Department of Ophthalmology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200093, People's Republic of China.
Exp Biol Med (Maywood). 2014 Feb;239(2):183-92. doi: 10.1177/1535370213514922. Epub 2014 Jan 23.
This study investigated a peripheral selective CB₁ antagonist 3,4,22-3-demethoxycarbonyl-3-hydroxylmethyl-4-deacetyl-vindoline 3,4-thionocarbonate (VD60) that efficiently inhibited hepatic fibrosis with lower psychological side effects. A competitive radiolabeled ligand binding experiment and 3'-5'-cyclic adenosine monophosphate (cAMP) response element-driven luciferase analysis were performed to evaluate the antagonistic activity of VD60. Cell viability and collagen production were examined in the human hepatic stellate cell (HSC) line LX-2 and primary cultured rat HSCs. The antifibrotic effects of VD60 were investigated in a CCl₄-induced liver fibrosis mouse model. The concentration of VD60 in the blood and the brain was determined by high-performance liquid chromatography-mass spectrum analysis. Furthermore, the potential underlying mechanisms of VD60 were investigated by Western blot. VD60 selectively competed with the radiolabeled CB1 agonist to bind to CB1. VD60 antagonized CB1 agonist-induced Akt phosphorylation and increased the accumulation of intracellular cAMP. VD60 strongly reduced the expression of α₂(I) pro-collagen mRNA and exerted potent antiproliferative effects on primary HSCs and LX-2 cells. The inhibition of reactive oxygen species production and phosphorylation of Akt, extracellular-signal-regulated kinase (ERK), and Smad3 may explain the underlying mechanisms behind the antiproliferative effect of VD60. Moreover, the in vivo antifibrotic activity of VD60 was confirmed in a CCl4-induced liver fibrosis mouse model. Most importantly, the concentration of VD60 in the peripheral blood was much higher than in the brain, suggesting that VD60 could act as a novel peripheral CB1 antagonist to efficiently inhibit hepatic fibrosis and could be used as a lead compound with low brain side effects in peripheral antifibrotic agents.
本研究考察了一种外周选择性 CB₁ 拮抗剂 3,4,22-3-去甲羰酰基-3-羟甲基-4-去乙酰基文朵灵 3,4-硫代碳酸盐（VD60），它能有效抑制肝纤维化，且心理副作用较低。通过竞争性放射性配体结合实验和 3'-5'-环磷酸腺苷（cAMP）反应元件驱动的荧光素酶分析评估了 VD60 的拮抗活性。在人肝星状细胞（HSC）系 LX-2 和原代培养的大鼠 HSCs 中检查了 VD60 的细胞活力和胶原产生。通过 CCl₄诱导的肝纤维化小鼠模型研究了 VD60 的抗纤维化作用。通过高效液相色谱-质谱分析测定 VD60 在血液和脑中的浓度。此外，通过 Western blot 研究了 VD60 的潜在作用机制。VD60 选择性地与放射性标记的 CB1 激动剂竞争结合 CB1。VD60 拮抗 CB1 激动剂诱导的 Akt 磷酸化并增加细胞内 cAMP 的积累。VD60 强烈降低α₂(I)前胶原 mRNA 的表达，并对原代 HSCs 和 LX-2 细胞发挥强大的抗增殖作用。抑制活性氧的产生和 Akt、细胞外信号调节激酶（ERK）和 Smad3 的磷酸化可能解释了 VD60 抗增殖作用的潜在机制。此外，在 CCl₄诱导的肝纤维化小鼠模型中证实了 VD60 的体内抗纤维化活性。最重要的是，VD60 在周围血液中的浓度远高于大脑中的浓度，表明 VD60 可作为一种新型外周 CB1 拮抗剂，有效抑制肝纤维化，并可用作外周抗纤维化药物中具有低脑副作用的先导化合物。
