Abstract:

Objective To investigate the protective effect of Troxerutin and Cerebroprotein Hydrolysate on neurovascular units after focal cerebral ischemia, and to explore the mechanism of its protective effects. Methods The middle cerebral artery occlusion (MCAO) was used to induce the focal cerebral ischemia model in rats. A total of 120 male SD rats were randomly divided into sham operation group (n =40), MCAO group (n =40), and MCAO + Troxerutin group (n =40). The MCAO + Troxerutin group was given Troxerutin 3 ml/kg, 1 time per day for 3 days after the operation by intraperitoneal injection. Modified Neurological Severity Score (mNSS) was used to evaluate the behavior changes of 3 d, 7 d and 14 d after embolization. Three days after embolization, the infarct area and blood flow was tested by T2 and ASL sequence of 7.0T high resolution MRI. Nissl staining was used to evaluate the survival rate and morphological changes of neurons. Immunofluores  staining was used to assess the expression of endothelial cells, astrocytes and tight junction markers in each group; furthermore, Western blot was used to evaluate the expression of 3-nitrotyrosine (3-NT), matrix metalloproteinase 9 (MMP-9) and inducible nitric oxide synthase (iNOS) in each group. Results Three days after MCAO, compared with the sham group, the ASL sequence of MCAO group showed a significant reduction of blood flow in the infarct area; nissl staining showed a reduction in neuronal survival, a large number of vacuoles, and pyknosis; immunofluorescence showed that endothelial cells and tight junction markers decreased and astrocytes increased; and Western blot demonstrated that iNOS, 3-NT and MMP-9 increased. While for MCAO + Troxerutin group, the blood flow of infarct area was significant decreased; the survival of neurons, the molecular marker of endothelial cells and tight junction were increased. Astrocytes and iNOS, 3-NT, MMP-9 were significant decreased compared with MCAO group. Conclusion Troxerutin and Cerebroprotein Hydrolysate could inhibit the expression of iNOS and MMP-9 and reduce the production of 3-NT, so as to play a role of protecting the neurovascular cells of MCAO rats.

Key words: Middle cerebral artery occlusion; Neurovascular unit; Blood-brain barrier;
Troxerutin

摘要：

目的 验证曲克芦丁脑蛋白水解物对局灶性脑缺血后神经血管单元的保护作用，探讨其保护作用的 机制。 方法 采用大脑中动脉栓塞（middle c erebral a rtery o cclusion，MCAO）制备大鼠局灶性脑缺血模型。 120只雄性SD大鼠，采用随机数字表法分成假手术（SHAM）组（n =40）、MCAO组（n =40）以及MCAO+曲 克芦丁治疗组（n =40）。MCAO+曲克芦丁治疗组于术后立即给予曲克芦丁3 ml/kg，1次/日腹腔注射 3 d。采用改良神经功能缺损评分（modified Neurological Severity Score，mNSS）分别评价栓塞后3 d、7 d、 14 d大鼠的行为改变；栓塞后3 d，采用7.0 T高分辨率磁共振的T2及动脉自旋标记（arterial spin label， ASL）序列评价梗死体积及梗死区血流变化；采用尼氏染色评价神经元存活率及形态学变化；采用 免疫荧光染色技术评价各组内皮细胞、星形胶质细胞及紧密连接标记分子表达的变化；采用蛋白质 印迹法评价各组3-硝基酪氨酸（3-nitrotyrosine，3-NT）、基质金属蛋白酶9（matrix metalloproteinase 9， MMP-9）及诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）表达的变化。 结果 MCAO后3 d，MCAO组与SHAM组相比，ASL序列显示梗死区域血流显著减少；尼氏染色结果显示 神经元存活率降低，大量空泡形成，核固缩；免疫荧光显示内皮细胞及紧密连接标记分子减少而星 形胶质细胞增多；蛋白质印迹法结果显示iNOS、3-NT、MMP-9增多。而MCAO+曲克芦丁治疗组与MCAO组 相比，梗死区域血流显著增多，神经元存活率升高，内皮细胞、紧密连接标记分子增多，星形胶质细 胞减少，i NOS、3-NT、MMP-9减少。 结论 曲克芦丁脑蛋白水解物可以通过抑制iNOS和MMP-9的表达，减少3-NT的产生，从而对MCAO后 大鼠神经血管单元起到保护作用。

关键词: 大脑中动脉栓塞; 神经血管单元; 血脑屏障; 曲克芦丁