톳 당에 의한 에탄올 위 손상 및 IEC-6 세포의 세포사멸

Abstract

옛 부터 이어져온 우리나라 고유의 식생활 습관과 최근 식생활의 서구화, 과식, 과음, 스트레스, 카페인의 섭취 및 아스피린, 부신피질호르몬제, 소염제 같은 약물(NSAID)의 섭취 증가로 인해 위장질환 또는 증세가 증가하고 있다. 위궤양으로 인한 위출혈이나 위천공 같은 병을 치료하기 위한 약들이 많이 개발되었으나 비싸고 다양한 부작용을 가지고 있어서 식물 중 생리 화학적 물질들을 분리하여 부작용 없는 여러 가지 효능을 확인하는 연구가 진행되어지고 있다. 갈조류의 대표적인 종인 톳은 최근생리활성에 대한 연구가 활발하게 이루어지고 있으나 위 손상에 대한 생리활성 연구는 잘 알려지지 않았다. 에탄올 섭취는 공격인자의 대표적인 요인으로 인체 내 산화적 스트레스(oxidative stress)를 유발하게 된다. 본 연구에서는 에탄올의 산화적 스트레스로 인한 위 손상 및 세포 독성에 대해 톳의 구성성분 중 가장 많은 탄수화물을 선택하여 정제한 Hf-PS-1의 생리활성 효과를 in vivo와 in vitro 실험을 통해 각각 살펴보았다.

Gastrointestinal disorders are important causes of human morbidity in non-industrialized countries. Many pharmaceutical products have been developed for the treatment of gastrointestinal symptoms. However, despite recent pharmaceutical advances, many pharmaceutical products are relatively expensive and are associated with various medical problems. Drugs that relieve pain, heal ulcers, delay ulcer recurrences, and even cure disease have been developed, but they generally have important side effects. Marine algae have provided great biological diversity for sampling in the discovery-phase of drug development. Some seaweeds contain high amounts of polysaccharides such as alginate, agarose, carrageenan and fucoidan. Various seaweed polysaccharides have diverse biological activities, including effects on the elicit a wide range of anti-tumor, anti-complimentary, anti-inflammatory, immunological and anti-viral activities. Among the various seaweeds, Hizikia fusiformis (Hf) is one of the blown algae consumed widely in Korea, Chines and Japanese. Brown seaweeds contain a various soluble polysaccharides as alginates, fucans and laminarins together with the insoluble fibers made of cellulose. This algae possesses a number of potential compounds, including antioxidants (Siriwardhana et al., 2004) and anticoagulants (Kim et al., 1998). It also contains inorganic arsenic, which is carcinogenic to humans (Watanabe et al., 1979; Nakamura et al., 2008). But the health effect of dietary Hf remains scientifically unclear. Especially, the mechanisms responsible for protective effect of Hf against ethanol-induced peptic injury is unparalleled. In this study, we extracted and purificated polysaccharide from the marine alga Hizikia fusiformis (Hf-PS-1) and examined its biological effects against ethanol-induced gastrointestinal injury, using in vitro and in vivo assay. In vitro assay, using a rat small intestine IEC-6 cells, we've destroyed the gut by ethanol, which is most frequently abused agent, and found that 5% ethanol treatment for 1 hr inhibited cell proliferation by 50% compared with untreated group. Since co-treatment of Hf-PS-1 and ethanol protect the ethanol-induced cell death, and we examined which signaling pathway is related with the effect of Hf-PS-1, focusing on IGF-IR signaling pathway, which is involved in cell growth, defferentiation, etc. The Hf-PS-1 inhibited Ethanol cytotoxicity through two IGF-IR signaling pathways: activation of the PI3K pathway and the MAPK pathway. The ethanol treatment induced IGF-IR and IRS-1 phosphorylation slightly. Furthermore, binding of IRS-1 and p85, and the phosphorylation of Akt markedly increased after Ethanol treatment. In contrast, co-treatment of Hf-PS-1 further phosphorylated IGF-IR, but decreased IRS-I phosphorylation and binding of p85 to IRS-1. Based on Western blotting analysis, treatment with ethanol alone induced an increase of Akt activation, whereas Hf-PS-1 co-treatment decreaased it. The Akt is generally activated by proliferative stimuli, such as growth factors, but in our system, Akt phosphorylation is induced ethanol treatment but decreased Hf-PS-1 co-treatment, which induced cell proliferation. Therefore, we suggest the Akt activation during the oxidative stress by Ethanol is induced to sustain cell survival. Furthermore, we examined the another pathway of IGF-IR signaling pathway, MAPK signaling pathway. Ethanol inhibited Shc activation and the its binding with Grb2. On the contrary, Hf-PS-1 co-treatment increased Shc activation and the binding of Grb2 to Shc. So we observed ERK, JNK, p38 activation, the downstream of Shc-Grb2 singaling. Ethanol induced phosphorylation of JNK compared control, but co-treated Hf-PS-1 is decreased. MAPKs mediate apoptosis and cell growth. Specially, JNK is activated by oxidative stress and one of the mitochondrial apoptosis pathway. Although p38 activation is not detected, ERK phosphorylation is increased in both of ethanol only or co-treatment of ethanol and Hf-PS-1. Therefore, the effect of Hf-PS-1 against ethanol-induced damage is related with JNK phosphorylation. In the results of in vivo assay, ethanol treatment induced gastric bleeding, surface epithelial cell destruction and loss of the surface mucosa layer compared to the control group, but co-treatment with Hf-PS-1 inhibited these damages. Ethanol treatment resulted in significant activation of caspases 3, 8, and 9, and DNA fragmentation, indicators of apoptosis, and co-treatment with Hf-PS-1 inhibited it. Furthermore, we examined GSH, which plays a major protective role as a scavenger of free radicals that combine with non-protein thiols at the GSH reactive center to abolish free radical toxicity. In the present study, GSH levels decreased by 34% in the ethanol-only group relative to the control group (100%), whereas they increased to 103.9% in the ethanol +Hf-PS-1 group. Based on Western blotting analysis, JNK activation in the ethanol-only group. Co-treatment with Hf-PS-1 and ethanol decreased JNK activation, but phospho-ERK levels were not significantly different among the three groups. These results suggested that the protective effect of Hf-PS-1 was primarily associated with the inhibition of JNK phosphorylation. These results are agreement with that of in vitro assay. Therefore, we suggest that Hf-PS-1 could provide a new, natural treatment option for gastric ulcers in humans.