Fig. 6

LIPUS treatment controlled the PAR4-PI3K/Akt-NF-κB signaling pathway after glial cells activation. A Nitrite, B MIP-2 and C IL-6 levels in the primary microglia culture (PRMC) were increased by thrombin and significantly decreased microglia activation by LIPUS treatment. D Immunofluorescence staining was used to morphologically analyze microglia. OX42 (microglia) is shown in green, and PAR4 (hallmarks of inflammation) is shown in red. Yellow labeling represents co-localization. LIPUS treatment reduced the intensity of PAR4 staining induced by thrombin. DAPI (blue) staining was used to show all nuclei. The scale bar is 50 μm. E Representative immunoblots and bar graphs show that LIPUS treatment reduced COX-2 levels and P65 phosphorylation levels and F Akt phosphorylation at both S473 and T308 sites induced by thrombin at 1 h and/or 3 h. G The PI3K inhibitor LY294002 significantly reduced thrombin-induced nitrite production. The results of the thrombin + LIPUS + LY294002 group were similar to those of the thrombin + LIPUS group (P = 0.262). The results suggest that LIPUS treatment ameliorates microglia activation by limiting the PAR4-PI3K/Akt-NF-κB signaling pathway. ∗ , #, denote significant difference from the control group and thrombin group, respectively (*, #P < 0.05, **, ##P < 0.01, ***, ###P < 0.001, n.s. = P > 0.05; n = 3–4)