KR20100037866A - A composition for preventing and treating hepatic damages containing glycoprotein from hizikia fusiformis - Google Patents
A composition for preventing and treating hepatic damages containing glycoprotein from hizikia fusiformis Download PDFInfo
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Abstract
Description
본 발명은 간 손상 예방 및 치료 활성을 갖는 톳 유래 당단백질에 관한 것으로, 보다 구체적으로는 톳 유래 당단백질을 유효성분으로 함유하는 간 손상 예방 및 치료용 약학적 조성물에 관한 것이다.The present invention relates to a sugar-derived glycoprotein having hepatic damage prevention and therapeutic activity, and more particularly to a pharmaceutical composition for preventing and treating liver damage, which contains sugar-derived glycoprotein as an active ingredient.
최근 해조류의 다양한 생리화학적 효과가 검증되면서 해조류 유래 생리활성물질에 대한 관심이 고도됨에 따라 톳, 다시마, 미역, 김, 등에서 추출한 추출물의 항암효과, 혈중지질대사 개선효과 등이 보고되고 있다. As various physiochemical effects of seaweeds have been verified recently, as the interest in seaweed-derived physiologically active substances increases, the anticancer effect of the extracts extracted from 톳, kelp, seaweed, seaweed, etc., and the improvement of blood lipid metabolism have been reported.
늦여름부터 초가을에 발아하여 가을의 중순경에는 육안으로 볼 수 있는 크기로 자라는 톳(Hizikia fusiformis)은 12월 말까지는 20cm 내외로 자라며 이듬해 3∼4월에는 급격히 생장한다. Hizikia fusiformis, which germinate in late summer and early autumn and grow to be visible to the naked eye in the middle of autumn, grows to around 20cm by the end of December and grows rapidly in March to April the following year.
톳은 칼슘 ·요오드 ·철 등의 무기염류가 많이 포함되어 있어, 혈액을 알카 리성으로 전환시키고 세포조직을 강화시킴으로써, 노화를 방지하고 모든 장기의 기능을 활발하게 해준다. 또한 혈관경화를 막아 주고, 상용으로 먹으면 칼슘의 대사가 순조로운 식품이므로 질병에 대한 저항력을 높여줄 뿐만 아니라 치아가 건강해지며 머리털이 윤택해지고, 임신부인 경우에는 태아의 뼈를 튼튼하게 해준다. 그 외에도 장의 유동작용을 활발히 하여 장내의 노폐물을 배설시키므로 변비예방에도 효과가 있는 등 예로부터 기호식품의 하나로서 특히 일본 사람들이 잘 먹는다. 따라서 톳에 대한 연구는 주로 일본에서 많이 이루어졌으며, 한국에서의 톳에 대한 연구는 양식을 위한 연구, 화학적 특성 등의 실험이 행해져왔다.It contains a lot of inorganic salts such as calcium, iodine and iron, which convert blood into alkaline and strengthen cell tissues, preventing aging and activating all organ functions. In addition, it prevents vascular hardening, and when eaten commercially, calcium metabolism is smooth, so it not only increases resistance to diseases, but also makes teeth healthy, hair is healthy, and in pregnant women, it strengthens the bones of the fetus. In addition, the intestinal fluid activity is actively excreted to excrete wastes in the intestine, which is effective in preventing constipation. Therefore, the research on 톳 was mainly done in Japan, and research on 톳 in Korea has been conducted for research on aquaculture and chemical properties.
아세토아미노펜(acetaminophen)은 세계적으로 널리 사용되어지고 있는 항염증, 항진통제로 과다복용 시 심각한 간손상을 야기한다. 실제로 미국과 영국에서는 약물에 의한 간질환원인 중에서 가장 우위를 차지하고 있는 것이 아세토아미노펜에 의한 간손상이라고 한다. 따라서 아세토아미노펜에 의해 야기되는 간 손상 메카니즘을 규명하고, 간손상을 예방, 혹은 회복하기위한 여려 연구들이 진행되어왔다. Acetoaminophen is an anti-inflammatory and analgesic drug that is widely used around the world, causing serious liver damage when overdose. In fact, in the United States and the United Kingdom, acetoaminophen is the leading cause of liver disease-causing liver disease. Therefore, various studies have been conducted to identify the liver damage mechanism caused by acetoaminophen, and to prevent or repair liver damage.
캐틀린 등(Cathleen et al.; Toxicological Sciences 84, 201-208, 2005)은 마우스에 아세토아미노펜투여 시 세포사멸현상중 하나인 PARP활성이 나타난다고 보고하였으며, 타마라 등(Tamara et al.; Toxicological Sciences 76, 229-236, 2003)은 아세토아미노펜이 간의 지질과산화를 일으켜 간손상을 야기한다고 보고하였다. Catlin et al. (Toxicological Sciences 84, 201-208, 2005) reported that PARP activity, one of apoptosis, was observed when acetoaminophen was administered to mice, and Tamara et al .; Toxicological Sciences 76, 229-236, 2003) reported that acetoaminophen causes hepatic lipid peroxidation and causes liver damage.
반면, 시드하르타 등(Sidhartha et al.; Archives of Biochemistry and Biophysics 369, 42-58, 1999)은 마우스에서 포도씨 추출물인 프로안토시아니딘(proanthocyanidin)을 경구투여하였을 때 아세토아미노펜에 의한 간세포사멸을 예방하였으며, 프란시스코 등(Francisco et al. Journal of Ethnopharmacology 98, 103-108, 2005)은 남미에서 민간요법으로 사용되어오던 프로티움 헵타필룸(Protium heptaphyllum)추출액을 투여한 결과, 아세토아미노펜에 의한 간손상을 예방할 수 있다고 보고하였다.On the other hand, Sidhartha et al. (Archives of Biochemistry and Biophysics 369, 42-58, 1999) showed that acetoaminophen induced hepatocyte death when oral administration of grape seed extract proanthocyanidin in mice. Francisco et al. Journal of Ethnopharmacology 98, 103-108, 2005, were treated with acetoaminophen-derived hepataphyllum extract, which had been used as a folk remedy in South America. It is reported that damage can be prevented.
정 등(Jeong et al.; "톳(Hizikia fusiformis) 추출물이 고지혈증 흰 쥐의 지질대사 및 간 효소활성에 미치는 영향" 한국식품영양과학회지 30, 1184-1189, 2001)은 톳 추출물이 생체 내 항 고지혈증, 항 고콜레스테롤 및 항 산화효과가 있음을 밝혀낸 바 있다.Jeong et al. ("Effect of Hizikia fusiformis Extract on Lipid Metabolism and Liver Enzyme Activity in Hyperlipidemic Rats" Journal of the Korean Society of Food Science and Nutrition 30, 1184-1189, 2001) It has been found to have hyperlipidemia, antihypercholesterol and antioxidant effects.
이에, 본 발명자들은 아세트아미노펜에 의해 유발된 간 손상을 예방 및 치료하고, 이러한 작용메커니즘을 제공하고자 예의 연구를 거듭한 결과 갈조류인 톳에서 분리한 당단백질 분리하고 이들의 상기 효과를 확인함으로써 본 발명에 이르게 되었다. Therefore, the present inventors have conducted extensive studies to prevent and treat liver damage caused by acetaminophen, and provide a mechanism of action. As a result, the present invention has been performed by separating glycoproteins isolated from brown algae and confirming their effects. Came to.
따라서 본 발명의 목적은 톳 유래 화합물의 간 손상을 예방 및 치료하기 위한 신규 용도를 제공하는 것이다.It is therefore an object of the present invention to provide novel uses for the prevention and treatment of hepatic damage of VII-derived compounds.
상기와 같은 본 발명의 목적을 톳으로부터 당단백질을 분리하고, 이들의 아미노아세토펜에 의한 간 손상 억제효과를 확인함으로써 달성되었다.The object of the present invention as described above was achieved by separating glycoproteins from bovine and confirming their inhibitory effect on liver damage by aminoacetophene.
본 발명은 톳 유래 당단백질을 유효성분으로 함유하는 간 손상 예방 및 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention and treatment of liver damage, which contains the 톳 derived glycoprotein as an active ingredient.
본 발명에 있어서, “유효성분”이라 함은 내재된 약리작용에 의해 그 의약품의 효능·효과를 직접 또는 간접적으로 발현한다고 기대되는 물질 또는 물질군(약리학적 활성성분 등이 밝혀지지 않은 생약 등을 포함한다)으로서 주성분을 포함하는 것을 의미한다.In the present invention, the term "active ingredient" refers to a substance or a group of substances (a medicinal agent whose pharmacologically active ingredient is not known, etc.) which is expected to express the efficacy or effect of the medicine directly or indirectly by inherent pharmacological action. It means containing a main component).
본 발명에서는 톳 분말로부터 당단백질을 추출, 분리하여 아세토아미노펜에 의한 간 손상 억제효과를 살펴보았다. 전기영동 및 염색결과에서 추출물은 당과 단백질로 구성되어 있었으므로 이를 당단백질이라 명명하였으며, 이들은 간세포에 아세토아미노펜을 투여하여 세포성장이 억제되었으며 당단백질 병행처리로 아세토아미노펜 독성에 의한 간세포손상이 저해되는 것을 확인하였다. In the present invention, the glycoprotein was extracted and separated from the powder, and the effect of inhibiting liver damage by acetoaminophen was examined. In electrophoresis and staining results, the extract was composed of sugar and protein, which is called glycoprotein.These cells were inhibited by the administration of acetoaminophen to hepatocytes and inhibited hepatocellular damage due to acetoaminophen toxicity by the glycoprotein combination treatment. It confirmed that it became.
본 발명에 따른 톳 유래 당단백질의 작용기전을 밝히기 위해 아세토아미노펜에의한 산화적 손상에 작용하는 MAPK 군 단백질들을 살펴본 결과에서는 ERK인산화가 관여하는 것으로 나타났다. 즉 아세토아미노펜에의해 ERK인산화가 유도되었다가 당단백질처리로 그 인산화가 더욱 증가되어 세포생존을 유도하였다. 아세토아미 노펜과 당단백질을 투여한 흰쥐의 간 손상정도를 살펴본 결과에서는 아세토아미노펜에 의해 혈액중의 GOT/GPT중 GPT가 증가되었으며, 당단백질이 이를 다시 정상수준으로 감소시키는 것으로 나타났다. 또한 간세포사멸에 직접적으로 관여하는 캐스파아제 활성측정 결과에서는 카스파아제-3/-9에는 의존적으로 아세토아미노펜 독작용이 일어났다가, 톳으로부터 추출한 당단백질이 아세토아미노펜에 의해 활성화되었던 카스파아제-3/-9활성을 감소시킴으로써 아세토아미노펜독작용을 저해하는 것이다. 이 결과를 바탕으로, 톳 유래 당단백질이 아세토아미노펜에 의한 흰쥐의 간손상과 간세포의 사멸을 억제하여 혈중 GPT농도를 정상수준으로 유지시킬 뿐 아니라 위 표피세포에서 세포생존과 관련한 단백질(PARP, ERK·) 및 캐스파아제활성에 영향을 주어 약물에 의한 간손상을 저해시키는 것으로 나타났다.ERK phosphorylation was found to be involved in the MAPK group proteins that act on the oxidative damage caused by acetoaminophene in order to elucidate the mechanism of action of the glycoprotein derived from the present invention. In other words, ERK phosphorylation was induced by acetoaminophen, and its phosphorylation was further increased by glycoprotein treatment to induce cell survival. Examination of liver damage in rats treated with acetoaminophene and glycoprotein showed that acetoaminophen increased GPT in GOT / GPT in blood and glycoprotein reduced it back to normal levels. In addition, caspase-3 / -9 showed a caspase-3 / -9-dependent caspase activity, and the acetoaminophen poisoning effect was dependent on caspase-3 / -9. Inhibition of acetoaminophen poisoning by reducing -9 activity. Based on these results, the glycoprotein derived from 톳 suppresses acetoaminophen-induced liver damage and hepatic cell death, thereby maintaining blood GPT levels at normal levels, as well as proteins related to cell survival in gastric epidermal cells (PARP, ERK). ·) And caspase activity has been shown to inhibit liver damage by drugs.
본 발명에 따른 톳 유래 당단백질은 아세토아미노펜에 의한 흰쥐의 간손상과 간세포의 사멸을 억제하여 혈중 GPT농도를 정상수준으로 유지시킬 뿐 아니라 간세포에서 세포생존과 관련한 단백질(PARP, ERK·) 및 캐스파아제활성에 영향을 주어 약물에 의한 간손상을 저해시키는 효과가 있다.The glycoprotein derived from 톳 according to the present invention inhibits liver damage and apoptosis of rats by acetoaminophen to maintain normal blood GPT levels, as well as proteins related to cell survival in hepatocytes (PARP, ERK ·) and Ca It affects the spatase activity and has the effect of inhibiting liver damage by drugs.
이하에서 본 발명의 바람직한 실시형태를 실시예를 참고로 보다 구체적으로 설명한다. 하지만 본 발명의 범위가 이러한 실시예에 한정되는 것은 아니다.Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.
실시예 1 : 톳으로부터 당단백질의 제조Example 1 Preparation of Glycoproteins from Sodium
매생이, 김, 톳 등과 같은 해조류 내 존재하는 성분들은 단순한 영양소 역할 외에 기능성 물질로서의 역할에 대한 관심이 높아짐에 따라 해조류 유용성분을 대상으로 항암, 항비만, 항콜레스테롤, 면역증강 효과 및 항염증 효과와 같은 각종 생리활성에 관한 연구가 활발히 진행되고 있다. 따라서 본 실시예에서는 해조류 당단백질을 추출, 분리하여 다음과 같은 효과 검증 실험을 행하고자 한다. Ingredients in seaweeds such as falcon, seaweed, and seaweed have increased interest in their role as functional substances in addition to mere nutrients, and have anti-cancer, anti-obesity, anti-cholesterol, immune enhancing and anti-inflammatory effects. Studies on the same physiological activity are being actively conducted. Therefore, in the present embodiment, the algae glycoprotein is extracted and separated to perform the following effect verification experiment.
우리나라 근해에서 채취한 톳(Hizikia fusiformis)을 세척, 열풍건조시킨 후, 분말화하고 -20℃에 보관한다. 톳 분말 80g을 물 2L에 넣고 80℃에서 3시간 열수 추출한다. 여과 후 나온 여액에 3배 볼륨의 에탄올을 넣고 다시 여과시킨다. 여기서 나온 여액을 농축하고, 물로 녹인 후 황산 암모늄을 80% 농도로 넣고 4℃에서 밤새 정치시켰다. 원심분리하여 나온 잔사에 물을 넣고 녹인 후, 투석막을 이용하여 염을 제거한다. 투석된 용액을 감압농축한 것을 당단백질시료로 사용하였다. After washing and hot-air drying 톳 ( Hizikia fusiformis) collected from the offshore of Korea, it is powdered and stored at -20 ℃. 80 80g of powder was put in 2L of water and extracted with hot water at 80 ℃ for 3 hours. Into the filtrate after filtration, three volumes of ethanol were added and filtered again. The filtrate was concentrated, dissolved in water, and ammonium sulfate was added at 80% concentration and left overnight at 4 ° C. Water is added to the residue obtained by centrifugation and dissolved, and then salt is removed using a dialysis membrane. The dialysis solution was concentrated under reduced pressure to use a glycoprotein sample.
실험예 1 : SDS-PAGE 및 아가로스 겔 전기영동 및 염색Experimental Example 1 SDS-PAGE and Agarose Gel Electrophoresis and Staining
상기 실시예 1에서 수득한 각각의 획분에 2X 래믈리 샘플 완충액(Laemmli sample buffer; 13.3% SDS, 0.4 M Tris, 0.013% 브로모페놀 블루, 40% 글리세롤, pH 6.5)를 1X의 농도가 되도록 첨가하고, 마이티 스몰 II 어패러투스(Mighty Small II Apparatus; Hofer Science Instrument, USA)를 사용하여 12.5% - 20% 사이의 SDS-폴리아크릴아미드 젤에 전기영동하였다. To each fraction obtained in Example 1 was added 2 × Laemmli sample buffer (13.3% SDS, 0.4 M Tris, 0.013% bromophenol blue, 40% glycerol, pH 6.5) to a concentration of 1 ×. And electrophoresed between 12.5% -20% SDS-polyacrylamide gel using a Mighty Small II Apparatus (Hofer Science Instrument, USA).
전기영동 후, 젤에 분리된 단백질은 쿠마씨 브릴리언트 블루(coomassie brilliant blue) R-250법과 실버 염색(Silver Staining)법으로 염색후 확인하고, 당의 존재여부는 SDS-PAGE 전기영동 후 당단백질 염색 키트(glycoprotein staining kit; Pierce)로 염색하거나, 아가로스 겔에 전기영동하여 톨루이딘 블루로 염색 후 확인하였다(도 1 참조). After electrophoresis, the protein isolated on the gel was confirmed after staining by Coomassie brilliant blue R-250 method and Silver Staining method, and the presence of sugar after the SDS-PAGE electrophoresis, glycoprotein staining kit (Glycoprotein staining kit; Pierce), or agarose gel electrophoresis was confirmed after staining with toluidine blue (see Fig. 1).
도 1에 나타낸 바와 같이, SDS-PAGE와 아가로스 갤 전기영동결과 이상에서 추출한 물질 내에 당과 단백질이 함께 존재함을 알 수 있다. 따라서 본 발명에서 톳 유래 물질을 당단백질(glycoprotein)이라 명명한다. As shown in FIG. 1, it can be seen that sugar and protein are present together in the material extracted from the SDS-PAGE and agarose gal electrophoresis results. Therefore, in the present invention 톳 derived material is called glycoprotein (glycoprotein).
실험예 2 : 아세토아미노펜 처리에 의한 간세포증식억제효과Experimental Example 2 Hepatocellular Proliferation Inhibitory Effect by Acetoaminophen Treatment
본 실시예에서는 대표적인 두 가지 인체 간세포인 Chang liver 세포와 HepG2세포를 이용하여 톳 유래 당단백질의 효능을 검증하였다. 그 중 Chang liver세포는 원래 정상 간세포로부터 유래한 세포이며, HepG2 세포는 정상 간세포와 동일한 대사기전으로 성장하기 때문에, 일반적으로 세포를 이용한 간 독성 및 회복에 관한 실험에 주로 사용된다. In this example, two human liver cells, Chang liver cells and HepG2 cells, were used to examine the efficacy of the K-derived glycoprotein. Among them, Chang liver cells are originally derived from normal hepatocytes, and HepG2 cells grow in the same metabolic mechanism as normal hepatocytes, so they are generally used for experiments on liver toxicity and recovery using cells.
Chang liver 세포와 HepG2 세포는 ATCC(American Type Culture Collection ; CCL13, HB8065)로부터 분양받아 사용하였다. MEM배지에 항생제(페니실린/스트렙토마이신) 100units/㎖을 첨가하여 0.22μm여과기로 여과한 후, 배양 전에 우태아혈청(Fetal bovine serum, FBS)을 최종농도가 10%가 되도록 첨가하여 사용하였고, 세 포는 37℃, 5% CO2배양기에서 배양하였다. 배양중인 세포를 일주일에 2번 배지를 교환하고, 컨플루언트(confluent)상태가 되면 PBS(phosphate buffered saline)로 세척한 뒤, 0.25% 트립신용액으로 부착된 세포를 분리하여 원심분리한 후 피펫으로 각각의 세포가 골고루 분산되도록 잘 혼합하여 동일하게 분주하였다. Chang liver cells and HepG2 cells were distributed from ATCC (American Type Culture Collection; CCL13, HB8065). 100units / ml of antibiotics (penicillin / streptomycin) was added to MEM medium and filtered through a 0.22μm filter, followed by adding fetal bovine serum (FBS) to a final concentration of 10% before incubation. Cells were incubated in a 37 ° C., 5% CO 2 incubator. Incubate the cells in culture twice a week, wash with PBS (phosphate buffered saline) when confluent, and centrifuge the cells attached with 0.25% trypsin solution. The cells were mixed well and evenly dispensed so that each cell was evenly distributed.
아세토아미노펜 처리 후 세포증식을 살펴보기 위해, 5×104 cells/well 농도의 Chang liver 세포와 HepG2 세포를 96웰플레이트에 분주하여 24시간동안 세포가 부착하도록 배양하였다. 24시간 후 무혈청배지로 24시간을 더 배양하고, 이후 24시간동안 무혈청배지에 아세토아미노펜을 농도별로 희석하여 세포에 첨가하였다. 아세토아미노펜 처리한 세포에 MTS/PMS 용액(CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay Kit, 프로메카 코퍼레이션, 미국)을 10㎕씩 첨가한 후, 30분후에 자동엘라이자 리더(Autometic ELIZA reader)를 이용하여 490nm에서 흡광도를 측정하였으며, 세포생존율은 계산식 1에 따라 산출하였다. 모든 실험은 3회 반복적으로 실시하였다.To examine cell proliferation after acetoaminophen treatment, Chang liver cells and HepG2 cells at 5 × 10 4 cells / well concentration were dispensed in 96-well plates and cultured for 24 hours. After 24 hours, the cells were further incubated with serum-free medium for 24 hours, and then diluted with acetoaminophene in serum-free medium for 24 hours and added to cells. Add 10 μl of MTS / PMS solution (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay Kit, Promeca Corporation, USA) to acetoaminophen treated cells, and then use the Autometic ELIZA reader within 30 minutes. Absorbance was measured at 490 nm, and cell viability was calculated according to Formula 1. All experiments were repeated three times.
<계산식 1><Calculation Formula 1>
도 2에 나타낸 바와 같이, 아세토아미노펜 농도증가와 배양시간에 따라 간세포증식(A ; Chang liver 세포, B ; HepG2 세포)이 저해되었다. As shown in Figure 2, hepatocellular proliferation (A; Chang liver cells, B; HepG2 cells) was inhibited with increasing acetoaminophen concentration and culture time.
세포사멸은 세포내에 본래부터 존재하고 있던 자살기작이 세포내부와 외부의 자극에 의해 활성화되어 계획한 대로 스스로 죽는 현상으로 세포의 괴사와는 달리 죽어가는 세포의 내용물이 세포외로 유리되지 않아 다른 세포에 손상을 주지 않는다. 형태학적으로는 세포의 비중감소와 세포막의 파괴 및 염색체의 응축과 더불어 사멸체(apoptotic body) 형성과 함께 식세포작용을 거치며, 생화학적으로는 염색체 DNA가 큰 조각에서 작은 조각으로 쪼개지는 DNA 단편화가 일어난다.Apoptosis is a phenomenon in which a suicide mechanism inherent in a cell is activated by internal and external stimuli and dies as planned. Unlike cell necrosis, the contents of a dying cell are not released to the outside of the cell. Does not damage. Morphologically, phagocytosis is accompanied by cell specific gravity reduction, cell membrane destruction, chromosome condensation, apoptotic body formation, and biochemically, DNA fragmentation, in which chromosomal DNA is split from large to small fragments. Happens.
따라서 아세토아미노펜에 의한 세포증식 저해효과가 세포사멸에 의한 현상임을 확인하기위해 세포핵내의 DNA에 특이적으로 염색되는 Hoechast 33342염색을 통해 세포형태를 관찰하였다. 구체적으로 세포를 PBS로 세척하고 3.7% 파라포름알데하이드로 상온에서 10분간 고정시킨 후, 형광물질인 Hoechast 33342를 30분간 처리한다. 30분 뒤, PBS로 세척하고, 100% 에탄올로 탈수시킨 다음, 형광현미경으로 관찰하였다. Therefore, in order to confirm that the cell proliferation inhibitory effect by acetoaminophen is a phenomenon caused by apoptosis, the cell morphology was observed through Hoechast 33342 staining which is specifically stained with DNA in the cell nucleus. Specifically, the cells were washed with PBS and fixed at room temperature for 10 minutes with 3.7% paraformaldehyde, followed by 30 minutes treatment of the fluorescent substance Hoechast 33342. After 30 minutes, washed with PBS, dehydrated with 100% ethanol and observed by fluorescence microscope.
도 3(A ; Chang liver 세포, B ; HepG2 세포)에 나타낸 것과 같이, 아세토아미노펜 처리군에서 세포사멸체가 발견되었으며, 세포수도 대조군에 비해 상당히 감소한 것을 확인하였다.As shown in FIG. 3 (A; Chang liver cells, B; HepG2 cells), apoptotic bodies were found in the acetoaminophen treatment group, and the number of cells was also significantly reduced compared to the control group.
DNA단편화를 조사하기 위해서는, 간세포에 아세토아미노펜을 24시간동안 처리한 후, 세포 파쇄액(nuclear lysis buffer, 100mM NaCl2, 40mM Tris-HCl, pH7.4, 20mM EDTA, 0.5% SDS)과 혼합하여 세포를 파쇄한 후 RNase A와 프로테이나제 K를 처리하여 RNA와 단백질을 제거하고 이소프로판올을 처리하여 염색체를 침전시킨 뒤 침전된 DNA를 70%에탄올로 세척, 건조하였다. 여기에 TE 완충용액(10mM Tris-HCl, 1mM EDTA, pH8.0)을 넣어 DNA 펠렛을 용해시킨 후, 260nm와 280nm파장의 분광광도 계(아머샴 파마테크)로 흡광도를 측정하여 DNA순도와 농도를 측정하였다. 10㎍의 DNA를 2%아가로즈젤에 전기영동(50V, 2시간)한 후, 에티디움 브로마이드로 염색하여 자외선 등으로 관찰하였다.To investigate DNA fragmentation, hepatocytes were treated with acetoaminophen for 24 hours, and then mixed with a cell lysate (nuclear lysis buffer, 100
도 4(A ; Chang liver 세포, B ; HepG2 세포)에 나타난 것과 같이, 아세토아미노펜에 의해 DNA단편화가 유도되었으며, 이로써 아세토아미노펜이 세포사멸을 유도하여 간손상을 일으킴을 알 수 있다.As shown in Figure 4 (A; Chang liver cells, B; HepG2 cells), DNA fragmentation was induced by acetoaminophen, it can be seen that the acetoaminophen induces cell death causing liver damage.
실험예 3 : 톳 유래 당단백질의 아세토아미노펜에 의한 간손상 억제효과 IExperimental Example 3 Inhibition Effect of Hepatic Glycoprotein from Hepatic Injury by Acetoaminophen I
아세토아미노펜 세포독성 메카니즘 연구에서는 아세토아미노펜 반응생성물에 의한 독작용을 억제시키는데 GSH가 결정적인 역할을 한다는 것이 대부분이었다. 많은 연구모델들을 통해 심각한 간손상 시 세포내 산화적 스트레스가 야기된다는 사실은 이미 잘 알려진 사실이다(Jaeschke and Mitchell, 1989). 아세토아미노펜 투여 4시간 이후에 활성산소 형성지표인 GSSG 농도가 증가(Jaeschke, 1990; Knight et al, 2001)되므로 산화적 스트레스는 간손상의 원인이라기보다는 결과라 하였다(Rogers et al., 2000). 최근 발표된 논문들에서는 내인성·외인성 활성산소종(reactive oxygen species ; ROS)이 세포증식, 분화, 사멸 등에 관여하는 mitogen-activated protein kinases (MAPKs) 경로를 조절한다고 보고하였다(Sano et al., 2001; Jang et al., 2002). 미토겐-활성화 단백질 키나아제(Mitogen-activated protein kinases)에는 세포외 신호-조절 키나아제(extracellular signal-regulated kinase; ERK), p38 키나아제와 c-Jun N-terminal 키나아제(JNK) 가 있다. 그 중 ERK는 세포분열과 증식에 작용하는 성장인자들에 의해 활성화되며 세포증식과 분화에 관여한다. Most studies of acetoaminophen cytotoxic mechanisms have shown that GSH plays a critical role in inhibiting the poisoning effect of acetoaminophen reaction products. It is well known that many liver models cause intracellular oxidative stress in severe liver injury (Jaeschke and Mitchell, 1989). After 4 hours of acetoaminophen administration, oxidative stress was the result rather than the cause of liver injury because the concentration of free radical formation GSSG (Jaeschke, 1990; Knight et al. , 2001) was increased (Rogers et al. , 2000). Recently published papers report that endogenous and exogenous reactive oxygen species (ROS) regulate mitogen-activated protein kinases (MAPKs) pathways involved in cell proliferation, differentiation and death (Sano et al ., 2001). Jang et al. , 2002). Mitogen-activated protein kinases include extracellular signal-regulated kinase (ERK), p38 kinase and c-Jun N-terminal kinase (JNK). Among them, ERK is activated by growth factors acting on cell division and proliferation and is involved in cell proliferation and differentiation.
한편, JNK와 p38 키나아제는 주로 UV조사, 염증, 사이토카인 등의 세포외 자극에 의해 활성화된다(Kyriakis and Avruch, 2001; Chung et al., 2003; Kang et al., 2003). 이들 단백질이 활성화되면 세포에 따라 다양한 세포내 반응들이 나타난다. On the other hand, JNK and p38 kinase are mainly activated by extracellular stimulation such as UV irradiation, inflammation, cytokines (Kyriakis and Avruch, 2001; Chung et al ., 2003; Kang et al. , 2003). When these proteins are activated, various intracellular reactions occur depending on the cell.
본 실시예에서는 위에서 설명한 아세토아미노펜에 의한 세포사멸 경로를 확인하기위해 Chang liver 세포와 HepG2 세포를 가지고 그 세포독작용을 관찰함과 동시에, S.D.계 흰쥐에 아세토아미노펜을 투여한 후 혈중 GOT/GPT 수준과 간조직내 카스파아제 활성측정을 통해 간손상 정도를 살펴보았다.In this example, in order to confirm the apoptotic pathway caused by acetoaminophen as described above, the cytotoxic activity was observed with Chang liver cells and HepG2 cells, and the blood GOT / GPT levels after administration of acetoaminophen to SD rats. The degree of liver damage was examined by measuring caspase activity in liver and liver tissue.
도 5에 나타낸 바와 같이, 아세토아미노펜에 의해 세포증식이 억제된 것을 Hoechast 33342염색한 세포사진에서 알 수 있었고, 여기에 당단백추출물을 첨가했을 때는 아세토아미노펜에 의해 억제되었던 세포증식이 다시 회복되었다. As shown in FIG. 5, cell proliferation was inhibited by acetoaminophen, which can be seen in the Hoechast 33342 stained cell photograph. When the glycoprotein extract was added thereto, cell proliferation inhibited by acetoaminophen was recovered.
먼저 아세토아미노펜 독성 저해효과가 어떤 메카니즘에 의해 일어나는 것인지를 확인하기위해 앞서 사용한 두가지 세포중 더 효과가 높은 것으로 나타난 HepG2 세포를 이용하여 MAPK 경로에 관여하는 단백질발현수준을 살펴보았다. First, we examined the level of protein expression involved in the MAPK pathway using HepG2 cells, which were shown to be the more effective of the two cells used earlier to determine which mechanism caused the acetoaminophen toxicity.
도 6에 나타낸 바와 같이, 아세토아미노펜에 의해 ERK인산화가 증가되었으며 여기에 톳단백질 추출물을 첨가했을 때 ERK인산화가 더욱 증가되는 것으로 나타났다. 즉, 아세토아미노펜에 의해 산화적 손상이 야기되면서 ERK인산화가 유발되었고, 톳 유래 당단백질에 의해 ERK인산화가 세포생존을 위해 더욱 활성화됨을 알 수 있다. As shown in FIG. ERK phosphorylation was increased by acetoaminophen, and it was shown that ERK phosphorylation was further increased by adding the protein extract. In other words, ERK phosphorylation was induced by oxidative damage caused by acetoaminophen, and it can be seen that ERK phosphorylation is more activated for cell survival by the glycoprotein derived from 톳.
실험예 4 : 톳 유래 당단백질의 아세토아미노펜에 의한 간손상 억제효과 ⅡExperimental Example 4 Inhibitory Effect of Glucose-Derived Glycoprotein on Liver Damage by Acetoaminophen Ⅱ
톳 유래 당단백질의 아세토아미노펜 독성 저해작용을 in vivo 수준에서 검증하기 위해 동물실험을 실시하였다. Animal experiments were performed to verify the acetoaminophene toxicity inhibitory activity of the γ-derived glycoprotein at the in vivo level.
(1) 혈청중의 GOT와 GPT(1) GOT and GPT in serum
간기능지표인 혈청 GOT와 GPT는 간 세포의 변성 및 괴사를 반영하는 효소로 간 조직 손상 시 혈중으로 다량 유출된다고 알려져 있다. 혈청 중의 GOT/GPT는 에리트만-프랑켈(Reitman-Frankel)의 방법에 따라 제조된 글루타믹 옥살로아세틱 트랜스아미나아제(Glutamic oxaloacetic transaminase, GOT) 및 클루타믹 피루빅 트랜스아미나아제(Glutamic pyruvic trasaminase, GPT) 활성측정용 혈청 트랜스아미나아제 측정용 키트(신양화학, 대한민국)을 써서 분광광도계(Ultrospec 2001 pro. 애머샴 파마시아 바이오테크, 영국)로 505nm에서 측정하고, 도 7에 그 결과를 도시하였다. Hepatic indicators, serum GOT and GPT, are enzymes that reflect the degeneration and necrosis of liver cells. GOT / GPT in serum is glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase prepared according to the method of Reitman-Frankel. measured at 505 nm using a spectrophotometer (Ultrospec 2001 pro. Amersham Pharmacia Biotech, UK) using a serum transaminase measurement kit (Shinyang Chemical, South Korea) for measuring trasaminase (GPT) activity, and the results are shown in FIG. 7. It was.
사염화탄소를 이용한 간 손상 시 혈청중의 GOT/GPT수치가 급격히 증가되었다는 보고가 있었다 (Cho et al., 2004 ; Cho et al., 2003 ; Kim et al., 2003). 그 외에도 아세토아미노펜 투여로 인한 간 손상 시 혈청중의 GOT/GPT 증가에 대한 보고가 있었다 (Lee et al., 1998 ; Gujral et al., 2002). 본 실험결과에서는 대조군에 비해 아세토아미노펜 단독투여 시 GPT가 유의적으로 증가 (p<0.05)했으며, 톳 유래 당단백질에 의해 GPT치의 감소가 대조군 이하로 감소됨을 알 수 있다(도 7). 따라서, 톳 유래 당단백질이 아세토아미노펜 투여로 인한 간손상을 보호하여 GPT 농도를 대조군수준으로 회복시킴으로써 간손상을 저해시킨 것으로 사료된다. Serum GOT / GPT levels have been reported to increase rapidly during liver damage using carbon tetrachloride (Cho et al ., 2004; Cho et al ., 2003; Kim et al ., 2003). In addition, there have been reports of increased GOT / GPT in serum upon liver injury due to acetoaminophen administration (Lee et al ., 1998; Gujral et al. , 2002). In the experimental results, GPT was significantly increased (p <0.05) when acetoaminophen alone was administered compared to the control group, and the decrease in GPT level was lowered below the control group by the G-derived glycoprotein (FIG. 7). Therefore, it is thought that the hepatocyte-derived glycoprotein inhibited the liver damage by restoring the GPT concentration to the control level by protecting the liver damage caused by acetoaminophen administration.
(2) 간조직중의 캐스파아제 활성 변화(2) Caspase activity change in liver tissue
세포사멸은 예정된 세포의 죽음으로 조직의 항상성 유지에 중요하다. 아세토아미노펜에 의한 세포사멸 유형에 관해 많은 연구들이 보고되었지만 세포의 자연사인 세포사멸과 괴사 사이에서 아직까지 명확한 결론이 나와 있지는 않다.Apoptosis is an important cell death that is important for maintaining homeostasis. Although many studies have been reported on the types of apoptosis caused by acetoaminophene, there is no clear conclusion yet between the natural death of cells and their death.
따라서 아세토아미노펜에 의한 간세포사멸을 확인하기 위해 캐스파아제활성을 측정하였다. 이를 위해, 간장조직을 단백질분해효소 억제제(protease inhibitor)가 든 라이시스 버퍼(lysis buffer, 25mM HEPES, pH7.5, 5mM EDTA, 2mM DTT, 0.1% CHAPS)로 균질화시킨 다음, 4℃, 14,000rpm으로 원심분리시켰다. 상층액을 취해 BCA 단백질 분석 키트로 단백질 농도를 측정하여 80㎍의 단백질을 96웰플레이트에 넣고 여기에 DEVD-pNA(카스파아제-3 기질), 그랜자임 B(Granzyme B; 카스파아제-8 기질)을 넣은 후 37℃에서 4시간 동안 반응시켰다. 4시간 후, 405nm에서 흡광도를 측정하였다.Therefore, caspase activity was measured to confirm apoptotic cell death by acetoaminophen. To this end, the liver tissue is homogenized with lysis buffer (25 mM HEPES, pH 7.5, 5 mM EDTA, 2 mM DTT, 0.1% CHAPS) containing protease inhibitor, and then 4 ° C., 14,000 rpm. Centrifugation. Take the supernatant and measure protein concentration with the BCA Protein Assay Kit, place 80 μg of protein in a 96-well plate and add DEVD-pNA (Caspase-3 Substrate) and Granzyme B (Caspase-8 Substrate). After the reaction was carried out for 4 hours at 37 ℃. After 4 hours, the absorbance was measured at 405 nm.
도 8에 나타낸 것처럼, 카스파아제-3 활성이 아세토아미노펜에 의해 증가되었다가, 톳단백질 병행투여로 다시 감소되는 것으로 나타났으나, 카스파아제-8 활성에는 아세토아미노펜이나 톳 단백질 병행투여에 의해 유의적인 차이가 보이지 않았다. 한편, 카스파아제-9 활성화를 알아보기 위해 웨스턴블롯을 실시하였다. 카스파아제-3 활성과 마찬가지로, 아세토아미노펜에 의해 카스파아제-9가 활성화되었다 가 톳 유래 당단백질 병행투여로 분열된 카스파아제-9밴드가 감소되는 것으로 나타났다. 이 결과를 종합해보면, 아세토아미노펜에 의한 세포사멸은 미토콘드리아를 통해 일어난다는 사실을 짐작할 수 있다. 즉 카스파아제-8에는 비의존적이나, 카스파아제-3/-9에는 의존적으로 아세토아미노펜 독작용이 일어났다가, 톳 유래 당단백질이 아세토아미노펜에 의해 활성화되었던 카스파아제-3/-9활성을 감소시킴으로써 아세토아미노펜독작용을 저해하는 것이다.As shown in FIG. 8, the caspase-3 activity was increased by acetoaminophen and decreased by the co-administration of the protein, but the caspase-8 activity was significantly increased by the acetoaminophen or co-protein administration. No difference was seen. Meanwhile, Western blot was performed to investigate caspase-9 activation. As with caspase-3 activity, caspase-9 was activated by acetoaminophen and the cleaved caspase-9 band was reduced due to the co-administration of o-derived glycoproteins. Taken together, it can be estimated that apoptosis by acetoaminophen occurs through mitochondria. In other words, acetoaminophen poisoning occurred independently of caspase-8, but caspase-3 / -9, and the glycoprotein derived from oocytes decreased caspase-3 / -9 activity, which was activated by acetoaminophen. It is to inhibit the acetoaminophen poisoning action.
도 1은 SDS-PAGE와 아가로스 겔 전기영동 결과를 나타낸 도이다.1 is a diagram showing the results of SDS-PAGE and agarose gel electrophoresis.
도 2는 아세토아미노펜 농도증가와 배양시간에 따른 간세포 증식 저해 효과를 나타낸 그래프이다.Figure 2 is a graph showing the effect of inhibiting hepatocyte proliferation according to the increase in acetoaminophen concentration and incubation time.
도 3은 아세토아미노펜에 의한 세포 증식 저해 효과를 Hoechast 33342 염색 결과를 통해 나타낸 형광 현미경 사진이다.Figure 3 is a fluorescence micrograph showing the effect of inhibiting cell proliferation by acetoaminophen through Hoechast 33342 staining.
도 4는 아세토아미노펜에 의한 DNA 단편화 유도 결과를 나타낸 도이다.4 is a diagram showing the results of DNA fragmentation induction by acetoaminophene.
도 5는 아세토아미노펜에 의한 세포 증식 저해 효과를 Hoechast 33342 염색 결과를 통해 나타낸 형광 현미경 사진다.5 is a fluorescence micrograph showing the effect of inhibiting cell proliferation by acetoaminophen through Hoechast 33342 staining.
도 6은 HepG2 세포를 이용하여 MARK 경로에 관여한 단백질 발현수준을 나타낸 도이다.Figure 6 is a diagram showing the protein expression level involved in the MARK pathway using HepG2 cells.
도 7은 혈청중의 GOT와 GPT 변화를 나타낸 도이다.7 is a diagram showing changes in GOT and GPT in serum.
도 8은 카스파아제 활성을 나타낸 도이다.8 is a diagram showing caspase activity.
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