肌醇磷酸酶-1与宫内感染小鼠海马神经炎性反应引起认知障碍的关系

摘要

Objective To investigate the relationship between cognitive impairment and SH2 domain-containing inositol phosphatase-1 (SHIP-1) induced by hippocampal neuritis in intrauterine infected mice.Methods Thirty C57BL/6 female mice and 15 male mice were caged in a ratio of 2 ∶ 1.After that,the pregnant mice were divided into 2 groups.A mice model of intrauterine infection was established that intrauterine infection group (lipopolysaccharides,LPS group) induced by LPS at the concentration of 350 μg/kg and control group treated with same volume of saline (9 g/L).At 3 days postpartum,15 mice in each group were killed for hippocampus,and the protein levels of SHIP-1,nuclear factor-κB(NF-κB) p65 and phosphorus NF-κB(NF-κBp) in the hippocampus of the newborn mice were detected by Western blott,while the levels of interleukin 1β (IL-1β) and tumor necrosis factor (TNF-α)were detected by using enzyme linked immunosoobent assay.When the remaining mice were 8 weeks old(10 in each group),Morris water maze experiments were performed respectively,which the mice were tested for evaluating learning and memory function by positioning navigation and space exploration experiments.Results The expression of SHIP-1 was significantly increased in control group (0.677 ± 0.074) compared with LPS group (0.317 ± 0.095,t =2.984,P =0.041),while the levels of NF-κB p65,and NF-κBp,were significantly lower in control group (0.630 ± 0.109,0.352 ± 0.084) than LPS group(0.630 ± 0.109,0.352 ± 0.084) (t =3.516,5.161,P =0.025,0.007).Moreover,LPS significantly enhanced the expression of IL-1β and TNF-α [(5.875 ± 0.349) pg/mg,(14.256 ± 0.784)pg/mg] compared with control group[(1.621 ± 0.151) pg/mg,(3.984 ± 0.255) pg/mg],and the differences were significant(t =11.190,12.460,P=0.000,0.000).By the average Escape Latency tests for6 days,LPS group [at 1-6 days (58.286±1.418) s,(56.036 ±2.252) s,(55.071 ±1.856) s,(50.071 ±3.251) s,(52.893 ±2.372) s,(46.929 ±3.761) s] markedly impaired the learning capacity compared with the control group[(53.679 ±2.413) s,(47.571 ±3.529) s,(54.071 ±2.777) s,(47.250 ±2.864) s,(45.107 ±3.447) s,(42.393 ±3.463) s],and the difference was significant (F =4.466,P =0.001).Concurrently,in probe trains LPS group increased the time of in zone southeast latency to first [(44.080 ± 6.313) s] compared with the control group [(25.900 ± 6.033) s],while shortened the period of in zone platform duration and in zone SE duration [(0.000 ± 0.040) s,(4.000 ± 1.693) s],decreased the times of in zone SE frequency and in zone platform frequency[(0.100 ±0.100) times,(1.000 ±0.394)times] compared with the control group [(0.400 ± 0.202) s,(14.360 ± 5.000) s,(0.600 ± 0.267) times,(3.400 ±0.763) times] (t=2.082,1.746,1.962,2.794,1.756,P=0.026,0.049,0.033,0.006,0.048).Conclusion The expression of SHIP-1 in hippocampus of newborn mice with intrauterine infection is decreased,and the inhibitory effect of SHIP-1 on the expression of downstream pro-inflammatory cytokines NF-κB,inflammatory cytokines IL-1β and TNF-α is decreased,along with cognitive impairments.%目的 探讨肌醇磷酸酶-1(SHIP-1)与宫内感染小鼠海马神经炎性反应引起认知障碍之间的关系.方法 30只C57BL/6雌鼠与15只雄鼠以2∶1合笼,孕鼠均分为宫内感染模型组脂多糖(LPS组)和对照组,LPS组于孕13.5 d尾静脉注射LPS350 μg/kg制备宫内感染模型,对照组注射等量9 g/L盐水.孕鼠产后3d,各组分别取15只新生鼠处死,分离海马组织提取蛋白,采用Western blot法检测海马组织中SHIP-1、核因子-κB(NF-κB) p65、NF-κB磷酸化(NF-κBp)的蛋白水平,采用酶联免疫吸附(ELISA)法检测海马组织中白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的水平.待剩余小鼠8周龄时(各15只),行Morris水迷宫实验、定位航行和空间探索实验,评估小鼠是否存在认知障碍.结果 对照组小鼠海马组织中SHIP-1蛋白表达(0.677±0.074)明显高于LPS组(0.317±0.095),而NF-κB p65及NF-KBp蛋白表达(0.630±0.109、0.352±0.084)则明显低于LPS组(1.028±0.029、1.080±0.113),差异均有统计学意义(t=2.984、3.516、5.161,P=0.041、0.025、0.007);LPS组海马组织中炎性因子IL-13和TNF-α的表达[(5.875±0.349) pg/mg、(14.256±0.784) pg/mg]明显高于对照组[(1.621±0.151) pg/mg、(3.984±0.255) pg/mg],差异均有统计学意义(t=11.190、12.460,P=0.000、0.000).LPS组小鼠学习能力[逃避潜伏期1～6d分别为(58.286±1.418)s、(56.036±2.252)s、(55.071±1.856)s、(50.071±3.251)s、(52.893±2.372)s、(46.929±3.761)s]明显高于对照组[逃避潜伏期1～6d分别为(53.679±2.413)s、(47.571±3.529)s、(54.071±2.777)s、(47.250±2.864)s、(45.107±3.447)s、(42.393±3.463)s],差异有统计学意义(F =4.466,P=0.001).空间探索实验结果:LPS组在第1次进入平台SE象限的潜伏时间[(44.080±6.313)s]明显长于对照组[(25.900±6.033)s],而停留在平台所在位置的时间、经过平台所在位置的次数及停留在平台东南象限的时间、进入平台所在象限的次数[(0.040±0.040)s、(0.100±0.100)次、(4.000±1.693)s、(1.000±0.394)次]均明显少于对照组[(0.400±0.202)s、(0.600±0.267)次、(14.360±5.000)s、(3.400±0.763)次],差异均有统计学意义(t=2.080、1.750、1.760、1.960、2.790,P=0.026、0.049、0.048、0.033、0.006).结论 宫内感染新生小鼠海马组织中SHIP-1表达降低,对其下游前炎性因子NF-κB和炎性因子IL-1β、TNF-α表达的抑制作用降低,同时伴认知功能障碍.