해양미세조류 Navicula incerta로부터 분리된 펩타이드의 간세포 손상에 미치는 효과 및 간섬유화 작용기전
- Alternative Title
- Hepatocyte Injury and Hepatic Fibrosis Inhibitory Effect of Peptides Isolated from Microalgae, Navicula incerta
- Abstract
- Nowadays, there are numerous commercial applications of microalgae. For example, (1) microalgae can be used to enhance the nutritional value of food and animal feed owing to their chemical composition, (2) they play a crucial role in aquaculture and (3) they can be incorporated into cosmetics. Moreover, they are cultivated as a source of highly valuable molecules. Many studies have shown the interest of marine microalgae protein hydrolysates as functional foods and flavour enhancers. Recently, among marine organism , microalgae was studied as a potent source for food additive, nutraceutical or pharmaceuticals. According to criteria of nutritional quality and cost, many marine organism sources have been investigated for the production of protein hydrolysates in functional foods. However, despite a variety of biological functions of microalgae, effective application of microalgae is still limited because of their inefficient cultivation and high production cost.
Alcohol abuse is related to a wide variety of medical complications including liver diseases, pancreatitis, cardiovascular diseases, immunological abnormalities, malignant neoplasms, endocrine disturbances, and kidney problems. Among them, the liver is the organ most severely affected by alcoholism. Alcoholic liver disease (ALD) remains a major cause of morbidity and mortality in Korea. However, the pathogenesis of alcohol-induced liver disease, despite intense research, and the implication of multiple factors is not yet completely understood. The liver response to alcohol or its metabolites includes at different stages such as: inflammation, fibrosis, and regeneration, which may finally lead to cirrhosis.
Accordingly, in the present study we discuss the cultivation of Navicula incerta in a cultivation system, with emphasis on the use of culture techniques. And also investigated the the hepatoprotective, anti-fibrosis effect and antioxidant properties of peptides obtained from enzymatic hydrolysis by microalge N. incerta, with the final aim of using these marine organism by-products as sources of antioxidant ingredients.
In the present study we discuss the cultivation of Navicula incerta in acultivation system, with emphasis on the use of culture techniques. And also investigated the hepatoprotective, anti-fibrosis effect and antioxidant properties of peptides obtained from enzymatic hydrolysis of microalge N.incerta, with the final aim of using these marine organism by-products as sources of antioxidant ingredients. Among the various conditions, the optimal growth condition determined in our experiments (culture condition in 20 ？C, 33 ？, pH 8.3 ？ 0.5, 12:12 L:D, and 250 ？mol m-2∙s-1, respectively). The growth constant and final cell concentration were steady at these ranges. To produce bioactive peptides from by-products of microaglae Navicula incerta was hydrolyzed using various enzymes (alcalase,？-chymotrypsin, neutrase, papain, pepsin, pronase-E and trypsin), and the hydrolysates were evaluated for antioxidant activity. Among hydrolysates, papain derived hydrolysate exhibited the highest antioxidant activities than those of other enzyme hydrolysates. Moreover, peptides having potent antihepatotoxic and antioxidative potencies were purified. The amino acid sequence of the purified peptides were analysed as; NIPP-1 (Pro-Gly-Trp-Asn-Gln-Trp-Phe-Leu), molecular mass was determined to be 1,171 Da, and NIPP-2 (Val-Glu-Val-Leu-Pro-Pro-Ala-Glu-Leu), molecular mass was determined to be 1,108 Da.
Furthermore, in this study, we have demonstrated that NIPP-1 and NIPP-2 peptides inhibited the ethanol-induced cytotoxicity in HepG2/CYP2E1 cells. Also, TGF-？1 activated fibrosis in LX-2 cells was examined in the presence or absence of purified peptides NIPP-1 and NIPP-2. Besides the mechanisms of liver cell injury, protective effects of NIPP-1 and NIPP-2 were studied to show the protective mechanism against TGF-？1 stimulated fibrogenesis. Our results showed that the core protein of NIPP-1 peptide prevented fibril formation of type I collagen, elevated the MMP level and inhibited TIMP production in a dose-dependent manner. The treatment of NIPP-1 and NIPP-2 peptides on TGF-？1 induced LX-2 cells alleviated liver fibrosis. Moreover, ？-SMA, TIMPs, collagen, PDGF and cytokine levels in the NIPP-1 treated group were significantly decreased. An decrease in NF-kB expression in the treated group were also observed in case of NIPP-1 but not NIPP-2.
It is concluded that NIPP-1 shows inhibitory effects on liver fibrosis, through inhibition of HSCs (LX-2) activation, in most cases more effective than NIPP-2.
- Issued Date
- 2011
- Awarded Date
- 2011. 2
- Type
- Dissertation
- Publisher
- 부경대학교
- Alternative Author(s)
- Kang, Kyong-Hwa
- Affiliation
- 부경대학교 일반대학원
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Chapter 1. RESEARCH BACKGROUND 1
1. Importance and Application of the Microalgae 2
2. The liver 3
3. Alcoholic Hepatocyte Injury 5
4. Hepatic Fibrosis 6
5. Research Objectives 8
Chapter 2. Characterization of cultivation and protein hydrolysation of marine microalgae, Navicula incerta (Bacillariophyceae) 10
1. INTRODICTION 11
2. MATERIALS AND METHODS 17
2.1. Materials 17
2. 2. Growth characteristics with laboratory cultures experiments 18
2.2.1. Determination of optimal growth conditions 18
2.2.1.1 Effect of temperature and salinity on growth 18
2.2.1.2 Effect of light intensity and pH on growth 19
2. 3. Growth measurements 19
2. 4. Mass Culture and Freeze-Drying 21
2. 5. Proximate Composition of N. incerta 23
2. 6. Amino Acid Analysis of N. incerta 23
2. 7. Preparation of Enzymatic Hydrolysates 23
2. 8. Antioxidant Assay by Electron Spin Resonance Spectrometer (ESR) 25
2. 8. 1. Scavenging effect on DPPH radical 25
2. 8. 2. Hydroxyl radical scavenging activity 25
2. 8. 3. Superoxide radical scavenging activity 26
2. 9. Cell culture and treatments 27
2. 10. Cell viability 28
2. 11. Genomic DNA isolation 29
2. 12. DNA oxidation assay 30
2. 13. Determination of intracellular formation of reactive oxygen species using fluorescence labeling 30
2. 14. GGT Activity 31
2. 15. Determination of Intracellular GSH Content 32
2. 16. Western Blot Analysis 33
2. 17. Statistical Analysis 34
3. RESULTS 35
3.1.The Optimum Culture Conditions of Navicula incerta 35
3.1.1. Effects of temperature and salinity on growth of N. incerta 35
3.1.2. Effect of light intensity and pH on growth rate of N. incerta 39
3.2. Mass Culture and Freeze-Drying 42
3.3. Proximate Composition of Navicula incerta 42
3.4. Preparation of N. incerta Protein Hydrolysates and Their Antioxidant Properties 46
3.5. Effect of N. incerta enzymatic hydrolysates on DNA oxidation 47
3.6. Scavenging of intracellular reactive oxygen species by N. incerta enzymatic hydrolysates 49
3.7. CYP2E1 levels in HepG2/CYP2E1 cells 50
3.8. Ethanol induces cytotoxicity and GGT release and effect of N. incerta enzymatic hydrolysates on cell viability 50
3.9. Effect of NEHs on Ethanol-induced GGT and GSH depletion in HepG2/CYP2E1 cells 53
3.10. Western Blot Analysis 60
4. DISCUSSION 64
Chapter 3. Purification and isolation of bioactive peptides from protein hydrolysate of microalgae Navicula incerta and their protective effects on ethanol-induced toxicity in HepG2/CYP2E1 cells 72
1. INTRODUCTION 73
2. MATERIALS AND METHODS 79
2. 1. Materials 79
2. 2. Purification of antioxidant peptide 79
2. 2. 1. Ion exchange chromatography 79
2. 2. 2. High-performance liquid chromatography (HPLC) 80
2. 2. 3. Determination of amino acid sequence 80
2.3. Cell culture and Cell viability assay 81
2.4. Determination of intracellular formation of reactive oxygen species (ROS) using fluorescence labeling 82
2.5. Genomic DNA isolation 83
2.6. DNA oxidation assay 84
2.7. GGT assay 85
2.8. Determination of intracellular GSH content 85
2.9. Western blot analysis 86
2.10. Statistical analysis 87
3. RESULTS 88
3. 1.Identification of peptide from N. incerta Protein Hydrolysates 88
3. 2. Cell viability 101
3. 3. Effect of NIPP-1 and NIPP-2 peptides on DNA oxidation 101
3. 4. Scavenging of intracellular reactive oxygen species by NIPP-1 and NIPP-2 104
3. 5. Effect of NIPP-1 and NIPP-2 peptides on ethanol-induced GGT and GSH depletion in HepG2/CYP2E1 cells 106
3. 6. Western Blot Analysis 109
3. 7. Cell Imaging 118
4. DISCUSSION 120
Chapter 4. Mechanism studies on the hepatic fibrosis by hepatotoxicants in Hepatic Stellate Cell Line, LX-2 cell 125
1. INTRODCUTION 126
2. MATERIALS AND METHODS 132
2. 1. Materials 132
2. 2. Culture of Hepatic Stellate Cells and TGF- β1 treatment 132
2. 3. TGF- β1 concentration measurement 133
2. 4. Cell proliferation, morphology and cytotoxicity 133
2. 5. Hepatic Stellate Cell (LX-2) activation 134
2. 6. Measurement of procollagen type I produced by fibroblasts 135
2. 7. Sircol Collagen assay 136
2. 8. Indirect Immunofluorescence Staining of Cultured Cells 137
2. 9. Western blot analysis 137
2. 10. Reverse transcription (RT)-PCR analysis 138
2. 11. Statistical analysis 140
3. RESULTS 142
3. 1. Determination of TGF- β1 concentration 142
3. 2. Cell proliferation, morphology and cytotoxicity 142
3. 3. TGF-β1 induced Hepatic Stellate Cell (LX-2) activation 143
3. 4. Measurement of procollagen type I produced by fibroblasts 147
3. 5. Sircol Collagen assay 149
3. 6. Indirect immunofluorescence 151
3. 7. Effects of NIPP-1 and NIPP-2 on α-SMA, TGF-β1 and PDGF expressions in LX-2 cells stimulated by TGF-β1 151
3. 8. Effects on NIPP-1 and NIPP-2 of TIMPs expression in LX-2 cells stimulated with TGF-β1 157
3. 9. Effects on NIPP-1 and NIPP-2 of MMPs expression in LX-2 cells stimulated by TGF-β1 160
3. 10. Effects on NIPP-1 and NIPP-2 on collagen expression in LX-2 cells stimulated with TGF-β1 161
3. 11. Effects of NIPP-1 and NIPP-2 on cytokine expression in LX-2 cells stimulated with TGF-β1 166
3. 12. Effects on NIPP-1 and NIPP-2 of NF-κB activation and MAPKs expression in LX-2 cells stimulated by TGF-β1 171
4. DISCUSSION 174
5. CONCLUSION 178
6. REFERENCES 180
- Degree
- Doctor
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