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目的研究洋葱黄酮类提取物对不同时间点脑出血模型大鼠血肿周围活化小胶质细胞数量及脑组织促炎性因子释放的影响,探讨洋葱黄酮类提取物治疗脑出血的可能机制。方法 100只Wistar大鼠用于制备脑出血动物模型,造模成功90只。其中脑出血组(40只)、洋葱黄酮类提取物干预组(简称干预组,40只)和假手术组(10只)。造模过程中脑出血组和干预组均向大鼠定位部位注射自体血100μL,同时干预组予洋葱黄酮类提取物(0.2 m L/10 g体重,每日2次)灌胃给药;脑出血组和假手术组未进行药物干预。造模后各组再以6、24、48、72 h和7 d为时间点分5个亚组,干预组和脑出血组每个亚组8只,共10组,假手术组每个亚组2只,共5组。采用Garcia JH评分法观察大鼠不同时间点的神经功能;采用HE染色观察各时间点大鼠脑组织的损伤程度;采用免疫组织化学染色法观察不同时间点大鼠血肿周围活化的小胶质细胞;采用ELISA法检测不同时间点大鼠血肿脑组织促炎因子TNF-α与IL-1β表达。结果脑出血组注入自体血后,血肿周围出现变性坏死区,细胞排列不整齐,核形态不整,部分核皱缩、髓质均片状间质水肿,且随着时间推移,变性坏死区域扩大外,细胞周围出现空白区,细胞分布不均匀,神经元细胞数减少,同时有淋巴细胞及中性粒细胞浸润;洋葱黄酮类提取物干预后,对应时间点的细胞坏死,细胞排列及核形态明显减轻,炎性细胞浸润减少。与假手术组比较,脑出血组5个时间点行为学评分均降低,活化的小胶质细胞数目增多,血肿脑组织中TNF-α及IL-1β表达升高,差异均有统计学意义(P<0.01)。与脑出血组比较,干预组大鼠出血后48、72 h和7 d时间点行为学评分升高,小胶质细胞的活化数目减少,TNF-α及IL-1β表达降低,差异均有统计学意义(P<0.01)。结论采用乙醇回流法从洋葱中提取的黄酮类物质可能通过抑制脑出血后血肿周围小胶质细胞的活化及促炎因子的释放,而改善脑出血模型大鼠的症状。
Objective To study the effect of onion flavonoids extract on the number of activated microglia around the hematoma and the release of proinflammatory cytokines in the brain of rats with intracerebral hemorrhage at different time points and to explore the possible mechanism of the onion flavonoid extract in the treatment of cerebral hemorrhage. Methods 100 Wistar rats were used to prepare the animal model of intracerebral hemorrhage. The intracerebral hemorrhage group (40 rats), the onion flavonoid extract intervention group (intervention group, 40 rats) and the sham operation group (10 rats). Cerebral hemorrhage and intervention groups were injected 100μL autologous blood into the locus of rats in the modeling process, while the intervention group was given orally the flavonoids extract (0.2 m L / 10 g body weight twice a day) Bleeding group and sham operation group without drug intervention. After modeling, the rats in each group were divided into 5 subgroups at 6, 24, 48, 72 and 7 d points respectively. There were 8 subgroups in each intervention group and 10 in the cerebral hemorrhage group, Group 2, a total of 5 groups. The Garcia JH score method was used to observe the neurological function of rats at different time points. HE staining was used to observe the degree of brain injury in rats at each time point. Immunohistochemical staining was used to observe the activation of microglial cells around the hematoma of rats at different time points The expression of pro-inflammatory cytokines TNF-α and IL-1β in rat hematoma at different time points were detected by ELISA. Results After hemorrhage of autologous blood was injected into autologous blood, there were degenerated and necrotic areas around the hematoma, irregular arrangement of cells, partial nuclear shrinkage, and medullary edema. As time went by, the area of degeneration and necrosis expanded , Blank area around cells, uneven distribution of cells, decreased number of neuronal cells, infiltration of lymphocytes and neutrophils; necrosis, cell arrangement and nuclear morphology at the corresponding time point after the intervention of onion flavonoids Reduce, reduce inflammatory cell infiltration. Compared with the sham-operation group, the scores of behavioral score, the number of activated microglia and the expression of TNF-α and IL-1β in hematoma brain tissue were significantly decreased at 5 time points after cerebral hemorrhage P <0.01). Compared with the intracerebral hemorrhage group, the behavioral score, the number of activated microglia and the expression of TNF-α and IL-1β in the intervention group were increased 48, 72 and 7 days after the hemorrhage Significance (P <0.01). Conclusion The flavonoids extracted from onion by ethanol reflux may improve the symptoms of intracerebral hemorrhage rats by inhibiting the activation of microglia around the hematoma and the release of pro-inflammatory cytokines after intracerebral hemorrhage.