【作者】 潘晓东；

【导师】 陈晓春；

【作者基本信息】 福建医科大学 ， 神经病学， 2009， 博士

【摘要】 随着人们对β-淀粉样蛋白(amyloid-β,Aβ)介导的小胶质细胞免疫异常在阿尔茨海默病(Alzheimer’s Disease, AD)发病机制中所起的关键性作用的认识,通过抑制脑内胶质细胞过度免疫激活所引起的Aβ神经毒性的策略已日益受到重视。AD脑内异常聚集的Aβ过度激活了小胶质细胞非特异性的免疫炎症反应并介导神经损伤。小胶质细胞表面相关的“受体”、细胞内丝裂原活化的蛋白激酶(MAPKs)、核因子-κB和细胞因子可能参与Aβ激活小胶质细胞的整个过程。同时,近年来人们对AD早期阶段的Aβ成分——可溶性Aβ寡聚体的研究也取得了重要的进展。研究Aβ寡聚体如何诱导小胶质细胞活化及其引发的神经毒性、明确其中的相应环节,对于探讨神经免疫炎症机制在AD发生、发展中的作用及寻找防治AD的药物靶标具有重要意义。天然植物减毒单体——雷公藤氯内酯醇(tripchlorolide, T4)具有很强的免疫抑制和抗炎活性。我们前期的研究发现,T4明显改善LPS诱导的小胶质条件培养基对皮层神经元的炎性损伤作用,并明显抑制LPS诱导的小胶质细胞产生炎症因子及细胞内氧自由基的产生。推测T4可能同样减轻寡聚态Aβ介导的胶质-炎症反应引起的神经毒性作用、并可能由此改善AD动物的认知能力。因此,本课题首先研究Aβ(1-42)对小胶质细胞活化、吞噬功能的影响及其机制；在此基础上观察T4对寡聚态Aβ(1-42)引发的胶质-炎症反应所产生的神经元毒性的影响,并探讨T4在Aβ介导小胶质细胞中MAPKs/NF-κB炎症信号通路的具体药理靶点和机制。最后选择快速老化小鼠P8(SAMP8)作为散发型AD的动物模型,从动物整体水平验证T4对SAMP8学习记忆功能的影响,并初步观察和评价药物的非临床安全性。结果总结如下：(一)Aβ(1-42)在AD炎症细胞模型中的作用及信号机制：1.Aβ(1-42)能够诱导小胶质细胞形态发生分枝化或阿米巴样改变,但凝聚态和寡聚态Aβ(1-42)诱导小胶质细胞形态的表型有所不同。2.寡聚态Aβ(1-42)激活小胶质细胞,能更早、更强烈地释放炎症介质(IL-1β、TNF-α、PGE2、NO)的产量、诱导细胞内氧自由基的产生,同时损害了小胶质细胞的吞噬功能。3.凝聚态Aβ(1-42)维持了小胶质细胞缓慢、相对缓和且持续性的炎症反应。4.寡聚态Aβ(1-42)损害了凝聚态Aβ(1-42)诱导的小胶质细胞的吞噬能力并与炎症介质水平的增高有关。早期抗炎和抗氧化治疗有助于改善AD炎症病理进程中小胶质细胞功能的损害。5.寡聚态Aβ(1-42)可通过激活JNK、ERK和NF-κB信号通路诱导小胶质细胞炎症反应,但不通过p38MAPK起作用。(二)T4在AD炎症细胞模型中的神经药理作用及其机制：1.T4对神经元及小胶质细胞活力的影响具有选择性：较高剂量的T4(≥20nM)对小胶质细胞的抑制效应远大于神经元。2.T4广谱、强效地抑制寡聚态Aβ(1-42)诱导小胶质细胞释放炎性介质(IL-1β、TNF-α、PGE2、NO)的产量及细胞内iNOS及COX-2蛋白的表达。3.T4减轻寡聚态Aβ(1-42)诱导的小胶质细胞-炎症反应引起的神经毒性并保护神经元,作用与其抑制小胶质细胞产生炎症介质水平的增高有关。4.一定剂量的T4(10nM)并无直接影响小胶质细胞的吞噬功能,但部分逆转寡聚态Ap对小胶质细胞吞噬的抑制。5.T4抑制寡聚态Aβ(1-42)诱导的小胶质细胞内JNK和NF-κB炎症信号的激活水平,但不影响p38MAPK及ERK活化水平。(三)T4对快速老化小鼠P8认知和行为障碍的影响及其非临床安全性观察1.低剂量(0.25～1.0μg/kg.d) T4长期腹腔注射给药25个月能够明显改善快速老化小鼠P8的视空间学习记忆及行为能力。2.低剂量T4长期慢性给药没有影响动物的生存率。3.T4长期慢性给药并不影响动物的外周血白细胞、血红蛋白、血小板水平,也未损害动物的心、肝、肾功能等生化指标。以上结果提示,早期AD病理事件中,β-淀粉样肽寡聚体诱导了小胶质细胞产生强烈的免疫炎症反应,随后抑制了细胞的吞噬功能；JNK、ERK和NF-κB是寡聚态Aβ激活小胶质细胞产生炎症反应的主要信号分子。雷公藤氯内酯醇(T4)可通过JNK及NF-κB信号通路抑制寡聚态Aβ诱导的胶质-炎症反应,并由此减轻炎症介导的神经毒性作用、保护神经元；同时T4改善了AD动物的认知和行为能力。因此,天然植物减单体T4有望成为防治AD有前途的候选药物。更多还原

【Abstract】 Recent research has focused on soluble oligomeric assemblies ofβ-amyloid peptides (Aβ) as the proximate cause of neuroinflammation, synaptic loss and the eventual dementia associated with Alzheimer’s disease (AD). Oligomeric AP (oAβ), which may be the major pathogenic form of Aβin the early stages of AD, can stimulate glia to induce a more profound inflammatory response and neurotoxicity than fibrillar AP (fAp). The inhibition of Ap-dependent activation of microglia could be an effective therapeutic approach to delay the progression of AD. Additionally, an aberrant expression of mitogen-activated protein kinases (MAPKs) and activation of nuclear factor-κB (NF-κB) in Aβ-induced microglia is directly correlated with pathogenic events of AD. Accordingly, strategies to suppress MAPKs and/or NF-κB activation may attenuate neuroinflammation and neuronal damage, which will be of benefit in treatment of AD.Extracts of natural herb Tripterygium wilfordii Hook. F (TWHF) have been found to have potent anti-inflammatory and immunosuppressive functions and widely used in China for treatment of rheumatoid arthritis. Our previous findings demonstrated that tripchlorolide (T4), an extract of TWHF, even at nanomolar level exerted potent anti-inflammatory effects on LPS-stimulated microglial cells and protected neuronal cells from neuroinflammatory toxicity. It suggest that T4 could be a potential neuroprotective agent against neuroinflammatory process, such as AD.Here we firstly tested the effects of oAβ(1-42) versus fAβ(1-42) on the cells viability, the expression of inflammatory mediators, as well as phagocytosis function in microglial cells, and explored the activations of MAPKs and NF-κB signaling pathways in oA(3-induced microglia. Secondly, we investigated whether T4 suppressed oAβ-induced microglial activation and protected against microglia-mediated oAβneurotoxicity. Also, we gained insights into the anti-inflammatory molecular mechanisms of T4 in oAβ-stimulated microglial cells. Lastly, we chose senescence-accelerated mouse Prone 8 (SAMP8) as a model of sporadic AD to testify if T4 ameliorated learning and memory deficits.The results showed that oAβincreased interleukin-1β(IL-1β) level in a manner that was rapid, potent and transient. Also, it induced higher levels of tumor necrosis factor-α(TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2) and intracellular SOA than fAβ. Interestingly, pretreatment of microglia with oAβfor 6～12h attenuated the capacity of microglial phagocytosis following fAβexposure. The further data was shown that a well inverse correlation between microglial phagocytosis and inflammatory mediators. Also, IL-1β, LPS and tert-butyl hydroperoxide (t-BHP) all decreased phagocytosis levels in fAβinduced-microglia, which could be relieved by NF-κB inhibitor, pyrrolidone dithiocarbamate (PDTC) and N-Acetyl-L-cysteine (NAC). Meanwhile, in microglia, the expressions of MAPKs (p38MAPK, ERK and JNK), I-κB and NF-κB were induced by oAβin a time-dependent manner. A peak activity of MAPKs and NF-κB was observed at 30-120min, and the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were significantly elevated after oAβstimulation for 24h. Inductions of inflammatory mediators, iNOS and COX-2 were attenuated by JNK inhibitor (SP600125), ERK inhibitor (U0126) and PDTC, but not by p38 inhibitor (SB203580), in oAβ-stimulated microglia.Further, the conditioned media from oAβ-treated microglial cells (Aβ-CM) was applied to treat Neuro-2A and primary cortical neurons. The findings confirmed that the neuronal toxic effect of oAp was preferentially mediated by microglia. Therefore, Aβ-CM was applied to the examination of the neuroprotective effect of T4. As expected, T4 protected neuronal cells from microglia-mediated oAβtoxicity. It significantly attenuated oAβ-induced release of inflammatory productions, including TNF-α, IL-1β, NO and PGE2, in a dose-dependent manner (P<0.05). It also reversed oAβ-mediated microglial phagocytosis injury, as well as down-regulated the protein levels of iNOS and COX-2 in microglia. Further molecular mechanism study demonstrated that T4 inhibited the nuclear translocation of NF-κB without affecting I-κBαphosphorylation. It repressed AP-induced JNK phosphorylation but not ERK or p38 MAPK.Lastly, in order to testify if T4 would ameliorate cognitive deficits, the 7-month-old SAMP8 mice were chronically treated with T4 (0.25,1.0,4.0μg/kg per day, injected intraperitoneally for 2.5 months, respectively). The SAMR1 mice with the same age were used as "normal aging" control. A NF-κB inhibitor, PDTC-treated SAMP8 mice (50mg/kg per day, i.p.) were used as the drug treatment controls. The beneficial role of T4 was manifested to improve learning and memory function in 10.5-month-old SAMP8 mice by Y-maze and Morris water maze behaviour tests. The optimal dose of T4 was 0.25～1.0μg/kg per day, which did not show significant side-effects of T4 on the blood routine test, blood biochemical assays, as well as the survival of mice.Collectively, these results suggest that Aβoligomers induces a potent inflammatory response, subsequently exacerbates microglial phagocytosis in the early AD pathological affairs, and that activation of microglia by Aβoligomers is through NF-κB, JNK and ERK signaling which subsequently lead to the upregulation of inflammatory mediators. T4 protects neuronal cells by blocking inflammatory responses of microglia to Aβoligomers and that it acts on the signaling of NF-κB and JNK which are involved in the modulation of this process. Also, T4 ameliorates cognitive deficits in senescence-accelerated AD mice. Therefore, our findings may provide new insight for developing the clinical application of tripchlorolide to Alzheimer’s disease.更多还原