Attenuation of Activated Enteric Glia Mediated Disruption of Intestinal Barrier Functions and Helicobacter pylori Associated Gastric Pathogenesis by Peptides Isolated from Microbial Hydrolysates of Chlamydomonas sp.
- Abstract
- With increasing demand for the natural substances with sustainable production as pharmaceutical agents, marine microalgae have been recognized to provide chemical and pharmacological novelty and diversity. Among them microalgae derived peptides produced with various hydrolysis techniques are more abundant owing to the rich protein content of microalgae. In this study marine microalga Chlamydomonas sp. was hydrolyzed with the combination of food-grade microbes, Candida utilis and Bacillus subtilis. The microbial hydrolysate of the Chlamydomonas sp. was purified using anion exchange chromatography and reverse phase high performance liquid chromatography to obtain bioactive peptides. The successful assay guided purification leads to isolation of three differently bioactive peptides. The accurate molecular mass and amino acid sequences of the purified peptides was determined using a hybrid quadrupole-TOF LC/MS/MS mass spectroscopy as RQALGAPG - 768.42 Da (H-P-2), ENLDDLE – 846.35 Da (H-P-3) and PQPKVLDS – 882.42 (H-P-6).
The activation of gastric enteric glial cells (EGC) was found to incur damages to the intestinal epithelial barriers. The isolated peptide H-P-3 showed a protective effect on bacterial lipopolysaccharide (1 µg/ml) and interferone-γ (10 ng/ml) activated enteric glial cells mediated disruption of the intestinal epithelial barriers. The peptide H-P-3 significantly attenuated the activated EGC mediated damage of tight junction proteins ZO-1 (Zonula occludens-1) and occludin with simultaneous suppression of enteric cell mortality. It was observed that the inflammatory cascade of the activated EGC create an oxidative stress condition in intestinal epithelial cells (IEC-6) which leads to DNA damage and cell cycle arrest. The molecular pathway studies showed that the peptide is protecting the intestinal epithelial cells by down-regulating the inflammatory signaling pathway NF-κB in EGC which suppress the activation of ATM/ATR dependent DNA damage in IEC-6 epithelial cells.
Helicobacter pylori infection is identified as one of the most critical causes of stomach cancer. The current study has focused on the suppressive effect of isolated peptides on the H. pylori associated carcinogenesis and macrophage apoptosis. The peptide H-P-6 has effectively attenuated the H. pylori mediated carcinogenic responses in gastric epithelial cells (AGS). It showed a down regulation in H. pylori induced AGS cell proliferation and migration. The molecular signaling studies indicated that H. pylori activate the epidermal growth factor receptor (EGFR) signaling and nuclear translocation of the β-catenin. Both consequences leading to increased cell proliferation and migration. The results indicated that the peptide H-P-6 down-regulated the EGFR signaling pathway and nuclear translocation of the β-catenin.
In further studies, the peptide H-P-2 was found as effective protector of H. pylori mediated macrophage apoptosis. The infection with H. pylori weakens the cellular defense system by down-regulating the production of anti-bacterial nitric oxide (NO). Moreover, the H. pylori infection induces the production of ornithine decarboxylase (ODC) mediated cytotoxic hydrogen peroxide production which leads to mitochondrial damage and subsequent apoptosis. The peptide has up-regulated the NO production via down regulating the arginase II enzyme. The down-regulation of arginase II has also lead to suppression of ODC mediated macrophage cell death. Collectively, this study has focused on identification of natural peptides from the microbial hydrolysates of marine microalgae Chlamydomonas sp. which are capable of attenuating activated enteric glial cells and H. pylori mediated gastric complications.
Key words: Chlamydomonas sp. enteric glia, epithelial barrier, Helicobacter pylori, EGFR, Argianse II, ODC
- Issued Date
- 2013
- Awarded Date
- 2013. 2
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학교
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Table of Contents
Page
Abstract i
Table of Contents iii
List of Tables viii
List of Figures ix
List of Abbreviations xiii
Chapter 1.
Culture Optimization, Microbial Fermentation and Peptide Purification from Marine Microalgae Chlamydomonas Sp. 1
1.1. Introduction
1.1.1. Potential of marine microalgae as a value added ingredient 2
1.1.2. Proteins and bioactive peptides from microalgae 3
1.1.3. Objectives 4
1.2. Materials and Methods
1.2.1. Materials 6
1.2.2. Determination of optimal growth conditions 6
1.2.3. Mass culture and freeze drying 7
1.2.4. Proximate composition analysis 7
1.2.5. Analysis of the amino acid composition 7
1.2.6. Culture of hydrolyzing microorganisms 13
1.2.7. Physical and enzymatic treatment prior to microbial hydrolysis 13
1.2.8. Setting up the conditions for microbial hydrolysis 13
1.2.9. Protein content analysis 14
1.2.10. Cell culture 14
1.2.11. Cell viability assay 14
1.2.12. Nitric oxide production assay 15
1.2.13. Flow cytometric analysis of reactive oxygen species 15
1.2.14. Purification and identification of active peptides
1.2.14.1. Ion exchange chromatography 16
1.2.14.2. Reverse phase HPLC 16
1.2.14.3. Determination of the amino acid sequence 17
1.2.15. Statistical analysis 17
1.3. Results
1.3.1. Optimal growth conditions 18
1.3.2. Yield of Chlamydomonas sp. from mass culture 19
1.3.3. Proximate composition and the amino acid composition 19
1.3.4. Selection of the microbial species for the hydrolysis 24
1.3.5. Cyto-compatibility, ROS and NO inhibitory activity of the hydrolysates 24
1.3.6. Purification of active fractions using FPLC 25
1.3.7. Purification of active peptides using HPLC 25
1.4. Discussion 36
1.5. Summary
39
CHAPTER 2
Protective Effect of The Isolated Peptide H-P-3 Against Activated Enteric Glia Mediated Disruption of Intestinal Barrier Functions
2.1. Introduction
2.1.1. Enteric glial cells; activation and pathogenesis 42
2.1.2. Intestinal epithelial barrier functions 42
2.1.3. Study objectives 44
2.2. Materials and methods
2.2.1. Cell culture and cytotoxicity analysis 47
2.2.2. Implementation of co-culture system 47
2.2.3. Assessment of NO production 48
2.2.4. Assessment of ROS production levels by flow cytometer 48
2.2.5. Western blot analysis 49
2.2.6. RT-PCR 49
2.2.7. Immunocytochemistry 50
2.2.8. Neutral Comet assay 51
2.2.9. Hoechst staining 51
2.2.10. Annexin V/PI staining 52
2.2.11. Cell cycle analysis by flow cytometer 52
2.2.12. Statistical analysis 52
2.3. Results
2.3.1. Selection of stimulator for the activation of EGC 54
2.3.2. Screening results of the active peptide 54
2.3.3. Selection of the optimal co-culture conditions 55
2.3.4. H-P-3 mediated suppression of EGC activation reduced the tight junction disruption in IEC 59
2.3.5. H-P-3 mediated suppression of EGC activation protects IEC-6 cells against oxidative stress damage 59
2.3.6. H-P-3 mediated suppression of EGC activation protects IEC-6 cells from apoptosis 65
2.3.7. H-P-3 mediated suppression of EGC activation reduced the cell cycle arrest in IEC-6 cells 65
2.3.8. H-P-3 mediated suppression of EGC activation regulated the cell signaling molecules related to cell cycle arrest and apoptosis 66
2.3.9. H-P-3 suppress the activation of EGC through blocking the nuclear translocation of NF-κB 71
2.4. Discussion 74
2.5. Summary 77
CHAPTER 3
A Peptide from Chlamydomonas Sp. Attenuates Helicobacter Pylori Mediated Hyper Proliferation in AGS Enteric Epithelial Cells via Blocking the Activation of EGFR
3.1. Introduction
3.1.1. Helicobacter pylori infection 81
3.1.2. CagA mediated pathogenesis 81
3.1.3. Cellular mechanisms of H. pylori mediated disruption of adherens junctions 82
3.1.4. Activation of EGFR and β-catenin in response to H. pylori infection 83
3.1.5. Objectives 84
3.2. Materials and methods
3.2.1. Reagents and materials 85
3.2.2. Helicobacter pylori strains and culture of bacteria 85
3.2.3. Cell culture and cellular proliferation analysis 85
3.2.4. Determination of anti-Helicobacter pylori activity of peptides 86
3.2.5. Determination Helicobacter pylori invasion into AGS cells 86
3.2.6. Radius cell migration assay 87
3.2.7. Scanning electron microscopic analysis 87
3.2.8. Western blot analysis 87
3.2.9. RT-PCR 88
3.2.10. Immunocytochemistry 89
3.2.11. Docking calculations 89
3.2.12. Statistical analysis 90
3.3. Results
3.3.1. All isolated peptides did not show any antibacterial effects against H. pylori 92
3.3.2. Peptide H-P-6 suppressed the H. pylori induced proliferation in AGS cells 92
3.3.3. H-P-6 treatment reduced the morphological change occurs due to H. pylori infection 92
3.3.4. H-P-6 suppressed the H. pylori mediated cell migration 93
3.3.5. H-P-6 peptide did not inhibit the H. pylori invasion into AGS cells 99
3.3.6. Effect of H-P-6 on H. pylori induced EGFR expression 100
3.3.7. Effect of H-P-6 on the downstream targets of EGFR 100
3.3.8. Peptide mediated down regulation of the β-catenin nuclear translocation 100
3.3.9. Peptide suppressed the proliferation and migration related gene expressions 104
3.3.10. EGFR activation contribute to H. pylori induced hyper proliferation of AGS cells 104
3.3.11. Docking results showing the binding sites of the peptide to EGFR 107
3.4. Discussion 109
3.5. Summary 112
CHAPTER 4
Peptide H-P-2 Protects against Helicobacter pylori Induced Macrophage Cell Death via Regulating Arginase II Activity
4.1. Introduction 115
4.2. materials and methods
4.2.1. Bacteria and cell culture 116
4.2.2. Cytotoxicity assessment 117
4.2.3. Nitric oxide production assay 117
4.2.4. Western blot analysis 118
4.2.5. RT-PCR 119
4.2.6. Immunocytochemistry analysis 119
4.2.7. Analysis of Annexin V/PI staining by flow cytometer 120
4.2.8. Analysis of mitochondrial membrane potential 120
4.2.10. Docking calculations 121
4.2.9. Statistical analysis 121
4.3. Results
4.3.1. Infection with H. pylori reduced the proliferation of macrophage cells 122
4.3.2. Effect of H. pylori infection on the NO and iNOS in RAW264.7 cells 122
4.3.3. Peptide H-P-2 protects the macrophage by up regulating the arginase II production 123
4.3.4. H-P-2 rescued RAW264.7 cells from H. pylori induced DNA damage and apoptosis via mitochondrial pathway 128
4.3.5. The effect of H-P-2 on the ODC expression levels 128
4.3.6. Docking of H-P-2 peptide with human arginase II enzyme 133
4.4. Discussion 135
4.5. Summary 137
References 139
Acknowledgements 151
List of Tables
Page
Table 01. Bioactive peptides isolated from marine microalgae 5
Table 02. The conditions tested for the different variables during the optimization of culture conditions 8
Table 03. Regression results of the Chlamydomonas sp. culture parameters 20
Table 04. Optimized variable responses for the culture of Chlamydomonas sp. 22
Table 05. Proximate composition of the dried Chlamydomonas sp. 22
Table 06. Amino acid content of the dried Chlamydomonas sp. 23
Table 07. Amino acid sequences of the isolated peptides 31
Table 08. Primer sequences used for RT-PCR (Chapter 2) 53
Table 09. Primer sequences used for RT-PCR (Chapter 3) 91
List of Figures
Page
Figure 1. Mass culture steps and collection methods of the Chlamydomonas sp. 9
Figure 2. (a) Culture plate of Candida utilis (KCCM 50045) and (b) culture plate of Bacillus subtilis (KCCM 3014). 10
Figure 3. Physical and enzymatic treatments for the breakdown of cell wall of Chlamydomonas sp. 11
Figure 4. The flow chart of obtaining the bacterial hydrolysate of Chlamydomonas sp. 12
Figure 5. Light microscopic picture of cultured Chlamydomonas sp. 21
Figure 6. Time and strain dependent variations in the protein content of microbial hydrolysate of Chlamydomonas sp. 26
Figure 7. Effect of the microbial hydrolysates on the relative cell viability and their ROS scavenging abilities 27
Figure 8. FPLC chromatogram of Candida utilis and Bacillus subtilis hydrolysate of Chlamydomonas sp. 28
Figure 9. The inhibitory effect of the FPLC fractions on NO production and iNOS expression in EGC 29
Figure 10. RP-HPLC chromatograms of the H-F-1 (a) and H-F-2 (b). 30
Figure 11. Quadrupole-TOF LC/MS/MS Mass spectroscopy of isolated peptides 32
Figure 12. Amino acid sequence alignment of the isolated peptides with Chlamydomonas protein sequences. 35
Figure 13. Schematic representation of chapter 1 summary 40
Figure 14. Schematized view of the enteric nervous system 45
Figure 15. Schematic representation of the study hypothesis 46
Figure 16. Selection of the optimal stimulator for the activation of EGC 56
Figure 17. Selection of the active peptide 57
Figure 18. Selection of the optimal co-culture condition 58
Figure 19. H-P-3 induced suppression of EGC activation reduced disruption of ZO-1 in IEC-6 cells 61
Figure 20. H-P-3 induced suppression of EGC activation reduced disruption of occluding in IEC-6 cells 62
Figure 21. H-P-3 induced suppression of EGC activation reduced the ROS production in IEC-6 cells 63
Figure 22. H-P-3 induced suppression of EGC activation reduced the DNA damage in IEC-6 cells 64
Figure 23. H-P-3 mediated suppression in EGC activation protects IEC-6 cells from apoptosis 67
Figure 24. H-P-3 mediated suppression of EGC activation reduced the IEC-6 cell death via down regulating the cell death signaling molecules 68
Figure 25. H-P-3 mediated suppression of EGC activation reduced the cell cycle arrest in IEC-6 cells 69
Figure 26. H-P-3 mediated suppression of EGC activation regulated the DNA damage signaling molecules in IEC-6 cells 70
Figure 27. H-P-3 suppresses the activation of EGC through blocking the nuclear translocation of NF-κB 72
Figure 28. The effect of the EGC activation on the proliferation of IEC-6 cells in the presence of specific inhibitors 73
Figure 29. Schematic representation of chapter 2 summary 79
Figure 30. Anti-Helicobacter pylori effect of the isolated peptide 94
Figure 31. Effect of peptides on H. pylori induced hyper proliferation in AGS cells 95
Figure 32. Morphological characteristics induced by H. pylori 96
Figure 33. Effect of H-P-6 on the H. pylori induced migration of AGS cells 97
Figure 34. The effect of H-P-6 on the invasion of H. pylori into AGS cells 98
Figure 35. The protein expression levels of EGFR in the presence of H-P-6 and EGFR inhibitor 101
Figure 36. Effect of H-P-3 on H. pylori induced PI3K signaling in AGS cells 102
Figure 37. The effect of H-P-6 on activation of β-catenin 103
Figure 38. Effect of the peptide H-P-6 on the m-RNA expression levels of proliferation and migration related genes. 105
Figure 39. Effect of the peptide and the EGFR inhibitor on the H. pylori induced AGS cell proliferation and migration. 106
Figure 40. Docking images of H-P-6 peptide with the tyrosine kinase domain of EGFR 108
Figure 41. Schematic representation of chapter 3 summary 113
Figure 42. Viability of H. pylori infected RAW264.7 cells in the presence of peptides 124
Figure 43. Effect of H-P-2 on the NO production and iNOS expression of H. pylori induced RAW264.7 cells. 125
Figure 44. Effect of H-P-2 on the arginase II expressions in RAW264.7 cells. 126
Figure 45. The comparative effect of the H-P-2 and arginase II inhibitor on the NO production level 127
Figure 46. The effect of the H-P-2 and the arginase II inhibitor on H. pylori induced apoptosis 129
Figure 47. Effect of H-P-2 and arginase II inhibitor on the H. pylori induced breakdown of mitochondrial membrane potential. 130
Figure 48. The effect of H-P-2 and the arginase II inhibitor on the ROS production level in H. pylori induced RAW264.7 cells 131
Figure 49. Effect of H-P-2 and arginase II inhibitor on the protein and m-RNA expression levels of ODC 132
Figure 50. Docking images of H-P-2 peptide with tyrosine kinase domain of Arginase II 134
Figure 51. Schematic representation of chapter 4 summary 138
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