Zhang Yu, Yu Chenxi, Feng Yi
Department of Critical Care Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200082, P.R. China.
Exp Ther Med. 2022 Jun 14;24(2):513. doi: 10.3892/etm.2022.11440. eCollection 2022 Aug.
Pinocembrin (PINO) is a natural flavonoid drug that possesses a range of biological activities, including antimicrobial, antioxidant and anti-inflammatory activities. The specific aim of the present study was to examine the pharmacological role of PINO in sepsis-mediated acute kidney injury (AKI), as well as to investigate the potential underlying mechanism. Human renal tubular epithelial cells (of the HK-2 cell line) were stimulated with lipopolysaccharide (LPS) for 24 h to simulate septic AKI , after which the experiments were repeated and the cells were pretreated with increasing concentrations of PINO (0, 50, 100 and 200 µg/ml). Using an MTT cell viability assay, PINO was revealed to be non-toxic to HK-2 cells. In LPS-treated HK-2 cells, PINO alleviated the loss of cell viability. Western blotting was used to analyze the expression levels of pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α, and the results revealed that PINO decreased the expression levels of these cytokines in a concentration-dependent manner. Furthermore, malondialdehyde (MDA) and glutathione (GSH) activities were assessed using MDA and GSH assay kits and it was revealed that PINO decreased the significantly increased level of malondialdehyde, while it also decreased the reduction in the level of GSH in LPS-challenged HK-2 cells. In addition, a TUNEL assay and western blotting were performed to examine cell apoptosis, and PINO was identified to significantly inhibit the level of apoptosis in LPS-induced HK-2 cells. Subsequently, the expression levels of endoplasmic reticulum stress (ERS)-associated factors, including activating transcription factor 4, C/EBP homologous protein and phosphorylated/total eukaryotic translation initiation factor 2 subunit 1 were examined by western blotting and it was demonstrated that ERS was triggered in HK-2 cells exposed to LPS, although this was partly circumvented through PINO treatment in a concentration-dependent manner. Furthermore, after the addition of tunicamycin, which acts as an agonist of ERS, the aforementioned experiments were performed again. Tunicamycin led to partial abolition of the protective function of PINO against inflammation, oxidative stress and apoptosis in LPS-challenged HK-2 cells. Overall, the results of the present study demonstrated that PINO was able to ameliorate the injuries sustained by LPS-challenged HK-2 cells via modulating ERS to reduce inflammation, oxidative stress and apoptosis; therefore, PINO may be a novel candidate drug for treating septic AKI.
白杨素（PINO）是一种天然黄酮类药物，具有一系列生物活性，包括抗菌、抗氧化和抗炎活性。本研究的具体目的是研究PINO在脓毒症介导的急性肾损伤（AKI）中的药理作用，并探讨其潜在的作用机制。用脂多糖（LPS）刺激人肾小管上皮细胞（HK-2细胞系）24小时以模拟脓毒症性AKI，之后重复实验并使用浓度递增的PINO（0、50、100和200μg/ml）对细胞进行预处理。使用MTT细胞活力测定法，发现PINO对HK-2细胞无毒。在LPS处理的HK-2细胞中，PINO减轻了细胞活力的丧失。采用蛋白质免疫印迹法分析促炎细胞因子白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的表达水平，结果显示PINO以浓度依赖性方式降低了这些细胞因子的表达水平。此外，使用丙二醛（MDA）和谷胱甘肽（GSH）检测试剂盒评估MDA和GSH活性，结果显示PINO降低了LPS刺激的HK-2细胞中显著升高的丙二醛水平，同时也减轻了GSH水平的降低。此外，进行了TUNEL检测和蛋白质免疫印迹法以检测细胞凋亡，发现PINO显著抑制LPS诱导的HK-2细胞中的凋亡水平。随后，通过蛋白质免疫印迹法检测内质网应激（ERS）相关因子激活转录因子4、C/EBP同源蛋白和磷酸化/总真核翻译起始因子2亚基1的表达水平，结果表明暴露于LPS的HK-2细胞中触发了ERS，尽管通过PINO处理以浓度依赖性方式部分规避了ERS。此外，在加入作为ERS激动剂的衣霉素后，再次进行上述实验。衣霉素导致PINO对LPS刺激的HK-2细胞的抗炎、抗氧化应激和抗凋亡保护作用部分丧失。总体而言，本研究结果表明，PINO能够通过调节ERS减轻LPS刺激的HK-2细胞所遭受的损伤，从而减轻炎症、氧化应激和细胞凋亡；因此，PINO可能是治疗脓毒症性AKI的新型候选药物。
