南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (3): 411-419.doi: 10.12122/j.issn.1673-4254.2024.03.01

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束缚应激通过激活Rho/ROCK通路诱导大鼠杏仁核血脑屏障的损伤

许光明，高安迪，丛 斌   

  1. 中央司法警官学院法医学教研室，河北 保定 071000；河北医科大学法医学院//河北省法医学重点实验室，河北 石家庄 050017
  • 出版日期:2024-03-20 发布日期:2024-04-03

Restraint stress induces blood-brain barrier injury in rat amygdala by activating the Rho/ROCK signaling pathway

XU Guangming, GAO Andi, CONG Bin   

  1. Department of Forensic Medicine, National Police University for Criminal Justice, Baoding 071000, China; College of Forensic Medicine, Hebei Medical University, Hebei Key Laboratory of Forensic Medicine, Shijiazhuang 050017, China
  • Online:2024-03-20 Published:2024-04-03

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摘要： 目的 探讨Rho/ROCK信号通路在束缚应激诱导大鼠杏仁核血脑屏障损伤过程中的作用及机制。方法 选取60只SPF级雄性 SD 大鼠建立束缚应激模型，将大鼠分为 4 组：Control 组（n=15，每日禁食水 6 h）；Stress 组（n=15，每日束缚 6 h）；Stress+Fasudil组（n=15，每日束缚6 h，束缚前0.5 h给予腹腔注射1 mg/100 g Fasudil溶液）；Fasudil组（n=15，每日给予腹腔注射1 mg/100 g Fasudil溶液）。用高架十字迷宫实验（EPM）检测各组大鼠行为学变化，ELISA检测血清CORT和S100B水平，检测脑组织伊文思蓝（EB）的渗漏情况以评估渗透性的改变，免疫荧光法和Western blotting方法检测紧密连接蛋白Claudin-5、Occludin、ZO-1的表达变化，Pull-down实验和Western blotting检测Rho/ROCK通路的激活情况，透射电镜观察脑微血管内皮细胞超微结构的形态变化。结果 与Control组相比，Stress组和Stress+Fasudil组大鼠表现出了明显的焦虑样行为；Stress组和Stress+Fasudil组血清CORT含量均显著高于Control组（P<0.001）；与Control组和Stress+Fasudil组相比，Stress组EB渗漏含量和S100B含量均显著升高（P<0.05）；Stress组紧密连接蛋白的表达显著低于Control组和Stress+Fasudil组（P<0.05）；Pull-down实验和Western blot分析证实，Stress组RhoA-GTP（P<0.001）、ROCK2（P<0.001）、p-MLC2（P<0.05）的表达显著高于Control组和Stress+Fasudil组；脑微血管内皮细胞超微结构显示，Stress组呈现显著的形态学改变。结论 束缚应激能够通过激活Rho/ROCK信号通路诱导大鼠杏仁核血脑屏障的损伤。

关键词: 束缚应激；血脑屏障；杏仁核；Rho/ROCK通路；紧密连接；渗透性

Abstract: Objective To investigate the role of Rho/ROCK signaling pathway in mediating restraint stress-induced blood-brain barrier (BBB) injury in the amygdala of rats. Methods Sixty male SD rats were randomized equally into control group (with food and water deprivation for 6 h per day), restraint stress group (with restraint for 6 h per day), stress + fasudil treatment (administered by intraperitoneal injection at 1 mg/100 g 30 min before the 6-h restraint) group, and fasudil treatment alone group. The elevated plus-maze test was used to detect behavioral changes of the rats, serum corticosterone and S100B levels were determined with ELISA, and Evans Blue leakage in the brain tissue was examined to evaluate the changes in BBB permeability. The changes in expression levels of tight junction proteins in the amygdala were detected using immunofluorescence assay and Western blotting, and Rho/ROCK pathway activation was detected by Pull-down test and Western blotting. Ultrastructural changes of the cerebral microvascular endothelial cells were observed using transmission electron microscopy. Results Compared with those in the control group, the rats in restrain stress group and stress + fasudil group showed obvious anxiety-like behavior with significantly increased serum corticosterone level (P<0.001). Compared with those in the control group and stress+fasudil group, the rat models of restrain stress showed more obvious Evans Blue leakage and higher S100B expression (P<0.01) but lower expressions of tight junction proteins in the amygdala. Pull- down test and Western blotting confirmed that the expression levels of RhoA-GTP, ROCK2 and P-MLC 2 were significantly higher in stress group than in the control group and stress + fasudil group (P<0.05). Transmission electron microscopy revealed obvious ultrastructural changes in the cerebral microvascular endothelial cells in the rat models of restrain stress. Conclusion Restraint stress induces BBB injury in the amygdala of rats by activating the Rho/ROCK signaling pathway.

Key words: restraint stress; blood-brain barrier; amygdala; Rho/ROCK signaling pathway; tight junction; permeability