Wang Canmin, Hu Yingfang, Song Yunfeng, Hu Xinyi
Intensive Care Unit, The Affiliated Guangdong Second Provincial General Hospital of Jinan University, Guangzhou City, Guangdong Province, 510317, China.
Int J Colorectal Dis. 2024 Dec 20;39(1):205. doi: 10.1007/s00384-024-04788-4.
Autophagy damage will aggravate intestinal damage caused by sepsis. Studies have shown that the activation of AQP3 and SIRT1 signals can reduce the inflammatory response of sepsis. However, their role and mechanism in intestinal injury in the late stage of sepsis are not deeply studied.
To explore whether AQP3 can mediate autophagy by regulating the SIRT1/P62 signaling pathway to alleviate intestinal epithelial cell damage caused by sepsis.
Caco-2 cells were transfected with plasmid to overexpress AQP3. Western blot and RT-qPCR were used to detect the expression of cell protein, ELISA was used to detect the level of cytokines, DCFH-DA probe was added to quantify the ROS level, and the integrity of cell barrier was evaluated by measuring the transepithelial resistance (TEER). The autophagy levels were observed by MDC staining, and the levels of ZO-1 and Occludin were detected by immunofluorescence.
AQP3 was down-regulated in the Caco-2 cell injury model induced by LPS in vitro. Overexpression of AQP3 inhibited the production of inflammatory factors and ROS, thus relieving LPS-induced intestinal epithelial cell damage; restored the TEER of cells; up-regulated the expression of claudin-1, TJP-1, Occludin, and ZO-1, thus alleviating the cell barrier injury; increased autophagy bodies in cells; and increased the expression of Beclin1 and the ratio of LC3-II/LC3-I while inhibiting the expression of p62, thus restoring the autophagy level of cells. However, autophagy inhibitor 3-MA and SIRT1 inhibitor EX 527 offset these effects of AQP3 overexpression.
AQP3 regulated the autophagy level of Caco-2 cells induced by LPS through SIRT1/p62 signal and relieved intestinal epithelial cell damage caused by sepsis.
自噬损伤会加重脓毒症所致的肠道损伤。研究表明，水通道蛋白3（AQP3）和沉默信息调节因子1（SIRT1）信号的激活可降低脓毒症的炎症反应。然而，它们在脓毒症晚期肠道损伤中的作用及机制尚未深入研究。
探讨AQP3是否可通过调节SIRT1/P62信号通路介导自噬，以减轻脓毒症所致的肠上皮细胞损伤。
将质粒转染至Caco-2细胞以过表达AQP3。采用蛋白质免疫印迹法和逆转录-定量聚合酶链反应检测细胞蛋白表达，酶联免疫吸附测定法检测细胞因子水平，加入2′,7′-二氯荧光黄双乙酸盐探针定量活性氧水平，通过测量跨上皮电阻评估细胞屏障完整性。采用单丹磺酰尸胺染色观察自噬水平，免疫荧光法检测紧密连接蛋白1（ZO-1）和闭合蛋白水平。
在体外脂多糖诱导的Caco-2细胞损伤模型中，AQP3表达下调。过表达AQP3可抑制炎症因子和活性氧的产生，从而减轻脂多糖诱导的肠上皮细胞损伤；恢复细胞的跨上皮电阻；上调闭合蛋白1、紧密连接蛋白1、闭合蛋白和ZO-1的表达，从而减轻细胞屏障损伤；增加细胞内自噬体数量；增加自噬相关蛋白1（Beclin1）的表达及微管相关蛋白轻链3-II（LC3-II）/微管相关蛋白轻链3-I（LC3-I）的比值，同时抑制p62蛋白的表达，从而恢复细胞的自噬水平。然而，自噬抑制剂3-甲基腺嘌呤和SIRT1抑制剂EX 527可抵消AQP3过表达的这些作用。
AQP3通过SIRT1/p62信号调节脂多糖诱导的Caco-2细胞自噬水平，减轻脓毒症所致的肠上皮细胞损伤。
