Keap1-Nrf2和NF-κB信号通路介导脂多糖诱导的脑损伤效应研究

Roles of Keap1-Nrf2 and NF-κB signaling pathways in lipopolysaccharide-induced brain injury

目的探讨Kelch样环氧氯丙烷相关蛋白1⁃红系衍生的核因子2相关因子2(Keap1⁃Nrf2)和核因子κB(NF⁃κB)信号通路在脓毒症相关脑病(SAE)中的作用。方法40只雄性SPF级C57BL/6J小鼠,随机分为4组(n=10):对照组和脂多糖(LPS)处理6、24、48 h组。根据小鼠一般状况和旷场实验评价小鼠的行为学变化。采用酶联免疫吸附法(ELISA)试剂盒检测小鼠外周血促炎性因子含量,采用抗氧化检测试剂盒检测脑组织抗氧化活性。实时定量PCR(qPCR)检测小鼠海马组织中Toll样受体4(Tlr4)、NF⁃κB、Keap1、Nrf2 mRNA含量,Western印迹分析NF⁃κB、Nrf2、Keap1和Tlr4蛋白的表达。结果与对照组相比,LPS 6 h组白细胞介素6(IL⁃6)血清浓度呈上升趋势,但差异无统计学意义,24、48 h达高峰(P<0.01);LPS 6 h和24 h组白细胞介素18(IL⁃18)血清浓度增加(P<0.01),但48 h组差异无统计学意义;脑组织超氧化物歧化酶(SOD)、谷胱甘肽(GSH)活性增加(P<0.05);除了LPS 24 h组旷场内中央区域时间差异无统计学意义,其他LPS组小鼠旷场内中央区域活动时间、活动路程及活动总路程均显著减少(P<0.01)。与对照组相比,LPS组小鼠海马区Tlr4、LPS 6 h、48 h组NF⁃κB和LPS 6 h组Keap1及Nrf2 mRNA表达增加(P<0.05);LPS组NF⁃κB、Keap1、Tlr4蛋白和LPS 24 h、48 h组Nrf2蛋白水平上升(P<0.05)。结论Keap1⁃Nrf2和NF⁃κB信号途径在脓毒症脑病中发挥一定作用。

Objective To investigate the role of Kelch⁃like⁃epichlorohydrin⁃associated protein1/nuclear factor erythroid⁃derived factor⁃2⁃related factor 2(Keap1⁃Nrf2)and nuclear factor⁃κB(NF⁃κB)signaling pathways in sepsis⁃associated encephalopathy(SAE).Methods Male C57BL/6J mice of SPF were randomly divided into four groups(n=10):the control group and LPS 6 h,24 h and 48 h groups.The behavioral changes of the mice were assessed based on their general conditions and open field test(OFT).ELISA was used to measure the levels of pro⁃inflammatory cytokines in mouse serum,and the antioxidant capacity assay kit to examine antioxidant activity in brain tissues of mice.Real⁃time quantitative PCR(qPCR)was adopted to detect the mRNA levels of toll⁃like receptor4(Tlr4),NF⁃κB,Keap1 and Nrf2 in the hippocampus,and to determine protein expressions of NF⁃κB、Nrf2、Keap1 and Tlr4 with Western blotting.Results Compared to the control group,the serum concentrations of interleukin 6(IL⁃6)in lipopolysaccharide(LPS)groups increased at 6 h,and reached the peak at 24 h and 48 h(P<0.01).The levels of serum interleukin 18(IL⁃18)in the LPS groups increased significantly at 6 h and 24 h(P<0.01)but there was no statistically significant difference compared with the 48h group.The results indicated the activity of superoxide dismutase(SOD)and glutathione(GSH)in brain tissues in LPS groups increased(P<0.01).OFT results showed the time spent in the center of the open field,the distance covered around the center,and total distance covered by mice in LPS groups were significantly reduced(P<0.01),except for the time spent in the center of the open field in the LPS 24 h group.The mRNA expressions of Tlr4 and(LPS 6 h,48 h)NF⁃κB in the hippocampus tissue of mice in LPS groups were elevated(P<0.05),so were the mRNA expressions of Keap1 and Nrf2 in LPS 6 h group.Additionally,the protein expressions of NF⁃κB,Keap1 and Tlr4 increased in LPS groups,so did the protein expression of Nrf2 in LPS 24 h and 48 h groups(P<0.05).Conclusion Keap1⁃Nrf2 and NF⁃κB signaling pathways may play a certain role in SAE.