DataSheet1_Echinocystic acid alleviated hypoxic-ischemic brain damage in neonatal mice by activating the PI3K/Akt/Nrf2 signaling pathway.pdf

Neonatal hypoxic-ischemic encephalopathy (HIE) is considered a major cause of death and long-term neurological injury in newborns. Studies have demonstrated that oxidative stress and apoptosis play a major role in the progression of neonatal HIE. Echinocystic acid (EA), a natural plant extract, shows great antioxidant and antiapoptotic activities in various diseases. However, it has not yet been reported whether EA exerts a neuroprotective effect against neonatal HIE. Therefore, this study was undertaken to explore the neuroprotective effects and potential mechanisms of EA in neonatal HIE using in vivo and in vitro experiments. In the in vivo study, a hypoxic-ischemic brain damage (HIBD) model was established in neonatal mice, and EA was administered immediately after HIBD. Cerebral infarction, brain atrophy and long-term neurobehavioral deficits were measured. Hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and dihydroethidium (DHE) staining were performed, and the contents of malondialdehyde (MDA) and glutathione (GSH) were detected. In the in vitro study, an oxygen-glucose deprivation/reperfusion (OGD/R) model was employed in primary cortical neurons, and EA was introduced during OGD/R. Cell death and cellular ROS levels were determined. To illustrate the mechanism, the PI3K inhibitor LY294002 and Nrf2 inhibitor ML385 were used. The protein expression levels of p-PI3K, PI3K, p-Akt, Akt, Nrf2, NQO1, and HO-1 were measured by western blotting. The results showed that EA treatment significantly reduced cerebral infarction, attenuated neuronal injury, and improved brain atrophy and long-term neurobehavioral deficits in neonatal mice subjected to HIBD. Meanwhile, EA effectively increased the survival rate in neurons exposed to OGD/R and inhibited oxidative stress and apoptosis in both in vivo and in vitro studies. Moreover, EA activated the PI3K/Akt/Nrf2 pathway in neonatal mice following HIBD and in neurons after OGD/R. In conclusion, these results suggested that EA alleviated HIBD by ameliorating oxidative stress and apoptosis via activation of the PI3K/Akt/Nrf2 signaling pathway.

新生儿缺氧缺血性脑病（Neonatal hypoxic-ischemic encephalopathy, HIE）是导致新生儿死亡及远期神经功能损伤的主要诱因。已有研究证实，氧化应激与细胞凋亡在新生儿缺氧缺血性脑病的病情进展中扮演核心角色。刺囊酸（Echinocystic acid, EA）作为一种天然植物提取物，在多种疾病模型中均展现出优良的抗氧化与抗细胞凋亡活性。但目前尚无研究报道刺囊酸是否对新生儿缺氧缺血性脑病具备神经保护作用。为此，本研究通过体内与体外实验，探究刺囊酸对新生儿缺氧缺血性脑病的神经保护作用及其潜在分子机制。
体内实验部分：本研究于新生小鼠体内构建缺氧缺血性脑损伤（hypoxic-ischemic brain damage, HIBD）模型，并于造模完成后即刻给予刺囊酸干预。本次实验检测指标包括脑梗死体积、脑萎缩程度及远期神经行为功能缺陷。依次开展苏木精-伊红（Hematoxylin and eosin, H&E）染色、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL）染色与二氢乙啶（dihydroethidium, DHE）染色，并检测丙二醛（malondialdehyde, MDA）与谷胱甘肽（glutathione, GSH）的含量。
体外实验部分：本研究于原代皮层神经元中构建氧糖剥夺/复氧（oxygen-glucose deprivation/reperfusion, OGD/R）模型，并在氧糖剥夺/复氧周期内加入刺囊酸干预。检测指标包括细胞死亡情况与细胞内活性氧水平。为阐明其作用机制，本研究使用磷脂酰肌醇3-激酶（PI3K）抑制剂LY294002与核因子E2相关因子2（Nrf2）抑制剂ML385进行干预。采用蛋白质印迹法检测p-PI3K、PI3K、p-Akt、Akt、Nrf2、NQO1及HO-1的蛋白表达水平。
实验结果显示，在缺氧缺血性脑损伤模型新生小鼠中，刺囊酸干预可显著缩小脑梗死体积、减轻神经元损伤，并改善脑萎缩与远期神经行为功能缺陷。与此同时，在氧糖剥夺/复氧模型的原代皮层神经元中，刺囊酸可有效提升神经元存活率，并同时抑制体内与体外实验中的氧化应激与细胞凋亡反应。此外，刺囊酸可在缺氧缺血性脑损伤模型新生小鼠及氧糖剥夺/复氧模型神经元中激活PI3K/Akt/Nrf2信号通路。
综上，本研究结果表明，刺囊酸可通过激活PI3K/Akt/Nrf2信号通路，改善氧化应激与细胞凋亡反应，从而减轻缺氧缺血性脑损伤。