Zhou Rui-Xi, Li Xi-Hong, Qu Yi, Li Shi-Ping, Huang Qu
Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China.
Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2019 Jul;50(4):512-519.
To investigate the role of p38 mitogen-activated protein kinase (MAPK) signaling pathway in autophagy of neurons in hippocampus of sepsis rats.
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.
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 ( < 0.05); immunofluorescence observation showed that localization and expression of LC3 and p62/SQSTM1 in NeuN were consistent with Western blot.
Inhibition of p38 MAPK signaling pathway in sepsis rats can further activate autophagy and protect neurons in the hippocampus.
探讨p38丝裂原活化蛋白激酶（MAPK）信号通路在脓毒症大鼠海马神经元自噬中的作用。
采用盲肠结扎穿孔术（CLP）建立脓毒症模型。将SD大鼠随机分为假手术组（假手术组）、模型组（CLP组）、溶剂处理组（CLP+Veh组）和抑制剂处理组（CLP+SB203580组），每组再分为3、6、12、24和48 h亚组。CLP+Veh组和CLP+SB203580组分别于侧脑室内注射1%二甲基亚砜5 μL和0.1 mmol/L SB203580 5 μL，注射后30 min建立CLP模型。假手术组仅翻转盲肠并关闭腹腔，无其他处理。监测大鼠生命体征，包括平均动脉压（MAP）和心率（HR）。采用神经行为评分法观察大鼠脑损伤情况。通过苏木精-伊红（HE）染色观察大鼠海马组织病理学变化。在透射电子显微镜（TEM）下观察大鼠海马神经元自噬过程。采用蛋白质免疫印迹法检测海马组织中微管相关蛋白1轻链3（LC3）Ⅱ、LC3Ⅰ、选择性自噬衔接蛋白p62/聚集体蛋白1（p62/SQSTM1）、MAPK激活的蛋白激酶2（MK-2）和磷酸化MK-2（p-MK-2）的表达。通过免疫荧光观察大鼠海马神经元中LC3和p62/SQSTM1的表达。
在不同时间点，CLP组MAP低于假手术组，HR高于假手术组，造模后12 h变化最明显；CLP组神经行为评分最低；海马组织病理学变化明显；透射电子显微镜下观察到许多自噬空泡；与CLP组相比，CLP+SB203580组神经行为评分升高；海马组织病理学变化改善；透射电子显微镜下自噬空泡内包涵体降解；蛋白质免疫印迹结果显示：与假手术组相比，大鼠CLP组海马组织中LC3Ⅱ/LC3Ⅰ、p-MK-2/MK-2表达升高，p62/SQSTM1表达降低，前者于12 h达到峰值，后者于12 h降至最低。与其他组相比，造模12 h时，大鼠CLP+SB203580组海马组织中LC3Ⅱ/LC3Ⅰ、p-MK-2/MK-2表达进一步升高，p62/SQSTM1表达进一步降低（P<0.05）；免疫荧光观察显示，NeuN中LC3和p62/SQSTM1的定位和表达与蛋白质免疫印迹结果一致。
抑制脓毒症大鼠p38 MAPK信号通路可进一步激活自噬，保护海马神经元。
