茯苓酸调节Nrf2/Keap1/ARE信号通路改善脂多糖诱导肺泡上皮细胞氧化应激损伤研究

Pachymic Acid Modulating Nrf2/Keap1/ARE Signaling Pathway to Ameliorate LPS-induced Oxidative Stress Injury in Alveolar Epithelial Cells

目的:研究茯苓酸(PA)对脂多糖(LPS)诱导的人肺泡上皮细胞氧化应激损伤的影响,以及核因子红细胞2相关因子2/Kelch样ECH关联蛋白1/抗氧化反应元件(Nrf2/Keap1/ARE)信号通路在此过程中的作用。方法:培养人肺泡上皮细胞HPAEpiC,使用CCK-8法筛选适合的PA处理浓度。将HPAEpiC细胞分为Control组、LPS组、LPS+PA组、LPS+PA+sh-NC组和LPS+PA+sh-Nrf2组。TUNEL染色法检测各组HPAEpiC细胞凋亡情况;检测各组HPAEpiC细胞中活性氧(ROS)的产生、丙二醛(MDA)水平以及总超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性;Western blot检测各组HPAEpiC细胞中p53、B淋巴细胞瘤-2基因相关X蛋白(Bax)、Nrf2、Keap1和血红素氧化酶1(HO-1)蛋白表达情况。结果:根据CCK-8结果选用4μmol·L^(-1)的PA用于后续研究。LPS诱导后HPAEpiC细胞中TUNEL阳性细胞率与p53和Bax蛋白水平均升高;ROS的产生、MDA水平均增高,SOD和GSH-Px活性降低;Nrf2、HO-1蛋白质水平减少,Keap1蛋白质水平增加(P<0.05)。使用PA处理可以减轻LPS诱导对以上指标的影响(P<0.05)。敲低Nrf2可部分逆转PA处理对LPS诱导的HPAEpiC细胞的影响(P<0.05)。结论:PA可抑制LPS诱导的肺泡上皮细胞氧化应激损伤和凋亡反应,其机制可能与激活Nrf2/Keap1/ARE信号通路有关。

Objective:To study the impact of pachymic acid(PA)on lipopolysaccharide(LPS)-induced oxidative stress injury of human alveolar epithelial cells,and explore the role of Nrf2/Keap1/ARE signaling pathway in this process.Methods:Human alveolar epithelial cells HPAEpiC were cultured,and the appropriate PA treatment concentration was screened by CCK-8 method.HPAEpiC cells were separated into control group,LPS group,LPS+PA group,LPS+PA+sh-NC group and LPS+PA+sh-Nrf2 group.TUNEL staining was performed to measure the apoptosis of HPAEpiC cells in each group;ROS production,MDA level,and SOD and GSH-Px activities in HPAEpiC cells of each group were measured according to the kit manufacturer’s instructions;Western blot was performed to measure the protein expression of p53,Bax,Nrf2,Keap1 and HO-1 in HPAEpiC cells in each group.Results:According to the results of CCK-8,4μmol·L^(-1) PA was selected as the treatment concentration of subsequent studies.After LPS induction,the TUNEL-positive cell rate,and p53 and Bax protein levels in HPAEpiC cells increased;the production of ROS and MDA levels increased,and the activities of SOD and GSH-Px decreased;the protein levels of Nrf2 and HO-1 decreased,and the protein level of Keap1 increased(P<0.05).The effects of LPS induction on the above indicators were able to be alleviated by PA treatment(P<0.05).Knockdown of Nrf2 partially reversed the effects of PA treatment on LPS-induced HPAEpiC cells(P<0.05).Conclusion:PA can inhibit LPS-induced oxidative stress injury and apoptosis of alveolar epithelial cells,and the mechanism may be related to the activation of Nrf2/Keap1/ARE signaling pathway.