丹龙醒脑方对血管性痴呆大鼠Notch信号通路及其下游靶基因的干预作用研究

Intervention of Danlong Xingnao Recipe on Notch Signaling Pathway and Its Downstream Target Genes in Vascular Dementia Rats

摘要: 目的：多层次地阐明丹龙醒脑方对血管性痴呆大鼠各指标的干预结果，从Notch信号通路及其下游靶基因角度探讨丹龙醒脑方治疗血管性痴呆(Vascular Dementia, VD)的作用机制。方法：60只大鼠通过改良双侧颈总动脉永久阻断法(2-VO)加去势手术法制备VD大鼠模型。将成模大鼠均分为四个大组，即模型组、阳性药物组、DAPT组、丹龙醒脑方组。分别运用干预措施后，光镜下观察HE染色的大鼠海马组织形态学变化，检测给药干预后大鼠血清T水平，及NO和ET-1水平。统计大鼠海马区、皮质区样本增殖活性细胞免疫荧光染色双阳性细胞数，使用免疫组织化学法、Western Blot法定性、定量检测VD大鼠海马组织Notch1、Hes1蛋白表达。结果：干预后各大组大鼠海马组织出现一定变化，与模型组比较，阳性药物组、丹龙醒脑方组T、NO水平均有明显上升，ET-1水平有明显下降(P < 0.01, P < 0.05)；与模型组比较，阳性药物组、丹龙醒脑方组大鼠海马区双阳性细胞数均有明显上升(P < 0.01, P < 0.05)；假手术组与DAPT组海马区可见Notch1、Hes1蛋白少许表达，模型组表达增强，丹龙醒脑方组、阳性药物组蛋白表达明显增强。结论：丹龙醒脑方能显著改善VD大鼠的学习能力、记忆能力、空间探索能力及脑组织病理损伤；丹龙醒脑方能通过积极调控VD大鼠Notch信号通路及其下游靶基因Hes1的表达，促进了神经干细胞的增殖、分化，从而发挥对VD大鼠的治疗作用。

Abstract: Objective: Explain the intervention results of Danlong Xingnao Recipe on various indexes of rats with vascular dementia at multiple levels, and explore the mechanism of Danlong Xingnao recipe in the treatment of VD from the perspective of Notch signal pathway and its downstream target genes. Method: In 60 rats, the VD rat model was prepared by modified bilateral common carotid artery permanent occlusion (2-VO) and castration. The model rats were divided into four groups: Model group, positive drug group, DAPT group and Danlong Xingnao formula group. After the intervention measures were applied respectively, the morphological changes of hippocampus of rats stained with he were observed under light microscope, The levels of serum T, no and ET-1 were detected after administration. Count the number of double positive cells in immunofluorescence staining of proliferative cells in hippocampus, and the expression of Notch1 and Hes1 protein in hippocampal tissue of VD rats was detected by immunohistochemistry and Western blot. Results: After the intervention, the hippocampal tissue of each group showed certain changes occurred in the hippocampus of rats in each group. Compared with the model group, the levels of T and NO in the positive drug group and the Danlong Xingnao formula group increased significantly, and and the level of ET-1 decreased significantly (P < 0.01, P < 0.05); compared with the model group, the number of double positive cells in the hippocampus of rats in the positive drug group and the Danlong Xingnao formula group increased significantly (P < 0.01, P < 0.05); in sham operation group and DAPT group, a little expression of Notch1 and Hes1 protein can be seen in hippocampus, the expression of model group is enhanced, and the expression of Danlong Xingnao formula group and positive drug histone is significantly enhanced. Conclusion: Danlong Xingnao recipe can significantly improve the learning ability, memory ability, space exploration ability and brain pathological damage of VD rats; Danlong Xingnao recipe can promote the proliferation and differentiation of neural stem cells by actively regulating the expression of Notch signal pathway and its down-stream target gene Hes1 in VD rats, so as to play a therapeutic role in VD rats.