p38 MAPK/CREB信号通路在川芎嗪减轻脓毒症相关性脑病小鼠海马炎症反应中的作用

Role of p38 MAPK/CREB signaling pathway in tetramethylpyrazine-induced reduction of hippocampal inflammatory responses in mice with sepsis-associated encephalopathy

目的评价p38丝裂原活化蛋白激酶/cAMP反应元件结合蛋白(p38 MAPK/CREB)信号通路在川芎嗪减轻脓毒症相关性脑病小鼠海马炎症反应中的作用。方法健康雄性C57BL6小鼠60只,体重24~27 g,采用随机数字表法分为4组(n=15):假手术组(Sham组)、脓毒症组(Sep组)、川芎嗪组(TMP组)和p38 MAPK抑制剂SB203580组(SB组)。采用盲肠结扎穿孔法制备小鼠脓毒症相关性脑病模型。于模型制备前3 d时TMP组腹腔注射川芎嗪10 mg/kg,1次/d,SB组于模型制备后30 min时腹腔注射SB2035802.0 mg/kg,Sham组和Sep组腹腔注射等容量生理盐水。于术后1 d时行Morris水迷宫实验测试认知功能,记录逃避潜伏期和靶象限活动时间比率。于水迷宫测试结束后处死小鼠取海马组织,采用ELISA法测定L-1β、TNF-α和IL-6含量;采用Western blot法测定p38 MAPK、GSK3和CREB磷酸化水平及BDNF的表达。结果与Sham组比较,Sep组、TMP组和SB组逃离潜伏期延长,靶象限活动时间比率降低,海马IL-1β、TNF-α和IL-6含量升高,p38 MAPK磷酸化水平升高,GSK3和CREB磷酸化水平降低,BDNF表达下调(P<0.05);与Sep组比较,TMP组和SB组逃离潜伏期缩短,靶象限活动时间比率升高,海马IL-1β、TNF-α和IL-6含量降低,海马p38 MAPK磷酸化水平降低,GSK3和CREB磷酸化水平升高,BDNF表达上调(P<0.05);与TMP组比较,SB组上述指标差异无统计学意义(P>0.05)。结论p38 MAPK/CREB信号通路参与了川芎嗪减轻脓毒症相关性脑病小鼠海马炎症反应的过程。

Objective To evaluate the role of p38 mitogen-activated protein kinase(MAPK)/cyclic adenosine monophosphate response element-binding protein(CREB)signaling pathway in tetramethylpyrazine-induced reduction of hippocampal inflammatory responses in mice with sepsis-associated encephalopathy(SAE).Methods Sixty healthy male C57BL6 mice,weighing 24-27 g,were divided into 4 groups(n=15 each)using a random number table method:sham operation group(group Sham),sepsis group(group Sep),tetramethylpyrazine group(group TMP)and p38 MAPK inhibitor SB203580 group(group SB).The model of SAE was established by cecal ligation and puncture in anesthetized mice.Tetramethylpyrazine 10 mg/kg was injected intraperitoneally once a day at 3 days before the establishment of the model in TMP group,and SB2035802.0 mg/kg was intraperitoneally injected at 30 min after the establishment of the model in SB group.The equal volume of normal saline was given intraperitoneally in Sham and Sep groups.At 1 day after operation,cognitive function was assessed by Morris water maze,and the escape latency and ratio of time spent in the target quadrant were recorded.The animals were sacrificed after the test,and hippocampal tissues were taken for determination of the contents of interleukin-1beta(IL-1β),tumor necrosis factor-alpha(TNF-α)and IL-6(by enzyme-linked immunosorbent assay)and for detection of the expression of phosphorylation of p38 MAPK,GSK3 and CREB and expression of brain-derived neurotrophic factor(BDNF)(by Western blot).Results Compared with group Sham,the escape latency was significantly prolonged,the ratios of time spent in the target quadrant were decreased,the contents of IL-1β,TNF-αand IL-6 were increased,the phosphorylation of hippocampus p38 MAPK was increased,the phosphorylation of GSK3 and CREB were decreased,and the expression of BDNF was down-regulated in Sep,TMP and SB groups(P<0.05).Compared with group Sep,the escape latency was significantly shortened,the ratios of time spent in the target quadrant were increased,the contents of IL-1β,TNF-αand IL-6 were decreased,the phosphorylation of hippocampus p38 MAPK was decreased,the phosphorylation of GSK3 and CREB were increased,and the expression of BDNF was up-regulated in TMP and SB groups(P<0.05).Compared with group TMP,no significant change was found in the parameters mentioned above in group SB(P>0.05).Conclusion p38 MAPK/CREB signaling pathway is involved in the process of tetramethylpyrazine-induced reduction of hippocampal inflammatory responses in mice with SAE.