成都市第五人民医院病理科
四川省科技厅科技支撑项目(项目编号：2013SZ0044)；成都市科技局重点研发支撑计划技术创新研发项目(项目编号：2022-YF05-01725-SN)
1.Chengdu Fifth People'2.'3.s Hospital
目的：研究厚朴酚(HKL)对脂多糖(LPS)引发的急性呼吸窘迫综合征(ARDS)中肺微血管内皮细胞的作用及其潜在机制。方法：小鼠肺微血管内皮细胞(PMVEC),于六孔板中以DMEM+10%FBS培养,分为对照组(Con组)、和厚朴酚组(HKL组)、脂多糖处理组(LPS组)、LPS+HKL治疗组组(HKL+LPS组),分别采用TBARS检测试剂盒与H2DCF-DA测定细胞裂解物中丙二醛水平与活性氧水平；采用TUNEL/DAPI双染检测细胞凋亡；采用VE-cadherin/DAPI、Claudin-5/DAPI双染检测细胞连接；采用Western印迹检测细胞中caspase-3、cleaved caspase-3、Sirt3、SOD2、乙酰化SOD2(Ac-SOD2)；32只小鼠随机分为对照组(Con组)、和厚朴酚组(HKL组)、脂多糖处理组(LPS组)、LPS+HKL治疗组组(HKL+LPS组),采用HE染色观察肺组织病理改变。结果：和厚朴酚预处理能明显减轻LPS诱导的ROS与MDA水平升高,同时降低LPS引起的SOD2乙酰化升高与SIRT3下调；TUNEL与caspase分析显示和厚朴酚能够保护LPS诱导的PMECs细胞凋亡；VE-cadherin荧光染色显示和厚朴酚的预处理能够阻止LPS对细胞粘附连接的破坏；Claudin-5荧光染色显示和厚朴酚的预处理能够阻止LPS对细胞紧密连接的破坏；动物实验中,HE染色显示和厚朴酚显著抑制LPS组小鼠肺组织中典型的ARDS病理改变。结论：HKL能够显著抑制LPS所致的肺微血管内皮细胞氧化应激与细胞凋亡以及细胞间隙破坏,从而减轻ARDS症状。
Objective: To study the effect of Honokiol (HKL) on pulmonary microvascular endothelial cells in lipopolysaccharide (LPS) induced acute respiratory distress syndrome (ARDS) and its potential mechanism.Methods: Mouse lung microvascular endothelial cells (PMVEC) were cultured with DMEM+10%FBS in a six-well plate and divided into control group (Con group), Honokiol group (HKL group), Lipopolysaccharide treatment group (LPS group) and LPS+HKL treatment group (HKL+LPS group). The levels of Malondialdehyde and Reactive Oxygen Species in cell lysates were determined by TBARS assay kit and H2DCF-DA, respectively. TUNEL/DAPI double staining was used to detect apoptosis. The cell junction was detected by VE-cadherin/DAPI and Claudin-5/DAPI double staining. Western blotting was used to detect caspase-3, cleaved caspase-3, Sirt3, SOD2, and acetylated SOD2 (Ac-SOD2). 32 mice were randomly divided into control group (Con group), Honokiol group (HKL group), Lipopolysaccharide treatment group (LPS group) and LPS+HKL treatment group (HKL+LPS group). HE staining was used to observe the pathological changes of lung tissue.Results: Honokiol pretreatment could significantly reduce LPS-induced increase of ROS and MDA levels, and decrease LPS-induced increase of SOD2 acetylation and SIRT3 down-regulation. TUNEL and Caspase analysis showed that Honokiol could protect apoptosis of PMECs induced by LPS. VE-cadherin fluorescence staining demonstrate thag Honokiol pretreatment could prevent LPS from damaging cell adhesion. Claudin-5 fluorescence staining show that Honokiol pretreatment could prevent LPS from disrupting tight connections of cells. In animal experiments, HE staining showed that Honokiol significantly inhibited the typical pathological changes of ARDS in the lung tissue of mice in LPS group.Conclusion: HKL can significantly inhibit LPS-induced oxidative stress, apoptosis and cell connection breakdown of pulmonary microvascular endothelial cells, thereby alleviating ARDS symptoms.
