基于JNK/AP-1信号通路探讨广西毛冬青在放射性脑损伤中的作用机制

Mechanism of Guangxi Ilex pubescens Hook.et Arn.in radiation brain injury based on JNK/AP-1 signaling pathway

目的:探讨广西毛冬青(IPH)对放射性脑损伤的改善作用及其机制。方法:将40只SPF级昆明小鼠随机分为对照组、IPH、放射组和放射+IPH组,每组10只。采用γ射线建立放射性脑损伤模型,放射前、后连续灌胃给药14 d。采用Morris水迷宫实验、避暗实验检测小鼠认知功能,苏木精—伊红(HE)染色和尼氏染色观察脑组织病理形态变化,电镜观察胶质细胞超微结构,酶联免疫吸附试验(ELISA)法检测血清白介素-6(IL-6)水平,免疫组织化学染色法检测脑组织c-Jun氨基末端激酶(JNK)、磷酸化(p-)JNK、激活蛋白-1(AP-1)和单核细胞趋化蛋白-1(MCP-1)蛋白表达,免疫荧光检测脑组织星型胶质细胞标记物GFAP和小胶质细胞标记物Iba-1蛋白表达,western blotting法检测脑组织JNK和p-JNK蛋白表达。结果:与对照组相比,放射组小鼠放射后7 d体重减低,血清IL-6含量升高,脑组织AP-1、MCP-1表达水平及p-JNK/JNK比值升高,小胶质细胞Iba-1和GFAP表达水平升高(均P<0.05)。与放射组比较,放射+IPH组小鼠放射后7 d体重增加,血清IL-6含量降低,脑组织AP-1、MCP-1表达水平及p-JNK/JNK比值降低,小胶质细胞Iba-1和GFAP表达水平降低(均P<0.05)。HE染色和尼氏染色显示,放射组大量细胞和神经元出现核固缩现象,小胶质细胞激活,呈圆形;电镜下可见细胞内溶酶体增多,星形胶质细胞细胞核及胞质肿胀;经IPH干预后,小鼠脑组织神经元变性情况和胶质细胞形态明显改善。结论:广西IPH能改善放射性脑损伤小鼠的认知功能,减少小胶质细胞和星形胶质细胞激活,其机制可能与减轻神经炎症反应、抑制JNK/AP-1信号通路有关。

Objective:To explore the protective effect and mechanism of Ilex pubescens Hook.et Arn.(IPH)in radiation brain injury.Methods:Forty SPF Kunming mice were randomly divided into control group,IPH group,radiation group,and radiation+IPH group,with 10 mice in each group.The model of radiation brain injury was established byγ-rays,and the mice were given intragastric administration for 14 d before and after radiation.Morris water maze test and dark avoidance test were used to detect the cognitive function of mice.Hematoxylin-eosin(HE)staining and Nissl staining were used to observe the morphological changes of brain tissue,the ultra-electron microscope was used to observe the changes of glial cells,enzyme-linked immunosorbent assay(ELISA)was used to detect the serum level of interleukin-6(IL-6),immunohistochemical method was used to detect the protein expression of c-Jun amino-terminal kinase(JNK),phosphorylated JNK(p-JNK),activator protein-1(AP-1),monocyte chemoattractant protein-1(MCP-1)in brain tissue,immunofluorescence was used to detect the protein expression of astrocyte marker GFAP and microglia marker Iba-1 in brain tissue,and western blotting was used to detect the protein expression of JNK and p-JNK in brain tissue.Results:Compared with the control group,the weight of the mice in the radiation group was reduced,and the serum IL-6 content,the expression levels of AP-1,MCP-1 and p-JNK/JNK ratio,as well as the expression levels of Iba-1 and GFAP in the microglia were increased 7 d after radiation(all P<0.05).Compared with the radiation group,the weight of the mice in the radiation+IPH group was increased,and the serum IL-6 content,the expression levels of AP-1 and MCP-1 in brain tissue and p-JNK/JNK ratio,as well as the expression levels of Iba-1 and GFAP in microglia were decreased 7 d after radiation(all P<0.05).HE staining and Nissl staining showed that a large number of cells and neurons in the radiation group appeared karyopyknosis,and microglia were activated and round;under the electron microscope,increased lysosomes were observed,with swelling of the nucleus and cytoplasm of astrocytes;after IPH intervention,the degeneration of neurons and the morphology of glial cells in the brain tissue of mice were significantly improved.Conclusion:Guangxi IPH can improve the cognitive function of mice with radiation brain injury and reduce the activation of microglia and astrocytes.The mechanism may be related to reducing neuroinflammatory response and inhibiting JNK/AP-1 signaling pathway.