炎症是机体对外界刺激的一种自身反应，适当的炎症反应可以修复受损组织，增强机体抗病机能，而过度的或持续的炎症反应均会引起组织损伤和诱发疾病，其本质就是机体内促炎/抗炎自稳失衡所致。巨噬细胞作为调控炎症反应的主要细胞，主要表现为活化后吞噬入侵的病原体并释放调节免疫应答的细胞因子。黄柏酮 (obacunone, OBA) 是一种柠檬苦素类三萜化合物，主要分布于芸香科柑橘属植物以及能清热解毒燥湿的黄柏、白鲜皮等中药中。黄柏和白鲜皮在临床常用于炎症疾患，作为主要有效成分，OBA是否具有抗炎作用迄今尚未见报道。因此，我们对其抗炎作用及分子机制展开了研究。

实验采用经典的细菌脂多糖 (LPS) 诱导小鼠巨噬细胞株RAW264.7细胞模型考察OBA抗炎作用。Griess试剂法测定一氧化氮 (NO)；酶联免疫法 (ELISA) 测定白介素-6 (IL-6)、IL-1β和单核细胞趋化蛋白-1 (MCP-1)；实时定量PCR (RT-PCR) 法分析一氧化氮合酶 (iNOS)、IL-1β、IL-6和MCP-1 mRNA变化；采用报告基因的方法测定与炎症相关的核转录因子-κB (NF-κB) 与活化蛋白-1 (AP-1) 激活；蛋白免疫印记 (Western blot) 法测定iNOS、p38分裂原活化蛋白激酶 (p-p38 / p38)、c-Jun氨基末端激酶 (p-JNK / JNK)、细胞外信号调节激酶 (p-ERK1/2 / ERK1/2)、丝裂原蛋白活化激酶磷酸酶-1 (MKP-1) 蛋白表达；在计算机上使用Seaware靶点预测软件进行OBA作用靶点分析，SEA (Similarity Ensemble Approach) 算法比较目标分子与已知作用靶点的小分子相似性，MOE软件模拟配体和受体之间分子对接情况。细胞计数法考察OBA对巨噬细胞迁移抑制因子 (MIF) 趋化巨噬细胞的作用；酶促反应法测定重组MIF活性；采用“基因静默”的方法得到稳定的MIF (-) RAW264.7细胞。

结果显示，OBA (25、50、100 μM) 能降低iNOS、IL-6、IL-1β、MCP-1转录与表达，抑制AP-1激活，降低p38 MAPK磷酸化水平，增加MKP-1表达；靶点预测结果显示OBA能对接到MIF (3l5t) 活性口袋A链和C链之间的位置。因此，我们聚焦于MIF，发现OBA能明显抑制MIF所致的巨噬细胞趋化，并且在非细胞系中OBA还能够直接抑制MIF酶活性。LPS刺激MIF (-) RAW264.7细胞，得到与OBA处理活化RAW264.7细胞后相似的结果。

以上结果说明：OBA通过与MIF (3l5t) 活性口袋A链和C链之间的位置对接抑制其功能，导致MKP-1 表达增多，MKP-1再促p-p38 MAPK脱磷酸化，从而减少了AP-1下游相关促炎基因如iNOS、 IL-6、IL-1β、MCP-1等的转录与表达，进而发挥抗炎作用。

Inflammation is an important component of innate immunity and the host response to pathogens. It must be precisely controlled at multiple levels, as excessive inflammation does not benefit organisms and may cause tissue impairment. Macrophages play important roles in inflammation by directly affecting microbial cell killing and releasing chemokines and cytokines to amplify acute inflammation. Obacunone (OBA) is a triterpenoids limonoids compound mainly in the Rutaceae citrus, Dictamnus dasycarpus Turcz and Phellodendron chinense Schneid. Although the latter two herbs have been clinically used for the treatment of inflammation, the anti-inflammatory activity of OBA has been little studied.

Herein, the anti-inflammatory effect of OBA on lipopolysaccharide (LPS)-stimulated RAW264.7 cells is demonstrated along with its underlying mechanisms. In LPS-activated RAW264.7 macrophages, the supernatant NO was determined by Griess method. Proteins (IL-6, IL-1β and MCP-1) in the supernatant were determined by ELISA, while other proteins (iNOS, p-JNK/JNK, ERK1/2/ERK1/2, p-p38/p38, MKP-1) in the cytosol were assayed by western blot. The mRNA levels of pro-inflammatory cytokines were measured by RT-PCR. Effects of OBA on the NF-κB and AP-1 activation were determined by luciferase assay. The results from Target-fishing and Docking indicates that OBA can directly bind with MIF (315t). Thus, we next evaluated the effects of OBA on MIF. Recombinant MIF activity assay was performed using L-dopachrome methyl ester as a substrate. The number of macrophages induced by MIF was counted in the chemotaxis assay. MIF kinase activity assay was performed using L-dopachrome as a substrate. MIF (-) RAW264.7 cells were obtained by using MIF shRNA plasmid.

Our results show that OBA (25, 50, 100 μM) potently represses the pro-inflammatory mediators, such as iNOS, MCP-1, IL-1β and IL-6 at the transcriptional and translational levels without cytotoxicity, indicating the excellent anti-inflammatory efficacy of OBA in vitro. Further study shows that OBA significantly suppresses the activation of activator protein-1 (AP-1) through inhibiting p38 kinase (p38) phosphorylation levels and increasing the mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) expression post-transcriptionally. Simultaneously, OBA can inhibit recombinant mouse MIF-induced chemotactic response of RAW264.7 cells, and suppress the activity of recombinant mouse MIF in a cell-free system. OBA was docked into the active pocket of 3l5t via locating between chain A and chain C. Hydrogen bonds are formed with Tyr36, Lys32 and Ile64 in chain A. We also confirmed that MIF is a negative regulator of MKP-1 expression. To verify whether the pro- and anti-inflammatory actions of OBA attribute to the MIF inhibition and exclude the off-target effects by MIF inhibitors, we conditionally knocked down the MIF gene in RAW264.7 cells using MIF shRNA plasmid and obtained stable MIF (-) cell, whose responses to LPS were similar to OBA-treated macrophages, suggesting that OBA exerted anti- and pro-inflammatory activities by directly targeting MIF inhibition.

In summary, our data indicates that OBA exert anti-inflammatory effects by suppressing production of IL-1β, IL-6 and MCP-1 at both the transcriptional and translational levels via MKP-1/p38 mediated AP-1 pathway. Further mechanism study showed that the molecular target of the anti-inflammatory activities of OBA is MIF. Our study provides a molecular mechanism for the anti-inflammatory of OBA by targeting MIF inhibition in macrophages for the first time.