基于PI3K/Akt-Nrf2/ARE通路探讨Astaxanthin对脑出血后脑损伤的保护作用和机制

Oxidative stress is one of the important pathological pathways associated with secondary brain injury after ICH. Astaxanthin (AST) is reported to have powerful antioxidant activity. Our previous research found that AST could ameliorate the secondary brain injury after ICH. Besides, we found that AST administration after intracerebral hemorrhage (ICH) significantly enhanced Nrf2 protein in the perihematoma tissue. Nrf2/ARE is recognized as major endogenous regulatory system against oxidative injury. It was reported that PI3K/Akt was essential for the activation of Nrf2/ARE pathway. Therefore, we hypothesized that AST could ameliorate the secondary brain injury after ICH through inducing the activation of Nrf2/ARE, which is mediated by the PI3K-Akt pathway. In this study, we would estimate the neuroprotective effect against ICH of AST in different concentration and survey the therapeutic time window to further confirm the neuroprotective role of AST on ICH. Besides, we would test our hypothesis in vivo and in vitro by using various techniques including immunohistochemistry, Western blot, RT-PCR, EMSA, Dual Luciferase Reporter Gene Assay with genetically knockout mice and RNA interference. This study could provide valuable information for the ICH therapy.

氧化应激反应在脑出血继发性损伤病理机制中起着重要作用。研究表明，Astaxanthin（AST）具有强大的抗氧化能力。我们前期研究发现，AST可以减轻脑出血继发性损伤，并发现AST治疗后，血肿周围脑组织Nrf2蛋白水平明显升高。Nrf2/ARE通路是机体抵抗氧化应激损伤的最主要内源性通路。研究表明，PI3K/Akt对诱导激活Nrf2/ARE通路有重要作用。据此，我们推测“AST通过激活PI3K/Akt-Nrf2/ARE通路，提升机体抗氧化能力，减轻脑出血后氧化应激损伤，发挥神经保护作用”。本项目拟在前期研究基础上，进一步明确AST的脑保护作用，摸索其治疗脑出血的量效关系及治疗时间窗。并通过体内、体外模型，结合基因敲除小鼠，RNA干扰技术，借助免疫组化，Western blot，RT-PCR，EMSA，双荧光素酶报告基因检测等多种技术反复验证此假说。本项目将为脑出血的治疗提供新的思路和策略。

氧化应激反应在脑出血继发性损伤病理机制中起着重要作用。研究表明，Astaxanthin (AST)具有强大的抗氧化能力。我们前期研究初步发现AST可以减轻脑出血继发性损伤。我们推测 “AST通过激活PI3K/Akt-Nrf2/ARE通路，提升机体抗氧化能力，减轻脑出血后氧化应激损伤，发挥神经保护作用”。本项目通过出血后24h检测脑水含量，开展神经功能评估，明确了AST的脑保护作用，并摸索了其治疗脑出血的量效关系及治疗时间窗。我们发现，术后给予50mg/kg与150mg/kg的AST均能显著减轻脑出血后24h脑水含量，改善Garcia评分。同时，术后0.5h给药的神经保护功能最显著。同时，通过脑出血后72h脑水含量、神经功能评估，进一步明确了AST的神经保护功能。本研究还通过Foot-Fault试验，Rotarod试验及水迷宫检测评估了AST对远期神经功能的保护作用，发现小鼠脑出血后0.5h给予50mg/kg的AST可以明显改善其远期功能。机制方面，通过检测氧化应激标志物蛋白水平，我们发现AST可以减轻脑出血后氧化应激水平。同时，脑出血后Nrf2表达量显著增加。进一步，我们通过PI3K选择性抑制剂，我们验证了PI3K/Akt对于Nrf2的调节作用。通过本项目的开展，发现AST可以减轻脑出血后继发性脑损伤，并初步探索了其潜在保护机制，为其进一步的转化提供了一定的理论基础和数据支撑。