【作者】 赵丹；

【导师】 郭莲军；

【作者基本信息】 华中科技大学 ， 药理学， 2016， 硕士

【摘要】 第一部分:α7nAchR激动剂DMXBA改善CRS小鼠学习记忆损伤和抑郁样行为目的:检测慢性束缚应激(Chronic restraint stress,CRS)模型对昆明小鼠认知功能及抑郁样行为的影响,烟碱型胆碱受体α7亚型(α7n Ach R)激动剂DMXBA对CRS小鼠行为学的影响。方法:采用慢性束缚应激(Chronic restraint stress,CRS)模型,造模后第11天给药,腹腔给予α7n Ach R激动剂DMXBA 4mg/kg/d,或联合给予拮抗剂α-BGT 1μg/kg/d,连续给药至第21天。Morris水迷宫实验(Morris Water Maze,MWM)和新事物认知(Novel-object recognition,NOR)实验观察小鼠的认知功能,糖水偏好(Sucrose preference test,SPT)实验,强迫游泳(Forced swim test,FST)和悬尾实验(Tail suspansion test,TST)实验观察小鼠的抑郁样行为。结果:(1)MWM结果显示,与对照组相比,前4天训练期CRS小鼠逃逸潜伏期第3、4天明显延长,第5天检测期目标象限时间显著缩短;NOR实验结果显示,对新事物的认知指数与对照组比CRS小鼠显著降低,以上结果提示CRS造成小鼠的学习记忆损伤。(2)SPT实验结果显示,CRS小鼠糖水摄入率与对照组比显著降低;FST和TST结果显示,CRS小鼠累计不动时间显著增加,以上结果提示CRS引发小鼠抑郁样行为。(3)给予DMXBA(4mg/kg),CRS小鼠逃逸潜伏期明显缩短,在目标象限停留的时间增加,新事物认知指数增加,α-BGT(1μg/kg)能逆转DMXBA(4mg/kg)的上述作用。(4)DMXBA(4mg/kg)显著增加CRS小鼠糖水摄入率及降低应激小鼠的累计不动时间;给予α-BGT(1μg/kg)能逆转DMXBA(4mg/kg)的上述作用。结论:DMXBA改善CRS小鼠的学习记忆损伤和抑郁样行为。第二部分:α7nAchR激动剂DMXBA对CRS小鼠神经元损伤的影响目的:观察慢性束缚应激对小鼠海马神经元形态和树突棘数目的的影响,观察DMXBA(4mg/kg)是否对神经元损伤有改善作用。方法:观察海马神经元的形态采用尼氏染色的方法,检测海马CA1区树突棘密度采用高尔基银染的方法。结果:(1)尼氏染色结果显示,CRS组与对照组相比,海马缩小,海马CA1区细胞皱缩,胞核变形,浓染。DMXBA(4mg/kg)显著缓解CRS所致的海马CA1区神经细胞损伤,而α-BGT(1μg/kg)能拮抗DMXBA(4mg/kg)对海马神经元损伤的改善作用。(2)高尔基结果显示,树突棘密度CRS小鼠与对照组相比,海马CA1区显著降低,DMXBA(4mg/kg)显著逆转树突棘的丢失,而α-BGT(1μg/kg)能拮抗DMXBA(4mg/kg)的保护作用。结论:DMXBA改善CRS小鼠神经元损伤。第三部分:α7n Ach R激动剂DMXBA对CRS小鼠脑组织炎症反应的调节作用及机制目的:观察CRS小鼠海马两条炎症通路ACh抗炎信号通路和TLR4/NF-κB促炎信号通路蛋白表达变化情况,检测下游促炎细胞因子释放情况,并探讨DMXBA(4mg/kg)对脑组织炎症反应的影响。方法:酶联免疫吸附法检测海马中促炎因子TNF-α和IL-1β的分泌情况。Western Blot检测α7n Ach R,CHAT,STAT3,TLR4,NF-κB,GFAP及Iba-1蛋白的表达规律,比色法检测海马组织ACh E活性。结果:(1)Western Blot检测了海马组织中蛋白变化,结果显示CRS小鼠TLR4、My D88蛋白表达增加,核蛋白NF-κB的表达降低,促炎因子TNF-α和IL-1β的释放增加,中枢Gail和AST增殖,DMXBA(4mg/kg)处理明显下调CRS小鼠海马的TLR4/NF-κB信号通路,海马促炎因子释放显著减少,中枢激活的Gail及AST被抑制,给予α-BGT(1μg/kg)能拮抗DMXBA(4mg/kg)的上述作用。(2)CRS小鼠转录因子STAT3核蛋白表达显著降低,海马组织中CHAT、α7n Ach R表达水平明显升高,ACh E表达水平无明显变化,给予DMXBA(4mg/kg)明显降低ACh E水平,但对应激小鼠海马组织中STAT3、CHAT、α7n Ach R水平无明显影响。结论:DMXBA(4mg/kg)下调CRS小鼠海马的TLR4/NF-κB信号通路,同时激活CRS小鼠海马的Ach抗炎信号通路,能抑制CRS诱导的小鼠脑组织炎症反应。第四部分:α7n Ach R激动剂DMXBA对CRS小鼠Treg细胞数目的影响目的:观察CRS小鼠脾脏及中枢调节性T细胞(Regulatory T cell,Treg)数目变化和外周血中促炎因子释放变化,探究DMXBA(4mg/kg)对外周炎症因子和Treg细胞数目的影响。方法:脾脏组织中Treg细胞数目的变化运用流式细胞术检测。Western Blot检测脑组织Treg细胞标志蛋白Foxp3+的变化。外周血促炎因子TNF-α和IL-1β的变化情况运用酶联免疫吸附法检测。结果:CRS21d小鼠脾脏组织Treg细胞数量显著降低,脑组织Foxp3+表达也显著降低,给予DMXBA(4mg/kg)能升高CRS21d小鼠脾脏组织Treg细胞数量,并促进脑组织Foxp3+表达,给予α-BGT(1μg/kg)能拮抗DMXBA(4mg/kg)的上述作用。CRS7d后小鼠外周血TNF-α和IL-1β水平增加,但CRS14d和CRS21d小鼠外周血NF-α和IL-1β水平没有显著改变,DMXBA(4mg/kg)对TNF-α和IL-1β的释放无明显影响。结论:DMXBA(4mg/kg)上调CRS21d小鼠外周和中枢Treg细胞含量。更多还原

【Abstract】 First part: DMXBA prevents CRS-induced learning and memory impairment and depression like behaviorAims: To explore whether the learning and memory function and depression behavior was changed after chronic restraint stress injury, and to evaluate the effect of DMXBA on mice behavior.Methods: We used the chronic restraint stress model to simulate chronic stress,during the restraint stress 11 d to 21 d, mice were injected with,DMXBA(4mg/kg,i.p.)combinded with α-BGT(1μg/kg, i.p.) or vehicle,until the end of the model, the spatial learning and memory was determined by the Morris water maze(MWM) test and the novel object recognition test(NOR), the depressive like behavior was determined using the sucrose preference test(SPT), forced swim test(FST) and tail suspension test(TST).Results:(1) In the Morris Water Maze test, the latency to find the platform was significantly longer in CRS mice compared with control group on days 3 and 4 of the training period, and CRS mice spent less time in target quadrant during test period. In the novel object recognition test, CRS mice showed the lower discrimination index.These results indicated that CRS induced learning and memory impairment.(2) In the sucrose preference test, CRS mice had the lower sucrose preference after chronic stress restraint, and the immobile time of CRS mice was significantly longer than control group. These results indicated that CRS induced depression like behavior.(3) CRS mice treated with DMXBA spent less time to find the platform and more time in target quadrant compared with CRS mice. DMXBA reversed the lower discrimination index after chronic stress restraint. And α-BGT treatment can reverse the protective effects of DMXBA.(4) In the sucrose preference test, DMXBA reversed the lower sucrose preference after chronic stress restraint. In the forced swim test and the tail suspension test,DMXBA alleviated depressive-like behavior. And α-BGT could reverse the therapeutic effects of DMXBA.Conclusions: DMXBA prevents CRS-induced learning and memory impairment and depression like behavior.Second part: DMXBA reverse CRS-induced neuron lossAims: To observe the morphology of neurons and dendritic spine density in hippocampus of mice after chronic restraint stress injury, and to evaluate the neuroprotection of DMXBA.Methods: Morphology of hippocampal neurons was examined by Nissal staining, and dendritic spine density was evaluated by Golgi silver staining.Results:(1) Nissl staining showed that neuronal damage in the hippocampus is characterized by shrunken cell bodies accompanied by shrunken and pyknotic nuclei. Significantly damaged neurons were observed in the hippocampus CA1 of CRS mice. DMXBA treatment alleviated CRS-induced neuronal damage in the CA1 region of hippocampus. And α-BGT could inhibit the therapeutic effects of DMXBA.(2) CRS mice decreased the dendritic spine density of hippocampus CA1. DMXBA increased the dendritic spine density in the hippocampus CA1 of CRS mice. Andα-BGT could reverse the therapeutic effects of DMXBA.Conclusions: Chronic treatment with DMXBA reverse CRS-induced the decrease in the dendritic spine density of hippocampus CA1 and neuron loss.Third Part: DMXBA regulate CRS-induced CNS inflammation and underlying mechanismAims: To observe the TLR4/NF-κB pro-inflammation and Ach anti-inflammation signaling pathway proteins expression pattern and the relationship with downstream inflammatory cytokines level. To explore the effect of DMXBA against inflammation in hippocampus induced by chronic restraint stress.Methods: Pro-inflammatory factor of TNF-α and IL-1β in the hippocampal homogenat were detected by the ELISA kit. α7n Ach R,CHAT,Ach E,STAT3,TLR4,NF-κB,GFAP and Iba-1 protein expression were also measured.Results:(1) The expression of TNF-α, IL-1β, Iba-1, GFAP, TLR4 and My D88 of CRS mice increased signifigantly, while the expression of NF-κB of CRS mice decreased.DMXBA depressed TLR4/NF-κB signaling pathway in the hippocampus of CRS mice and decreased pro-inflammatory factor express in the hippocampal homogenat of CRS mice. DMXBA inhibited microglia and astrocyte activation in the hippocampus of CRS mice. And α-BGT could reverse the effects of DMXBA.(2) The expression of STAT3 of CRS mice decreased significantly, and the expression of CHAT, α7n Ach R increased dramatically, but level of ACh E in hippocampus had no difference between all groups. DMXBA treatment reversed the CRS-induced elevation of Ach E, but have no effect on the expression of STAT3, CHAT andα7n Ach R.Conclusions: DMXBA depressed TLR4/NF-κB signaling pathway in the hippocampus of CRS mice, activated Ach signaling pathways meanwhile.Fourth part: The effect of DMXBA on the amount of Treg cell of CRS miceAims: To observe the discipline of Treg-cell in spleen and hippocampus of CRS mice,to detect the leve of TNF-α and IL-1β in the peripheral blood and explore the effect of DMXBA on Treg-cell and pro-inflammatory factor.Methods: The amount of Treg cell in spleen was examined by flow cytometry; and the expression of Treg cell marker Foxp3+ in hippocampus was examined by Western blot. Pro-inflammatory factor of TNF-α and IL-1β in the peripheral blood were detected by ELISA.Results: The amount of Treg cell in spleen and hippocampus of CRS mice was decreased significantly, DMXBA treatment upregulated Treg-cell in the spleen and hippocampus of CRS mice. And α-BGT can reverse the effects of DMXBA in hippocampus. DMXBA treatment have no effect on the expression of TNF-α and IL-1β.Conclusions: DMXBA treatment reverse the CRS-induced decline of Treg.更多还原