Hepatoprotective effect of dieckol on the tacrine-induced HepG2 cells

Abstract

해조류는 최근 서구화된 식습관에 의해 증가하고 있는 비만, 당뇨, 고혈압 등의 혈행 장해성 질환과 같은 생활 습관병에 대해 예방 효과를 가지는 높은 함량의 비타민, 무기질은 물론 섬유질이 풍부한 식품으로 알려져 전통적인 웰빙 식품으로 추천 되어 왔다. 또한, 해조류에는 2차 대사 산물로서 phenyl과 phenoxyl기를 가지는 polyphenol류인 phlorotannin 성분이 다량 함유되어 있으며, 그 생리 활성 기능에 대한 관심이 더해지고 있다. 특히 Ecklonia 종의 대표적인 해조류 중 하나인 곰피는 (Ecklonia stolonifera OKAMURA) 다년생 다시마과 (Laminaricese)에 속하는 갈조류로 아시아에서 미역, 다시마, 톳 등과 같이 식용으로 많이 이용되어 왔다. 최근의 연구를 통하면 곰피의 2차대산물인 phlorotannin류들이 항 산화 활성, 항 돌연 변이 활성 등을 비롯한 여러 가지 생리 활성 기능이 있는 것으로 알려지고 있다. 본 연구에서는 곰피의 phlorotannin류 중 항 산화능이 높은 것으로 확인된 dieckol을 분리한 후, Alzheimer병의 치료제 중 하나인 tacrine을 처리하여 간암 세포의 손상을 유도한 후, dieckol의 간보호 효과를 조사하였다. Tacrine은 acetylcholinesterase의 기능을 억제시켜 Alzheimer병에 대해 치료 효과를 보였으나, 간 독성을 비롯한 여러 가지 부작용으로 인해 현재는 사용이 중단 되어져 있다. 간은 물질 대사에 관여하여 외부로부터 유입된 물질 뿐 아니라 대사 생성물에 의한 독성으로부터 보호와 해독 기능에 깊이 관여함으로, 여러 질병에 대해 치료 목적으로 사용되는 화학 제제들에 의한 손상들도 적지 않게 보고되고 있다. 이에 항 산화능이 높은 것으로 알려진 dieckol을 천연물인 곰피로부터 분리한 후 tacrine과 함께 처리하여 간세포 독성에 대한 보호 효과를 확인하고자 하였다. 곰피의 MeOH 추출물 중, EtOAc 분획물이 높은 DPPH radical 소거능을 가진 것을 확인하고, silica gel chromatography와 HPLC를 반복적으로 수행하여 항 산화 활성을 가진 dieckol을 분리하였다. 본 실험에서 Tacrine 처리(EC_(50): 0.3 mM)는 HepG2 세포의 손상을 유발시켰고, dieckol의 첨가는 손상된 세포의 보호 효과를 가지는 것이 확인 되었다 (EC_(50): 43.6 M). Dieckol은 또한 tacrine에 의하여 유발되는 세포 내 ROS의 생성을 강하게 억제시키는 것으로 나타났으며, Western blot으로 확인한 결과, tacrine은 Fas와 관련된 apoptosis 신호 전달 체계를 활성화시켰다. 반면 dieckol은 Fas 단백질들의 발현을 억제시킴으로써 연차적으로 capsase 8, bid, caspase 3, PARP 등의 단백질의 활성을 저해하여 세포 사멸 기전을 통한 세포 사멸을 막는 것으로 나타났다. 최종적으로, Dieckol은 ROS의 생성을 억제하는 항 산화기전과 함께 이와 연관된 Fas 관련 단백질들에 의한 apoptosis를 억제하여 tacrine으로 유도되는 세포 사멸로부터 간세포를 보호하는 것으로 확인되었다.

Our life has been threatened by so many kinds of antibiotics, drugs, and chemical additives etc. They were originally developed to cure and improve the health conditions in human life, but also have had negative effects which could cause damages in living organs. Especially liver is the representatively protective organ in metabolic digestion and detoxification of ingested agents such as foods, xenobiotics and drugs etc. Thus liver or hepatic disorders are recognized to be severe problem. Tacrine was developed as a drug to treat Alzheimer's disease, as acting an acetylcholinesterase inhibitor. Tacrine was suggested to increase Central Nerve System (CNS) acetylcholine levels of Alzheimer's disease patients (Mesulam et al., 2002; Darvesh and Hopkins, 2003; Johnson et al., 2004). And tacrine has been reported to have function of antioxidant in vitro and it protects from hydrogen peroxide-induced rat pheochromocytoma line PC12 cells by regulating expression of apoptosis-related genes. Its neuroprotective effects of cholinesterase inhibitor by antioxidation might partly contribute to the clinical efficacy in AD treatment (Xiao et al., 2000; Zhang and Tang. 2000; Wang et al., 2002). However, tacrine had been also reported to cause hepatic injury in 30-50% patients, as indicated by the reversible increasing level of transaminase (Watkins et al., 1994). In recent reports, tacrine is thought to alter intracellular glutathione concentrations in hepatocytes (Lagadic-Gossmann et al., 1998). Moreover tacrine affects on intracellular ROS generation (Osseni et al., 1999), lipid peroxidation, membrane fluidity (Galisteo et al., 2000) and mtDNA synthesis (Mansouri et al., 2003), indicating that those effects of tacrine might affect on liver cells proliferation through increasing necrosis and/or apoptosis in liver cells. Figure 6 shows that tacrine(0-0.5 M) induces cell death on HepG2 cells as cell viability was reduced in dose dependent manner. When dieckol, a kind of phlorotannins which had been isolated from E. stolonifera, was co-treated with tacrine on HepG2 cells, the cell viability was recovered (Fig 7) and the intracellular ROS (reactive oxygen species) generation was completely blocked compared with only tacrine treated groups (Fig 8). From this result, we confirm that Tacrine increases ROS level, standing for significant damages on hepatic cells(Osseni RA et al ., 1999), and dieckol recovers them by eliminating it. We have also found the expression of Fas/FasL system proteins was induced by tacrine, and was decreased by treatment with dieckol for 24 hr. (Fig 9) Fas, activated as a stimulus of tacrine in HepG2 cells, was decreased by co-treatment with dieckol in dose-dependent manner. Activation of Fas on the cell membrane by Fas ligand or agonist antibody results to the activation of caspase-8, the capital caspase in this pathway, and then caspase-8 activates caspase-3 through direct or indirect pathway. (Boldin et al.,1996; Muzio et al., 1996) Bid, one of the BH3-only Bcl-2 family members, is also activated post-translationally via cleavage by caspase-8/FLICE in response to Fas or TNF receptor activation. (Li et al.,1998) The truncated Bid (t-Bid) cleavage product, p15, acts in mitochondria to release cytochrome C to cytosol. (Luo et al.,1998) The released cytochrome c to cytosol activates procaspase-3, which usually presents as inactive proenzymes form in cytosol, to active form and the active caspase-3 hydrolyzes PARP. (Zhuang et al.,1999; Reed, 1997; Cohen, 1997; Enari et al., 1995) The specific cleavage of PARP at the sequence Asp-Glu-Val-Asp (DEVD) has been reported to be a sensitive marker of caspase-3 activation in apoptosis. (Janicke et al., 1998; Kaufmann et al., 1993; Um et al.,1996) In our results, the expression of Fas-related apoptotic signals including caspase-8, Bid, and PARP fragmentation, was ascertained to be down-regulated by dieckol with dose-dependent manner. JUN NH2-terminal kinases(JNKs) are known to be involved in apoptosis, neurodegradation, cell differentiation and proliferation, inflammatory conditions etc,(Liu et al., 2005) to confirm whether dieckol can block the activation of JNK, SP600125, an antibody of JNK, was treated with tacrine to compare with dieckol. 50 M of dieckol suppressed the phosphorylation and activation of JNK, as much as SP600125, as an inhibitor of JNK. (Fig 10) It is important because the phosphorylated JNK modifies the activity of numerous proteins in mitochondria and nucleus, and regulates several important cellular functions by changing the levels of intracellular ROS. (Xia et al., 1995) Some compounds extracted from brown alga have been reported to reduce intercellular ROS levels effectively. Choi et al. (1997) reported that methanolic extractions of E. stolonifera, its subsequent fractions and its components, phloroglucinol and phlorotannin A, are useful as nitrite scavangers. Brown alga polyphenols have strong antioxidant effects, and phlorotannins, extracted from E. cava, were performed as scavengers of DPPH radical in dose-dependent manner. (Kim et al., 2004; Kang et al ., 2003) (Shibata et al., 2002) Our evidence certified that the intracellular events started from generation of ROS by tacrine lead to hepatic cell death sequentially. In the initial step, there is generation of ROS and activation of Fas-mediated protein signaling transduction. At this stage, dieckol suppresses the production of ROS and prevents cell death by blocking these cell signals in apoptosis. Based on our observation with other latest studies, compounds having antioxidant activities are considered to protect hepatocytes from tacrine-induced cytotoxicity, as its antioxidant function results to reduce formation of ROS or to scavenge free radicals. Therefore, we suppose that tacrine stimulates hepatic cells death through Fas/FasL signal transduction activated by ROS, and dieckol protects the cells from tacrine by inhibiting Fas down-stream proteins with reducing ROS level. It seems that the reductive effect of dieckol is caused both directly and indirectly by redox-reaction, and it triggers the expression of scavenging enzymes such as catalase and superoxide dismutase (SOD) in signaling transduction. Therefore, dieckol should be studied more with the heaptic damages caused by other factors such as acetaminophen or alchol, as they also have the similar side effect of tacrine. (Mitchell et al., 1973) Although, in our report, the effect of dieckol on expression of intracellular scavenging enzymes has not covered yet, these findings will provide a biochemical basis for the influence of dieckol on the hepatoprotective effect.