盐酸戊乙奎醚对脑出血大鼠血脑屏障及脑组织ROCK2、CLDN5和AQP-4表达的影响

doi:10.3969/j.issn.1673-5765.2024.10.011

摘要/Abstract

摘要： 目的探究盐酸戊乙奎醚（penehyclidine hydrochloride，PHC）对脑出血（intracerebral hemorrhage，ICH）大鼠血脑屏障（blood-brain barrier，BBB）及脑组织Rho相关卷曲螺旋蛋白激酶2（Rho associated coiled-coil containing protein kinase 2，ROCK2）、闭合蛋白5（claudin 5，CLDN5）和水通道蛋白4（aquaporin-4，AQP-4）表达的影响。
方法将100只SD大鼠按20只/组分为假手术组、ICH组、PHC-L组（1 mg/kg PHC）、PHC-M组（2 mg/kg PHC）和PHC-H组（4 mg/kg PHC）；采用自体血（50 μL）注入法构建ICH大鼠模型（假手术组不注入自体血）；造模成功后PHC-L组、PHC-M组和PHC-H组大鼠分别腹腔注射相应剂量PHC，假手术组和ICH组注射等量的0.9%氯化钠溶液，连续7 d，1次/日；通过Longa评分法对大鼠神经功能损伤进行评分，使用透射电镜观察BBB超微结构，通过伊文思蓝（Evans blue，EB）法评估大鼠BBB通透性，检测大鼠脑组织含水量，并利用免疫组织化学染色及免疫印迹试验检测脑组织ROCK2、CLDN5、AQP-4表达情况。
结果与假手术组相比，ICH组大鼠神经功能损伤评分[0分 vs.（2.45±0.48）分]、脑组织EB含量[（9.75±1.01）μg/g vs.（32.07±3.22）μg/g]、脑组织含水量[（77.21±0.33）% vs.（80.96±0.45）%]、ROCK2累积光密度（integrated optical density，IOD）[（1.02±0.14）×103 vs.（11.05±0.71）×103]和相对表达量（0.39±0.03 vs. 1.43±0.24）、AQP-4 IOD[（1.67±0.18）×103 vs.（10.85±0.58）×103]和相对表达量（0.60±0.07 vs. 1.69±0.21）均显著增加，CLDN5 IOD[（10.83±0.64）×103 vs.（3.02±0.33）×103]和相对表达量（1.53±0.20 vs. 0.42±0.06）显著降低；与ICH组相比，PHC-L组、PHC-M组、PHC-H组不同程度地逆转了上述各指标的变化趋势。
结论 PHC可改善ICH大鼠BBB结构损伤，可能与上调CLDN5水平、抑制ROCK2和AQP-4表达有关。

文章导读： 本研究探索了盐酸戊乙奎醚对脑出血大鼠的潜在治疗效果和可能机制，为寻找治疗脑出血的新型药物提供了参考。

关键词: 盐酸戊乙奎醚; 脑出血; 血脑屏障; 水通道蛋白4

Abstract: Objective To explore the effects of penehyclidine hydrochloride (PHC) on the blood-brain barrier (BBB) and the expression of Rho associated coiled-coil containing protein kinase 2 (ROCK2), claudin 5 (CLDN5), and aquaporin-4 (AQP-4) in rats with intracerebral hemorrhage (ICH).
Methods A total of 100 SD rats were divided into 20 rats/group as the sham group, the ICH group, the PHC-L group (1 mg/kg PHC), the PHC-M group (2 mg/kg PHC), and the PHC-H group (4 mg/kg PHC). The rat model of ICH was established by autologous blood (50 μL) injection method (except for the sham group). After successful modeling, the rats in the PHC-L, PHC-M, and PHC-H groups were intraperitoneally injected with the corresponding doses of PHC. The sham group and the ICH group were injected with an equal amount of 0.9% sodium chloride solution for 7 consecutive days, once a day. The Longa scoring method was used to score the neurological impairment in rats. A transmission electron microscope was used to observe the ultrastructure of BBB. The Evans blue (EB) method was used to evaluate the permeability of BBB and the water content in rat brain tissue was detected. Immunohistochemical staining and western blot were used to detect the expressions of ROCK2, CLDN5, and AQP-4 in brain tissue.
Results Compared with the sham group, the neurological impairment score [0 score vs. (2.45±0.48) score], EB content [(9.75±1.01) μg/g vs. (32.07±3.22) μg/g], brain tissue water content [(77.21±0.33) % vs. (80.96±0.45) %], ROCK2 integrated optical density (IOD) [(1.02±0.14)×103 vs.(11.05±0.71)×103] and relative expression (0.39±0.03 vs. 1.43±0.24), AQP-4 IOD [(1.67±0.18)×103 vs. (10.85±0.58)×103] and relative expression (0.60±0.07 vs. 1.69±0.21) increased significantly in the ICH group, while the CLDN5 IOD [(10.83±0.64)×103 vs. (3.02±0.33)×103] and relative expression (1.53±0.20 vs. 0.42±0.06) decreased significantly. Compared with the ICH group, the PHC-L, PHC-M, and PHC-H groups reversed the trend of changes in the aforementioned indicators to varying degrees according to different doses of PHC.
Conclusions PHC can ameliorate structural damage to the BBB in ICH rats, potentially through up-regulating the level of CLDN5 and inhibiting the expression of ROCK2 and AQP-4.

Key words: Penehyclidine hydrochloride; Intracerebral hemorrhage; Blood-brain barrier; Aquaporin-4

中图分类号:

李达宇, 郭少雷, 张波, 黄志鹏, 叶巍巍, 姚亮. 盐酸戊乙奎醚对脑出血大鼠血脑屏障及脑组织ROCK2、CLDN5和AQP-4表达的影响[J]. 中国卒中杂志, 2024, 19(10): 1180-1187.

LI Dayu, GUO Shaolei, ZHANG Bo, HUANG Zhipeng, YE Weiwei, YAO Liang. Effects of Penehyclidine Hydrochloride on the Blood-Brain Barrier and ROCK2, CLDN5, and AQP-4 Expression of Brain Tissue in Rats with Intracerebral Hemorrhage[J]. Chinese Journal of Stroke, 2024, 19(10): 1180-1187.

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