【作者】 张斌；

【导师】 张岫美；

【作者基本信息】 山东大学 ， 医学基础药理学， 2008， 博士

【摘要】 研究目的:创伤性脑损伤(traumatic brain injury,TBI)能够产生大量病理损害包括炎症反应。炎症级链效应以小胶质细胞激活和致炎因子的产生为特征,进而加重其他病理损伤。能够干预致炎因子产生和胶质细胞活化的药物对TBI具有潜在的治疗价值。目前大量研究显示组蛋白脱乙酰酶(histone deacetylase,HDAC)抑制剂的抗炎作用以及在多种中枢神经系统疾病中的应用前景。然而在TBI领域尚无报道。最近一项研究发现在实验性儿科TBI后海马CA3区的乙酰化组蛋白表达降低,这种变化与TBI导致的兴奋性毒性等损伤密切相关。因此HDAC抑制剂可能会对TBI的防治产生积极作用。研究方法:本研究利用成年大鼠液压冲击损伤(fluid percussion injury,FPI)模型评价新型HDAC抑制剂DMA-PB对TBI的保护作用。采用乙酰组蛋白免疫组织化学方法检测海马CA2/3区组蛋白乙酰化程度,通过测量相对光密度评价乙酰化组蛋白表达水平。以OX-42免疫组织化学方法检测海马CA2/3区小胶质细胞的变化。通过对四种不同形态的小胶质细胞(静息型、肥大型、活化型、吞噬型)的定量分析鉴别炎症反应程度。以Fluoro-Jade B组织荧光法观察TBI后海马CA2/3区早期神经元变性。酶联免疫吸附实验分别检测海马组织中肿瘤坏死因子α(tumor necrosis factor-alpha,TNFα)以及白介素1β(interlukin-1β,IL-1β)的含量。Morris水迷宫评价大鼠的空间记忆及认知功能。结晶紫染色分析海马CA2/3区延迟性神经元损伤。兴趣区域(region of interest,ROI)为海马组织。结果:乙酰组蛋白免疫染色后,相对光密度的测量结果显示损伤侧海马CA2/3区乙酰化组蛋白表达降低,新型HDAC抑制剂DMA-PB在0.25mg/kg、2.5mg/kg、25mg/kg的剂量下均明显提高了组蛋白乙酰化程度,但各剂量组之间无显著差异。OX-42免疫染色后,四种不同形态的小胶质细胞的定量分析结果显示假手术组以静息型占绝对优势,显著的高于其他各TBI组,无活化型及吞噬型,各TBI组以活化型及吞噬型为主,与模型组相比,HDAC抑制剂DMA-PB显著的降低了TBI后吞噬型小胶质细胞的密度,抑制了小胶质细胞炎症反应程度,但各剂量组之间无显著差异。Fluoro-Jade B组织荧光的定量分析结果显示,假手术组无荧光标记细胞,提示无神经元变性发生,各TBI组变性神经元在背景的衬托下发亮荧光,与模型组相比,HDAC抑制剂DMA-PB有减少变性神经元的趋势,但无统计学差异,其中以高剂量组的抑制效果最明显。酶联免疫吸附实验结果显示,与假手术组相比,模型组损伤侧海马内两种炎症因子的含量显著增加,而HDAC抑制剂DMA-PB则明显降低了TNFα及IL-1β的含量。Morris水迷宫结果显示,与假手术组相比,模型组大鼠发现隐藏平台的潜伏期明显延长,提示记忆缺损,而HDAC抑制剂DMA-PB高剂量组(25mg/kg)的潜伏期显著缩短,改善了TBI所致的认知损害,中低剂量组(2.5mg/kg、0.25mg/kg)只呈现缩短趋势无统计学差异。结晶紫染色结果显示,假手术组海马神经元形态正常,细胞边界清晰,胞膜完整,胞核规则,核仁清晰可见,损伤后细胞皱缩明显,形态不规则,神经元典型的多层排列结构被破坏,且细胞数量明显减少,与模型组相比,HDAC抑制剂DMA-PB高剂量(25mg/kg)显著增加了神经元密度,而中低剂量(2.5mg/kg、0.25mg/kg)对细胞数量的影响无统计学意义。结论:我们的实验证实TBI可致成年大鼠海马组蛋白脱乙酰化。新型HDAC抑制剂DMA-PB可能以一种间接作用逆转组蛋白脱乙酰化。并且DMA-PB各剂量均可抑制小胶质细胞炎症反应及炎症因子TNFα、IL-1β的产生,显示出强大的抗炎作用。对TBI后早期神经元变性,DMA-PB具有减少趋势,但对延迟性神经元损伤及空间记忆认知功能的损坏,高剂量组呈现明显的改善作用。可见,DMA-PB对大鼠创伤性脑损伤具有保护作用,尤以抗炎作用显著,并抑制神经元变性而改善神经功能。意义:本课题首次研究了新型组蛋白脱乙酰酶抑制剂DMA-PB对创伤性脑损伤的作用,发现DMA-PB可抑制早期小胶质细胞活化等炎症反应,并改善延迟性神经元损伤及记忆认知功能,对组蛋白脱乙酰酶抑制剂的抗炎性能及在创伤性脑损伤中的应用提供了实验依据。同时首次发现成年大鼠液压冲击性创伤性脑损伤后海马CA2/3区组蛋白乙酰化降低,DMA-PB可能通过间接作用提高组蛋白乙酰化,为创伤性脑损伤的病理生理机制增加了新的内容,也为深入阐明DMA-PB的作用机制提供了切入点。更多还原

【Abstract】 Objective:Traumatic brain injury(TBI) produces a number of pathologies including inflammation.The inflammatory cascade is characterized by activation of microglia and proinflammatory cytokines which can exacerbate other pathologies.Pharmacological therapy that can modulate pro-inflammatory cytokines or microglia activation could have potential efficacy in TBI.A number of studies have focused on histone deacetylase(HDAC) inhibitors as novel therapeutics in a variety of central nervous system(CNS) diseases and showed anti-inflammatory effects.However no data have been reported on TBI.A recent study in experimental pediatric TBI reported a decrease in hippocampal CA3 histone H3 acetylation after injury.These changes were attributed to documented upstream excitotoxic and stress cascades associated with TBI.HDAC inhibitors may contribute to prevention and treatment of TBI.The aim of this study was to evaluate the possible protective effects of DMA-PB,a novel HDAC inhibitor,on TBI.Methods:Drug was administered intraperitoneally(i.p.) immediately after TBI.Coronal brain sections were stained with acetyl-histone H3 antibody,OX-42 antibody or Fluoro-Jade B.Measurement of proinflammatory cytokines(TNFα and IL-1β),acquisition function and delayed neuronal degeneration was assessed with Enzyme-Linked ImmunoSorbent Assay(ELISA),Morris Water Maze(MWM) and cresyl violet staining techniques.The region of interest (ROI) was focused on hippocampus.Results:Histone acetylation was assessed by Acetyl-Histone H3 immunohistochemistry and measured by relative optical density(OD).The result showed that lower ROD was observed in the hippocampus CA2/3 region ipsilateral to FPI compared to sham group.The ROD among DMA-PB dosage treatment groups was not significantly different from the sham group.Microglia changes was carried out by using OX-42 immunohistochemistry and assessed by quantification of four classifications of microglia to identify inflammatory response.The sham group had a preponderance of resting microglia density that was significantly greater than any of the injury groups.No activated or phagocytic microglia was detected in the sham group.There were no significant differences in mean density of activated microglia between the injury groups.The density of phagocytic microglia was significantly different between injury groups.The DMA-PB treatments had significantly lower density of phagocytic microglia compared to the vehicle-treated TBI group.Neuronal degeneration was analyzed by using Fluoro-Jade B histofluorescence.The results showed that there was a trend for the DMA-PB treatment to reduce the number of ipsilateral degeneration neurons compared to vehicle with the highest dose(25 mg/kg) producing the largest reduction in neurodegeneration.Detection of TNFαand IL-1βwas made using enzyme-linked immunosorbent assay(ELISA).In vehicle-treated injured rats,the ipsilateral hemisphere showed a significant increase in TNFαand IL-1βlevels.The profound increase in pro-inflammatory cytokines associated with trauma was significantly reduced by DMA-PB treatment compared with levels in vehicle-treated injured rats.We further investigated more chronic outcome after TBI.Acquisition of a spatial memory was measured by using Morris water maze.The vehicle-treated TBI group showed a significant increase in the latencies(time to find a hidden platform) across testing days compared to sham control.However,DMA-PB treated group(25mg/kg) demonstrated significant improvement in decreased latencies. Delayed neuronal injury was analyzed by using Cresyl violet staining.In the sham group the neurons were normal,their borders were distinct and cell membrane integrity was preserved.Nucleus borders were regular and nucleolus was distinctively observed.In the injury groups,marked shrinkage was observed in the pyramidal neurons of CA2/3 region and increased irregularity was determined in cell and nucleus borders.Typical multiple row cell arrangements were disturbed and the number of cells was decreased.The effect of DMA-PB treatment on neuron density was examined by quantitative histopathological analysis.In vehicle group,hippocampal neuron density was significantly decreased in comparison with the sham group.However,treatment with DMA-PB (25mg/kg) significantly preserved the neurons of hippocampal regions as compared to vehicle group.Conclusions:the present study provided further evidence for the deacetylation of histone H3 by TBI and demonstrated that the novel HDAC6 inhibitor,DMA-PB,reversed the deacetylation measured in the hippocampus likely through an indirect action and robustly attenuated the inflammatory microglia response and production of inflammatory cytokines,produced a trend toward reduced neuronal degeneration in the hippocampus at early time after injury.DMA-PB significantly exhibited more chronic protective outcome characterized by efficiently decreasing delayed neuronal injury and improving spatial memory function.Our findings may provide a primary evidence for the possible benefits of HDAC inhibitors in TBI.We also provide some basic materials for novel HDAC inhibitor,DMA-PB.Further studies of HDAC inhibitors appear warranted for clarification of the underlying mechanisms of action for potential development of therapeutics for patients suffering from TBI.更多还原