罗格列酮通过抑制小胶质细胞活化而保护多巴胺能神经元的研究
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摘要
帕金森病(Parkinson's disease, PD)是一种常见的神经退行性疾病,在65岁以上人群中PD的患病率为1%-3%,其临床表现为静止性震颤、运动迟缓、肌强直和姿势不稳。PD由英国医师James B. Parkinson于1817年首先报导,但至今其发病机制仍未完全明了,目前普遍认为与遗传、年龄、环境、氧化应激、线粒体功能异常、兴奋性毒性、免疫异常等因素有关。PD的病理特征为中脑黑质致密部多巴胺能神经元逐渐变性死亡和纹状体多巴胺(Dopamine, DA)递质减少,同时伴随胶质细胞的增生活化。在目前已报道的各种PD动物模型中,中脑黑质也均出现了显著的小胶质细胞活化,这提示由小胶质细胞介导的神经炎症与PD的发病关系密切。
小胶质细胞属单核吞噬细胞系统,生理情况下,在中枢神经系统内发挥清除病原微生物等外源物质及自身坏死细胞组织的作用,维持神经组织内环境的稳定；病理情况下,小胶质细胞在各种病原物质的刺激下持续激活,不仅分泌多种炎性因子,如IL-1β、IL-6、TNF-α、 INF-y等,还会产生大量一氧化氮(Nitric oxide,NO)、超氧阴离子、谷氨酸和其他神经毒素,各种毒性因素长期共同作用可导致神经元损伤甚至死亡。有学者研究发现中脑黑质部位小胶质细胞的分布密度较其他脑区高,提示在病理情况下,中脑黑质会发生更强烈的炎症反应。另外,由于多巴胺能神经元需要进行多巴胺的合成分解代谢,自身氧化负荷较其他类型神经元重,更容易受到各种炎症介质的损伤而退变死亡。尽管炎症作为帕金森病发病的启动因素这一假说还存在争议,但局部炎症持续存在可导致多巴胺能神经元损伤,引起多巴胺能神经元加速丢失进而促进PD病情发展已成共识。
因此,基于抑制小胶质细胞介导的炎症反应治疗PD的设想,许多学者开展了大量实验研究,证实应用地塞米松、美满霉素及吲哚美辛等药物可以抑制小胶质细胞的活化及其炎症介质的产生,减轻多巴胺能神经元的炎症损伤,缓解多种PD动物模型的症状。另外,在体外细胞培养PD模型中,应用纳洛酮等药物可以抑制小胶质细胞分泌各种毒性因子,从而保护多巴胺能神经元。流行病学调查也发现,长期服用非固醇类抗炎药的病人患PD的几率显著低于正常人群。以上证据进一步表明由小胶质细胞介导的神经炎症参与了多巴胺能神经元的损伤过程,通过药物抑制炎症反应有助于阻止或延缓PD的发展。
过氧化物酶体增殖物激活受体γ (Peroxisome proliferator-activated receptor gamma, PPARy)属于核激素受体超家族,是配体激活的转录因子,此受体激活后可以调控多种基因的表达,调节细胞增殖分化和糖类脂类代谢,并可以调控炎症反应。在外周系统,PPARy激活后对单核细胞系细胞介导的炎症反应有明显抑制作用；在中枢神经系统,神经元和小胶质细胞都表达PPARy,提示PPARy在这两种细胞可能具有重要生理功能。噻唑烷二酮类化合物(Thiadiazolidinones, TZDs)是人工合成的PPARy激动剂,包括罗格列酮、匹格列酮、环格列酮和曲格列酮等,其中罗格列酮对PPARy的亲和力最大。由于噻唑烷二酮类药物能增强机体对胰岛素的敏感性并具有降低血糖的作用,目前临床上主要用于治疗Ⅱ型糖尿病。近期研究发现,TZDs可以抑制小胶质细胞的活化及其炎性因子的释放,提示其在中枢神经退行性疾病治疗中的应用前景,但其详细作用机制需要进一步研究予以阐明。
基于上述研究基础,本研究利用脂多糖(Lipopolysaccharide, LPS)激活小胶质细胞诱发炎症损伤制作体外PD模型,用罗格列酮(Rosiglitazone, RGZ)处理治疗,观察其抑制小胶质细胞激活与保护多巴胺能神经元的作用,并深入探讨其作用机制,为应用TZDs类药物治疗PD提供实验依据。本研究分为三部分,内容如下：
第一部分罗格列酮在原代细胞培养环境下对多巴胺能神经元的保护作用
目的：
在原代细胞培养环境下,研究罗格列酮能否抑制小胶质细胞活化而保护多巴胺能神经元,并检测罗格列酮能否抑制小胶质细胞炎症介质的释放。
方法：
1.建立体外多巴胺能神经元炎症损伤模型,首先进行小胶质细胞的分离纯化并与中脑神经元联合培养,用LPS(1μg/ml)激活小胶质细胞,OX-42免疫组织化学染色观察小胶质细胞的形态变化并计数活化小胶质细胞的数量,酪氨酸羟化酶(Tyrosine Hydroxylase,TH)免疫组化染色检测多巴胺能神经元形态和数量变化,分析小胶质细胞活化与多巴胺能神经元退变丢失的关系。
2.检测罗格列酮抑制小胶质细胞活化的作用,用不同浓度的罗格列酮处理多巴胺能神经元炎症损伤模型,同样方法染色后计数活化小胶质细胞和多巴胺能神经元的数量变化,分析罗格列酮抑制小胶质细胞活化与保护多巴胺能神经元的关系。
3.检测罗格列酮抑制炎症介质释放的作用,罗格列酮处理LPS激活的小胶质细胞,用ELISA的方法检测小胶质细胞培养液中肿瘤坏死因子(TNF一α)的变化,用Griess法检测一氧化氮(NO)的变化,用专用试剂盒检测超氧化物(Superoxide)的变化。
结果：
1.小胶质细胞由LPS激活后,形态发生显著变化：OX-42阳性染色显著增强,胞体变大,形态不规则,周边细胞质呈半透明状。另外,活化的小胶质细胞可大量分泌TNF-α、NO和Superoxide三种炎症介质。
2.在小胶质细胞和多巴胺能神经元的联合培养体系中,LPS作用3天后,多巴胺能神经元数量显著减少,残存多巴胺能神经元突起减少、变短或断裂。而无小胶质细胞存在条件下,LPS对多巴胺能神经元无损伤作用,提示多巴胺能神经元损伤与小胶质细胞的活化密切相关。
3.罗格列酮明显降低了活化小胶质细胞的数量和培养液中炎症介质的浓度,并显著提高了联合培养体系中多巴胺能神经元的存活数量。
结论：
小胶质细胞激活后形态发生明显变化,并分泌大量毒性炎症介质,进而损伤多巴胺能神经元；罗格列酮可以抑制小胶质细胞的活化,并减少多种炎症介质的产生,从而保护多巴胺能神经元。
第二部分炎症介质损伤多巴胺能神经元的机制及罗格列酮的保护作用
目的：研究活化BV-2细胞分泌的炎症介质造成MN9D细胞退变凋亡的机制,探讨罗格列酮能否抑制炎症损伤导致的MN9D细胞凋亡及其机制。
方法：
1.BV-2细胞是小鼠小胶质细胞系,MN9D细胞是小鼠多巴胺能神经元细胞系,实验以LPS激活后的BV-2细胞培养液作为炎症条件培养液,用ELISA和Greiss法检测条件培养液中TNF-α和NO的浓度变化,用专用试剂盒检测培养液中Superoxide的浓度。
2.用炎症条件培养液培养MN9D细胞,首先用MTT法检测MN9D的活力变化,台盼蓝染色检测MN9D细胞的生存率,然后为阐明MN9D的损伤机制,用比色法检测MN9D细胞中丙二醛(氧化应激标志物)的含量,JC-1染色检测MN9D细胞线粒体膜电位(线粒体功能的指标)的变化,最终用TUNEL和Hoechst33258染色检测炎症条件培养液能否引起MN9D细胞的凋亡。
3.以罗格列酮处理LPS激活的BV-2细胞,检测培养液中TNF-α, NO和Superoxide的浓度变化,并用此培养液上清培养MN9D细胞,同样方法检测MN9D细胞的活力、生存率、丙二醛的含量和线粒体膜电位变化情况,分析MN9D细胞中的氧化应激和线粒体功能的变化以及由此变化能否减轻MN9D细胞的凋亡。
结果：
1.LPS处理后BV-2细胞形态略微增大,但培养液中TNF-α, NO和Superoxide的含量明显升高。
2.经炎症条件培养液孵育后,MN9D细胞的存活率降低,细胞丙二醛含量上升,细胞线粒体膜电位降低,导致凋亡增多,提示MN9D细胞在炎症环境下发生了氧化应激和线粒体功能障碍,二者共同作用可能是导致凋亡的重要原因。
3.罗格列酮处理后的培养液提高了MN9D的存活率,减少了细胞内丙二醛的含量,部分恢复了线粒体膜电位,并通过抑制氧化应激和恢复线粒体功能减少了MN9D细胞的凋亡。
结论：
多巴胺能神经元细胞系MN9D细胞在炎症介质的作用下出现了氧化应激损伤和线粒体功能障碍,进而导致凋亡增多；罗格列酮可以通过抑制BV-2细胞炎症介质的分泌减轻以上两种机制的损伤,减少MN9D细胞的凋亡,提高其生存率。
第三部分罗格列酮抑制小胶质细胞活化的分子机制研究
目的：
在小胶质细胞系BV-2细胞中,从基因转录、蛋白表达和信号转导三个方面深入研究罗格列酮抑制BV-2细胞活化的机制。
方法：
1.在转录水平研究罗格列酮对炎症介质生成的影响,用RT-PCR的方法检测BV-2细胞内TNF-a和诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)的基因表达变化。
2.在蛋白水平探讨罗格列酮对炎症反应标志性蛋白的影响,用Western blot的方法检测LPS和罗格列酮作用前后iNOS的表达变化。
3.在转录因子水平探讨罗格列酮的抑制作用是否与NF-κB的活化有关,用Western的方法检测LPS和罗格列酮作用前后IκBα (NF-κB抑制蛋白)和磷酸化NF-κB p65的表达变化,并用免疫荧光染色的方法检测NF-κB p65核移位的变化。
4.在信号转导通路水平探讨罗格列酮对丝裂原活化蛋白激酶(Mitogen-activated protein kinases, MAPKs)三条信号通路(p38MAPK、 JNK和ERK)激活的影响,用Western的方法检测LPS和罗格列酮作用前后三条通路的磷酸化情况,分析罗格列酮发挥抑制作用可能的途径。
结果：
1.LPS激活BV-2细胞后, TNF-a和iNOS的基因表达显著上调,炎症反应的标志性蛋白iNOS表达增多；应用罗格列酮后,TNF-a和iNOS的基因表达明显下降,TNF-a和iNOS蛋白表达受到抑制,以上结果表明罗格列酮可以在转录水平和蛋白水平抑制炎症介质的产生。
2. NF-κB是机体调控免疫炎症反应的重要转录因子。Western检测发现：LPS激活BV-2细胞后,IKBα表达降低,罗格列酮可以部分恢复IKBα的表达：LPS可以诱导磷酸化NF-κB p65的水平显著升高,罗格列酮作用后可以抑制其升高。另外,NF-κB激活后其p65亚基需进入细胞核与相应DNA特定序列结合而启动炎症介质的表达,免疫荧光染色显示提示罗格列酮可抑制NF-κB的核移位,以上实验结果提示罗格列酮减少炎症介质的释放与抑制NF-κB通路的激活关系密切。
3. MAPKs是与炎症反应密切相关的信号通路,LPS激活BV-2细胞后,p38MAPK、 JNK和ERK通路均发生激活,罗格列酮抑制了p38MAPK和JNK通路的激活,而对ERK的激活无显著影响,说明在信号转导水平,罗格列酮可能通过抑制p38MAPK和JNK通路而发挥抗炎作用。
结论：
罗格列酮可以抑制活化BV-2细胞炎症介质的基因转录,并抑制炎症反应蛋白的表达；在转录因子水平罗格列酮可通过抑制NF-κB活化与核移位而发挥作用；在信号转导水平罗格列酮可通过抑制p38MAPK和JNK的激活而发挥抑制BV-2细胞活化的作用。
总结
本研究利用体外PD炎症模型,系统研究了PPARγ激动剂罗格列酮对小胶质细胞活化的抑制作用及对多巴胺能神经元的保护作用,首次阐明罗格列酮通过抑制NF-κB、 p38MAPK和JNK通路的激活而发挥抗炎作用。另外,本研究首次利用小胶质细胞系BV-2细胞的条件培养液在MN9D细胞上模拟了炎症损伤,直观显示了炎症介质对多巴胺能神经元造成的氧化应激和线粒体功能障碍,两者是导致多巴胺能神经元退变凋亡的主要机制,并证实罗格列酮抑制炎性介质释放与保护多巴胺能神经元的高度相关。本研究深化了罗格列酮抑制小胶质细胞活化药理机制的认识,为TZDs类药物治疗PD提供了新的实验依据。
Parkinson's disease (PD) is a common neurodegenerative disorder among the old people, which affects approximately1-3%population. PD was originally described by James Parkinson in1817and its etiology is still unknown up to date. PD is characterized by clinical symptoms, namely bradykinesia, resting tremor, rigidity and postural instability. The primary pathological changes of the disease are the progressive loss of dopaminergic neurons within the substantia nigra pars compacta and the reduction of dopamine in the striatum. So far, researchers proposed that heredity, aging, environment, oxidative stress, mitochondrial dysfunction and apoptosis were probably of the main mechanisms. Recently, extensive evidences suggest the involvement of microglia in the degenerative process of PD.
Microglia, the resident immune cells in the central nervous system (CNS), are considered to be the major cell type underlying the inflammatory process. Although microglia have beneficial effects in protective immunity and tissue repair, uncontrolled microglia activation has been implicated in contributing to host cell damage. In response to the pathological stimuli, microglia readily become activated and release various neurotoxic mediators, such as TNF-α, interleukin-1β (IL-1β), prostaglandin E2(PGE2), nitric oxide (NO) and reactive oxygen species (ROS), which work in concert to trigger neurodegeneration. The substantia nigra has an extremely high density of microglia which readily leads to intensive inflammation upon pathological stimuli. Since McGeer et al. described a large number of activated microglia in the substantia nigra of PD patient brains in1988, increasing studies have demonstrated that activated microglia may be associated with the development of PD and inhibition of microglia activation can protect dopaminergic neurons. On this basis, researchers have extensively searched for new agents to inhibit microglia activation for therapeutic intervention against neurodegeneration in PD.
Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily which regulates transcription of distinct genes through heterodimerization with the retinoid X receptors (RXR). The receptor has been proven to play a critical role in glucose and lipid metabolism. In addition to adipocytes, cells of the monocyte/macrophage lineage also express PPARγ, suggesting the involvement of PPARγ in the function of these cells. Recently, PPARγ ligands have been reported to inhibit the expression of proinflammatory molecules from microglia in several in vitro and in vivo models of neurodegenerative diseases.
Thiadiazolidinones (TZDs) are synthetic PPARγ ligands. They are small heterocyclic compounds with favorable pharmaceutic properties, such as oral bioavailability. Recently, it has been shown that TZDs have potent anti-inflammatory effects, such as inhibition of macrophage lineage activation, along with suppressing the expressions of TNF-α, IL-1β, NO and cyclooxygenase type2(COX-2). In the CNS, TZDs have similar anti-inflammatory effects on microglia. More recently, since several studies have shown that pioglitazone, a member of TZDs, exhibits a neuroprotective effect in various models of neuroinflammation, it is hypothesized that the TZDs could be used as a novel treatment approach to reduce the neuroinflammation in PD. Therefore, we sought to investigate the effect of rosiglitazone on microglia activated by lipopolysaccharide (LPS) and the protective effect on dopaminergic neurons. Along with this, we also examined its molecular mechanism regulating microglial production of inflammtory mediators such as TNF-α and NO. All the experiments were divided into3parts as following:
Part1:Rosiglitazone protects dopaminergic neurons in primary cell culture
Objective:
To study the protective effect of rosiglitazone on dopaminergic neurons through the inhibition of microglia activation in primary ventral mesencephalic cultures.
Methods:
1. Microglia were prepared from whole brains of1～2day-old Wistar rat pups. The enriched microglia were>98%in purity, as determined by immunostaining for microglia specific marker (OX-42). To establish an in vitro PD model, microglia were co-cultured with primary mesencephalic neuron-enriched cultures and then treated with LPS (1μg/ml) for72h. Dopaminergic neurons were detected with the anti-TH antibody and counted to assess the viability upon the insult of LPS.
2. The above in vitro PD model was treated with rosiglitazone at indicated concentrations and with the same amount of vehicle as controls. To assess the protective effect of rosiglitazone on dopaminergic neurons through the inhibition of microglia activation, the dopaminergic neurons and activated microglia were stained with their respective antibodies and counted. Microglia activation was determined by the obvious morphological changes with the staining of the OX-42, including an irregular shape with an expanded transparent extension from the soma and intensified OX-42staining.
3. To explore the effect of rosiglitazone on the release of inflammatory mediators in microglia, the levels of TNF-α, NO and Superoxide in the supernatants of microglia culture were measured with different detection kits according to the manufacture's instructions.
Results:
1. After48h of LPS exposure,70%～80%of the microglia were activated. The generation of TNF-α, NO and Superoxide are significantly increased in microglia-enriched cultures after LPS treatment for24h.
2. In neuron-enriched cultures treated with LPS, no dopaminergic neuronal loss was found. In contrast, in neuron-microglia co-culture, LPS (1μg/ml) induced～45%loss of dopaminergic neurons after a72h treatment. Pretreatment with rosiglitazone (50μM)1h prior to LPS treatment significantly increased the viability of dopaminergic neuron compared with that LPS alone. These indicated that the loss of dopaminergic neuron was associated with the microglia activation.
3. Rosiglitazone significantly decreased the number of activated microglia and the subsequent release of TNF-a, NO and Superoxide.
Conclusion:
Activated microglia secrete several neurotoxic mediators which have detrimental effects on dopaminergic neurons. Rosiglitazone protects dopaminergic neurons against LPS-induced neurotoxicity through the inhibition of microglia activation and subsequent release of inflammatory mediators.
Part2:Neurotoxic mechanisms of inflammatory mediators in dopaminergic neurons and rosiglitazone-mediated reduction of the neurotoxicity
Objective:
1. To study the mechanisms that inflammatory mediators are toxic to dopaminergic neurons.
2. To explore whether rosiglitazone can protect dopaminergic neurons through reducing the release of inflammatory mediators.
Methods:
1. The supernatant of BV-2cells treated with LPS was used as inflammation-conditioned media and the level of TNF-a, NO and Superoxide was measured with ELISA, Griess and Superoxide assay kits.
2. The viability of MN9D cell cultured in the inflammation-conditioned media was measured by MTT and Trypan Blue staining. To clarify the toxicity of inflammatory mediators secreted by activated BV-2cells, the amount of MDA (an indicator of oxidative stress) in MN9D cells was measured by colorimetry and the mitochondrial membrane potential were measured with JC-1mitochondrial membrane potential assay kit. The apoptosis of BV-2cell was detected with TUNEL and Hoechst33258staining.
3. After the administration of rosiglitazone in BV-2cells, viability, level of MDA, mitochondrial membrane potential and apoptosis of MN9D cells were measured with same methods.
Results:
1. Stimulation of BV-2cells with LPS led to a significant increase in the TNF-a, NO and Superoxide levels although the morphology of BV-2cells had no obvious changes upon LPS exposure.
2. Mitochondrial membrane potential was greatly decreased and the amount of MDA was obviously increased when MN9D cells were cultured in inflammation-conditioned media. These indicated that MN9D cells underwent oxidative stress and mitochondrial dysfunction. Consistent with the above results, survival of MN9D cells cultured in inflammation-conditioned media was greatly decreased, and the apoptosis of MN9D cells was significantly increased.
3. After the administration of rosiglitazone in BV-2cells, the viability of MN9D cells was obviously increased, mitochondrial membrane potential was partly restored, the amount of MDA was greatly decreased and the apoptosis was significantly decreased, when compared with that in MN9D cells cultured in inflammation-conditioned media, indicating the protective effects of rosiglitazone.
Conclusion:
MN9D cells cultured in inflammatory conditioned-media exhibit oxidative stress and mitochondrial dysfunction which lead to apoptosis. Rosiglitazone reduces apoptosis in MN9D cells through the attenuation of oxidative stress and restoration of mitochondrial membrane potential. In summary, rosiglitazone protects MN9D cells through the attenuation of oxidative stress and mitochondrial dysfunction which were derived from the insults of inflammatory mediators.
Part3:The molecular mechanisms of rosiglitazone-mediated inhibitory effect on microglia activation
Objective:
To elucidate the mechanism responsible for the inhibitory effect on microglia of rosiglitazone.
Methods:
1. To elucidate the mechanism responsible for the inhibitory effect of rosiglitazone on TNF-a and NO production, we examined TNF-a and iNOS mRNA expression levels with RT-PCR.
2. To study the influence of rosiglitazone on the inflammatory marker protein, iNOS expression was measured with Western blot.
3. To assess whether the inhibitive effect of rosiglitazone on gene expression occurred via blockade of NF-κB activity in BV-2cells, the expression of IKBa and the phosphorylation levels of NF-κB p65was detected by Western blot. The translocation of NF-κB p65from cytoplasm to the nucleus was determined with immunofluorescence analysis.
4. To determine whether the repressive effect of rosiglitazone on synthesis and release of inflammatory mediators occurred via MAPK signaling pathway, the expression of p38MAPK, JNK and ERK were measured with Western blot.
Results:
1. Consistent with the results obtained from the cytokine production assays, LPS-upregulated mRNA levels of TNF-a and iNOS were inhibited by rosiglitazone, suggesting that rosiglitazone negatively regulated the production of TNF-a and NO at the transcriptional level in the LPS-stimulated BV-2cells.
2. Rosiglitazone significantly inhibited LPS-induced phosphorylation of NF-κB p65subunit and cytosol-unclear translocation of NF-κB p65in BV-2cells. The results indicated the NF-κB might potentially involved in suppressing TNF-a and NO production by rosiglitazone.
3. The signal pathways of p38MAPK, JNK and ERK were activated in BV-2cells upon LPS treatment. Administration of rosiglitazone exhibited inhibitory effects on p38MAPK and JNK activation and no effect on ERK activation. These findings indicated that rosiglitazone inhibited the generation of TNF-a and NO involving the suppression of p38MAPK and JNK signal pathway.
Conclusion:
Rosiglitazone inhibits LPS-induced gene expression of inflammatory mediators in microglia. In addition, the anti-inflammatory property of rosiglitazone is associated with the inhibition of NF-κB, p38MAPK and JNK activation in LPS-stimulated BV-2cells.
Summary
Using in vitro inflammatory PD model, we systematically studied the inhibitive effect of PPARy agonist rosiglitazone on microglia activation which is involved in the degenerative process of PD. For the first time we demonstrated that rosiglitazone inhibited the activation of microglia through the suppression of NF-κB, p38MAPK and JNK pathway. Additionally, our study simulated the inflammatory insult on MN9D cells with conditioned media from activated BV-2cells and visually showed the inhibitive effect of rosiglitazone on oxidative stress, mitochondrial dysfunction and apoptosis via reducing the release of inflammatory mediators. Our study provided more data to understand the mechanism underlying the inhibition of rosiglitazone on microglia activation and to discuss the potential of TZDs compounds for the treatment of PD.
小胶质细胞属单核吞噬细胞系统,生理情况下,在中枢神经系统内发挥清除病原微生物等外源物质及自身坏死细胞组织的作用,维持神经组织内环境的稳定；病理情况下,小胶质细胞在各种病原物质的刺激下持续激活,不仅分泌多种炎性因子,如IL-1β、IL-6、TNF-α、 INF-y等,还会产生大量一氧化氮(Nitric oxide,NO)、超氧阴离子、谷氨酸和其他神经毒素,各种毒性因素长期共同作用可导致神经元损伤甚至死亡。有学者研究发现中脑黑质部位小胶质细胞的分布密度较其他脑区高,提示在病理情况下,中脑黑质会发生更强烈的炎症反应。另外,由于多巴胺能神经元需要进行多巴胺的合成分解代谢,自身氧化负荷较其他类型神经元重,更容易受到各种炎症介质的损伤而退变死亡。尽管炎症作为帕金森病发病的启动因素这一假说还存在争议,但局部炎症持续存在可导致多巴胺能神经元损伤,引起多巴胺能神经元加速丢失进而促进PD病情发展已成共识。
因此,基于抑制小胶质细胞介导的炎症反应治疗PD的设想,许多学者开展了大量实验研究,证实应用地塞米松、美满霉素及吲哚美辛等药物可以抑制小胶质细胞的活化及其炎症介质的产生,减轻多巴胺能神经元的炎症损伤,缓解多种PD动物模型的症状。另外,在体外细胞培养PD模型中,应用纳洛酮等药物可以抑制小胶质细胞分泌各种毒性因子,从而保护多巴胺能神经元。流行病学调查也发现,长期服用非固醇类抗炎药的病人患PD的几率显著低于正常人群。以上证据进一步表明由小胶质细胞介导的神经炎症参与了多巴胺能神经元的损伤过程,通过药物抑制炎症反应有助于阻止或延缓PD的发展。
过氧化物酶体增殖物激活受体γ (Peroxisome proliferator-activated receptor gamma, PPARy)属于核激素受体超家族,是配体激活的转录因子,此受体激活后可以调控多种基因的表达,调节细胞增殖分化和糖类脂类代谢,并可以调控炎症反应。在外周系统,PPARy激活后对单核细胞系细胞介导的炎症反应有明显抑制作用；在中枢神经系统,神经元和小胶质细胞都表达PPARy,提示PPARy在这两种细胞可能具有重要生理功能。噻唑烷二酮类化合物(Thiadiazolidinones, TZDs)是人工合成的PPARy激动剂,包括罗格列酮、匹格列酮、环格列酮和曲格列酮等,其中罗格列酮对PPARy的亲和力最大。由于噻唑烷二酮类药物能增强机体对胰岛素的敏感性并具有降低血糖的作用,目前临床上主要用于治疗Ⅱ型糖尿病。近期研究发现,TZDs可以抑制小胶质细胞的活化及其炎性因子的释放,提示其在中枢神经退行性疾病治疗中的应用前景,但其详细作用机制需要进一步研究予以阐明。
基于上述研究基础,本研究利用脂多糖(Lipopolysaccharide, LPS)激活小胶质细胞诱发炎症损伤制作体外PD模型,用罗格列酮(Rosiglitazone, RGZ)处理治疗,观察其抑制小胶质细胞激活与保护多巴胺能神经元的作用,并深入探讨其作用机制,为应用TZDs类药物治疗PD提供实验依据。本研究分为三部分,内容如下：
第一部分罗格列酮在原代细胞培养环境下对多巴胺能神经元的保护作用
目的：
在原代细胞培养环境下,研究罗格列酮能否抑制小胶质细胞活化而保护多巴胺能神经元,并检测罗格列酮能否抑制小胶质细胞炎症介质的释放。
方法：
1.建立体外多巴胺能神经元炎症损伤模型,首先进行小胶质细胞的分离纯化并与中脑神经元联合培养,用LPS(1μg/ml)激活小胶质细胞,OX-42免疫组织化学染色观察小胶质细胞的形态变化并计数活化小胶质细胞的数量,酪氨酸羟化酶(Tyrosine Hydroxylase,TH)免疫组化染色检测多巴胺能神经元形态和数量变化,分析小胶质细胞活化与多巴胺能神经元退变丢失的关系。
2.检测罗格列酮抑制小胶质细胞活化的作用,用不同浓度的罗格列酮处理多巴胺能神经元炎症损伤模型,同样方法染色后计数活化小胶质细胞和多巴胺能神经元的数量变化,分析罗格列酮抑制小胶质细胞活化与保护多巴胺能神经元的关系。
3.检测罗格列酮抑制炎症介质释放的作用,罗格列酮处理LPS激活的小胶质细胞,用ELISA的方法检测小胶质细胞培养液中肿瘤坏死因子(TNF一α)的变化,用Griess法检测一氧化氮(NO)的变化,用专用试剂盒检测超氧化物(Superoxide)的变化。
结果：
1.小胶质细胞由LPS激活后,形态发生显著变化：OX-42阳性染色显著增强,胞体变大,形态不规则,周边细胞质呈半透明状。另外,活化的小胶质细胞可大量分泌TNF-α、NO和Superoxide三种炎症介质。
2.在小胶质细胞和多巴胺能神经元的联合培养体系中,LPS作用3天后,多巴胺能神经元数量显著减少,残存多巴胺能神经元突起减少、变短或断裂。而无小胶质细胞存在条件下,LPS对多巴胺能神经元无损伤作用,提示多巴胺能神经元损伤与小胶质细胞的活化密切相关。
3.罗格列酮明显降低了活化小胶质细胞的数量和培养液中炎症介质的浓度,并显著提高了联合培养体系中多巴胺能神经元的存活数量。
结论：
小胶质细胞激活后形态发生明显变化,并分泌大量毒性炎症介质,进而损伤多巴胺能神经元；罗格列酮可以抑制小胶质细胞的活化,并减少多种炎症介质的产生,从而保护多巴胺能神经元。
第二部分炎症介质损伤多巴胺能神经元的机制及罗格列酮的保护作用
目的：研究活化BV-2细胞分泌的炎症介质造成MN9D细胞退变凋亡的机制,探讨罗格列酮能否抑制炎症损伤导致的MN9D细胞凋亡及其机制。
方法：
1.BV-2细胞是小鼠小胶质细胞系,MN9D细胞是小鼠多巴胺能神经元细胞系,实验以LPS激活后的BV-2细胞培养液作为炎症条件培养液,用ELISA和Greiss法检测条件培养液中TNF-α和NO的浓度变化,用专用试剂盒检测培养液中Superoxide的浓度。
2.用炎症条件培养液培养MN9D细胞,首先用MTT法检测MN9D的活力变化,台盼蓝染色检测MN9D细胞的生存率,然后为阐明MN9D的损伤机制,用比色法检测MN9D细胞中丙二醛(氧化应激标志物)的含量,JC-1染色检测MN9D细胞线粒体膜电位(线粒体功能的指标)的变化,最终用TUNEL和Hoechst33258染色检测炎症条件培养液能否引起MN9D细胞的凋亡。
3.以罗格列酮处理LPS激活的BV-2细胞,检测培养液中TNF-α, NO和Superoxide的浓度变化,并用此培养液上清培养MN9D细胞,同样方法检测MN9D细胞的活力、生存率、丙二醛的含量和线粒体膜电位变化情况,分析MN9D细胞中的氧化应激和线粒体功能的变化以及由此变化能否减轻MN9D细胞的凋亡。
结果：
1.LPS处理后BV-2细胞形态略微增大,但培养液中TNF-α, NO和Superoxide的含量明显升高。
2.经炎症条件培养液孵育后,MN9D细胞的存活率降低,细胞丙二醛含量上升,细胞线粒体膜电位降低,导致凋亡增多,提示MN9D细胞在炎症环境下发生了氧化应激和线粒体功能障碍,二者共同作用可能是导致凋亡的重要原因。
3.罗格列酮处理后的培养液提高了MN9D的存活率,减少了细胞内丙二醛的含量,部分恢复了线粒体膜电位,并通过抑制氧化应激和恢复线粒体功能减少了MN9D细胞的凋亡。
结论：
多巴胺能神经元细胞系MN9D细胞在炎症介质的作用下出现了氧化应激损伤和线粒体功能障碍,进而导致凋亡增多；罗格列酮可以通过抑制BV-2细胞炎症介质的分泌减轻以上两种机制的损伤,减少MN9D细胞的凋亡,提高其生存率。
第三部分罗格列酮抑制小胶质细胞活化的分子机制研究
目的：
在小胶质细胞系BV-2细胞中,从基因转录、蛋白表达和信号转导三个方面深入研究罗格列酮抑制BV-2细胞活化的机制。
方法：
1.在转录水平研究罗格列酮对炎症介质生成的影响,用RT-PCR的方法检测BV-2细胞内TNF-a和诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)的基因表达变化。
2.在蛋白水平探讨罗格列酮对炎症反应标志性蛋白的影响,用Western blot的方法检测LPS和罗格列酮作用前后iNOS的表达变化。
3.在转录因子水平探讨罗格列酮的抑制作用是否与NF-κB的活化有关,用Western的方法检测LPS和罗格列酮作用前后IκBα (NF-κB抑制蛋白)和磷酸化NF-κB p65的表达变化,并用免疫荧光染色的方法检测NF-κB p65核移位的变化。
4.在信号转导通路水平探讨罗格列酮对丝裂原活化蛋白激酶(Mitogen-activated protein kinases, MAPKs)三条信号通路(p38MAPK、 JNK和ERK)激活的影响,用Western的方法检测LPS和罗格列酮作用前后三条通路的磷酸化情况,分析罗格列酮发挥抑制作用可能的途径。
结果：
1.LPS激活BV-2细胞后, TNF-a和iNOS的基因表达显著上调,炎症反应的标志性蛋白iNOS表达增多；应用罗格列酮后,TNF-a和iNOS的基因表达明显下降,TNF-a和iNOS蛋白表达受到抑制,以上结果表明罗格列酮可以在转录水平和蛋白水平抑制炎症介质的产生。
2. NF-κB是机体调控免疫炎症反应的重要转录因子。Western检测发现：LPS激活BV-2细胞后,IKBα表达降低,罗格列酮可以部分恢复IKBα的表达：LPS可以诱导磷酸化NF-κB p65的水平显著升高,罗格列酮作用后可以抑制其升高。另外,NF-κB激活后其p65亚基需进入细胞核与相应DNA特定序列结合而启动炎症介质的表达,免疫荧光染色显示提示罗格列酮可抑制NF-κB的核移位,以上实验结果提示罗格列酮减少炎症介质的释放与抑制NF-κB通路的激活关系密切。
3. MAPKs是与炎症反应密切相关的信号通路,LPS激活BV-2细胞后,p38MAPK、 JNK和ERK通路均发生激活,罗格列酮抑制了p38MAPK和JNK通路的激活,而对ERK的激活无显著影响,说明在信号转导水平,罗格列酮可能通过抑制p38MAPK和JNK通路而发挥抗炎作用。
结论：
罗格列酮可以抑制活化BV-2细胞炎症介质的基因转录,并抑制炎症反应蛋白的表达；在转录因子水平罗格列酮可通过抑制NF-κB活化与核移位而发挥作用；在信号转导水平罗格列酮可通过抑制p38MAPK和JNK的激活而发挥抑制BV-2细胞活化的作用。
总结
本研究利用体外PD炎症模型,系统研究了PPARγ激动剂罗格列酮对小胶质细胞活化的抑制作用及对多巴胺能神经元的保护作用,首次阐明罗格列酮通过抑制NF-κB、 p38MAPK和JNK通路的激活而发挥抗炎作用。另外,本研究首次利用小胶质细胞系BV-2细胞的条件培养液在MN9D细胞上模拟了炎症损伤,直观显示了炎症介质对多巴胺能神经元造成的氧化应激和线粒体功能障碍,两者是导致多巴胺能神经元退变凋亡的主要机制,并证实罗格列酮抑制炎性介质释放与保护多巴胺能神经元的高度相关。本研究深化了罗格列酮抑制小胶质细胞活化药理机制的认识,为TZDs类药物治疗PD提供了新的实验依据。
Parkinson's disease (PD) is a common neurodegenerative disorder among the old people, which affects approximately1-3%population. PD was originally described by James Parkinson in1817and its etiology is still unknown up to date. PD is characterized by clinical symptoms, namely bradykinesia, resting tremor, rigidity and postural instability. The primary pathological changes of the disease are the progressive loss of dopaminergic neurons within the substantia nigra pars compacta and the reduction of dopamine in the striatum. So far, researchers proposed that heredity, aging, environment, oxidative stress, mitochondrial dysfunction and apoptosis were probably of the main mechanisms. Recently, extensive evidences suggest the involvement of microglia in the degenerative process of PD.
Microglia, the resident immune cells in the central nervous system (CNS), are considered to be the major cell type underlying the inflammatory process. Although microglia have beneficial effects in protective immunity and tissue repair, uncontrolled microglia activation has been implicated in contributing to host cell damage. In response to the pathological stimuli, microglia readily become activated and release various neurotoxic mediators, such as TNF-α, interleukin-1β (IL-1β), prostaglandin E2(PGE2), nitric oxide (NO) and reactive oxygen species (ROS), which work in concert to trigger neurodegeneration. The substantia nigra has an extremely high density of microglia which readily leads to intensive inflammation upon pathological stimuli. Since McGeer et al. described a large number of activated microglia in the substantia nigra of PD patient brains in1988, increasing studies have demonstrated that activated microglia may be associated with the development of PD and inhibition of microglia activation can protect dopaminergic neurons. On this basis, researchers have extensively searched for new agents to inhibit microglia activation for therapeutic intervention against neurodegeneration in PD.
Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily which regulates transcription of distinct genes through heterodimerization with the retinoid X receptors (RXR). The receptor has been proven to play a critical role in glucose and lipid metabolism. In addition to adipocytes, cells of the monocyte/macrophage lineage also express PPARγ, suggesting the involvement of PPARγ in the function of these cells. Recently, PPARγ ligands have been reported to inhibit the expression of proinflammatory molecules from microglia in several in vitro and in vivo models of neurodegenerative diseases.
Thiadiazolidinones (TZDs) are synthetic PPARγ ligands. They are small heterocyclic compounds with favorable pharmaceutic properties, such as oral bioavailability. Recently, it has been shown that TZDs have potent anti-inflammatory effects, such as inhibition of macrophage lineage activation, along with suppressing the expressions of TNF-α, IL-1β, NO and cyclooxygenase type2(COX-2). In the CNS, TZDs have similar anti-inflammatory effects on microglia. More recently, since several studies have shown that pioglitazone, a member of TZDs, exhibits a neuroprotective effect in various models of neuroinflammation, it is hypothesized that the TZDs could be used as a novel treatment approach to reduce the neuroinflammation in PD. Therefore, we sought to investigate the effect of rosiglitazone on microglia activated by lipopolysaccharide (LPS) and the protective effect on dopaminergic neurons. Along with this, we also examined its molecular mechanism regulating microglial production of inflammtory mediators such as TNF-α and NO. All the experiments were divided into3parts as following:
Part1:Rosiglitazone protects dopaminergic neurons in primary cell culture
Objective:
To study the protective effect of rosiglitazone on dopaminergic neurons through the inhibition of microglia activation in primary ventral mesencephalic cultures.
Methods:
1. Microglia were prepared from whole brains of1～2day-old Wistar rat pups. The enriched microglia were>98%in purity, as determined by immunostaining for microglia specific marker (OX-42). To establish an in vitro PD model, microglia were co-cultured with primary mesencephalic neuron-enriched cultures and then treated with LPS (1μg/ml) for72h. Dopaminergic neurons were detected with the anti-TH antibody and counted to assess the viability upon the insult of LPS.
2. The above in vitro PD model was treated with rosiglitazone at indicated concentrations and with the same amount of vehicle as controls. To assess the protective effect of rosiglitazone on dopaminergic neurons through the inhibition of microglia activation, the dopaminergic neurons and activated microglia were stained with their respective antibodies and counted. Microglia activation was determined by the obvious morphological changes with the staining of the OX-42, including an irregular shape with an expanded transparent extension from the soma and intensified OX-42staining.
3. To explore the effect of rosiglitazone on the release of inflammatory mediators in microglia, the levels of TNF-α, NO and Superoxide in the supernatants of microglia culture were measured with different detection kits according to the manufacture's instructions.
Results:
1. After48h of LPS exposure,70%～80%of the microglia were activated. The generation of TNF-α, NO and Superoxide are significantly increased in microglia-enriched cultures after LPS treatment for24h.
2. In neuron-enriched cultures treated with LPS, no dopaminergic neuronal loss was found. In contrast, in neuron-microglia co-culture, LPS (1μg/ml) induced～45%loss of dopaminergic neurons after a72h treatment. Pretreatment with rosiglitazone (50μM)1h prior to LPS treatment significantly increased the viability of dopaminergic neuron compared with that LPS alone. These indicated that the loss of dopaminergic neuron was associated with the microglia activation.
3. Rosiglitazone significantly decreased the number of activated microglia and the subsequent release of TNF-a, NO and Superoxide.
Conclusion:
Activated microglia secrete several neurotoxic mediators which have detrimental effects on dopaminergic neurons. Rosiglitazone protects dopaminergic neurons against LPS-induced neurotoxicity through the inhibition of microglia activation and subsequent release of inflammatory mediators.
Part2:Neurotoxic mechanisms of inflammatory mediators in dopaminergic neurons and rosiglitazone-mediated reduction of the neurotoxicity
Objective:
1. To study the mechanisms that inflammatory mediators are toxic to dopaminergic neurons.
2. To explore whether rosiglitazone can protect dopaminergic neurons through reducing the release of inflammatory mediators.
Methods:
1. The supernatant of BV-2cells treated with LPS was used as inflammation-conditioned media and the level of TNF-a, NO and Superoxide was measured with ELISA, Griess and Superoxide assay kits.
2. The viability of MN9D cell cultured in the inflammation-conditioned media was measured by MTT and Trypan Blue staining. To clarify the toxicity of inflammatory mediators secreted by activated BV-2cells, the amount of MDA (an indicator of oxidative stress) in MN9D cells was measured by colorimetry and the mitochondrial membrane potential were measured with JC-1mitochondrial membrane potential assay kit. The apoptosis of BV-2cell was detected with TUNEL and Hoechst33258staining.
3. After the administration of rosiglitazone in BV-2cells, viability, level of MDA, mitochondrial membrane potential and apoptosis of MN9D cells were measured with same methods.
Results:
1. Stimulation of BV-2cells with LPS led to a significant increase in the TNF-a, NO and Superoxide levels although the morphology of BV-2cells had no obvious changes upon LPS exposure.
2. Mitochondrial membrane potential was greatly decreased and the amount of MDA was obviously increased when MN9D cells were cultured in inflammation-conditioned media. These indicated that MN9D cells underwent oxidative stress and mitochondrial dysfunction. Consistent with the above results, survival of MN9D cells cultured in inflammation-conditioned media was greatly decreased, and the apoptosis of MN9D cells was significantly increased.
3. After the administration of rosiglitazone in BV-2cells, the viability of MN9D cells was obviously increased, mitochondrial membrane potential was partly restored, the amount of MDA was greatly decreased and the apoptosis was significantly decreased, when compared with that in MN9D cells cultured in inflammation-conditioned media, indicating the protective effects of rosiglitazone.
Conclusion:
MN9D cells cultured in inflammatory conditioned-media exhibit oxidative stress and mitochondrial dysfunction which lead to apoptosis. Rosiglitazone reduces apoptosis in MN9D cells through the attenuation of oxidative stress and restoration of mitochondrial membrane potential. In summary, rosiglitazone protects MN9D cells through the attenuation of oxidative stress and mitochondrial dysfunction which were derived from the insults of inflammatory mediators.
Part3:The molecular mechanisms of rosiglitazone-mediated inhibitory effect on microglia activation
Objective:
To elucidate the mechanism responsible for the inhibitory effect on microglia of rosiglitazone.
Methods:
1. To elucidate the mechanism responsible for the inhibitory effect of rosiglitazone on TNF-a and NO production, we examined TNF-a and iNOS mRNA expression levels with RT-PCR.
2. To study the influence of rosiglitazone on the inflammatory marker protein, iNOS expression was measured with Western blot.
3. To assess whether the inhibitive effect of rosiglitazone on gene expression occurred via blockade of NF-κB activity in BV-2cells, the expression of IKBa and the phosphorylation levels of NF-κB p65was detected by Western blot. The translocation of NF-κB p65from cytoplasm to the nucleus was determined with immunofluorescence analysis.
4. To determine whether the repressive effect of rosiglitazone on synthesis and release of inflammatory mediators occurred via MAPK signaling pathway, the expression of p38MAPK, JNK and ERK were measured with Western blot.
Results:
1. Consistent with the results obtained from the cytokine production assays, LPS-upregulated mRNA levels of TNF-a and iNOS were inhibited by rosiglitazone, suggesting that rosiglitazone negatively regulated the production of TNF-a and NO at the transcriptional level in the LPS-stimulated BV-2cells.
2. Rosiglitazone significantly inhibited LPS-induced phosphorylation of NF-κB p65subunit and cytosol-unclear translocation of NF-κB p65in BV-2cells. The results indicated the NF-κB might potentially involved in suppressing TNF-a and NO production by rosiglitazone.
3. The signal pathways of p38MAPK, JNK and ERK were activated in BV-2cells upon LPS treatment. Administration of rosiglitazone exhibited inhibitory effects on p38MAPK and JNK activation and no effect on ERK activation. These findings indicated that rosiglitazone inhibited the generation of TNF-a and NO involving the suppression of p38MAPK and JNK signal pathway.
Conclusion:
Rosiglitazone inhibits LPS-induced gene expression of inflammatory mediators in microglia. In addition, the anti-inflammatory property of rosiglitazone is associated with the inhibition of NF-κB, p38MAPK and JNK activation in LPS-stimulated BV-2cells.
Summary
Using in vitro inflammatory PD model, we systematically studied the inhibitive effect of PPARy agonist rosiglitazone on microglia activation which is involved in the degenerative process of PD. For the first time we demonstrated that rosiglitazone inhibited the activation of microglia through the suppression of NF-κB, p38MAPK and JNK pathway. Additionally, our study simulated the inflammatory insult on MN9D cells with conditioned media from activated BV-2cells and visually showed the inhibitive effect of rosiglitazone on oxidative stress, mitochondrial dysfunction and apoptosis via reducing the release of inflammatory mediators. Our study provided more data to understand the mechanism underlying the inhibition of rosiglitazone on microglia activation and to discuss the potential of TZDs compounds for the treatment of PD.
引文
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