吴茱萸次碱对棕榈酸诱导的脂质损伤肝细胞的影响

目的:研究吴茱萸次碱(Rut)对棕榈酸(PA)诱导的脂质损伤肝细胞(LO2)的细胞活力、活性氧(ROS)以及缝隙连接蛋白32(Cx32)和缝隙连接蛋白26(Cx26)表达水平的影响。方法:加入不同浓度的Rut溶液预处理LO2细胞20 min后,每个孔加入PA(0.2 mmol/L)溶液,共同孵育24 h,分别应用细胞增殖及毒性检测试剂盒(CCK-8)检测细胞活力,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)荧光探针检测细胞内活性氧的表达、划痕负载实验检测缝隙连接细胞间通讯(GJIC)的功能变化及免疫印迹实验(Western blotting)检测缝隙连接蛋白Cx32和Cx26的表达水平。结果:CCK-8实验结果显示,Rut(0.1μmol/L,0.5μmol/L,1μmol/L)能抑制PA诱导的LO2细胞活力的下降;DCFH-DA荧光探针实验显示PA诱导细胞后ROS的表达增强,Rut预处理细胞后能够减少ROS的表达;划痕负载实验结果显示,PA诱导细胞后能够抑制细胞的GJIC功能,预先给予Rut处理能够改善GJIC的功能障碍;Western blotting显示PA诱导细胞后缝隙连接蛋白Cx32和Cx26的表达水平显著降低,给予Rut预处理后能够恢复Cx32和Cx26的表达。结论:PA能够诱导LO2细胞的脂质损伤,导致细胞活性降低、细胞内活性氧增加以及GJIC的功能障碍;Rut预处理LO2细胞后可减轻PA诱导的细胞损伤,并且恢复细胞的缝隙连接蛋白Cx32和Cx26的表达和GJIC的功能。

Effect of rutaecarpine on lipid injury induced by palmitic acid in hepatocytes

Objective:To investigate the effects of Rutaecarpine(Rut)on cell viability,active oxygen(ROS),and connexin 32(Cx32)and connexin 26(Cx26)expression levels of lipid-damaged liver cells(LO2)induced by palmitic acid(PA).Methods:After LO2 cells were pretreated with Rut solutions of different concentrations for 20 min,PA(0.2 mmol/L)solution was added to each hole and co-incubated for 24 h.Cell proliferation and toxicity detection kit(CCK-8)experiment was applied to detect cell viability,and dichlorodihydrofluorescein-acetoacetate(DCFH-DA)fluorescence probe was used to detect the expression of ROS in cells.The functional changes of Gap Junction Intercellular Communication(GJIC)were detected by the scratch load experiment and the expression levels of Cx32 and Cx26 were detected by Western blotting.Results:CCK-8 experiment results showed that Rut(0.1μmol/L,0.5μmol/L,1μmol/L)could inhibit the decline of the activity of LO2 cells induced by PA.DCFH-DA fluorescence probe experiment showed that the expression of ROS was enhanced after PA induction,and the ROS expression could be reduced after Rut pretreatment.The results of the scratch load experiment showed that the GJIC function of cells could be inhibited after PA induction,and the dysfunction of GJIC could be improved by Rut treatment in advance.Western blotting showed that the expression levels of gap junction proteins Cx32 and Cx26 were significantly reduced after PA induction,and the expressions of Cx32 and Cx26 could be recovered after Rut pretreatment.Conclusion:Palmitic acid can induce lipid damage in LO2 cells,leading to decreased cell activity,increased intracellular ROS and dysfunction of GJIC.Pretreatment with Rutaecarpine can reduce PA-induced cell damage in LO2 cells and restore the expression of gap junction proteins Cx32 and Cx26 as well as the function of GJIC.