Today’s results show that luteolin promotes neurite outgrowth in PC12 cells and improved cholinergic activities through the service of ERK1/2 and Akt signaling pathways. Our results suggest the potential usage of luteolin as neuroprotective agent to stop infection in-which cholinergic deficiency is involved. Luteolin, NGF 7s, radioimmunoprecipitation assay buffer and r ERK1/2 antibody were obtained from Sigma Aldrich Co., Ltd., and acetylcholine iodide was from Wako. ERK1/2 antibody and goat anti rabbit IgG HRP were purchased selective FAAH inhibitor from Santa Cruz Biotechnology Inc.,, and 1,4 diamino 2,3 dicyano 1,4 bis butadiene was purchased from Promega. Goat anti mouse IgG HRP was from Bethyl Laboratories Inc., and r Akt and 2-8 phenyl 4H 1 benzopyran 4 one were obtained from Cell signaling Technology Inc.. Dulbeccos altered Eagle medium was from Sigma Aldrich Co., Ltd.. Fetal bovine serum was from Biowest SAS. Heat inactivated horse serum was from Invitrogen. Penicillin?streptomycin was from Lonza Inc.. MTT 2, 5 diphenyltetrazolium bromide) was from. PC12 cells were maintained in DMEM supplemented with 50 U/ml penicillin, ten percent HS and 5% FBS, and 50 ug/ml streptomycin in a humidified incubator at 37 C, 5% CO2. Cell passages were completed in 75 cm2 flask and cells were detached by pipetting. Just before each test, cells were washed with 10 ml of DMEM. The tests Plastid were done between pathways 3 and 8. 4. 3. Trial therapy NGF was dissolved in medium, and luteolin, U0126 and LY294002 were dissolved in dimethyl sulfoxide. NGF and luteolin were stored at?80 C, and LY294002 and U0126 were stored at 20 C. MTT was stored at 4 and dissolved in PBS at 5mg/ml C in the dark. Cell viability, choline/ acetylcholine quantification and cell differentiation, AChE activity, were performed in poly Llysine 96 well lined microplate. Cells were seeded at a of 1?105 cells/ml in 100 ul of medium and incubated for 24 h in a humidified incubator at 37 C, five minutes CO2. Then, cells were treated with Docetaxel Microtubule Formation inhibitor luteolin or NGF at 50 ng/ml. U0126, a inhibitor and LY294002, a inhibitor were pre treated at 50 uM for 1 h and 10 uM for 30 min, respectively before treatment with luteolin or NGF. 4. 4. Analysis of cell viability and cell differentiation Cell viability was measured from the dependent reduction of MTT to pink formazan. PC12 cells were treated with luteolin or NGF at 50 ng/ml for 48 h with or without pretreatment with 10 uM U0126 for 30 min and 50 uM LY294002 for 1 h. Then cells were washed once with 100 ul of DMEM, and incubated over night with ten percent MTT in culture medium. The come formazan was dissolved in 100 ul of 10% SDS solution after 24 h incubation in the same circumstances.