苦碟子对急性脑皮质下梗死大鼠ephrin-A5信号通路神经重塑作用

Neural Remodeling Mechanism of Ephrin-A5 Signaling Pathway in Rats with Acute Subcortical Infarction by Kudiezi(Ixeris Sonchifolia)

目的研究苦碟子注射液对急性脑皮质下梗死大鼠缺血侧皮质ephrin-A5和EphB2的调节作用,从蛋白分子水平探讨苦碟子对急性脑皮质下梗死大鼠ephrin-A5信号通路的神经重塑作用机制。方法将雄性Wistar大鼠随机分为假手术组(6只)、模型组(36只)和治疗组(36只),后两组采用腹腔注射角叉菜胶,线栓法致左侧大脑中动脉闭塞制备急性脑皮质下梗死大鼠模型。术后当天治疗组腹腔注射苦碟子注射液8.4 g/(kg·d),假手术组和模型组腹腔注射等体积的0.9%生理盐水,频率均为1次/d,直至取材前。分别在造模后1、3、6、24、72 h和7 d取材,并采用Western Blotting和Q-PCR方法检测各组大鼠缺血侧皮质ephrin-A5和EphB2的表达。结果急性脑缺血后6、72 h和7 d,治疗组ephrin-A5 mRNA表达低于模型组(P<0.01或P<0.05),其中6 h治疗组与假手术组差异无统计学意义(P>0.05),72 h、7 d治疗组低于假手术组(P<0.01);缺血6、24、72 h和7 d治疗组EphB2 mRNA表达低于模型组(P<0.01或P<0.05),其中6 h治疗组与假手术组差异无统计学意义(P>0.05),24、72 h和7 d治疗组低于假手术组(P<0.01);缺血后24、72 h和7 d治疗组ephrin-A5蛋白表达低于对应时间点模型组(P<0.01),并且低于假手术组(P<0.01);缺血后1、24、72 h和7 d治疗组EphB2蛋白表达低于对应时间点模型组(P<0.01或P<0.05),其中72 h治疗组与假手术组差异无统计学意义(P>0.05),7 d治疗组表达低于假手术组(P<0.05)。结论急性脑缺血后ephrin-A5、EphB2蛋白在24 h变化最明显,为急性脑皮质下梗死最严重的时间点;应用苦碟子治疗后24 h起效,72 h、7 d显著改善,表明苦碟子可以通过调节急性脑皮质下梗死大鼠缺血侧皮质ephrin-A5信号通路的过表达起到神经重塑作用。

Objective To investigate the regulation of ephrin-A5 and Eph B2 in the ischemic side of rats with acute subcortical infarction by Kudiezi Injection(苦碟子注射液),exploring the neural remodeling mechanism of ephrin-A5 signaling pathway in rats with acute subcortical infarction at the protein molecular level. Methods 100 male Wistar rats were randomly divided into sham operation group(6 rats),model group(36 rats)and treatment group(36 rats). In order to establish the acute subcortical infarction model,rats in the latter two groups was prepared by subcutaneous injection of Carrageenan into the toes and occlusion of the left middle cerebral artery. The treatment group received intraperitoneal injection of Kudiezi Injection,the sham operation group and the model group received intraperitoneal injection of 0.9% saline of the same volume. All three groups received injections once a day until brain removal. The samples were collected at 1 h,3 h,6 h,24 h,72 h and 7 d after ischemia,respectively. Then Western Blotting and Q-PCR were used to research the dynamic changes of ephrin-A5 and Eph B2 expression in the ischemic cerebral cortex of rats. Results The expressions of ephrin-A5 m RNA in the treatment group were lower than those in the model group at 6 h,72 h and 7 d after ischemia(P<0.05 or P<0.01);There was no significant difference between the treatment group and the sham operation group at 6 h(P>0.05);The treatment group were lower than the sham group at 72 h and 7 d(P<0.01). The expressions of Eph B2 m RNA in the treatment group were lower than those in the model group at 6 h,24 h,72 h,7 d(P<0.05 or P<0.01);There was no significant difference between the treatment group and the sham operation group at 6 h(P>0.05);The treatment group were lower than the sham group at 24 h,72 h and 7 d(P<0.01). The expression of ephrin-A5 proteins in the treatment group were lower than those in the model group and the sham group at 24 h,72 h and 7 d(P<0.01);The expression of Eph B2 proteins in the treatment group were lower than those in the model group at 1 h,24 h,72 h and 7 d(P<0.05 or P<0.01);There was no significant difference between the treatment group and the sham operation group at 72 h(P>0.05),while the treatment group showed lower expression than the sham operation group at 7 d(P<0.05). Conclusion Ephrin-A5 and Eph B2 proteins changed most dramatically at 24 h after ischemia,which was the most serious time point of acute subcortical infarction. The effect of Kudiezi Injection started at 24 h and was significant at 72 h and 7 d,indicating that Kudiezi(Ixeris Sonchifolia)could modulate the ephrin-A5 signaling pathway in the ischemic side of rats with acute subcortical infarction and play a role in neural remodeling.