p38MAPK信号通路在脓毒症大鼠海马区神经元自噬中的作用

  目的  探讨p38丝裂原激活的蛋白激酶(mitogen-activated protein kinase, MAPK)信号通路在脓毒症大鼠海马区神经元自噬中的作用。  方法  通过盲肠结扎穿孔术(cecal ligation and puncture, CLP)建立大鼠脓毒症模型。SD大鼠随机分为假手术组(sham组)、模型组(CLP组)、溶媒组(CLP+Veh组)、p38MAPK抑制剂组(CLP+SB203580组)，每组分为3、6、12、24和48 h亚组；CLP+Veh组和CLP+SB203580组分别侧脑室注射1% DMSO 5 μL和0.1 mmol/L SB203580 5 μL，注射后30 min建立CLP模型，sham组仅翻看盲肠后关腹，未做其他处理。监测大鼠生命体征，包括平均动脉压(mean arterial pressure, MAP)和心率(heart rate, HR)；神经行为学评分了解大鼠的脑损伤情况；大鼠脑海马区组织HE染色观察病理改变；透射电镜下观察大鼠脑海马区神经元自噬过程；Western blot检测海马区微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3, LC3)Ⅱ、LC3Ⅰ选择性自噬接头蛋白(p62/sequestosome-1, p62/SQSTM1)、MAPK激活蛋白激酶2(MAPK-activated protein kinase 2, MK-2)、磷酸化MK-2(phosphorylation MK-2, p-MK-2)的蛋白表达；免疫荧光染色观察大鼠海马区神经元LC3和p62/SQSTM1的表达。  结果  在不同时间点，CLP组大鼠MAP低于、HR高于sham组，以造模后12 h变化最明显；CLP组大鼠神经行为学评分最低，海马区组织病理改变明显，透射电镜下观察有较多自噬空泡形成。与CLP组比较，CLP+SB203580组大鼠神经行为学评分回升，海马区病理改变好转，透射电镜下自噬空泡中的包裹物降解；Western blot结果显示，与sham组比较，造模后CLP组大鼠海马组织LC3Ⅱ/ LC3Ⅰ和p-MK-2/MK-2表达升高，p62/SQSTM1表达下降，前者至12 h达到高峰，后者至12 h达到谷底，CLP+SB203580组与其它组相比，在造模12 h时，大鼠海马组织LC3Ⅱ/LC3Ⅰ和p-MK-2/MK-2表达升高，而p62/SQSTM1表达进一步下降(P＜0.05)；免疫荧光法观察结果显示，海马区LC3和p62/SQSTM1在NeuN的定位及表达与Western blot所测得的结果一致。  结论  脓毒症大鼠抑制p38 MAPK信号通路，可以使自噬进一步激活，对海马区神经元起到保护作用。

Role of p38 Mitogen-activated Protein Kinase Signaling Pathway in the Hippocampal Neurons Autophagy of Rats with Sepsis

  Objective  To investigate the role of p38 mitogen-activated protein kinase (MAPK) signaling pathway in autophagy of neurons in hippocampus of sepsis rats.  Methods  A sepsis model was established by cecal ligation and puncture (CLP). SD rats were randomly divided into sham-operated group (sham group), model group (CLP group), vehicle-treated group (CLP+Veh group) and inhibitor-treated group (CLP+SB203580 group), and each group was divided into 3, 6, 12, 24 and 48 h subgroups. CLP+Veh group and CLP+SB203580 group were injected with 1% DMSO 5 μL and 0.1 mmol/L SB203580 5 μL respectively in the lateral ventricle, and CLP was established 30 min after injection. The sham group only turned over the cecum and closed the abdomen without other treatments. The vital signs of rats were monitored, including mean arterial pressure (MAP) and heart rate (HR). Neurobehavioral score was used to investigate the brain injury in rats. Histopathological changes in hippocampus of rats were observed by HE staining. The process of neuronal autophagy in hippocampal of rats was observed under transmission electron microscope (TEM). Western blot assay was performed to detect the expression of microtubule associated protein 1 light chain 3 (LC3)Ⅱ, LC3Ⅰ, selective autophagy adaptor protein p62/sequestosome-1 (p62/SQSTM1), MAPK-activated protein kinase 2 (MK-2) and phosphorylation MK-2 (p-MK-2) in the hippocampus. The expressions of LC3 and p62/SQSTM1 in hippocampal neurons of rats were observed by immunofluorescence.  Results  At different time points, MAP of CLP group was lower than sham group, while HR was higher than sham group, the change was most obvious at 12 h after molding; the neurobehavioral score of CLP group was the lowest; the histopathological changes in the hippocampus were obvious; and many autophagy vacuoles were observed under transmission electron microscope; compared with CLP group, the neurobehavioral score of CLP+SB203580 group increased; the pathological changes in the hippocampus improved; the inclusions in autophagy vacuoles were degraded under transmission electron microscopy; Western blot results showed:compared with sham group, expression of-LC3Ⅱ/LC3Ⅰ, p-MK-2/MK-2 increased, and p62/SQSTM1 decreased in hippocampal tissue of CLP group in rat, the former reaches its peak at 12 h, the latter bottomed out at 12 h. Compared with the other groups, at 12 h of modeling, the expression of LC3Ⅱ/LC3Ⅰ, p-MK-2/MK-2 was further increased, the expression of p62/SQSTM1 decreased further in hippocampal tissue of CLP+SB203580 group in rat (P < 0.05); immunofluorescence observation showed that localization and expression of LC3 and p62/SQSTM1 in NeuN were consistent with Western blot.  Conclusion  Inhibition of p38 MAPK signaling pathway in sepsis rats can further activate autophagy and protect neurons in the hippocampus.