Figure 5. The CCL20/CCR6 axis in microglia is involved in microglial activation and microglia-mediated neuronal apoptosis in vitro. (A) Cultured microglia were transfected with the negative control siRNA (NC), CCL20 siRNA or CCR6 siRNA. After incubation for 72 h, microglia were exposed to OxyHb for 24 h. Next, Western blotting was used to measure the levels of CCL20, CCR6 and Iba1 (a microglial activation marker) (n = 3 per group). (B, C) The effects of CCL20 siRNA and CCR6 siRNA on OxyHb-induced IL-1β and TNF-α expression in microglia were analyzed by RT-qPCR (B) and ELISA (C) (n = 3 per group). (D) Cultured neurons were subjected to OxyHb-untreated conditioned medium (non-CM), OxyHb-treated conditioned medium (OxyHb CM), OxyHb CM from microglia pretreated with NC for 72 h (OxyHb+NC CM), OxyHb CM from microglia pretreated with CCL20 siRNA for 72 h (OxyHb+CCL20 siRNA CM), and OxyHb CM from microglia pretreated with CCR6 siRNA for 72 h (OxyHb+CCR6 siRNA CM) for 24 h. The apoptotic neurons were stained with TUNEL (red) (n = 3 per group). Scale bar = 50 μm. Data were presented as means ± SD. **P < 0.01 vs. the control group (con) or the non-CM group. Data were presented as mean ± SD. ##P < 0.01 vs. the OxyHb+NC group or the OxyHb+NC CM group.