长链非编码RNA-核富集转录本1对创伤性脑损伤小鼠的神经保护作用及其可能机制

Neuroprotective effect of long noncoding RNA-nuclear-enriched abundant transcript 1 on traumatic brain injury in mice and its possible mechanism

目的探讨长链非编码RNA(lncRNA)核富集转录本1(NEAT1)的表达对创伤性脑损伤(TBI)后神经功能和神经元凋亡的影响及其可能机制。方法将90只C57BL/6J小鼠按随机数字表法分为假手术组、空白对照组、空载病毒1组和2组及NEAT1上调组和NEAT1下调组,每组15只。构建小鼠控制性皮层撞击(CCI)模型模拟TBI状态,并通过侧脑室内注射腺病毒载体对NEAT1水平进行干预。采用神经功能缺失评分(NSS)及Morris水迷宫试验评估空白对照组、NEAT1上调组和NEAT1下调组小鼠伤后1周内及14~21d时神经功能变化。Western blot检测空白对照组小鼠皮层含死亡结构域的p53诱导蛋白1(Pidd 1)、含半胱氨酸的天冬氨酸蛋白水解酶(caspase)-2、caspase-9和caspase-3在伤后6 h、1,3,7 d的表达。TUNEL法检测空白对照组、NEAT1上调组和NEAT1下调组伤后第3天小鼠神经元凋亡情况及免疫荧光半定量分析Pidd1的表达及定位。Western blot检测假手术组、空白对照组、空载病毒组、NEAT1上调组及NEAT1下调组小鼠伤后第3天Pidd1、caspase-2、细胞色素c(Cyt c)和caspase-3蛋白表达量。结果与空白对照组[(4.9±1.0)分]比较,NEAT1上调组伤后第1天NSS[(3.5±0.7)分]显著降低(P<0.01),NEAT1下调组[(5.0±1.5)分]显著升高(P<0.01)。Morris水迷宫试验显示,与空白对照组[(20.1±5.6)s]比较,伤后第19天NEAT1上调组寻找平台时间[(10.9±2.8)s]缩短(P<0.05),NEAT1下调组寻找平台时间[(30.7±6.2)s]延长(P<0.01)。Western blot提示,空白对照组Pidd1、caspase-2、caspase-9和caspase-3在伤后第3天表达明显升高(P<0.01)。通过TUNEL得到伤后第3天NEAT1上调组神经元凋亡率[(18.0±2.7)%]明显下降,而NEAT1下调组[(63.0±8.6)%]明显升高(P<0.01)。伤后第3天免疫荧光提示,与空白对照组比较,NEAT1上调组神经元胞质中Pidd1蛋白表达[(22.7±2.2)%]减少(P<0.01),而NEAT1下调组[(72.7±7.0)%]显著增加(P<0.01)。伤后第3天Western blot显示,与空白对照组比较,NEAT1上调组Pidd1(0.5±0.0)、capsase-2(0.3±0.0)、Cyt c(0.5±0.0)及caspase-3(0.4±0.0)的表达明显减少(P<0.01);而NEAT1下调组结果则完全相反。结论TBI后NEAT1通过调控Pidd1-caspase-2-Cyt c,减少caspase-3活化,抑制神经元凋亡,这可能是TBI后NEAT1保护神经功能的机制之一。

To explore the impact of the expression of long noncoding RNA-nuclear-enriched abundunt transcript 1(NEAT1)on neurological function and neuronal apoptosis after traurmatie brain injury(TBI)in mice and the possible mechanism.Methods According to the random number table,90 C57BL/6J mice were divided into sham group,blank control group,empty vinus goup 1,empty vins group 2,NEATI over-expression group and NEATI knockdowrn group,with 15 mice per group.The traumatie brain injury(TBI)was simulated by contolldl cortical injury(CCI)model,and NEATI was regulated by intracerebrorvnticular injection with recombinant adenovinus.The neurological severiy score(NSS)and Morris water maze test were used to evaluate the neurological function in blank control goup,NEATI over expression group and NEATI knokdown group within 1 week and 14-21 days ater injury.The Westem blot was used to observe the expressions of P53-induced protein with a death domain 1(Pidd1),easpase-2,caspuse-9 and caspuse-3 in blamk control group at 6 hour and 1,3,7 days afer injury.The TUNEL method and immunoluorescence were used to observe the neurological apoptosis and expression of Pidd1 in blank control group,NEATI over expression group and NEAT1 knockdown group at 3 days after injury.The Western blot analysis was used to detect protein expressions of Piddl,caspase-2,cytochrome c(Cyt c)and caspase-3 in sham group,blank control group,empty virus groups,NEATI over expression group und NEAT1 knockdowmn group a 3 days alter injuy.Results The NSS was significantly reduced in NEATI over-expression group[(3.5±0.7)points],and was signifcanly inceased in NEATI knockdown group[(5.0±1.5)poins]at day 1 after injury,when compared with blank control group[(4.9±1.0)point](P<0.01).The Mrris water maxe test showed that the time to find platforom was decreased in NEATI over exression group[(10.9±2.8)seconds],and was prolongod in NEATI knoekdown group[(30.7±6.2)seconds]at day 19 after injury(P<0.05 or 0.01),when compared with blank control goup[(20.1±5.6)scconds].The Westem blot analysis showed that the expressions of Pidd1,caspase-2,ceaspase-9 and easpase-3 had signifcant increase at day 3 after injuy(P<0.01).The TUNEL test showed that the apoptosis rnte of neurons was sigifcanty dereased in NEATI overexpression group[(18.0±2.7)%],and the apoptosis rate was signifcantl'y inereased in NEATI knockdown goup[(63.0±8.6)%]at day 3 after injury(P<0.01).Immunofluorescence showed that the expression of Piddl protein in eytoplasm of the neurons was decreased in NEATI over expression goup[(22.7±2.2)%](P<0.01),and was increased in NEAT1 knockdowmn group[(72.7±7.0)%](P<0.01)at day 3 after injury,wrhen comparred with blank control group.The Western blot analysis showed that the expressions of Pidd1,eapsase-2,Cyt。and caspase-3 were sigifeanly reduced in NEATI over-expression group(0.5±0.0,0.3±0.0,0.5±0.0,0.4±0.0)at day 3 afere injuy,when compured with blank control group.However,the results were opposite in NEATI knockdown group.Conclusion After TBI,the NEAT1 can reduce the activation of caspase-3 through the Pidd1-aspas-2-Cy.e pathway ater TBI,regulate neuronal apoptosis,and ulimately play a protective role in neurological funetion.