地黄饮子对缺血性脑卒中的血管新生作用及机制研究

Exploring the Vasculogenesis Effects and Mechanisms of Dihuangyinzi in Ischemic Stroke

目的研究地黄饮子对氧糖剥离再灌注(OGD/R)损伤PC12细胞和对缺血/再灌注损伤大鼠的神经保护作用及作用机制。方法(1)采用PC12细胞制备OGD/R细胞损伤模型。MTT法检测不同浓度地黄饮子对PC12细胞活性的影响;MTT法检测OGD/R细胞模型制备的最佳时间;荧光倒置显微镜下观察各组细胞的形态;EDU染色检测OGD/R损伤以及地黄饮子给药处理对PC12细胞增殖的影响;Western blot检测PC12细胞内p-PI3K/PI3K、p-AKT/AKT、p-eNOS/eNOS相关通路蛋白的表达。(2)健康SPF级SD雄性大鼠50只,随机分为假手术组,模型组,地黄饮子低、中、高剂量组(8、16、32 g·kg^(-1)),每组10只。除假手术组外其余4组均建立双侧颈总动脉夹闭再灌注损伤(BCCAO/R)模型,缺血再灌注模型制备结束后,于次日灌胃给药,1次/天,连续给药7 d后,采用Western blot检测大鼠脑组织中p-PI3K/PI3K、p-AKT/AKT、p-eNOS/eNOS相关通路蛋白以及血管内皮生长因子(VEGF)的表达。结果地黄饮子能显著提高细胞存活率(P<0.01);OGD/R处理4 h能显著降低细胞增殖率,为最佳缺氧时间(P<0.01);OGD/R损伤显著降低细胞的增殖率,而地黄饮子能显著提高细胞的增殖率(P<0.01);地黄饮子给药处理后,PC12细胞内p-PI3K、p-AKT、p-eNOS水平显著上升(P<0.05);脑缺血再灌注损伤大鼠血管新生通路调节蛋白p-PI3K、p-AKT、p-eNOS以及VEGF水平显著上升(P<0.05,P<0.01)。结论地黄饮子可能通过调节PI3K/AKT/eNOS信号通路表达,对经OGD/R损伤的PC12细胞产生保护作用,并调节VEGF蛋白表达水平从而促进脑缺血再灌注损伤大鼠血管新生,发挥脑保护作用,为临床应用地黄饮子治疗脑缺血疾病提供了理论基础。

Objective Study of the neuroprotective effects and mechanisms of Dihuangyinzi on PC12 cells after oxygen gluoose stripping reperfusion(OGD/R)injury and SD rate after ischemia/reperfusion injury.Methods(1)A model of OGD/R cell injury was prepared using PC12 cells;the effect of different concentrations of Dihuangyinzi on the activity of PC12 cells was examined by MTT;the optimal time for the preparation of OGD/R cell model was examined by MTT;the morphology of each group of cells was observed under fluorescence inverted microscope;the proliferation of PC12 cells under OGD/R injury and Dihuangyinzi administration was examined by EDU staining;the effect of PC12 cell proliferation under Dihuangyinzi administration was examined by Western blot.Western blot was performed to detect the expression of p-PI3K/PI3K,p-AKT/AKT and p-eNOS/eNOS related pathway proteins in PC12 cells.(2)Fifty healthy SPF-grade SD male rats were randomly divided into the sham-operated group,the model group and Dihuangyinzi low,medium and high dose groups(8,16 and 32 g·kg^(-1)),with 10 rats in each group.The ischemic/reperfusion injury(bilateral common carotid artery occlusion reperfusion ischemic injury,BCCAO/R)model was established in all four groups except the sham-operated group,and the ischemic/reperfusion model was prepared and administered by gavage on the next day,once/day.After 7 days of continuous administration with once a day,the expression of p-PI3K/PI3K,p-AKT/AKT,p-eNOS/eNOS-related pathway proteins and vascular endothelial growth factor(VEGF)in rat brain tissues were detected by Western blot.Results Dihuangyinzi significantly increased the cell survival rate(P<0.01);OGD/R treatment for 4 h significantly decreased the cell proliferation rate,which was the optimal time for hypoxia(P<0.01);OGD/R injury significantly decreased the cell value-added rate,while Dihuangyinzi significantly increased the cell proliferation rate(P<0.01);The levels of p-PI3K,p-AKT and p-eNOS in PC12 cells increased significantly after the treatment with Dihuangyinzi(P<0.05).The levels of p-PI3K,p-AKT,p-eNOS and VEGF were significantly increased in rats with cerebral ischemia/reperfusion injury(P<0.05,P<0.01).Conclusion Dihuangyinzi may have a protective effect on OGD/R danaged PC12 cells by regulating the expression of PI3K/AKT/eNOS signaling pathway,and regulate the expression level of VEGF protein to promote cerebral neovascularization in rats with cerebral ischemia/reperfusion injury,providing a theoretical basis for the clinical application of Dihuangyinzi in the treatment of cerebral ischemic diseases.