DHA调控PPARγ介导NFκB/TGF-β1/Smad通路减轻实验性脑缺血损伤

Intravenous thrombolysis and intra-arterial thrombectomy are now the standard therapies for patients with acute ischemic stroke. Immune mechanisms play a key role in the early stages of ischemic stroke. Clinical studies and cellular and / or animal models have demonstrated that DHA (docosahexaenoic acid) has anti-inflammatory and immunomodulatory effects, and it could prevent and cure cerebral infarction. the neuroprotective effect of DHA may be related to PPAR-γ signaling,but the exact mechanism is still not clear. Our previous studies found that DHA could reduce infarct volume of cerebral ischemia injury in rats by regulating the inflammatory and anti-inflammatory factors axis which associated with Treg and Th17 cells,and NF-κB pathway. Our previous study also found that TGF-β1/Smad pathway involved in inflammatory / proinflammatory factor axis, reduce brain damage and improve neurological recovery. Therefore, we propose a scientific hypothesis: DHA as a potential mediator of PPARγ via NFκB / TGF-β1 / Smad signaling pathway attenuates cerebral ischemic injury in rats. This project aims to investigate the protective effects and mechanisms of DHA in MACO model, which should be clearly understood with immunology, pathology,molecular and cell biology techniques at the different organ, cell, and molecular levels. Implementation of this project will provide sufficient theoretical basis for the prophylaxis and treatment of cerebral infarction, and have major significance in public health.

脑梗死急性期，除溶栓和血管内治疗外，特效的治疗手段非常有限。免疫机制在其发病早期中起关键作用。研究证实PPARγ参与了DHA在脑梗死治疗中的抗炎及免疫调节作用，但具体机制不明确。我们预实验显示Treg与Th17相关致炎/抑炎因子与NF-κB及TGF-β1/Smad通路参与缺血性脑损伤早期发病过程；DHA调控致炎/抑炎因子轴负变化及介导TGF-β1/Smad通路减少动物模型梗死体积和改善病情。据此，我们提出假设：DHA可调控PPARγ介导的NFκB/TGF-β1/Smad通路发挥对脑梗死的治疗作用。本课题以构建模拟缺血性卒中的大鼠MCAO模型为研究对象，利用免疫学、分子、细胞生物学手段明确MCAO模型早期病变过程中Treg与Th17细胞及其因子与PPARγ介导NFκB/TGF-β1/Smad通路的关系；阐明DHA对脑梗死治疗的分子机制，为临床应用DHA治疗脑梗死干预新靶点的建立提供科学依据。

脑梗死急性期，除溶栓和血管内治疗外，特效的治疗手段非常有限。究其原因缺血性卒中的病理生理机制仍未明确。研究证实免疫炎性机制在其发病早期中起关键作用。本项目采用线栓法建立MCAO模型，应用mNSS评估神经功能，TTC染色评估脑梗死体积，HE、Nissl染色评估梗死区病理形态学变化，激光散斑血流成像监测大脑皮层血流变化，伊文思蓝评估血脑屏障通透性，免疫荧光技术检测血脑屏障、血-脑脊液屏障完整性、评估微血管、线粒体功能状态，利用ELISA与PR-PCR 技术检测细胞及转录因子、mRNA，West-blot技术鉴定调控机制中信号蛋白。结果发现DHA通过NF-kB、Smad、ERK/Akt、PI3K/Akt/NF-κB发挥免疫炎症调节的保护作用；PPAR-γ、TNF-α、NF-κB参与BBB损伤；1,25-D3激活 PPAR-γ，降低TNF-α和NF-κB，增加BDNF；VDR 介导TGF-β/Smad2/3通路，增加 VEGF 释放；缺血再灌注急性期小胶质细胞M1 型极化，NF-κB、TNF-α、IL-1β炎性通路活化， TGF-β、AMPK、Akt、GSK-3β参与线粒体功能损伤；1,25- D3激活AMPK/Akt/GSK-3β信号传导，增加VEGF；激活PPARγ下调NF-κB和TNF-α，调节TGF-β、Smad蛋白表达；1,25-D3减轻炎性细胞向脉络丛浸润，与激活抗炎PPARγ/NF-κB/TNF-α与抑制TGF-β/Smad通路有关。本项目证实上述细胞因子及信号蛋白参与了MCAO早期缺血再灌注脑损伤的病理过程，丰富了缺血性脑损伤致病机制；提示调控其基因及蛋白可减少梗死体积、减少神经细胞死亡，改善线粒体功能，减轻神经功能损伤，保护血脑屏障、血脑脊液屏障，促进血管新生，减轻缺血性脑损伤，提供了可干预的靶点及研究方向。