Neuroprotective effects of peptides derived from Seahorse Hippocampus trimaculatus mediated through PI3K/Akt pathway
- Abstract
- Neurodegenerative diseases are estimated to surpass cancer as the second most common cause of death among elderly by the 2040s. For this reason, a great deal of attention has been expressed by the scientists regarding safe and effective neuroprotective agents. Even though many categories of natural and synthetic neuroprotective agents have been reported, the use is limited due to wide array of side effects. Hence, researchers have focused on studying natural bioactive substances that can act in neuroprotection.
Seahorses are one of the most important components of Chinese traditional medicine. Among seahorse species, Hippocampus trimaculatus are highly valued and the most heavily traded species for traditional medicine purposes in many Asian countries. One of the main interesting features in this species is their richness in protein content (75.75 ？ 0.13%) which can be hydrolized into bioactive peptides. By using central composite design (CCD) based response surface methodology (RSM), the present study investigated the effects of the four parameters (temperature, hydrolysis time, enzyme to substrate ratio and pH) on H. trimaculatus hydrolysis using Pronase E and analyzed their optimal hydrolysis conditions to attain bioactive peptides which able to protect cholinergic neuron (PC12) cells from A？42-induced cell death. For the selection of active fractions during the purifications steps, MTT, Bcl-2 gene expression and DNA oxidation assays were performed as marker of the cell survival. Following several purification steps, the peptides responsible for neuroprotective activity were purified and identified as HTP-1 (GTEDELDK; 906.4 Da; +25.39 Kcal/mol hydrophobicity) and HTP-2 (FLHDVQ; 997.4 Da; +11.22 Kcal/mol hydrophobicity).
In vitro co-culture system of PC12 cells and A？42-stimulated murine microglia (BV2) cells were used in this study. BV2 cells are the resident immune cells in the brain which play an important role in neuronal cells. These coculture systems of BV2 and PC12 cells closely mimic the conditions of Alzheimer？s diseases. The result of coculture system showed that HTP-1 and HTP-2 protect PC12 cells from neurotoxic responses-induced cell death. Notably, HTP-1 was found to be more effective in protecting PC12 cells from A？42-induced microglia-mediated neurotoxic responses. In addition, the activation of PI3K/Akt signaling pathway by HTP-1 was confirmed. The PI3K/Akt activation was found to be mediated through TGF-？ induction by HTP-1 in both BV2 and PC12 cells. Through this signaling pathway, the expression of pro-survival protein was up-regulated. Not only restricted to that activity, HTP-1 also attenuated the release of neurotoxic mediators by BV2 cells which inversely correlate with their cytoprotective effects in PC12 cells.
Amyotrophic Lateral Sclerosis (ALS), a disabling and fatal neurodegenerative disease, is characterized by the progressive loss of muscle power as a result of the selective loss of motor neurons. Approximately 2？3% of observed ALS cases are related to a mutation in the antioxidant enzyme. Therefore, anti-neurooxidative stress may be the most realistic approach of neuroprotection for ALS. The result in this study, demonstrated that HTP-1 is able to protect BV2 and PC12 cells from neurooxidative stress and also increase the expression of antioxidant enzyme such as SOD, GSH and catalase. Collectively, HTP-1 has a great potential to be used in pharmaceuticals and nutraceuticals as neuroprotective agents as they protect neuronal cells from death and neurooxidative stress.
- Issued Date
- 2012
- Awarded Date
- 2012. 8
- Type
- Dissertation
- Publisher
- Pukyong National University
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 화학과
- Advisor
- Prof. Se-Kwon Kim
- Table Of Contents
- Chapter 1. Research Background 1
1. Marine organisms 2
2. Central nervous systems 3
3. Neurodegenerative diseases 7
4. Neuroprotection 9
4.1. Antioxidant (free radical trapper or scavenger) 10
4.2. Anti-neuroinflammatory 11
4.3. Inhibition of apoptosis 12
4.4. Antineurotoxicity 12
5. Research Objectives 13
Chapter 2. Optimization and isolation of bioactive peptides derived from Hippocampus trimaculatus 15
1. Introduction 16
2. Material and methods 21
2.1. Seahorses sample 21
2.2 Reagents 21
2.3. NGF-Induced Differentiation of PC12 Cells 22
2.4. Preparation of A?42 oligomers 22
2.5. Enzyme selection 22
2.6. MTT assay 23
2.7.Experimental design and hydrolysis optimization 23
2.8. Preparation of H. trimaculatus enzymatic hydrolysate 25
2.9. Purification of active peptide from fermented H. trimaculatus 25
2.9.1.Ion exchange chromatograph 25
2.9.2.High-performance liquid chromatography (HPLC) 26
2.9.3. Determination of amino acid sequen 26
2.10.Quantification of degree of hydrolysis 27
2.11.Proximate analysis 27
2.12.Amino acid analysis of H. trimaculatus 29
2.13.Genomic DNA isolation 29
2.14.Determination of radical-mediated DNA damage 30
2.15.Reverse Transcription Polymerase Chain Reaction (RT-PCR) 30
2.16.Statistical analysis 32
3.Results 32
3.1.Proximate analysis of H. trimaculatus 32
3.2.NGF-induced PC12 cells differentiation 32
3.3.A?42-induced neurotoxicity in PC12 cell 33
3.4.Preparation of H. trimaculatus protein hydrolysates and neuroprotective properties 33
3.5. Optimization of the H. trimaculatus hydrolysis conditions by RSM 34
3.6.H. trimaculatus enzymatic hydrolysates protect PC12 cell from A?42 induced cell death 46
3.7.Preparation and identification of peptide from H. trimaculatus enzymatic hydrolysates 56
4.Discussion 68
5.Conclusion 76
Chapter 3. Effect of bioactive peptides derived from H. trimaculatus on A?42 induced-microglia mediated neurotoxicity in neuronal cells 78
1.Introduction 79
2.Materials and methods 88
2.1.Reagents 88
2.2.Cell culture 88
2.3.Co-culture experiment 89
2.4.Assessment of cell viability 89
2.5.Cell cycle analysis for apoptosis determination ? PI- Annexin V method 90
2.6.Hoecst/PI double staining 90
2.7.Determiation of ROS production 91
2.8.NO production assay 91
2.9. Enzyme immuno assay of PGE2 92
2.10.Enzyme immune assay of TNF-?, IL-6 and IL-1? 92
2.11. Enzyme immune assay of cathepsin B and D 93
2.12.Reverse Transcription Polymerase Chain Reaction (RT-PCR) 94
2.13Western blot analysis 96
2.14. Statistical analysis 96
3. Results 97
3.1.Effect of HTP-1 and HTP-2 on BV2 cells viability 97
3.2.Effects of A?42 on BV2 cell viability and neurotoxic responses 97
3.3.Effects of HTP-1 and HTP-2 on microglial neurotoxic mediators mediated PC12 cell death 98
3.4.Effects of HTP-1 on A?42-induced neurotoxic responses in BV2 cell 99
3.5.Effects of HTP-1 on anti-inflammatory cytokines in A?42- induced BV2 cells 113
3.6.Effects of HTP-1 on mitogen activated protein kinases in A?42- induced BV2 cells 116
3.7. Effects of HTP-1 on PC12 cell survival through PI3K/Akt signaling pathway 118
3.8. Effects of HTP-1 on pro-survival gene and protein expressions 122
3.9.Effects of HTP-1 on microglial neurotoxic mediators mediated caspase activation 124
3.10. Effects of HTP-1 on pro-apoptotic gene and protein expression 126
3.11.Effects of HTP-1 on cell survival were mediated through TGF-? induced PI3K/Akt signaling pathway 130
4.Discussion 133
5.Conclusion 138
Chapter 4. Effect of bioactive peptides derived from H. trimaculatus on free radical induced-neurooxidative stress 140
1.Introduction 141
2.Material and methods 148
2.1.Reagents 148
2.2.Cell culture 148
2.3. Determination of intracellular formation of ROS using fluorescence labeling 149
2.4.Determination of ROS production by FACs analysis 149
2.5.Intracellular ROS by DCFH-DA staining 150
2.6.Genomic DNA isolation 150
2.7.Determination of radical-mediated DNA damage 151
2.8.Protein oxidation 152
2.9.Membrane lipid peroxidation assessment by DPPP fluorescence method 153
2.10.RNA isolation and RT-PCR analysis 154
2.11.Western blot analysis 155
2.12.Statistical analysis 155
3.Results 156
3.1.Cellular radical scavenging effect and DNA protection activity of HTP-1 156
3.2.Inhibition of cell membrane protein oxidation by HTP-1 164
3.3.Inhibition of membrane lipid peroxidation by HTP-1 164
3.4.Effect of HTP-1 on antioxidant enzymes 170
4.Discussion 173
5. Conclusion 177
References 179
Acknowledgement 202
- Degree
- Doctor
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