Attenuated severity and reduced and a serum NO concentration of PIA caused by RNAi of TLR3 in vivo. Four TLR3-shRNA plasmids and a negative control plasmid were transfected into NR8383 cells. mRNA ((a), 24 hours after transfection) and protein ((b), 48 hours after transfection) expression levels of TLR3 were detected by RT-PCR and FACS respectively, and shRNA3-TLR3 (shRNA-TLR3) shows the best RNAi efficacy. The plasmids of shRNA-TLR3 and shRNA-NC were used to treat PIA rats, and GFP mRNA expression (c) in plasmid treatment groups except in the saline group was successfully amplified by RT-PCR. (d), TLR3 expression of the spleen at d20 was detected by Real-time quantitative PCR. Relative mRNA expression was compared with the housekeeping gene β-actin. The arthritis clinical indexes (e) including clinical score, max clinical score and onset day; and serum NO concentration at d26 (f) were compared among three group PIA rats treated with saline, shRNA-NC and shRNA-TLR3, respectively. Data are presented as means ± SEM. * represents the comparison between the shRNA treated group and the saline group, and # between the shRNA-TLR3 and shRNA-NC groups. Levels of significance were calculated by using Mann-Whitney U test (n = 8 for each group, */# P < 0.05).