School of Medicine, Zhejiang University City College, Hangzhou 310015, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2023 Feb 25;52(1):77-87. doi: 10.3724/zdxbyxb-2022-0567.
To investigate the protective effect and mechanism of total flavonoids from extracts (TFC) on oxygen-glucose deprivation (OGD) of primary neurons and chronic ischemia-induced cerebral injury in mice.
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.
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 (<0.01), whereas treatment with TFC significantly reversed anxiety and cognitive deficits (<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 <0.01). However, after treatment with medium dose of TFC, the number of Nissl bodies and dendritic spines (all <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 (<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly decreased (<0.05), and the relative content ratio of G-actin/F-actin was significantly increased (<0.05). After administration of TFC, the phosphorylation level of ROCK2 in brain tissue of each group was significantly decreased (<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly up-regulated (<0.05) and the relative content ratio of G-actin/F-actin was significantly decreased (<0.05).
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.
研究 提取物总黄酮（TFC）对原代神经元氧葡萄糖剥夺（OGD）及慢性缺血诱导的脑损伤的保护作用及机制。
分离培养 18 日龄胎鼠海马神经元，培养 1 周后，分别用 0.25、0.50 和 1.00 mg/mL TFC 处理，OGD 1 h 后再灌注 6 和 24 h，分别用鬼笔环肽染色观察细胞骨架。在动物研究中，将 6 周龄 ICR 雄性小鼠随机分为假手术组、模型组、低剂量（10 mg/kg）、中剂量（25 mg/kg）和高剂量（50 mg/kg）TFC 治疗组，每组 20 只。除假手术组外，其余各组均采用单侧颈总动脉结扎法诱导慢性脑缺血。3 周后，各组小鼠分别用不同浓度 TFC 治疗 4 周。采用旷场试验、新物体识别试验和 Morris 水迷宫试验评价各组小鼠的焦虑、学习和记忆能力。尼氏染色、HE 染色和高尔基染色检测皮质和海马区神经元变性和树突棘变化。Western blot 检测各组小鼠海马组织 Rho 相关激酶（ROCK）2、LIM 激酶（LIMK）1、丝切蛋白及其磷酸化、球状肌动蛋白（G-actin）和丝状肌动蛋白（F-actin）蛋白的表达水平。
OGD 后神经元的轴突出现缩短和断裂；而 TFC 处理可逆转 OGD 诱导的轴突损伤，尤其是在 0.50 mg/mL TFC 组。与假手术组相比，模型组小鼠的焦虑和认知能力显著下降（均<0.01），而 TFC 治疗则显著逆转了焦虑和认知缺陷（均<0.05）。中剂量 TFC 组的改善最明显。组织病理学分析表明，模型组海马和皮质内的尼氏小体和树突棘数量减少（均<0.01）。然而，经中剂量 TFC 治疗后，尼氏小体和树突棘数量（均<0.05）明显恢复。与假手术组相比，模型组脑组织中 ROCK2 的磷酸化水平显著升高（均<0.05），而 LIMK1 和丝切蛋白的磷酸化水平显著降低（均<0.05），G-actin/F-actin 的相对含量比值显著升高（均<0.05）。TFC 给药后，各组脑组织中 ROCK2 的磷酸化水平均显著降低（均<0.05），而 LIMK1 和丝切蛋白的磷酸化水平显著上调（均<0.05），G-actin/F-actin 的相对含量比值显著降低（均<0.05）。
TFC 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，防止慢性脑缺血，提示 TFC 可能是治疗慢性缺血性脑损伤的潜在候选药物。
