KR20180052080A - Composition to prevent the side effect of NSAID and pharmaceutical anti-inflammatory composition having no side effects - Google Patents
Composition to prevent the side effect of NSAID and pharmaceutical anti-inflammatory composition having no side effects Download PDFInfo
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- KR20180052080A KR20180052080A KR1020170121429A KR20170121429A KR20180052080A KR 20180052080 A KR20180052080 A KR 20180052080A KR 1020170121429 A KR1020170121429 A KR 1020170121429A KR 20170121429 A KR20170121429 A KR 20170121429A KR 20180052080 A KR20180052080 A KR 20180052080A
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Abstract
Description
본 발명은 비스테로이드 항염증제(NSAID)의 부작용을 방지 및 부작용이 나타나지 않은 항염용 조성물에 관한 것이다.The present invention relates to anti-inflammatory compositions which prevent the side effects of non-steroidal anti-inflammatory drugs (NSAIDs) and which exhibit no side effects.
비스테로이드 항염증제(NSAID)는 통증이나 염증 관리용으로 전세계적으로 널리 애용되는 약품이다. 하지만 가벼운 침식적 변화에서부터 심각한 궤양에 이르기까지 위장관(GI) 손상을 일으켜 고령 환자의 경우 위 출혈과 위궤양 같은 치명적인 합병증을 일으키는 것으로 알려져 있다. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used worldwide for pain and inflammation management. However, gastrointestinal (GI) damage from light erosion to severe ulcers is known to cause fatal complications such as gastric bleeding and gastric ulcers in older patients.
또한 선택적 COX-2 억제제(coxib) 투여로 GI 손상이 감소하지만 GI에 안전한 coxib도 COX-1 발현에 따라 심혈관(CV)계에 대한 위험이 있다. coxib은 비선택적으로 NSAID만 투여 또는 양성자펌프억제제(PPI)와 함께 투여한 것보다 GI 위험을 감소시키는데 더 큰 효과를 나타내는 것으로 밝혀졌으나, 신장 합병증과 CV 위험은 여전이 이 약품과 관련하여 나타나는 흔한 문제다.In addition, selective COX-2 inhibitor ( coxib ) reduces GI damage, but GI-safe coxib also poses a risk for cardiovascular (CV) system due to COX-1 expression. c oxib has been shown to have a greater effect in reducing GI risk than non-selective NSAID alone or with PPI, but renal complications and CV risk are still associated with this drug This is a common problem.
그리고 산화질소(NO)를 배출하는 NSAID, phosphatidylcholine(PC)이 결합된 NSAID, 황화수소(H2S)를 배출하는 NSAID와 같이 더 안전한 NSAID를 개발하기 위한 연구가 진행되고 있으나 뚜렷한 진전은 없다.Studies are underway to develop safer NSAIDs, such as NSAIDs that release nitric oxide (NO), phosphatidylcholine (PC) coupled NSAIDs, and hydrogen sulfide (H 2 S) releasing NSAIDs, but no significant progress has been made.
따라서 본 발명이 이루고자 하는 과제는 가벼운 침식적 변화에서부터 심각한 궤양에 이르기까지 위장관(GI) 손상을 일으켜 고령 환자의 경우 위 출혈과 위궤양 같은 치명적인 합병증을 일으키는 것으로 알려져 있는 비스테로이드 항염증제(NSAID)의 투여 문제를 해결하는 것이다. Therefore, the object of the present invention is to provide a nonsteroidal anti-inflammatory drug (NSAID) which is known to cause gastrointestinal (GI) injury from light erosion change to severe ulcer and causes fatal complications such as gastric bleeding and gastric ulcer in elderly patients .
상기 기술적 과제를 달성하기 위하여,According to an aspect of the present invention,
본 발명은 ω-3 불포화지방산(ω-3 PUFA)을 활성 성분으로 포함하는 것을 특징으로 하는 비스테로이드 항염증제(NSAID) 부작용 방지용 조성물을 제공한다.The present invention provides a composition for preventing side effects of non-steroidal anti-inflammatory drug (NSAID), which comprises an omega-3 unsaturated fatty acid (omega-3 PUFA) as an active ingredient.
또한 본 발명은 비스테로이드 항염증제(NSAID) 및 ω-3 불포화지방산(ω-3 PUFA)을 활성 성분으로 포함하는 것을 특징으로 하는 비스테로이드 항염증제(NSAID)의 부작용이 방지되는 항염증용 조성물을 제공한다.The present invention also provides a composition for anti-inflammation wherein a side effect of a non-steroidal anti-inflammatory drug (NSAID) is prevented, which comprises a nonsteroidal anti-inflammatory drug (NSAID) and an omega-3 unsaturated fatty acid (? -3 PUFA) .
또 다르게 본 발명은 비스테로이드 항염증제(NSAID) 및 ω-3 불포화지방산(ω-3 PUFA)가 화학적으로 결합되어 활성 성분으로 포함하는 것을 특징으로 하는 비스테로이드 항염증제(NSAID)의 부작용이 방지되는 항염증용 조성물을 제공한다.Alternatively, the present invention provides an anti-inflammatory agent (NSAID) which is non-steroidal anti-inflammatory agent (NSAID) which is chemically combined with non-steroidal anti-inflammatory agent (NSAID) and? -3 unsaturated fatty acid ≪ / RTI >
본 발명에 있어서, 상기 ω-3 불포화지방산이 에이코사펜타에콘산(EPA) 또는 도코사헥사에콘산(DHA)일 수 있다.In the present invention, the ω-3 unsaturated fatty acid may be eicosapentaenoic acid (EPA) or docosahexaeconic acid (DHA).
본 발명에 있어서, 상기 비스테로이드 항염증제(NSAID)가 아스피린, 이부프로펜, 덱시부프로펜, 나프록센, 페노프로펜, 케토프로펜, 덱스케토프로펜, 플루비오프로펜, 옥사프로진, 록소프로펜, 인도메타신, 톨메틴, 술리닥, 에토돌락, 케토록락, 디클로페낙 및 아세클로페낙으로 이루어진 군에서 선택되는 어느 하나 이상일 수 있다.In the present invention, the nonsteroidal anti-inflammatory drug (NSAID) is selected from the group consisting of aspirin, ibuprofen, dexibupropene, naproxen, fenoprofen, ketoprofen, dexketopropene, Penicillin, indomethacin, tolmetin, sulindac, etodolac, ketoroxac, diclofenac, and aceclofenac.
본 발명에 있어서, 활성 성분으로서 상기 ω-3 불포화지방산이 위에 대한 부작용 방지일 경우 1일 60kg 당 0.5g 투여되고 소장 내지 대장에 대한 방지일 경우 1일 60kg 당 3.0g 투여되는 것이 바람직하다.In the present invention, it is preferable that 0.5 g of the ω-3 unsaturated fatty acid as the active ingredient is administered per 60 kg per day when the ω-3 unsaturated fatty acid is prevented from being adversely affected, and 3.0 g per 60 kg per day if the intestine is protected against small intestine.
본 발명에 따르면 ω-3 PUFA가 화학적으로 결합된 NSAID 또는 ω-3 PUFA와 NSAID의 조합을 도입하여 심혈관계 위험을 낮추면서 GI 안전을 향상할 수 있으며, ω-3 PUFA가 세포 독성을 약화시키고 지질뗏목 형성을 억제하고 산화적 스트레스를 완화하고 항염증성 효과를 낸다는 것이 밝혀졌으므로 ω-3 PUFA 기반 NSAID를 차세대 GI 안전 NSAID로 개발이 가능하여 졌다.According to the present invention, it is possible to improve the safety of GI by lowering the risk of cardiovascular diseases by introducing a combination of NSAID or ω-3 PUFA chemically bound with ω-3 PUFA and NSAID, and ω-3 PUFA weakens cytotoxicity 3-PUFA-based NSAIDs could be developed as the next generation GI-safe NSAIDs, as it has been shown to inhibit lipid raft formation, alleviate oxidative stress, and produce anti-inflammatory effects.
도 1은 인도메타신(IND, indomethacin) 500μM 투여 후 위 상피 세포에서 지질뗏목(lipid raft) 조직에 대한 EPA(eicosapentaenoic acid)의 억제 효과에 대한 도면이다.
도 2는 IND 단독 또는 IND와 EPA 병용에 따른 NOX, 세포자멸(Apoptosis) 및 산화적 손상(Oxidative stress)의 변화에 대한 도면이다.
도 3은 IND로 유도된 위 손상에 대한 ω-3 불포화 지방산(ω-3 PUFA)의 완화 효과에 대한 도면이다.
도 4는 IND로 유도된 위 손상에 대한 ω-3 불포화 지방산(ω-3 PUFA)의 완화 효과를 설명하는 분자 메카니즘 도면이다.
도 5는 IND로 유도된 소장 손상에 대한 ω-3 불포화 지방산(ω-3 PUFA)의 완화 효과에 대한 도면이다.
도 6 및 도 7은 위와 소장에서 DHA, EPA 농도와 ω-6, ω-3 PUFA 비율을 비교한 도면이다.
도 8은 비스테로이드 항염증제(NSAID)의 위장관(GI)에 손상에 대한 ω-3 불포화 지방산(ω-3 PUFA)의 완화 효과에 대한 요약 도면이다.Figure 1 is a plot of inhibitory effect of EPA (eicosapentaenoic acid) on lipid raft tissue in gastric epithelial cells after administration of 500 μM of indomethacin (IND).
Figure 2 is a plot of changes in NOX, apoptosis and oxidative stress according to IND alone or in combination with IND and EPA.
Figure 3 is a plot of mitigation effects of omega-3 unsaturated fatty acids (omega-3 PUFAs) on gastric damage induced by IND.
Figure 4 is a molecular mechanism diagram illustrating the mitigating effect of omega-3 unsaturated fatty acids (omega-3 PUFAs) on gastric damage induced by IND.
Figure 5 is a plot of mitigation effects of omega-3 unsaturated fatty acids (omega-3 PUFAs) on intestinal damage induced by IND.
6 and 7 are graphs comparing DHA and EPA concentrations with ω-6 and ω-3 PUFA ratios in stomach and small intestine.
Figure 8 is a summary drawing of the mitigating effect of omega-3 unsaturated fatty acids (omega-3 PUFAs) on injury to the gastrointestinal tract (GI) of non-steroidal anti-inflammatory drugs (NSAIDs).
비스테로이드 항염증제(NSAID)가 위장관(GI)에 유발하는 손상 기전은 다양하다. 하지만 1차적 기전은 cyclooxygenase(COX) 억제와 추후 mucoprotective prostaglandins(PGs)의 감소다. 선택적 COX-2 억제제(coxib) 또는 합성 PG와의 조합 또는 양성자펌프억제제(PPI)가 부작용을 줄이는 데는 일반적인 NSAID보다 우수하다고 알려졌으나, 이런 약품들도 아직 개선이 필요하다. Nonsteroidal anti-inflammatory drugs (NSAIDs) cause a variety of damage mechanisms in the gastrointestinal tract (GI). However, the primary mechanism is cyclooxygenase (COX) inhibition and subsequent reduction of mucoprotective prostaglandins (PGs). Combinations with selective COX-2 inhibitors ( coxib ) or synthetic PGs or proton pump inhibitors (PPIs) are known to be superior to generic NSAIDs in reducing side effects, but these drugs still need improvement.
NSAID는 합성막, 생체막 모두에 분산되는 성질이 있다. 막 인지방질 분자와 상호작용하며 유동성, 휨 강도, 투과성 같이 세포의 ‘gatekeeping' 기능을 변경시킬 수 있는 막의 소수성을 현저히 변화시켜 침식, 궤양, 출혈 같은 필연적인 GI 점막 손상을 유발하여 막 구멍이 형성된다. 따라서 지질뗏목(lipid raft) 조직과 관련된 변화를 예방하여, 결과로 발생한 NSAID로 인한 GI 손상을 최소화할 수 있다. NSAIDs are dispersed in both synthetic and biological membranes. It interacts with membrane or water-repellent molecules and changes the hydrophobicity of the membrane which can change the 'gatekeeping' function of cells such as fluidity, flexural strength and permeability, thus causing inevitable GI mucosal damage such as erosion, ulceration and hemorrhage, do. Thus, it is possible to prevent changes associated with lipid raft tissue and to minimize GI damage due to the resulting NSAIDs.
NSAID가 막 안정성을 변형시키면서 불안정한 구멍 형성을 유도하고 막 파열 후 루미날 산을 역확산시킬 때 ω-3 고도불포화 지방산(ω-3 PUFA)은 멤브레인 간의 연계(liaison)을 형성하여 gatekeeping 기능을 강화시켜서 NSAID의 역할을 줄이게 된다. When ω-3 polyunsaturated fatty acids (ω-3 PUFAs) form a liaison between the membranes to enhance gatekeeping function, when NSAIDs modify membrane stability and induce unstable pore formation and reverse diffusion of luminal acid after membrane rupture To reduce the role of NSAIDs.
ω-3 PUFA는 막 구조 수정 및 막 지질뗏목 조직 배분, 세포소멸과 관련된 신호를 보내어 세포를 비활성시켜서 막을 안정시키고, 이에 따라 ω-3 PUFA가 NSAID의 세포독성 효과에 대해 gatekeeper로 작용한다는 가설을 설정했다. The ω-3 PUFA hypothesized that ω-3 PUFA acts as a gatekeeper for the cytotoxic effects of NSAIDs by signaling membrane structure modification, membrane lipid raft tissue distribution, and cell extinction signals to inactivate cells to stabilize the membrane Setting.
이전 연구들의 한계를 극복하기 위해 ω-3 PUFA의 gatekeeping 작용이 GI 안전을 향상시킬 수 있다는 전제 하에 fat-1 유전자 이식(TG) 쥐와 쥐들의 in vitro 기전에서의 indomethacin (IND)과 관련된 GI 손상에 대한 내생적으로 합성된 ω-3 PUFA의 효과를 기록했다. To overcome the limitations of previous studies, GI injury associated with indomethacin (IND) in the in vitro mechanism of fat -1 gene transplantation (TG) mice and rats, assuming that the gatekeeping action of ω-3 PUFA can improve
n-6 PUFA를 n-3 PUFA14로 변환시킬 수 있는 벌레인 예쁜꼬마선충(Caenorhabditis elegans)으로부터 n-3 불포화화효소를 부호화하는 fat-1 유전자를 운반하도록 조작된 fat-1 TG 쥐를 야생형 쥐와 비교했다. pretty little the n-6 PUFA in insect that can be converted to n-3 PUFA14 nematode (Caenorhabditis elegans) from the n-3 unsaturated enzyme the fat -1 TG mice engineered to carry the fat-1 gene encoding a wild-type mice to .
본 연구는 gate-keeping 기능을 향상시켜 GI 안전성을 개선할 수 있는 ω-3 PUFA와 ω-3 PUFA가 결합된 NSAID 개발에 관한 것으로서, 본 연구에 대하여 상세하게 설명하기로 한다.This study describes the development of NSAIDs combined with ω-3 PUFAs and ω-3 PUFAs to improve GI safety by improving the gate-keeping function.
Ⅰ시험 물질 및 방법Ⅰ Test substance and method
1. 시약1. Reagents
NSAID와ω-3 PUFA 중에서 IND과 EPA를 각각 Cayman 및 Sigma Aldrich(St. Louis, MO)에서 구입했다. RT-PCR용 Primer는 Macrogen(한국 서울)에서 합성했다. 항체는 Cell Signaling Technology (Beverly, MA)와 Santa Cruz Biotechnology (Santa Cruz, CA)에서 구입했다.Among the NSAID and omega-3 PUFAs, IND and EPA were purchased from Cayman and Sigma Aldrich (St. Louis, Mo.). Primer for RT-PCR was synthesized in Macrogen (Seoul, Korea). Antibodies were purchased from Cell Signaling Technology (Beverly, MA) and Santa Cruz Biotechnology (Santa Cruz, CA).
Horeseradish peroxidase 결합 anti-mouse/rabbit/goat IgG는 Santa Cruz Biotechnology에서 구입했다.Horeseradish peroxidase conjugated anti-mouse / rabbit / goat IgG was purchased from Santa Cruz Biotechnology.
2.세포와2. Cells 세포 독성 분석 Cytotoxicity analysis
쥐 위 점막 세포인 RGM1은 37℃에서 CO2 5%를 함유한 습한 대기에서 유지됐으며 fetal bovine serum 10%(v/v)와 penicillin 100U/ml가 함유된 Dulbecco의 수정된 Eagle의 medium으로 배양됐다. 세포는 각각 DMSO 또는 에탄올에서 IND 또는 EPA로 처리됐고 본문과 도면에 기재된 최종 농도에서 사용됐다. 세포의 세포 독성은 MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] 분석으로 측정됐다.RGM1, a mouse gastric mucosal cell, was maintained in a humid atmosphere containing 5% CO 2 at 37 ° C and cultured in Dulbecco's modified Eagle medium containing 10% ( v / v ) fetal bovine serum and 100 U / ml penicillin . Cells were treated with IND or EPA in DMSO or ethanol, respectively, and used at the final concentrations described in the text and drawings. Cell cytotoxicity was measured by MTT, [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] assay.
3. 3. DichlorofluorescinDichlorofluorescin diacetatediacetate ( ( DCFDCF -DA) 측정-DA) measurement
RGM1 세포에서 ROS의 축적을 파악하기 위해 fluorescence 생성 probe DCF-DA를 사용하여 모니터링 했다. 세포는 HBSS 용액으로 헹궜고 10 μM DCF-DA로 채워졌다. 37℃에서 30분 동안 둔 후 세포를 유동 세포 분석법으로 분석했다. The accumulation of ROS in RGM1 cells was monitored using a fluorescence-generating probe DCF-DA. Cells were rinsed with HBSS solution and filled with 10 μM DCF-DA. After incubation at 37 ° C for 30 minutes, the cells were analyzed by flow cytometry.
4. Hydroxyl radical 측정을 위한 4. For Hydroxyl radical measurement electroelectro spin resonance ( spin resonance ( ESRESR ) 분광학 ) Spectroscopy
SAC의 여러 농축액은 실내온도에서 0.05 mM FeSO4, 1 mM H2O2, 1 mM 5,5-dimethylpyrroline-Noxide (DMPO, Sigma), 5-tert-Butoxycarbonyl-5-methyl-1-pyrroline-Noxide(BMPO, Enzo, Plymouth Meeting, PA, USA), 50 mM, pH 7.4 sodium phosphate를 함유한 총 200㎕에 추가됐다. H2O2를 추가하여 반응을 유발했다. 1분 동안 둔 후 반응을 보이는 부분 표본을 quartz cell로 옮겼으며 DMPO-OH 및 BMPO-OH의 스펙트럼을 ESR 분광 광도계(JES-TE300, JEOL, Tokyo, Japan)를 사용하여 다음과 같은 조건 하에 조사했다. 자기장: 338.0 ±5.0 mT, 마이크로파 전력: 4.95 mW, 주파수: 9.421700 GHz, 진폭 변조: 5mT, 소인 시간: 0.5분, 시상수: 0.03초.Several concentrates of SAC were incubated at room temperature with 0.05 mM FeSO 4 , 1 mM H 2 O 2 , 1
5. 총 세포 5. Total cells 용해물로부터From the melt 지질뗏목Geological raft 격리 Isolation
RGM1 세포는 10cm 세포배양 10 cm에서 2-7 X 107로 seed 됐고 하룻밤동안 그대로 뒀다. 1일 후 IND 또는 EPA를 사용했다. 그런 다음 세포를 모아서 Triton X-100의 Cell Lysis buffer로 용해했다. Caveolae/뗏목 격리 kit는 Sigma (Saint Louis, Missouri, USA)에서 구입했으며 제조사 지침에 따라 사용했다. 변화도로부터의 각 분수의 대푯값은 caveolae 및 뗏목 표시자로 anti-caveolin1 및 Cholera Toxin B Subunit-Peroxidase (CTB-HRP)를 사용하는 면역 화학 및 면역 blotting으로 분석했다. RGM1 cells were seeded at 2-7 × 10 7 at 10 cm in 10 cm cell culture and left overnight. One day later, IND or EPA was used. Cells were then collected and lysed with Cell Lysis buffer in Triton X-100. The caveolae / raft isolation kit was purchased from Sigma (Saint Louis, Missouri, USA) and used according to the manufacturer's instructions. Representative values of each fraction from the degree of variance were analyzed by immunochemistry and immunoblotting using anti-caveolin1 and Cholera Toxin B Subunit-Peroxidase (CTB-HRP) as caveolae and raft indicators.
6. 동물6. Animals
C57BL/6 쥐는 Orient bio(한국 서울)에서 구입했고 순수 C57BL/6 배경의 fat-1 TG 쥐는 Dr. Kang JX (Harvard Medical School, Boston, MA)14에게서 구입했다. 쥐들은 프로젝트를 위한 시설에서 길렀고 유전자형이 형성됐다. fat-1 TG 쥐는 추출된 tail DNA를 사용하여 PCR에 의해 검증됐다. PCR primer의 시퀀스는 Jackson Laboratories와 Dr. Kang JX에서 추천됐다. 동물은 실험 동물 관리 국제 평가 및 인증 협회(가천대학교 AALAC) 정책에 따라 공인된 동물 시설에서 관리했다. 동물 연구는 가천대학교 동물 관리 및 사용 센터(CACU) 위원회의 승인을 받았다(승인번호: LCDI-2010-0038).C57BL / 6 mice were purchased from Orient bio (Seoul, Korea) and fat-1 TG mice with pure C57BL / 6 background Kang JX (Harvard Medical School, Boston, MA). Rats were raised in facilities for the project and genotyped. Fat-1 TG mice were verified by PCR using extracted tail DNA. Sequences of PCR primers were obtained from Jackson Laboratories & Recommended by Kang JX. The animals were administered at accredited animal facilities in accordance with the International Association for Testing and Management of Animals (AACAC). Animal studies have been approved by the CACU Committee of Gachon University (approval number: LCDI-2010-0038).
7. 동물 실험 절차7. Animal test procedure
5주된 암컷 C57BL/6과 fat-1 TG쥐를 23℃로 유지되는 우리에서 특정한 무병 조건 하에 낮밤 각각 12시간 주기로 하여 길렀다. 1주일 후 6주된 1822g 체중의 쥐를 실험에 사용했다. 위 궤양과 소장 손상은 IND의 위장 투여로 유도됐다. 그룹을 4개로 나눴다. C57BL/6 WT와 fat-1 TG 쥐는 24시간 동안 금식됐고 구강 투여로 식염수나 IND를 공급했다. 위궤양 손상과 소장 손상 모델의 쥐는 16, 48시간 후 각각 희생되어 위 및 소장 조직을 수집하여 상세한 분자 연구로 분석됐다.Five-week-old female C57BL / 6 and fat-1 TG rats were raised at 23 ℃ for 12 hours each night under specific disease-free conditions. One week later, a 6-week-old rats weighing 1822 g were used in the experiment. Gastric ulcer and small bowel injury were induced by gastric administration of IND. I divided the group into four. C57BL / 6 WT and fat- 1 TG rats were fasted for 24 hours and either oral saline or IND was administered via oral administration. The mice in the gastric ulcer lesions and small intestine lesions were sacrificed at 16 and 48 hours, respectively, and gastric and small intestine tissues were collected and analyzed for detailed molecular studies.
8. 8. HematoxylinHematoxylin 및 Eosin (H&E) staining 그리고 면역 조직 화학 And Eosin (H & E) staining and immunohistochemistry
조직병리학적 분석을 위해 위와 소장는 10% neutralized buffered formalin으로 처리되고 표준방법으로 처리되어 paraffin에 넣어졌다. 4-㎛ 두께의 단면은 H&E로 stain 됐다. Corpus와 antrum의 선 점막은 조직학적으로 검사됐다. 병리학적 지수는 조건에 따라 결정됐다. 병리학적 데이터와 슬라이드는 3개의 개별 GI 전문가가 맹검으로 검토했다. 면역조직화학의 경우, paraffin unstained 슬라이드를 탈 파라핀화하고 재수하여 각 5분 동안 850 W 전자레인지에서 5% 요소가 함유된 100 mM Tris-buffered saline (pH 7.6)에 넣어 끓였다. 단면들은 Claudin 1로 배양하고 0.1 % bovine serum albumin이 있는 ZO1 항체로 배양하여 최종적으로 4℃에서 24시간 동안 배양했다. 단면들은 hematoxylin으로 counterstain 했다. For histopathological analysis, stomach and intestine were treated with 10% neutralized buffered formalin, treated with standard methods and placed in paraffin. The 4-μm thick section was stained with H & E. Corpus and antrum were examined histologically. The pathological index was determined by the conditions. Pathological data and slides were reviewed blindly by three individual GI experts. For immunohistochemistry, paraffin unstained slides were de-paraffinized and lyophilized and boiled in 100 mM Tris-buffered saline (pH 7.6) containing 5% urea in an 850 W microwave oven for 5 min each. The sections were incubated with
9. Terminal Terminal 9 deoxynucleotidyldeoxynucleotidyl transferasetransferase -mediated -mediated dUTPdUTP nick end labeling (TUNEL) 분석 nick end labeling (TUNEL) analysis
in situ 세포 세포 소멸 감지 kit(Promega, Madison, WI)를 사용하여 TUNEL 방법으로 세포 독성 파악. Paraffin block 조직 슬라이드는 탈 파라핀화 되어 paraformaldehye로 투과됐다. 슬라이드는 TdT와 fluorescein-dUTP 또는 TMR-dUTP를 함유한 UNEL 반응혼합물로 배양됐다. 이 배양 단계 동안 TdT는 DNA의 free 3’OH end에 fluorescein-dUTP가 붙는 것을 촉진했고 형광현미경으로 병합된 fluorescein을 시각화 했다. Detection of cytotoxicity by TUNEL method using in situ cell apoptosis detection kit (Promega, Madison, WI). Paraffin block tissue slides were deparaffinized and permeated with paraformaldehyde. Slides were incubated with UNEL reaction mixture containing TdT and fluorescein-dUTP or TMR-dUTP. During this incubation step, TdT promoted the attachment of fluorescein-dUTP to the free 3 'OH end of DNA and visualized the fluorescein incorporated by fluorescence microscopy.
10. 10. CytokineCytokine 분석 및 ELISA Analysis and ELISA
쥐의 cytokine 항체 분석은 R&D Systems에서 구입했고 제조사 지침에 철저히 따르면서 시행됐다. 각 그룹 대표적 사례의 300 microgram을 이 분석에 사용했다. 쥐 IL1β 및 IL-6 (R&D Systems)에 대한 ELISA kit를 구입했고 제조사 지침에 철저히 따라 사용했다.The cytokine antibody analysis of rats was purchased from R & D Systems and followed strictly according to the manufacturer's instructions. We used 300 micrograms of each representative group for this analysis. An ELISA kit for mouse IL1 [beta] and IL-6 (R & D Systems) was purchased and used thoroughly according to the manufacturer's instructions.
11. 통계 분석11. Statistical Analysis
데이터는 ± SD로 나타냈다. Unpair된 결과에 대한 Tukey test 또는 Student t를 사용하여 3개 그룹 이상 또는 2개 그룹 간에 차이를 각각 평가했다. 차이는 P < 0.05 수치에 대하여 유의한 것으로 나타났다.Data are presented as ± SD. Tukey test or Student t for unpaired outcomes were used to assess differences between groups of three or more groups or between two groups, respectively. The difference was significant for P <0.05 values.
Ⅱ 결과Ⅱ Results
1. One. EicosapentaenoicEicosapentaenoic acid(EPA)는 gatekeeper로 작용하여 acid (EPA) acts as a gatekeeper IND로With IND 인한 Due to 지질뗏Geological raft 목 조직형성을 막으며 GRP120에 결합 후 caveolin-1의 비활성화를 막는다. Block the formation of cervical tissue and block the inactivation of caveolin-1 after binding to GRP120.
NSAID로 인한 막 구조의 변화로 표면 장력, 막 partition, 지질 형태가 변하고 막 이중층 안정성에 변화되어 불안정한 구멍이 형성된다. 아스피린 또는 NSAID 분자의 partitioning으로 막 두께가 얇아지고 휨 강도가 떨어져서 막에 구멍이 생기게 된다. NSAID의 직접적인 세포 독성을 확인하기 위해 전자를 집중 빔으로 스캔하여 샘플 이미지를 생성하는 전자현미경 유형의 전자 현미 기술(SEM)로 IND 투여 16시간 후에 스캔을 실시하였고 도 1a는 IND(500μM, 16시간) 노출로 세포 표면에 수포가 발생하고 막에 구멍이 생기고 기저막이 분리되는 등 세포 손상이 분명하게 드러났다. 하지만 EPA와 IND를 함께 투여하여 이러한 변화가 유의하게 완화되어 EPA가 IND에 대한 gate-keeping 기능을 하는 것을 알 수 있다. Changes in membrane structure due to NSAIDs change the surface tension, membrane partition, and lipid morphology, resulting in changes in membrane bilayer stability resulting in unstable pores. Partitioning of aspirin or NSAID molecules results in thinning of the membrane and loss of bending strength resulting in pores in the membrane. Scanning was performed 16 h after administration of IND with electron microscopy (SEM) of the type of electron microscope, in which electrons were scanned with a focused beam to produce a direct cytotoxicity of the NSAID, and IND (500 μM, 16 h ) Exposure resulted in blisters on the cell surface, pores in the membrane, and separation of the basement membrane. However, administration of both EPA and IND significantly alleviates these changes, indicating that the EPA performs a gate-keeping function for the IND.
도 1a는 RGM-1 세포 표면에서 가교결합된 GM1 지질 극소범위를 보여준다. 이 범위는 콜레라독소 subunit B (CTB)-FITC로 시각화되었다. Glycosphingolipid인 GM1 ganglioside가 지질뗏목과 포낭 내에서만 partitioning 하는 것으로 밝혀졌고 ganglioside GM1에 대한 CTB의 결합이 지질뗏목의 표시로 수용되었으므로 그룹들의 확산 패턴을 비교했다. 극소범위를 형성하는 500μM IND로 처리된 세포와 비교했을 때 콜레라 독소 가교에 의한 지질 극소범위 clustering은 처리되지 않은 세포에 비교하여 10μM EPA가 있을 때 유의하게 약화됐다. IND는 지질뗏목 clustering을 유도하여 지질뗏목 조직을 형성하고, IND와 EPA를 동시에 사용하면 이 효과가 완화되어 지질뗏목이 방해를 받게 된다. 따라서 EPA로 인한 gatekeeping 기능은 즉각적인 NSAID 손상으로부터 유의하게 보호하는 작용을 한다. GM1 염색은 CTB-HRP 활동을 반영하기 위해 GM1 dotblot 분석으로 추후 평가됐다 (도 1b 참조). IND가 지질뗏목 극소범위에서 caveolin-1 localization에 영향을 미치는지 여부를 판단하고자, IND 또는 IND와 10μM EPA를 함께 사용한 RGM-1 세포의 Triton X-100 용해물을 설탕 밀도 기울기에 대하여 분류했다. 기울기 원심법의 직렬 분수로 지질뗏목 표시자로 자주 사용되는 caveolin-1은 통제 세포에서 분수 3-5로 강하게 축적됐고 IND로 처리된 세포에서 조직형성이 파악됐다(도 1b 참조). Figure 1 A shows the GM1 lipid microcapsules cross-linked at the RGM-1 cell surface. This range was visualized as cholera toxin subunit B (CTB) -FITC. GM1 ganglioside, a glycosphingolipid, was found to partition only within lipid rafts and cysts, and the association of CTBs with ganglioside GM1 was accepted as an indication of lipid rafts, so the diffusion patterns of the groups were compared. Lipid minimal range clustering by cholera toxin crosslinking was significantly attenuated when 10 μM EPA was present compared to untreated cells when compared to cells treated with 500 μM IND forming a minimal range. IND induces lipid raft clustering to form lipid raft tissue, and using IND and EPA simultaneously alleviates this effect, interfering with the lipid raft. Therefore, the EPA-induced gatekeeping function protects against immediate NSAID damage. GM1 staining was subsequently assessed by GM1 dotblot analysis to reflect CTB-HRP activity (see FIG. 1b). To determine whether IND affects caveolin-1 localization in a lipid raft subspecies, Triton X-100 solubles of RGM-1 cells using either IND or IND and 10 μM EPA were sorted for the sugar density gradient. Caveolin-1, which is frequently used as a lipid raft marker as a tandem fractional tandem fraction, was strongly accumulated in control cells at fractions of 3-5 and tissue formation was determined in IND-treated cells (see FIG.
EPA가 IND로 인한 지질뗏목 조직 형성을 유의하게 억제하지만, 이들 실험은 IND로 인한 지질뗏목 형성을 일관되게 보여준다. 기능적으로 Fyn , Abl, Src 같은 비수용체 타이로신 키나아제를 위한 기질인 caveolin-1은 구조 단백질로 작용하고, 활성산소(ROS) 같은 외부 자극에 반응할 때 이런 키나아제에 의해 tyrosine 14에서 인산화될 수 있다. 이런 타이로신 인산화는 downstream signalling target을 활성화 시켜 세포 내 신호를 위한 중요한 단계로 작용하여 세포독성을 유발한다. Although EPA significantly inhibited the formation of lipid raft tissue due to IND, these experiments consistently show lipid raft formation due to IND. Functionally, caveolin-1, a substrate for non-receptor tyrosine kinases such as Fyn , Abl , and Src , acts as a structural protein and can be phosphorylated by
따라서, IND만 투여 또는 EPA와 IND를 함께 투여한 후 caveolin-1로 면역침강반응 시험을 시행했고 인산화 티로신 항체로 단백질흡입법을 시행했다. 도 1c에서 보는 바와 같이 EPA는 NOX-1 발현 뿐 아니라 IND로 인한 caveolin 인산화도 유의하게 비활성화 했는데 이것은 막 결합 효소 복합체인 NOX-1의 발현이 IND 치료 후 증가했으며(도 1c 참조), 이것이 막 포낭에서 지질뗏목 해체의 일부인 것을 의미한다. 또한 IND이 지질뗏목을 형성하여 신호를 보내어 세포사 및 염증을 일으키므로 ω-3 PUFA는 IND로 인한 지질뗏목 형성을 차단하여 세포사 신호를 방해하고 염증 증식을 막을 수 있다. G단백질공역수용체 120(GPR120)은 ω-3 PUFA 수용체로 강한 소염, 인슐린 민감성 효과와 같은 여러 기능을 완화한다21. 따라서, 이러한 지질뗏목 해체가 EPA에 의한 것인지 판단하기 위해 야생형 GRP120과 GRP120-knockout 세포에 있는 세포로 실험을 반복했다. RGM-1 세포가 불특정 및 특정 GPR120-siRNA로 세포감염된 후 세포성장에서는 유의한 변화가 관찰되지 않았는데 이것은 GPR120-knockout 세포가 정상적으로 성장했으며 24시간 동안의 증식이 GPR-120-siRNA에 영향을 미치지 않았기 때문이다. GPR120-knockout 세포는 EPA의 보호 효과를 상실했으며(도 1d 참조), 이것은 EPA 작용이 EPA가 결합된 GRP120을 통해 이루어진다는 의미다. EPA를 투여했음에도 EPA로 인한 전형적인 지질뗏목 해체는 GPR120-siRNA 세포에서 보이지 않았는데(도 1e 참조), 이는 EPA 이후 지질뗏목 해체가 IND로 인한 세포 독성으로부터 유의한 보호를 제공했음을 의미한다. Therefore, the IND was administered alone or the EPA and IND were administered together, followed by the immunoprecipitation test with caveolin-1, and the protein inhalation test was carried out with the phosphorylated tyrosine antibody. As shown in FIG. 1c, EPA significantly inactivated NOX-1 expression as well as caveolin phosphorylation due to IND, indicating that expression of NOX-1, a membrane-bound enzyme complex, was increased after IND treatment (see FIG. 1c) Which is part of the decommissioning of the geological raft. In addition, IND forms lipid rafts and sends signals to cause cell death and inflammation, so ω-3 PUFAs block the formation of lipid rafts due to IND, which can interfere with cell death signals and prevent inflammation proliferation. G protein-coupled receptor 120 (GPR120), an omega-3 PUFA receptor, alleviates several functions such as strong anti-inflammatory and insulin-sensitizing effects. Thus, to determine if this lipid raft disruption was due to EPA, the experiment was repeated with cells in wild-type GRP120 and GRP120-knockout cells. There was no significant change in cell growth after RGM-1 cells were infected with specific and specific GPR120-siRNAs, indicating that normal growth of GPR120-knockout cells and 24-hour proliferation did not affect GPR-120-siRNA Because. GPR120-knockout cells lost the protective effect of EPA (see Fig. 1d), which means that EPA action is mediated through EPA bound GRP120. Although EPA was administered, typical lipid raft disruption due to EPA was not seen in GPR120-siRNA cells (see FIG. 1e), which implies that lipid raft decay after EPA provided significant protection against cytotoxicity due to IND.
2. EPA의 Gatekeeper 작용으로 세포 소멸 및 산화 스트레스 완화2. EPA's Gatekeeper action reduces cell death and oxidative stress
이전 보고에서 IND가 ROS를 생성하여 위에서 세포소멸이나 산화적 스트레스를 유발했음을 보여줬으므로, ω-3 PUFA의 gatekeeping 작용으로 ROS를 소기할 수 있고 이로 인해 표적 세포에서 세포소멸이 일어나지 않는다는 가설을 설정했다.Previous reports have shown that IND induces apoptosis or oxidative stress on ROS, leading to the hypothesis that the gatekeeping action of ω-3 PUFA can scavenge ROS and cause cell death in target cells .
IND로 인한 세포 독성이 산화적 스트레스를 일으키므로, MTT 분석으로 500 μM IND 투여 후 세포 생존능을 측정했으며 세포 생존능이 유의하게 감소한 것을 발견했다 (도 2a 참조). Since cytotoxicity due to IND causes oxidative stress, cell viability was measured after administration of 500 [mu] M IND by MTT assay and it was found that the cell viability was significantly reduced (see Fig. 2a).
또한 IND만 투여 후 또는 EPA와 함께 투여 후 Bcl-2, Bax, PARP 같은 세포소멸 관련 유전자의 발현을 측정했다 (도 2b 참조). IND(500 μM)는 Bax 발현을 유의하게 증가시켰고 RGM-1 세포에서 PARP 분할을 증가시켰다. 하지만 RGM-1 세포는 IND와 함께 사용됐으며 10 μM EPA는 Bax와 PARP 분할 발현에서 유의한 감소를 보였다 (p < 0.01). 또한 10 μM EPA는 항세포소멸 단백질인 Bcl-2의 발현을 증가시켰다. IND로 인한 산화물 생성의 원인 대부분이 막 관련 촉매 부분과 세포질 규제 성분을 가진 철저하게 통제되는 다성분 효소인 NOX-1이므로, IND만 투여 후, EPA와 함께 IND 투여 후, EPA 만 투여 후 NOX의 subunit(Nox -1, Nox -2, Nox -3, Nox -4 mRNA)의 변화를 분석했다. 도 2c에서 보는 바와 같이, EPA는 Nox1 mRNA의 발현을 억제했다 (도 2c 참조). Expression of apoptosis-related genes such as Bcl-2, Bax, and PARP was measured after administration of IND only or with EPA (see FIG. 2B). IND (500 μM) significantly increased Bax expression and increased PARP cleavage in RGM-1 cells. However, RGM-1 cells were used with IND and 10 μM EPA showed a significant decrease in Bax and PARP cleavage ( p <0.01). In addition, 10 μM EPA increased the expression of Bcl-2, an anti-apoptotic protein. Since most of the causes of the IND-induced oxidative production are NOX-1, a fully controlled multicomponent enzyme with a membrane-associated catalytic moiety and cytoplasmic regulatory components, only the IND is administered, then the IND is administered together with the EPA, subunit ( Nox- 1, Nox- 2, Nox- 3, Nox- 4 mRNA) were analyzed. As shown in FIG. 2C, the EPA contains Nox1 mRNA (Fig. 2C).
또한 DCF-DA 형광을 사용한 유세포 분석으로 EPA가 변형되지 않는 위점막 세포, RGM-1 세포에서(도 2d 참조) IND 관련 ROS 생성을 감소시키는지 분석했다. IND를 RGM-1 세포에 사용한 후, DCF-DA 형광이 증가했다. 하지만 10 μM EPA와 500 μM IND를 함께 투여한 경우, DCF-DA 형광이 유의하게 억제됐다. DMPO가 없는 급진적인 내전을 사용한 전자 스핀 공명 (ESR)은 분광을 통해 hydroxyl radical을 측정하는 가장 좋은 방법 중 하나다(도 2e 참조). DMPO를 내전기로 사용한 ESR 측정에서는 Fenton 반응으로 생성된 hydroxyl radical의 경우 유의한 peak가 나타났다. 하지만 1 nM 이상의 EPA 농도는 DMPO 내전으로 설명된 대로 hydroxyl radical의 유의한 소기를 보였다. In addition, flow cytometry analysis using DCF-DA fluorescence analysis was performed to determine whether the decrease of IND-related ROS production in gastric mucosal cells, RGM-1 cells (see Fig. 2d), in which EPA was not modified. After using IND in RGM-1 cells, DCF-DA fluorescence increased. However, when 10 μM EPA and 500 μM IND were administered together, DCF-DA fluorescence was significantly inhibited. Electron spin resonance (ESR) using radical adduction without DMPO is one of the best ways to measure hydroxyl radicals through spectroscopy (see Figure 2e). In the ESR measurements using DMPO as internal electric power, hydroxyl radicals generated by Fenton reaction showed a significant peak. However, EPA concentrations above 1 nM showed significant scavenging of hydroxyl radicals as described by DMPO adduction.
3. 3. IND로With IND 인한 위 손상에서 ω-3 Of ω-3 PUFA의Of PUFA 효과 effect
IND로 인한 위 점막 손상을 예방하는 ω-3 PUFA의 효용성을 평가하기 위해 C57BL/6 야생형(WT)과 fat-1 TG 쥐에서 IND로 인한 위 손상을 유도했다 (도 3a 참조). fat-1 TG 쥐가 위에서 ω-3 PUFA를 생성했는지 여부를 확인하기 위해, ω-6 PUFA는 포함됐지만 ω-3 PUFA는 부족한 정상 식단으로 쥐에게 먹이를 먹인 후 WT와 fat-1 TG 쥐의 위 조직에서 지질 지방산의 profile을 IND 투여 후 GC/MS 분석을 사용하여 측정했다. 도 3b에서 보는 바와 같이 ω-6 지방산의 대표적인 arachidonate peak가 WT와 fat-1 TG 쥐에게서 모두 파악됐으나 높은 수치의 α-linolenic acid (ALA), docosa hexaenoic acid (DHA), EPA는 fat-1 TG 쥐의 위에서만 파악됐다. WT와 fat-1 TG 쥐의 모든 수치의 지방산을 측정 후, fat-1 TG 쥐의 ω-3 지방산 수치는 WT 쥐의 ω-3 지방산 수치보다 유의하게 더 높은 것으로 나타났다 (도 3c 참조). IND를 50 mg/kg 투여한 후, WT 쥐와 CMC가 사용된 WT 쥐 그룹 모두에게서 위 손상이 발생했는데 이것은 빠른 위장 출혈을 동반한 위궤양임을 확실히 보여줬다 (도 3d). 하지만 fat-1 TG 쥐 그룹에서는 유의한 병변이 파악되지 않았으며 가벼운 홍반과 작은 침식만 나타났다. 현미경을 봤을 때 16시간 동안 IND에 노출되면 궤양, 침식, 위 점막 염증 같은 위 점막 손상이 분명이 나타났다. 총 병리조직점수(도 3e 참조)는 IND로 인한 WT 그룹에서 증가했으나 IND를 사용한 fat-1 TG 그룹에서는 유의하게 감소했다. 위 점막 침식 증가가 세포소멸적 세포사와 관련이 있는지 평가하기 위해 TUNEL 분석을 시행했다 (도 3f 참조). TUNEL에 양성반응을 보인 상피세포 수는 각 10 section으로 세었고 총 상피세포 비율로 나타냈다. IND 투여 후 WT 쥐의 위 점막에서 TUNEL에 양성반응을 보인 세포 수의 증가가 관찰됐으나 그 수는 fat-1 TG 쥐에서는 더 낮았다. 또한 조직 샘플로 ELISA 분석을 실시하여 IND로 인한 위 손상에 대한 ω-3 PUFA의 예방 효과가 IND로 인한 위 손상에 관여하는 것으로 알려진 interleukin (IL)-1βand IL-6의 억제와 연관이 있는지 파악하고자 했다. 도 3g에서 보는 바와 같이 IND 투여 후 조직 내 IL-1β 와 IL-6의 수치는 모든 그룹에서 높아졌는데 단 fat-1 TG 쥐의 경우에는 더 낮아졌다. IND가 COX 효소의 비활성화로 소염작용을 하지만 COX-2 발현의 증가는 GI 손상을 위한 대표적인 염증성 매개체다. IND was induced in C57BL / 6 wild-type (WT) and fat- 1 TG rats to assess the efficacy of omega-3 PUFAs to prevent gastric mucosal damage due to IND (see Fig. to determine whether the fat-1 TG mice that produce the ω-3 PUFA on, ω-6 PUFA including dwaetjiman ω-3 PUFA are fed after feeding mice a normal diet, lack of WT and fat -1 TG mice The profile of lipid fatty acids in the stomach tissues was measured using IND / GC / MS analysis. As shown in FIG. 3b ω-6 dwaeteuna a representative peak of the fatty acid arachidonate identify all from WT and TG mice fat -1 high levels of α-linolenic acid (ALA), docosa hexaenoic acid (DHA), EPA is fat -1 TG It was identified only on the top of the mouse. After the measurement of all the fatty acid levels in WT and TG mice fat- 1, ω-3 fatty acid levels of the fat- 1 TG mice was found to significantly higher levels than the ω-3 fatty acids in WT mice (see Fig. 3c). After administration of IND at 50 mg / kg, the stomach lesions occurred in both the WT rats and in the WT rats group using CMC, which clearly showed a gastric ulcer with rapid gastric bleeding (Fig. 3d). However, significant lesions were not observed in the fat -1 TG mouse group, and only mild erythema and small erosion were observed. When exposed to IND for 16 hours under microscope, gastric mucosal damage such as ulceration, erosion, gastric mucosal inflammation was evident. Total pathology scores (see FIG. 3e) were increased in the WT group due to IND but significantly decreased in the fat -1 TG group using IND. TUNEL analysis was performed to assess whether increased gastric mucosal erosion is associated with apoptotic cell death (see FIG. 3f). The number of epithelial cells positive for TUNEL was counted in 10 sections and expressed as total epithelial cell ratio. An increase in the number of TUNEL-positive cells in the gastric mucosa of WT rats after IND treatment was observed, but the number was lower in fat- 1 TG rats. In addition, ELISA analysis was performed on tissue samples to determine whether the protective effect of ω-3 PUFAs on gastric damage due to IND was associated with the inhibition of interleukin (IL) -1βand IL-6, which is known to be involved in gastric damage due to IND I want to. As shown in FIG. 3g, the levels of IL-1β and IL-6 in tissues after administration of IND were increased in all groups but lower in fat- 1 TG rats. Induced by the inactivation of the COX enzyme, IND increases the expression of COX-2, a typical inflammatory mediator for GI injury.
도 4a에서 보는 바와 같이, IND 투여로 WT 쥐에서 COX-2 단백질 발현이 증가했으나, fat-1 TG 쥐에서의 COX-2 발현은 IND 투여 후에도 감소했다. Core proteome이 fat-1 TG 쥐에서 IND로 인한 위 손상이나 ω-3 PUFA의 예방 효과와 관련됐음을 명확히 밝히기 위해, WT와 fat-1 TG 쥐의 위에서 고립된 균질화된 단백질로 cytokine 항체 분석을 실시했다 (도 4b 참조) IND 사용 후 다음 유전자들의 발현이 유의하게 증가했다: B lymphocyte chemoattractant (BLC), granulocyte-colony 촉진인자 (G-CSF, 호중성의 전구 물질), GM-CSF, 세포간 접합분자-1 (ICAM-1. CD54라고도 알려짐), interferon gamma(IFN-γ), IL-3, IL-5, IL-7, IL-12, IL-13, IL-21, keratinocyte chemoattractant(KC), lipopolysaccharide으로 인한 CXC chemokine(LIX), 염증단백질-1(MIP-1), 대식세포증식자극인자(MCSF), gamma interferon으로 인한 monokine(MIG, CXCL9), MIP-1α, MIP-1γ thymus, 활성화 통제된 chemokine(TARC, CCL17), metalloproteinase-1의 조직억제제(TIMP1). 하지만 이러한 유전자들의 발현은 fat-1 TG 쥐에서는 감소했다 (도 4b 참조). 또한 IND는 몇몇 in vivo 모델에서 세포소멸 유발 매개체 인식됐다. IND는 Bcl-2계 요소들을 하향 조절하고 FAS 발현을 상향 조절하여 세포소멸을 유도한다. fat-1 TG 쥐에게 IND를 사용하면 FAS 발현이 억제됐고 IND를 사용한 WT 쥐와 비교했을 때 Bcl-2 발현을 유도했는데(도 4c) 이는 fat-1 TG 쥐의 위 손상 예방이 세포소멸 억제를 통한 IND로 유도된다는 것을 의미한다. 숙주는 heme oxygenase-1 (HO-1) 같은 항산화 효소를 활성화 시켜 산화적 변화에 반응한다. 분자샤프롱으로서의 HSP계 요소들은 mitochondria를 보호하고 스트레스로 인한 세포소멸에 간섭하여 위보호 작용을 한다는 의견이 제기됐었다. 도 4d에서 보는 바와 같이, fat-1 TG 쥐에서의 HO-1과 HSP 70의 발현은 IND가 사용된 WT 쥐에서보다 더 높았다.As shown in FIG. 4A, IND administration increased COX-2 protein expression in WT rats, but COX-2 expression in fat- 1 TG rats also decreased after IND administration. Core proteome is subjected to the above damage or ω-3 PUFA to say you that clearly related to the protective effect, cytokine antibodies with a homogeneous protein isolated from the top of WT and fat -1 TG mice in the analysis of fat -1 TG mice due to IND The expression of the following genes was significantly increased after the use of IND: B lymphocyte chemoattractant (BLC), granulocyte-colony stimulating factor (G-CSF, precursor of neutrophil), GM-CSF, intercellular junction IL-13, IL-21, keratinocyte chemoattractant (KC), IL-2, IL-3, IL-5, , monocyte (MIG, CXCL9) due to lipopolysaccharide, CXC chemokine (LIX), inflammatory protein-1 (MIP-1), macrophage proliferative factor (MCSF), gamma interferon, MIP-1α, MIP-1γ thymus, Controlled chemokine (TARC, CCL17), metalloproteinase-1, tissue inhibitor (TIMP1). However, the expression of these genes was decreased in fat- 1 TG rats (see Fig. 4b). In addition, IND was recognized in several in vivo models as a cell destruction-inducing medium. IND induces apoptosis by downregulating Bcl-2 elements and upregulating FAS expression. Using IND in fat- 1 TG rats inhibited the expression of FAS and induced Bcl-2 expression compared to WT rats using IND (Fig. 4c), suggesting that prevention of stomach injury in fat- 1 TG mice inhibited cell death Lt; RTI ID = 0.0 > IND. ≪ / RTI > The host reacts to oxidative changes by activating antioxidant enzymes such as heme oxygenase-1 (HO-1). The HSP elements as molecular chaperone have been suggested to protect the mitochondria and to interfere with the cell death caused by stress, thus acting as a gastric protective effect. As shown in Figure 4d, the expression of HO-1 and HSP70 in fat -1 TG rats was higher than in the WT rats using IND.
4. 4. IND로With IND 인한 장 손상에 대한 ω-3 Ω-3 for intestinal damage PUFA의Of PUFA 효과 effect
IND는 침식과 궤양을 유도하여 크고 소장에서 독성 효과를 나타낸다24. 이전 위장 손상과 비슷한 IND로 인한 창자 손상에 대한 ω-3 PUFA의 임상적 효과를 평가하기 위해, WT와 fat-1 TG 쥐에서 IND로 소장에 손상을 유도했다 (도 5a 참조). ω-3 PUFA, ALA, EPA, DHA가 창자에 있는지 확인하기 위해 WT와 fat-1 TG 쥐의 소장 조직의 IND 투여 전후의 지질 지방산 profile을 GC/MS 분석으로 측정했다. 도 5b에서 보는 바와 같이 fat-1 TG 쥐의 ω-3 PUFA 수치가 WT 쥐의 수치보다 유의하게 더 높았다. 그러나 fat-1 TG 쥐는 ω-6 PUFA 수치가 유의하게 낮아졌다 (도 5b 참조). IND로 인한 소장 손상 모델에서는 일부 출혈을 동반한 선형 궤양, 공장, 회장의 장간막 경계를 따라 확장되는 홍반성 및 부종 장 점막이 나타났다 (도 5c 참조). IND를 투여한 WT 쥐에서 총병변지수 또는 병적 점수가 유의하게 증가했으나 fat-1 TG 쥐에서는 점수가 낮아진 것을 발견했다. 조직 샘플을 이용하여 실험군들을 비교해서 IND로 인한 소장 손상에 ω-3 PUFA가 예방효과가 염증과 세포소멸 억제에 영향을 미치는지 파악하고자 했을 때, IND를 사용한 치료가 COX-2 발현을 현저하게 유도했으나 IND 치료 후 fat-1 TG 쥐에서 COX-2 발현은 IND로 치료한 WT 쥐의 경우보다 더 낮은 것을 발견했다 (도 5d 참조). 소장에서의 세포소멸 세포사를 평가하고자 TUNEL 분석을 시행했다 (그림 5e). TUNEL에 양성반응을 보이는 세포가 IND 치료 후 WT 쥐의 소장 점막에서 증가했으나 TUNEL 양성반응 세포의 수는 IND 치료 후 fat-1 TG 쥐의 경우에는 현저히 낮아진 것을 발견했다. IND는 Bcl-2 계열 요소를 하향조절하고 FAS 발현을 상향조절하여 (도 5f 참조) 세포소멸을 유도하고 fat-1 TG 쥐의 경우에 이러한 수치가 변화됐다. 밀착접합점이 약한 것은 주로 NSAID로 인한 장 질환의 병인 때문인 것으로 보이며 따라서 각 그룹의 밀착접합점 단백질, claudin-1, ZO-1에서의 변화를 측정했다. 밀착접합점은 NSAID 독성을 고려했을 때 세포구조에서 아주 중요한 요소이기도 하다. 여러 연구에 따르면 ZO-1와 claudin-1 같은 핵심 밀착접합점 단백질에서 감소가 나타났고 장누수증후군이 증가했으며 상피전위 저항이 감소한 것이 나타났다. 도 5g에서 보는 바와 같이 IND 투여 이전에 WT 또는 fat-1 쥐의 소장 점막에서 claudin-1과 ZO-1이 풍부한 것으로 나타났다. 하지만 WT 쥐의 경우 IND 투여 후 claudin-1과 ZO-1 수치가 유의하게 감소했으나 at-1 TG 쥐의 경우 변화가 없었다. 이러한 모든 in vivo 동물모델 연구는 ω-3 PUFA가 IND로 인한 위장 또는 창자 손상에 대해 강력한 항염증, 항세포 소멸 및 상피 항상성 작용이 복합적으로 일어나 유의한 예방 작용을 하는 것으로 나타났다. IND induces erosion and ulceration, which is a large and toxic effect in the
5. 5. fatfat -1 TG 쥐와 외인성 ω-3 -1 TG rat and exogenous omega-3 PUFA를PUFA 투여한 쥐 간에 위 및 창자에서의 ω-6 / ω-3 The ω-6 / ω-3 PUFAPUFA 비교 compare
위의 in vivo 데이터는 ω-3 PUFA가 NSAID로 인한 GI 손상을 유의하게 완화한다는 사실을 뒷받침한다. 이에 근거하여 두 가지 의문을 제기할 수 있다. Above in vivo Data confirm that omega-3 PUFAs significantly alleviate GI damage due to NSAIDs. Based on this, two questions can be raised.
(i) NSAID로 인한 GI 손상을 개선하기 위해 외인성 ω-3 PUFA가 얼마나 필요한가? (i) How much exogenous ω-3 PUFA is needed to improve GI damage due to NSAIDs?
(ii) NSAID 투여로 인한 막 변화는 무슨 작용으로 설명할 수 있는가? (ii) What is the effect of membrane changes caused by NSAID administration?
실험에서 각기 다른 양의 ω-3 PUFA를 WT 쥐에 투여했고 위와 소장에서의 n ω-6 / ω-3 PUFA 비율을 측정하여 fat-1 TG 쥐에서의 비율 수치와 비교했다. WT와 fat-1 TG 쥐의 위와 소장 ω-6 / ω-3 PUFA 비율에서 유의한 차이가 나타났다. In the experiment, different amounts of ω-3 PUFAs were administered to WT rats and the ratio of n ω-6 / ω-3 PUFAs in the stomach and small intestine was measured and compared with the ratio values in fat- 1 TG rats. WT and fat- 1 TG rats showed a significant difference in the proportion of ω-6 / ω-3 PUFA in the stomach and small intestine.
이에 대하여 본 발명자들은 여러 양의 ω-3 PUFA(0.5g/60kg ~ 10g/60kg)를 투여하여 위에서의 eicosapentaenoic acid(EPA), docosahexaenoic acid(DHA) 수치를 비교했다. 도 6의 A와 B에서 보는 바와 같이, 0.5g/60kg이상의 ω-3 PUFA를 공급받은 쥐에서 ω-6 PUFAs/ω-3 PUFA 비율이 파악됐으므로, 0.5g/60kg 이상의 ω-3 PUFA는 Fat-1 TG 쥐에서 유사한 패턴을 보였다(도 6의 B). 따라서 위에 대한 부작용을 예방하는 경우에는 0.5g/60kg 이상의 ω-3 PUFA가 바람직하다.In contrast, the present inventors compared the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels of the above by administering various amounts of omega-3 PUFA (0.5 g / 60 kg to 10 g / 60 kg). As shown in Figs. 6A and 6B, omega-6 PUFAs / omega-3 PUFAs were found in rats fed with omega-3 PUFA of at least 0.5 g / 60 kg, Fat- 1 TG mice showed a similar pattern (Fig. 6B). Therefore, when preventing adverse effects on the above, omega-3 PUFAs of 0.5 g / 60 kg or more are preferable.
또한, 본 발명자들은 C57BL/6 WT 쥐에게 다양한 농도의 ω-3 PUFA를 투여한 후 소장의 DHA와 EPA 수치를 측정하여 이 수치들을 fat-1 쥐의 수치들과 비교했다(도 7의 a, b). 본 발명자들은 총 32마리의 쥐(각 그룹 당 n=8)를 사용하였고, 쥐는 8개의 그룹으로 나눠졌으며, fat-1 그룹과 C57BL/6 쥐들은 체중 60kg당 0, 0.5, 1, 2, 3, 5, 10g의 ω-3 PUFA를 1주일 동안 투여 받았다. 외인성 ω-3 PUFA를 1주일 동안 위관 삽입법으로 투여했고 쥐를 희생시켜 소장 조직을 GC/MS/MS로 분석했다. fat-1 TG 쥐의 DHA와 EPA 수치는 ω-3 PUFA를 투여 받지 않은 야생형 쥐보다 유의하게 높았다(p<0.005; 도 7의 a, b). In addition, the present inventors measured DHA and EPA levels in the small intestine after administering various concentrations of omega-3 PUFAs to C57BL / 6 WT rats and compared these values with those of fat-1 rats (Fig. 7a, b). We used a total of 32 rats (n = 8 per group), rats were divided into 8 groups, fat-1 and C57BL / 6 rats weighing 0, 0.5, 1, 2, 3 , 5, and 10 g of omega-3 PUFAs were administered for one week. Exogenous omega-3 PUFAs were administered by gavage for 1 week and the small intestine tissues were sacrificed by GC / MS / MS. DHA and EPA levels in fat-1 TG rats were significantly higher than wild-type rats not receiving ω-3 PUFAs (p <0.005; Fig. 7a, b).
그 결과, fat-1 쥐는 7.5㎍의 DHA와 1.7㎍의 EPA를 소장 10mg 조직 당 가졌다. ω-3 PUFA 3g/60kg이상을 투여 받은 야생형 쥐는 6.3㎍의 DHA와 1.7㎍의 EPA를 소장 조직 10mg당 갖게 됐는데 이것은 fat-1 쥐의 경우와 수치가 비슷하다. ω-3 PUFA의 절대 수치뿐 아니라 ω-6 : ω-3 PUFA의 비율도 중요하다. 건강한 사람의 경우 비율은 4:1~10:1로 유지된다. 하지만, 서양음식 섭취로 ω-3 PUFA가 부족해지면 비율이 15-20:1이 된다. 본 발명자들은 ω-6 : ω-3 PUFA의 비율이 fat-1 쥐에서는 5:1, 야생형 쥐의 경우에는 24:1이 최적이었음을 파악했다(도 7의 c).As a result, fat-1 rats had 7.5 mg of DHA and 1.7 g of EPA per 10 mg of tissue in the small intestine. Wild-type rats receiving ω-3 PUFA 3 g / 60 kg or more received 6.3 μg of DHA and 1.7 μg of EPA per 10 mg of the small intestine, which is similar to that of fat-1 rats. The ratio of ω-6: ω-3 PUFA as well as the absolute value of ω-3 PUFA is also important. For healthy people the ratio is maintained between 4: 1 and 10: 1. However, when omega-3 PUFAs are lacking due to ingestion of Western foods, the ratio is 15-20: 1. We found that the ratio of omega-6: omega-3 PUFAs was 5: 1 in fat-1 rats and 24: 1 in wild-type rats (FIG.
하지만 야생형 쥐에게 ω-3 PUFA를 투여하면 비율이 6-10:1로 최적값이 변했다(도 7의 c). ω-3 PUFA 섭취로 효과를 보려면 최소 3g/60kg의 ω-3 PUFA를 투여해야 한다는 결론을 내렸으며, 따라서 소장 내지 대장의 부작용을 방지하는 경우에는 최소 3g/60kg의 ω-3 PUFA을 투여하는 것이 바람직하다.However, when the ω-3 PUFA was administered to the wild-type mice, the optimum value was changed to 6-10: 1 (FIG. 7C). We concluded that a minimum of 3 g / 60 kg of omega-3 PUFAs should be administered to achieve efficacy with omega-3 PUFA ingestion. Therefore, omega-3 PUFAs of at least 3 g / 60 kg should be administered to prevent small bowel or intestinal side effects .
결론적으로 ω-3 PUFA의 gatekeeper 기능은 지질뗏목 형성, NSAID의 caveolin-1 인산화를 통한 NOX-1 활성화 같은 NSAID관련 세포 독성을 판단하는데 아주 중요한 것으로 보인다. 이 in vivo 데이터를 바탕으로 ω-3 PUFA와 NSAID의 조합은 NSAID로 인한 GI 손상을 완화할 수 있다. In conclusion, the gatekeeper function of ω-3 PUFA appears to be crucial in determining NSAID-related cytotoxicity, such as lipid raft formation, NOX-1 activation via caveolin-1 phosphorylation of NSAIDs. This in vivo Based on the data, the combination of ω-3 PUFA and NSAID can mitigate GI damage due to NSAIDs.
Ⅲ. 고찰Ⅲ. Review
이 연구에서 ω-3 PUFA를 사용하여 ω-3 PUFA와 NSAID의 조합은 NSAID로 인한 GI 손상과 유의한 caveolin-1 비활성화 및 NOX-1 억제를 동반한 지질뗏목 극소범위 저해를 완화할 수 있는지에 대해 증명하였다(도 8 참조). Using the ω-3 PUFA in this study, the combination of ω-3 PUFAs and NSAIDs could reduce the GI impairment due to NSAIDs and mitigate the caveolin-1 inactivation and lipid-lowering inhibition with NOX-1 inhibition (See FIG. 8).
ω-3 PUFA를 3g/60kg 이상 투여하면 fat-1 TG 쥐에서와 같은 지질농도가 나타나고 NSAID로 인한 GI 손상에 대해 보호 효과가 있었음을 알 수 있었으므로 외인성 ω-3 PUFA 투여를 연구한 이전의 결과들과는 다른 결과를 얻었다. When ω-3 PUFA was administered at a dose of 3 g / 60 kg or more, lipid concentrations similar to those in fat- 1 TG mice were observed and protective effects against GI injury due to NSAID were observed. Results were different from the results.
결론적으로 ω-3 PUFA가 결합된 NSAID가 NSAID 투여 시 GI 안전성 향상에 사용될 수 있으며 NO 배출 NSAID, H2S 배출 NSAID, PC 결합 NSAID 같은 다른 결합 NSAID 또는 현재 coxib의 효용성을 능가한다. In conclusion, the combination of the NSAID ω-3 PUFA can be used in the improved GI safety when administered NSAID and surpasses other combination NSAID or utility of present coxib, such as NO emissions NSAID, H2S emissions NSAID, NSAID combination PC.
coxib가 아닌 일반적인 NSAID가 GI에서의 부작용에도 불구하고 신경 또는 CV 보호에 대한 추가적인 효과를 제공하고 PPI가 NSAID로 인한 장 질환을 확대시킨다는 것이 밝혀졌으므로, 이번 결과는 NSAID 관련 GI 손상 완화와 관련하여 중요하다. It has been shown that general NSAIDs other than coxib provide additional effects on nerve or CV protection despite side effects in GI and that PPIs exacerbate enteric disease caused by NSAIDs, so this result is important for NSAID- Do.
이 연구에서 fat-1 TG 쥐에서 합성된 ω-3 PUFA가 유사한 항염증성, 항세포소멸 기전으로 IND로 인한 위장 손상 또는 소장 손상에 효과가 있다는 것을 발견했다. 또한 IND로 인한 chemokine과 다른 유형의 cytokine을 억제하면서 위 손상 시 HO-1 농도를 높이고 손상으로 인한 밀착접합, claudin-1, ZO-1을 강화했다. 임상적 적용 가능성을 고려하면서 이전 연구에서의 식이성 ω-3 PUFA 투여로 장기 내에 쌓이는 ω-3 PUFA의 최적 농도를 측정했다.In this study, we found that ω-3 PUFAs synthesized in fat- 1 TG mice were effective against similar anti-inflammatory, anti-cytotoxic, gastrointestinal or intestinal damage by IND. In addition, inhibition of chemokine and other types of cytokines caused by IND increased HO-1 levels and enhanced tight junctions, claudin-1 and ZO-1, in the gastric lesions. Taking into consideration the clinical applicability, we measured the optimum concentration of ω-3 PUFA accumulated in the organ by the administration of the dietary ω-3 PUFA in the previous study.
NSAID로 인한 GI 손상은 유동성, 역학 및 세포 투과성과 막 인지질 소수성에 대한 변화 등 COX 억제 그 이상이다. 따라서 NSAID와 접촉한 막에서 이런 변화를 안정시키는 성분은 불안정한 구멍 형성을 억제하고 루미날산의 역확산을 방지하며 막 파열을 예방할 수 있어야 한다. ω-3 PUFA acyl chain을 포함한 막 인지질이 막 뗏목 구조와 구성을 변화시키고 세포 신호에 영향을 주어 세포 독성 변화를 예방하고 막 유동성, 단계 행동, 침투성, 융합, 상주 막의 단백질 활동을 유지하기 때문에 ω-3 PUFA가 이에 알맞은 성분일 것으로 가설을 설정했다. GI impairment due to NSAIDs is more than COX inhibition, including changes in fluidity, mechanics, and cellular permeability and membrane phospholipid hydrophobicity. Therefore, a component that stabilizes this change in the membrane in contact with the NSAID should be able to inhibit unstable pore formation, prevent reverse diffusion of ruminal acid, and prevent membrane rupture. Because the membrane phospholipid containing the ω-3 PUFA acyl chain changes the structure and composition of membrane rafts and prevents cell toxicity changes by affecting cellular signals and maintains membrane fluidity, step behavior, permeability, fusion, -3 PUFAs would be a suitable component.
지질뗏목의 구조, 크기, 기능이 파악됐더라도 유리한 지질-지질 및 지질-단백질 상호작용으로 형성된 원형질막의 nano 크기 범위는 여전히 조사 중이다. 건강질병연구에서 막 극소범위는 지질뗏목이 식단, 특히 식이성 FA36로 변할 수 있기 때문에 의미가 커지고 있다. 식이성 PUFA가 지질뗏목에 실제로 병합될 수 있는지는 아직 확실히 밝혀지지 않았으나 NSAID 관련 세포 소멸 및 염증에서의 지질뗏목의 역할은 NSAID 투여 후 지질뗏목관련의 부정적인 신호를 방해하는 것이다. 이 기전들을 조사하기 위해서 한 연구에서 고체상태 NMR 분광학으로 스핑고지질 또는 콜레스테롤 혼합물의 분자구성을 조사했으며 ω-3 PUFA가 지질뗏목 범위 구성을 방해했다는 확실한 결과가 나왔다. 이것은 연구를 확증하는 결과이며 NSAID로 인한 손상에 대한 유의한 항염증, 항세포 소멸 작용이 ω-3 PUFA의 지질뗏목 해체 작용과 연관이 있음을 보여주는 것이다. Even though the structure, size, and function of lipid rafts are known, the nano size range of plasma membranes formed by favorable lipid-lipid and lipid-protein interactions is still under investigation. In the study of health illness, the microscopic extent of the lipid raft is becoming more significant because it can be changed into a diet, especially dietary FA36. It is not yet clear whether dietary PUFAs can actually be incorporated into lipid rafts, but the role of lipid rafts in NSAID-related cell destruction and inflammation is to interfere with the negative signals associated with lipid rafting after NSAID administration. To investigate these mechanisms, one study investigated the molecular composition of a sphingolipid or cholesterol mixture by solid-state NMR spectroscopy, and there was a clear result that ω-3 PUFA interfered with the geological range configuration. This is the result of confirmation of the study and demonstrates that significant anti-inflammatory and anti-apoptotic effects on NSAID-induced injury are associated with lipid-raft dissolution of ω-3 PUFA.
간략하게 설명하자면, 막 지질뗏목과 단백질의 분자조직을 방해하는 과정은 다음과 같다. ω-PUFA가 뗏목에 병합되고 콜레스테롤이 뗏목이 아닌 것에 재분포되고 뗏목이 병렬되고 뗏목이 아닌 단백질이 병렬된 뗏목으로 격리된다. 지질뗏목은 막 기하학의 주요한 조절자로 작용하여 분자의 수평적 움직임, 이동, 신호 전달, NSAID 관련 세포 소멸을 유발하고 세포 소멸 신호를 전달하는 경로를 만드는데 이것은 뗏목 영역으로부터 시작된다. 이 과정 동안 ω-3 PUFA chain이 세포막 스핑고-지질-콜레스테롤이 풍부한 지질뗏목의 분자구조를 재구성하므로 소위 ω-3 PUFA를 포함하는 ‘방해받은 지질뗏목’은 세포 소멸 신호를 억제하고 IND로 인한 세포 독성을 완화하며 사멸 수용체 단백질, 단백질 kinase, 세포 소멸 관련 칼슘 channel 같은 뗏목관련 단백질을 비활성화할 수 있다. Briefly, the process of interfering with the molecular organization of membrane lipids and proteins is as follows. The ω-PUFAs are incorporated into the raft, the cholesterol is redistributed to the non-raft, the rafts are lined up, and the non-raft protein is sequestered by a parallel raft. The lipid raft acts as a major regulator of membrane geometry, resulting in horizontal movement of molecules, migration, signal transduction, NSAID-related cell death, and a pathway for cellular extinction signals, which begins in the raft area. During this process, the ω-3 PUFA chain reconstitutes the molecular structure of lipid rafts rich in cell membrane sphingo-lipid-cholesterol, so the 'interrupted lipid raft' containing the so-called ω-3 PUFA inhibits the extinction signal, It can mitigate cytotoxicity and inactivate raft-related proteins such as death receptor protein, protein kinase, and apoptosis related calcium channel.
지질뗏목 범위는 막에서 신호 단백질의 clustering을 최적화하여 효율적인 세포 signaling을 도모하는데 이것은 CD4+ T 세포 활성화 및 분화에 필요한 것이고 ω-3 PUFA는 항염증 지질 매개체, resolvin라 불리는 EPA 및 DHA에서 나온 지질 매개체 수치를 증가시킨다. 이에 반해 여러 유형의 염증성 매개체에서 유의한 감소를 파악했다. ω-3 PUFA는 GPR120을 통해 매개되는 것으로 밝혀졌는데 GPR120는 GPR의 rhodopsin 계열 중 하나다. 5개의 희귀수용체인 GPR 40, GPR41, GPR43, GPR84, GPR 120은 FA로 활성화되며 GPR120은 caveolae 막의 주요 성분인 필수 막 단백질 계열인 caveolin-1에 결합된다. 이것들은 특별한 유형의 지질뗏목들로 GPCR를 위한 signaling platform , 특정 tyrosine kinase 수용체 등 세포 기능을 통제하며 이들 뗏목/caveolae는 redox signaling(산화환원 신호전달)에 영향을 미칠 수 있다. 연구에서 ω-3 PUFA 투여 후 이들 지질뗏목 해체가 GRP120을 통해 이루어지는 것을 파악했다.The lipid raft range optimizes clustering of signal proteins in the membrane to promote efficient cell signaling, which is required for CD4 + T cell activation and differentiation, and ω-3 PUFA is a lipid mediator, derived from the EPA and DHA called anti-inflammatory lipid mediators, resolvin . On the other hand, we found a significant decrease in inflammatory mediators of various types. The ω-3 PUFA has been shown to be mediated through GPR120, which is one of the rhodopsin family of GPRs. Five rare receptors, GPR40, GPR41, GPR43, GPR84 and GPR120, are activated by FA, and GPR120 binds to caveolin-1, the essential membrane protein family, a key component of the caveolae membrane. These are special types of lipid rafts that control cellular function, such as the signaling platform for GPCRs, specific tyrosine kinase receptors, and these rafts / caveolae can affect redox signaling. Studies have shown that disintegration of these lipid rafts occurs via GRP120 after ω-3 PUFA administration.
결론적으로 atherosclerosis 예방용으로 이미 병원에 소개된 omega-3-acid ethyl ester capsule (Lovaza, Omarcor)처럼, ω-3 PUFA가 화학적으로 결합된 NSAID 또는 ω-3 PUFA와 NSAID의 조합을 도입하여 심혈관계 위험을 낮추면서 GI 안전을 향상할 수 있을 것이다. ω-3 PUFA가 세포 독성을 약화시키고 지질뗏목 형성을 억제하고 산화적 스트레스를 완화하고 항염증성 효과를 낸다는 것이 밝혀졌으므로 ω-3 PUFA 기반 NSAID를 차세대 GI 안전 NSAID로 개발이 가능하여 졌다.In conclusion, the combination of ω-3 PUFA chemically bound NSAID or ω-3 PUFA and NSAID, such as the omega-3-acid ethyl ester capsule (Lovaza, Omarcor) already introduced in the hospital for the prevention of atherosclerosis, You will be able to improve your GI safety while lowering your risk. It has been shown that omega-3 PUFAs weaken cytotoxicity, inhibit lipid raft formation, alleviate oxidative stress, and exert an anti-inflammatory effect, making it possible to develop ω-3 PUFA-based NSAIDs as the next generation GI-safe NSAIDs.
본 연구 결과를 최종적으로 정리하면 다음과 같다.The results of this study are summarized as follows.
본 연구는 ω-3 불포화지방산(ω-3 PUFA)을 활성 성분으로 포함하는 것을 특징으로 하는 비스테로이드 항염증제(NSAID) 부작용 방지용 조성물을 제공한다.The present invention provides a composition for preventing side effects of nonsteroidal anti-inflammatory drugs (NSAIDs), which comprises an ω-3 unsaturated fatty acid (ω-3 PUFA) as an active ingredient.
또한 본 연구는 비스테로이드 항염증제(NSAID) 및 ω-3 불포화지방산(ω-3 PUFA)을 활성 성분으로 포함하는 것을 특징으로 하는 비스테로이드 항염증제(NSAID)의 부작용이 방지되는 항염증용 조성물을 제공한다.The present invention also provides a composition for anti-inflammation wherein a side effect of a nonsteroidal anti-inflammatory drug (NSAID) is prevented, which comprises a nonsteroidal anti-inflammatory drug (NSAID) and an omega-3 unsaturated fatty acid (? -3 PUFA) as an active ingredient .
본 연구에 있어서, 상기 ω-3 불포화지방산이 에이코사펜타에콘산(EPA) 또는 도코사헥사에콘산(DHA)일 수 있다.In this study, the ω-3 unsaturated fatty acid may be eicosapentaenoic acid (EPA) or docosahexaeconic acid (DHA).
본 연구에 있어서, 상기 비스테로이드 항염증제(NSAID)가 아스피린, 이부프로펜, 덱시부프로펜, 나프록센, 페노프로펜, 케토프로펜, 덱스케토프로펜, 플루비오프로펜, 옥사프로진, 록소프로펜, 인도메타신, 톨메틴, 술리닥, 에토돌락, 케토록락, 디클로페낙 및 아세클로페낙으로 이루어진 군에서 선택되는 어느 하나 이상일 수 있다.In the present study, the non-steroidal anti-inflammatory drug (NSAID) was administered in combination with aspirin, ibuprofen, dexibupropene, naproxen, fenoprofen, ketoprofen, dexketopropene, Penicillin, indomethacin, tolmetin, sulindac, etodolac, ketoroxac, diclofenac, and aceclofenac.
본 연구에 있어서, 활성 성분으로서 상기 ω-3 불포화지방산이 위에 대한 부작용 방지일 경우 1일 60kg 당 0.5g 투여되고 소장 내지 대장에 대한 방지일 경우 1일 60kg 당 3.0g 투여되는 것이 바람직하다.In the present study, it is preferable that 0.5 g of the omega-3 unsaturated fatty acid as an active ingredient is administered per 60 kg per day when the side effect is prevented, and 3.0 g per 60 kg per day when the omega-3 is protected against small intestine.
Claims (7)
A composition for preventing side effects of non-steroidal anti-inflammatory drugs (NSAIDs), which comprises an? -3 unsaturated fatty acid (? -3 PUFA) as an active ingredient.
The composition according to claim 2, wherein the ω-3 unsaturated fatty acid is eicosapentaenoic acid (EPA) or docosahexaeconic acid (DHA).
3. The composition according to claim 1 or 2, wherein 0.5 g of the ω-3 unsaturated fatty acid as an active ingredient is administered per 60 kg per day when the adverse effect on the ω-3 unsaturated fatty acid is inhibited, and 3.0 g per 60 kg per day (NSAID) as an anti-inflammatory agent.
A composition for anti-inflammation wherein the side effect of a non-steroidal anti-inflammatory drug (NSAID) is prevented, which comprises a nonsteroidal anti-inflammatory drug (NSAID) and an omega-3 unsaturated fatty acid (? -3 PUFA) as an active ingredient.
The anti-inflammatory composition according to claim 4, wherein the ω-3 unsaturated fatty acid is eicosapentaenoic acid (EPA) or docosahexaeconic acid (DHA).
5. The method of claim 4 wherein the nonsteroidal anti-inflammatory drug (NSAID) is selected from the group consisting of aspirin, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketopropene, An antiinflammatory composition characterized by comprising at least one selected from the group consisting of lophene, indomethacin, tolmetin, sulindac, etodolac, ketoroxac, diclofenac, and aceclofenac
The method according to any one of claims 4, 5, and 6, wherein 0.5 g of the ω-3 unsaturated fatty acid is administered per 60 kg per day when the ω-3 unsaturated fatty acid as an active ingredient is prevented from adversely affecting the stomach, Wherein the composition is administered in an amount of 3.0 g per 60 kg per day.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020160148151 | 2016-11-08 | ||
| KR20160148151 | 2016-11-08 |
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| Publication Number | Publication Date |
|---|---|
| KR20180052080A true KR20180052080A (en) | 2018-05-17 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020170121429A Ceased KR20180052080A (en) | 2016-11-08 | 2017-09-20 | Composition to prevent the side effect of NSAID and pharmaceutical anti-inflammatory composition having no side effects |
Country Status (2)
| Country | Link |
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| KR (1) | KR20180052080A (en) |
| WO (1) | WO2018088689A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210002037A (en) | 2019-06-28 | 2021-01-06 | 주식회사 엘지생활건강 | A composition for preventing, improving or treating pain comprising olive leaf and citrus pericarp extract |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110071090A1 (en) * | 2009-03-11 | 2011-03-24 | Stable Solutions Llc | Method of mitigating adverse drug events using omega-3-fatty acids as a parenteral therapeutic drug vehicle |
-
2017
- 2017-09-20 KR KR1020170121429A patent/KR20180052080A/en not_active Ceased
- 2017-09-20 WO PCT/KR2017/010347 patent/WO2018088689A1/en active Application Filing
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210002037A (en) | 2019-06-28 | 2021-01-06 | 주식회사 엘지생활건강 | A composition for preventing, improving or treating pain comprising olive leaf and citrus pericarp extract |
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| WO2018088689A1 (en) | 2018-05-17 |
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