本文關鍵詞： 硫化氫 糖尿病 認知障礙 多胺 凋亡　出處：《南華大學》2016年碩士論文　論文類型：學位論文

【摘要】：目的：糖尿病是一種由于胰島素分泌不足或胰島素抵抗引起的血糖升高的代謝性疾病,糖尿病引起的認知障礙并發(fā)癥逐年增高,并嚴重影響患者日常生活。硫化氫(Hydrogen sulfide, H2S)是一種新型的內源性氣體信號分子,具有抗凋亡、抗氧化、抗內質網應激和增強海馬神經元細胞可塑性等神經保護作用。本課題組以往研究發(fā)現(xiàn)H2S具有拮抗糖尿病大鼠認知障礙和海馬損傷的作用,但其具體機制尚未明確。多胺廣泛存在生物體內,是維持細胞正常生理功能不可缺少的低分子脂肪族含氮化合物,具有調節(jié)神經遞質受體、神經的生長發(fā)育、增強突觸的可塑性和增強學習記憶能力等神經保護作用。因此,我們將從認知行為學和海馬損傷兩個方面探討H2S對糖尿病大鼠認知障礙的拮抗作用是否依賴于多胺信號通路。方法：將雄性SD大鼠給予一次性的腹腔注射鏈脲佐菌素(Streptozotocin, STZ,55 mg/kg),3天后尾靜脈采血測血糖,血糖"g16.7 mmol/L,視為糖尿病模型造模成功；分別用新物體識別實驗、Y迷宮、穿梭實驗、水迷宮檢測評價各組SD大鼠認知功能；通過HE染色觀察海馬形態(tài)學改變；利用Tunel法檢測海馬細胞凋亡情況；Western blot檢測海馬內相關凋亡蛋白Bcl-1、Bax的表達。結果：1.H2S可改善糖尿病大鼠的認知功能。當糖尿病大鼠予以NaHS(H2S供體,30,100 pmol/kg/d, ip)處理30天后：(1)新物體識別實驗結果顯示,NaHS (100 μmol/kg)明顯改善糖尿病大鼠新物體識別指數；(2)Y-迷宮實驗結果顯示,NaHS (100 μmol/kg)明顯提高糖尿病大鼠交替正確率；(3)穿梭實驗結果顯示,NaHS (100 μmol/kg)明顯提高糖尿病大鼠主動逃避率和縮短糖尿病大鼠平均主動逃避時間；(4)水迷宮實驗結果顯示,NaHS (100 pmol/kg)明顯縮短糖尿病大鼠尋找平臺的潛伏期,增加糖尿病大鼠穿越原平臺的的次數以及進入目標象限(原來放平臺的象限)的時間。上述結果表明,H2S對糖尿病大鼠認知功能具有改善作用。2.H2S可拮抗糖尿病大鼠的海馬損傷。當糖尿病大鼠予以NaHS (30,100 μmol/kg, ip)處理30天后：(1)HE染色結果顯示,NaHS (100 μmol/kg)明顯使糖尿病大鼠海馬CA3區(qū)細胞排列得更整齊,減少神經細胞核溶解和神經細胞缺失；(2) Tunel染色結果顯示,NaHS (100 μmol/kg)明顯減少糖尿病大鼠海馬CA3區(qū)細胞凋亡；(3) Western blot檢測結果顯示,NaHS (100 μmol/kg)明顯提高糖尿病大鼠海馬內Bcl-2蛋白的表達,減少Bax蛋白的表達。以上結果提示,H2S對糖尿病大鼠海馬損傷有拮抗作用。3.二氟甲基鳥氨酸(Difluoromethylomithine, DFMO)逆轉H2S對糖尿病大鼠認知功能的改善作用。在糖尿病大鼠予以NaHS (100 pmol/kg/d, ip)和DFMO (5 μg/d, icv)共同處理30天后：(1)新物體識別實驗結果顯示,DFMO逆轉NaHS (100 μmol/kg)對糖尿病大鼠新物體識別指數的增加作用；(2)Y-迷宮實驗結果顯示,DFMO逆轉NaHS(100 μmol/kg)對糖尿病大鼠交替正確率的提高作用；(3)穿梭實驗結果顯示,DFMO逆轉NaHS (100 μmol/kg)對糖尿病大鼠主動逃避率的增加作用和對糖尿病大鼠平均主動逃避時間的縮短作用；(4)水迷宮實驗結果顯示,DFMO逆轉NaHS (100 μmol/kg)對糖尿病大鼠尋找平臺潛伏期的縮短作用,以及對糖尿病大鼠穿越原平臺次數和進入目標象限(原來放平臺的象限)時間的減少作用。上述結果表明,阻斷多胺信號通路能逆轉H2S對糖尿病大鼠認知功能的改善作用。4. DFMO逆轉H2S對糖尿病大鼠海馬損傷的改善作用。在糖尿病大鼠予以NaHS (100 μmol/kg/d, ip);和DFMO (5 μg/d, icv)共同處理30天后：(1)HE染色結果顯示,DFMO逆轉NaHS (100μmol/kg)對糖尿病大鼠海馬CA3區(qū)細胞排列的改善作用和對神經細胞核溶解、神經細胞缺失的減少作用；(2) Tunel染色結果顯示,DFMO逆轉NaHS (100 μmol/kg)對糖尿病大鼠CA3區(qū)凋亡細胞的減少作用；(3) Western blot檢測結果顯示,DFMO逆轉NaHS (100 μmol/kg)對糖尿病大鼠海馬內Bcl-2蛋白表達的增加作用和Bax蛋白表達的減少作用。以上結果提示,阻斷多胺信號通路能逆轉H2S對糖尿病大鼠海馬損傷的拮抗作用。結論：H2S對糖尿病大鼠認知障礙和海馬損傷的拮抗作用依賴于多胺信號通路。
[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.

【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R587.2;R749.2

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