从炎症的角度研究FLZ防治神经退行性疾病的作用机制及胞壁酰二肽衍生物MDP-C免疫活性研究
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摘要
本论文包括两部分内容,第一部分:从炎症的角度研究FLZ防治神经退行性疾病的作用机制。第二部分:胞壁酰二肽衍生物MDP-C免疫活性研究
第一部分:从炎症的角度研究FLZ防治神经退行性疾病的作用机制
FLZ是一种人工合成的番荔枝酰胺衍生物,以往研究显示具有很强的抗氧化、抗凋亡和神经保护作用,对多种实验性帕金森氏病动物模型的运动障碍和实验性痴呆小鼠的学习记忆障碍都具有改善作用。炎症在神经退行性疾病的发病过程中起重要的作用,可能是其诱发因素之一,控制疾病过程中的炎症反应,有益于神经退行性疾病的预防与治疗。因此,本研究从炎症的角度对FLZ防治神经退行性疾病的作用及作用机理进行了研究。
一、FLZ对侧脑室注射LPS小鼠学习记忆能力的影响及相关抗炎作用研究
脂多糖(Lipopolysaccharides,LPS)是革兰氏阴性细菌细胞膜的结构成分,是一种强的炎症刺激物,可引起动物学习记忆障碍,神经元发生损伤,可以较好的模拟阿尔茨海默病(Alzheimer's disease,AD)脑内炎症的改变。我们的研究结果显示,侧脑室注射LPS(2.5μg/只)小鼠在水迷宫实验中表现出了学习记忆能力的障碍,给予FLZ(150 mg/kg、75mg/kg)可明显改善动物的学习记忆能力。病理和尼氏染色结果显示FLZ对LPS引起的海马神经元的损伤具有明显的保护作用。生化检测结果显示,FLZ能降低海马内LPS诱导产生的肿瘤坏死因子-α(Tumor necrosisfactor-α,TNF-α)、白介素1β(Interleukin 1β,IL-1β),并降低LPS引起的诱导型一氧化氮合酶(Inducible nitric oxide synthase,iNOS)活性的升高。免疫组化实验结果发现FLZ能抑制LPS引起的小胶质细胞和星形胶质细胞的活化和增殖,对LPS诱导的iNOS、环氧合酶2(Cyclooxygenase-2,Cox-2)的表达有明显的抑制作用,实验还观察了炎症状态下与AD密切相关的β-淀粉样蛋白(β-amyloid,Aβ)和β-分泌酶(β-Secretase,BACE1)的变化,结果显示,LPS刺激可以促进BACE1的表达,并增加Aβ的产生,FLZ对此有明显的抑制作用。本部分实验结果提示FLZ能显著抑制LPS侧脑室注射引起的小胶质细胞和星形胶质细胞的活化,减少神经毒性炎症介质的产生,并显著改善炎症反应引起的学习记忆障碍和神经元损伤。
二、FLZ对巨噬细胞炎症反应的抑制作用及相关机制研究
上部分研究内容提示FLZ能通过抑制小胶质细胞和星形胶质细胞的活化、减少神经毒性炎症介质的分泌从而改善炎症反应引起的小鼠学习记忆障碍。为进一步探讨其作用机制,我们应用RAW264.7小鼠巨噬细胞株,检测了FLZ对LPS诱导的炎症介质一氧化氮(Nitric oxide,NO)的释放,TNF-α的产生,以及iNOS、TNF-α和Cox-2表达的影响。结果显示,FLZ在1、5、10μM剂量明显抑制LPS引起的NO的产生和iNOS、Cox-2 mRNA的转录与蛋白表达,在10gM剂量,显著减少TNF-α的分泌,对TNF-αmRNA的转录也有抑制,此外FLZ对LPS和聚集态Aβ_(1-40)诱导BV-2小鼠小胶质瘤细胞株释放的炎症介质NO和TNF-α也有明显的抑制作用。随后我们从核转录因子B(Nuclear factor-kappa B,NF-κB)和丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号通路探讨了FLZ抗炎作用机制,FLZ对LPS引起的IκB激酶(IκB kinase,IKK)和抑制性κB(InhibitoryκB,IκB)的磷酸化、IBB的降解、NF-κB的转位入核及NF-κB与特异DNA序列的结合均有明显抑制作用。对转录因子(Activator protein 1,AP-1)与特异DNA序列的结合、c-Jun氨基末端激酶(c-Jun NH2-terminal kinase,JNK)与p38的磷酸化也有显著的抑制作用,但对细胞外信号调节激酶(Extracellular signal regulated kinase,ERK)的磷酸化没有明显的影响。TGF-β活化激酶1(Transforming growth factor-βactivatedkinase 1,TAK1)是IKK和JNK、p38的上游激酶,FLZ能剂量依赖性的抑制TAK1的磷酸化。此外,FLZ对LPS和H_2O_2诱导产生的活性氧(Reactive oxygen species,ROS)有明显的抑制作用。以上结果提示FLZ对LPS引起的炎症反应具有抑制作用,这种作用可能与其抑制LPS引起的TAK1-IKK-NF-κB信号通路和TAK1-JNK/p38MAPK/AP-1信号通路活化的抑制有关,此外,FLZ对ROS的抑制可能也是其抗炎作用的重要机制之一。
综上所述,FLZ能显著抑制小鼠侧脑室注射LPS引起的炎症反应,对LPS引起的学习记忆障碍和神经元损伤有明显的改善作用。FLZ对LPS诱导的炎症反应的抑制作用可能与其抑制TAK1-IKK-NF-κB信号通路和TAK1-JNK/p38MAPK-AP-1信号通路有关,对ROS的抑制作用也参与其中。对炎症反应的抑制作用可能是FLZ防治神经退行性疾病的机制之一。
第二部分:胞壁酰二肽衍生物MDP-C免疫活性研究
N-乙酰胞壁酸-L-丙氨酸-D-异谷胺酰胺,简称胞壁酰二肽(Muramyl dipeptide,MDP),是分枝杆菌细胞壁中具有免疫佐剂活性的最小的结构单位,具有广泛的生物学活性,可以作为免疫调节剂在一定程度上增强机体对感染和肿瘤的非特异抵抗力。但是MDP难以穿透细胞膜,在体内迅速消除,以及体内实验可以引起发热、嗜睡、厌食和新陈代谢亢进等不良反应影响了其应用。因此,对MDP进行结构改造,合成活性好的结构类似物做为疫苗的佐剂或免疫调节剂用于感染和肿瘤的治疗成为药物研究领域内的一项重要课题。
N~2-[a-O-苄基-N-(乙酰胞壁酰)-L-丙胺酰-D-异谷胺酰胺酰-N~6-反式-(间-硝基肉桂酰基)-L赖胺酸酰胺简称MDP-C,是一种新合成的MDP衍生物。以往的研究显示MDP-C是一个潜在的免疫增强剂,本研究进一步观察了MDP-C的免疫活性,并对其在肿瘤免疫治疗方面的应用价值进行了初步探讨。
首先应用小鼠Lewis肺癌模型检测了MDP-C对肿瘤生长和转移有无抑制作用,结果显示MDP-C单独应用对小鼠Lewis肺癌原发瘤和肺转移都没有明显的影响,MDP-C 1.25 mg/kg、2.5 mg/kg剂量与低剂量(15 mg/kg)异环磷酰胺合用时,肺转移结节数减少,但是与正常对照组相比没有明显的差异。随后,应用酶联免疫斑点分析(Enzyme-linked immunospot assay,ELISPOT)方法检测了MDP-C对CTL表位多肽TRP-2_(180-188)诱导小鼠特异性细胞毒性T淋巴细胞(Cytotoxic Tlymphocyte,CTL)活化产生IFN-γ的佐剂作用,结果显示,MDP-C与TRP-2_(180-188)多肽合用,在一定程度上增强了TRP-2_(180-188)多肽诱导的CTL反应,但是作用较弱,统计学差异不显著。
树突细胞(Dendritic cells,DCs)是重要的专职抗原提呈细胞,在启动机体特异性、非特异性免疫应答的过程中起着关键的作用。促使DCs成熟,增强其抗原提呈功能是佐剂作用的重要机制。本研究在体外应用人单核细胞来源的树突细胞(Monocyte derived dendritic cells,MoDCs)探讨了MDP-C对树突细胞成熟和功能的影响。经重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF,1000U/ml)和重组人白细胞介素-4(rhIL-4,500U/ml)诱导培养的方法得到了不成熟树突细胞(iDCs),iDCs在含有MDP-C的完全培养液中继续培养24h后,采用流式细胞仪检测MoDCs表型,ELISA法检测培养上清中细胞因子IL-6、IL-12(p40+70)浓度,以MoDCs为刺激细胞,异体同种T淋巴细胞为效应细胞观察MDP-C对MoDCs抗原提呈和淋巴细胞增殖的影响。结果显示,与溶剂对照组相比,10μM MDP-C能显著增加MoDCs细胞表面HLA-DR、CD80、CD86以及CD83的表达,并能够增强MoDCs刺激异体T淋巴细胞增殖的能力,但是MDP-C不能诱导细胞因子IL-6、IL-12(p40+70)的产生,而阳性对照药Romurtide 1μM剂量对上述表面分子和细胞因子均有明显的诱导作用。随后我们应用RAW264.7小鼠巨噬细胞系检测了MDP-C对TNF-α有无作用,结果显示,经1μM MDP刺激的RAW264.7巨噬细胞和小鼠腹腔巨噬细胞分泌TNF-α的能力明显增强,而MDP-C各剂量组对两种巨噬细胞均未表现出增强作用。
综合本部分研究,MDP-C在体内对Lewis肺癌小鼠肿瘤生长和转移均未显示明显的治疗作用,对多肽引起的CTL活化也未显示明显的增强。体外实验中MDP-C能够促进MoDCs表面分子HLA-DR、CD80、CD86以及CD83的表达,增强抗原提呈功能,但是不能诱导MoDCs产生细胞因子IL-6、IL-12(p40+70),也不能诱导巨噬细胞产生TNF-α。提示,MDP-C有一定的免疫增强活性,但是作用较弱,其作用机制需进一步探讨。
The thesis is consist of two sections:"Mechanism of FLZ controlling neurodegenerative disease by modulation of inflammation" and "The immunocompetence of a synthetic muramyl dipeptide analogue MDP-C"
Section 1.Mechanism of FLZ controlling neurodegenerative disease by modulation of inflammation
Compound FLZ is a synthetic novel derivative of squamosamide.Previous studies in our laboratory demonstrated that FLZ not only had strong anti-oxidation, anti-apoptosis and neuroprotective effect,but also improved experimental learning and memory deficit and dyskinesia in animal models.This thesis first explored the ability of FLZ modulating inflammation and focused on mechanism studies controlling neurodegenerative disease.
Part 1.FLZ improvement of learning and memory dysfunction that is caused by LPS-induced inflammation in hippocampus
Lipopolysaccharide(LPS) is the major component of the outer surface of gram-negative bacteria and be considered as a potent inflammatory stimulator. Experimental evidences indicated that intracerebroventricular(i.c.v.) injection of LPS to rodents induced learning and memory impairments as well as neurodegeneration in brain areas related to cognitive function.In the present study,we explored the effects of FLZ on the LPS i.c.v.injected mice models.In water maze test,we found that i.c.v.injection of 2.5μg LPS induced learning and memory impairment.FLZ(150 mg/kg,75mg/kg) significantly improved the learning and memory ability and inhibited the damage of hippocampus neurons as assessed by histopathology method.To further investigate the mechanism of FLZ in improving the learning and memory impairments induced by LPS, biochemistry and immunohistochemistry methods were used.The results revealed that FLZ decreased the levels of TNF-αand IL-1βand inhibited the activity of inducible nitric oxide synthase(iNOS) which were induced by LPS.The immunohistochemistry results showed that the increased activation of microglia and astrocyte and the over expression of iNOS,cyclooxygenase-2(Cox-2) andβ-Secretase(BACE1) by LPS were all significantly inhibited by FLZ.Aβproduction induced by LPS was decreased either. In summary,FLZ significantly inhibited the inflammatory reaction in hippocampus caused by i.c.v.injection of LPS and improved the learning and memory impairment and neuron damage.
2.Inhibition effect of FLZ on the inflammatory reaction in macrophages and the mechanism of action
The above results indicated that FLZ inhibited the inflammatory reaction in hippocampus caused by i.c.v.injection of LPS.To further study the mechanism of FLZ, the RAW264.7 macrophages were used to detect the effects of FLZ on the production of inflammatory mediators-induced by LPS.The results showed that FLZ dose-dependently inhibited the LPS induced production of nitric oxide(NO) as well as the expression of iNOS,cyclooxygenase 2(Cox-2) and tumor necrosis factor(TNF)-αat both mRNA and protein levels.Furthermore,the production of NO and secretion of TNF-αinduced by LPS or aggregated Aβ_(1-40) in BV-2 microglias were also suppressed by FLZ.The anti-inflammatory mechanism of FLZ was investigated subsequently.In RAW264.7 cells, the LPS-induced DNA binding activation of Nuclear factor kappa-B(NF-κB) and activator protein 1(AP-1),the nuclear translocation of NF-κB p65,the degradation of inhibitoryκBαprotein(IκBα) and the phosphorylation of IκBα,IκB kinase(IKK)α/β, c-Jun NH_2-terminal kinase(JNK) and p38 mitogen-activated protein kinases(MAPKs) were all suppressed by FLZ.But the phosphorylation of extracellular signal-regulated kinase(ERK) was not impacted.Further study revealed that FLZ dose dependently inhibited the phosphorylation of transforming growth factor-β(TGF-β)-activated kinase 1(TAK1) which is the upstream signal of IKKα/β,JNK and p38 activation.In addition, reactive oxygen species(ROS) induced by LPS or H_2O_2 were suppressed by FLZ either. The results suggested that FLZ inhibited the production of inflammatory mediators at least partly via downregulation of TAK1-IKK-NF-κB and TAK1-JNK/p38MAPK/AP-1 pathways.In addition,the suppression of ROS was also one of the important mechanisms for the anti-inflammatory activity of FLZ.
In summary,FLZ significantly inhibited the inflammatory reaction in hippocampus caused by i.c.v,injection of LPS and improved the learning and memory impairment and neuron damage.The production of inflammatory mediators induced by LPS was inhibited by FLZ at least partly via the downregulation of TAK1-IKK-NF-κB and TAK1-JNK/p38MAPK-AP-1 pathways and the inhibition of ROS.The protection against neurodegenerative diaseases of FLZ may be partially accounts for its anti-inflammation activity.
Section 2.The immunocompetence of a synthetic muramyl dipeptide analogue MDP-C
N-acetylmuramyl-L-alany-D-isoglutamin(muramyl dipeptide,MDP),which was identified as the minimal active component of mycobacterium cell wall,possesses extensive biological activities on host immune systems and improves the nonspecific resistance against infections and tumors.However,the characters of poor penetration of membranes and rapid elimination in vivo,as well as the induction of toxic responses, such as pyrogenicity,lethargy,anoresy and hypermetabolism limit the application of MDP.Therefore,structure modification and synthesis of MDP derivatives possess good activitie is extremely needed.MDP-C(N2-[R-Obenzyl-N-(acetylmuramyl) -L-alanyl -D-isoglutaminyl]-N6-trans-(m-nitrocinnamoyl)-L-lysine),a novel chemically modified MDP,has been reported to be an potential immunostimulator.In this study,we investigated the immunocompetence of MDP-C.
First of all,we detected the effects of MDP-C on the growth and metastasis of tumors in a Lewis lung cancer mouse model.The results showed that MDP-C has no inhibitory activity on both the primarily tumor and pulmonary metastasis.Under tested condition,at the dose of 1.25 mg/kg and 2.5mg/kg,MDP-C combined with low dose isofosfamide(15 mg/kg) decreased the numbers of pulmonary metastasis nodes.But the data have no statistical significance compared with control group.In the experiment of ELISPOT,MDP-C slightly augmented the TRP-2_(180-188) peptide-specific cytotoxic T lymphocytes(CTLs) reaction in spleen cells of C57BL/6J mice immunized with the TRP-2_(180-188) peptide and MDP-C,but have no statistical significance compared with TRP-2_(180-188) peptide group.
Dendritic cells(DCs) are highly specialized antigen-presenting cells(APC) that play a central role in the initiation of both innate and adaptive immune responses.The effects of MDP-C on the differentiation and activation of human monocyte derived dendritic cells(MoDCs) were investigated.The combined action of GM-CSF and IL-4 could direct monocytes to differentiate into DCs displaying features of immature DC(iDCs).After cultured with various concentration of MDP-C for 24h,the surface molecules human leucocyte antigen-DR(HLA-DR),CD80,CD86 and CD83 were detected by flow cytometry.The allogeneic mixed lymphocyte reaction and the levels of IL-6 and IL-12(p40+70) were investigated with ATP bioluminescent assay kit and ELISA kit, respectively.The results showed that MDP-C up-regulated the expression of HLA-DR, CD80,CD86 and CD83,as well as the allogeneic mixed lymphocyte reaction.But the secretion of IL-6 and IL-12(p40+70) were not affected.While the positive control compound romurtide induced the expression of surface molecules and cytokines strongly. Further study revealed that MDP-C has no effect on the secretion of TNF-αin both RAW264.7 and mouse peritoneal macrophages.
In summary,MDP-C has no obvious inhibitory effect on the growth and metastasize of tumors in the Lewis lung cancer mouse model and has no obvious adjuvant activity on the TRP-2_(180-188) peptide induced cytotoxic T lymphocytes(CTLs) activition under tested condition.MDP-C increased the expression of surface molecules and enhanced the antigen presenting activity of MoDCs,but has no effect on the cytokine expression in both MoDCs and macrophages.The results indicated that MDP-C possesses immunocompetence to some extent.The mechanism of action needs further investigation.
第一部分:从炎症的角度研究FLZ防治神经退行性疾病的作用机制
FLZ是一种人工合成的番荔枝酰胺衍生物,以往研究显示具有很强的抗氧化、抗凋亡和神经保护作用,对多种实验性帕金森氏病动物模型的运动障碍和实验性痴呆小鼠的学习记忆障碍都具有改善作用。炎症在神经退行性疾病的发病过程中起重要的作用,可能是其诱发因素之一,控制疾病过程中的炎症反应,有益于神经退行性疾病的预防与治疗。因此,本研究从炎症的角度对FLZ防治神经退行性疾病的作用及作用机理进行了研究。
一、FLZ对侧脑室注射LPS小鼠学习记忆能力的影响及相关抗炎作用研究
脂多糖(Lipopolysaccharides,LPS)是革兰氏阴性细菌细胞膜的结构成分,是一种强的炎症刺激物,可引起动物学习记忆障碍,神经元发生损伤,可以较好的模拟阿尔茨海默病(Alzheimer's disease,AD)脑内炎症的改变。我们的研究结果显示,侧脑室注射LPS(2.5μg/只)小鼠在水迷宫实验中表现出了学习记忆能力的障碍,给予FLZ(150 mg/kg、75mg/kg)可明显改善动物的学习记忆能力。病理和尼氏染色结果显示FLZ对LPS引起的海马神经元的损伤具有明显的保护作用。生化检测结果显示,FLZ能降低海马内LPS诱导产生的肿瘤坏死因子-α(Tumor necrosisfactor-α,TNF-α)、白介素1β(Interleukin 1β,IL-1β),并降低LPS引起的诱导型一氧化氮合酶(Inducible nitric oxide synthase,iNOS)活性的升高。免疫组化实验结果发现FLZ能抑制LPS引起的小胶质细胞和星形胶质细胞的活化和增殖,对LPS诱导的iNOS、环氧合酶2(Cyclooxygenase-2,Cox-2)的表达有明显的抑制作用,实验还观察了炎症状态下与AD密切相关的β-淀粉样蛋白(β-amyloid,Aβ)和β-分泌酶(β-Secretase,BACE1)的变化,结果显示,LPS刺激可以促进BACE1的表达,并增加Aβ的产生,FLZ对此有明显的抑制作用。本部分实验结果提示FLZ能显著抑制LPS侧脑室注射引起的小胶质细胞和星形胶质细胞的活化,减少神经毒性炎症介质的产生,并显著改善炎症反应引起的学习记忆障碍和神经元损伤。
二、FLZ对巨噬细胞炎症反应的抑制作用及相关机制研究
上部分研究内容提示FLZ能通过抑制小胶质细胞和星形胶质细胞的活化、减少神经毒性炎症介质的分泌从而改善炎症反应引起的小鼠学习记忆障碍。为进一步探讨其作用机制,我们应用RAW264.7小鼠巨噬细胞株,检测了FLZ对LPS诱导的炎症介质一氧化氮(Nitric oxide,NO)的释放,TNF-α的产生,以及iNOS、TNF-α和Cox-2表达的影响。结果显示,FLZ在1、5、10μM剂量明显抑制LPS引起的NO的产生和iNOS、Cox-2 mRNA的转录与蛋白表达,在10gM剂量,显著减少TNF-α的分泌,对TNF-αmRNA的转录也有抑制,此外FLZ对LPS和聚集态Aβ_(1-40)诱导BV-2小鼠小胶质瘤细胞株释放的炎症介质NO和TNF-α也有明显的抑制作用。随后我们从核转录因子B(Nuclear factor-kappa B,NF-κB)和丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号通路探讨了FLZ抗炎作用机制,FLZ对LPS引起的IκB激酶(IκB kinase,IKK)和抑制性κB(InhibitoryκB,IκB)的磷酸化、IBB的降解、NF-κB的转位入核及NF-κB与特异DNA序列的结合均有明显抑制作用。对转录因子(Activator protein 1,AP-1)与特异DNA序列的结合、c-Jun氨基末端激酶(c-Jun NH2-terminal kinase,JNK)与p38的磷酸化也有显著的抑制作用,但对细胞外信号调节激酶(Extracellular signal regulated kinase,ERK)的磷酸化没有明显的影响。TGF-β活化激酶1(Transforming growth factor-βactivatedkinase 1,TAK1)是IKK和JNK、p38的上游激酶,FLZ能剂量依赖性的抑制TAK1的磷酸化。此外,FLZ对LPS和H_2O_2诱导产生的活性氧(Reactive oxygen species,ROS)有明显的抑制作用。以上结果提示FLZ对LPS引起的炎症反应具有抑制作用,这种作用可能与其抑制LPS引起的TAK1-IKK-NF-κB信号通路和TAK1-JNK/p38MAPK/AP-1信号通路活化的抑制有关,此外,FLZ对ROS的抑制可能也是其抗炎作用的重要机制之一。
综上所述,FLZ能显著抑制小鼠侧脑室注射LPS引起的炎症反应,对LPS引起的学习记忆障碍和神经元损伤有明显的改善作用。FLZ对LPS诱导的炎症反应的抑制作用可能与其抑制TAK1-IKK-NF-κB信号通路和TAK1-JNK/p38MAPK-AP-1信号通路有关,对ROS的抑制作用也参与其中。对炎症反应的抑制作用可能是FLZ防治神经退行性疾病的机制之一。
第二部分:胞壁酰二肽衍生物MDP-C免疫活性研究
N-乙酰胞壁酸-L-丙氨酸-D-异谷胺酰胺,简称胞壁酰二肽(Muramyl dipeptide,MDP),是分枝杆菌细胞壁中具有免疫佐剂活性的最小的结构单位,具有广泛的生物学活性,可以作为免疫调节剂在一定程度上增强机体对感染和肿瘤的非特异抵抗力。但是MDP难以穿透细胞膜,在体内迅速消除,以及体内实验可以引起发热、嗜睡、厌食和新陈代谢亢进等不良反应影响了其应用。因此,对MDP进行结构改造,合成活性好的结构类似物做为疫苗的佐剂或免疫调节剂用于感染和肿瘤的治疗成为药物研究领域内的一项重要课题。
N~2-[a-O-苄基-N-(乙酰胞壁酰)-L-丙胺酰-D-异谷胺酰胺酰-N~6-反式-(间-硝基肉桂酰基)-L赖胺酸酰胺简称MDP-C,是一种新合成的MDP衍生物。以往的研究显示MDP-C是一个潜在的免疫增强剂,本研究进一步观察了MDP-C的免疫活性,并对其在肿瘤免疫治疗方面的应用价值进行了初步探讨。
首先应用小鼠Lewis肺癌模型检测了MDP-C对肿瘤生长和转移有无抑制作用,结果显示MDP-C单独应用对小鼠Lewis肺癌原发瘤和肺转移都没有明显的影响,MDP-C 1.25 mg/kg、2.5 mg/kg剂量与低剂量(15 mg/kg)异环磷酰胺合用时,肺转移结节数减少,但是与正常对照组相比没有明显的差异。随后,应用酶联免疫斑点分析(Enzyme-linked immunospot assay,ELISPOT)方法检测了MDP-C对CTL表位多肽TRP-2_(180-188)诱导小鼠特异性细胞毒性T淋巴细胞(Cytotoxic Tlymphocyte,CTL)活化产生IFN-γ的佐剂作用,结果显示,MDP-C与TRP-2_(180-188)多肽合用,在一定程度上增强了TRP-2_(180-188)多肽诱导的CTL反应,但是作用较弱,统计学差异不显著。
树突细胞(Dendritic cells,DCs)是重要的专职抗原提呈细胞,在启动机体特异性、非特异性免疫应答的过程中起着关键的作用。促使DCs成熟,增强其抗原提呈功能是佐剂作用的重要机制。本研究在体外应用人单核细胞来源的树突细胞(Monocyte derived dendritic cells,MoDCs)探讨了MDP-C对树突细胞成熟和功能的影响。经重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF,1000U/ml)和重组人白细胞介素-4(rhIL-4,500U/ml)诱导培养的方法得到了不成熟树突细胞(iDCs),iDCs在含有MDP-C的完全培养液中继续培养24h后,采用流式细胞仪检测MoDCs表型,ELISA法检测培养上清中细胞因子IL-6、IL-12(p40+70)浓度,以MoDCs为刺激细胞,异体同种T淋巴细胞为效应细胞观察MDP-C对MoDCs抗原提呈和淋巴细胞增殖的影响。结果显示,与溶剂对照组相比,10μM MDP-C能显著增加MoDCs细胞表面HLA-DR、CD80、CD86以及CD83的表达,并能够增强MoDCs刺激异体T淋巴细胞增殖的能力,但是MDP-C不能诱导细胞因子IL-6、IL-12(p40+70)的产生,而阳性对照药Romurtide 1μM剂量对上述表面分子和细胞因子均有明显的诱导作用。随后我们应用RAW264.7小鼠巨噬细胞系检测了MDP-C对TNF-α有无作用,结果显示,经1μM MDP刺激的RAW264.7巨噬细胞和小鼠腹腔巨噬细胞分泌TNF-α的能力明显增强,而MDP-C各剂量组对两种巨噬细胞均未表现出增强作用。
综合本部分研究,MDP-C在体内对Lewis肺癌小鼠肿瘤生长和转移均未显示明显的治疗作用,对多肽引起的CTL活化也未显示明显的增强。体外实验中MDP-C能够促进MoDCs表面分子HLA-DR、CD80、CD86以及CD83的表达,增强抗原提呈功能,但是不能诱导MoDCs产生细胞因子IL-6、IL-12(p40+70),也不能诱导巨噬细胞产生TNF-α。提示,MDP-C有一定的免疫增强活性,但是作用较弱,其作用机制需进一步探讨。
The thesis is consist of two sections:"Mechanism of FLZ controlling neurodegenerative disease by modulation of inflammation" and "The immunocompetence of a synthetic muramyl dipeptide analogue MDP-C"
Section 1.Mechanism of FLZ controlling neurodegenerative disease by modulation of inflammation
Compound FLZ is a synthetic novel derivative of squamosamide.Previous studies in our laboratory demonstrated that FLZ not only had strong anti-oxidation, anti-apoptosis and neuroprotective effect,but also improved experimental learning and memory deficit and dyskinesia in animal models.This thesis first explored the ability of FLZ modulating inflammation and focused on mechanism studies controlling neurodegenerative disease.
Part 1.FLZ improvement of learning and memory dysfunction that is caused by LPS-induced inflammation in hippocampus
Lipopolysaccharide(LPS) is the major component of the outer surface of gram-negative bacteria and be considered as a potent inflammatory stimulator. Experimental evidences indicated that intracerebroventricular(i.c.v.) injection of LPS to rodents induced learning and memory impairments as well as neurodegeneration in brain areas related to cognitive function.In the present study,we explored the effects of FLZ on the LPS i.c.v.injected mice models.In water maze test,we found that i.c.v.injection of 2.5μg LPS induced learning and memory impairment.FLZ(150 mg/kg,75mg/kg) significantly improved the learning and memory ability and inhibited the damage of hippocampus neurons as assessed by histopathology method.To further investigate the mechanism of FLZ in improving the learning and memory impairments induced by LPS, biochemistry and immunohistochemistry methods were used.The results revealed that FLZ decreased the levels of TNF-αand IL-1βand inhibited the activity of inducible nitric oxide synthase(iNOS) which were induced by LPS.The immunohistochemistry results showed that the increased activation of microglia and astrocyte and the over expression of iNOS,cyclooxygenase-2(Cox-2) andβ-Secretase(BACE1) by LPS were all significantly inhibited by FLZ.Aβproduction induced by LPS was decreased either. In summary,FLZ significantly inhibited the inflammatory reaction in hippocampus caused by i.c.v.injection of LPS and improved the learning and memory impairment and neuron damage.
2.Inhibition effect of FLZ on the inflammatory reaction in macrophages and the mechanism of action
The above results indicated that FLZ inhibited the inflammatory reaction in hippocampus caused by i.c.v.injection of LPS.To further study the mechanism of FLZ, the RAW264.7 macrophages were used to detect the effects of FLZ on the production of inflammatory mediators-induced by LPS.The results showed that FLZ dose-dependently inhibited the LPS induced production of nitric oxide(NO) as well as the expression of iNOS,cyclooxygenase 2(Cox-2) and tumor necrosis factor(TNF)-αat both mRNA and protein levels.Furthermore,the production of NO and secretion of TNF-αinduced by LPS or aggregated Aβ_(1-40) in BV-2 microglias were also suppressed by FLZ.The anti-inflammatory mechanism of FLZ was investigated subsequently.In RAW264.7 cells, the LPS-induced DNA binding activation of Nuclear factor kappa-B(NF-κB) and activator protein 1(AP-1),the nuclear translocation of NF-κB p65,the degradation of inhibitoryκBαprotein(IκBα) and the phosphorylation of IκBα,IκB kinase(IKK)α/β, c-Jun NH_2-terminal kinase(JNK) and p38 mitogen-activated protein kinases(MAPKs) were all suppressed by FLZ.But the phosphorylation of extracellular signal-regulated kinase(ERK) was not impacted.Further study revealed that FLZ dose dependently inhibited the phosphorylation of transforming growth factor-β(TGF-β)-activated kinase 1(TAK1) which is the upstream signal of IKKα/β,JNK and p38 activation.In addition, reactive oxygen species(ROS) induced by LPS or H_2O_2 were suppressed by FLZ either. The results suggested that FLZ inhibited the production of inflammatory mediators at least partly via downregulation of TAK1-IKK-NF-κB and TAK1-JNK/p38MAPK/AP-1 pathways.In addition,the suppression of ROS was also one of the important mechanisms for the anti-inflammatory activity of FLZ.
In summary,FLZ significantly inhibited the inflammatory reaction in hippocampus caused by i.c.v,injection of LPS and improved the learning and memory impairment and neuron damage.The production of inflammatory mediators induced by LPS was inhibited by FLZ at least partly via the downregulation of TAK1-IKK-NF-κB and TAK1-JNK/p38MAPK-AP-1 pathways and the inhibition of ROS.The protection against neurodegenerative diaseases of FLZ may be partially accounts for its anti-inflammation activity.
Section 2.The immunocompetence of a synthetic muramyl dipeptide analogue MDP-C
N-acetylmuramyl-L-alany-D-isoglutamin(muramyl dipeptide,MDP),which was identified as the minimal active component of mycobacterium cell wall,possesses extensive biological activities on host immune systems and improves the nonspecific resistance against infections and tumors.However,the characters of poor penetration of membranes and rapid elimination in vivo,as well as the induction of toxic responses, such as pyrogenicity,lethargy,anoresy and hypermetabolism limit the application of MDP.Therefore,structure modification and synthesis of MDP derivatives possess good activitie is extremely needed.MDP-C(N2-[R-Obenzyl-N-(acetylmuramyl) -L-alanyl -D-isoglutaminyl]-N6-trans-(m-nitrocinnamoyl)-L-lysine),a novel chemically modified MDP,has been reported to be an potential immunostimulator.In this study,we investigated the immunocompetence of MDP-C.
First of all,we detected the effects of MDP-C on the growth and metastasis of tumors in a Lewis lung cancer mouse model.The results showed that MDP-C has no inhibitory activity on both the primarily tumor and pulmonary metastasis.Under tested condition,at the dose of 1.25 mg/kg and 2.5mg/kg,MDP-C combined with low dose isofosfamide(15 mg/kg) decreased the numbers of pulmonary metastasis nodes.But the data have no statistical significance compared with control group.In the experiment of ELISPOT,MDP-C slightly augmented the TRP-2_(180-188) peptide-specific cytotoxic T lymphocytes(CTLs) reaction in spleen cells of C57BL/6J mice immunized with the TRP-2_(180-188) peptide and MDP-C,but have no statistical significance compared with TRP-2_(180-188) peptide group.
Dendritic cells(DCs) are highly specialized antigen-presenting cells(APC) that play a central role in the initiation of both innate and adaptive immune responses.The effects of MDP-C on the differentiation and activation of human monocyte derived dendritic cells(MoDCs) were investigated.The combined action of GM-CSF and IL-4 could direct monocytes to differentiate into DCs displaying features of immature DC(iDCs).After cultured with various concentration of MDP-C for 24h,the surface molecules human leucocyte antigen-DR(HLA-DR),CD80,CD86 and CD83 were detected by flow cytometry.The allogeneic mixed lymphocyte reaction and the levels of IL-6 and IL-12(p40+70) were investigated with ATP bioluminescent assay kit and ELISA kit, respectively.The results showed that MDP-C up-regulated the expression of HLA-DR, CD80,CD86 and CD83,as well as the allogeneic mixed lymphocyte reaction.But the secretion of IL-6 and IL-12(p40+70) were not affected.While the positive control compound romurtide induced the expression of surface molecules and cytokines strongly. Further study revealed that MDP-C has no effect on the secretion of TNF-αin both RAW264.7 and mouse peritoneal macrophages.
In summary,MDP-C has no obvious inhibitory effect on the growth and metastasize of tumors in the Lewis lung cancer mouse model and has no obvious adjuvant activity on the TRP-2_(180-188) peptide induced cytotoxic T lymphocytes(CTLs) activition under tested condition.MDP-C increased the expression of surface molecules and enhanced the antigen presenting activity of MoDCs,but has no effect on the cytokine expression in both MoDCs and macrophages.The results indicated that MDP-C possesses immunocompetence to some extent.The mechanism of action needs further investigation.
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