米诺环素对海湾战争综合征大鼠模型海马CA1区神经细胞凋亡的保护作用

发布时间：2018-07-31 10:55

【摘要】： 背景与目的：参加1990～1991年海湾战争的近70万美国军人中,至少四分之一在战后出现了持续的、难以处理的健康问题,包括认知困难、持续广泛的疼痛、疲劳、头痛、慢性腹泻、皮疹等,被称为海湾战争综合征(Gulf War Syndrome,GWS)或海湾战争疾病。GWS的病因可能包括贫铀、油井火焰烟雾、神经毒气、溴化吡啶斯的明(PB)及杀虫剂(DEET)、疫苗接种、心理应激、感染性疾病等多种因素。海湾战争流行病学研究一致性发现,溴化吡啶斯的明及杀虫剂是与GWS明显有关的唯一两种接触,较长时间服用PB或多量使用杀虫剂者,GWS发生率增高。有关研究表明,GWS患者大脑海马、扣带回等脑区体积缩小。动物实验也提示海马神经细胞死亡。米诺环素(盐酸二甲胺四环素,minoeyeline hydroehloride)为第二代人工半合成四环类抗生素。研究表明,米诺环素是一种Caspase-3抑制剂,具有神经保护作用。本实验目的,联合应用PB及DEET建立海湾战争大鼠模型,观察海马CA1区神经细胞凋亡、凋亡蛋白酶激活因子-1(Apaf-1)及Caspase-3的表达,并探讨米诺环素的神经保护机制。方法：采用Abdel-Rahman法建立海湾战争综合征大鼠模型。实验设立对照组、束缚组、PB/DEET组、模型组、低剂量米诺环素组、高剂量米诺环素组。束缚组大鼠给与固定束缚,置于暗环境中,每次20分钟。模型组：参照Abdel-Rahman文献建立海湾战争大鼠模型,大鼠每天给与溴化吡啶斯的明(1.3mg/kg/d)口服,DEET (40mg/kg/d)以70%乙醇皮下注射,暗环境固定束缚20分钟。PB/DEET组：每天给予PB及DEET皮下注射。低剂量米诺环素组：米诺环素15mg/kg/d,腹腔注射,余同模型组。高剂量米诺环素组：米诺环素45mg/kg/d,腹腔注射,余同模型组。对照组大鼠自由取水进食,给与等量的酒精溶液皮下注射。实验周期为28天。在0天、14天及28天测定大鼠体重。实验结束后,行多聚甲醛灌流固定、取脑、石蜡包埋、切片、采用免疫组化检测大鼠海马CA1区神经细胞Apaf-1及Caspase-3的表达,采用TUNEL法检测海马CA1区神经细胞的凋亡情况。结果：各组大鼠体重均有不同程度的增加,4周后对照组大鼠体重增加了79%,束缚组为75%,PB/DEET组为49%,模型组为47%,低高剂量米诺环素组为70%,高剂量米诺环素组为71%。PB/DEET组与对照组、模型组与束缚组相比,体重增幅减少,P0.01,有统计学差异；束缚组与对照组相比,模型组与PB/DEET组比较,体重增加相似,P0.05,无统计学差异；低、高剂量米诺环素组与模型组相比较,体重增加明显,P0.01,有统计学差异。束缚组与对照组相比较以及PB/DEET组与模型组相比较,海马CA1区神经细胞凋亡数量、Caspase-3及Apaf-1积分光密度无统计学意义(P0.05)。模型组与对照组相比,海马CA1区凋亡细胞数增多,Caspase-3及Apaf-1积分光密度明显增高,统计学差异非常显著(P0.01)。低、高剂量米诺环素组与对照组比较,海马CA1区凋亡细胞数增多,Caspase-3及Apaf-1积分光密度明显增高,统计学差异非常显著(P0.01)。低、高剂量米诺环素组与模型组比较,海马CA1区凋亡细胞数减少,Caspase-3积分光密度降低,统计学差异非常显著(P0.01)。两个剂量米诺环素对海马CA1区神经细胞凋亡数目、Caspase-3及Apaf-1积分光密度的影响并未显示明显差异(P0.05)。结论：1、束缚应激对大鼠体重增幅无明显影响,对海马CA1区神经细胞未见明显损害。2、海湾战争综合征大鼠模型体重增幅减少,海马CA1区神经细胞凋亡增多,Caspase-3及Apaf-1表达增加,考虑与PB及DEET有关。3、米诺环素通过抑制Caspase-3的表达,减少神经细胞凋亡,对海马CA1区神经细胞具有神经保护作用。4、低、高剂量米诺环素对海湾战争大鼠模型的海马神经细胞保护作用未见明显区别。
[Abstract]:Background and purpose: of the nearly 700 thousand American soldiers who participated in the 1990~1991 year Gulf War, at least 1/4 of the post-war health problems, including cognitive difficulties, persistent and extensive pain, fatigue, headache, chronic diarrhea, rash, are known as the Gulf War Syndrome (GWS) or the Gulf War. The causes of disease.GWS may include depleted uranium, oil well flame smoke, nerve gas, PB and DEET, vaccination, psychological stress, infectious diseases and many other factors. The consensus of the Gulf War epidemiological studies found that pyridine bromide and insecticides are the only two contacts associated with GWS The incidence of GWS in the brain hippocampus and cingulate gyrus in GWS patients was reduced. The animal experiments also suggested that the hippocampal neurons died. Minocycline (two methylamine tetracycline hydrochloride, minoeyeline hydroehloride) was the second generation of artificial semi synthetic four ring antibiotics. Minocycline (minocycline) is a kind of Caspase-3 inhibitor, which has neuroprotective effect. The aim of this experiment was to establish a rat model of Gulf War with PB and DEET, to observe the apoptosis of neurons in the hippocampus CA1, the expression of -1 (Apaf-1) and Caspase-3, and to explore the neuroprotective mechanism of minocycline.
Methods: the rat model of Gulf War syndrome was established by Abdel-Rahman. The experiment set up the control group, the binding group, the PB/DEET group, the model group, the low dose minocycline group and the high dose minocycline group. The bound group rats were given the fixed shackles and placed in the dark environment for 20 minutes each time. The model group established the Gulf War with reference to the Abdel-Rahman literature. Rat model, rats were given oral 1.3mg/kg/d per day, DEET (40mg/kg/d) was subcutaneously injected with 70% ethanol, and dark environment was bound for 20 minutes in group.PB/DEET: PB and DEET were given subcutaneously every day. Low dose minocycline group: minocycline 15mg/kg/d, abdominal cavity injection, residual model group. High dose minocycline group: Mino 45mg/kg/d, intraperitoneal injection, and Yu Tong model group. The rats in the control group were free to take water to feed and give the same amount of alcohol solution subcutaneously. The experimental period was 28 days. The rats' weight was measured at 0 days, 14 days and 28 days. After the experiment, the paraformaldehyde was perfused and fixed, the brain, paraffin embedded and sliced, and the hippocampus CA1 region nerve was detected by immunohistochemistry. The expression of Apaf-1 and Caspase-3 was detected by TUNEL, and the apoptosis of CA1 neurons in hippocampus was detected.
Results: the weight of the rats in each group increased in different degrees. After 4 weeks, the weight of the control group increased by 79%, the bound group was 75%, the PB/DEET group was 49%, the model group was 47%, the low dose minocycline group was 70%, the high dose minocycline group was 71%.PB/DEET and the control group. The model group was compared with the restraint group, the weight gain decreased, P0.01, statistically significant. Compared with the control group, the model group and the control group compared with the PB/DEET group, the weight increased similar, P0.05, no statistical difference, low, high dose minocycline group compared with the model group, the weight increased significantly, P0.01, there was statistical difference.
Compared with the control group and the control group, the number of apoptotic neurons in the hippocampal CA1 region, the Caspase-3 and the Apaf-1 integral light density were not statistically significant (P0.05). Compared with the control group, the number of apoptotic cells in the hippocampal CA1 area increased and the integral light density of Caspase-3 and Apaf-1 increased significantly, and the statistical difference was very significant. (P0.01) low, high dose minocycline group compared with the control group, the number of apoptotic cells in the hippocampal CA1 area increased, the Caspase-3 and Apaf-1 integral light density increased significantly, the statistical difference was very significant (P0.01). Low dose minocycline group compared with the model group, the number of apoptotic cells in the hippocampus CA1 area decreased, the Caspase-3 integral light density decreased, statistical difference. It was very significant (P0.01). The effect of two doses of minocycline on the number of apoptotic neurons in the hippocampal CA1 region, the effect of Caspase-3 and Apaf-1 integral light density did not show significant difference (P0.05).
Conclusion: 1, there is no obvious effect of restraint stress on the weight gain of rats, no significant damage to.2 in the CA1 region of the hippocampus, the decrease of the weight gain of the rat model of the Bay War syndrome, the increase of neuronal apoptosis in the hippocampal CA1 region, the increase of the expression of Caspase-3 and Apaf-1, considering the.3 of PB and DEET, and the expression of minocycline by inhibiting the expression of Caspase-3, The neuronal apoptosis was reduced and the neuroprotective effect of the hippocampal CA1 region nerve cells was.4, low, and the protective effect of high dose of minocycline on the hippocampal neurons in the rat model of the Gulf War was not significantly different.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R82

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