에탄올에 의한 흰쥐 위 점막 손상 및 인간 IEC-6 세포 사멸에 대한 파래 당단백질의 보호효과

Abstract

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. such as Capsosiphon, Chlorella and Yezoensis. Various seaweed 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, Enteromorpha linza is one of the green algae consumed widely in Korea, and Japanese. Green seaweeds contain a various nutrition as protein, polysaccaride and vitamin. 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 EGP remains scientifically unclear. Especially, the mechanisms responsible for protective effect of EGP aginst EtOH-induced peptic injury is unparalleled. In this study, we extracted and purificated glycoprotein from the marine alga Enteromorpha linza and examined its biological effects against EtOH-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 EtOH, which is most frequently abused agent, and found that 8% EtOH treatment for 1 hr inhibited cell proliferation by 20% compared with untreated group. Since co-treatment of EGP and EtOH protect the EtOH induced cell death, and we examined which signaling pathway is related with the effect of EGP, focusing on IGF-1R signaling pathway, which is involved in cell growth, differentiation, etc. The EGP inhibited EtOH cytotoxicity through two IGF-1R signaling pathways: activation of the Akt and MAPK pathway, The EtOH treatment induced IGF-1R and phosphorylation of Akt increased after EtOH treatment. In contrast, co-treatment of EGP futher phosphorylated IGF-1R, but decreased IRS-1 phosphorylation. based on western blotting analysis, treatment with EtOH alone induced an increase of Akt activation, whereas EGP co-treatment decrease it. The Akt is generally activated by proliferative stimuli, such as growth factors, but in our system, Akt phosphorylation is induced EtOH treatment but decreased EGP co-treatment, which induced cell proliferation. Therefore, we suggest the Akt activation during the oxidative stress by EtOH is induced to sustain cell survival. Futhermore, we examined the another pathway of IGF-1R signaling pathway, MAPK signaling pathway. EtOH inhibited Shc activation and the its binding with Grb2. On the contrary, EGP co-treatment increased Shc activation and the binding of Grb2 to Shc. So we observed ERK, JNK, p38 activation, the downstream of Shc-Grb2 signaling. EtOH induced phosphorylation of JNK compared control, but co-treated EGP 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 EtOH only or co-treatment of EtOH and EGP. Therefore, the effect of EGP against EtOH-induced damage is related with JNK phosphorylation.

In the results of in vivo assay, EtOH treatment induced gastric bleeding, surface epithelial cell destruction and loss of the surface mucosa layer compared to the control group, but co-treatment with EGP inhibited these damages. EtOH treatment resulted in significant activation of caspases-3, -8 indicators of apoptosis and co-treatment with EGP inhibited it. Futhermore 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 22% in the EtOH-only group relative to the control group(100%), whereas they increased to 106.7% in the EtOH + EGP group. Based on western blotting analysis, JNK activation in the EtOH-only group. Co-treatment with EGP and EtOH decreased JNK activation but phospho-ERK levels were not significantly different among the three groups. These results suggested that the protective effect of EGP was primarily associated with the inhibition of JNK phosphorylation. These results are agreement with that of in vitro assay. Therefore, we suggest that EGP could provide a new, natural treatment option for gastric ulcers in human.