Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
J Gastroenterol Hepatol. 2010 May;25(5):991-1001. doi: 10.1111/j.1440-1746.2009.06122.x. Epub 2010 Jan 4.
Anandamide (AEA), the most extensively studied endocannabinoid, and its putative cannabinoid receptors, CB1 and CB2, exert a variety of physiological and pharmacological effects in chronic liver diseases, such as hyperdynamic circulation. Anandamide selectively blocks proliferation and induces cell death in hepatic stellate cells (HSC), the key cell type of liver fibrogenesis. However, its precise molecular mechanism in rat HSC has not been fully elucidated.
CB1 and CB2 mRNA transcriptions were evaluated by reverse transcription polymerase chain reaction; CB1, CB2, phosphoinositide 3-kinases (PI3K) and protein kinase B (PKB) protein expressions were investigated by western blot and/or immunofluorescence. Cell death was detected by Annexin V-PE/7AAD flow cytometry, lipid raft content by confocal microscopic analysis, cell viability by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, nuclear morphological changes by Hoechst 33258 fluorochrome, and inflammatory cytokines interleukin (IL)-2 and IL-6, and tumor necrosis factor-alpha (TNF-alpha) by enzyme-linked immunosorbent assay.
CB1 and CB2 receptors were detectable in HSC. AEA caused HSC growth inhibition in a concentration-dependent manner. Furthermore, a high concentration of AEA (20 micromol/L) triggered potent cell death-induced necrosis but not apoptosis. None of these effects were blocked by CB1 or CB2 receptor antagonist, but by methyl-beta-cyclodextrin (MCD; 10 mmol/L), a cholesterol depletory agent. AEA significantly inhibited PI3K/PKB activity, and increased IL-2, IL-6 and TNF-alpha release.
These results demonstrated that AEA induced HSC necrosis through lipid rafts: a possible role of PI3K/PKB signaling pathway downregulation and inflammatory factors production. Cholesterol depletion abolished the effects of AEA on HSC necrosis.
内源性大麻素（AEA）是研究最为广泛的内源性大麻素，其假定的大麻素受体 CB1 和 CB2 在慢性肝病（如高动力循环）中发挥着多种生理和药理作用。AEA 选择性地阻断肝星状细胞（HSC）的增殖并诱导其死亡，而 HSC 是肝纤维化的关键细胞类型。然而，其在大鼠 HSC 中的精确分子机制尚未完全阐明。
通过反转录聚合酶链反应评估 CB1 和 CB2 mRNA 转录；通过 Western blot 和/或免疫荧光法检测 CB1、CB2、磷酸肌醇 3-激酶（PI3K）和蛋白激酶 B（PKB）的蛋白表达。通过 Annexin V-PE/7AAD 流式细胞术检测细胞死亡，通过共聚焦显微镜分析检测脂筏含量，通过 3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-四唑溴盐（MTT）测定法检测细胞活力，通过 Hoechst 33258 荧光染料检测核形态变化，通过酶联免疫吸附试验检测白细胞介素（IL）-2 和 IL-6 以及肿瘤坏死因子-α（TNF-α）等炎症细胞因子。
在 HSC 中可检测到 CB1 和 CB2 受体。AEA 以浓度依赖性方式引起 HSC 生长抑制。此外，高浓度的 AEA（20 μmol/L）引发强烈的细胞死亡诱导坏死而不是凋亡。这些作用均不能被 CB1 或 CB2 受体拮抗剂阻断，但可被胆固醇耗竭剂甲基-β-环糊精（MCD；10 mmol/L）阻断。AEA 显著抑制了 PI3K/PKB 活性，并增加了 IL-2、IL-6 和 TNF-α 的释放。
这些结果表明，AEA 通过脂筏诱导 HSC 坏死：PI3K/PKB 信号通路下调和炎症因子产生可能发挥作用。胆固醇耗竭消除了 AEA 对 HSC 坏死的作用。
