Part Ⅰ: Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4-MD2 complex
Reactive oxygen species (ROS) contribute to the development of non-alcoholic fatty liver disease (NAFLD). ROS generation by infiltrating macrophages involves multiple mechanisms including Toll-like receptor 4 (TLR4)-mediated NADPH oxidase (NOX) activation. However, the mechanisms by which palmitate stimulates TLR4-mediated NOX2 activation are not fully understood. Here, we report that palmitate-stimulated $CD11b^+F4/80^{low}$ hepatic infiltrating macrophages, but not $CD11b^+F4/80^{high}$ Kupffer cells, generate ROS via dynamin-mediated endocytosis of TLR4 and NOX2, without the involvement of MYD88 and TRIF. Using size exclusion chromatography, we demonstrate that unlike LPS-mediated dimerization of the TLR4-MD2 complex, palmitate binds a monomeric TLR4-MD2 complex that triggers endocytosis, ROS generation and increased pro-interleukin-1$\beta$ expression in macrophages. Moreover, palmitate-induced ROS generation in human $CD68^{low}CD14^{high}$ macrophages is strongly suppressed by inhibition of dynamin. Furthermore, NOX2-deficient mice are protected against high-fat diet-induced hepatic steatosis and insulin resistance. Therefore, endocytosis of TLR4 and NOX2 into macrophages might be a novel therapeutic target for NAFLD
Part Ⅱ. Exosomes derived from palmitic acid-treated hepatocytes induce fibrotic activation of hepatic stellate cells
Non-alcoholic fatty liver disease (NAFLD) is a dominant cause of chronic liver disease, but the exact mechanism of progression from simple steatosis to nonalcoholic steatohepatitis (NASH) remains unknown. Here, we investigated the role of exosomes in NAFLD progression. Exosomes were isolated from a human hepatoma cell line treated with palmitic acid (PA) and their miRNA profiles examined by microarray. The human hepatic stellate cell (HSC) line (LX-2) was then treated with exosome isolated from hepatocytes. Compared with controls, PA-treated hepatocytes displayed significantly increased CD36 and exosome production. The microarray analysis showed there to be distinctive miRNA expression patterns between exosomes from vehicle- and PA-treated hepatocytes. When LX-2 cells were cultured with exosomes from PA-treated hepatocytes, the expression of genes related to the development of fibrosis were significantly amplified compared to those treated with exosomes from vehicle-treated hepatocytes. In conclusion, PA treatment enhanced the production of exosomes in these hepatocytes and changed their exosomal miRNA profile. Moreover, exosomes derived from PA-treated hepatocytes caused an increase in the expression levels of fibrotic genes in HSCs. Therefore, exosomes may have important roles in the crosstalk between hepatocytes and HSCs in the progression from simple steatosis to NASH.
서구화된 식습관과 에너지 소비 불균형으로 인해 비만, 당뇨병, 고지혈증, 고혈압 등의 대사질환의 발병이 꾸준히 증가하는 동시에, 비알코올성 지방간의 발병도 함께 증가하는 추세이다. 지방간은 위 모든 간 손상의 시발점으로서, 식이 조절과 규칙적인 운동에 의해 정상적인 상태로 돌아올 수 있으므로 간질환/간손상의 진행의 중요한 전환점이라 볼 수 있다. 그러므로 지방간의 치료와 예방에 있어 비알코올성 지방간 발병의 메커니즘, 또는 단순 지방간에서 지방간염으로 발전하는 기전에의 연구가 매우 중요하다.
하여 Part 1에서는 ‘지방간 발병에 있어 간 대식세포에의 영향; 간 대식세포의 TLR4-MD2 복합체와 포화지방산의 단량체 세포내섭취로 인한 NOX2 유래 활성산소의 역할’에 대해 연구하였고, Part 2에서는 ‘단순 지방간에서 지방간염으로의 진행에서 간세포와 간성상세포에의 영향; 간세포 유래 엑소좀에 의해 간성상세포가 간섬유화를 촉진하는 메커니즘’을 연구하였다.
