Yeshan Zhu1, Xueqing Li2, Jianquan Chen3, Tongjun Chen4, Zhimin Shi5, Miaona Lei6, Yanjun Zhang7, Pengfei Bai8, Yifang Li9, Xuan Fei10. 1. Department of Digestive Internal Medicine, Tangshan Hospital of Traditional Chinese Medicine, Tangshan, Hebei Province, China. Electronic address: zhuyeshan1965@163.com. 2. Department of Acupuncture, North China University of Science and Technology School of Traditional Chinese Medicine, Tangshan, Hebei Province, China. Electronic address: 15531558238@163.com. 3. Department of Traditional Chinese Medicine, Yutian Hospital, Tangshan, Hebei Province, China. Electronic address: 741094776@qq.com. 4. Department of Digestive Internal Medicine, Tangshan Hospital of Traditional Chinese Medicine, Tangshan, Hebei Province, China. Electronic address: tongjunchen@sina.com. 5. Department of Digestive Internal Medicine, Tangshan Hospital of Traditional Chinese Medicine, Tangshan, Hebei Province, China. Electronic address: shizhimin0613@163.com. 6. Department of Digestive Internal Medicine, Tangshan Hospital of Traditional Chinese Medicine, Tangshan, Hebei Province, China. Electronic address: eimiaona001@sina.com. 7. Department of Digestive Internal Medicine, Tangshan Hospital of Traditional Chinese Medicine, Tangshan, Hebei Province, China. Electronic address: zhangyj1312@126.com. 8. Department of Digestive Internal Medicine, Tangshan Hospital of Traditional Chinese Medicine, Tangshan, Hebei Province, China. Electronic address: 854951455@qq.com. 9. Department of Digestive Internal Medicine, Tangshan Hospital of Traditional Chinese Medicine, Tangshan, Hebei Province, China. Electronic address: liyifang02@163.com. 10. Department of Digestive Internal Medicine, Tangshan Hospital of Traditional Chinese Medicine, Tangshan, Hebei Province, China. Electronic address: 1tsfuyou@126.com.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Lupeol ameliorates experimental inflammatory bowel disease through, at least in part, inhibiting M1 and promoting M2 macrophages.
BACKGROUND: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 pentacyclictriterpene 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. METHODS: 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. RESULTS: 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 colitismice 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. CONCLUSION:Lupeol ameliorates experimental inflammatory bowel disease through, at least in part, inhibiting M1 and promoting M2 macrophages.