Effects of andrographolide and 14-deoxy-11,12-didehydroandrographolide on cultured primary astrocytes and PC12 cells Yew-Min Tzeng, Yi-Ching Lee, Wen-Tai Cheng, Hui-Nung Shih, Huang-chi Wang, Yerra Koteswara Rao, Meng-Jen Lee ⁎ Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Wufeng, Taiwan, ROC abstract article info Article history: Received 13 April 2011 Accepted 18 November 2011 Keywords: Andrographolide 14-Deoxy-11,12-didehydroandrographolide CSPG Reactive oxygen species Astrocytes Aims: To test the effects of andrographolide (AP1) and 14-deoxy-11,12-didehydroandrographolide (AP2) on pheochromocytoma cell line 12 (PC12) cells in an astrocyte-rich environment. Main methods: The abilities of AP1 and AP2 to reduce the secretion of pro-inflammatory cytokines Interleukin (IL)-1, IL-6, and Tumor necrosis factor (TNF)-α from stimulated astrocytes were tested. In addition, the abil- ities of AP1 and AP2 to reduce oxidative stress in astrocytes were tested using an oxidative-sensitive fluores- cent dye. The reduction of chondroitin sulfate proteoglycan (CSPG) in stimulated astrocytes was tested using the dot blot method. Reduction of H 2 O 2 -induced death was tested in PC12 cells. Astrocyte-conditioned me- dium (ACM) and TNF-α-stimulated astrocyte-conditioned medium (SACM) were used to assess the effects of AP2 on PC12 cells treated with H 2 O 2 . Key findings: AP1 and AP2 reduced pro-inflammatory cytokines, reactive oxygen species (ROS), and CSPG in TNF-α stimulated astrocytes. AP1 protected H 2 O 2 -treated PC12 cells cultured in ACM. Co-incubation of PC12 cells in H 2 O 2 , and ACM collected from AP1 treated astrocytes did not prevent cell death. Significance: AP1 and AP2 effectively ameliorated astrocytic pro-inflammatory reactions and prevented PC12 cell death with different efficacies. These compounds may be candidates for treatment of spinal-cord injury and neurodegeneration. © 2011 Elsevier Inc. All rights reserved. Introduction White-matter degeneration after spinal-cord injury (SCI) blocks nerve impulses, resulting in paralysis. Following SCI, surviving neu- rons in long tracts are capable of sprouting, but this process halts in the presence of inhibitory proteoglycans (Silver and Miller, 2004). An inflammatory response at the injury site stimulates astrocytes to form glial scars and secrete high concentrations of chondroitin sulfate proteoglycan (CSPG), which has been shown to be involved in axonal growth inhibition (McKeon et al., 1995; Rudge and Silver, 1990). Di- gestion of CSPG or blocking its synthesis results in targeted axonal re-growth and improved function (Bradbury et al., 2002; Grimpe and Silver, 2004), suggesting that CSPG is an important target for white-matter regeneration. Macrophages rapidly infiltrate the central nervous system (CNS) injury site and secrete pro-inflammatory cytokines and reactive oxy- gen species to minimize injury and kill microbes. However, these ac- tions also cause further inflammation and additional damage (Fitch et al., 1999). Free radicals and oxidative species generated by macrophages result in lipid peroxidation-mediated cell death (Hall, 2003). High doses of methylprednisolone (MP) that reduce reactive oxygen-induced lipid peroxidation have been used for treating SCI (Genovese et al., 2006; Hall, 2003). Administration of anti- inflammatory drugs to inhibit secondary degenerative reactions after SCI results in improved locomotion and reduced histopatholog- ical changes (Genovese et al., 2006; Koopmans et al., 2009; Lee et al., 2003; Mulcahy et al., 2003; Nesic et al., 2001). One way to improve axonal regeneration is to reduce macrophage-induced inflammatory reactions. However, SCI often cannot be treated within 24 h, which is the window when many macrophage-induced molecules are re- leased. Several important astrocytic reactions, however, occur down- stream of macrophage-released factors (Asher et al., 2000; Giulian et al., 1988). These delayed astrocytic reactions may be easier to arrest following SCI; therefore, agents that inhibit reactive gliosis and pro- teoglycans in astrocytes are of particular interest. Andrographolide (AP1) and 14-deoxy-11,12-didehydroandrogra- pholide (AP2) are derived from Andrographis paniculata. Both com- pounds are structurally similar, although AP1 present in larger quantities and contains a hydroxyl group and two additional hydro- gen atoms. Besides its anticancer and antiviral effects (Geethangili et al., 2008), AP1 inhibits the innate immune response via its actions on macrophages, pro-inflammatory cytokines, and chemotaxis (Burgos et al., 2005; Qin et al., 2006; Tsai et al., 2004). It ameliorates hypoxia Life Sciences 90 (2012) 257–266 ⁎ Corresponding author at: Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, 168 Gifong East Road, Wufeng, Taichung County, Taiwan, ROC. Tel.: + 886 4 23323000; fax: + 886 4 23304921. E-mail address: mjlee@cyut.edu.tw (M.-J. Lee). 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.11.004 Contents lists available at SciVerse ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie