硫化氢对糖尿病大鼠认知障碍的拮抗作用：依赖于海马多胺信号通路

发布时间：2018-01-24 10:19

本文关键词： 硫化氢糖尿病认知障碍多胺凋亡　出处：《南华大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的：糖尿病是一种由于胰岛素分泌不足或胰岛素抵抗引起的血糖升高的代谢性疾病,糖尿病引起的认知障碍并发症逐年增高,并严重影响患者日常生活。硫化氢(Hydrogen sulfide, H2S)是一种新型的内源性气体信号分子,具有抗凋亡、抗氧化、抗内质网应激和增强海马神经元细胞可塑性等神经保护作用。本课题组以往研究发现H2S具有拮抗糖尿病大鼠认知障碍和海马损伤的作用,但其具体机制尚未明确。多胺广泛存在生物体内,是维持细胞正常生理功能不可缺少的低分子脂肪族含氮化合物,具有调节神经递质受体、神经的生长发育、增强突触的可塑性和增强学习记忆能力等神经保护作用。因此,我们将从认知行为学和海马损伤两个方面探讨H2S对糖尿病大鼠认知障碍的拮抗作用是否依赖于多胺信号通路。方法：将雄性SD大鼠给予一次性的腹腔注射链脲佐菌素(Streptozotocin, STZ,55 mg/kg),3天后尾静脉采血测血糖,血糖"g16.7 mmol/L,视为糖尿病模型造模成功；分别用新物体识别实验、Y迷宫、穿梭实验、水迷宫检测评价各组SD大鼠认知功能；通过HE染色观察海马形态学改变；利用Tunel法检测海马细胞凋亡情况；Western blot检测海马内相关凋亡蛋白Bcl-1、Bax的表达。结果：1.H2S可改善糖尿病大鼠的认知功能。当糖尿病大鼠予以NaHS(H2S供体,30,100 pmol/kg/d, ip)处理30天后：(1)新物体识别实验结果显示,NaHS (100 μmol/kg)明显改善糖尿病大鼠新物体识别指数；(2)Y-迷宫实验结果显示,NaHS (100 μmol/kg)明显提高糖尿病大鼠交替正确率；(3)穿梭实验结果显示,NaHS (100 μmol/kg)明显提高糖尿病大鼠主动逃避率和缩短糖尿病大鼠平均主动逃避时间；(4)水迷宫实验结果显示,NaHS (100 pmol/kg)明显缩短糖尿病大鼠寻找平台的潜伏期,增加糖尿病大鼠穿越原平台的的次数以及进入目标象限(原来放平台的象限)的时间。上述结果表明,H2S对糖尿病大鼠认知功能具有改善作用。2.H2S可拮抗糖尿病大鼠的海马损伤。当糖尿病大鼠予以NaHS (30,100 μmol/kg, ip)处理30天后：(1)HE染色结果显示,NaHS (100 μmol/kg)明显使糖尿病大鼠海马CA3区细胞排列得更整齐,减少神经细胞核溶解和神经细胞缺失；(2) Tunel染色结果显示,NaHS (100 μmol/kg)明显减少糖尿病大鼠海马CA3区细胞凋亡；(3) Western blot检测结果显示,NaHS (100 μmol/kg)明显提高糖尿病大鼠海马内Bcl-2蛋白的表达,减少Bax蛋白的表达。以上结果提示,H2S对糖尿病大鼠海马损伤有拮抗作用。3.二氟甲基鸟氨酸(Difluoromethylomithine, DFMO)逆转H2S对糖尿病大鼠认知功能的改善作用。在糖尿病大鼠予以NaHS (100 pmol/kg/d, ip)和DFMO (5 μg/d, icv)共同处理30天后：(1)新物体识别实验结果显示,DFMO逆转NaHS (100 μmol/kg)对糖尿病大鼠新物体识别指数的增加作用；(2)Y-迷宫实验结果显示,DFMO逆转NaHS(100 μmol/kg)对糖尿病大鼠交替正确率的提高作用；(3)穿梭实验结果显示,DFMO逆转NaHS (100 μmol/kg)对糖尿病大鼠主动逃避率的增加作用和对糖尿病大鼠平均主动逃避时间的缩短作用；(4)水迷宫实验结果显示,DFMO逆转NaHS (100 μmol/kg)对糖尿病大鼠寻找平台潜伏期的缩短作用,以及对糖尿病大鼠穿越原平台次数和进入目标象限(原来放平台的象限)时间的减少作用。上述结果表明,阻断多胺信号通路能逆转H2S对糖尿病大鼠认知功能的改善作用。4. DFMO逆转H2S对糖尿病大鼠海马损伤的改善作用。在糖尿病大鼠予以NaHS (100 μmol/kg/d, ip);和DFMO (5 μg/d, icv)共同处理30天后：(1)HE染色结果显示,DFMO逆转NaHS (100μmol/kg)对糖尿病大鼠海马CA3区细胞排列的改善作用和对神经细胞核溶解、神经细胞缺失的减少作用；(2) Tunel染色结果显示,DFMO逆转NaHS (100 μmol/kg)对糖尿病大鼠CA3区凋亡细胞的减少作用；(3) Western blot检测结果显示,DFMO逆转NaHS (100 μmol/kg)对糖尿病大鼠海马内Bcl-2蛋白表达的增加作用和Bax蛋白表达的减少作用。以上结果提示,阻断多胺信号通路能逆转H2S对糖尿病大鼠海马损伤的拮抗作用。结论：H2S对糖尿病大鼠认知障碍和海马损伤的拮抗作用依赖于多胺信号通路。
[Abstract]:Objective: diabetes is a due to insulin secretion due to elevated blood glucose or insulin resistance metabolic disorders, cognitive disorders caused by complications of diabetes increased year by year, and seriously affected the daily life of the patients. Hydrogen sulfide (Hydrogen sulfide H2S) is a kind of new gasotransmitter, anti apoptosis, antioxidant, anti ER stress and enhancement of hippocampal neuron plasticity of neural protection. Previous study of our group found that H2S has a cognitive impairment in diabetic rats and antagonism of hippocampal damage, but the mechanism is not clear. Polyamines widely exist in organisms, low molecular aliphatic is indispensable to maintain the normal physiological function of the cells containing compounds with neural regulation neurotransmitter receptors, nerve growth, enhance synaptic plasticity and reinforcement learning and memory ability of neural protection. Therefore, we will from the cognitive behavior and hippocampal damage in two aspects to explore the antagonistic effects of H2S on cognitive dysfunction of diabetic rats is dependent on the polyamine pathway. Methods: male SD rats were given a one-time intraperitoneal injection of streptozotocin (Streptozotocin, STZ, mg/ 55 kg), blood sugar in 3 days tail vein blood glucose, "g16.7 mmol/L, as the diabetic models were successful; respectively with new object recognition test, Y maze, through experiment, the cognitive function of SD rats were detected to evaluate water maze; morphological changes of hippocampus were observed by HE staining; detecting the apoptosis of hippocampal cells by Tunel method; Bcl-1 Western blot related apoptosis protein detection in the hippocampus, the expression of Bax. Results: 1.H2S can improve the cognitive function of diabetic rats. The diabetic rats treated with NaHS (H2S donor, 30100 pmol/kg/d, IP) 30 days after treatment: (1) the new object recognition. The experiment results show that NaHS (100 mol/kg) significantly improved in diabetic rats, the new object recognition index; (2) Y- maze test showed that the NaHS (100 mol/kg) significantly improved the correct rate of diabetic rats alternately; (3) through the experimental results show that NaHS (100 mol/kg) significantly increased in diabetic rats active escape rate and shorten the average time of active avoidance of diabetic rats; (4) the water maze test showed that the NaHS (100 pmol/kg) significantly shortened the latency to find the platform in diabetic rats, diabetic rats increased the frequency of crossing the original platform and enter the target quadrant (original platform quadrant). The results of the time show that H2S can improve the function of.2.H2S can antagonize diabetic rats hippocampus damage on cognitive function in diabetic rats. The diabetic rats treated with NaHS (30100 mol/kg, IP) 30 days after treatment: (1) HE staining showed that NaHS (100 m Ol/kg) was the hippocampus CA3 in diabetic rats are arranged more regularly, reduce the loss of nerve cells and nerve cell lysis; (2) Tunel staining showed that NaHS (100 mol/kg) significantly reduced cell apoptosis in hippocampal CA3 region of diabetic rats; (3) Western blot detection results show that NaHS (100 mol/kg) significantly increased the expression of Bcl-2 protein in hippocampus of diabetic rats, decrease the expression of Bax protein. These results suggest that H2S antagonist.3. two DFMO on hippocampal injury in diabetic rats (Difluoromethylomithine, DFMO) to improve the reversal effect of H2S on cognitive function in diabetic rats. The NaHS in diabetic rats (100 pmol/kg/d, IP) and DFMO (5 g/d, ICV) to 30 days of treatment: (1) the new object recognition experiment results show that DFMO reverse NaHS (100 mol/kg) on diabetic rat novel object recognition index increase (2; ) Y- maze test showed that the reversal of DFMO NaHS (100 mol/kg) function to enhance the correct rate of the diabetic rats alternately; (3) through the experimental results show that DFMO reverse NaHS (100 mol/kg) on diabetic rats and increase the rate of active avoidance effect on diabetic rats the average active escape time shortening effect (4); water maze test showed that DFMO reversed NaHS (100 mol/kg) in order to shorten the latency of platform of diabetic rats and diabetic rats, the frequency of crossing the original platform and enter the target quadrant (original platform quadrant) time reducing effect. The results show that blocking polyamine signaling pathways improve the reversal effect of H2S on cognitive function in diabetic rats the effect of.4. DFMO H2S on reversing hippocampal injury in diabetic rats. The NaHS in diabetic rats (100 mol/kg/d, IP); and DFMO (5 g/d, ICV) in common 30 days: (1) HE staining showed that DFMO reversed NaHS (100 mol/kg) to improve the function of neurons in hippocampal CA3 region of diabetic rats and the arrangement of nucleus dissolved, nerve cell loss reducing effect; (2) Tunel staining showed that DFMO reversed NaHS (100 mol/kg) on the apoptosis of cells CA3 region of diabetic rats reduced; (3) Western blot assay showed that DFMO reversed NaHS (100 mol/kg) to increase the role of expression of Bax protein and Bcl-2 protein expression in hippocampus of diabetic rats decreased. These results suggest that blocking polyamine pathway can reverse H2S of hippocampal injury in diabetes the antagonistic effect of rat. Conclusion: H2S antagonistic effects on cognitive impairment in diabetic rats and hippocampus dependent on polyamine pathway.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2;R749.2

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