摘要：
Objective To study the effects of discoidin domain receptor 1 (DDR1) mediated phosphorylation of protein Tau on hypoxic-ischemic brain damage (HIBD) in neonatal rats and its possible mechanism.Methods Sixty-four seven-day-old male specific-pathogen-free Wistar rats were randomly divided into four groups with sixteen in each: Sham, HIBD, HIBD with normal saline (HIBD+NS) and HIBD with DDR1 inhibitor (HIBD+DI) groups. A rat model of HIBD was established by subjecting the rats to left common carotid artery ligation, followed by exposing them to hypoxia for two hours. In HIBD+DI group, the inhibitor of DDR1 was immediately injected into lateral cerebroventricles of the rats following modeling. Forty-eight hours after injection, tissues of left cerebral cortex were collected from each rat to evaluate histopathological changes with HE staining. Western-blotting was used to assess the phosphorylation levels of DDR1 and protein Tau. Enzyme-linked immunosorbent assay was performed to detect the concentrations of acetylcholine. Analysis of variance ort test were used for statistical analysis.Results (1) Damages in cerebral cortex: Percentages of abnormal neurons in the rats of HIBD group were higher than those in Sham group [(80.28±4.51)% vs (10.40±2.17)%,t=39.491,P<0.01]. Pyknotic or necrotic neurons in the rats of HIBD+DI group were less than those in HIBD+NS group [(31.91±3.05)% vs (82.01±7.20)%,t=18.123,P<0.01]. (2) Phosphorylation of DDR1 and protein Tau: Levels of phosphorylated DDR1 in the cerebral cortexes of rats in HIBD group were higher than those in Sham group (0.922±0.199 vs 0.095±0.023,t=10.379,P<0.01), and those levels in HIBD+NS group were higher than those in HIBD+DI group (1.200±0.171 vs 0.255±0.111,t=11.901, P<0.01). The phosphorylation of protein Tau was similar to that of DDR1 (0.919±0.228 vs 0.194±0.224 in HIBD and Sham groups,t=7.347; 1.100±0.167 vs 0.291±0.210 in HIBD+NS and HIBD+DI groups,t=9.447;bothP<0.01). (3) Levels of acetylcholine: Levels of acetylcholine in cerebral cortexes of rats in HIBD group were lower than those in Sham group [(3.685±0.472) vs (7.429±0.861) ng/g protein,t=10.781,P<0.01], and that levels in HIBD+DI group were higher than those in HIBD+NS group [(7.058±0.915) vs (2.521±0.723) ng/g protein,t=10.989,P<0.01].Conclusions Activation of DDR1 plays a key role in enhancing the phosphorylation of protein Tau and in reducing the secretion of acetylcholine in cerebral cortexes of rats with HIBD. Inhibitor of DDR1 could protect neonatal rats from HIBD through the decreasing of protein Tau phosphorylation and increasing of acetylcholine release by inhibiting the activation of DDR1.%目的 研究盘状结构域受体1(discoidin domain receptor 1,DDR1)调控微管相关蛋白Tau(简称Tau蛋白)表达及其磷酸化在新生大鼠缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)中的作用及其机制.方法 将64只7日龄无特定病原体级雄性Wistar大鼠随机分为假手术组(Sham组)、HIBD组、生理盐水(normal saline,NS)治疗组(HIBD+NS组)及DDR1抑制剂(DDR1 inhibitor,DI)治疗组(HIBD+DI组),每组16只.通过结扎左侧颈动脉后置于低氧箱内2 h,复制新生大鼠HIBD模型.HIBD+DI组造模后,立即给予DDR1抑制剂侧脑室注射治疗.造模后48 h收集左侧皮层组织,HE染色观察大脑皮层的组织病理学改变;蛋白质印迹法检测皮层组织中DDR1及Tau蛋白的磷酸化水平;酶联免疫吸附试剂盒法检测乙酰胆碱含量.用方差分析和t检验对数据进行统计学分析.结果 (1)皮层损伤情况:与Sham组相比,HIBD组异常神经元所占比例明显增多[(80.28±4.51)%与(10.40±2.17)%,t=39.491,P<0.01].HIBD+DI组动物大脑皮质区核固缩及坏死细胞比例较和HIBD+NS组显著降低[(31.91±3.05)%和(82.01±7.20)%,t=18.123,P<0.01)].(2)DDR1和Tau蛋白磷酸化水平比较:DDR1磷酸化水平方面,HIBD组高于Sham组(0.922±0.199与0.095±0.023,t=10.379,P<0.01),HIBD+NS组高于HIBD+DI组(1.200±0.171与0.255±0.111,t=11.901,P<0.01).Tau磷酸化方面也显示相似的趋势(HIBD组和Sham组分别为,0.194±0.224与0.919±0.228,t=7.347;HIBD+DI组和HIBD+NS组分别为1.100±0.167与0.291±0.210,t=9.447,P值均<0.01).(3)乙酰胆碱含量:HIBD组低于Sham组[(3.685±0.472)与(7.429±0.861)ng/g蛋白,t=10.781,P<0.01].而HIBD+DI组高于HIBD+NS组[(7.058±0.915)与(2.521±0.723)ng/g蛋白,t=10.989,P<0.01].结论 DDR1激活引起HIBD新生大鼠皮层组织中Tau过度磷酸化,进而导致神经递质乙酰胆碱释放减少.抑制DDR1活化能够降低HIBD新生鼠皮层组织中Tau的磷酸化水平,增加乙酰胆碱的释放.DDR1抑制剂对HIBD新生鼠脑损伤具有保护作用.
