한수지 51(2), 127-134, 18 Original Article Korean J Fish Aquat Sci 51(2),127-134,18 혈관내피세포에서 TNF-α 로유도되는혈관염증에대한쏙 (Upogebia major) 효소가수분해물의억제효과 김소연 양지은 송재희 1 맹상현 이지현 윤나영 국립수산과학원식품위생가공과, 1 서해수산연구소갯벌연구센터 Inhibition Effect of Enzymatic Hydrolysate from Japanese Mud Shrimp Upogebia major on TNF-α-induced Vascular Inflammation in Human Umbilical Vein Endothelial Cells (HUVECs) So-Yeon Kim, Ji-Eun Yang, Jae-Hee Song 1, Sang-Hyun Maeng, Ji-Hyun Lee and Na-Young Yoon Food Safety and Processing Research Division, National Institute of Fisheries Science, Busan 483, Korea 1 Tidal Flat Research Center, National Institute of Fisheries Science, Gunsan 514, Korea Arteriosclerosis is the major cause of coronary artery and cerebrovascular disease, which are leading causes of death. Pro-inflammatory cytokines induce injury to vascular endothelial cells by increasing cell adhesion molecules, leading to vascular inflammation, a major risk factor for the development of arteriosclerosis. In the current study, we investigated the inhibitory effect of enzymatic hydrolysate from Japanese mud shrimp Upogebia major on the inflammation of tumor necrosis factor-α (TNF-α)-stimulated human umbilical vein endothelial cells (HUVECs). We first evaluated the antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of eight U. major enzymatic hydrolysates: alcalase, papain, α-chymotrypsin (α-chy), trypsin, pepsin, neutrase, protamex and flavourzyme. Of these, α-chy exhibited potent antioxidant and ACE inhibitory activities. The α-chy hydrolysate was fractionated by two ultrafiltration membranes of 3 and 1 kda. The α-chy hydrolysate of U. major and its molecular weight cut-off fractions resulted in a significant reduction in NO production and a decrease in cell adhesion molecules [vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and endothelial-selectin (E-selectin)] and pro-inflammatory cytokines [interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1)] in TNF-α-stimulated HUVECs. These results suggest that enzymatic hydrolysate from U. major can be used in the control and prevention of vascular inflammation and arteriosclerosis. Key words: Upogebia major, Enzymatic hydrolysate, Atherosclerosis, HUVEC, TNF-α 서론,,,,, (Lee et al., 12a). (Packard and Libby, 8).,,,,,,,, (Lee et al., 12b; Cho et al., 16).. (Upogebia major), https://doi.org/1.5657/kfas.18.127 Korean J Fish Aquat Sci 51(2) 127-134, April 18 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3./) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 6 February 18; Revised 27 February 18; Accepted 2 March 18 Corresponding author: Tel: +82. 82. 51. 7. 2652 Fax: 82. 51. 7. 2669 E-mail address: 82. 51. 7. 2652 Copyright 18 The Korean Society of Fisheries and Aquatic Science 127 pissn:374-8111, eissn:2287-8815
128 김소연ㆍ양지은ㆍ송재희ㆍ맹상현ㆍ이지현ㆍ윤나영,,,.,,,, (Hong, 13).,, carotenoids,,,, (Pan et al., ; Rosa and Barracco, 1; Sindhu and Sherief, 11; Oh and Jung, 15).,., human umbilical vein endothelial cells (HUVECs) tumor necrosis factor- (TNF- ), NO, [vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and endothelial-selectin (E-selectin)] [interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1)]. 재료및방법 재료 (U. major) 16 3 5 kg, - C. Alcalase 2.4L (Alc), papain (Pa), -chymotrypsin ( -Chy), trypsin (Try), pepsin (Pep), serine, o-phthaldialdehyde (OPA) Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), Neutrase (Neu), Protamex (Pro), Flavourzyme (Fla) Novo Co. (Novozyme Laboratories, Copenhagen, Denmark). 2, 2 -azino-bis [3-ethylbenzothiazoline-6-sulfonicacid] (ABTS + ), potassium persulfate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium phosphate, potassium ferricyanide [K 3 Fe(CN) 6 ], trichloroacetic acid (TCA), L-ascorbic acid, hippuryl-l-histidyl-lleucine (HHL), sodium borate, angiotensin I-converting enzyme (ACE), hydrochloric acid, pyridine, benzene sulfonyl chloride (BSC), captopril, tumor necrosis factor- (TNF- ), Griess reagent Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), iron (III) chloride (FeCl 3 ) Junsei Chemical Co. (Tokyo, Japan), cell lysis buffer Cell signaling technology, Inc (Danvers, MA, USA), nitrite standard Promega (Madison, WI, USA). human umbilical vein endothelial cells (HUVECs) Endothelial Growth Medium-2 (EGM TM -2 Medium) Lonza (Walkersville, MD, USA), penicillin-streptomycin solution Cellgro (Herndon, VA, USA), trypsin ethylenediaminetetraacetic acid (Trypsin-EDTA) Gibco (Grand Island, NY, USA), Cell Proliferation Reagent water-soluble tetrazolium salt (WST-1) Roche (Mannheim, Germany) ELISA kit R&D systems, Inc. (Minneapolis, MN, USA). 쏙효소가수분해 1 g 8 (Alc, Pa, -Chy, Try, Pep, Neu, Pro, Fla), 1%. 41.39 g/ g ( ). 8 Table 1 8., 1. 4,45 g. -. Table 1.The conditions of enzymatic hydrolysis and degree of hydrolysis of the enzymatic hydrolysates of Japanese mud shrimp Upogebia major Enzyme Buffer ph Temperature ( ) DH 1 (%) Alcalase (Alc).1 M Na 2 8. 5 63.82±.4 α-chymotrypsin (α-chy).1 M Na 2 8. 37 67.91±.2 Flavourzyme (Fla).1 M Na 2 7. 5 67.84±.3 Neutrase (Neu).1 M Na 2 8. 5 65.76±.8 Papain (Pa).1 M Na 2 6. 37 65.9±.5 Pepsin (Pep).1 M Glycine-HCl 2. 37 67.47±.6 Protamax (Pro).1 M Na 2 8. 45 67.32±.9 Trypsin (Try).1 M Na 2 8. 37 63.13±.4 DH 1, degree of hydrolysis. Enzymatic hydrolysates were obtained from 8 hour under the optical conditions.
쏙효소가수분해물의혈관염증억제효과 129 가수분해도 (Degree of hydrolysis, DH) Adler-Nissen (1979), o-phthaldialdehyde (OPA) free amino groups (Adler-Nissen, 1986). DH = h / h tot h tot protein equivalent peptide bonds, h. 가수분해물분획 8 1 ml, 3 kda 1 kda ultrafiltration membranes (Amicon Ultrafilter devices; Millipore, Billerica, MA, USA) 9 g (<3 kda, 3-1 kda, >1 kda).. ABTS + 라디칼소거활성 ABTS + Roberta et al. (1999). 7.4 mm ABTS 2.6 mm potassium persulfate buffer 1:1 4. ABTS + 734 nm.7-1 1 L ABTS + 19 L 1 734 nm. L-ascorbic acid, ABTS + 5% IC 5 (mg/ ml). DPPH 라디칼소거활성 DPPH Blois (1958)..2 mm DPPH 1 L L, 37 3, 5 nm. L-ascorbic acid, DPPH 5% IC 5 (mg/ml). Reducing power 측정 Reducing power Oyaizu (1986). L.2 M sodium phosphate buffer (ph 6.6) 1% K 3 Fe(CN) 6 L, 5. 1% TCA L, 2, g 1. L.1% FeCl 3 L, 7 nm. L-ascorbic acid, 5% EC 5 (mg/ml). ACE 억제효과측정 ACE Li et al. (5). L 5 L 5 mm HHL,.1M sodium borate buffer (ph 8.3) 37 5. 1 U ACE 1 L 37 3. 1 M HCl L sodium borate buffer (3 L), pyridine ( L), BSC ( Table 2. The antioxidant and ACE inhibitory activities of eight enzymatic hydrolysates of Japanese mud shrimp Upogebia major (mg/ml) ABTS + (IC 51 ) DPPH (IC 5 ) Reducing power (EC 52 ) ACE (IC 5 ) Alcalase (Alc).9±. c,3 3.23±.22 c 3.1±.18 b,c.7±.6 c α-chymotrypsin (α-chy).9±. c 2.87±.18 d 1.77±.13 d.8±.5 c Flavourzyme (Fla).9±. c 4.28±.21 b 2.81±.19 c.17±.8 a Neutrase (Neu).8±. c 5.61±.69 a 3.38±.42 b.18±. a Papain (Pa).13±. b 3.56±.61 c 2.±.9 c.11±.28 b Pepsin (Pep).18±. a 4.72±.28 b 5.93±.66 a.17±.5 a Protamax (Pro).9±. c 4.59±.17 b 2.64±.32 c.2±.5 d Trypsin (Try).12±. b 3.68±.38 c 3.62±.22 b.11±.8 b A L-Ascorbic acid (μg/ml) 5.61±.9 d 4.46±.27 e 4.71±. e B Captopril (ng/ml).25±.5 e 1 IC 5 (5% inhibition concentration) values of ABTS + and DPPH radical scavenging, and ACE inhibitory activities were expressed as a mean ±SD. 2 The reducing power was expressed as an EC 5 (concentration of the.5 absorbance) value. 3 Mean within the same row with different superscripts are significantly different by Duncan s multiple range test (P<.5). A L-ascorbic acid was used as a positive control of ABTS + and DPPH radicals scavenging and reducing power. B Captopril was used as a positive control of ACE inhibitory activity. ABTS +, 2, 2 -azinobis [3-ethylbenzothiazoline-6-sulfonicacid]; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ACE, angiotensin I-converting enzyme.
13 김소연ㆍ양지은ㆍ송재희ㆍ맹상현ㆍ이지현ㆍ윤나영 Cell viability (%) 1 1 Nitric oxide (%) Control α-chy <3 kda 3-1 kda >1 kda Blank Control α-chy <3 kda 3-1 kda >1 kda Fig. 1. The cell viability of the α-chymotrypsin hydrolysates of Japanese mud shrimp Upogebia major and its molecular weight cut-off fractions in HUVEC. HUVEC, human umbilical vein endothelial cell. L) 37 3, 41 nm. captopril, ACE ACE 5% IC 5 (mg/ml). 세포배양 HUVEC 2% fetal bovine serum (FBS) vascular endothelial growth factor (VEGF) Endothelial Growth Medium-2 (EGM TM -2 Medium) 37, 5% CO 2. 세포독성측정 HUVEC WST-1 cell viability. 96 well plate HU- VEC 5 1³ cells/well,, well WST-1 1 L 2, ELISA reader (US/MQX ; Bio-Tek Instruments Inc., Winooski, VT, USA) 45 nm. Nitric oxide (NO) 생성억제능측정 HUVEC 6 well plates 24, 2 TNF- ( ng/ml)., lysis buffer L cell, 14, rpm 1. L Griess reagent L, 5 nm. Nitrite standard NO. Fig. 2. The inhibition effect of the α-chymotrypsin hydrolysates of Japanese mud shrimp Upogebia major and its molecular weight cut-off fractions on the production of nitric oxide (NO) in TNF-αstimulated HUVEC. All data are presented as means±sd of three independent experiments. P<.5 compared with TNF-α treatment only. P<.1, compared with the TNF-α-nontreated group. TNF-α, tumor necrosis factor-α; HUVEC, human umbilical vein endothelial cell. 세포부착인자 (Cell adhesion molecules, CAMs) 생성량측정 HUVEC 6 well plates 24, 2 TNF- ( ng/ml). ELISA kit (R&D systems, Inc., Minneapolis, MN, USA) VCAM-1, ICAM- 1, E-selectin ELISA reader (US/MQX ; Bio- Tek Instruments Inc., Winooski, VT, USA) 45 nm. 사이토카인 (Cytokines) 생성량측정 HUVEC 6 well plates 24, 2 TNF- ( ng/ml). ELISA kit (R&D systems, Inc., Minneapolis, MN, USA) IL-6, IL- 8, MCP-1 ELISA reader (US/MQX ; Bio-Tek Instruments Inc., Winooski, VT, USA) 45 nm. 통계 (SD), SAS (SAS Institute, Cary, NC, USA), Duncan multiple-comparison test Student t-test P<.5.
쏙효소가수분해물의혈관염증억제효과 131 결과및고찰 효소가수분해및가수분해도 8, (Table 1). -Chy (67.91.2%)>Fla (67.84.3%)>Pep (67.47.6%)>Pro (67.32.9%)>Neu (65.76.8%) >Pa (65.9.5%)>Alc (63.82.4%) >Try (63.13.4%). 8 -Chy. 항산화및 ACE 억제활성 DNA,,, (Nardin, 1). cytokine, growth factor,,, apoptosis (Kunsch and Medford, 1999).. angiotensin-ii, superoxide anion, VCAM-1, IL-6, MCP-1. ACE angiotensin-i angiotensin-ii,. ACE (Parish and Miller, 1992; Paul et al., 6; Takahashi et al., 11). ACE 8 (ABTS + DPPH, reducing power) ACE (Table 2). ABTS + IC 5.8-.18 mg/ml, Alc, -Chy, Fla, Neu, Pro. DPPH 8 -Chy IC 5 2.87.18 mg/ml, Reducing power -Chy EC 5 1.77.13 mg/ml. ACE Alc, -Chy, VCAM-1 (%) ICAM-1 (%) E-selectin (%) Fig. 3. The inhibition effect of the α-chymotrypsin hydrolysates of Japanese mud shrimp Upogebia major and its molecular weight cut-off fractions on the expression of adhesion molecules (VCAM- 1, ICAM-1 and E-selectin) in TNF-α-stimulated HUVEC. All data are presented as means±sd of three independent experiments. P<.5 compared with TNF-α treatment only. P<.1, compared with the TNF-α-nontreated group. VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; E- selectin, endothelial-selectin; TNF-α, tumor necrosis factor-α; HUVEC, human umbilical vein endothelial cell. Blank Control α-chy <3 kda 3-1 kda >1 kda Blank Control α-chy <3 kda 3-1 kda >1 kda Blank Control α-chy <3 kda 3-1 kda >1 kda
132 김소연ㆍ양지은ㆍ송재희ㆍ맹상현ㆍ이지현ㆍ윤나영 Pro. 8 ACE -Chy. HUVEC 세포에대한독성 HUVEC -Chy (Fig. 1). -Chy (.125-1 mg/ml), -Chy.125- %,. NO 생성억제효과 NO (Korhonen et al., 5). TNF- HUVEC -Chy NO (Fig. 2). -Chy HUVEC TNF- NO. 세포부착인자 (Cell adhesion molecules, CAMs) 의발현에미치는영향,, (Peter et al., 1997; Koo et al., 14). (VCAM-1, ICAM-1, E-selectin) (IL-6, IL-8, MCP-1), (Bae et al., 8). TNF- HUVEC -Chy (Fig. 3). TNF- VCAM-1 -Chy,,., -Chy 3, <3 kda, 3-1 kda, >1 kda (.125-1 mg/ ml) VCAM-1, >1 kda. HUVEC ICAM-1 -Chy, IL-6 (%) IL-8 (%) MCP-1 (%) 1 Blank Control α-chy <3 kda 3-1 kda >1 kda Blank Control α-chy <3 kda 3-1 kda >1 kda Blank Control α-chy <3 kda 3-1 kda >1 kda Fig. 4. The inhibition effect of the α-chymotrypsin hydrolysates of Japanese mud shrimp Upogebia major and its molecular weight cut-off fractions on the expression of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) in TNF-α-stimulated HUVEC. All data are presented as means±sd of three independent experiments. P<.5 compared with TNF-α treatment only. P<.1, compared with the TNF-α -nontreated group. IL-6, interleukin-6; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; TNF-α, tumor necrosis factor-α; HUVEC, human umbilical vein endothelial cell.
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