Abstract: Objective To observe the expression of α7nAChR, P2X4R and BDNF in BV-2 cells after nicotine treatment, and to investigate the molecular mechanism of the involvement of microglia in the nicotine induced pain hypersensitivity. Methods (1)BV-2 microglial cells were seeded on 12hole plate that containing 12mm circular sterile coverslips; The cells in the logarithmic phase were randomly divided into 2 groups: α7nAChR group and P2X4R group. Expression of α7nAChR and P2X4R in BV-2 cells was detected by immunofluorescence staining.(2) When BV-2 cells of 12 well plate in the logarithm growth phase, the cells were randomly divided into 4 groups: Nicotine treatment group (Group N ), nicotine at a final concentration of 100μmol /L ; Serum free medium was used as a control group (Group C); Nicotine antagonists group (Group NM ), pretreatment cells with MLA 30min at a final concentration of10nmol/L,then use nicotine treatment cells; Simple antagonist group (group M), pretreatment cells with MLA 30min at a final concentration of10nmol/L,then use Serum free medium treatment cells; After 72h ,Real-time PCR was used to detect the expression of α7nAChR mRNA and P2X4R mRNA, and the expression of α7nAChR and P2X4R protein was detected by Western-bloting.(3) After treatment with nicotine72h, cells were treated with, ATP, DMEM, 5-BDBD. Normal cultured cells as control. BDNF levels were measured in culture media by ELISA after 24h. Results (1)The expression of α7nAChR and P2X4R was founded in BV-2 cells by immunofluorescence staining. (2)Real-time PCR results showed that nicotine can up-regulate the expression of α7nAChR mRNA and P2X4R mRNA in BV-2 cells, and this up-regulation could be inhibited by MLA, a specific antagonist of α7nAChR; Western-bloting results showed that nicotine can up-regulate the expression of α7nAChR and P2X4R protein in BV-2 cells, and this up-regulation could be inhibited by MLA. (3)ELISA results showed that the content of BDNF in the culture media, the ATP group were significantly increased compared with the normal control group and the DMEM group, the DMEM group increased significantly compared with the normal control group, the 5-BDBD group was lower than that in the DMEM group and the normal control group. Conclusions Nicotine may increase the expression of P2X4R through α7nAChR, and then through the release of BDNF to cause hypersensitivity.
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