丝裂原和应激激活蛋白激酶1参与丙泊酚降低小鼠缺血性脑损伤

Involvement of Mitogen-and stress-activated protein kinase 1 in propofol-induced neuroprotection against cerebral ischemic injuries in mice

目的探讨丝裂原和应激激活蛋白激酶1(MSK1)在丙泊酚保护小鼠缺血脑组织中的作用。方法50只健康成年雄性BALB/c小鼠采用随机数字表法分为5组,分别为假手术组(Sham);大脑中动脉阻塞(MCAO)+生理盐水组;MCAO+25 mg/kg丙泊酚组(小剂量组);MCAO+50 mg/kg丙泊酚组(中剂量组);MCAO+100 mg/kg丙泊酚组(大剂量组),每组10只,各组术后30 min经腹腔注射0.2 ml生理盐水或是等体积生理盐水稀释的丙泊酚。双盲断头收集缺血核心区(Ic);半影区(P);C,对侧皮层(C)皮层组织,利用小鼠大脑中动脉阻塞模型,结合神经行为学评分表观察(n=10)不同剂量丙泊酚对小鼠神经缺陷的影响,利用蛋白印迹(Western blot)(n=6)检测小鼠脑皮层不同缺血区域磷酸化MSK1(P-MSK1)和总MSK(T-MSK1)的表达;利用免疫荧光染色、共聚焦显微镜等技术(n=4)明确小鼠MCAO 6 h缺血皮层P-MSK1阳性细胞类型及数目的改变。组间比较进行单因素方差、t检验。结果各实验组行为学评分差异有统计学意义(MCAO:7.90±0.35,MCAO+Propofol 25组:7.10±0.28,MCAO+Propofol 50组:6.40±0.27,MCAO+Propofol 100组:5.70±0.26,F=10.56,P<0.05)。各实验组皮层半影区P-MSK1差异有统计学意义(F=15.22,P<0.05)。与MCAO比较,MCAO+Propofol 25组差异无统计学意义(53.06±3.09比48.65±2.86,t=0.99,P>0.05);MCAO+Propofol 50组差异有统计学意义(5.40±0.27比7.60±0.27,t=-5.83,P<0.05);MCAO+Propofol 100组差异有统计学意义(78.13±3.28比48.65±2.86,t=8.053,P<0.05)。而MSK1总蛋白表达量在缺血核心区和半影区对侧皮层均无明显改变。缺血皮层P-MSK1阳性细胞和神经元核特异性标记物NeuN共定位,不同实验组小鼠皮层缺血半影区高倍镜视野下NeuN阳性和P-MSK1阳性共定位细胞数差异有统计学意义(MCAO:18.53±1.31,MCAO+Propofol 25组:21.57±1.66,MCAO+Propofol 50:36.72±3.13,MCAO+100 mg/kg组:48.3±3.86,F=26.39,P<0.05),MCAO+Propofol 25组与MCAO组比较(21.57±1.66比18.53±1.31,t=1.44,P>0.05),MCAO+Propofol 50组与MCAO组比较(36.72±3.13比18.53±1.31,t=5.36,P<0.05),MCAO+Propofol 100组与MCAO组比较(48.30±3.86比18.53±1.31,t=7.31,P<0.05)。结论丙泊酚可通过提高小鼠缺血皮层半影区内MSK1磷酸化水平,提高神经元存活数来减轻小鼠缺血性脑损伤。

Objective To explore the role of mitogen-and stress-activated protein kinase 1(MSK1)in propofol-induced neuroprotection against cerebral ischemic injuries.Methods 50 healthy male BALB/c mice were divided into 5 groups by random number table:sham-operation;sham operation group,middle cerebral artery occlusion(MCAO)+normal saline group,MCAO+25mg/kg propofol(low dose)group,MCAO+50 mg/kg propofol(medium dose)group,MCAO+100 mg/kg propofol(high dose)group,with 10 mice in each group.30 min after MCAO operation,propofol diluted with 0.2 ml normal saline or equal volume of normal saline was injected intraperitoneally into each mouse.After double blind decapitation the tissues from the ischemic core area(IC),penumbra(P)and contralateral cortex(c)were sampled.With the MCAO model in mice,neurobehavioral scale was used to assess the effects of different doses of propofol on neurological deficits in mice.Western blot was used to detect the expression of phosphorylated MSK1(p-msk1)and total MSK1(t-msk1)in different ischemic regions of cerebral cortex.Immunofluorescence staining and confocal microscopy were used to detect the type and the change in the number of p-msk1 positive cells in the ischemic cortex 6 hours after MCAO.Software Spss23 was used for one-way variance analysis and t test.Results There were significant differences in neurological scores among these groups(MCAO:7.9±0.35,MCAO+25:7.1±0.28,MCAO+50:6.4±0.27,MCAO+Propofol 100:5.7±0.26,F=10.56,P<0.05).MCAO+Propofol 25 groups vs.MCAO groups(7.1±0.28 vs.7.9±0.35,t=-1.80,P>0.05),MCAO+Propofol 50 groups vs.MCAO groups(6.4±0.27 vs.7.9±0.35 t=-3.42,P<0.05),MCAO+Propofol 100 groups vs.MCAO groups(5.7±0.26 vs.7.9±0.35 t=-5.06,P<0.05).There was significant difference in p-MSK1 in penumbra area of cortex among these groups(F=15.22,P<0.05).There was no significant difference between the MCAO+propofol 25 group and the MCAO group(53.06±3.09 vs.48.65±2.86,t=0.99,P>0.05);whereas there were statistically significant differences between the MCAO+propofol 50 group and the MCAO group MCAO+Propofol 50 groups vs.MCAO groups(66.29±3.87 vs.48.65±2.86,t=5.83,P<0.05);and between the MCAO+propofol 100 group and the MCAO group.MCAO+Propofol 100 groups vs.MCAO groups(78.13±3.28 vs.48.65±2.86,t=8.05,P<0.05).However,there was no significant change in the total expression of MSK1 in the contralateral cortex of ischemic core and penumbra.Co-localization assay of P-MSK1 positive cells in ischemic cortex and NeuN(a specific marker of neuronal nucleus)positive cells showed that the differences in the number of cells with NeuN and P-MSK1 colocalized in ischemic penumbra of mice among different groups were statistically significant.MCAO:18.53±1.31,MCAO+25:21.57±1.66,MCAO+50:36.72±3.13 MCAO+Propofol 100 groups:48.3±3.86,F=26.39,P<0.05 MCAO+25 mg/kg groups vs.MCAOgroups(21.57±1.66 vs.18.53±1.31,t=1.44,P>0.05),MCAO+Propofol 50 groups vs.MCAO groups(36.72±3.13 vs.18.53±1.31,t=5.36,P<0.05),MCAO+Propofol 100 groups vs.MCAO groups(48.3±3.86 vs.18.53±1.31,t=7.31,P<0.05).Conclusion Propofol can alleviate the ischemic brain injury in mice via elevating the phosphorylation level of MSK1 in the ischemic cortical penumbra and increasing the number of survived neurons.