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Anti-Inflammatory Effects of Sulfatide in Macrophage through Inhibition of HMGB1 Secretion

 College of Medicine (의과대학) 
 Others (기타) 
Issue Date
The high mobility group box 1 (HMGB1) is a well-known late mediator of sepsis secreted by multiple stimuli. Such stimuli involve multiple pathways, such as the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways, and reactive oxygen species (ROS) under inflammation. Sulfatide, on the other hand, is a sphingolipid commonly found in myelin sheets with a disputed immunological role where it is reported to be pro-inflammatory in the central nervous system, whereas it shows protective effect in the periphery. Such discrepancy regarding the immunological characteristics of sulfatide led me to seek to determine the immunological characteristics of sulfatide in the periphery. Immunological characteristics of sulfatide was analyzed through studying the secretion of HMGB1 triggered by lipopolysaccharide (LPS) stimulation in Raw 264.7 cells after pre-treatment or post-treatment of sulfatide. Suppression of HMGB1 secretion by inhibiting its cytosolic translocation was observed after both pre-treatment with sulfatide before LPS stimulation, and post-treatment with sulfatide after LPS stimulation. Further analysis of the downstream molecules of toll-like receptor (TLR) signaling revealed suppression of c-Jun N-terminal kinase (JNK) phosphorylation and p65 translocation. LPS-mediated ROS production was also decreased when sulfatide pre-treatment was provided, caused by the down-regulation of the phosphorylation of activators, such as IRAK4 and TBK1. Investigation of the upstream mechanism that encompasses all the aforementioned inhibitory characteristics unveiled the involvement of lipid rafts. In addition to the co-localization of biotinylated sulfatide and monosialotetrahexosylganglioside, a decrease in LPS-induced co-localization of TLR4 and lipid raft markers was observed when sulfatide treatment was given before LPS stimulation. Overall, pre-treated sulfatide was found to exert its anti-inflammatory properties by hindering the co-localization of TLR4 and lipid rafts, nullifying the effect of LPS on TLR4 signaling. On the other hand, post-treatment of sulfatide exhibited anti-inflammatory characteristics via modifying metabolic characteristics of macrophage. Similar effects of sulfatide were also confirmed in the LPS-mediated murine experimental sepsis model, showing decreased levels of serum HMGB1, TNF-α, IL-6, increased survivability, and reduced pathological severity. High mobility group box 1 (HMGB1)은 패혈증 후반부에 나타나는 패혈증 중재 물질로 미토겐활성화단백질키나아제 (MAPK)와 NF-κB, 그리고 활성산소 (ROS)와 같은 경로를 통해 염증 시 분비되어 진다고 알려져 있다. Sulfatide는 HMGB1과 달리 myelin sheath에 다량 존재하는 스핑고지질로 면역학적 역할에 대한 의견이 분분하다. 중추신경계에서는 sulfatide가 염증을 일으킨다고 보고되었고, 반대로 말초면역계에서는 sulfatide가 세포를 보호하는 역할을 한다고 보고되었다. Sulfatide가 가지고 있는 면역학적 역할에 대한 이견을 말초면역계에서 확인하기 위해 sulfatide가 말초에서 보이는 면역학적 특징을 Raw 264.7 세포주가 sulfatide를 전/후처리와 LPS 자극을 주었을 때 분비하는 HMGB1양을 비교함으로써 연구해보고자 하였다. Sulfatide의 전처리와 후처리 모두 LPS 자극에 의한 HMGB1의 세포질로의 이동과 분비량이 줄어는 것을 확인하였으며, 톨유사수용체 4 (TLR4)의 하위 신호 전달 경로 분자인 c-Jun N-terminal kinase (JNK)의 인산화와 p65 분자의 핵내이동량이 감소하였다. LPS에 의한 활성산소 생성은 sulfatide의 전처리에 의해 줄었으며, 이는 IRAK4나 TBK1과 같은 활성체의 인산화가 감소하여 일어난 것임을 확인하였다. 상위 신호 전달 과정을 찾고자 지질 라프트 (lipid raft)를 연구하였다. 지질 라프트와 비오틴화 sulfatide가 공존하고, LPS 자극으로 인한 지질 라프트 내로 TLR4의 이동 역시 감소하였다. 이를 통해 전처치 된 sulfatide는 TLR4이 지질 라프트 내로 이동하는 것과 신호 전달을 방해하고, 그 결과로 소염 효과를 내는 것을 알 수 있었다. 한편, 후처치 된 sulfatide는 대사적 특징을 바꿈으로써 소염 효과를 내는 것 또한 확인 할 수 있었다. 실험쥐에서 LPS 매개 실험적 패혈증 모델에서도 sulfatide 처리로 인해 혈장 내 HMGB1, TNF-α, 그리고 IL-6의 수치가 낮아짐에 따라 생존율이 증가하고 패혈증 질병 척도 점수가 낮아짐을 확인하였다.
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