Protective Effects of Cirsium japonicum and Pectolinarin on Cognitive Impairment by Regulation of Neuronal Oxidative Stress = 엉겅퀴와 pectolinarin의 neuronal oxidative stress 조절을 통한 인지능 개선 효과

다국어 초록 (Multilingual Abstract)

In the development of neurodegenerative diseases, the excessive accumulation of amyloid beta (Aβ) causes neuronal oxidative stress, leading to cognitive impairment. Cirsium japonicum var. maackii (CJM) is a widely used medicinal material, and pectolinarin is an active compound of CJM. This study investigated the protective effects of ethyl acetate (EtOAc) fraction of CJM (ECJM) and pectolinarin on cognitive impairment by regulation of neuronal oxidative stress. In the in vivo study, ECJM showed higher spatial memory and object recognition compared with Aβ25-35-induced control group. Besides, ECJM-administered mice showed higher learning and memory abilities than Aβ25-35-induced control mice. ECJM attenuated the oxidative stress by reduced the reactive oxygen species (ROS) production, nitrite oxide (NO) production, and lipid peroxidation in the brain. In addition, administration of ECJM attenuated neuronal oxidative stress by regulation of oxidative stress-related protein heme oxygenase-1 (HO-1) in the brain. The ECJM-administrated mice showed down-regulations of inflammation-related protein nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 1 beta (IL-1β) , apoptosis-related protein Bcl-2 associated X protein (Bax)/B-cell lymphoma 2 (Bcl-2), and amyloidogenic pathway related protein beta-secretase 1 (BACE), presenilin1 (PS1), and presenilin2 (PS2) in Aβ25-35–induced mice brain. In addition, pectolinarin concentration-dependently increased ·OH and NO radical scavenging ability. The treatment of pectolinarin showed significantly increased cell viability, decreased the ROS production and lactate dehydrogenase (LDH) release level compared with H2O2-induced SH-SY5Y neuronal cells. Furthermore, to evaluate the neuroprotective mechanisms of pectolinarin, we measured the protein expression. Treatment of pectolinarin showed up-regulation of oxidative stress-related proteins such as nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1, while down-regulation of kelch-like ECH-associated protein 1 (Keap1) in H2O2-induced SH-SY5Y cells. The cells treated with pectolinarin down-regulated inflammation-related proteins such as iNOS, COX-2, IL-1β and apoptosis-related protein, Bax/Bcl-2 ratio in H2O2-induced SH-SY5Y cells. The present results demonstrated that ECJM had a protective effect on cognitive impairment in Aβ25-35–induced mice via attenuation of neuronal oxidative stress and pectolinarin had neuroprotective effects against H2O2-induced oxidative stress in SH-SY5Y cells. Therefore, we suggeste that CJM and pectolinarin may used as therapeutic agents in Alzheimer’s disease treatment.

목차 (Table of Contents)

• PART 1. Cognitive Improvement Effects of Cirsium japonicum under Aβ25-35-induced Alzheimer's Disease Mouse 1
• Abstract 1
• 1. Introduction 2
• 2. Materials and methods 3
• 2.1. Reagents 3
• 2.2. Sample preparation 3
• 2.3. Animals and experimental designs 3
• 2.4. Aβ25-35 injection 4
• 2.5. T-maze test 6
• 2.6. Novel object recognition test 6
• 2.7. Morris water maze test 7
• 2.8. Statistical analysis 7
• 3. Results 8
• 3.1. Effect of ethyl acetate fraction from Cirsium japonicum on body weight change 8
• 3.2. Effect of ethyl acetate fraction from Cirsium japonicum on space perceptive ability 8
• 3.3. Effect of ethyl acetate fraction from Cirsium japonicum on object recognition ability 8
• 3.4. Effect of ethyl acetate fraction from Cirsium japonicum on learning and memory ability 12
• 4. Discussion 16
• 5. References 20
• PART 2. Protective Effects and Mechanisms of Cirsium japonicum on Cognitive Impairment Induced by Aβ25-35 in Mice 26
• Abstract 26
• 1. Introduction 27
• 2. Materials and methods 28
• 2.1. Materials 28
• 2.2. Sample preparation 29
• 2.3. Animals and experimental protocols 29
• 2.4. Aβ25-35 injection 29
• 2.5. Measurement of reactive oxygen species (ROS) production 30
• 2.6. Measurement of lipid peroxidation 30
• 2.7. Measurement of nitric oxide (NO) scavenging activity 30
• 2.8. Western blot analysis 31
• 2.9. Biochemical analysis 32
• 2.10. Statistical analysis 32
• 3. Results 32
• 3.1. Effects of ethyl acetate fraction from Cirsium japonicum on ROS production induced by Aβ25-35 32
• 3.2. Effect of ethyl acetate fraction from Cirsium japonicum on lipid peroxidation induced by Aβ25-35 34
• 3.3. Effect of ethyl acetate fraction from Cirsium japonicum on NO production induced by Aβ25-35 36
• 3.4. Protective effects of ethyl acetate fraction from Cirsium japonicum on oxidative stress-related protein expressions 36
• 3.5. Protective effects of ethyl acetate fraction from Cirsium japonicum on inflammation-related protein expressions 39
• 3.6. Protective effects of ethyl acetate fraction from Cirsium japonicum on apoptosis-related protein expressions 39
• 3.7. Protective effects of ethyl acetate fraction from Cirsium japonicum on amyloidogenic pathway-related protein expressions 42
• 3.8. Effects of ethyl acetate fraction from Cirsium japonicum on serum ALT and AST 42
• 4. Discussion 45
• 5. References 51
• PART 3. Protective Effects and Mechanisms of Pectolinarin agianst H2O2-induced Oxidative Stress in SH-SY5Y Neuronal Cells 60
• Abstract 61
• 1. Introduction 63
• 2. Materials and methods 62
• 2.1. Reagents 62
• 2.2. Hydroxyl radical (OH) radical scavenging assay 63
• 2.3. Nitric oxide (NO) radical scavenging assay 64
• 2.4. Cell culture 64
• 2.5. Cell viability 64
• 2.6. Measurement of ROS production 65
• 2.7. Measurement of LDH release activity 65
• 2.8. Western blot analysis 65
• 2.9. Statistical analysis 66
• 3. Results 67
• 3.1. Hydroxyl radical (OH) radical scavenging activity 67
• 3.2. Nitric oxide (NO) radical scavenging activity 67
• 3.3. Effects of pectolinarin on cell viability in H2O2-induced SH-SY5Y cells . 67
• 3.4. Effects of pectolinarin on ROS formation in H2O2-induced SH-SY5Y cells 71
• 3.5. Effects of pectolinarin on LDH release in H2O2-induced SH-SY5Y cells 71
• 3.6. Effects of pectolinarin on oxidative stress-related protein expression in H2O2-induced SH-SY5Y cells 71
• 3.7. Effects of pectolinarin on inflammation-related protein expression in H2O2-induced SH-SY5Y cells 74
• 3.8. Effects of pectolinarin on apoptosis-related protein expression in H2O2-induced SH-SY5Y cells 74
• 4. Discussion 77
• 5. References 82
• Summary and conclusion 89
• Abstract (in korean) 91