基于内质网应激-自噬探讨龙蛭汤促进缺血性脑损伤大鼠血管新生作用

Some research results demonstrated that the regulation of endoplasmic reticulum stress(ERS)- autophagy affected the angiogenesis significantly during the cerebral ischemic injury process. The project preliminary studies showed that the Longzhi decoction with the effect of invigorating qi and promoting blood circulation could promote the angiogenesis of ischemic cerebral injury,so the hypothesis held that the Longzhi decoction promote the angiogenesis of ischemic cerebral injury through regulating the ERS-autophagy. The project would down-regulate or up-regulate the ERS- autophagy in rat model with cerebral ischemic injury by 4-phenyl butyric acid (4-PBA), 3-methlyadenine (3-MA), Rap respectively through intracerebroventricular microinjection.Further more, cell autophagy will be induced with palmitic acid (PA), and to close down the Beclin-1 gene by shRNA or 3-MA down-regulate and the Rap up-regulate the autophagy,and then the cell will be treated with Longzhi decoction.To compare the microvessel density through detection of vWF in the ischemic area by immunofluorescence,and the cell migration as well as tube formation ability by scratch test and Matrigel test will be tested,in the same time the ERS associated factors, such as GRP78/Bip, IRE-1，CHOP will be checked，and the autophagy related factors ,such as Beclin-1, LC3I, LC3II in the upper culture fluid will be tested through ELISA, Western-blot and laser scanning confocal microscopy,the GRP78/Bip，IRE-1，CHOP，Beclin-1，LC3 mRNA through quantitative real - time polymerase chain reaction (qRT-PCR), in order to reveal the mechanism of Longzhi Decoction promoting angiogenesis in ischemic brain injury.

研究表明缺血性脑损伤过程中，调控内质网应激（ERS）-自噬水平显著影响其血管新生过程。本项目的前期研究表明，补气活血的龙蛭汤具有促进缺血性脑损伤血管新生作用，因而假说龙蛭汤通过调节ERS-自噬促进血管新生。项目通过复制缺血性脑损伤大鼠气虚血瘀证模型，采用 Rap 、3-MA、shRNA 下调或上调ERS-自噬，shRNA沉默Beclin-1基因、3-MA、Rap调控自噬，并以龙蛭汤含药血清干预ERS细胞。免疫荧光检测缺血区vWF以比较微血管密度，同时检测细胞的细胞迁移能力及管形形成能力，ELISA、western-blot及激光共聚焦显微镜检测ERS相关因子GRP78/Bip、IRE-1、CHOP及自噬相关因子Beclin-1、LC3I、LC3II，qRT-PCR检测GRP78/Bip、IRE-1、CHOP、Beclin-1、LC3，以揭示龙蛭汤促进缺血性脑损伤血管新生机理。

研究表明缺血性脑损伤过程中调控内质网应激（ERS）-自噬水平显著影响其血管新生过程。本项目的前期研究表明，具有补气活血作用的龙蛭汤具有促进缺血性脑损伤血管新生作用，因而假说龙蛭汤通过调节ERS-自噬促进血管新生。项目通过复制缺血性脑损伤大鼠模型，采用雷帕霉素（Rap）、3-甲基腺嘌呤（3-MA）、shRNA 调节自噬水平，shRNA沉默Bcl-2 同源结构域蛋白 (Beclin-1)基因、3-MA、Rap调控自噬，并以龙蛭汤含药血清干预ERS-HUVEC。免疫荧光检测缺血区vWF以比较微血管密度，同时检测细胞的细胞迁移能力及管形形成能力，ELISA、western-blot及激光共聚焦显微镜检测ERS相关因子GRP78/Bip、IRE-1、CHOP及自噬相关因子Beclin-1、LC3I、LC3II，qRT-PCR检测GRP78/Bip、IRE-1、CHOP、Beclin-1、LC3，以揭示LZD促进缺血性脑损伤血管新生机理。研究结果表明：龙蛭汤可通过下调ERS，提高HUVEC细胞活力，上调自噬同时下调RA所致的过度自噬，促进HUVECs的增殖、迁移和管型形成，同时LZD通过上调自噬并促进VEGF的表达，从而HUVEC细胞管型形成。LZD通过双向调节自噬水平，促进血管内皮细胞管型形成，可能是其促进血管新生的作用机制之一，为LZD促进缺血性脑损伤血管新生阐明了机理。