The Role Of Raf/MEK/ERK Signalling Pathway Regulated By RKIP In The Apoptosis Of PC12 Cells Induced By Microwave Exposure

Objective: With the economic and scientific technology growing fast, the microwave technology has wide application of communication, information, medicine and military domain. Microwave is widely used in our life. Companied with great benefits, it bring us increasingly serious threats to people's health. The bioeffects and mechanism of microwave are the forward subject in bioelectromagnetic. It has been elucidated that central nervous system is the sensitive target to microwave radiation, and hippocampus is the important sensitive district. However, the injury mechanism is unclear, In present study, we have found that the Raf/MEK/ERK signaling pathway regulated by RKIP may play an important role in the process of hippocampus injury induced by microwave radiation. Therefore, we investigated the role and mechanism of Raf/MEK/ERK signaling pathway regulated by RKIP in the apoptosis of PC12 cells induced by microwave exposure is important to make clear the injury effects, clarify the mechanism, find out the sensitive target, establish standard and provide measures for prevention and treatment.Materials and methods: (1) In order to establish the apoptosis model of nerve cells induced by radiation, PC12 cells after differentiation were exposed to microwave radiation with the average power density at 30mw/cm2for 5 min. The percentage of apoptosis and necrosis, the membrane potential of mitochondria were both detected by FCM, the fragmentation of DNA in the early time of apoptosis was observed by TUNEL, the ultramicrostructure and apoptotic body of PC12 cells was observed by the TEM and Hoechest dyeing, the expression of apoptotic gene product was detected by immuncytochemistry. (2) The expression of RKIP and its mRNA, the phosphorylated changes of key molecules in Raf/MEK/ERK signaling pathway such as Raf-1, MEK, ERK and its downstream nuclear transcriptional factor CREB, also the interaction of RKIP and Raf-1 in PC12 cells were detected by Western Blot, Real-time PCR, , Immunfluorescence and image analysis. (3) To investigate the role of Raf/MEK/ERK signaling pathway in cell apoptosis induced by radiation, U0126 as the specific inhibitor of MEK was used. (4) Construct the sense and antisense recombinant plasmid of RKIP respectively. With gene transfection and RNA interference method, the excessive expression and gene silencing of RKIP was used to study the role of Raf/MEK/ERK signaling pathway in cell apoptosis induced by radiation. The antagonistic action of RKIP gene silencing and U0126 was also investigated.Results: (1) Establishment of PC12 cells apoptosis model induced by microwave radiation: After the exposure of 30 mW/cm2 for 5min, Nucleus of PC12 cells showed karyopycnosis, anachromasis, and many apoptotic bodies formed,The ultramicrostructure of PC12 cells showed chromatinic concentrated, gathering along the karyolemma and apoptosis occurred. The percentages of apoptosis and necrosis significantly increased during 6h~3d after exposure (P<0.01), and the membrane potential of mitochondria significantly decreased (P<0.05 or P<0.01). At 6h and 1d, the rates of DNA fragmentation significantly increased (P<0.01), the expression of apoptotic gene product of Bcl-2 decreased, Bax and Caspase-3 increased, and the ratio of Bcl-2/Bax significantly decreased (P<0.01). (2) The rule of expression of RKIP and the key molecules in Raf/MEK/ERK pathway: RKIP expressed in the membrane and plasma of differentiated PC12 cells, especially strong expressed in the membrane. The expression and distribution significantly reduced after radiation. During 6h~3d after radiation, the expression of RKIP showed progressive decrease trend, but the radiation group still significantly decreased than sham radiation group(P<0.05 or P<0.01), especially at 6h (P<0.01). The expression of RKIP mRNA decreased at 6h (P<0.01), but increased at 1d (P<0.01). The signal of interaction of RKIP and Raf-1 reduced. No obvious difference was seen between the experimental groups for Raf-1 expression, but the expression of p-Raf-1 up-regulated significantly at 2d and 3d after radiation(P<0.05 or P<0.01). There were no obvious difference between the experimental groups for ERK expression, but the expression of p-ERK increased significantly during 6h~3d after radiation(P<0.05 or P<0.01), and its nuclear translocation occurred. Also the expression of p-CREB up-regulated significantly (P<0.05 or P<0.01). (3) The influence of U0126 on the expression of RKIP and key molecules in Raf/MEK/ERK signaling pathway: The expression of RKIP was significantly enforced in radiation group at 6h~1d by U0126 (P<0.01), and RKIP expressed more than the control group (P<0.01). U0126 inhibited significantly the expression of p-MEK p-ERK and p-CREB (P<0.01), but had no influence on ERK expression. (4) The influence of U0126 on the expression of apoptotic gene product after radiation There was no obvious effect on Bcl-2 expression, but the expression of Caspase-3 was obviously decreased at 6h and 1d, and the expression of Bax was decreased at 6h (P<0.01) by U0126. (5) Construction of the recombinant plasmid of RKIP and gene transfection in PC12 cells: With pcDNA3.0 and pGPU6/GFP/Neo vectors, we successfully constructed pcDNA3.0-RKIP and RKIP shRNA recombinant plasmid based on the root ganglion neurons of rats, and acquired the stable transfection PC12 cells with the sense and antisense recombinant plasmid of RKIP. Using non-lipid transfection reagent VigoFect, PC12 cells can be transfected with high efficiency until after differentiation (about 80%). The reliable technique platform of gene transfection into PC12 nerve cells was provided. (6) The regulation of ERK signaling pathway by RKIP: At 6h and 1d, RKIP excessive expression inhibited the up-regulation of p-MEK, p-ERK and p-CREB significantly(P<0.01).However, RKIP gene silencing promoted the up-regulation of p-MEK, p-ERK and p-CREB. No obvious effects were seen on ERK expression by RKIP. (7) The influence of RKIP on cell apoptosis induced by radiationThe impact of apoptosis in the induced PC12 cells: At 6h and 1d, RKIP excessive expression obviously reduced cell apoptosis after radiation (P<0.01), but cell apoptosis was increased by RKIP gene silencing. RKIP excessive expression inhibited the up-regulation of Bax and Caspase-3, and down-regulation of Bcl-2 significantly. However, RKIP gene silencing had the reverse effects. For cell apoptosis induced by radiation, expressive changes of Bcl-2, Bax and Caspase-3, obvious antagonism was found between RKIP gene silencing and U0126 (P<0.05 or P<0.01).Conclusion: (1) 30 mW/cm2 microwave radiation for 5min induced the percentage of apoptosis and necrosis increased, karyopycnosis, anachromasis, DNA fragmentation, formation of apoptotic bodies and decrease of mitochondria membrane potential. Nerve cell apoptosis model induced by microwave radiation was established successfully. (2) The down-regulation of RKIP attenuated the inhibition to Raf/MEK/ERK signaling pathway after microwave exposure, and the activation of this pathway was enforced. On the other hand, changes of post-translated modification of RKIP may participate in the radiation injury. (3) The activation of Raf/MEK/ERK signaling pathway may promote PC12 cells apopotosis induced by microwave radiation. The mechanism related to the decrease of Bcl-2/Bax ratio and up-regulation of Caspase-3. U0126 can reduce cell apoptosis caused by radition, and have protective effect on radiation injury. (4) The successive construction of RKIP sense and antisense plasmid and their effective expression in PC12 cells provide experimental basis for deeply research on the neurobiological function of RKIP. Also, the reliable technical platform was provided. To succeed construction the plus sense and antisense recombinant plasmid of RKIP and the effect expression of in the PC12 cells had established good experiment fundament and provided a reliability technology platform for the neurobiology of RKIP. (5) The activation of Raf/MEK/ERK signaling pathway after radiation was mainly regulated by RKIP. The regulation take part in the induction of cell apoptosis caused by radiation, and play an important role. RKIP excessive expression can reduce cell apoptosis after radiation obviously. However, RKIP gene silencing caused the increase of cell apoptosis. (6)The down-regulation of RKIP caused excessive activation of CREB, decrease of Bcl-2/Bax ratio and increase of Caspase-3 expression, which is the important regulation mechanism of cell apopotosis induced by radiation. In addition, the regulation mechanism of cell apoptosis by RKIP may refer to the crosstalk of several signaling pathways.