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Toll-like receptor 4 mediated inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells

Title
Toll-like receptor 4 mediated inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells
Other Titles
Bacterial lipopolysaccharide가 사람 간성상세포에서 Toll-like receptor 4를 매개로
Issue Date
2004
Publisher
Graduate School, Yonsei University
Description
Dept. of Medical Science/박사
Abstract
[한글] 그람음성 세균 내독소는 간내에서 Kupffer 세포를 자극하여 염증성 사이토카인을 분비시키고 활성산소를 유리시키는 등의 작용으로 알코올성 간질환의 중요한 병인으로 간주되고 있다. 간성상세포는 간내 섬유화 및 염증반응에 중심적인 역할을 하는 세포이다. 그러나 현재까지 세균 내독소가 간성상세포에 직접적으로 작용하는지, 작용한다면 어떤 영향을 나타내는지 알려져 있지 않다. 본 연구에서는 세균 내독소가 간성상세포에 직접 작용하여 일으키는 염증성 반응과 이에 관계하는 신호전달체계를 규명하고자 하였다. 1. 배양에 의해 활성화되거나, C형 간염에 의한 간경변증으로 생체내에서 활성화된 사람 간성상세포에서 세균 내독소의 수용체인 TLR4, CD14, MD2 가 발현되었다. 2. 내독소로 사람 간성상세포를 자극했을 때 내독소의 용량과 자극시간에 비례하여 NF-B가 현저히 활성화되었다. 3. 내독소와 lipid A에 의한 NF-B 활성화는 TLR4 차단항체와 내독소길항제인 Polymyxin B에 의해 억제되었다. 4. 간성상세포에서 Lipid A에 의한 NF-B 활성화는 TLR4 유전자 변이로 기능이 상실된 C3H HeJ 마우스에서는 관찰되지 않았으며 TLR4 기능이 정상인 C3H/OuJ 마우스에서만 관찰되었다. 5. 사람 간성상세포에서 내독소는 c-Jun N-termianl Kinase (JNK)를 활성화 시켰다. 6. 사람 간성상세포에서 내독소는 IL-8과 MCP-1의 발현을 현저히증가시켰으며 이러한 효과는 NF-B 억제물질인 IB super-repressor (Ad5IB)에 의해 완전히 억제되었고 선택적 JNK 억제제인 SP600125에 의해서 부분적으로 억제되었다. 7. 사람 간성상세포에서 내독소는 세포표면에 발현된 ICAM-1과 VCAM-1의 발현을 유의하게 증가시켰다. 결론적으로 활성화된 사람 간성상세포는 그람음성 세균 내독소에반응하여 내독소 수용체인 TLR4 관련 신호전달체계를 이용하여 NF-B 및 JNK가 활성화되고 뚜렷한 염증성 반응을 나타내었다. 따라서 간성상세포는 내독소 유발성 간손상에서 중요한 역할을 하는 것으로 생각된다.
[영문]Bacterial lipopolysaccharide (LPS) stimulates Kupffer cells and participates in the pathogenesis of alcohol-induced liver injury. However, it is unknown whether LPS directly affects hepatic stellate cells (HSCs), the main fibrogenic cell type in the injured liver. This study characterizes LPS-induced signal transduction and pro-inflammatory gene expression in activated human HSCs. Human HSCs were isolated and activated in culture. Expressions of CD14, Toll-like receptor (TLR) 4, and MD2 mRNA were assessed by RT-PCR. IkB and phospho-c-Jun were assessed by Western blot analysis. IkB kinase (IKK) and c-Jun N-terminal kinase (JNK) activity were measured by in vitro kinase assay using a GST-IkB(1-54) or GST-c-Jun substrates, respectively. LPS-induced NF-kB transcriptional activation was assessed by a luciferase reporter gene assay in response to various concentrations of purified LPS (1-1000 ng/ml). Nuclear translocation of NF-kB was assessed by immunofluorescent staining for p65 and electrophoretic mobility shift assay (EMSA). IL-8 and MCP-1 expression were assessed by an RNase protection assay and enzyme-linked immunosorbent assay (ELISA). ICAM-1 and VCAM-1 expression were assessed by flow cytometry. Culture-activated HSCs and HSCs isolated from patients with hepatitis C virus-induced cirrhosis express LPS-associated signaling molecules including CD14, TLR4, and MD2. Stimulation of culture-activated HSCs with LPS results in a rapid and marked activation of NF-kB. Lipid A induces NF-kB activation in a similar manner. Both LPS- and lipid A-induced NF-kB activation is blocked by preincubation with either anti-TLR4 blocking antibody (HTA125) or Polymyxin B. Lipid A induces NF-kB activation in HSCs from C3H/OuJ (TLR4- sufficient mice) but not from C3H/HeJ (TLR4-deficient mice). LPS also activates c-Jun N-terminal Kinase (JNK). LPS upregulates gene expression and secretion of IL-8 and MCP-1. LPS-induced IL-8 secretion is completely inhibited by the IkB super-repressor (Ad5IkB) and partially inhibited by a specific JNK inhibitor, SP600125. LPS also upregulates cell surface expression of ICAM-1 and VCAM-1. Human activated HSCs utilize components of TLR4 signal transduction cascade to stimulate NF-kB and JNK, and upregulate chemokines and adhesion molecules. Thus, HSCs are a potential mediator of LPS-induced liver injury.
URI

http://ir.ymlib.yonsei.ac.kr/handle/22282913/128644
Appears in Collections:
2. 학위논문 > 1. College of Medicine (의과대학) > 박사
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