【作者】 雷德亮；

【导师】 罗学港；

【作者基本信息】 中南大学 ， 神经生物学， 2007， 博士

【摘要】 研究背景阿尔茨海默病(Alzheimer’s disease，AD)的脑内病变是一个慢性炎症的观点，已经普遍被人们接受。AD脑内慢性炎症反应的特征包括：与β-淀粉样蛋白(β-amyloid，Aβ)沉积和老年斑(senile plaques，SP)形成有关的小胶质细胞激活，反应性星形胶质细胞增生，以及补体成份和致炎性细胞因子的表达增加等。非甾体类抗炎药(non-steroidal anti-inflammatory drugs，NSAIDs)不仅可以降低AD发生的危险性，而且可以延缓AD的进程。动物实验研究证实，NSAIDs可以抑制AD转基因模型小鼠脑内Aβ的沉积和SP的形成，并抑制小胶质细胞活化和反应性星形胶质细胞增生以及IL-1β等致炎性细胞因子的表达与分泌。因此，抗炎治疗AD具有重要的价值。但是，由于NSAIDs严重的毒副作用，可能限制其在临床上的使用。雷公藤甲素(triptolide，T10)是中药雷公藤提取物的有效成份之一，具有抗炎和免疫抑制的作用，临床上被应用于治疗风湿和类风湿关节炎，系统性红斑狼疮等自身免疫和炎症反应性疾病。不少的体外研究证实，雷公藤甲素的抗炎和免疫抑制效应是通过抑制炎症介质如IL-1β、TNFα等的表达释放和抑制诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)和环氧化酶-2(Cyclooxygenase-2，COX-2)诱导表达实现的。最近的研究表明，雷公藤甲素具有神经保护作用，可以保护中脑黑质多巴胺能神经元免受脂多糖(lipopolysaccharide，LPS)的损伤，其机制可能与雷公藤甲素抑制LPS所致的小胶质细胞的活化以及抑制LPS所致的炎症反应有关。T10对AD脑内炎症和免疫反应影响的研究，到目前为止还少见有文献报道。本研究首次用T10处理β-淀粉样前体蛋白(β-Amyloid precursor protein，APP)基因和早老素Ⅰ(Presinilin 1，PS1)基因双转基因AD模型小鼠(APP／PS1 dtg)，研究T10对APP／PS1 dtg的影响，探讨T10治疗AD的可能性及其可能机制，为AD的防治提供新的方法。目的研究雷公藤甲素(T10)对APP／PS1双转基因AD模型小鼠海马内Aβ沉积与SP形成、小胶质细胞和星形胶质细胞的活化以及COX-2诱导表达的影响及其可能机制，为T10防治AD提供新的理论依据。方法1．动物实验取18只4.5月龄健康雄性APP／PS1双转基因AD模型小鼠，随机分为3组，分别腹腔注射5μg／kg．d T10(T10 H组)、1μg／kg．d T10(T10 L组)和等容量的溶媒(PLC组)共计45天；另取5只野生型非转基因同龄雄性小鼠，不作任何处理作为对照。45天后取材，左侧半大脑用于组织学染色，右侧半大脑用于蛋白分析。组织学研究：按无偏性体视学(Unbiased stereology)SUR原则(Systematic-Uniform-Random principle)，每10片取1片收集切片，6E10、GFAP、MAC1、COX-2免疫组织化学方法染色结合无偏性体视学定量分析研究Aβ、星形胶质细胞、小胶质细胞和COX-2等的变化，刚果红染色显示老年斑形成的改变，6E10与GFAP、6E10与MAC1、COX-2与GFAP、COX-2与MAC1等免疫荧光双标记显示星形胶质细胞和小胶质细胞与Aβ和COX-2等的关系。Western Blot蛋白分析海马Aβ和COX-2等蛋白水平的变化。2．细胞培养实验：(1)培养A172人星形胶质细胞，分别用0.2、1和5μg／L T10预处理A172细胞1h，随后用1mg／L LPS处理细胞3h和6h，逆转录-PCR(Reverse transcription-PCR，RT-PCR)、Western blot研究不同浓度T10对LPS所致COX-2表达的影响；电泳迁移率检测(Electrophoretic Mobility Shift Assay，EMSA)研究T10对LPS所致的NF-κB／DNA结合活性的影响。(2)培养高表达人类COX-2蛋白(hCOX-2)的sf-9细胞系，分别用T10、COX-2酶活性抑制剂NS398和吲哚美辛处理细胞，放射免疫(Radioimmunoassay，RIA)检测培养液中PGE2的含量，研究T10对COX-2酶活性的影响。结果1动物实验结果：(1)T10处理APP／PS1 dtg 45 d，①可以抑制Aβ在海马的沉积，减少SP的形成。无偏性体视学定量分析发现，与PLC组的6E10阳性的Aβ斑的总面积比较，T10 H组海马Aβ斑的总面积减少了35％(P＜0.001)，T10 L组海马Aβ斑的总面积减少了18％(P＜0.05)。此外，T10 H组较T10 L组减少了21％，差异有统计学意义(P＜0.05)。②Western Blot蛋白分析也显示，海马内T10 H组Aβ蛋白水平最低，PLC组Aβ蛋白水平最高，T10 L组Aβ蛋白水平介于二者之间；③T10还可以减少6E10阳性的神经元的数量；④T10可以抑制沉积的Aβ聚集和纤维化，减少SP的形成。无偏性体视学定量分析发现，与PLC组的刚果红阳性SP比较，T10 H组海马SP总面积减少了32％(P＜0.001)，T10 L组海马SP总面积减少了21％(P＜0.05)。(2) T10可以抑制神经胶质细胞的活化与增生：①T10可以抑制小胶质细胞的活化与增生，减少小胶质细胞团簇的面积，无偏性体视学定量分析发现，与PLC组比较，T10 H组海马小胶质细胞总数减少了30％(P＜0.01)，T10 L组减少18％(P＜0.05)；T10 H组较T10 L组减少17％(P＜0.05)。②T10可以抑制星形胶质细胞活化，减少GFAP的表达以及抑制星形胶质细胞的增生，无偏性体视学定量分析发现，与PLC组比较，T10 H组海马星形胶质细胞总数减少了20％(P＜0.01)，T10 L组减少13％(P＞0.05)。(3) T10可以抑制APP／PS1 dtg AD模型小鼠海马内COX-2的表达／活化：①T10抑制COX-2在APP／PS1 dtg海马神经元内的表达／活化，减少COX-2阳性神经元的数量；②野生型小鼠海马内星形胶质细胞不表达COX-2，但在APP／PS1 dtg小鼠海马内星形胶质细胞表达COX-2，T10处理45天后，海马内COX-2阳性星形胶质细胞总数分别减少28％(T10 H，P＜0.01)和12％(T10 L，P＞0.05)；③Western blot蛋白分析显示，PLC组海马COX-2蛋白水平最高，其次为野生型小鼠，T10 H组海马内COX-2蛋白水平最低。2．细胞培养结果：(1) LPS刺激培养的A172人星形胶质细胞，可以导致COX-2表达增加，PGE2产生增加，T10呈剂量依赖性降低COX-2的表达和PGE2的产生；(2) T10对高表达hCOX-2的sf-9细胞系COX-2酶活性没有影响，提示T10对COX-2的抑制发生在转录水平；(3) LPS刺激培养的A172人星形胶质细胞可以引起NF-κB／p50／P65 DNA结合活性明显增高，T10呈剂量依赖性抑制NF-κB结合活性。结论本实验的结果表明，T10可以抑制APP／PS1 dtg小鼠海马内Aβ的沉积与纤维化形成SP，并且抑制小胶质细胞和星形胶质细胞的活化与增殖以及抑制活化的星形胶质细胞诱导表达COX-2。说明T10具有NSAIDs的部分特性，对该模型小鼠或AD脑内慢性炎症反应具有一定的抑制作用，或可用于AD的预防和治疗。由于T10没有NSAIDs的毒副作用，因而可能在一定程度上优于NSAIDs。更多还原

【Abstract】 BACKGROUNDThe pathological changes seen in Alzhemier’s disease(AD) brains are a series of chronic inflammation processes. The chronic inflammation response is characterized by the presence of abundant activated microglia, and reactive astrocytes proliferation associated with deposition of fibrillarβ-amyloid(Aβ), and increases expression of pro-inflammatory cytokines and complement components. Non-steroidal anti-inflammatory drugs(NSAIDs) may not only reduce the risk of AD, moreover, they may delay AD progression. Animal experimental studies confirmed that NSAIDs may 1. suppress Aβdeposition and plaque formation, 2. Inhibit microglia activation and reactive astrocytes proliferation, and 3. decrease the expression and secretion of pro-inflammatory cytokines such as TNFα、and IL-1βin the AD brain. Therefore, the anti-inflammation effect of NSAIDs in AD seems to have an important value. However, as a result of the NSAIDs treatment there is serious and poisonous side effects, which limits its clinical usage.The Triptolide(T10) compound is one of the active components from an extract of Traditional Chinese Medicine Tripterygium wilfordii hook F. It possesses anti-inflammation and immuno-suppression functionality and has been used to treat rheumatism and rheumatism arthritis, systematic lupus erythematosus and other auto-immunity and inflammatory responsive diseases. Previous in vitro research has indicated that T10 anti-inflammation and immunosuppression effects is via it’s suppression of the expression and releasing of pro-inflammatory cytokines like IL-1β, TNFα, and as well as suppression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2(COX-2) induction. Current research suggested that T10 has a neurotrophic function and may protect the DOPAminergic neurons in the mesencephalon from lipopolysaccharide (LPS)injury. The suggested T10’s neuronal protective mechanism is involved in inhibiting microglia activation and suppresses the inflammatory reaction. There has been found no literature that reports on the effect of T10 on the inflammatory and immune cascade seen in the AD brain. The present research is the first that used T10 to treat APP/PS1 double transgenic mice model of AD(APP/PS1 dtg). This is a preliminary study to assess the effects of T10 on APP/PS1 dtg mice and discusses the possibility and the possible mechanism for T10 to treat or prevent AD.OBJECTIVESTo examine the effects of T10 using the APP/PS1 dtg mouse model of AD on: 1. Aβdeposition and senile plaques formation. 2. Microglia and astrocytes activation and proliferation. 3. COX-2 induction and possible mechanism of T10 inhibits expression of COX-2. METHODS1. Research in vivo: 18 Male APP/PS1 dtg mice aged at 4.5 months were used in this study. The mice were divided into 3 groups randomly, High-5μg/kg.d T10(T10 H, n=6), Low-1μg/kg.d T10(T10 L, n=6) and placebo(PLC, n=6). Intraperitoneal administration of T10 and capacity solvents (PLC group) was in a period of a total of 45 days. In addition, five male wild type non-transgenic mice of comparable age and receiving no treatment were used for comparison. After 45 days of treatment, the mice were sacrificed and the brains removed for processing. The brains were separated along the middle sagittal sulcus. The left side was used for the histological study and the right half was used for protein analysis. Histological research: According to unbiased stereological system SUR (Systematic-Uniform-Random) principle, every tenth section was collected for immunohistochemistry. 6E10, GFAP, MAC1, COX-2 Immunohistochemical staining combined with unbiased stereological methods was used to quantitative analyze Aβ、astrocytes、microglia cells and COX-2 as well as changes of Congo red positive senile plaques(SP) in hippocampus. Double immunofluorescence staining to showed the relationship between microglia cells and astrocytes with Aβand COX-2. Western blot protein analysis assessed changes of protein level for Aβand COX-2.2. Research in vitro: (1) Cultured human A172 astrocytes cell lines were pretreated with a low, middle and high dose of T10(0.2, 1 and 5μg/L T10) for 1h subsequent to use of 1mg/L LPS processing cells for 3 h and 6 h. Reverse transcription PCR(RT-PCR), and Western blot methods were used to analyze the effects of the different concentrations of T10 on the COX2 expression induced by LPS; electrophoretic mobility shift assay(EMSA) studied the effects of T10 on the NF-κB/DNA binding activity in the cells. (2) Cultured high expresses the human COX-2 protein(hCOX-2) sf-9 cell lines were processed with T10, COX-2 activity inhibitor NS398 and indomethine. Radioimmunoassay method was used to analyses PGE2 level in the media to study the effects of T10 on the COX-2 enzyme activity.RESULTS1. Results in vivoA. Findings indicate(1) T10 may suppress Aβdeposition in the hippocampus, and reduces senile plaques formation. Unbiased stereology quantitative analysis showed that the total area of 6E10 positive Aβplaques in the hippocampus compared with the PLC group’s, Aβplaques of T10 H was reduced 35％(P＜0.001), and that of T10 L reduced 18％(P＜0.05). In addition, the T10 H group compared with the T10 L group showed a reduction of 21％(P＜0.05). (2) Western blot protein analysis also displayed that T10 H group Aβprotein levels in the hippocampus was lowest, Aβprotein levels in PLC group was highest, and Aβprotein levels in T10 L group was moderate; (3) T10 also may reduce the number of 6E10 positive neuron in the CA1 region of hippocampus; (4) T10 may suppress Aβaggregation and fibrils, and reduces SP formation. Compared with PLC group’s Congo red positive SP, the total area of SP in the hippocampus of T10 H group was reduced 32％(P＜0.001), and in the T10 L reduced 27％(P＜0.05).B. T10 may also inhibit neuroglia cell activation and proliferation: (1) T10 may suppress the microglia cell activation by reducing the cluster area of microglia cell. Compared with the PLC group, the total number of microglia cells in hippocampus of T10 H was reduced 30％(P＜0.01), the T10 L was reduced 18％(P＜0.05). When comparing T10 H with the T10 L a reduction of 17％(P＜0.05) was found. These results indicate that T10 assumes a dosage dependent inhibition of microglia proliferation. (2) T10 may suppress astrocytes activation, reduces GFAP expression as well as the suppression of astrocytes proliferation. Compared with the PLC group, the total number of astrocytes in the hippocampus of T10 H was reduced 20％(P＜0.01), the T10 L reduced by 13％(P＞0.05).Co T10 may suppress COX-2 expression/activation in the hippocampus of APP/PS1 dtg mice model of AD: (1) T10 may suppresses COX-2 expression/activation in hippocampus of APP/PS1 dtg; reduces the total number of COX-2 positive neurons in the CA2-4 region of hippocampus; (2) In the hippocampus of the wild type non-transgenic controls astrocytes was not seen to express COX-2, but astrocytes in the hippocampus of APP/PS1 dtg mice showed COX-2 expression. After 45 days T10 treatment, the total number of COX-2 positive astrocytes in hippocampus was reduced by 28％(T10H, P＜0.01) and 12％(T10L, P＞0.05) compared to the group PLC respectively; (3) Western blot protein analysis showed that COX-2 protein level was lowest in the hippocampus of wild type non-transgenic control, followed by the T10 H, the T10 L, and highest in the PLC group.2. Results in vitroA. LPS stimulated the A172 human astrocytes resulting in COX-2 expression enhancement; the level of PGE2 in the media was significant increased, and T10 showed the dosage-dependent manner to reduce COX-2/PGE2 production;B. T10 when administered to the high expressing human COX-2 sf-9 cell lines didn’t change the COX-2 enzyme activity which suggested that T10 may have an effect on the transcription level for COX-2 expression.C. LPS stimulates cultured A172 human astrocytes line resulting in the increased binding of NF-kB/p50/P65; T10 assumes the dosage dependence response to suppress the NF-kB active binding.CONCLUSIONS:The results in present research indicated that T10 suppresses Aβproduction and deposition and inhibits its aggregation and plaques formation in the hippocampus of APP/PS1 dtg mouse, and inhibits microglia and astrocytes activation and proliferation. And T10 may also suppress activated astrocytes and neurons COX-2 induction. Theses results suggested that T10 possibly possesses some of NSAIDs identities and may suppress the chronic inflammation in the AD brain and/or may be useful in AD prevention and treatment. Because of T10 therapy there is no serious and poisonous side effects of NSAIDs, which may has advantage over the NSAIDs to be used for AD patients.更多还原