RKIP调控的Raf/MEK/ERK信号通路在微波辐射诱导PC12细胞凋亡中的作用
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摘要
目的和意义：随着经济与科学技术的迅猛发展，微波技术已经广泛应用于通讯、广播、医药、军事等各个领域且深入到人们日常生活的各个方面，微波给人类带来极大益处的同时，也给人类健康造成了日益严重的威胁。微波的生物效应及其机制研究是目前生物电磁学的前沿课题。研究表明，中枢神经系统是微波辐射的敏感靶部位，海马是微波辐射损伤的敏感靶区，但其致伤机制尚未阐明。我们前期的研究发现，RKIP调控的Raf/MEK／ERK信号通路可能在微波辐射致海马损伤中发挥重要作用。因此，探讨RKIP对Raf/MEK／ERK信号通路的调控在微波辐射诱导PCI2细胞凋亡中的作用及其机制，对于明确微波辐射损伤效应及阐明其损伤机制，寻找敏感靶标，制定防护标准和提供防治措施部具有重要意义。
材料和方法：(1)采用平均功率密度为30mw／cm的微波辐射诱导分化后的PCI2细胞5 nljn，建立辐射诱导神经细胞的凋亡模型。通过流式细胞术检测细胞凋亡与坏死率及线粒体膜电位，原位末端标记检测凋亡早期DNA断裂；透射电镜和Hoec]nest染色观察细胞超微结构改变及凋亡小体形成；免疫细胞化学检测凋亡基因产物表达。(2)通过实时荧光定量PCR、免疫印迹、免疫荧光、免疫共定位和图像分忻等技术，动态检测PCI2细胞中RKIP及其mRNA、Ra/MEK／ERK信号通路中关键分子Raf_1、：MEtK、ERK及其下游核转录因子CREB的磷酸化改变，以及RKIP与Raf_1的相互作用。(3)应用MEK的特异性抑制剂u0126研究Ra/MEK／ERK信号通路在辐射诱导细胞凋亡中的作用。(4)分别构建RKIP的正义和反义重组质粒，利用基因转染和RNA干涉技术研究RKIP过表达和基因沉默对Ra/MEK／ERK信号通路的调控在辐射诱导细胞凋亡中的作用，并研究RKIP基因沉默和u0126的拮抗作用。
实验结果：(1)微波辐射诱导Pcl2细胞凋亡模型建立：30mw／cm。微波辐射5min后导致PCI2细胞胞核固缩、深染、较多凋亡小体形成；超微结构呈现部分细胞核染色质浓缩、沿核膜边集，凋亡发生；辐射后6h～3d，细胞凋亡与坏死率较假辐射组均显著升高P<0．01)，线粒体膜电位明显下降P<0．05或尸l<0．01)；辐射后6h和1d，DNA断裂发生率明显升高P<0．01)，凋亡基因产物Bcl—2表达减少、B“及Caspase一3表达增加，Bcl—2／Bax的H,J比值明显降低(尸l<0．01)；(2)RKIP及Raf／MEI(／ERK通路关键分子的表达变化规律：RKIP于分化后的Pcl2细胞胞膜与胞浆均呈阳性表达，其中胞膜呈强阳性表达，辐射后其表达分布明显减少；辐射后6h～3d，假辐射组和辐射组PCI2细胞中RKIP表达变化均呈持续减少趋势，但辐射组较假辐射组始终显著降低P<0．05或P<0．01)，以6h最为显著(P<0．01)；RKIP mRNA表达于6h时较假辐射组明显下调P<0．01)，而1d时明显上调P<0．01)；同时RKIP与Ral：1的细胞共定位信号明显减弱；Raf．1的表达在假辐射组和辐射组两组之间均未见显著性差异；但辐射组p—Ral：1表达于2d和3d时均显著上调P<0．05或P<0．01)；两组ERK表达均未见统计学差异，但p—ERK于辐射组611~3d均显著上调(P<0．05或P<0．01)，并可见核移位发生；辐射组p—CREB表达于611~3d较假辐射组亦显著上调P<0．05或P<0．01)；(3)u0126对RKIP及ERK信号通路关键分子表达的影响：辐射后6h～1d，u0126可明显增强辐射组RKIP的表达P<0．01)，且显著高于假辐射组P<0．01)；u0126均能显著抑制辐射后p—MEK、p—ERK及p—CREB的上调(P<0．01)；而对ERK的表达没有影响；(4)u0126对辐射后凋亡基因产物表达的影响：u0126对辐射后Bcl一2的表达未见明显影响；u0126可明显减弱辐射后6h和1d Caspase一3的表达以及辐射后6h Bax的表达P<0．01)；(5)RKIP重组质粒构建与Pcl2细胞基因转染：分别采用pcDNA3．0及pGPu6／GFP／Neo载体，以大鼠背根神经节为模扳，成功构建了pc~)NA3．0一RKIP及RKIP shRNA重组质粒，并筛选获得了稳定转染RKIP正义和反义质粒的PC2细胞株；采用非脂质体转染试剂～~igoI，ect介导的重组质粒可高效转染未分化的PCI2细胞，诱导分化后细胞转染效率较高(约80％)，从而为开展Pcl2神经细胞基因转染实验提供了可靠技术平台；(6)RKIP对辐射后ERK信号通路的调控作用：辐射后6h和1d，RKIP过表达显著抑制辐射后p—MEK、p—ERK及p—CREB上调P<0．01)；而RKIP基因沉默对辐射后p—MEK、p—ERK及p—CREB上调具有促进作用；RKIP对辐射后ERK的表达未见明显影响；(7)RKIP对辐射诱导细胞凋亡的影响：辐射后6h和1d，RKIP过表达可明显抑制辐射后细胞凋亡增多P<0．01)，RKIP基因沉默则导致凋亡进一步增多P<0．05)；RKIP过表达显著抑制辐射后：Bcl一2下调、Bax及Caspase-一3的上调，RKIP基因沉默则与其作用相反；RKIP基因沉默和u0126对于辐射诱导的细胞凋亡、Bcl一2、Bax及Caspase一3表达改变均具有明显的拮抗作用(P<0．01)。
结论：
一、平均功率密度30mw／cm。的微波辐射5min导致PCI2细胞凋亡和坏死率明显升高，细胞核染色质浓缩、DNA断裂、凋亡小体形成，线粒体膜电位下降，建立了良好的微波辐射诱导神经细胞凋亡模型。
二、微波辐射后RKIP表达下调，对Ral7MEK／ERK信号通路的抑制作用减弱，从而导致Rar／MEK／ERK信号通路活化增强。另一方面，RKIP蛋白质翻译后修饰水平的变化可能参与了微波辐射损伤。
三、Ra~7MEK／ERK信号通路活化可促进微波辐射诱导的PCI2神经细胞凋亡，其作用机制与Bcl一2／Bax比值下降及C{aspase一3表达上调有关；u0126可减少辐射所致细胞凋亡，对微波辐射损伤具有一定防护作用。
四、RKIP正义与反义质粒的成功构建及其在PCI2细胞的有效表达为深入研究RKIP的神经生物学功能奠定了良好实验基础，并提供了可靠技术平台。
五、RKIP对微波辐射后Ral7MEK／ERK信号通路的活化具有主要调控作用，这一调控参与诱导辐射所致PCI2神经细胞凋亡，并在其中发挥重要调节作用。RKIP过表达明显减少辐射后细胞凋亡，RKIP基因沉默则导致辐射后细胞凋亡增加。
六、RKIP下调导致CREB活化增强，Bcl一2／：Bax比值下降，Gaspase-一3表达增加，是RKIP调控微波辐射所致细胞凋亡的重要机制。另一方面，RKIP对辐射诱导细胞凋亡的调控机制可能涉及多条细胞信号通路之间的相互对话。
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.
材料和方法：(1)采用平均功率密度为30mw／cm的微波辐射诱导分化后的PCI2细胞5 nljn，建立辐射诱导神经细胞的凋亡模型。通过流式细胞术检测细胞凋亡与坏死率及线粒体膜电位，原位末端标记检测凋亡早期DNA断裂；透射电镜和Hoec]nest染色观察细胞超微结构改变及凋亡小体形成；免疫细胞化学检测凋亡基因产物表达。(2)通过实时荧光定量PCR、免疫印迹、免疫荧光、免疫共定位和图像分忻等技术，动态检测PCI2细胞中RKIP及其mRNA、Ra/MEK／ERK信号通路中关键分子Raf_1、：MEtK、ERK及其下游核转录因子CREB的磷酸化改变，以及RKIP与Raf_1的相互作用。(3)应用MEK的特异性抑制剂u0126研究Ra/MEK／ERK信号通路在辐射诱导细胞凋亡中的作用。(4)分别构建RKIP的正义和反义重组质粒，利用基因转染和RNA干涉技术研究RKIP过表达和基因沉默对Ra/MEK／ERK信号通路的调控在辐射诱导细胞凋亡中的作用，并研究RKIP基因沉默和u0126的拮抗作用。
实验结果：(1)微波辐射诱导Pcl2细胞凋亡模型建立：30mw／cm。微波辐射5min后导致PCI2细胞胞核固缩、深染、较多凋亡小体形成；超微结构呈现部分细胞核染色质浓缩、沿核膜边集，凋亡发生；辐射后6h～3d，细胞凋亡与坏死率较假辐射组均显著升高P<0．01)，线粒体膜电位明显下降P<0．05或尸l<0．01)；辐射后6h和1d，DNA断裂发生率明显升高P<0．01)，凋亡基因产物Bcl—2表达减少、B“及Caspase一3表达增加，Bcl—2／Bax的H,J比值明显降低(尸l<0．01)；(2)RKIP及Raf／MEI(／ERK通路关键分子的表达变化规律：RKIP于分化后的Pcl2细胞胞膜与胞浆均呈阳性表达，其中胞膜呈强阳性表达，辐射后其表达分布明显减少；辐射后6h～3d，假辐射组和辐射组PCI2细胞中RKIP表达变化均呈持续减少趋势，但辐射组较假辐射组始终显著降低P<0．05或P<0．01)，以6h最为显著(P<0．01)；RKIP mRNA表达于6h时较假辐射组明显下调P<0．01)，而1d时明显上调P<0．01)；同时RKIP与Ral：1的细胞共定位信号明显减弱；Raf．1的表达在假辐射组和辐射组两组之间均未见显著性差异；但辐射组p—Ral：1表达于2d和3d时均显著上调P<0．05或P<0．01)；两组ERK表达均未见统计学差异，但p—ERK于辐射组611~3d均显著上调(P<0．05或P<0．01)，并可见核移位发生；辐射组p—CREB表达于611~3d较假辐射组亦显著上调P<0．05或P<0．01)；(3)u0126对RKIP及ERK信号通路关键分子表达的影响：辐射后6h～1d，u0126可明显增强辐射组RKIP的表达P<0．01)，且显著高于假辐射组P<0．01)；u0126均能显著抑制辐射后p—MEK、p—ERK及p—CREB的上调(P<0．01)；而对ERK的表达没有影响；(4)u0126对辐射后凋亡基因产物表达的影响：u0126对辐射后Bcl一2的表达未见明显影响；u0126可明显减弱辐射后6h和1d Caspase一3的表达以及辐射后6h Bax的表达P<0．01)；(5)RKIP重组质粒构建与Pcl2细胞基因转染：分别采用pcDNA3．0及pGPu6／GFP／Neo载体，以大鼠背根神经节为模扳，成功构建了pc~)NA3．0一RKIP及RKIP shRNA重组质粒，并筛选获得了稳定转染RKIP正义和反义质粒的PC2细胞株；采用非脂质体转染试剂～~igoI，ect介导的重组质粒可高效转染未分化的PCI2细胞，诱导分化后细胞转染效率较高(约80％)，从而为开展Pcl2神经细胞基因转染实验提供了可靠技术平台；(6)RKIP对辐射后ERK信号通路的调控作用：辐射后6h和1d，RKIP过表达显著抑制辐射后p—MEK、p—ERK及p—CREB上调P<0．01)；而RKIP基因沉默对辐射后p—MEK、p—ERK及p—CREB上调具有促进作用；RKIP对辐射后ERK的表达未见明显影响；(7)RKIP对辐射诱导细胞凋亡的影响：辐射后6h和1d，RKIP过表达可明显抑制辐射后细胞凋亡增多P<0．01)，RKIP基因沉默则导致凋亡进一步增多P<0．05)；RKIP过表达显著抑制辐射后：Bcl一2下调、Bax及Caspase-一3的上调，RKIP基因沉默则与其作用相反；RKIP基因沉默和u0126对于辐射诱导的细胞凋亡、Bcl一2、Bax及Caspase一3表达改变均具有明显的拮抗作用(P<0．01)。
结论：
一、平均功率密度30mw／cm。的微波辐射5min导致PCI2细胞凋亡和坏死率明显升高，细胞核染色质浓缩、DNA断裂、凋亡小体形成，线粒体膜电位下降，建立了良好的微波辐射诱导神经细胞凋亡模型。
二、微波辐射后RKIP表达下调，对Ral7MEK／ERK信号通路的抑制作用减弱，从而导致Rar／MEK／ERK信号通路活化增强。另一方面，RKIP蛋白质翻译后修饰水平的变化可能参与了微波辐射损伤。
三、Ra~7MEK／ERK信号通路活化可促进微波辐射诱导的PCI2神经细胞凋亡，其作用机制与Bcl一2／Bax比值下降及C{aspase一3表达上调有关；u0126可减少辐射所致细胞凋亡，对微波辐射损伤具有一定防护作用。
四、RKIP正义与反义质粒的成功构建及其在PCI2细胞的有效表达为深入研究RKIP的神经生物学功能奠定了良好实验基础，并提供了可靠技术平台。
五、RKIP对微波辐射后Ral7MEK／ERK信号通路的活化具有主要调控作用，这一调控参与诱导辐射所致PCI2神经细胞凋亡，并在其中发挥重要调节作用。RKIP过表达明显减少辐射后细胞凋亡，RKIP基因沉默则导致辐射后细胞凋亡增加。
六、RKIP下调导致CREB活化增强，Bcl一2／：Bax比值下降，Gaspase-一3表达增加，是RKIP调控微波辐射所致细胞凋亡的重要机制。另一方面，RKIP对辐射诱导细胞凋亡的调控机制可能涉及多条细胞信号通路之间的相互对话。
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.
引文
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