FK506与米诺环素联合用药对朊病毒感染金黄地鼠治疗作用研究
发布时间：2020-11-01 02:59
研究背景:朊蛋白疾病,也称为传染性海绵状脑病(transmissible spongiform encephalopathies,TSEs),是一类全球性的人畜共患的致死性神经退行性疾病。近期研究表明错误折叠的朊蛋白的蓄积会导致内质网应激,最终使内质网和线粒体激活以缓解细胞损伤。在内质网应激背后,最主要的是钙离子的失调和其下游对线粒体的影响。。朊病毒感染中枢神经系统(CNS)以最初的反应性胶质细胞增生和随后显著的神经元凋亡为特点。胶质细胞增生可能是由于早期脑内细胞性朊蛋白(PrPc)的具有蛋白酶K抗性的错误折叠亚型(PrPsc)的形成引起的。激活的胶质细胞和应激的神经元释放出前炎症细胞因子和趋化因子可能通过增强胶质细胞增生和引发神经元毒性,而直接或间接的推进病程的发展。最近的研究表明,早期的神经炎症激活活化T细胞核因子(NFAT)信号级联,导致NF-kB核转位,引起细胞凋亡。因此,延缓朊蛋白病病程的有效治疗方法应该通过控制早期炎症来抑制NFAT信号通路。在本文中,我们使用朊蛋白病金黄地鼠模型进行实验,对比了使用米诺环素这种新型半合成四环素与能够抑制NFAT通路的磷酸酶抑制剂FK506,对动物模型神经元的保护和NFAT通路抑制作用的差异。第一部分结果:在实验的第一部分中,我们在金黄地鼠朊蛋白病模型的不同时期,分别使用FK506和米诺环素。该研究的宗旨在于观察上述两种药物对朊蛋白疾病早期的神经炎症和突触失调,以及晚期神经元退变的影响。结果表明,从疾病的亚临床期开始,延长米诺环素的使用时间,相比于在亚临床期或临床期使用FK506更加有效。结果还显示FK506对疾病早期的治疗有导致体重下降和细胞周期阻滞的副作用。而在其应用于疾病晚期治疗时,并没有观察到这些副作用。使用米诺环素,能够减少星形胶质细胞活化标志物GFAP和小胶质细胞活化标志物IBA-1的合成,继而降低前炎症因子白介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)的水平。此外,我们还发现使用米诺环素和FK506能够抑制丝裂原活化蛋白激酶(MAPK)p38磷酸化和胱天蛋白酶(caspase)依赖性的NF-kB的核转位,并提高磷酸化环腺苷酸反应序列结合蛋白(pCREB)和磷酸化Bcl2相关死亡启动子(pBAD)水平,减轻记忆损伤和细胞凋亡。第二部分结果:在实验的第二部分中,基于我们对于在朊蛋白疾病不同时期使用FK506和米诺环素的实验结果,我们在金黄地鼠朊蛋白病模型的临床期混合使用了 FK506和米诺环素。实验结果表明,在该疾病模型中联合使用FK506和米诺环素能够有效地减轻疾病引起的胶质细胞增生和细胞毒性。联用FK506和米诺环素后,对空泡的形成并没有表现出显著的影响,但是我们观察到相比于阳性对照组,联用FK506和米诺环素的实验组金黄地鼠齿状回神经元的完整性得到改善。此外,联合使用FK506和米诺环素能够降低前炎症因子IL-1β和TNF-α水平,提升抗炎症因子IL-10和IL-27水平。联用FK506和米诺环素能够通过caspase依赖性途径调节MAPK-p38通路。联用FK506和米诺环素通过减少胞浆内细胞色素C的转位和抑制胞质中促凋亡bcl2家族蛋白4(BAX)向线粒体转位,显著缓解了受感染金黄地鼠的线粒体损伤。组合药物的使用能够通过稳定线粒体膜的完整性,减轻疾病模型中由阮病毒引起的线粒体碎裂;通过MAPK-NRF2-HO-1通路,维持pBAD和pCREB水平,能够提高动物的存活率和认知能力。综上所述,我们的实验结果表明,FK506和米诺环素的联合用药是治疗朊蛋白病长期用药的更佳选择,有望作为一种可能的临床治疗方案,得到进一步的发展。结论:第一部分实验表明在朊病毒感染的临床期使用FK506有效,而在亚临床期使用米诺环素更有效。此外,FK506在提高细胞存活率和生长中是必须的,如果在朊病毒感染的亚临床期使其受到抑制会导致细胞周期停滞和生长受阻。根据该结果,我们进行了第二部分FK506和米诺环素联合用药的实验,并发现临床期的联合用药对金黄地鼠的朊蛋白病是有益的。本实验证明联用FK506和米诺环素通过MAPK-NF-kB-NRF2-HO-1通路减轻了由朊病毒引起的神经元和突触失调,并通过pBAD和pCREB通路增强了模型鼠的记忆和存活率。
【学位单位】：中国农业大学
【学位级别】：博士
【学位年份】：2018
【中图分类】：S859.7
【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
CHAPTER NUMBER: 1 INTRODUCTION
1.1 MISFOLDED PRION PROTEINS AND ENDOPLASMIC RETICULUM STRESS
1.1.1 Endoplasmic Reticulum and UPR
1.1.2 Stress-Related Chaperones and UPR
1.1.3 Unfolded Protein Response （UPR） in Neurodegenerative Diseases
1.2 ENDOPLASMIC RETICULUM RESIDENT CALCIUM RECEPTORS
1.3 CALCINEURIN ACTIVATION IN PROTEIN MISFOLDING DISORDERS
1.3.1 Calcium dysregulation and calcineurin-mediated signaling in neurodegenerative diseases
1.3.2 Endoplasmic Reticulum Stress and Mitochondria Crosstalk in Neurodegenerative Diseases
1.4 BASIC STRUCTURE OF CALCINEURIN
1.5 THE ROLE OF CALCINEURIN IN NEURODEGENERATION
1.5.1 Mantaining Neuronal Integrity and Homeostasis
1.5.2 Mantaining Synaptic Integrity
1.5.3 Calcineurin and Protein Misfolding/Unfolding Neurodegenerative Diseases
1.5.4 Inhibition of CaN by Immunophilins and Immunosuppressants
1.5.5 Calcineurin Inhibition and NFAT Suppression as Therapy in Prion Diseases
1.6 MINOCYCLINE AND NFAT SUPPRESSION AS THERAPY IN PRION DISEASES
1.7 AIMS AND OBJECTIVES
CHAPTER NUMBER: 2 MATERIALS AND METHODS
2.1 ETHICAL STATEMENT
2.2 ANIMAL MODEL OF PRIONS
2.3 FK506 PREPARATION AND ADMINISTRATION
2.4 MINOCYCLINE PREPARATION AND ADMINISTRATION
2.5 FK506 AND MINOCYCLINE COCKTAIL PREPARATION AND ADMINISTRATION
2.6 REAGENTS
2.7 ENZYME-LINKED IMMUNOSORBENT ASSAY （ELISA）
2.8 CAN ACTIVITY ASSAY
2.9 ANIMAL BEHAVIORAL STUDIES
SC DETECTION ASSAY'> 2.10 PRPSC DETECTION ASSAY
2.11 WESTERN BLOTTING
2.12 TISSUE PREPARATION AND IMMUNOHISTOCHEMISTRY ANALYSIS
2.13 FLUORO-JADE C STAINING PROCEDURE
2.14 THIOFLAVIN-S STAINING PROTOCOL
2.15 PREPARATION OF THE CYTOPLASMIC AND MITOCHONDRIAL EXTRACTS
2.16 PREPARATION OF THE CYTOPLASMIC AND NUCLEAR EXTRACTS
2.17 TRANSMISSION ELECTRON MICROSCOPY
2.18 STATISTICAL ANALYSIS
CHAPTER NUMBER: 3 RESULTS AND DISCUSSION PART: 1 FK506 AND MINOCYCLINE TREATMENT ALONE IN DIFFERENT STAGES OF PRIONINFECTION
3.1 BEHAVIORAL ANALYSIS IN PRION INFECTED HAMSTERS
3.1.1 Introduction
3.1.2 Aims and Objectives
3.1.3 Early minocycline treatment enhanced nesting behavior, locomotor function, novel object findingand prolonged survival in prion infected hamsters
3.1.4 Discussion
3.1.5 Conclusions
Sc'> 3.2 RELATIONSHIP OF CALCINEURIN ACTIVITY AND PRPSc
3.2.1 Introduction
3.2.2 Aims and Objectives
3.2.3 FK506 and minocycline partially reduced the calcineurin activity in prion infected hamstersindependent of the misfolded prion accumulation
3.2.4 Discussion
3.2.5 Conclusions
3.3 ASTROGLYOSIS IN PRION INFECTED HAMSTERS
3.3.1 Introduction
3.3.2 Aims and Objectives
3.3.3 Minocycline efficiently reduced astroglyosis in prion infected hamsters
3.3.4 Discussion
3.3.5 Conclusions
3.4 SPONGIFORM DEGENERATION IN PRION INFECTED HAMSTERS
3.4.1 Introduction
3.4.2 Aims and objectives
3.4.3 Minocycline rescues prion infected hamsters from spongiform degeneration
3.4.4 Discussion
3.4.5 Conclusions
3.5 SYNAPTIC DYSFUNCTION AND NEURODEGENERATION IN PRION INFECTED HAMSTERS
3.5.1 Introduction
3.5.2 Aims and objectives
3.5.3 Minocycline rescues prion infected hamsters from synaptic dysfunction and neurodegeneration
3.5.4 Discussion
3.5.5 Conclusions
3.6 POST ASTROGLIAL INFLAMMATORY EVENTS IN PRION INFECTED HAMSTERS
3.6.1 Introduction
3.6.2 Aims and objectives
3.6.3 Minocycline modulates caspase dependent MAPKpathway
3.6.4 Discussion
3.6.5 Conclusions
3.7 NF-κB NUCLEAR TRANSLOCATION IN PRION INFECTED HAMSTERS
3.7.1 Introduction
3.7.2 Aims and objectives
3.7.3 Minocycline effectively leads to reduced NFkB p65 nuclear translocation
3.7.4 Discussion
3.7.5 Conclusions
3.8 SURVIVAL AND MEMORY ENHANCING PATHWAYS IN PRION INFECTED HAMSTERS
3.8.1 Introduction
3.8.2 Aims and objectives
3.8.3 Minocycline increases cognition and survival via CREB and BAD phosphorylation
3.8.4 Discussion
3.8.5 Conclusions
GENERAL DISCUSSION PART: 1
GENERAL CONCLUSION PART: 1
CHAPTER NUMBER: 4 RESULTS AND DISCUSSION PART: 2 FK506 AND MINOCYCLINE COMBINATORY TREATMENT AT CLINICAL STAGE OF PRIONINFECTION
4.1 COMBINATORY FK506+MINOCYCLINE TREATMENT ENHANCES SURVIVAL
4.1.1 Introduction
4.1.2 Aims and objectives
4.1.3 Combinatory FK506+minocycline therapy prolonged survival ofprion infected hamsters
4.1.4 Discussion
4.1.5 Conclusions
4.2 ENHANCED BEHAVIORAL FUNCTIONS AFTER FK506+MINOCYCLINE TREATMENT
4.2.1 Introduction
4.2.2 Aims and objectives
4.2.3 FK506+minocycline treatment enhanced nesting behavior, locomotor function and novel objectfinding in prion infected hamsters
4.2.4 Discussion
4.2.5 Conclusion
4.3 CALCINEURIN ACTIVITY AND FK506+MINOCYCLINE THERAPY
4.3.1 Introduction
4.3.2 Aims and objectives
4.3.3 FK506+minocycline treatment partially reduced calcineurin activity in prion infected hamsters 984.3.4 Discussion
4.3.4 Discussion
4.3.5 Conclusion
4.4 EFFECT OF FK506+MINOCYCLINE ON BRAIN RESIDENT CELLS
4.4.1 Introduction
4.4.2 Aims and objectives
4.4.3 FK506+minocycline treatment efficiently reduced astroglyosis in prion infected hamsters
4.4.4 Discussion
4.4.5 Conclusion
4.5 NEURONAL TOXICITY AND FK506+MINOCYCLINE THERAPY
4.5.1 Introduction
4.5.2 Aims and objectives
4.5.3 FK506+ minocycline treatment rescues prion infected hamsters from synaptic dusfunction andneurodegeneration
4.5.4 Discussion
4.5.5 Conclusion
4.6 INFLAMMATORY PATHWAYS AND FK506+MINOCYCLINE THERAPY
4.6.1 Introduction
4.6.2 Aims and objectives
4.6.3 FK506+minocycline modulates caspase-dependent MAPKpathway in prion infected hamsters
4.6.4 Discussion
4.6.5 Conclusion
4.7 MITOCHONDRIAL DYSFUNCTION AND FK506+MINOCYCLINE THERAPY
4.7.1 Introduction
4.7.2 Aims and objectives
4.7.3 FK506+minocycline treatment reduced mitochondrial dysfunction in prion infected hamsters
4.7.4 Discussion
4.7.5 Conclusion
4.8 EFFECT OF FK506+MINOCYCLINE ON NF-κB AND NRF2 PATHWAYS
4.8.1 Introduction
4.8.2 Aims and objectives
4.8.3 FK506+minocycline treatment effectively leads to reduced NF-kB p65 and increased NRF2nuclear translocation
4.8.4 Discussion
4.8.5 Conclusion
4.9 FK506+MINOCYCLINE IN COGNITION AND SURVIVAL
4.9.1 Introduction
4.9.2 Aims and objectives
4.9.3 FK506+minocycline treatment increases cognition and survival via CREB and BADphosphorylation
4.9.4 Discussion
4.9.5 Conclusion
GENERAL DISCUSSION PART: 2
GENERAL CONCLUSION PART: 2
INNOVATIONS
RECOMMENDATIONS AND FUTURE DIRECTIONS
REFERENCES
ACKNOWLEDGEMENTS
AUTHOR CV
本文编号：2864933
【学位单位】：中国农业大学
【学位级别】：博士
【学位年份】：2018
【中图分类】：S859.7
【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
CHAPTER NUMBER: 1 INTRODUCTION
1.1 MISFOLDED PRION PROTEINS AND ENDOPLASMIC RETICULUM STRESS
1.1.1 Endoplasmic Reticulum and UPR
1.1.2 Stress-Related Chaperones and UPR
1.1.3 Unfolded Protein Response （UPR） in Neurodegenerative Diseases
1.2 ENDOPLASMIC RETICULUM RESIDENT CALCIUM RECEPTORS
1.3 CALCINEURIN ACTIVATION IN PROTEIN MISFOLDING DISORDERS
1.3.1 Calcium dysregulation and calcineurin-mediated signaling in neurodegenerative diseases
1.3.2 Endoplasmic Reticulum Stress and Mitochondria Crosstalk in Neurodegenerative Diseases
1.4 BASIC STRUCTURE OF CALCINEURIN
1.5 THE ROLE OF CALCINEURIN IN NEURODEGENERATION
1.5.1 Mantaining Neuronal Integrity and Homeostasis
1.5.2 Mantaining Synaptic Integrity
1.5.3 Calcineurin and Protein Misfolding/Unfolding Neurodegenerative Diseases
1.5.4 Inhibition of CaN by Immunophilins and Immunosuppressants
1.5.5 Calcineurin Inhibition and NFAT Suppression as Therapy in Prion Diseases
1.6 MINOCYCLINE AND NFAT SUPPRESSION AS THERAPY IN PRION DISEASES
1.7 AIMS AND OBJECTIVES
CHAPTER NUMBER: 2 MATERIALS AND METHODS
2.1 ETHICAL STATEMENT
2.2 ANIMAL MODEL OF PRIONS
2.3 FK506 PREPARATION AND ADMINISTRATION
2.4 MINOCYCLINE PREPARATION AND ADMINISTRATION
2.5 FK506 AND MINOCYCLINE COCKTAIL PREPARATION AND ADMINISTRATION
2.6 REAGENTS
2.7 ENZYME-LINKED IMMUNOSORBENT ASSAY （ELISA）
2.8 CAN ACTIVITY ASSAY
2.9 ANIMAL BEHAVIORAL STUDIES
SC DETECTION ASSAY'> 2.10 PRPSC DETECTION ASSAY
2.11 WESTERN BLOTTING
2.12 TISSUE PREPARATION AND IMMUNOHISTOCHEMISTRY ANALYSIS
2.13 FLUORO-JADE C STAINING PROCEDURE
2.14 THIOFLAVIN-S STAINING PROTOCOL
2.15 PREPARATION OF THE CYTOPLASMIC AND MITOCHONDRIAL EXTRACTS
2.16 PREPARATION OF THE CYTOPLASMIC AND NUCLEAR EXTRACTS
2.17 TRANSMISSION ELECTRON MICROSCOPY
2.18 STATISTICAL ANALYSIS
CHAPTER NUMBER: 3 RESULTS AND DISCUSSION PART: 1 FK506 AND MINOCYCLINE TREATMENT ALONE IN DIFFERENT STAGES OF PRIONINFECTION
3.1 BEHAVIORAL ANALYSIS IN PRION INFECTED HAMSTERS
3.1.1 Introduction
3.1.2 Aims and Objectives
3.1.3 Early minocycline treatment enhanced nesting behavior, locomotor function, novel object findingand prolonged survival in prion infected hamsters
3.1.4 Discussion
3.1.5 Conclusions
Sc'> 3.2 RELATIONSHIP OF CALCINEURIN ACTIVITY AND PRPSc
3.2.2 Aims and Objectives
3.2.3 FK506 and minocycline partially reduced the calcineurin activity in prion infected hamstersindependent of the misfolded prion accumulation
3.2.4 Discussion
3.2.5 Conclusions
3.3 ASTROGLYOSIS IN PRION INFECTED HAMSTERS
3.3.1 Introduction
3.3.2 Aims and Objectives
3.3.3 Minocycline efficiently reduced astroglyosis in prion infected hamsters
3.3.4 Discussion
3.3.5 Conclusions
3.4 SPONGIFORM DEGENERATION IN PRION INFECTED HAMSTERS
3.4.1 Introduction
3.4.2 Aims and objectives
3.4.3 Minocycline rescues prion infected hamsters from spongiform degeneration
3.4.4 Discussion
3.4.5 Conclusions
3.5 SYNAPTIC DYSFUNCTION AND NEURODEGENERATION IN PRION INFECTED HAMSTERS
3.5.1 Introduction
3.5.2 Aims and objectives
3.5.3 Minocycline rescues prion infected hamsters from synaptic dysfunction and neurodegeneration
3.5.4 Discussion
3.5.5 Conclusions
3.6 POST ASTROGLIAL INFLAMMATORY EVENTS IN PRION INFECTED HAMSTERS
3.6.1 Introduction
3.6.2 Aims and objectives
3.6.3 Minocycline modulates caspase dependent MAPKpathway
3.6.4 Discussion
3.6.5 Conclusions
3.7 NF-κB NUCLEAR TRANSLOCATION IN PRION INFECTED HAMSTERS
3.7.1 Introduction
3.7.2 Aims and objectives
3.7.3 Minocycline effectively leads to reduced NFkB p65 nuclear translocation
3.7.4 Discussion
3.7.5 Conclusions
3.8 SURVIVAL AND MEMORY ENHANCING PATHWAYS IN PRION INFECTED HAMSTERS
3.8.1 Introduction
3.8.2 Aims and objectives
3.8.3 Minocycline increases cognition and survival via CREB and BAD phosphorylation
3.8.4 Discussion
3.8.5 Conclusions
GENERAL DISCUSSION PART: 1
GENERAL CONCLUSION PART: 1
CHAPTER NUMBER: 4 RESULTS AND DISCUSSION PART: 2 FK506 AND MINOCYCLINE COMBINATORY TREATMENT AT CLINICAL STAGE OF PRIONINFECTION
4.1 COMBINATORY FK506+MINOCYCLINE TREATMENT ENHANCES SURVIVAL
4.1.1 Introduction
4.1.2 Aims and objectives
4.1.3 Combinatory FK506+minocycline therapy prolonged survival ofprion infected hamsters
4.1.4 Discussion
4.1.5 Conclusions
4.2 ENHANCED BEHAVIORAL FUNCTIONS AFTER FK506+MINOCYCLINE TREATMENT
4.2.1 Introduction
4.2.2 Aims and objectives
4.2.3 FK506+minocycline treatment enhanced nesting behavior, locomotor function and novel objectfinding in prion infected hamsters
4.2.4 Discussion
4.2.5 Conclusion
4.3 CALCINEURIN ACTIVITY AND FK506+MINOCYCLINE THERAPY
4.3.1 Introduction
4.3.2 Aims and objectives
4.3.3 FK506+minocycline treatment partially reduced calcineurin activity in prion infected hamsters 984.3.4 Discussion
4.3.4 Discussion
4.3.5 Conclusion
4.4 EFFECT OF FK506+MINOCYCLINE ON BRAIN RESIDENT CELLS
4.4.1 Introduction
4.4.2 Aims and objectives
4.4.3 FK506+minocycline treatment efficiently reduced astroglyosis in prion infected hamsters
4.4.4 Discussion
4.4.5 Conclusion
4.5 NEURONAL TOXICITY AND FK506+MINOCYCLINE THERAPY
4.5.1 Introduction
4.5.2 Aims and objectives
4.5.3 FK506+ minocycline treatment rescues prion infected hamsters from synaptic dusfunction andneurodegeneration
4.5.4 Discussion
4.5.5 Conclusion
4.6 INFLAMMATORY PATHWAYS AND FK506+MINOCYCLINE THERAPY
4.6.1 Introduction
4.6.2 Aims and objectives
4.6.3 FK506+minocycline modulates caspase-dependent MAPKpathway in prion infected hamsters
4.6.4 Discussion
4.6.5 Conclusion
4.7 MITOCHONDRIAL DYSFUNCTION AND FK506+MINOCYCLINE THERAPY
4.7.1 Introduction
4.7.2 Aims and objectives
4.7.3 FK506+minocycline treatment reduced mitochondrial dysfunction in prion infected hamsters
4.7.4 Discussion
4.7.5 Conclusion
4.8 EFFECT OF FK506+MINOCYCLINE ON NF-κB AND NRF2 PATHWAYS
4.8.1 Introduction
4.8.2 Aims and objectives
4.8.3 FK506+minocycline treatment effectively leads to reduced NF-kB p65 and increased NRF2nuclear translocation
4.8.4 Discussion
4.8.5 Conclusion
4.9 FK506+MINOCYCLINE IN COGNITION AND SURVIVAL
4.9.1 Introduction
4.9.2 Aims and objectives
4.9.3 FK506+minocycline treatment increases cognition and survival via CREB and BADphosphorylation
4.9.4 Discussion
4.9.5 Conclusion
GENERAL DISCUSSION PART: 2
GENERAL CONCLUSION PART: 2
INNOVATIONS
RECOMMENDATIONS AND FUTURE DIRECTIONS
REFERENCES
ACKNOWLEDGEMENTS
AUTHOR CV
本文编号：2864933
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