| Objective The mechanism of TLR4 regulating pro-inflammatory cytokines TNF-α and IL-1β through MyD88-dependent pathway was observed in cells.To explore the changes of TLR4,MyD88,TNF-α and IL-1β in microglia activation mediated inflammation and cognitive dysfunction induced by ionizing radiation in animals.Methods 1.BV-2 microglia cell were divided into blank Control group(Control),Irradiated group(IR),TLR4 negative Control virus irradiation group(Lv-NC),TLR4 knockdown irradiation group(Lv-TLR4-shRNA),TLR4 agonist(LPS)irradiation group(LPS+IR),MyD88 inhibitor irradiation group(ST2825+IR),MyD88 inhibitor+TLR4 agonist irradiation group(ST2825+LPS+IR),cells were given 6MV X-ray irradiation(10Gy)after 1 day cultured.BV-2 microglia was verified by immunofluorescence staining,and the expression levels of TLR4,TNF-α and IL-1β were determined by RT-PCR.TLR4 and MyD88 protein expression were determined by Western blot.2.4-week-old Sprague Dawley(SD)rats were randomly divided into the Control group,the Irradiated group,the Sham+NS group,the Irradiated+NS group,the Lv-TLR4-shRNA group,and the Lv-NC group.There were 10 rats in each group.In the Sham+NS group,the Irradiated+NS group,the Lv-TLR4-shRNA group,and the Lv-NC group,stereotaxic injection was used to inject into the dentate gyrus of the hippocampus.In the irradiation group,the whole brain was irradiated 30 Gy at the age of 8 weeks by 6Me V electron wire.Morris water maze test was used to test the learning,memory and spatial exploration ability of rats 1 month after irradiation.The expression levels of TNF-α and IL-1β in rat hippocampus were determined by reverse transcription-polymerase chain reaction(RT-PCR),and the protein expressions of TLR4,MyD88,TNF-α and IL-1β in rat hippocampal dentate gyrus(DG)were determined by Western blot.Immunofluorescence staining was used to analyze the changes of hippocampal microglia,TLR4 and MyD88.Results 1.TLR4 mRNA expression of microglia in the Irradiated group(IR)was higher than that in the blank Control group(non-irradiation group,Control)after 10 Gy X-ray irradiation(P<0.01).The expression of TLR4 mRNA in LPS+IR group and ST2825+LPS+IR group was significantly higher than that in IR group and non-irradiated group(P<0.001).The expression of MyD88 in BV-2 microglia was significantly higher in the IR group than in the non-irradiated group(P<0.001).The expression of MyD88 protein in LPS+IR group was significantly higher than that in IR group and non-irradiated group(P<0.01,P<0.001,respectively).Compared with the IR group,the expression of MyD88 protein was significantly inhibited in the ST2825+IR group(P<0.05),and the MyD88 inhibitor ST2825 significantly inhibited the expression of MyD88 protein after the addition of TLR4 agonist LPS(P<0.001).The expression of TNF-α mRNA and IL-1β mRNA in b V-2 microglia after 10 Gy irradiation was significantly higher in the IR group than in the non-irradiation group(P<0.001).TNF-α mRNA and IL-1β mRNA expression were significantly increased in LPS+IR group compared with the IR group and non-irradiated group(P<0.001).Compared with the IR group,the expression of TNF-α mRNA and IL-1β mRNA in ST2825+IR group was significantly decreased(P<0.001).In addition,MyD88 inhibitor ST2825 could significantly inhibit the expression of TNF-α mRNA and IL-1β mRNA after adding TLR4 agonist LPS(P<0.001).The expression of TLR4 protein in Lv-TLR4-shRNA irradiated group was significantly lower than that in Lv-NC irradiated group and IR group(P<0.05),and the expression of MyD88 protein was significantly decreased compared with Lv-NC and IR group(P<0.001).The expression of TNF-α mRNA and IL-1β mRNA in Lv-TLR4-shRNA irradiated group was significantly lower than that in Lv-NC irradiated group and IR group(P<0.001).2.In the water maze test,between rats in the normal control group and the Irradiated group,there was statistically significant difference in escape latency at different test time points(day 1-5)(P<0.001).On the fifth day,compared with the normal control group,the escape latency of rats in the Irradiated group was longer one month after 30 Gy whole brain irradiation.The difference was statistically significant(P=0.003).Compared with the normal group,the expression of TLR4 protein in hippocampal tissue of rats in the Irradiated group was significantly increased one month after 30 Gy whole brain irradiation(P<0.01).The expression of TNF-α mRNA and IL-1β mRNA in the hippocampus of the irradiated group was significantly higher than that of the normal group(P<0.01).Compared with the normal control group,the microglia cells in the 30 Gy irradiation group were significantly activated and proliferation(Iba-1 marker),and TLR4 immunofluorescence staining of microglia nuclei was significantly enhanced.Compared with the Lv-NC and the Irradiated+NS group,the expression of TLR4 protein in hippocampus of Lv-TLR4-shRNA group was significantly decreased(P<0.01).Results of Immunofluorescence showed that TLR4 knockdown significantly inhibited the activation of hippocampal microglia proliferation(Iba-1 marker).This result was confirmed by immunostaining with antibodies specific for CD68(labeled activated microglia).The expression of TNF-α and IL-1β in hippocampus of Lv-TLR4-shRNA group was significantly lower than that of the Lv-NC irradiated group and the Irradiated+NS group(P<0.001).There were statistically significant differences in escape latency on day 5 between the Sham+NS group,the Irradiated+NS group,the Lv-TLR4-shRNA irradiated group,and the Lv-NC irradiated group(P<0.001).The escape latency on day 5 in Irradiated+NS group was significantly higher than that in the Sham+NS group,the difference was statistically significant(P=0.002).Compared with the Sham+NS group,the escape latency of the Lv-NC irradiated group was significantly increased on the fifth day,and the difference was statistically significant(P=0.009).The escape latency of the Lv-TLR4-shRNA irradiated group was significantly decreased on the fifth day compared with the Irradiated+NS group and the Lv-NC irradiated group.The differences were statistically significant(P=0.005,P=0.022).Compared with the Sham+NS group,the time ratio in the target quadrant of the Irradiated+NS group and the Lv-NC irradiated group was significantly increased(P=0.001,P=0.002,respectively),while there was no significant difference between the Lv-TLR4-shRNA irradiated group and the Sham+NS group(P=0.083).Compared with the normal control group,the expression of MyD88 protein in the hippocampus of the Irradiated group was significantly increased one month after 30 Gy irradiation(P<0.01).Immunofluorescence method was used to co-locate MyD88 and IBA-1.Compared with the normal control group,immunofluorescence staining of MyD88 microglia nucleus in hippocampal tissue of rats in the Irradiated group was significantly enhanced.The expression of MyD88 protein in hippocampus of the Lv-TLR4-shRNA Irradiated group was significantly decreased compared with the Lv-NC irradiated group and the Irradiated+NS group(P<0.01).Conclusion 1.TLR4/Myd88 signaling pathway involved in the regulation of ionizing radiation microglia-mediated inflammation2.Cognitive impairment was induced 1 month after 30 Gy whole brain irradiation in 8-week-old SD rats.3.TLR4 regulates microglia-mediated inflammation through MyD88 and participates in ionizing radiation induced cognitive impairment in rats. |
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