Figure 7. Effects of TIIA on the expression of iNOS at 24 h after SCI. RT-PCR results demonstrated that the expression of iNOS was increased after SCI. Treatment of TIIA decreased the mRNA level of iNOS compared to the SCI group (A). Western blot analysis demonstrated that the protein expression of iNOS was increased in the tissues from the SCI group, but the treatment of TIIA significantly reduced the level of iNOS (B). The analyzed data for relative iNOS protein content are shown (C). Bars represent means 6 SEM. *p,0.05, **p,0.001 vs. sham group; #p,0.05 vs. SCI group. doi:10.1371/journal.pone.0038381.g007 Effects of TIIA on histological changes after SCI To evaluate the protective effect of TIIA on SCI, we observed histological changes in the spinal cord using electronic light microscopy at 24 h after operation. There were normal structures in both the white and grey matter in the sham and the sham/TIIA groups (Fig. 2A and 2B). The weight-drop rod was used to generate a mechanical insult, including edema, hemorrhage, neutrophil infiltration and loosened tissue structure in the SCI group (Fig. 2C). Compared with the SCI group, the morphologic changes were significantly alleviated in the TIIA-treated group (Fig. 2D), and some areas of the spinal cord even exhibited normal histological features. The calculated SCI scores are shown in Fig. 2E. TIIA exhibited a significant protective effect against SCI. Effects of TIIA on neutrophil infiltration after SCI As previously described, the histological changes of SCI were associated with the influx of leukocytes into the spinal cord. Therefore, we studied the effect of TIIA on neutrophil infiltration Effects of Tanshinone IIA in Adult Rats after SCI using an MPO activity assay at 24 h after SCI. The results showed no significant difference in MPO activity between the sham and sham/TIIA groups (Fig. 2F), but the MPO activity was increased in the SCI group. On the contrary, the MPO activity was markedly decreased in the SCI/TIIA group (Fig. 2F). Effects of TIIA on the activation of astrocytes after SCI To test whether the activation of astrocytes in the injured tissues was changed, we measured GFAP positive cells under electronic light microscopy at 24 h after SCI. In both the sham and sham/ TIIA groups, few GFAP positive cells were stained with GFAP antibody (Fig. 3A and 3B). The sections from the SCI group exhibited a significant increase in positive staining for astrocytes (Fig. 3C). However, as shown in Fig. 3D, TIIA treatment reduced the amount of GFAP-positive cells. The number of GFAP-positive cells was calculated in Fig. 3E. Effects of TIIA on NF-kB activation after SCI As the NF-kB pathway plays a key role in inflammation-related injury, we investigated the level of phosphorylated NF-kB (p-NF- kB) and total NF-kB using western blot analysis to assess the therapeutic effect of TIIA at 24 h after SCI (Fig. 4A and 4B). In the sham and sham/TIIA groups, we detected a basal level of p- NF-kB and total NF-kB. The p-NF-kB was significantly increased and the total NF-kB was up-regulated in rats that were subjected to SCI. Moreover, we found that the expression of both forms of NF-kB was reduced in the SCI/TIIA group. As a result, we concluded that TIIA could inhibit the activation of the NF-kB pathway after SCI. Effects of TIIA on the activation of MAPK (p38, ERK, JNK) pathway after SCI Our study also measured the expression of p38, ERK and JNK in spinal cord tissue to fully investigate the mechanisms involved in the TIIA-mediated protective effects against SCI. The result showed that there were no differences in the activation of those pathways between the sham and sham/SCI groups at 24 h after operation (Fig. 5A and F). There was more p-p38 in the lesion site from the SCI group, but the up-regulation of p-p38 was significantly attenuated upon TIIA treatment (Fig. 5A and 5B). The changes in p-ERK and p-JNK expression were similar to that of p-p38 (Fig. 5C to 5F). Collectively, these data showed that TIIA could attenuate the activation of MAPK pathway after SCI. Effects of TIIA on the expression of pro-inflammatory cytokines after SCI In our study, we evaluated the effect of TIIA on the expression of pro-inflammatory cytokines, such as TNF-a, IL-1b, and IL-6. In our preliminary experiment, we analyzed these cytokines at 6, 12 and 24 h after SCI. We found that the mRNA level of TNF-a, IL-1b, and IL-6 peaked at 6 h, and the concentration of these pro- inflammatory factors quickly decreased at 24 h after SCI (data not shown). Therefore, in this study, we measured the mRNA and protein expression of these cytokines from the damaged site at 6 h after SCI. As shown in Fig. 6, there were low levels of these cytokines in the sham and sham/TIIA groups. A significant increase in the mRNA and protein expression of TNF-a, IL-1b, and IL-6 was observed in the spinal cords of the SCI group (Fig. 6). Furthermore, compared with the SCI group, treatment with TIIA attenuated the expression of TNF-a, IL-1b, and IL-6 in the spinal cord. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38381