Effect And Mechanism Of PIDD On Microglia Activation In Radiation-induced Brain Injury

Part one: Over expressed the PIDD-C, PIDD-CC gene and targeted silencing the PIDD gene in mouse microglia stably transfected cell line after ionizing radiation, the release of inflammatory factors and changes in the state of activation?Objective?To construct and identify PIDD-C,PIDD-CC overexpression-Retrovirus system and PIDD-sh RNA-lentivirus system in BV-2 cells. To explore the radiation induced microglia activation and inflammatory cytokines release by regulating the PIDD gene.?Method?(1) Constructed PIDD-sh RNA-lentivirus system and BV-2 cells as LV-PIDD-ih group was infected with PIDD-sh RNA-lentivirus silencing the PIDD gene expression. PIDD-C overexpression-Retrovirus system was contructed and infected BV-2 cells as LV-PIDD-C-oe group. PIDD-CC overexpression-Retrovirus system was contructed and infected BV-2 cells as LV-PIDD-CC-oe group. Negative control lentivirus infected with BV-2 cells as LV-PIDD-ih. And the cells that infected with lentivirus were selected by puromycin.(2) Cells of all groups were treated with 10 Gy irradiation. Detected the PIDD, TNF-? and IL-? m RNA and protein level of each group at different time points(3h, 6h, 24 h, 48h) after irradiation.(3) To detected the level of apoptosis-related protein(caspase-3, BAX, bcl-2, caspase-9, p53, PARP1) by western blot in different groups after 24 h irradiation.(4) Immunofluorescence experiment was used to observe the expression level of Iba-1, F4/80, TNF-? and caspase-3 in different treated goups.(5) Co-immunoprecipitation test was used to detect the relationship between PIDD-C, PIDD-CC and NEMO.(6) The scratch assay and transwell migration assay was used to observe the change of cell's migration ability in different groups after 10 Gy irradiation.(7) Clone formation assay was used to detect effects of PIDD gene on radiation tolerance.(8) The flow cytometry was used to detect the change of apoptosis in different groups after irradiation.(9) Transmission electron microscopy(TEM) observed the changes of cell ultrastructure after radiation?Results?(1) The stable PIDD silencing cell lines, the stable PIDD-C overexpression cell lines and the stable PIDD-CC overexpression cell lines were set uo successfully. Experiments were divided into six groups:(1) Blank control group(Control): without radiation;(2) radiotherapy group(RT): with 10 Gy radiaton;(3) silencing PIDD gene(LV-PIDD-ih): infected silencing PIDD gene lentivirus and received 10 Gy irradiation;(4)the overexpressed PIDD-C gene group(LV-PIDD-PIDD-C-oe): infected with PIDD-C overexpression gene lentivirus and accepted 10 Gy of ionizing radiation.(5) the overexpressed PIDD-CC gene group(LV-PIDD-PIDD-CC-oe): infected with PIDD-CC overexpression gene lentivirus and accepted 10 Gy of ionizing radiation.(6) negative control group(LV-NC): infection in negative lentivirus and accepted 10 Gy of ionizing radiation.(2) Real-time PCR and western blot were used to detect the level of PIDD, TNF-? and IL-1? after 10 Gy irradiaton in different group. The result showed that silencing PIDD gene can down-regulated the level of PIDD, TNF-? and IL-1? after 10 Gy irradiation. Up-regulated PIDD-C gene can increase the level of PIDD-C and PIDD-CC, but decrease the expression of TNF-? and IL-1?. Up-regulated PIDD-CC gene can increase the level of PIDD-CC, TNF-? and IL-1?.(3) Western blot showed the expression of apoptosis-related protein(caspase-3, BAX, caspase-9, PARP1) in RT cell, LV-NC cell and LV-PIDD-CC-oe cell were significantly increase compared with control cell. The level of caspase-3, BAX, caspase-9, p53, PARP1 in LV-PIDD-ih cell and LV-PIDD-CC cells were down-regulation compared with RT and LV-NC cells, and the difference was statistically significant(P<0.05).(4) Immunofluorescence assay revealed that Iba-1, F4/80, TNF-a and caspase-3 expression in RT cells, LV-NC cells and LV-PIDD-CC-oe cells were increased compared with control cell. There is no significant difference in LV-PIDD-ih cells and LV-PIDD-C-oe cells between control cells.(5) Co-immunoprecipitation test showed PIDD-Cand PIDD-CC both interacts with NEMO.(6) The scratch assay and transwell migration assay showed that, after ionizing radiation, RT cells, the LV-NC cells, LV-PIDD-C-oe cells and LV-PIDD-CC-oe cell migration rate is higher than that of control cells, and the difference is statistically significant(P < 0.05). After iradiation, the migration rate of LV-PIDD-ih cells had no significant difference with control cells.(8)The clone formation assay showed the radiation tolerance enhancement enhanced in BV-2 cells after PIDD gene silenced and overexpression of PIDD-CC gene reducted radiation tolerance of microglia cell(P<0.05).(9) The flow cytometry shows down-regulated PIDD can inhibite the radiation-induced apoptosis and up-regulated PIDD-CC can promote the radiation-induced apoptosis.(P<0.05), the difference has statistically significant).(10) Transmission electron microscopy(TEM) displayed that silencing PIDD can inhibit the radiation-induced cell ultrastructure injury and up-regulated PIDD-C can promote the repair after radiation.?Conclusion?The silence of PIDD gene can induce microglial activation after ionizing radiation, and reduce the release of inflammatory cytokines, inhibition of microglial cell apoptosis and enhance cell radiation tolerance. Overexpression of PIDD-C gene promotes repair of microglial cells after ionizing radiation, inhibit the activation of microglia, and reduce the release of inflammatory factors. Overexpression of PIDD-CC gene can promote the activation of microglial cells after ionizing radiation, increased apoptosis induced by radiation, and reduce the radiation tolerance of cells. According to the interaction between PIDD-C and PIDD-CC two fragments with NEMO Co-immunoprecipitation.Part two: The effects of lentivirus mediated overexpress PIDD-C, PIDD-CC gene and silence PIDD gene on radiation-induced brain injury in mice?Objective?: The high titer lentivirus that overexpressed PIDD-C, PIDD-CC gene and silenced PIDD gene were injected into mice lateral ventricle. Constructed the radiation-induced brain injury models that overexpressed PIDD-C, PIDD-CC gene or silenced PIDD gene. After treated with 10 Gy radiation, compared the expression of PIDD, TNF-? and IL-1? in different groups' brain tissues, and detected the activation state of microglia and changes of mice learning and memory ability to further explore the PIDD gene on the effects in RIBI.?Methods?(1) 60 C57BL/6J mice were divided into 6 groups randomly, each group had 10 mice.(1) control group;(2) RT group;(3) LV-NC group;(4) LV-PIDD-ih group;(5)LV-PIDD-C-oe group;(6)LV-PIDD-CC-oe(pidd-cc gene in expression) group.(2) The mouse brain stereotactic technology to inject the lentivirus into lateral ventricle by microsyrine. After 5 days of operation, the mice of(2)-(6) group treated with 10 Gy radiation and tested the PIDD, TNF-? and IL-1? expression of different time points in each groups' mice brain tissues by using western blot and real-time PCR.(3) Western blot was used to dected the expression of apoptosis-related protein(caspase-3, BAX, bcl-2, p53, PARP1) after radiation.(4) Tissue immunofluorescence double staining was used to observe the activated state and apoptosis state by staining the Iba-1, F4/80, PIDD, TNF-?and caspase-3 of different groups' brain tissues.(5) Morris water maze test was performed on 7 mice in each group 6 weeks after radiotherapy, and the changes of learning and spatial memory ability were detected after 10 Gy whole brain radiotherapy in each group.?Results?(1) Successfully established PIDD silencing and stable overexpression PIDD-C and PIDD-CC in mice radiation-induced injury brain model. Blank control group as control group, simple irradiation group as RT group, negative control vector infection group as LV-NC group and PIDD gene silencing infection group as LV-PIDD-ih group, overexpression PIDD-C retrovirus infection group as LV-PIDD-C-oe group, and overexpression PIDD-CC retrovirus infection group as LV-PIDD-CC-oe group. Mice received 10 Gy irradiation, and western blot results showed the expression of LV-PIDD-C-oe group, the PIDD-C protein was significantly higher than that in the control group and the lv-nc group(P<0.05), the expression of LV-PIDD-CC-oe group protein PIDD-CC was obviously higher than that in control group(P<0.05), but between RT group and LV-NC group no significant difference. The expression of PIDD protein in LV-PIDD-ih group's brain tissue was significantly lower than other groups(P<0.05), but no significant difference with Control group.(2) TNF- Real-time PCR Western and inflammatory factor alpha blot assay after radiotherapy of mice brain tissue, expression of IL-1 beta. Experimental results showed that the expression of inflammatory factor in each group after the radiotherapy changes with time and change, RT group, LV-NC group, LV-PIDD-C-oe group and LV-PIDD-CC-oe group brain tissue TNF-? and expression after irradiated 3h reached the highest, and IL-1? expression reached the peak at 6 h. The inflammatory factors level of LV-PIDD-C-oe group is lower than LV-PIDD-CC-oe group(P<0.05).(3) Caspase-3, Bax, caspase-9, p53, PARP1 protein expression levels in RT group, LV-NC group, LV-PIDD-C-oe group and LV-PIDD-CC-oe group were significantly higher than control group(P<0.05) and bcl-2 protein expression level was significantly lower than control group. In LV-PIDD-ih group and LV-PIDD-C-oe group, ccspase-3, BAX, caspase-9, PARP1 protein expression in brain tissue was lower than in RT group and Lv-NC group, the protein expression of Lv-PIDD-CC were higher than RT and Lv-NC group, the difference was statistically significant(P<0.05).(4) Tissue immunofluorescence assay showed that the RT group, LV-NC group and LV-PIDD-CC-oe group received ionizing radiation Iba-1 and F4 / 80, TNF-? and caspase-3 fluorescent expression amount than in the control group increased significantly, and LV-PIDD-ih and LV-PIDD-C-oe group brain tissue Iba-1 and F4 / 80 fluorescence expression amount compared with the control group no significant difference.(5) Morris water maze test, PIDD gene silencing and overexpression of PIDD-C gene can improve the memory ability of mice after radiation brain injury.?Conclusion? The expression of PIDD gene silencing can inhibit NF-?B pathway activation, inhibiting the activation of microglia, decreing the release of inflammatory factors, and may be a target for the treatment of radiation-induced brain injury.