基于神经细胞外基质探讨健脾补土方抗大鼠脑缺血再灌注神经细胞失巢凋亡机制

Mechanism of Jianpi Butu Formula against Anoikis and Apoptosis of Neurocytes in Rats with CIRI Based on Extracellular Matrix of Nerve Cells

目的:探讨健脾补土方通过调控神经细胞外基质抗大鼠脑缺血再灌注神经细胞失巢凋亡的机制。方法:SD雄性大鼠随机分为假手术组、模型组、依达拉奉组、健脾补土方低剂量组、健脾补土方中剂量组和健脾补土方高剂量组。采用线栓法制备大鼠右侧大脑中动脉阻塞(MCAO)模型,缺血2 h后再灌注7 d。对神经功能缺损进行评分;原位末端标记法(TUNEL)检测神经细胞凋亡指数(AI);免疫组织化学染色检测细胞外基质相关蛋白(FN、LN)的表达;采用蛋白质印迹法检测凋亡因子(Bcl-2、Bax)的表达;实时荧光定量PCR检测Caspase-3 mRNA的表达。结果:与假手术组相比,模型组神经功能缺损评分、神经细胞AI、Bax和Caspase-3表达显著增加(P<0.01),LN、FN和Bcl-2表达水平显著降低(P<0.01)。与模型组相比,健脾补土方各剂量组神经功能缺损评分、神经细胞AI、Bax和Caspase-3表达水平显著降低(P<0.05),LN、FN和Bcl-2表达显著增加(P<0.05)。结论:健脾补土方能有效改善脑缺血再灌注损伤,其机制可能与调控FN、LN表达,稳定神经细胞ECM,抑制神经细胞失巢凋亡有关。

Objective:To explore the mechanism of Jianpi Butu Formula against anoikis and apoptosis of neurocytes in rats with cerebral ischemia-reperfusion injury(CIRI)by regulating the extracellular matrix of nerve cells.Methods:Male SD rats were randomly divided into the sham operation group,the model group,the Edaravone group,the Jianpi Butu Formula groups of low-dose,medium-dose and high-dose.The thread suture method was used to prepare the right MCAO model in rats.After 2 hours of ischemia and reperfusion for 7 days,neurological deficit score of rats was evaluated by Zea-Longa,the TUNEL method was applied to detect the apoptosis index of neurocytes;the expressions of the extracellular matrix related proteins(FN,LN)were conducted by immunohistochemical method;the protein expressions of Bcl-2 and Bax were detected by Western blot;the expression of Caspase-3 mRNA was measured by the real-time fluorescent quantitative PCR.Results:Compared with those in the sham operation group,the neurological deficit score,the apoptosis index of nerve cells,as well as the protein expressions of Bax and Caspase-3 were significantly increased(P<0.01),the expressions of LN,FN and Bcl-2 were reduced remarkably in the model group(P<0.01).Compared with those in the model group,the neurological deficit score,the apoptosis index of nerve cells,as well as the protein expressions of Bax and Caspase-3 were significantly decreased(P<0.05),the expressions of LN,FN and Bcl-2 were significantly increased in the Jianpi Butu Formula groups(P<0.05).Conclusion:Jianpi Butu Formula can effectively improve CIRI,which mechanism may be related to regulating the protein expressions of FN and LN to stabilize extracellular matrix of nerve cells and inhibit anoikis and apoptosis of neurocytes.