Zhu Yeshan, Li Xueqing, Chen Jianquan, Chen Tongjun, Shi Zhimin, Lei Miaona, Zhang Yanjun, Bai Pengfei, Li Yifang, Fei Xuan
Department of Digestive Internal Medicine, Tangshan Hospital of Traditional Chinese Medicine, Tangshan, Hebei Province, China.
Department of Acupuncture, North China University of Science and Technology School of Traditional Chinese Medicine, Tangshan, Hebei Province, China.
Int Immunopharmacol. 2016 Jan;30:74-84. doi: 10.1016/j.intimp.2015.11.031. Epub 2015 Dec 3.
Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a chronic inflammatory disease in the lower gastrointestinal tract. Mounting evidence suggests that the predominance of the classically activated (M1) macrophages versus the alternatively activated (M2) macrophages plays a role in the progression of IBD. Thus, agents able to shift pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages may be beneficial to IBD. The pentacyclic triterpene Lup-20(29)-en-3β-ol (Lupeol), a potent anti-inflammatory natural product, has been shown to inhibit pro-inflammatory cytokine production, suggesting it is potentially able to modulate macrophage polarization, thereby beneficial to IBD.
CD4(+) monocytes were differentiated to M1 or M2 macrophages, which were cocultured with epithelial cell lines, T84 and Caco-2, in the absence or presence of Lupeol (10μM). Experimental colitis was induced with dextran sodium sulfate (DSS), with or without oral administration of Lupeol (50mg/kg, q.d.). Cytokines were measured with Luminex kits. M1/M2 genes were measured with real-time polymerase chain reaction. Macrophage phenotypes were defined by measuring M1 and M2 markers with confocal microscopy. Proteins were measured with Western blotting, while cell surface markers were measured with confocal microscopy or flow cytometry. Histology was evaluated with H&E staining.
Treatment of M1 macrophages with Lupeol resulted in a marked decrease in the production of pro-inflammatory cytokines, including IL-12, IL6, IL-1β and TNFα, and a marked increase in the production of IL-10, an anti-inflammatory cytokine. This was associated with a down-regulation of CD86, a typical marker of M1 macrophages, and an up-regulation of CD206, a typical M2 macrophage marker. IRF5, a transcription factor that is critically involved in M1 polarization, was down-regulated in M1 macrophages after being incubated with Lupeol, associated with a marked decrease in the phosphorylation of p38 mitogen activated protein kinase. Coculture of epithelial cells with M1 macrophages resulted in down-regulation of the tight junction protein ZO-1 and disruption of epithelial integrity, which were blocked by Lupeol treatment of the M1 macrophages. Moreover, oral administration of Lupeol to dextran sulfate sodium (DSS)-induced colitis mice resulted in mitigated intestinal inflammation and increased survival from lethal colitis, associated with decreased expression of M1-related genes and increased expression of M2-related genes.
Lupeol ameliorates experimental inflammatory bowel disease through, at least in part, inhibiting M1 and promoting M2 macrophages.
炎症性肠病（IBD），包括溃疡性结肠炎和克罗恩病，是下消化道的一种慢性炎症性疾病。越来越多的证据表明，经典活化（M1）巨噬细胞与交替活化（M2）巨噬细胞的优势状态在IBD的进展中起作用。因此，能够将促炎性M1巨噬细胞转变为抗炎性M2巨噬细胞的药物可能对IBD有益。五环三萜类化合物羽扇豆醇（Lupeol）是一种有效的抗炎天然产物，已被证明可抑制促炎细胞因子的产生，表明它可能能够调节巨噬细胞极化，从而对IBD有益。
将CD4(+)单核细胞分化为M1或M2巨噬细胞，在不存在或存在羽扇豆醇（10μM）的情况下，将其与上皮细胞系T84和Caco-2共培养。用葡聚糖硫酸钠（DSS）诱导实验性结肠炎，同时给予或不给予口服羽扇豆醇（50mg/kg，每日一次）。用Luminex试剂盒检测细胞因子。用实时聚合酶链反应检测M1/M2基因。通过共聚焦显微镜测量M1和M2标志物来定义巨噬细胞表型。用蛋白质印迹法检测蛋白质，用共聚焦显微镜或流式细胞术检测细胞表面标志物。用苏木精和伊红（H&E）染色评估组织学。
用羽扇豆醇处理M1巨噬细胞导致促炎细胞因子（包括IL-12、IL-6、IL-1β和TNFα）的产生显著减少，抗炎细胞因子IL-10的产生显著增加。这与M1巨噬细胞的典型标志物CD86的下调以及M2巨噬细胞典型标志物CD206的上调有关。IRF5是一种在M1极化中起关键作用的转录因子，在用羽扇豆醇孵育后，M1巨噬细胞中的IRF5下调，这与p38丝裂原活化蛋白激酶的磷酸化显著减少有关。上皮细胞与M1巨噬细胞共培养导致紧密连接蛋白ZO-1下调和上皮完整性破坏，而羽扇豆醇处理M1巨噬细胞可阻断这种情况。此外，对葡聚糖硫酸钠（DSS）诱导的结肠炎小鼠口服羽扇豆醇可减轻肠道炎症并提高致死性结肠炎的存活率，这与M1相关基因表达降低和M2相关基因表达增加有关。
羽扇豆醇至少部分通过抑制M1巨噬细胞和促进M2巨噬细胞来改善实验性炎症性肠病。
