KR20010044175A - Method for inhibiting synthesis and secretion of cell activating substances using chitosan and Food composition comprising chitosan for the inhibition - Google Patents
Method for inhibiting synthesis and secretion of cell activating substances using chitosan and Food composition comprising chitosan for the inhibition Download PDFInfo
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- KR20010044175A KR20010044175A KR1020000080624A KR20000080624A KR20010044175A KR 20010044175 A KR20010044175 A KR 20010044175A KR 1020000080624 A KR1020000080624 A KR 1020000080624A KR 20000080624 A KR20000080624 A KR 20000080624A KR 20010044175 A KR20010044175 A KR 20010044175A
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- alcohol
- chitosan
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- tnf
- soluble chitosan
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/334—Foods, ingredients or supplements having a functional effect on health treating the effects of consuming alcohol, narcotics or other addictive behavior, e.g. treating hangover or reducing blood alcohol levels
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
본 발명은 키토산을 이용하여 세포활성물질의 합성 및 분비를 억제하는 방법에 관한 것이다. 또한 본 발명은 키토산을 함유하는 식품 조성물에 관한 것이다. 더욱 상세하게는 본 발명은 분자량이 10,000 내지 1,500,000인 고분자 수용성 키토산을 이용하여 간세포의 알코올-매개성 세포활성물질의 합성 및 분비를 억제하는 방법과, 분자량이 10,000 내지 1,500,000인 고분자 수용성 키토산을 함유하는 식품 조성물에 관한 것이다.The present invention relates to a method of inhibiting the synthesis and secretion of cellular active materials using chitosan. The present invention also relates to a food composition containing chitosan. More specifically, the present invention provides a method for inhibiting the synthesis and secretion of alcohol-mediated cell activator of hepatocytes using a polymer soluble chitosan having a molecular weight of 10,000 to 1,500,000, and a polymer soluble chitosan having a molecular weight of 10,000 to 1,500,000. It relates to a food composition.
알코올은 약한 전하를 띠는 분자로서 세포막 사이를 쉽게 이동하며, 혈액과 조직 사이에서 빠르게 평형을 이룬다. 알코올은 기본적으로 뉴런의 활성도를 감소시키는 중추신경 억제제이다. 국내에서는 간질환 중 바이러스성 간염이 가장 높은 사망률의 원인이지만, 최근에는 알코올성 간질환이 급증하고 있다. 미국과 같은 선진국에서는 바이러스성 간질환보다 알코올성 간질환에 의한 사망률이 5 내지 10배 정도로 높게 나타나고 있는 것으로 보고되고 있다. 따라서 바이러스성 간질환은 최근에 백신 사용 등으로 인해 현저히 감소하는 추세이나, 알코올성 간질환에 의한 사망률은 꾸준히 증가하고 있다. 알코올성 간질환으로는 알콜성 지방간, 알코올성 간염, 알코올성 간경화 등을 들 수 있다.Alcohol is a weakly charged molecule that moves easily between cell membranes and quickly balances blood and tissue. Alcohol is basically a CNS inhibitor that reduces neuronal activity. Viral hepatitis is the leading cause of mortality among liver diseases in Korea, but alcoholic liver disease is increasing rapidly in recent years. In developed countries such as the United States, the mortality rate from alcoholic liver disease is reported to be 5 to 10 times higher than that of viral liver disease. Therefore, viral liver disease has recently decreased considerably due to the use of vaccines, but the mortality rate from alcoholic liver disease is steadily increasing. Alcoholic liver disease includes alcoholic fatty liver, alcoholic hepatitis, alcoholic cirrhosis and the like.
간에서 소량의 지방 변화는 일과성에 그치며 임상 증상으로는 거의 나타나지 않지만, 지속성 또는 고도의 지방 침윤이 있는 경우에는 임상 증상으로 나타난다. 알콜성 지방간, 급성 간염 및 만성 활동성 간염 등에 있어서 증가된 과산화지질은 지질과 단백질로 구성된 세포막에 자유기(free radical)가 작용하여 불포화지방산을 과산화시켜 형성된 것으로서, 그 과산화지질은 세포막의 투과성을 변화시키고, 이로인해 세포막의 파괴를 초래하여 세포독성을 유발시키는 것으로 알려져 있다.Small amounts of fat change in the liver are transient and rarely present in clinical symptoms, but in the presence of persistent or high fat infiltration, they appear as clinical symptoms. Increased lipid peroxide in alcoholic fatty liver, acute hepatitis, and chronic active hepatitis is formed by the peroxidation of unsaturated fatty acids by the action of free radicals on cell membranes composed of lipids and proteins. It is known to cause cytotoxicity, thereby causing destruction of the cell membrane.
상기와 같은 알코올에 의한 독성을 감소시킴으로써 숙취를 제거하고 간을 보호하기 위한 많은 방법들이 개발되어 왔다. 그 중에서도 천연물을 이용하는 방법으로서, 천궁, 감초, 갈근, 진피 등의 생약, 모과, 다시마, 녹차, 매실, 더덕 등의 자연 식품을 이용하는 방법들이 알려져 왔다. 그러나 종래의 이러한 방법들은 알코올에 의한 독성을 감소시키는 특정 성분을 이용한 것이 아니라, 식품 자체를 이용한 것이므로, 그것의 독성 감소 효과는 미미하였다. 따라서 구체적으로 어떠한 특정 성분이 알코올 자체, 아세트알데하이드와 같은 대사물질 또는 알코올 대사과정 중에 발생한 물질의 분비를 억제하거나, 알코올에 의한 면역반응을 조절할 수 있는 지에 대한 연구가 절실히 요구되고 있었다.Many methods have been developed for eliminating hangovers and protecting the liver by reducing such toxicity by alcohol. Among them, as a method of using natural products, methods of using natural foods such as medicinal herbs such as cheonggung, licorice, brown root, dermis, quince, kelp, green tea, plum, and deodeok have been known. However, these conventional methods are not using specific ingredients to reduce the toxicity by alcohol, but using the food itself, so the effect of reducing the toxicity is insignificant. Therefore, in particular, there is an urgent need for a study on whether certain components can suppress the secretion of alcohol itself, metabolites such as acetaldehyde or substances generated during alcohol metabolism, or modulate the immune response by alcohol.
키토산은 부분적으로 탈아세틸화된 키틴으로서, 무독성인 천연 고분자 양이온이다. 키토산은 그 분자 구조로 볼 때 인체 조직에 친화성이 우수하여 거부반응이 일어나지 않기 때문에 식품 및 의약품으로써 우수한 물질이다. 그러나 키토산은 그 물성인 분자량과 탈아세틸화도에 따라 그 사용 용도가 매우 다르게 적용될 수 있다. 분자량과 탈아세틸화도에 따라서 의약 및 식품뿐만 아니라 환경 폐수 응집제, 중금속 흡착제, 생산량 증대를 위한 씨앗 코팅제, 단백질 회수제 및 창상 치료제 등의 다양한 분야에서 이용되고 있다. 그러나 종래의 어떤 문헌도 키토산을 알코올 매개 세포활성물질의 합성 및 분비를 억제시키는 데에 이용하는 것에 관련된 기술을 언급한 적이 없었다. 뿐만 아니라, 알코올 매개 세포활성물질의 합성 및 분비의 억제에 이용되기 위한 키토산의 물성, 즉 키토산의 분자량과 탈아세틸화도가 제시된 적이 없었다.Chitosan is a partially deacetylated chitin, a non-toxic natural polymeric cation. Chitosan is an excellent material for food and medicine because its molecular structure is excellent in affinity with human tissues and no rejection occurs. However, chitosan may be used very differently depending on its physical molecular weight and degree of deacetylation. Depending on the molecular weight and degree of deacetylation, it is used in various fields such as pharmaceutical and food as well as environmental wastewater flocculant, heavy metal adsorbent, seed coating agent for increasing production, protein recovery agent and wound healing agent. However, no prior literature mentions techniques related to the use of chitosan to inhibit the synthesis and secretion of alcohol mediated cell activators. In addition, the physical properties of chitosan, that is, the molecular weight and deacetylation degree of chitosan, have not been suggested for use in the synthesis and suppression of secretion of alcohol mediated cell activators.
본 발명은 상기와 같은 문제점을 해결하기 위한 것으로서, 본 발명의 목적은 간세포에서 알코올에 의해 매개되는 세포활성물질의 합성 및 분비를 억제함으로써, 알코올에 의한 독성을 감소시키고 숙취를 제거하며 나아가 알코올성 간질환 환자의 간을 보호하는 것이다.The present invention is to solve the above problems, an object of the present invention by inhibiting the synthesis and secretion of alcohol-mediated cytoactive substances in hepatocytes, reducing alcohol toxicity and eliminating hangover and further alcoholic liver It is to protect the liver of patients with the disease.
도1은 Hep G2 세포에서 본 발명에 의한 고분자 수용성 키토산이 알코올 매개성 NF-κB/Rel A 합성에 미치는 영향을 나타내는 웨스턴 블롯이다.1 is a western blot showing the effect of the polymer-soluble chitosan according to the present invention on alcohol-mediated NF-κB / Rel A synthesis in Hep G2 cells.
도2는 Hep G2 세포에서 본 발명에 의한 고분자 수용성 키토산이 알코올 매개성 IκB 분해에 미치는 영향을 나타내는 웨스턴 블롯이다.Figure 2 is a Western blot showing the effect of the polymer water-soluble chitosan on alcohol-mediated IκB degradation in Hep G2 cells.
상기와 같은 목적을 달성하기 위해서, 본 발명에 의한 방법은 분자량이 10,000 내지 1,500,000인 고분자 수용성 키토산을 이용하여 간세포의 알코올-매개성 세포활성물질의 합성 및 분비를 억제하는 것을 특징으로 한다.In order to achieve the above object, the method according to the invention is characterized by inhibiting the synthesis and secretion of alcohol-mediated cell activator of hepatocytes using a polymer-soluble chitosan having a molecular weight of 10,000 to 1,500,000.
상기 방법에 있어서, 상기 고분자 수용성 키토산은 90%이상이 탈아세틸화된 것임을 특징으로 한다.In the method, the polymer water-soluble chitosan is characterized in that more than 90% deacetylated.
상기 방법에 있어서, 상기 세포활성물질은 TNF-α, IL-1α 및 IL-6 중 어느 하나인 것을 특징으로 한다.In the method, the cell activator is any one of TNF-α, IL-1α and IL-6.
또한 본 발명에 의한 키토산 함유 식품 조성물은, 간세포의 알코올-매개성 세포활성물질의 합성 및 분비를 억제하기 위한 식품 조성물로서, 분자량이 10,000 내지 1,500,000인 고분자 수용성 키토산을 0.01 내지 90중량% 포함하는 것을 특징으로 한다.In addition, the chitosan-containing food composition according to the present invention is a food composition for inhibiting the synthesis and secretion of alcohol-mediated cell-active substance of hepatocytes, which contains 0.01 to 90% by weight of a polymer water-soluble chitosan having a molecular weight of 10,000 to 1,500,000. It features.
상기 키토산 함유 식품 조성물에 있어서, 상기 고분자 수용성 키토산은 90%이상이 탈아세틸화된 것임을 특징으로 한다.In the chitosan-containing food composition, the polymer water-soluble chitosan is characterized in that more than 90% deacetylated.
상기 키토산 함유 식품 조성물에 있어서, 상기 세포활성물질은 TNF-α, IL-1α 및 IL-6 중 어느 하나인 것을 특징으로 한다.In the chitosan-containing food composition, the cytoactive substance is any one of TNF-α, IL-1α and IL-6.
이하 본 발명에 따른 세포활성물질의 합성 및 분비 억제 방법, 및 그 억제를 위한 키토산 함유 식품 조성물에 대하여 구체적으로 살펴본다.Hereinafter, the synthesis and secretion inhibition method of the cell active material according to the present invention, and the chitosan-containing food composition for the inhibition will be described in detail.
알코올의 대사Metabolism of alcohol
에탄올은 혈중 알코올 농도가 2%(저 혈중 알코올 농도) 내지 10%(고 혈중 알코올 농도)인 경우, 직접 폐, 소변 또는 땀을 통해 배설되기도 하지만, 대부분은 간에서 아세트알데히드로 대사된다. 간에서는 3가지의 주요 경로를 통해 알코올의 대사가 이루어지는데, 그 주요 경로들은 각기 다른 에탄올 적정농도(Km)를 가지며, 결과적으로 시간당 한 잔의 알코올이 대사된다.Ethanol is sometimes excreted directly through the lungs, urine or sweat when the blood alcohol concentration is between 2% (low blood alcohol) and 10% (high blood alcohol), but most are metabolized to acetaldehyde in the liver. In the liver, alcohol is metabolized through three major pathways, each of which has a different ethanol titer (Km), which results in one metabolism of alcohol per hour.
3가지 주요 경로 중 첫째는, 임상적으로 중요한 경로로서 세포질에서 20밀리몰의 Km값에서 알코올 탈수소효소(ADH)에 의해 일어나는 경로이다. 이 반응에 의해 아세트알데히드가 생성된다. 생성된 아세트알데히드는 세포질 또는 미토콘드리아에서 알데히드 탈수소효소(ALDH)에 의해 급속히 분해된다. 상기 각 단계는 조효소로서 니코틴아미드 아데노신 디뉴클레오티드(NAD)를 필요로 한다. 음주 후에 나타나는 대사 혼란상태는 조효소인 NADH가 NAD로 변환하기 때문이다.The first of the three major pathways is a clinically important pathway that is caused by alcohol dehydrogenase (ADH) at a Km value of 20 mmol in the cytoplasm. This reaction produces acetaldehyde. The resulting acetaldehyde is rapidly degraded by aldehyde dehydrogenase (ALDH) in the cytoplasm or mitochondria. Each step requires nicotinamide adenosine dinucleotide (NAD) as a coenzyme. The metabolic disruption after drinking alcohol is due to the conversion of coenzyme NADH to NAD.
둘째, Km값이 10밀리몰인 활면소포체의 마이크로좀이 마이크로조말 에탄올 산화 체계(Microsomal ethanol oxidation system, MEOS)에 따라 총 혈중 알코올농도의 약 10% 또는 그 이상의 산화를 수행한다. 셋째, 퍼옥시좀과 미토콘드리아에 존재하는 카탈라제 등이 에탄올의 산화를 수행한다.Second, the microsomes of the surface vesicles having a Km value of 10 mmol perform oxidation of about 10% or more of the total blood alcohol concentration according to the microsomal ethanol oxidation system (MEOS). Third, peroxysomes and catalase present in the mitochondria perform oxidation of ethanol.
알코올에 의한 간손상Liver damage caused by alcohol
알코올에 의한 간손상 기전은 크게 알코올 자체에 의한 경우, 상기와 같은 대사과정 중에 생성되는 아세트알데히드와 같은 대사물질에 의한 경우, 알코올 대사과정에서 발생한 물질에 의한 경우, 면역반응에 의한 경우 등으로 분류할 수 있다.The mechanism of liver damage caused by alcohol is largely classified as being caused by alcohol itself, by metabolites such as acetaldehyde produced during metabolic processes as described above, by substances caused by alcohol metabolism, or by immune responses. can do.
특히, 아세트알데히드는 세로토닌, 도파민, 아드레날린과 반응하여 약리학적으로 활성을 갖는 물질을 생성시키고, 그 물질은 세포에서 제1 프로콜라겐 및 피브로넥틴의 합성을 촉진시키고, 섬유아세포를 자극하여 기질 단백질, 콜라겐-α1, 콜라겐-α2를 분비하게 하고, 근육섬유아세포 콜라겐의 합성을 촉진함으로써, 간 섬유화를 초래한다. 또한 단백질, 지질 및 DNA와 공유결합을 형성하여 항원으로 작용하므로, 면역원성 손상을 유발하여 간질환을 발생시킨다.In particular, acetaldehyde reacts with serotonin, dopamine, and adrenaline to produce pharmacologically active substances, which promote the synthesis of first procollagen and fibronectin in cells and stimulate fibroblasts to stimulate matrix proteins, collagen By secreting -α1 and collagen-α2 and promoting the synthesis of myofibroblast collagen, liver fibrosis is caused. In addition, it forms covalent bonds with proteins, lipids and DNA to act as an antigen, causing immunogenic damage to cause liver disease.
또한 아세트알데히드는 산화적 스트레스를 증가시키고 세포내 글루타치온(glutathion)을 고갈시킴으로써, 간손상을 더욱 심화시킨다.Acetaldehyde also aggravates liver damage by increasing oxidative stress and depleting intracellular glutathione.
알코올 섭취를 중단한 이후에도 간질환이 계속 진행되는 이유는, 면역학적 측면에서 설명될 수 있다. 알코올성 간질환에서, 혈청내 면역단백질이 상승하는 것과 간내 동양구조(sinusoid) 벽을 따라 IgA가 침착되는 것을 통해서, 체액성 면역의 손상이 입증된다. 또한 알코올에 의해 변형된 토끼의 간 세포막 항원에 대한 항체의 생성으로서 세포매개성 면역의 손상이 확인된다.The reason why liver disease continues even after stopping alcohol consumption can be explained in terms of immunological aspects. In alcoholic liver disease, impairment of humoral immunity is demonstrated by elevated serum protein and deposition of IgA along the hepatic sinusoid wall. Impairment of cell mediated immunity is also confirmed by the production of antibodies to rabbit liver membrane antigens modified by alcohol.
알코올성 간질환의 발생과 병의 진행에 세포독성 T림프구 및 간세포의 상호작용이 관여한다는 가설은, 활동성 알코올성 간질환에서 CD4, CD8 림프구의 구성비, 림프구들의 지속적인 분포, 간세포의 주조직적합복합체(MHC; Major Histocompatibility Complex)의 발현, 알코올성 유리질(hyalin) 및 세포괴사와의 관계 등에 의해 뒷받침되고 있다. 항원성 물질의 실체는 확실하지는 않지만 말로리 알코올성 유리질이 주목받고 있고, 아세트알데히드 콜라겐 복합물이 알코올성 간염의 간조직에서 발견되고 있는데, 이는 병의 활성도와 관련이 있으므로 세포매개성 면역 기전의 손상을 반영하는 것이다.The hypothesis that the interaction of cytotoxic T lymphocytes and hepatocytes is involved in the development and progression of alcoholic liver disease, suggests that the composition of CD4 and CD8 lymphocytes, the continuous distribution of lymphocytes, and the major histocompatibility complex (MHC) in active alcoholic liver disease. It is supported by the expression of Major Histocompatibility Complex, the relationship with alcoholic hyalin, and cell necrosis. Although the substance of the antigenic substance is not certain, malory alcoholic glass is attracting attention, and acetaldehyde collagen complexes are found in the liver tissue of alcoholic hepatitis, which is associated with disease activity and thus reflects impaired cell-mediated immune mechanisms. will be.
하기 실시예에 나타난 바와 같이, 알코올은 Hep G2 세포에서 TNF-α, IL-1α, IL-6의 합성 및 분비량을 현저히 증가시킨다. 뿐만 아니라, 알코올성 간질환을 가지고 있는 환자들의 혈액의 단핵구는 정상인과 비교하여, TNF-α, IL-1α 및 IL-6의 생성이 3 내지 6배 높은 것으로 나타난다. 따라서 세포활성물질인 TNF-α, IL-1α, IL-6 등이 알코올에 의한 간손상에 관여한다는 것을 알 수 있다. 그러므로 TNF-α, IL-1α 및 IL-6의 발현 및 분비를 억제하는 물질을 찾는다면 이를 이용하여 알코올에 의한 간 손상을 억제할 수 있을 것이다. 본 발명은 그 물질로서 키토산을 제시하고 있다. 또한 본 발명자는 본 발명에 의한 고분자 수용성 키토산이 상기 TNF-α, IL-1α 및 IL-6의 발현 및 분비를 억제한다는 것을 입증하기 위해 하기 실시예의 실험들을 행하였다.As shown in the examples below, alcohol significantly increases the synthesis and secretion of TNF-α, IL-1α, IL-6 in Hep G2 cells. In addition, monocytes in the blood of patients with alcoholic liver disease appear to have three to six times higher production of TNF-α, IL-1α and IL-6, compared to normal individuals. Therefore, it can be seen that TNF-α, IL-1α, IL-6 and the like, which are cytoactive substances, are involved in liver damage caused by alcohol. Therefore, if you look for a substance that inhibits the expression and secretion of TNF-α, IL-1α and IL-6 will be able to suppress the liver damage caused by alcohol. The present invention proposes chitosan as the substance. The inventors also conducted experiments of the following examples to demonstrate that the polymeric water soluble chitosan according to the present invention inhibits the expression and secretion of the TNF-α, IL-1α and IL-6.
세포활성물질Cell activator
세포활성물질은 다양한 기능을 가진 단백질 그룹으로서, 일부는 림프구 계통의 세포로부터 생성되며, 면역조절을 하고 조혈작용을 관장한다. TNF-α, IL-6 및 IL-1α를 포함하는 대부분의 세포활성물질은 표적세포의 세포표면 수용체에 결합함으로써 특정 반응을 유도하며 그 작용기전은 호르몬과 같다. 각 세포활성물질은 고유의 독성이 있으며 이를 사람에게 외부에서 주입시켰을 경우에는, 열을 발생시키며 염증 및 조직파괴를 유발하고, 드물게는 쇼크와 사망을 초래하기도 한다.Cytoactive substances are a group of proteins with a variety of functions, some of which are produced from cells of the lymphocyte lineage and regulate immune regulation and regulate hematopoiesis. Most cell activators, including TNF-α, IL-6 and IL-1α, induce specific responses by binding to cell surface receptors of target cells and their mechanism of action is like hormones. Each cytoactive substance is inherently toxic, and when injected externally into humans, it generates heat, causes inflammation and tissue destruction, and rarely causes shock and death.
염증성 세포활성물질로서 잘 알려져 있는 TNF-α는 세포내 활성산소(ROS; Reactive Oxygen Species)를 유도하는 기능이 있으며 세포자멸사(apoptosis)에도 관여한다. T 림프구 및 NK-세포(Natural Killer cell) 또한 TNF-α를 생성하는 것으로 알려져 있다. TNF-α는 생성된 수준에 따라 그것이 세포 또는 조직에 미치는 영향이 전혀 다른 양면성을 지니고 있다. 예컨대, 간에서 낮은 수준의 TNF-α는 급성기 반응(acute phase reponse)을 활성화시키고 간세포의 증식을 유도하며 망간의 초산화물 불균등화효소(superoxide dismutase) 생성의 촉진 등의 방어기작에 관여한다. 그러나 이와는 반대로, 높은 수준의 TNF-α는 간독성 반응 및 간 섬유화에 관여한다. 정상 상태에서는 TNF-α가 낮은 수준으로 유지되다가 알코올에 의해 높은 수준으로 합성 및 분비되면 간독성 반응 및 간 섬유화가 일어나 간이 손상을 입는다.TNF-α, which is well known as an inflammatory cell activator, functions to induce intracellular reactive oxygen (ROS) and is also involved in apoptosis. T lymphocytes and NK-cells (Natural Killer cells) are also known to produce TNF-α. TNF-α has a bilateral difference in how its effect on cells or tissues varies depending on the level produced. For example, low levels of TNF-α in the liver are involved in defense mechanisms such as activating acute phase reponse, inducing proliferation of hepatocytes, and promoting the production of superoxide dismutase. In contrast, however, high levels of TNF-α are involved in hepatotoxic reactions and liver fibrosis. Under normal conditions, TNF-α remains at a low level, but is synthesized and secreted at high levels by alcohol, resulting in hepatotoxic reactions and liver fibrosis, resulting in liver damage.
TNF-α와 IL-6의 증가는 서로 관련성이 없으나 TNF-α와 IL-1α는 서로 관련이 있다. 즉 TNF-α가 증가하면 IL-1α의 분비도 증가한다. 최근의 연구들은 간의 급성기 반응을 조절하는 IL-1α 활성과 TNF-α활성과의 관계에 관심이 집중되고 있다.Increases in TNF-α and IL-6 are not related to each other, but TNF-α and IL-1α are related to each other. In other words, as TNF-α increases, the secretion of IL-1α also increases. Recent studies have focused on the relationship between IL-1α activity and TNF-α activity, which regulate the acute phase of the liver.
Hep G2 세포Hep G2 Cells
사람의 간모세포종 세포주(human hepato-blastoma cell line)인 Hep G2 세포는 알코올에 의한 간의 손상을 연구하는 데에 아주 적합한 모델이다. 그 이유는 상기 세포에서 알코올에 의한 TNF-α, IL-6 및 IL-1α의 발현 및 생성과, 간 손상과의 밀접한 관련성 등이 밝혀졌기 때문이다. Hep G2 세포의 손상은 알코올의 처리 시간 및 농도에 의존적이다. Hep G2 세포에 에탄올을 장시간 처리하면, 그 세포의 생화학적 및 형태학적 특성이 알코올에 의한 간질환 환자에게서 나타나는 특성과 유사하게 나타난다. 에탄올은 장시간 처리시 세포의 면역 조절력과 해독력을 저해한다.Hep G2 cells, a human hepato-blastoma cell line, are a good model for studying liver damage caused by alcohol. The reason for this is that the expression and production of TNF-α, IL-6 and IL-1α by alcohol in the cells and the close association with liver damage have been found. Damage to Hep G2 cells is dependent on the treatment time and concentration of alcohol. After prolonged treatment of ethanol with Hep G2 cells, the biochemical and morphological properties of the cells are similar to those seen in alcoholic liver disease patients. Ethanol inhibits cellular immune regulation and detoxification after prolonged treatment.
세포활성물질의 전사 억제Inhibition of Transcription of Cellular Active Materials
본 발명자는 본 발명에 의한 고분자 수용성 키토산이 TNF-α, IL-1α 및 IL-6의 분비를 억제할 뿐만 아니라, TNF-α, IL-1α 및 IL-6 유전자의 전사를 억제한다는 것 또한 입증하였다. 구체적으로, 본 발명에 의한 고분자 수용성 키토산은 TNF-α, IL-1α 및 IL-6 유전자의 전사에 관여하는 전사인자인 NF-κB의 활성을 조절한다.The present inventors demonstrate that the polymer water-soluble chitosan according to the present invention not only inhibits the secretion of TNF-α, IL-1α and IL-6, but also inhibits the transcription of TNF-α, IL-1α and IL-6 genes. It was. Specifically, the polymer water-soluble chitosan according to the present invention modulates the activity of NF-κB, a transcription factor involved in the transcription of TNF-α, IL-1α and IL-6 genes.
NF-κB/Rel A는 유전자 내의 일정한 결합 자리에 결합함으로써, 염증 반응에 관여하는 여러 유전자들, 예컨대, TNF-α, IL-6, IL-1, IL-8, 과립구 집락 자극인자 등과 같은 많은 세포활성물질, 막간 접착분자, 내피성 백혈구 접착분자-1, 혈관세포 접착분자-1 등의 세포접착단백질, MHC 유전자, 그리고 산화질소 합성효소, 사이클로옥시게나제, 초산화 마그네슘 불균등화효소 등의 각종 효소의 유전자들의 전사를 조절한다. 간의 쿠퍼(Kufpper) 세포에서 알코올에 의해 NF-κB/Rel A가 활성화되는 것이 알려져 있다.NF-κB / Rel A binds to a constant binding site within a gene, thereby allowing many of the genes involved in the inflammatory response, such as TNF-α, IL-6, IL-1, IL-8, granulocyte colony stimulating factors, etc. Cell adhesion proteins such as cell activator, intercellular adhesion molecule, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1, MHC gene, and nitric oxide synthase, cyclooxygenase, magnesium acetate disproportionase, etc. Regulates transcription of genes of various enzymes. It is known that NF-κB / Rel A is activated by alcohol in Kufpper cells of the liver.
NF-κB/Rel A가 평상시에는 억제인자인 IκB에 의해 불활성화 상태로 세포질내에 존재하다가 어떤 자극이 주어졌을 때 IκB가 인산화되면서 분해되어 NF-κB/Rel A가 자유로운 형태로 방출되어 핵속으로 이동한다. 알코올은 IκB를 인산화시켜 분해하는 자극 중의 하나이다. 본 발명자는 TNF-α, IL-1α 및 IL-6의 전사를 억제하는 기전이 NF-κB/Rel A 및 IκB의 활성 조절과 관련이 있는 지를 입증하기 위하여 웨스턴 블롯팅 방법으로 하기 실시예와 같이 실험하였다.NF-κB / Rel A is normally present in the cytoplasm in an inactivated state by the inhibitory factor IκB, and when given a stimulus, IκB is phosphorylated to break down, releasing NF-κB / Rel A in a free form and moving into the nucleus. do. Alcohol is one of the stimuli that phosphorylates and decomposes IκB. In order to demonstrate whether the mechanism of inhibiting the transcription of TNF-α, IL-1α and IL-6 is related to the regulation of activity of NF-κB / Rel A and IκB, the western blotting method is as follows. Experiment.
이하, 하기 실시예를 들어 본 발명을 더욱 구체적으로 설명한다.Hereinafter, the present invention will be described in more detail with reference to the following examples.
실시예Example
본 발명자는 발명에 의한 고분자 수용성 키토산이 Hep G2 세포에서 알코올-매개 TNF-α, IL-1α 및 IL-6 분비를 억제하는 효과를 입증하기 위해서 각각 하기와 같이 실험하였다.The present inventors experimented as follows to demonstrate the effect of the polymer-soluble chitosan according to the invention to inhibit the alcohol-mediated TNF-α, IL-1α and IL-6 secretion in Hep G2 cells.
실험방법Experiment method
1. 시약1. Reagent
에탄올은 독일의 Merck KGaA사로부터 구입하였고, TNF-α, IL-1α 및 IL-6 및 각각에 대한 항체는 미국의 R＆D사로부터 구입하였다. 세포배양용 배지(DMEM)는 Gibco BRL에서 구입한 것을 사용하였고, 3-(4,5-디메틸티아졸-2-일)-2,5-디페닐-테트라졸륨 브롬화물(MTT)은 시그마 케미칼사(Sigma chemical co.)로부터 구입한 것을 사용하였다. 또한 세포배양용 플레이트, 효소면역측정법(ELISA; enzyme linked immunosorbent assay) 용 플레이트 및 디쉬는 Nunc(Naperville, MD)로부터 구입한 것을 사용하였다. 그리고 본 발명에 의한 분자량이 10,000 내지 1,500,000인 고분자 수용성 키토산은 본 발명자가 통상의 방법에 따라 제조한 것을 사용하였다.Ethanol was purchased from Merck KGaA, Germany, and antibodies against TNF-α, IL-1α and IL-6, and each, were purchased from R & D, USA. Cell culture medium (DMEM) was purchased from Gibco BRL, and 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) was sigma chemical. The one purchased from Sigma chemical co. Was used. In addition, the plate for cell culture, the plate for enzyme linked immunosorbent assay (ELISA), and the dish were purchased from Nunc (Naperville, MD). The polymer water-soluble chitosan having a molecular weight of 10,000 to 1,500,000 according to the present invention was prepared by the inventors according to a conventional method.
2. Hep G2 세포의 준비2. Preparation of Hep G2 Cells
사람의 간모세포종 세포주인 Hep G2 세포는 10% 태아 소 혈청(FBS) 및 1% 페니실린/스트렙토마이신이 첨가된 DMEM 배지를 사용하여 37℃, 5% CO2의 인큐베이터에서 배양하였다. 4웰에 2×105셀/웰을 플레이팅하고 12시간 이상 안정화시킨 후에 실험에 이용하였다.Hep G2 cells, a human hepatoblastoma cell line, were cultured in an incubator at 37 ° C., 5% CO 2 using DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. 2 × 10 5 cells / well were plated into 4 wells and used for experiments after stabilization for at least 12 hours.
3. 고분자 수용성 키토산3. Polymer Water Soluble Chitosan
분자량이 10,000 내지 1,500,000인 고분자 수용성 키토산은 1mg/ml를 최대 농도로 하여 3차 증류수에 녹여 0.2㎛ 필터에 걸러 사용하였다.The polymer water-soluble chitosan having a molecular weight of 10,000 to 1,500,000 was dissolved in tertiary distilled water at a maximum concentration of 1 mg / ml and used in a 0.2 μm filter.
4. ELISA4. ELISA
세포활성물질의 정량은 ELISA 방법을 사용하여, 96웰 ELISA 플레이트에서 중복 실행하였다. 세포활성물질에 대한 단클론 항체 1㎍/ml을 PBS(pH 7.4)로 희석하여 96웰 플레이트에 100㎕씩 각각 입힌 다음 4℃에서 12시간동안 방치하였다. 이 플레이트를 0.05% 트윈(Tween-20)이 포함된 PBS로 세척한 다음 1% BSA, 5% 수크로즈 및 0.05% NaN3가 포함된 PBS로 1시간동안 블록킹하였다. 수회 세척한 후 샘플을 첨가한 후 37℃에서 2시간동안 방치하였다. 플레이트 웰을 다시 세척한 후 비오틴이 결합된 항체 0.2㎍/ml을 첨가한 후 다시 37℃에서 2시간동안 방치하였다. 웰을 세척한 후 토끼 IgG-접합된 알칼라인 포스파타제 및 아비딘 퍼옥시다제를 첨가하고 37℃에서 20분동안 방치하였다. 웰을 다시 세척한 후 ABTS 기질을 첨가하였다. 발색반응은 ELISA 판독기를 사용하여 405nm에서 측정하였고, 표준 곡선은 순차적으로 희석된 재조합 세포활성물질을 사용하여 각각의 정량에 적용하였다.Quantification of cell activators was performed in 96-well ELISA plates in duplicate using the ELISA method. 1 μg / ml of the monoclonal antibody against the cell activator was diluted with PBS (pH 7.4), coated in 100 μl each in a 96 well plate, and left at 4 ° C. for 12 hours. The plate was washed with PBS containing 0.05% Tween-20 and then blocked for 1 hour with PBS containing 1% BSA, 5% sucrose and 0.05% NaN 3 . After washing several times, the sample was added and then left at 37 ° C. for 2 hours. The plate wells were washed again, followed by addition of 0.2 μg / ml of the biotin-bound antibody, followed by another 2 hours at 37 ° C. The wells were washed and then rabbit IgG-conjugated alkaline phosphatase and avidin peroxidase were added and left at 37 ° C. for 20 minutes. The wells were washed again and then ABTS substrate was added. The color reaction was measured at 405 nm using an ELISA reader, and standard curves were applied to each quantitation using sequentially diluted recombinant activators.
5. 웨스턴 블롯 분석5. Western Blot Analysis
처리된 세포를 용해 버퍼로 수확한 다음, BCA 용액으로 단백질을 정량하였다. 50㎍ 단백질을 10% 겔에서 전기영동한 후, 니트로셀룰로스 막으로 이동시켜 10% 탈지유로 1시간동안 블록킹시켰다. 항-NF-κB 및 항-IκB에 대한 항체를 1시간 처리하고 PBS(트윈-20)로 세척하였다. 각각에 대한 2차 항체를 30분동안 처리한 다음 세척한 후, ECL 용액 키트로 검출하였다.Treated cells were harvested with lysis buffer and then protein quantified with BCA solution. 50 μg protein was electrophoresed on a 10% gel, then transferred to nitrocellulose membrane and blocked for 1 hour with 10% skim milk. Antibodies against anti-NF-κB and anti-IκB were treated for 1 hour and washed with PBS (Tween-20). Secondary antibodies to each were treated for 30 minutes and then washed, then detected with an ECL solution kit.
6. 통계학적 분석6. Statistical Analysis
모든 자료는 평균들±S.E.로 나타내었으며, 통계학적 분석은 스투던트 t-테스트로 행하였다. 유의 수준은 P〈0.05로 하였다.All data are expressed as means ± S.E. And statistical analysis was done by Student's t-test. The significance level was P <0.05.
상기 실험방법을 이용하여 본 발명자는 본 발명에 의한 고분자 수용성 키토산이 Hep G2 세포에서 알코올-매개 TNF-α, IL-1α 및 IL-6 합성 및 분비를 억제하는 효과에 대해서 하기와 같이 각각 실험하여 다음과 같은 결과를 얻을 수 있었다.Using the above experimental method, the present inventors experimented on the effects of the polymer-soluble chitosan according to the present invention to inhibit the synthesis and secretion of alcohol-mediated TNF-α, IL-1α and IL-6 in Hep G2 cells. The following results were obtained.
〈실험예1〉Experimental Example 1
먼저, 상기와 같은 실험 방법을 이용하여 알코올-매개 TNF-α분비에 대해 다음과 같이 실험하였다.First, the following experiment was performed on alcohol-mediated TNF-α secretion using the above experimental method.
분자량이 100,000 및 1,000,000인 고분자 수용성 키토산을 상기 처리방법에 따라 처리하여 준비하였다. 준비된 상기 각 분자량의 고분자 수용성 키토산을 상기 준비된 Hep G2 세포 배양액에 각각 0.1, 1, 10 및 100㎍/ml로 처리하여 실험군 1-4를 준비하였다. 그리고 고분자 수용성 키토산으로 처리하지 않은 대조군인 두개의 Hep G2 세포배양액을 또한 준비하였다. 상기 고분자 수용성 키토산을 처리하고 1시간이 경과한 후 준비된 4개의 실험군과 1개의 대조군(대조군2)에 4%의 에탄올을 24시간동안 처리하였다. 처리된 배양액의 상층액 중의 TNF-α의 수준을 측정하기 위해 상기와 같이 ELISA법을 수행하였다.Polymeric water-soluble chitosans having molecular weights of 100,000 and 1,000,000 were prepared by treatment according to the above treatment method. Experimental groups 1-4 were prepared by treating each of the prepared polymer water-soluble chitosans with 0.1, 1, 10 and 100 µg / ml of the prepared Hep G2 cell culture. And two Hep G2 cell cultures were also prepared as controls not treated with polymer water soluble chitosan. After 1 hour of treatment with the polymer water-soluble chitosan, 4% of ethanol was treated in 4 experimental groups and 1 control group (control group 2) prepared for 24 hours. ELISA was performed as above to measure the level of TNF-α in the supernatant of the treated culture.
그 결과는 다음과 같다.the results are as follow.
*P〈0.01* P <0.01
상기 표 1에 나타난 바와 같이, 알코올을 Hep G2 세포에 처리하면 TNF-α가 많이 분비됨을 알 수 있었다. 또한 Hep G2 세포에서 본 발명에 의한 고분자 수용성 키토산을 처리하였을 때, 알코올에 의한 TNF-α 분비는 고분자 수용성 키토산의 농도에 따라 감소하였음을 알 수 있었다. 특히, 100㎍/ml의 농도(실험군1)에서는 약 70%까지 현저하게 억제되었다. 따라서 본 발명에 의한 고분자 수용성 키토산이 알코올에 의한 TNF-α의 분비를 효과적으로 억제함으로써 간세포를 보호한다는 것이 입증되었다.As shown in Table 1, it can be seen that TNF-α is secreted much when the alcohol is treated to Hep G2 cells. In addition, when the polymer water-soluble chitosan of the present invention was treated in Hep G2 cells, it was found that the secretion of TNF-α by alcohol decreased with the concentration of the polymer-soluble chitosan. In particular, the concentration of 100 μg / ml (Experimental Group 1) was significantly suppressed by about 70%. Therefore, it was demonstrated that the polymer water-soluble chitosan according to the present invention protects liver cells by effectively inhibiting the secretion of TNF-α by alcohol.
〈실험예2〉Experimental Example 2
상기와 같은 실험 방법을 이용하여 아세트알데히드-매개 TNF-α분비에 대해 다음과 같이 실험하였다.Using the same experimental method as described above for the acetaldehyde-mediated TNF-α secretion was performed as follows.
분자량이 100,000 및 1,000,000인 고분자 수용성 키토산을 상기 처리방법에 따라 처리하여 준비하였다. 준비된 상기 각 분자량의 고분자 수용성 키토산을 상기 준비된 Hep G2 세포 배양액에 각각 0.1, 1, 10 및 100㎍/ml로 처리하여 실험군1-4를 준비하였다. 그리고 고분자 수용성 키토산으로 처리하지 않은 대조군인 두개의 Hep G2 세포배양액을 또한 준비하였다. 상기 고분자 수용성 키토산을 처리하고 1시간이 경과한 후 준비된 모든 4개의 실험군과 1개의 대조군(대조군2)에 500μM의 아세트알데히드를 24시간동안 처리하였다. 처리된 배양액의 상층액 중의 TNF-α의 수준을 측정하기 위해 상기와 같이 ELISA법을 수행하였다.Polymeric water-soluble chitosans having molecular weights of 100,000 and 1,000,000 were prepared by treatment according to the above treatment method. Experimental groups 1-4 were prepared by treating the prepared polymer water-soluble chitosan of each molecular weight with 0.1, 1, 10 and 100 µg / ml, respectively. And two Hep G2 cell cultures were also prepared as controls not treated with polymer water soluble chitosan. After 1 hour of treatment with the polymer water-soluble chitosan, 500 μM of acetaldehyde was treated for 24 hours in all four experimental groups and one control group (control group 2) prepared. ELISA was performed as above to measure the level of TNF-α in the supernatant of the treated culture.
그 결과는 다음과 같다.the results are as follow.
*P〈0.01* P <0.01
상기 표 2에 나타난 바와 같이, 아세트알데히드를 Hep G2 세포에 처리하면 TNF-α가 많이 분비됨을 알 수 있었다. 또한 Hep G2 세포에서 본 발명에 의한 고분자 수용성 키토산을 처리하였을 때, 아세트알데히드에 의한 TNF-α 분비는 고분자 수용성 키토산의 농도에 따라 감소하였음을 알 수 있었다. 특히, 100㎍/ml의 농도(실험군1)에서 가장 많이 억제되었다. 따라서 본 발명에 의한 고분자 수용성 키토산이 아세트알데히드에 의한 TNF-α의 분비를 효과적으로 억제함으로써 간세포를 보호한다는 것이 입증되었다.As shown in Table 2, the treatment of acetaldehyde in Hep G2 cells was found to secrete a lot of TNF-α. In addition, when Hep G2 cells were treated with the polymer-soluble chitosan according to the present invention, it was found that the secretion of TNF-α by acetaldehyde decreased with the concentration of the polymer-soluble chitosan. In particular, the most inhibited at a concentration of 100 μg / ml (Experimental Group 1). Therefore, it has been demonstrated that the polymer water-soluble chitosan according to the present invention protects liver cells by effectively inhibiting the secretion of TNF-α by acetaldehyde.
〈실험예3〉Experimental Example 3
상기와 같은 실험 방법을 이용하여 알코올-매개 IL-1α분비에 대해 다음과 같이 실험하였다.Using the same experimental method as described above for the alcohol-mediated IL-1α secretion was tested as follows.
분자량이 100,000 및 1,000,000인 고분자 수용성 키토산을 상기 처리방법에 따라 처리하여 준비하였다. 준비된 상기 각 분자량의 고분자 수용성 키토산을 상기 준비된 Hep G2 세포 배양액에 각각 0.1, 1, 10 및 100㎍/ml로 처리하여 실험군1-4를 준비하였다. 그리고 고분자 수용성 키토산으로 처리하지 않은 대조군인 두개의 Hep G2 세포배양액을 또한 준비하였다. 상기 고분자 수용성 키토산을 처리하고 1시간이 경과한 후 준비된 실험군 4개와 대조군 1개(대조군2)에 4%의 알코올을 24시간동안 처리하였다. 처리된 배양액의 상층액 중의 IL-1α의 수준을 측정하기 위해 상기와 같이 ELISA법을 수행하였다.Polymeric water-soluble chitosans having molecular weights of 100,000 and 1,000,000 were prepared by treatment according to the above treatment method. Experimental groups 1-4 were prepared by treating the prepared polymer water-soluble chitosan of each molecular weight with 0.1, 1, 10 and 100 µg / ml, respectively. And two Hep G2 cell cultures were also prepared as controls not treated with polymer water soluble chitosan. After 1 hour of treatment with the polymer water-soluble chitosan, 4% alcohol was treated in 4 experimental groups and 1 control group (control group 2) prepared for 24 hours. ELISA was performed as above to measure the level of IL-1α in the supernatant of the treated culture.
그 결과는 다음과 같다.the results are as follow.
*P〈0.01* P <0.01
상기 표 3에 나타난 바와 같이, 알코올을 Hep G2 세포에 처리하면 IL-1α 가 많이 분비됨을 알 수 있었다. 또한 Hep G2 세포에서 본 발명에 의한 고분자 수용성 키토산을 처리하였을 때, 알코올에 의한 IL-1α 분비는 고분자 수용성 키토산의 농도에 따라 감소하였음을 알 수 있었다. 특히, 100㎍/ml의 농도(실험군1)에서는 약 50%까지 현저하게 억제되었다. 따라서 본 발명에 의한 고분자 수용성 키토산이 알코올에 의한 IL-1α의 분비를 효과적으로 억제함으로써 간세포를 보호한다는 것이 입증되었다.As shown in Table 3, it was found that IL-1α was secreted much when the alcohol was treated to Hep G2 cells. In addition, when the polymer water-soluble chitosan according to the present invention was treated in Hep G2 cells, IL-1α secretion by alcohol was found to decrease with the concentration of the polymer water-soluble chitosan. In particular, the concentration of 100 μg / ml (Experimental Group 1) was significantly inhibited by about 50%. Therefore, it has been demonstrated that the polymer water-soluble chitosan according to the present invention protects liver cells by effectively inhibiting the secretion of IL-1α by alcohol.
〈실험예4〉Experimental Example 4
상기와 같은 실험 방법을 이용하여 알코올-매개 IL-6의 분비에 대해 다음과 같이 실험하였다.Using the same experimental method as described above for the secretion of alcohol-mediated IL-6.
분자량이 100,000 및 1,000,000인 고분자 수용성 키토산을 상기 처리방법에 따라 처리하여 준비하였다. 준비된 상기 각 분자량의 고분자 수용성 키토산을 상기 준비된 Hep G2 세포 배양액에 각각 0.1, 1, 10 및 100㎍/ml로 처리하여 실험군1-4 준비하였다. 그리고 고분자 수용성 키토산으로 처리하지 않은 대조군인 두개의 Hep G2 세포배양액을 또한 준비하였다. 상기 고분자 수용성 키토산을 처리하고 1시간이 경과한 후 준비된 4개의 실험군과 1개의 대조군(대조군2)에 4%의 알코올을 24시간동안 처리하였다. 처리된 배양액의 상층액 중의 IL-6의 수준을 측정하기 위해 상기와 같이 ELISA법을 수행하였다.Polymeric water-soluble chitosans having molecular weights of 100,000 and 1,000,000 were prepared by treatment according to the above treatment method. Each of the prepared polymer water-soluble chitosans of each molecular weight was treated with 0.1, 1, 10 and 100 µg / ml in the prepared Hep G2 cell culture, respectively, to prepare Experimental Groups 1-4. And two Hep G2 cell cultures were also prepared as controls not treated with polymer water soluble chitosan. After 1 hour of treatment with the polymer water-soluble chitosan, 4% alcohol was treated in 4 experimental groups and 1 control group (control group 2) prepared for 24 hours. ELISA was performed as above to measure the level of IL-6 in the supernatant of the treated culture.
그 결과는 다음과 같다.the results are as follow.
*P〈0.01* P <0.01
상기 표 4에 나타난 바와 같이, 알코올을 Hep G2 세포에 처리하면 IL-6이 많이 분비됨을 알 수 있었다. 또한 Hep G2 세포에서 본 발명에 의한 고분자 수용성 키토산을 처리하였을 때, 알코올에 의한 IL-6 분비는 고분자 수용성 키토산의 농도에 따라 감소하였음을 알 수 있었다. 특히, 100㎍/ml의 농도(실험군1)에서는 약 25%까지 억제되었다. 따라서 본 발명에 의한 고분자 수용성 키토산이 알코올에 의한 IL-6의 분비를 효과적으로 억제함으로써 간세포를 보호한다는 것이 입증되었다.As shown in Table 4, it can be seen that the IL-6 is secreted when the alcohol is treated to Hep G2 cells. In addition, when the polymer water-soluble chitosan according to the present invention was treated in Hep G2 cells, IL-6 secretion by alcohol was reduced according to the concentration of the polymer water-soluble chitosan. In particular, the concentration of 100 μg / ml (Experimental Group 1) was suppressed to about 25%. Therefore, it has been demonstrated that the polymer water-soluble chitosan according to the present invention protects liver cells by effectively inhibiting the secretion of IL-6 by alcohol.
또한, 본 발명자는 본 발명에 의한 고분자 수용성 키토산의 알콜-매개 세포활성물질의 합성 억제 기전이 유전자의 전사단계에서의 전사인자인 NF-κB/Rel A과 그 억제자인 IκB의 활성 변화에 기인한 것인지를 규명하기 위하여 각각 하기와 같이 실험하였다.The present inventors also found that the mechanism of inhibiting the synthesis of the alcohol-mediated cell activator of the polymer water-soluble chitosan according to the present invention is due to the change in the activity of the transcription factor NF-κB / Rel A and its inhibitor IκB in the transcriptional phase of the gene. Each experiment was carried out as follows to determine whether or not.
〈실험예5〉<Experiment 5>
분자량이 100,000 및 1,000,000인 고분자 수용성 키토산을 상기 처리방법에 따라 처리하여 준비하였다. 준비된 상기 각 분자량의 고분자 수용성 키토산을 상기 준비된 Hep G2 세포 배양액에 각각 10 및 100㎍/ml(도1의 4 및 3)로 처리하였다. 그리고 고분자 수용성 키토산으로 처리하지 않은 두개(도1의 1, 2)의 Hep G2 세포배양액을 또한 준비하였다. 상기 고분자 수용성 키토산을 처리하고 1시간이 경과한 후, 준비된 상기 키토산 처리된 배양액 2개(3, 4)와 배양액 1개(2)에 4%의 알코올을 24시간동안 처리하였다. 본 실험을 위해 총 네개의 배양액에 행해진 처리는 다음과 같이 표 5로 정리할 수 있다.Polymeric water-soluble chitosans having molecular weights of 100,000 and 1,000,000 were prepared by treatment according to the above treatment method. The prepared polymer water-soluble chitosan of each molecular weight was treated with the prepared Hep G2 cell culture at 10 and 100 μg / ml (4 and 3 of FIG. 1), respectively. And two Hep G2 cell cultures were also prepared which were not treated with polymer water soluble chitosan (1, 2 in Fig. 1). After 1 hour of treatment with the polymer water-soluble chitosan, 4% alcohol was treated for 2 hours (3, 4) and 1 medium (2) of the prepared chitosan treated solution for 24 hours. The treatments performed on a total of four cultures for this experiment can be summarized in Table 5 as follows.
처리된 세포를 용해 버퍼로 수확한 다음, BCA 용액으로 단백질을 정량하였다. 50㎍ 단백질을 10% 겔에서 전기영동한 후, 니트로셀룰로스 막으로 이동시켜 10% 탈지유로 1시간동안 블록킹시켰다. 항-NF-κB에 대한 항체를 1시간 처리하고 PBS(트윈-20)로 세척하였다. 각각에 대한 2차 항체를 30분동안 처리한 다음 세척한 후, ECL 용액 키트로 검출하였다.Treated cells were harvested with lysis buffer and then protein quantified with BCA solution. 50 μg protein was electrophoresed on a 10% gel, then transferred to nitrocellulose membrane and blocked for 1 hour with 10% skim milk. Antibodies against anti-NF-κB were treated for 1 hour and washed with PBS (Tween-20). Secondary antibodies to each were treated for 30 minutes and then washed, then detected with an ECL solution kit.
그 결과는 도1에 나타난 바와 같다. 도1에 나타난 바와 같이, 4% 알코올을 처리(2)함으로써 NF-κB의 합성이 현저히 증가된 것을 알 수 있었다. 또한 고분자 수용성 키토산 100㎍/ml를 처리한 다음 4% 알코올을 처리한 3의 경우에 NF-κB의 양이 가장 작은 것을 알 수 있었다. 따라서 고분자 수용성 키토산은 간 손상의 원인인 세포활성물질을 발현하는 유전자의 전사인자인 NF-κB의 합성을 억제함으로써 간을 보호할 수 있다는 것이 입증되었다.The result is as shown in FIG. As shown in FIG. 1, it was found that the synthesis of NF-κB was significantly increased by treating (2) 4% alcohol. In addition, it was found that the amount of NF-κB was the smallest in the case of 3 treated with 100 μg / ml of polymer water-soluble chitosan and then treated with 4% alcohol. Therefore, it has been demonstrated that the polymer water-soluble chitosan can protect the liver by inhibiting the synthesis of NF-κB, a transcription factor of genes expressing cytoactive substances that cause liver damage.
〈실험예6〉<Experiment 6>
분자량이 100,000 및 1,000,000인 고분자 수용성 키토산을 상기 처리방법에 따라 처리하여 준비하였다. 준비된 상기 각 분자량의 고분자 수용성 키토산을 상기 준비된 Hep G2 세포 배양액에 각각 10 및 100㎍/ml(도2의 4 및 3)로 처리하여 실험군을 준비하였다. 그리고 고분자 수용성 키토산으로 처리하지 않은 두개(도2의 1, 2)의 Hep G2 세포배양액을 또한 준비하였다. 상기 고분자 수용성 키토산을 처리하고 1시간이 경과한 후, 준비된 상기 키토산 처리된 배양액 2개(3, 4)와 처리되지 않은 배양액 1개(2)에 4%의 알코올을 24시간동안 처리하였다. 본 실험을 위해 총 네개의 배양액에 행해진 처리는 다음과 같이 표 6으로 정리할 수 있다.Polymeric water-soluble chitosans having molecular weights of 100,000 and 1,000,000 were prepared by treatment according to the above treatment method. The experimental groups were prepared by treating the prepared polymer water-soluble chitosan of each molecular weight at 10 and 100 µg / ml (4 and 3 of FIG. 2) to the prepared Hep G2 cell culture, respectively. And two Hep G2 cell culture solutions (1, 2 in Fig. 2) that were not treated with polymer water soluble chitosan were also prepared. After 1 hour of treatment with the polymer water-soluble chitosan, 4% alcohol was treated for 2 hours (3, 4) of the prepared chitosan-treated culture medium and 1 (2) of the untreated culture solution for 24 hours. The treatments performed on a total of four cultures for this experiment can be summarized in Table 6 as follows.
처리된 세포를 용해 버퍼로 수확한 다음, BCA 용액으로 단백질을 정량하였다. 50㎍ 단백질을 10% 겔에서 전기영동한 후, 니트로셀룰로스 막으로 이동시켜 10% 탈지유로 1시간동안 블록킹시켰다. 항-IκB에 대한 항체를 1시간 처리하고 PBS(트윈-20)로 세척하였다. 각각에 대한 2차 항체를 30분동안 처리한 다음 세척한 후, ECL 용액 키트로 검출하였다.Treated cells were harvested with lysis buffer and then protein quantified with BCA solution. 50 μg protein was electrophoresed on a 10% gel, then transferred to nitrocellulose membrane and blocked for 1 hour with 10% skim milk. Antibodies against anti-IκB were treated for 1 hour and washed with PBS (Tween-20). Secondary antibodies to each were treated for 30 minutes and then washed, then detected with an ECL solution kit.
그 결과는 도 2에 나타난 바와 같다. 도 2에 나타난 바와 같이, 4% 알코올을 처리(2)함으로써 IκB의 합성이 현저히 감소하는 것을 알 수 있었다. 또한 고분자 수용성 키토산 100㎍/ml를 처리한 다음 4% 알코올을 처리한 3의 경우에 NF-κB의 양이 가장 많은 것을 알 수 있었다. 따라서 고분자 수용성 키토산은 간 손상의 원인인 세포활성물질을 발현하는 유전자의 전사인자인 NF-κB의 억제인자인 IκB의 분해를 억제함으로써 간을 보호할 수 있다는 것이 입증되었다.The result is as shown in FIG. As shown in FIG. 2, it was found that the synthesis of IκB was significantly reduced by treating 2% of 4% alcohol. In addition, it was found that the amount of NF-κB was the highest in the case of 3 treated with 100 μg / ml of polymer water-soluble chitosan and then treated with 4% alcohol. Therefore, it has been demonstrated that the polymer water-soluble chitosan can protect the liver by inhibiting the degradation of IκB, an inhibitor of NF-κB, a transcription factor of genes expressing cytoactive substances that cause liver damage.
본 발명에 의한 세포활성물질의 합성 및 분비 억제 방법, 및 그 억제를 위한 식품 조성물을 이용하면, 간세포에서 알코올에 의해 매개되는 세포활성물질의 합성 및 분비를 억제함으로써, 알코올에 의한 독성을 감소시키고 숙취를 제거하며 나아가 알코올성 간질환 환자의 간을 보호할 수 있다.By using the method of inhibiting the synthesis and secretion of the cell activator according to the present invention, and the food composition for the inhibition, by inhibiting the synthesis and secretion of the cell activator mediated by alcohol in hepatocytes, it is possible to reduce the toxicity by alcohol Eliminating hangovers can further protect the liver of patients with alcoholic liver disease.
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| KR20020090087A (en) * | 2001-05-25 | 2002-11-30 | 장태순 | Method for Inhibiting Injuries of Liver cells, Method for solving alcohol-hangover Method and Food Composition for Treating Hepatoma cell and Kit for Treating Hepatoma |
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| JPH0622725A (en) * | 1992-05-11 | 1994-02-01 | Nippon Kayaku Co Ltd | Enteric metabolism improving food and enteric metabolism improver |
| KR19990026532A (en) * | 1997-09-25 | 1999-04-15 | 오천금 | Prevention and improvement agent of liver function disorder caused by chitooligosaccharide |
| KR100213868B1 (en) * | 1995-02-21 | 1999-08-02 | 박영광 | Beverage containing kitosan |
| KR20010026947A (en) * | 1999-09-09 | 2001-04-06 | 이충삼 | Sub-food manufecturing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH0622725A (en) * | 1992-05-11 | 1994-02-01 | Nippon Kayaku Co Ltd | Enteric metabolism improving food and enteric metabolism improver |
| KR100213868B1 (en) * | 1995-02-21 | 1999-08-02 | 박영광 | Beverage containing kitosan |
| KR19990026532A (en) * | 1997-09-25 | 1999-04-15 | 오천금 | Prevention and improvement agent of liver function disorder caused by chitooligosaccharide |
| KR20010026947A (en) * | 1999-09-09 | 2001-04-06 | 이충삼 | Sub-food manufecturing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20020090087A (en) * | 2001-05-25 | 2002-11-30 | 장태순 | Method for Inhibiting Injuries of Liver cells, Method for solving alcohol-hangover Method and Food Composition for Treating Hepatoma cell and Kit for Treating Hepatoma |
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