damaged tissue. Moreover, we found that TIIA significantly reduced the levels of TNF-a, IL-1b, IL-6 and iNOS and markedly inhibited the activation of NF-kB and MAPKs pathways in the SCI model. After injury, cell death occurs, and positive apoptotic cells are found at the lesion area, especially in the white matter [37]. Bcl-2 family proteins play an important role in mediating the intrinsic apoptotic pathway. Bcl-2 is an anti-apoptosis membrane protein, and prevents cell apoptotic processes through the inhibition of cytochrome c release from the mitochondria following the activation of caspase [38]. Caspases are a family of proteases that initiate apoptosis. Caspase-3 is a key factor in apoptosis. Various stimulators, such as Bcl-2 and Bax, regulate caspase activation [10,39]. Caspase-3 participates in the downstream functions of the mitochondrial pathway, and the activation of caspase-3 might result in irreversible apoptosis. We found that cleaved caspase-3 was over-expressed in the SCI group, and TIIA could alleviate the effects of caspase-3. In addition, we found that treatment with TIIA increased Bcl-2 expression in our animal SCI model, suggesting that protection against apoptosis might be a prerequi- site for regenerative processes in SCI. Oxidative stress after SCI plays a pivotal role in secondary damage, and contributes to tissue injury during a post-traumatic inflammatory response and cell death [39]. During this process, the balance between anti-oxidants and pro-oxidants is disrupted. For example, SOD, which is a scavenger for reactive oxygen species (ROS), is consumed during oxidative stress. However, MDA, a lipid peroxidation end product, is increased during stress. Consistent with previous studies [40], we showed that SCI induced References 1. Young W (1993) Secondary injury mechanisms in acute spinal cord injury. J Emerg Med 11 Suppl 1: 13–22. 2. Taoka Y, Okajima K (2000) Role of leukocytes in spinal cord injury in rats. J Neurotrauma 17: 219–229. 3. Blight AR (1992) Macrophages and inflammatory damage in spinal cord injury. J Neurotrauma 9 Suppl 1: S83–S91. 4. Nakamura M, Houghtling RA, MacArthur L, Bayer BM, Bregman BS (2003) Differences in cytokine gene expression profile between acute and secondary injury in adult rat spinal cord. Exp Neurol 184: 313–325. 5. Akira S, Hirano T, Taga T, Kishimoto T (1990) Biology of multifunctional cytokines: IL 6 and related molecules (IL 1 and TNF). FASEB J 4: 2860–2867. 6. Carlson SL, Parrish ME, Springer JE, Doty K, Dossett L (1998) Acute inflammatory response in spinal cord following impact injury. Exp Neurol 151: 77–88. 7. Barnes PJ, Karin M (1997) Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336: 1066–1071. 8. Li Q, Verma IM (2002) NF-kappaB regulation in the immune system. Nat Rev Immunol 2: 725–734. 9. Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J 326 (Pt 1): 1–16. 10. Esposito E, Mazzon E, Paterniti I, Impellizzeri D, Bramanti P, et al. (2010) Olprinone attenuates the acute inflammatory response and apoptosis after spinal cord trauma in mice. PLoS One 5: e12170. 11. Liu XZ, Xu XM, Hu R, Du C, Zhang SX, et al. (1997) Neuronal and glial apoptosis after traumatic spinal cord injury. J Neurosci 17: 5395–5406. 12. Fu J, Huang H, Liu J, Pi R, Chen J, et al. (2007) Tanshinone IIA protects cardiac myocytes against oxidative stress-triggered damage and apoptosis. Eur J Pharmacol 568: 213–221. 13. Yang R, Liu A, Ma X, Li L, Su D, et al. (2008) Sodium tanshinone IIA sulfonate protects cardiomyocytes against oxidative stress-mediated apoptosis through inhibiting JNK activation. J Cardiovasc Pharmacol 51: 396–401. 14. Lam BY, Lo AC, Sun X, Luo HW, Chung SK, et al. (2003) Neuroprotective effects of tanshinones in transient focal cerebral ischemia in mice. Phytomedicine 10: 286–291. 15. Zhou L, Zuo Z, Chow MS (2005) Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J Clin Pharmacol 45: 1345–1359. 16. Gao J, Yang G, Pi R, Li R, Wang P, et al. (2008) Tanshinone IIA protects neonatal rat cardiomyocytes from adriamycin-induced apoptosis. Transl Res 151: 79–87. 17. Zhao BL, Jiang W, Zhao Y, Hou JW, Xin WJ (1996) Scavenging effects of salvia miltiorrhiza on free radicals and its protection for myocardial mitochondrial Effects of Tanshinone IIA in Adult Rats after SCI an increase in the expression of oxidative stress markers and reduced the expression of anti-oxidant markers. We also showed that TIIA treatment after SCI significantly reversed the changes of SOD activity and MDA levels, thereby restoring the balance of the redox state. Taken together, we demonstrated that TIIA confers neuronal protection against apoptosis and attenuates the inflammatory and oxidative stress response after SCI. These data suggest that combination therapy against multiple targets might promote recovery from traumatic injury in the spinal cord. Although the mechanisms involved in TIIA protection against SCI in rats requires further elucidation, our data suggest that the application of TIIA might be an effective treatment for SCI and that TIIA could be widely used to alleviate SCI in humans in the future. Acknowledgments The authors would like to thank Dr. Scott D. Boden (Emory University, School of Medicine, Atlanta, GA, USA) for many fruitful discussions and helpful suggestions concerning the manuscript. We also thank Prof. Jielai Xia (The Department of Health Statistics of the Fourth Military Medical University, Xi’an, China) for his advice concerning the statistical analysis. Author Contributions Conceived and designed the experiments: F-LC XY Y-FC. Performed the experiments: XY F-LC S-KS. Analyzed the data: XY YP. Contributed reagents/materials/analysis tools: F-LC S-KS W-GH. Wrote the paper: Z- JL XY YY. membranes from ischemia-reperfusion injury. Biochem Mol Biol Int 38: 1171–1182. 18. Fang ZY, Lin R, Yuan BX, Yang GD, Liu Y, et al. (2008) Tanshinone IIA downregulates the CD40 expression and decreases MMP-2 activity on atherosclerosis induced by high fatty diet in rabbit. J Ethnopharmacol 115: 217–222. 19. Lee B, Butcher GQ, Hoyt KR, Impey S, Obrietan K (2005) Activity-dependent neuroprotection and cAMP response element-binding protein (CREB): kinase coupling, stimulus intensity, and temporal regulation of CREB phosphorylation at serine 133. J Neurosci 25: 1137–1148. 20. Beattie MS, Hermann GE, Rogers RC, Bresnahan JC (2002) Cell death in models of spinal cord injury. Prog Brain Res 137: 37–47. 21. Li J, Xu M, Fan Q, Xie X, Zhang Y, et al. (2011) Tanshinone IIA ameliorates seawater exposure-induced lung injury by inhibiting aquaporins (AQP) 1 and AQP5 expression in lung. Respir Physiol Neurobiol 176: 39–49. 22. Gruner JA (1992) A monitored contusion model of spinal cord injury in the rat. J Neurotrauma 9: 123–126, 126–128. 23. Genovese T, Mazzon E, Esposito E, Muia C, Di Paola R, et al. (2007) N- benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone reduces severity of experi- mental spinal cord injury. Shock 27: 258–265. 24. Tian DS, Xie MJ, Yu ZY, Zhang Q, Wang YH, et al. (2007) Cell cycle inhibition attenuates microglia induced inflammatory response and alleviates neuronal cell death after spinal cord injury in rats. Brain Res 1135: 177–185. 25. Renault-Mihara F, Okada S, Shibata S, Nakamura M, Toyama Y, et al. (2008) Spinal cord injury: emerging beneficial role of reactive astrocytes’ migration. Int J Biochem Cell Biol 40: 1649–1653. 26. Merrill JE, Benveniste EN (1996) Cytokines in inflammatory brain lesions: helpful and harmful. Trends Neurosci 19: 331–338. 27. Yang L, Jones NR, Blumbergs PC, Van Den Heuvel C, Moore EJ, et al. (2005) Severity-dependent expression of pro-inflammatory cytokines in traumatic spinal cord injury in the rat. J Clin Neurosci 12: 276–284. 28. Xu J, Kim GM, Chen S, Yan P, Ahmed SH, et al. (2001) iNOS and nitrotyrosine expression after spinal cord injury. J Neurotrauma 18: 523–532. 29. Tak PP, Firestein GS (2001) NF-kappaB: a key role in inflammatory diseases. J Clin Invest 107: 7–11. 30. Chen F, Castranova V, Shi X, Demers LM (1999) New insights into the role of nuclear factor-kappaB, a ubiquitous transcription factor in the initiation of diseases. Clin Chem 45: 7–17. 31. Uto T, Fujii M, Hou DX (2005) 6-(Methylsulfinyl)hexyl isothiocyanate suppresses inducible nitric oxide synthase expression through the inhibition of Janus kinase 2-mediated JNK pathway in lipopolysaccharide-activated murine macrophages. Biochem Pharmacol 70: 1211–1221. PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e38381