乳香药效成分β-乳香酸通过PKCε/Nrf2通路改善缺血性脑卒中的作用及机制研究

Ischemic stroke is a major disease which has seriously jeopardized human health. Oxidative stress is the main pathogenesis and injury mechanism of ischemic stroke. At present, there are no ideal clinical treatments or measures. “Removing blood stasis and detoxifying” is the main method of treating ischemic stroke in traditional Chinese medicine. “Removing blood stasis” means improving hemorheology and protecting vascular endothelial function. “Detoxification” mainly refers to the removal of reactive oxygen species in the body. Frankincense is a natural drug for the treatment of brain diseases. Studies have shown that it can reduce nerve cell apoptosis through antioxidant stress. β-BA is the main medicinal ingredients of the frankincense，has a high blood-brain barrier permeability, and can reduce reactive oxygen species in some cells, suggesting that it may improve ischemic stroke through antioxidant stress. PKCε/Nrf2 and its downstream proteins are critical pathways regulating oxidative stress and play a crucial role in maintaining the redox balance in nerve cells. Our study also confirmed the role of PKCε/Nrf2 pathway in protecting nerves and improving ischemic stroke. Thus, on the basis of preliminary work, we plan to reveal the role and mechanism of β-BA in anti-ischemic stroke on the animal, cellular and molecular levels through the PKCε/Nrf2 pathway and by means of immunoblot, immunofluorescence and flow cytometry et al. For the treatment of ischemic stroke provides the theory basis for clinical application.

缺血性脑卒中是严重危害人类健康的重大疾病，氧化应激是其主要发病和损伤机制，目前临床尚无理想治疗药物或措施。“化瘀解毒”是中医治疗缺血性脑卒中的主要方法。“化瘀”指改善血液流变学、保护血管内皮功能，“解毒”主要指清除体内活性氧自由基。乳香是脑疾病治疗的天然药物，研究显示其可通过抗氧化应激降低神经细胞凋亡，β-BA是其主要药效成分，具有较高血脑屏障渗透率，可降低某些细胞中活性氧，提示其可能通过抗氧化应激保护神经细胞、改善缺血性脑卒中。PKCε/Nrf2及下游蛋白是调节氧化应激的关键通路，在维持神经细胞内氧化还原平衡发挥至关重要作用，我们研究亦证实了其保护神经细胞、改善缺血性脑卒中的作用。因此，本项目在前期工作基础上，拟通过PKCε/Nrf2通路，采用免疫印迹、免疫荧光、流式细胞术等方法，从整体、细胞、分子水平揭示β-BA改善缺血性脑卒中的作用及机制，为其在防治缺血性脑卒中的临床应用提供理论依。

缺血性脑卒中是一种具有高死亡率和高致残率的疾病，严重危害人类健康。脑缺血再灌注(Cerebral ischemia/reperfusion, I/R)损伤是缺血性脑卒中后发生的一种与氧化应激相关的脑损伤。β-乳香酸（β-boswellic acid, β-BA) 是乳香的主要药效成分具有抗氧化和神经保护作用。但其在脑I/R损伤中的作用尚不清楚。为探讨β-BA对脑I/R损伤的保护作用及其机制，我们采用大鼠大脑中动脉闭塞(MCAO)模型，神经元细胞氧糖剥夺(OGD)模型，分别作为脑缺血再灌注损伤的体内和体外模型。通过神经功能评分、TTC染色、HE染色、TUNEL染色及脑内相关抗氧化酶水平的检测来评估β-BA的作用。采用流式细胞术检测细胞凋亡和活性氧水平。Western blot和免疫荧光染色检测蛋白水平。结果显示β-BA能显著改善大鼠神经功能评分，减少梗死体积和坏死神经元。体外实验结果表明，β-BA可促进OGD损伤神经元细胞的存活，减少神经元细胞凋亡，降低神经元细胞Cleaved-caspase 3、Bax表达，提高Bcl2表达。此外，β-BA可显著促进Nrf2核转移、上调HO-1的表达，并提高p-PKCε (S729)的磷酸化水平。为进一步研究PKCε在β-BA发挥脑保护中的作用，我们敲低PKCε进行试验，结果显示敲低PKCε减弱了β-BA诱导的Nrf2/HO-1的激活，降低了其神经元的保护作用。基于此可以得出结论，β-BA可通过激活PKCε/Nrf2通路降低神经元氧化应激改善大鼠脑I/R损伤，为其在防治缺血性脑卒中的临床应用提供理论依。