Anti-inflammatory and foam cell formation inhibitory activities of ark shell (Scapharca subcrenata) hydrolysates in murine macrophages
- Alternative Title
- 마우스 대식세포에서 꼬막 단백질 가수분해물의 항 염증 및 거품세포 형성 억제 활성
- Abstract
- Inflammation, complex biological response of body tissue to defense against harmful stimuli, leads to chronic diseases in uncontrolled conditions and threat to mankind due to its high-risk factor coupled with morbidity and mortality. It has given much attention worldwide in identifying novel anti-inflammatory agents using natural bioactive compounds with minimal side effects on human health. The present study investigates the potential anti-inflammatory effect and foam cell formation inhibitory effect of ark shell hydrolysates. Enzymatic hydrolysis was performed with alcalase and pepsin enzymes to prepare ark shell alcalase hydrolysate (ASAH) and ark shell pepsin hydrolysate (ASPH) respectively and prepared ASAH was used to examine anti-inflammatory activity in lipopolysaccharides (LPS) induced RAW264.7 cell line and foam cell formation inhibition activity was investigated in oxidized low density lipo-proteins (oxLDL) stimulated RAW264.7 macrophages using ASPH. ASAH showed promising anti-inflammatory effect by minimizing the nitric oxide (NO) production, reactive oxygen species (ROS) production, and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) production dose-dependently in LPS stimulated murine macrophages. Further analysis revealed that ASAH increases the heme- oxygenase-1 (HO-1) expression and suppresses the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS stimulated RAW264.7. In addition, ASAH enhances the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and mitogen-activated protein kinases (MAPK) including ERK1/2. JNK1/2 and p-38 phosphorylation in RAW264.7 macrophages. Three MAPK inhibitors attenuate the nuclear translocation of Nrf2 and HO-1 protein expression. Taken together these results suggest that protective effect of ASAH may be directed via activating the Nrf2/HO-1 pathway. ASPH showed a potential inhibitory effect on foam cell formation. ASPH decreased pro-inflammatory cytokines production (TNF-α, IL-1β and IL-6), cholesterol influx and SR-A1 and CD-36 protein expression level and increased cholesterol efflux as well as ABCA-1 and ABCG-1 protein expression level in a dose dependent manner in ox-LDL stimulated RAW264.7 macrophages. Furthermore, ASPH treatment dose dependently suppresses the nuclear factor-kappa B (NF-kB) nuclear translocation in oxLDL induced RAW264.7 macrophages. These results suggest that ASPH has a potential foam cell formation inhibitory effect.
- Issued Date
- 2021
- Awarded Date
- 2021. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Alternative Author(s)
- Marasinghe Pathiranalage Chathuri Kaushalya Marasinghe
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 식품생명과학과
- Advisor
- JeJaeYoung
- Table Of Contents
- 1. Introduction 1
2. Materials and methods 10
2.1 Anti-inflammatory activities of ASAH 10
2.1.1 Materials 10
2.1.2 Preparation of ark shell alcalase hydrolysate (ASAH) 10
2.1.3 Determination of molecular weight distribution 11
2.1.4 Determination of amino acid composition 11
2.1.5 Cell culture 12
2.1.6 MTT assay 12
2.1.7 Nitric oxide (NO) quantification assay 12
2.1.8 ROS measurement 13
2.1.9 Determination of TNF-α, IL-1β, and IL-6 levels 13
2.1.10 Determination of ZnPP (HO-1 inhibitor) activity 13
2.1.11 Western blot analysis 13
2.1.12 Statistical analysis 14
2.2 Foam cell formation inhibition of ASPH 15
2.2.1 Materials 15
2.2.2 Preparation of ark shell pepsin hydrolysates (ASPH) 15
2.2.3 Determination of molecular weight distribution 15
2.2.4 Determination of amino acid composition 15
2.2.5 Cell culture 15
2.2.6 MTT assay 16
2.2.7 Oxidation of LDL and determination of thiobarbituric acid-reactive substances (TBARS) 16
2.2.8 Oil red O staining assay 16
2.2.9 Determination of TNF-α, IL-1β, and IL-6 levels 17
2.2.10 Determination of intracellular cholesterol content 17
2.2.11 Cholesterol influx assay 17
2.2.12 Cholesterol efflux assay 18
2.2.13 Western blot analysis 18
2.2.14 Statistical analysis 19
3. Results 20
3.1 Anti-inflammatory activity of ASAH 20
3.1.1 Molecular weight distribution of ASAH 20
3.1.2 Amino Acid Composition ASAH 22
3.1.3 Effect of ASAH on cytotoxicity in RAW264.7 macrophages 24
3.1.4 Effect of ASAH on NO production in LPS stimulated RAW264.7 macrophages 26
3.1.5 Effect of ASAH on HO-1, iNOS, and COX-2 28
3.1.6 Effect of ASAH on pro-inflammatory cytokine production 30
3.1.7 Effect of ASAHs on NO, ROS, pro-inflammatory cytokines, HO-1, iNOS, and COX-2 in the presence of ZnPP 32
3.1.8 Effect of ASAH on Nrf2 nuclear translocation 37
3.1.9 Effect of ASAH on MAPK expression 39
3.1.10 Effect of ASAH on Nrf2 mediated HO-1 expression through MAPK pathway 41
3.2 Foam cell formation inhibition of ASPH 43
3.2.1 Molecular weight distribution of ASAH 43
3.2.2 Amino acid composition of ASPH 45
3.2.3 Effect of ASPH on cytotoxicity in RAW264.7 macrophages 47
3.2.4 Effect of ASPH on Oil Red O staining 49
3.2.5 Effect of ASPH on pro-inflammatory cytokine production 51
3.2.6 Effect of ASPH on total cholesterol, free cholesterol and cholesterol ester content 53
3.2.7 Effect of ASPH on cholesterol influx and cholesterol efflux 55
3.2.8 Effect of ASPH on CD-36, SR-A, ABCA-1, and ABCG-1 57
3.2.9 Effect of ASPH on NF-kB p-65 subunit nuclear translocation 59
4. Discussion 61
5. Conclusion 69
6. Abstract (Korean) 70
7. References 72
Acknowledgement 82
- Degree
- Master
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