咯利普兰对糖尿病脑病认知障碍和神经炎症改善作用的研究

发布时间：2018-07-13 07:55

【摘要】：第一部分目的观察糖尿病脑病（DE）大鼠认知功能及海马组织肿瘤坏死因子α（TNF-α）、白细胞介素10（IL-10）表达的变化，探讨炎症在DE发病机制中的作用。方法30只雄性SD大鼠随机分为对照组和糖尿病组，糖尿病组给予高脂饮食4周后按30mg/kg腹腔注射链脲佐菌素（STZ）建立2型糖尿病（T2DM）大鼠模型，并在STZ注射前和实验末测量大鼠体重、空腹血糖和空腹胰岛素。实验末行水迷宫测试评价两组大鼠认知功能。酶联免疫吸附测定法检测大鼠海马组织的β-淀粉样蛋白（Aβ）的浓度，蛋白质印迹法检测TNF-α和IL-10的表达，免疫组织化学法观察Aβ、TNF-α和IL-10的表达。结果糖尿病组大鼠空腹血糖和空腹胰岛素高于对照组，体重低于对照组（均P＜0.001）。糖尿病组大鼠在目标象限的探索时间比对照组短（P＜0.01），原平台穿越次数相对对照组也较少（P＜0.01），糖尿病组Aβ和TNF-α表达较对照组高（均P＜0.01），IL-10表达较对照组低（P＜0.01），免疫组织化学染色观察糖尿病组Aβ、TNF-α阳性表达明显，IL-10阳性表达较少。结论T2DM能够诱发DE，DE大鼠表现为空间学习记忆能力下降，，海马组织Aβ沉积增多，炎症反应增强，认知的下降可能与炎症因子表达失衡有关。第二部分目的观察磷酸二酯酶4（PDE4）抑制剂咯利普兰对DE大鼠的认知下降是否具有改善作用以及药物对海马组织炎症的影响。方法45只雄性SD大鼠随机分为对照组、糖尿病组和咯利普兰组。糖尿病组和咯利普兰组给予高脂饮食加小剂量STZ（30mg/kg，腹腔注射）诱导T2DM模型，并在STZ注射前和实验末测量大鼠体重、空腹血糖和空腹胰岛素。STZ注射2个月后开始给予咯利普兰治疗23天（0.5mg/kg），治疗2周后行水迷宫实验评估大鼠空间学习记忆能力。实验末采用酶联免疫吸附测定法检测海马组织Aβ1-40和Aβ1-42，蛋白质印迹法分析海马组织TNF-α、IL-10、环磷酸腺苷反应原件结合蛋白（CREB）和磷酸化CREB（pCREB）的表达。结果糖尿病大鼠空腹血糖和空腹胰岛素高于对照组，体重低于对照组(均P＜0.001)，咯利普兰组和糖尿病组比较，差异没有统计学意义（P＞0.05）。隐形平台实验中糖尿病组大鼠较对照组和咯利普兰组，逃避潜伏期延长，目标象限探索时间缩短，穿越平台次数减少。与对照组比较，糖尿病组和咯利普兰组大鼠海马组织Aβ1-40和Aβ1-42表达增多（均P＜0.01），咯利普兰组与糖尿病组相比较没有统计学意义（均P＞0.05）。糖尿病组TNF-α表达增多，咯利普兰组比糖尿病组TNF-α表达减少（均P＜0.01）；糖尿病组IL-10、CREB、pCREB表达减少，咯利普兰组表达升高(均P＜0.01)。结论PDE4抑制剂咯利普兰可通过增加2型糖尿病大鼠海马组织CREB的表达、CREB磷酸化水平，从而恢复炎症因子表达的失平衡，进而改善DE大鼠的认知功能。
[Abstract]:Objective to observe the changes of cognitive function, tumor necrosis factor- 伪 (TNF- 伪) and interleukin-10 (IL-10) expression in hippocampus of diabetic encephalopathy (DE) rats and to explore the role of inflammation in the pathogenesis of DE. Methods Thirty male Sprague-Dawley rats were randomly divided into two groups: control group and diabetic group. After 4 weeks of high-fat diet, diabetic rats were injected with streptozotocin (STZ) intraperitoneally to establish type 2 diabetes mellitus (T2DM) model, and their body weight was measured before and at the end of the experiment. Fasting blood glucose and fasting insulin. At the end of the experiment, water maze test was performed to evaluate the cognitive function of the two groups. The concentration of 尾 -amyloid protein (A 尾) in rat hippocampus was detected by enzyme-linked immunosorbent assay (Elisa), the expression of TNF- 伪 and IL-10 was detected by Western blotting, and the expression of TNF- 伪 and IL-10 was detected by immunohistochemistry. Results the fasting blood glucose and fasting insulin in diabetic group were higher than those in control group, and their body weight was lower than that in control group (P < 0.001). The exploration time in target quadrant of diabetic rats was shorter than that of control group (P < 0.01), and the number of crossing of original platform was also less than that of control group (P < 0.01). The expression of A 尾 and TNF- 伪 in diabetic group was higher than that in control group (P < 0.01), and the expression of IL-10 in diabetic group was lower than that in control group (P < 0.01). The positive expression of A 尾 -TNF- 伪 in diabetic group was significantly lower than that in control group. Conclusion T2DM can induce the decrease of spatial learning and memory ability, increase of A 尾 deposition in hippocampus, and increase of inflammatory response. The decrease of cognition may be related to the imbalance of inflammatory factor expression. In the second part, we investigated whether pyripran, an inhibitor of phosphodiesterase 4 (PDE4), could ameliorate the cognitive decline in DE rats and the effect of PDE4 on hippocampal inflammation. Methods 45 male Sprague-Dawley rats were randomly divided into control group, diabetic group and Rolipram group. T2DM model was induced by high-fat diet and low-dose STZ (30 mg / kg, intraperitoneal injection) in diabetic group and Rolipram group. The weight of rats was measured before and after STZ injection. Two months after injection of fasting blood glucose and fasting insulin. STZ was administered with Rolipram for 23 days (0.5mg/kg). After 2 weeks of treatment, the spatial learning and memory ability of rats was evaluated by water maze test. At the end of the experiment, A 尾 1-40 and A 尾 1-42 were detected by enzyme-linked immunosorbent assay (Elisa). The expression of TNF- 伪 -IL-10, the original binding protein (CREB) and phosphorylated CREB (pCREB) in hippocampal tissues were analyzed by Western blot. Results the fasting blood glucose, fasting insulin and body weight of diabetic rats were higher than those of control group (all P < 0.001). There was no significant difference between Rolipram group and diabetes group (P > 0.001). In the invisible platform experiment, compared with the control group and the Rolipram group, the escape latency was prolonged, the target quadrant exploration time was shortened, and the number of crossing the platform was decreased in the diabetic group. Compared with the control group, the expression of A 尾 1-40 and A 尾 1-42 in the hippocampus of diabetic group and Rolipram group were increased (P < 0.01), but there was no significant difference between rolipran group and diabetic group (all P > 0.05). The expression of TNF- 伪 in diabetic group was higher than that in control group (all P < 0.01), and the expression of IL-10 CREB-pCREB was lower in the group of rolipran than that in the group of diabetes mellitus (P < 0.01), and the expression of IL-10 CREB-pCREB was higher in the group of clolipram (all P < 0.01). Conclusion Rolipram, a PDE4 inhibitor, can restore the imbalance of the expression of inflammatory factors by increasing the expression of CREB in hippocampal tissue of type 2 diabetic rats and improve the cognitive function of DE rats.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R742

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