However, we observed that ibrutinib alone did not reduce BV2 microglial cell migration compared with vehicle (Additional?file?1: Determine S7), suggesting that ibrutinib itself may affect other unknown functions (e.g., phagocytosis) in the absence of LPS in BV2 microglial cells. immunocytochemistry, and subcellular fractionation were performed to examine the effects of ibrutinib on neuroinflammatory responses. In addition, wild-type mice were sequentially injected with ibrutinib (10?mg/kg, i.p.) or vehicle (10% DMSO, i.p.), followed by LPS (10?mg/kg, i.p.) or PBS, and microglial and astrocyte activations were assessed using immunohistochemistry. Results Ibrutinib significantly reduced LPS-induced increases in proinflammatory cytokine levels in BV2 microglial and main microglial cells but not in main astrocytes. Ibrutinib regulated TLR4 signaling to alter LPS-induced proinflammatory cytokine levels. In addition, ibrutinib significantly decreased BH3I-1 LPS-induced increases in p-AKT and p-STAT3 levels, suggesting that ibrutinib attenuates LPS-induced neuroinflammatory responses by inhibiting AKT/STAT3 signaling pathways. Interestingly, ibrutinib also reduced LPS-induced BV2 microglial cell migration by inhibiting AKT signaling. Moreover, ibrutinib-injected wild-type mice exhibited significantly reduced microglial/astrocyte activation and COX-2 and IL-1 proinflammatory cytokine levels. Conclusions Our data provide insights around the mechanisms of a potential therapeutic strategy for neuroinflammation-related diseases. Electronic supplementary material The online version of this article (10.1186/s12974-018-1308-0) contains supplementary material, which is available to authorized users. O111:B4 was purchased from Sigma-Aldrich (St. Louis, MO, USA). MTT assays Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. BV2 microglial cells were seeded in 96-well plates and treated with numerous concentrations of ibrutinib (100?nM to 1?M at lesser doses and 1?M to 50?M at higher doses) for 24?h in the absence of FBS. The cells were then treated with 0.5?mg/ml MTT and incubated for 3?h at 37?C in a 5% CO2 incubator. Absorbance was measured at 580?nm. Rat main microglial and astrocyte cultures Rat main mixed glial cells were cultured from your BH3I-1 cerebral cortices of 1-day-old Sprague-Dawley rats. Briefly, the cortices were triturated into single cells in high-glucose DMEM made up of 10% FBS/penicillin-streptomycin answer (5000?models/ml penicillin, 5?mg/ml streptomycin, Corning, Mediatech Inc., Manassas, VA, USA) and plated into 75 T culture flasks (0.5 hemisphere/flask) for 2?weeks. To harvest rat main microglial cells, the flask were shaken constantly at 120?rpm for 2?h to facilitate microglial detachment from your flask. The fluid medium was subsequently collected BH3I-1 and centrifuged at 1500?rpm for 15?min, and the cell pellets were resuspended to plate 1??105 cells per well. The remaining cells in the flask were harvested using 0.1% trypsin to obtain primary astrocytes. These main astrocytes and main microglial cells were cultured in 12-well plates (35?mm) pre-coated with poly-d-lysine (Sigma). Reverse transcription polymerase chain reaction Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturers instructions. Total RNA was reverse transcribed into cDNAs using a Superscript cDNA Premix Kit II with oligo (dT) primers (GeNetBio, Korea). RT-PCR was performed using Prime Taq Premix (GeNetBio, Korea). RT-PCR was performed using the following primers for BV2 microglial cells: IL-1: forward (F), AGC TGG AGA GTG TGG ATC CC, and reverse (R) , CCT GTC TTG GCC GAG GAC TA; IL-6: F, CCA BH3I-1 CTT CAC AAG TCG GAG GC, and R, GGA GAG CAT TGG AAA TTG Rabbit Polyclonal to CG028 GGG T; IL-18: F, TTT CTG GAC TCC TGC CTG CT, and R, ATC GCA GCC ATT GTT CCT GG; COX-2: F, GCC AGC AAA GCC TAG AGC AA, and R, GCC TTC TGC AGT CCA GGT TC; iNOS: F, CCG GCA AAC CCA AGG TCT AC, and R, GCA TTT CGC TGT CTC CCC AA; TNF-: F, CTA TGG CCC AGA CCC TCA CA, and R, TCT TGA CGG CAG AGA GGA GG; and GAPDH: F, CAG GAG CGA GAC CCC Take action AA, and R, ATC ACG CCA CAG CTT TCC AG. For rat main microglia and astrocytes, the following primers were used for.