Objectives: To investigate the protective effect and mechanism of total flavonoids from Citrus paradise cv. Changshan-huyou extracts (TFC) on oxygen-glucose deprivation (OGD) of primary neurons and chronic ischemia-induced cerebral injury in mice.
Methods: Primary hippocampal neurons of 18-day fetal rats were isolated and cultured for 1 week, then treated with 0.25, 0.50 and 1.00 mg/mL TFC. After oxygen-glucose deprivation for 1 h, cells were reperfused for 6 and 24 h, respectively. The cytoskeleton was observed by phalloidin staining. In animal study, 6-week ICR male mice were randomly divided into sham operation group, model group, low-dose (10 mg/kg), medium-dose (25 mg/kg) and high-dose (50 mg/kg) TFC treatment groups, with 20 mice in each group. After 3 weeks, chronic cerebral ischemia was induced by unilateral common carotid artery ligation in all groups except sham operation group. Mice were treated with different concentrations of TFC in the three TFC treatment groups for 4 weeks. Open field test, novel object recognition test and Morris water maze test were used to evaluate anxiety, learning and memory of these mice. Nissl, HE and Golgi stainings were used to detect neuronal degeneration and dendritic spine changes in the cortex and the hippocampus. The expression levels of Rho-associated kinase (ROCK) 2, LIM kinase (LIMK) 1, cofilin and its phosphorylation, as well as the expression of globular actin (G-actin) and filamentous actin (F-actin) protein in hippocampus of mice were detected by Western blotting.
Results: Neurons subjected to OGD showed that neurites displayed shortening and breakage; while treatment with TFC reversed OGD-induced neurite injury, especially in the 0.50 mg/mL TFC group. Compared with the sham operation group, the mice in the model group showed a significant decline in anxiety and cognitive ability (P<0.01), whereas treatment with TFC significantly reversed anxiety and cognitive deficits (P<0.05). Improvement in the medium-dose TFC group was the most obvious. Histopathological analysis indicated that the number of Nissl bodies and dendritic spines in hippocampus and cortex were decreased in the model group (all P<0.01). However, after treatment with medium dose of TFC, the number of Nissl bodies and dendritic spines (all P<0.05) was significantly recovered. Compared with the sham operation group, the phosphorylation level of ROCK2 in the brain tissue of the model group was significantly increased (P<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly decreased (P<0.05), and the relative content ratio of G-actin/F-actin was significantly increased (P<0.05). After administration of TFC, the phosphorylation level of ROCK2 in brain tissue of each group was significantly decreased (P<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly up-regulated (P<0.05) and the relative content ratio of G-actin/F-actin was significantly decreased (P<0.05).
Conclusions: TFC protects from ischemia-induced cytoskeletal damage, reduces neuronal dendritic spine injury and protects mice against chronic cerebral ischemia through RhoA-ROCK2 signaling pathway, indicating that TFC might be a potential candidate for treatment of chronic ischemic cerebral injury.
目的: 研究胡柚黄酮对原代神经元氧糖剥夺和小鼠慢性脑缺血的保护作用及其机制。方法: 分离胎龄18 d胎鼠的原代海马神经元，培养1周后分组分别加入0.25、0.50和1.00 mg/mL胡柚黄酮，经氧糖剥夺处理1 h后，各组分别复灌6、24 h。运用鬼笔环肽着色镜下观察细胞骨架。6周龄ICR雄性小鼠随机分为手术对照组、模型对照组和胡柚黄酮小剂量（10 mg/kg）、中剂量（25 mg/kg）、大剂量（50 mg/kg）组，每组20只。给药3周后，除手术对照组外，其余各组单侧颈总动脉结扎诱导小鼠慢性脑缺血，其中胡柚黄酮各组术后再以胡柚黄酮干预4周。采用旷场实验、新事物认知实验、莫里斯水迷宫实验评估小鼠的焦虑情绪和学习记忆能力；采用尼氏染色、苏木精-伊红染色、高尔基染色检测小鼠皮层、海马的神经元变性及树突棘变化；采用蛋白质印迹法检测小鼠海马Rho相关激酶（ROCK）2、LIM激酶（LIMK）1、丝切蛋白及其磷酸化蛋白的表达水平和球状肌动蛋白（G-actin）、纤维状肌动蛋白（F-actin）的表达水平。结果: 原代海马神经元氧糖剥夺后，细胞骨架出现断裂、缩短等结构异常，胡柚黄酮处理后可逆转上述改变，尤以0.50 mg/mL胡柚黄酮为佳。与手术对照组比较，模型对照组表现出焦虑和认知能力显著下降（均P<0.01），而胡柚黄酮治疗可显著改善焦虑和认知功能障碍（均P<0.05），其中胡柚黄酮中剂量组改善最为明显。组织病理学检查结果显示，模型对照组海马和皮层尼氏小体和树突棘数减少（均P<0.01）；经中剂量胡柚黄酮处理后，尼氏小体和树突棘数明显恢复（均P<0.05）。与手术对照组比较，模型对照组脑组织ROCK2蛋白磷酸化水平显著升高（P<0.05），而LIMK1蛋白和丝切蛋白磷酸化水平明显降低（P<0.05），G-actin/F-actin相对含量比值明显上升（P<0.05）；胡柚黄酮给药后，各组脑组织ROCK2蛋白磷酸化水平显著降低（P<0.05），LIMK1蛋白和丝切蛋白磷酸化水平显著上调（P<0.05），G-actin、F-actin相对含量比值显著下降（P<0.05）。结论: 胡柚黄酮通过RhoA-ROCK2信号通路保护缺血诱导的细胞骨架损伤，减少神经元树突棘损伤，以抵抗慢性脑缺血损伤。.
Keywords: Chronic cerebral ischemia; Citrus paradise cv. Changshan- huyou; Flavonoids; Mice; Oxygen-glucose deprivation; RhoA-ROCK2 signaling pathway.