e then immunohistochemically labeled with the avidin-biotin complex technique using an ABC kit. Finally, 23237488 the sections were treated with 3,39diaminobenzidine tetrahydrochloride for 15 min. All slides for immunohistochemistry were analyzed under a light microscope with microscope digital image output system. 2449244 Statistical analysis All data are presented as mean 6 SEM. For the immunoreactivity data, each test band is shown as the integrated order Relebactam optical density computed with respect to the average optical density of the corresponding control band. The data were analyzed using 1-way analysis of variance followed by Student-Newman-Keuls post hoc test. Differences resulting in P values less than 0.05 were considered significant. Results The inhibitory effects of Ya-s11 on iNOS and COX-2 protein expression Effect of Ya-s11 in Adjuvant-Induced Arthritis relative protein expression compared to the cells treated only with LPS. Levels of b-actin protein demonstrated no significant difference between concentrations of 1, 10, 25, and 50 mM Ya-s11 or compared with LPS only. Thus, Ya-s11 demonstrated concentration-dependent inhibition of LPSinduced iNOS and COX-2 protein expression in RAW 264.7 murine macrophages. Additionally, at the experimental concentrations, Ya-s11 did not induce cytotoxicity as determined through trypan blue staining and Annexin-V/PI double-staining assay. or arthritis score was observed in the group treated with Ya-s11 . The effect of Ya-s11 on histological features of AIA in the ankle joint Rats were sacrificed on day 28 after immunization, and paraffin sections of ankle joints were subjected to H&E staining for histopathological analysis. Representative photographs of ankle joint sections are shown from control, AIA, AIA plus Ya-s11 and AIA Plus Yas11 rats, respectively. Similar to previous 
studies, in the AIA group, synovial tissue demonstrated synovial hyperplasia, cartilage and bone erosion, and moderate to severe infiltration of immune cells into subchondral bone marrow. The AIA plus Ya-s11 group did not show attenuation of the synovial hyperplasia or cartilage and bone erosion, but moderated bone resorption in bone marrow. By contrast, the AIA plus Ya-s11 group demonstrated significantly inhibited AIA-induced joint destruction and synovial hyperplasia, cartilage and bone erosion, pannus formation, and bone resorption. Histological damage scores were significantly higher in the AIA group compared to the naive group. Those of the AIA plus Ya-s11 group were significantly lower than in the AIA and AIA plus Ya-s11 groups. No significant differences were observed between the AIA and AIA plus Ya-s11 groups. Effect of Ya-s11 on the clinical features of AIA AIA developed rapidly in rats immunized with heat-killed M. butyricum, and 4 Effect of Ya-s11 in Adjuvant-Induced Arthritis Effect of Ya-s11 on infiltrating cells in synovial tissue Pannus formation is accompanied by the infiltration of inflammatory cells, including lymphocytes, monocytes/macrophages, neutrophils, and synovial fibroblasts, into synovial tissue. Representative photographs show synovial tissue stained with H&E from the naive group, AIA group, AIA plus Ya-s11 group, and AIA plus Ya-s11 group. The synovial tissue of the naive group demonstrated synovial fibroblasts with few immune cells, and marked upregulation of infiltrating cells was apparent in synovial tissue from the AIA group. Ya-s11 significantly inhibited AIA-induced upregulation of infiltrating cells in synovia