Figure 4. The effect of cofilin knockdown on LPS induced microglial cells activation.

A and B. Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM concentration for 72 h and then treated with LPS (100 ng/ml) or OGD for 1 h. Nitrite release assay was done after that by mixing 100 μl of cell culture medium with 100 μl of griess reagent. Relative to LPS treated control, NO release was significantly reduced by cofilin knockdown. Furthermore, no significant difference in NO release was detected between LPS untreated groups (scrambled and cofilin) and LPS treated cofilin transfected group (A). In OGD treated groups, no significant increase in NO release was detected (B). C–F. Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM concentration for 72 h and then treated with LPS (100 ng/ml) for further 24 h. Cell culture medium was used for ELISA assay of TNF-α and IL-1β (E and F) and cell lysate was used for WB analysis of iNOS and COX2 expression (C and D). Relative to LPS stimulated control, cofilin knockdown reduced LPS induced iNOS (C) and COX2 (D) expression significantly. Furthermore, no significant differences in iNOS and COX2 levels was detected between LPS untreated groups (scrambled and cofilin) and LPS treated cofilin transfected group. α-tubulin was used as a loading control. (E and F). In ELISA assay, LPS treatment increased TNF-α level significantly in both groups (scrambled and cofilin transfected), compared to unstimulated scrambled control group. In LPS treated groups, TNF-α level was significantly less in cofilin transfected group (E). Similarly, IL-1β release was also reduced by cofilin knockdown but the reduction did not reach the level of significance (P = 0.2) (F). Data are represented as Mean±SEM of three independent experiments. **P < 0.01, ***P < 0.001 relative to scrambled siRNA group without LPS treatment. #P < 0.05, ###P < 0.001 relative to scrambled transfected and LPS treated group.