Effects of Tanshinone IIA in Adult Rats after SCI Figure 4. Effects of TIIA on NF-kB activation at 24 h after SCI. A basal expression of p-NF-kB and total NF-kB were detected by Western blot in the sham and sham/TIIA groups at 24 h after SCI, whereas NF-kB levels were increased after SCI. Treatment of TIIA (20 mg/kg) reduced the expression of NF-kB (A). The relative content of two types of NF-kB was counted (B). 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.g004 benzidine and 0.1 mM peroxide (H2O2). The rate of change in absorbance was measured spectrophotometrically at 460 nm. The MPO activity was defined as the quantity of enzyme required to degrade 1 mmol of H2O2 per min at 37uC, expressed as units of MPO/g wet tissue. Immunohistochemistry Animals were sacrificed at 24 h after injury (n = 4 for each group) in all groups. Subsequently, tissue blocks were cut into 5- mm slices as mentioned above. After dewaxing and hydration, the tissue slices were incubated with citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) for 10 min at 121uC to facilitate GFAP antigen retrieval. The sections were incubated in 1% H2O2 for 30 min at room temperature to inhibit endogenous peroxidase activity. After washing in PBS, the sections were preincubated with goat serum albumin for 30 min at 37uC followed by an incubation with a primary antibody against GFAP (Millipore, Bedford, MA, USA) for 18 h at 4uC. Subsequently, the sections were washed with PBS and stained with biotin-labeled rabbit anti-mouse IgG for 30 min at 37uC. The slices were washed with PBS and incubated in horseradish peroxidase-labeled streptavidin working solution for 30 min at 37uC. Finally, the slides were washed with PBS before application of diaminobenzidine (DAB), covered with a coverslip and analyzed under a light microscope. TUNEL staining The rats were anesthetized with an intraperitoneal injection of 4% sodium pentobarbital (40 mg/kg) in all groups (n = 4 for each group) on Day 3 after SCI. The paraffin-embedded tissue slices were dewaxed, washed with PBS, and digested with proteinase K Figure 5. Effects of TIIA on MAPK pathway at 24 h after SCI. The expression of p-p38 was up-regulated in the SCI group compared with the sham and sham/TIIA groups. The level of p-p38 was attenuated with TIIA treatment as compared to the SCI group (A). The analyzed data for p-p38 are shown (B). The content of p-ERK was significantly increased in the SCI group at 24 h compared with the sham and sham/TIIA groups. TIIA prevented the expression of p-ERK (C). The analyzed data for p-ERK are shown (D). p-JNK was up-regulated after SCI, but a significant reduction of p-JNK in the tissues from the SCI/TIIA group was observed (E). The analyzed data for p-JNK are shown (F). Bars represent means 6 SEM. **p,0.001 vs. sham group, #p,0.05 vs. SCI group. doi:10.1371/journal.pone.0038381.g005 in a wet box for 30 min at 37uC. After washing with PBS, the slides were dipped in TUNEL reaction mixture, and incubated for 1 h at 37uC. After washing, the sections were incubated with converter-AP for 30 min at 37uC, and washed with PBS. Subsequently, the sections were stained with NBT/BCIP substrate solution for 1 h, and the signals were observed under a microscope. The cells with purple nuclei were considered dead. The number of TUNEL positive cells was counted. Extraction of RNA and Quantitative Real-time PCR The rats were anesthetized with an intraperitoneal injection of 4% sodium pentobarbital (40 mg/kg) in all groups (n = 4 for each group) at 6 h after SCI. The lesioned tissue was dissected and homogenized in a solution of TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). The RNA extraction was performed according to the manufacturer’s instructions. The total RNA concentration was determined spectrophotometrically. A total of 1 mg of RNA was incubated with a reverse transcription mixture at 42uC for 50 min, followed by PCR amplification with the specific primers (synthesized by Invitrogen). We used the following primers: for TNF-a, P1 59-TGATCGGTCCCAACAAGGA -39 and P2 59- TGCTTGGTGGTTTGCTACGA -39; for IL-1b, P1 59- TGCTGATGTACCAGTTGGGG -39 and P2 59-CTCCAT- GAGCTTTGTACAAG -39; for IL-6, P1 59- GCCCTTCAG- GAACAGCTATG -39 and P2 59- CAGAATTGCCATTGCA- CAAC -39; for iNOS, P1 59- GCAAGT CCA PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38381