基于MAPK信号通路研究脑震宁对三重脑震荡模型大鼠海马神经元损伤的影响

Effect of Naozhenning on hippocampal neuron injury in multiple cerebral concussion rats model based on MAPK signaling pathway

目的 研究脑震宁对三重脑震荡模型(multiple cerebral concussion,MCC)大鼠海马神经元损伤的影响及其机制。方法 采用自由落体法制备MCC大鼠模型,将造模成功的大鼠随机分为吡拉西坦(0.16 g/kg)组和脑震宁低、中、高剂量(6.68、13.36、26.73 g/kg)组,各给药组ig相应药物,2次/d,连续给药14 d。造模前后进行水迷宫平台实验,观察大鼠学习记忆能力变化;采用ELISA法检测大鼠血清中白细胞介素-1β(interleukin-1β,IL-1β)、IL-6水平,WST-1法检测血清超氧化物歧化酶(superoxide dismutase,SOD)活性,硫代巴比妥酸法检测血清丙二醛(malondialdehyde,MDA)水平;qRTPCR法检测大鼠海马组织p38、细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)、c-Jun氨基末端激酶(cJun N-terminal kinase,JNK)m RNA表达;Western blotting法检测大鼠海马组织p38、p-p38、ERK、p-ERK、JNK和p-JNK蛋白表达;采用苏木素-伊红(HE)、尼氏体染色以及透射电镜(TEM)法分别观察大鼠海马神经元病理、尼氏体及超微结构的变化。结果 与对照组相比,模型组大鼠逃避潜伏时间增加(P<0.01),固定象限停留时间百分比减少(P<0.01);血清中IL-1β、IL-6和MDA水平升高(P<0.05、0.01),SOD活性下降(P<0.01);海马组织p38、JNK和ERK mRNA表达水平均升高(P<0.01);海马组织p38、p-p38、JNK、p-JNK、ERK和p-ERK蛋白表达水平均升高(P<0.05、0.01)。与模型组相比,脑震宁组大鼠逃避潜伏时间减少(P<0.05、0.01),固定象限停留时间百分比增加(P<0.05、0.01);血清中IL-1β、IL-6、MDA水平降低(P<0.05、0.01),SOD活性升高(P<0.01、0.001);海马组织p38、JNK和ERK m RNA表达水平降低(P<0.05、0.01);脑震宁各剂量组海马组织p38、p-p38和p-ERK蛋白表达水平降低(P<0.01),脑震宁低、中剂量组JNK和ERK蛋白表达水平降低(P<0.05),脑震宁中、高剂量组p-JNK蛋白表达水平升高(P<0.01)。HE、尼氏染色结果显示,模型组神经元排列稀疏,数量减少,部分细胞核消失或核仁破裂,胞质内混浊,尼氏体颜色变浅;脑震宁组大部分神经元细胞形态较完整,核大而圆,着色浅,部分细胞出现核仁消失,尼氏体颜色增加。TEM结果显示,模型组神经元细胞体积缩小,核仁破裂分散,微丝排列紊乱,板层崩解、断裂甚至消失;脑震宁组神经元细胞核大,核膜完整,核仁偏移,线粒体内嵴结构清晰,微丝排列较模型组整齐,部分崩解。结论 脑震宁可能通过影响丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的激活改善MCC模型大鼠海马神经元损伤。

Objective To study the effect and mechanism of Naozhenning(脑震宁)on hippocampal neuron injury in multiple cerebral concussion(MCC)model rats.Methods Free fall method was processed to establish MCC models,MCC rats were randomly divided into piracetam(0.16 g/kg)group and low-,medium-,high-dose Naozhenning(6.68,13.36,26.73 g/kg)groups,each administration group was ig given corresponding drugs,twice a day for 14 d.Water maze platform experiment was carried out before and after modeling to observe the changes of learning and memory ability of rats;Levels of interleukin-1β(IL-1β)and IL-6 in serum of rats were detected by ELISA;Superoxide dismutase(SOD)content was detected by WST-1 method;Malondialdehyde(MDA)content was detected by thiobarbituric acid method;mRNA expressions of p38,extracellular regulated protein kinases(ERK)and c-Jun N-terminal kinase(JNK)in hippocampus were detected by qRT-PCR;p38,p-p38,ERK,p-ERK,JNK and p-JNK protein expressions in hippocampus were detected by Western blotting;HE,Nissl staining and transmission electron microscopy were used to observe the changes of pathology,Nissl body and ultrastructure of hippocampal neurons.Results Compared with control group,escape latency in model group was increased(P<0.01),percentage of residence time in fixed quadrant was decreased(P<0.01);IL-1β,IL-6 and MDA levels in serum were increased(P<0.05,0.01),SOD activity was decreased(P<0.01);mRNA expressions of p38,JNK and ERK in hippocampus were increased(P<0.01);p38,p-p38,JNK,p-JNK,ERK and p-ERK protein expressions in hippocampus were increased(P<0.05,0.01).Compared with model group,escape latency of rats in Naozhenning groups was decreased(P<0.05,0.01),percentage of fixed quadrant residence time was increased(P<0.05,0.01);IL-1β,IL-6 and MDA levels were decreased(P<0.05,0.01),SOD activity was increased(P<0.01,0.001);mRNA expressions of p38,JNK and ERK in hippocampus were decreased(P<0.05,0.01);p38,p-p38 and p-ERK protein expressions of hippocampus in Naozhenning groups were decreased(P<0.01),JNK and ERK protein expressions were decreased in Naozhenning low-and medium-dose groups(P<0.05),p-JNK protein expression was increased in Naozhenning medium-and high-dose groups(P<0.01).The results of HE and Nissl staining showed that neurons in model group were sparse,numbers of neurons were decreased,some nuclei disappeared or nucleoli ruptured,cytoplasm was turbid,and Nissl body color became lighter;Most neurons in Naozhenning groups had complete morphology,large and round nuclei,light staining,disappearance of nucleoli in some cells and increase of Nissl body color.The results of electron microscope showed that volume of neurons in model group was decreased,nucleoli broke and dispersed,microtubules and microfilaments disintegrated,broke or even disappeared;In Naozhenning groups,nucleus were large,nuclear membrane were complete,nucleolus were offset,cristae structure in mitochondria were clear,microtubules and microfilaments were arranged orderly and partially depolymerized.Conclusion Naozhenning may improve the injury of hippocampal neurons in MCC rats model by inhibiting the activation of mitogen-activated protein kinase(MAPK)signaling pathway.