Effects of Tanshinone IIA in Adult Rats after SCI Figure 6. Effects of TIIA on the expression of pro-inflammatory cytokines at 6 h after SCI. The mRNA expression of TNF-a, IL-1b and IL-6 was enhanced in the tissues from the SCI group, but treatment of TIIA reduced their mRNA expression (A–C). Compared to the sham and sham/TIIA groups, the protein level of TNF-a, IL-1b and IL-6 was significantly up-regulated, whereas TIIA reduced the level of these proteins (D–F). 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.g006 AGTCTTGCTTGG-39 and P2 59-GGT TGATGAACT- CAATGGCAT G-39; for GAPDH, P1 59- CCCCCAATG- TATCCGTTGTG -39 and P2 59-TAGCCCAGGATGCCCTT- TAGT -39. The PCR conditions were 95uC for 15 s followed by 30 cycles of amplification consisting of 95uC for 5 s and 60uC for 30 s. The reactions were performed in triplicate, and the results were averaged. Each value was normalized to GAPDH mRNA levels to control for variations in the amount of input cDNA. The relative expression level of the genes compared with the control group was calculated using the DDCt method. Extraction of protein and western blot The tissues (n = 4 for each group) were homogenized in lysis buffer (10 mM Tris-HCl, 250 mM sucrose, 5 mM MgCl2, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate, 1 mM NaF, 1 mM leupeptin, 2 mM pepstatin A, 1 mM dithiothreitol)) and centrifuged at 4uC for 15 min at 900 g. Subsequently, the supernatant (total protein) was collected and the protein levels were quantified using a Coomassie brilliant blue assay. Total protein (50 mg) was separated on 12% SDS- polyacrylamide gels and transferred to nitrocellulose filter mem- branes. The membranes were labeled with primary antibodies (Millipore, Bedford, MA, USA) for phosphorylated or total NF-kB (1:1000) and MAPKs (p38, ERK and JNK) (1:1000), iNOS (inducible Nitric Oxide Synthase) (1:400), caspase-3 (1:200), Bcl-2 (1:200), and b-actin (1:5000) overnight at 4uC. The membranes were incubated with the appropriate horseradish peroxidase- coupled secondary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted at 1:5000 for 1 h at 37uC followed by enhanced chemiluminescence detection of the proteins using an ECL kit (BestBio Inc, Shanghai, China). The protein density was quantified using imaging software (BioSens Digital Imaging 5, Shanghai Bio-Tech Inc, Shanghai, China). For the western blot analysis, caspase-3 and Bcl-2 were measured at 1, 3 and 7 days after SCI, and the other proteins were measured at 24 h after SCI. Biochemical analysis To determine the cytokine levels (n = 4 for each group), the lesion site was rapidly dissected and homogenized in PBS at 24 h after SCI. After centrifugation at 4uC for 15 min at 900 g, the supernatants were used to measure the concentrations of TNF-a, IL-1b and IL-6 using corresponding ELISA kits (R&D systems, Minneapolis, MN), and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) using corresponding kits (Jiancheng Bioengineering Institute, Nanjing, China). The data are presented as the mean 6 SEM from three replicate experiments. Statistical analysis The data are expressed as the mean 6 SEM, and the statistical analysis was performed with two-way analysis of variance (ANOVA) using the SPSS 13.0 software package (SPSS Inc., Chicago, IL), in which surgery (SCI vs. sham) and treatment (TIIA vs. vehicle) were the two fixed factors. A post-hoc analysis was performed using Dunnett’s test for multiple comparisons. A significant difference was accepted as significant if p,0.05. Results Effects of TIIA on functional recovery after SCI The hindlimb movements were abolished immediately after SCI. A spontaneous functional recovery after SCI in all groups was observed. As shown in Fig. 1, the rats in the SCI group gradually improved from Day 3, and rats in the SCI/TIIA group improved more rapidly. The statistical analysis showed a significantly greater increase in the BBB score from Day 3 to Day 10 in the SCI/TIIA group than in the SCI group. Ten days after SCI, the BBB average scores were 2.860.6 and 660.8 in the SCI and the SCI/TIIA groups, respectively (Fig. 1, p,0.05), indicating that TIIA treatment could promote the movement of rats with SCI. PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e38381