创伤性颅脑损伤后脑组织中TLR4表达的实验研究

Expression of Toll like receptor 4 in injured brain tissue after traumatic brain injury

目的研究创伤性脑损伤（Tin）后损伤灶周围脑组织Toll样受体4（TLR4）的表达，探讨TLR4／NF-κB信号通路在TBI中的作用机制。方法SD大鼠36只按随机数字表法分为对照组（n=12）、TBI后1d组（n=6）、TBI后3d组（n=12）和TBI后7d组（n=6），后3组采用Feeney自由落体撞击法制作TBI模型，对照组仅行右侧顶部开窗而无TBI。应用RT—PCR、凝胶电泳迁移率实验fEMSA）、ELISA分别检测4组大鼠挫伤脑组织TLR4mRNA、NF—κB活性、TNF-α和IL-6浓度的变化；免疫组化染色检测对照组和TBI后3d组大鼠挫伤脑组织TLR4的表达。结果与对照组比较，TBI后1d、3d、7d组TLR4mRNA表达、NF—κB活性、TNF-α和IL-6浓度均增加，差异有统计学意义（P〈0．05）；对照组脑组织TLR4表达较少，ribI后3d组创伤灶周围可见大量TLR4阳性细胞，主要表达在皮层胶质细胞、神经元中；NF-κB活性与TLR4mRNA的表达呈正相关关系（r=0．786，P=0．000）。TNF—α、IL-6与TLR4的表达也呈正相关关系（r=0．517，P=0．010；r=0．503，P=0．012）。结论TBI可引起损伤区脑组织TLR4的表达和下游NF-κB、促炎症因子水平的增加，TLR4／NF-κB信号通路可能在脑组织的继发性损害中起重要作用。

Objective To investigate the expression of Toll like receptor 4 （TLR4） in the injured brain tissue after traumatic brain injury （TBI） and explore the potential role of TLR4/NF-κB in the secondary brain injury. Methods Thirty-six SD rats were randomly divided into control group （n=12）, TBI inducement for 1 d group （n=6）, TBI inducement for 3 d group （n=12） and TBI inducement for 7 d group （n=6）. TBI models of the later 3 groups were induced by Feendy＇s free-falling, and rats of the control group are only performed exposure ofdura of the right parietal lobe. TLR4 mRNA expression in the injured brain tissue was studied by RT-PCR, NF-KB binding activity was detected by electrophoretic mobility shift assay （EMSA）, and the TNF-α and IL-6 levels were detected by enzyme linked immunosorbent assay （ELISA）. Immunohistochemistry was employed to determine the protein expression of TLR4 in the brain tissues of control group and TBI inducement for 3 d group. Results The TLR4 mRNA expression, NF-KB binding activity, and the levels of TNF-α and IL-6 in rats of the TBI inducement for 1, 3 and 7 d groups were significantly increased as compared with those in the control group （P〈0.05）. The protein expression level of TLR4 in the brain tissue of control group was rare, and a large number of TLR4-positive immunostained cells, including cortical glial cells and neurons, were noted in the brain tissue of TBI inducement for 3 d group. TLR4 mRNA level was positively correlated to the NF-κB activity （r=0.786, P=-0.000）, and positive relations were also noted between TLR4 mRNA level and both TNF-α and IL-6 levels （r=0.517, P=-0.010; r=0.503, P=0.012）. Conclusion TBI could induce concomitant and persistent up-regulation of TLR4 expression and NF-κB binding activity in the injured brain tissue. TLR4/NF-κB might play a central role in the secondary injury after TB1.