. In the probe trial study, the time spent in the target quadrant was significantly shorter in diabetes group as compared to the control group. The mice in aspirin (4W) and aspirin (8W) spent more time in the target quadrant than the diabetes group. There was no significant difference in the percentage of time spent in the target quadrant between aspirin (1W) group and diabetes group. There was no difference in the total path lengths between any two groups in the probe trial.Compared with control mice, diabetic mice had higher levels of fasting blood glucose, accompanied by lower body weight. It was showed that the serum insulin levels of diabetic mice significantly decreased after streptozotocin injection. Compared with the mice in diabetes group, both long-term and short-term treatment with aspirin had no effect on body weight, fasting blood glucose and insulin levels.TNF-a and IL-1β contents significantly increased in the hippocampus of diabetic mice as compared to that of control group. Compared with diabetes group, the TNF-a and IL-1β contents in aspirin (4W) and aspirin (8W) reduced, while the TNF-a and IL-1β contents in aspirin (1W) did not decrease remarkedly.The acetylcholinesterase activity in hippocampus of diabetes group significantly increased as compared to that of control group. Treatment with aspirin for4or8weeks reduced the acetylcholinesterase activity in hippocampus.2. Primary hippocampus neuron-glia undergoes degeneration when exposed to AGEs. Survival of cells was decreased dose dependently. While LDH in the supernatant significantly increased. Compared with control, Sodium Danshensu increased the survival of cells and the numbers of NeuN-positive neurons, inhibited the increase of LDH in supernatant. Sodium Danshensu also reduced the levels of NO, PGE2, or TNF-a in the supernatant.3. At15min,30min and60min after Sodium Danshensu treatment, Danshensu concentrations in the brain of the verapamil group were significantly higher than that of the control group. Compared with control, pre-treatment with verapamil had no effect on Danshensu concentrations in plasma. At15min,30min and60min after cerebral ischemia/reperfusion, Danshensu concentrations in the ischemic hemisphere of I/R+Sodium Danshensu group increased significantly when compared with that in non-ischemic hemisphere of I/R+Sodium Danshensu group and in the brain of sham group. There was no difference observed in Danshensu concentration between the non-ischemic hemisphere of I/R+Sodium Danshensu group and in the brain of sham group. There was no significant difference observed in Danshensu concentrations of I/R+Sodium Danshensu group in plasma when compared with that in sham group. In sham group, Danshensu had no effect on the expression of P-gp. However, the expression of P-gp in the ischemic hemisphere of I/R and I/R+Sodium Danshensu groups were lower than that of sham group. Furthermore, at60min after Sodium Danshensu treatment, the P-gp level in the ischemic hemisphere of I/R+Sodium Danshensu group decreased significantly as compared with the sham group or I/R group.4. The ANOVA for repeated measures followed by least significant difference testing revealed that treatment with Sodium Danshensu resulted in a significant reduction in escape latency as compared with the diabetes group. There was no difference in average swim speed in the five-day trials. In the retrieval trial study, the time spent in the target quadrant was significantly shorter in the diabetes group than in the control group. The mice in the Sodium Danshensu groups spent more time in the target quadrant than the diabetes group. There was no difference in the total path lengths between any two groups in the probe trial.Compared with the control mice, the diabetic mice had higher levels of fasting blood glucose and GHb, accompanied by lower body weight. The serum insulin levels of the diabetic mice significantly decreased after streptozotocin injection. Sodium Danshensu treatment had no effect on body weight, fasting blood glucose, insulin, and GHb levels. The AGEs level in the hippocampus of the diabetes group was significantly increased in comparison with the control group. Treatment with Sodium Danshensu did not reduce the AGEs level in the hippocampus. RAGE protein expression was significantly higher in the diabetic group than in the control group. However, the Sodium Danshensu groups had lower RAGE expression than the diabetic group. There were no significant differences in the protein expression of p38between any two groups. The level of p-p38in the diabetic group significantly increased. Sodium Danshensu treatment markedly attenuated the diabetes-induced phosphorylation of p38. NF-κB activation in nuclear extracts from the hippocampus of diabetic mice was significantly increased. Treatment with Sodium Danshensu abolished the p38-phosphorylation-induced increase of NF-κB activation. COX-2protein was up-regulated in the diabetes group. The increase of COX-2expression was significantly blocked by treatment with Sodium Danshensu.The levels of TNF-a, IL-6, and PGE2significantly increased in the hippocampus of diabetic mice compared to those in the control group. Compared with the diabetes group, the TNF-a, IL-6, and PGE2levels in the Sodium Danshensu groups were reduced. The acetylcholinesterase activity in hippocampus of diabetes group significantly increased as compared to that of control group. Treatment with Sodium Danshensu had no effect on the acetylcholinesterase activity in hippocampus.Conclusions:1. Hyperglycemia-associated learning and memory decline were attenuated by long-term aspirin treatment in mice, and that the effect of aspirin was related to its anti-inflammatory potency.2. AGEs can result in the injury of hippocampus cells and the mechanism is related to the neuroinflammation induced by AGEs.3. Danshensu can across the blood-brain barrier and P-glycoprotein plays an important role in Danshensu transportation in brain.4. The hyperglycemia-induced cognitive dysfunction is associated with a signaling cascade in the hippocampus in which AGEs-bound RAGE leads to the phosphorylation of p38MAPK and causes increased NF-κB activation and secretion of pro-inflammatory cytokines5. The cognitive decline in diabetic mice was both prevented and reversed by treatment with sodium danshensu, and the mechanism was associated with the anti-inflammatory potency that followed the decreased expression of RAGE caused by Danshensu.
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