虎杖苷对脂多糖介导的肺泡上皮细胞线粒体损伤的影响

Effects of polydatin on lipopolysaccharide-induced mitochondrial injury in alveolar epithelial cells

摘要:

目的 探讨虎杖苷对脂多糖(LPS)介导的肺泡上皮线粒体损伤的影响及可能机制。方法 A549细胞分为4组:对照组为细胞仅接受0.1% DMSO处理7h；模型组为细胞给予DMSO预处理,1h后与LPS(5mg/L)共孵育6h；治疗组为细胞接受虎杖苷(50μmol/L)预处理,1h后与LPS(5mg/L)共孵育6h；抑制剂组为细胞接受沉默信息调节因子2相关酶3(SIRT3)抑制剂(50μmol/L 3-TYP)及虎杖苷50μmol/L预处理,1h后与LPS(5mg/L)共孵育6h。细胞计数试剂盒－8(CCK-8)检测细胞活性；荧光素－荧光素酶法检测细胞ATP水平；钙黄绿素－氯化钴探针检测线粒体通透性转变孔(mPTP)状态；DCFH-DA荧光探针检测细胞活性氧(ROS)水平；荧光探针JC-1检测线粒体膜电位(MMP)；Western blotting检测细胞SIRT3表达。应用SPSS 20.0软件进行统计分析。组间比较采用单因素方差分析、LSD两两比较或Tamhane′s T2检验。结果 与对照组比较,模型组中SIRT3表达、JC-1红色/绿色荧光、钙黄绿素荧光、ATP水平及细胞活力分别显著下降至(73.3±4.5)%、(54.0±6.5)%、(2035±217)U、(72.2±4.8)%及(73.7±3.7)%,ROS水平显著增加为(218.0±21.7)%；与模型组比较,治疗组中SIRT3表达、JC-1红色/绿色荧光、钙黄绿素荧光、ATP水平及细胞活力分别显著上升为(86.7±7.6)%、(75.8±7.6)%、(2571±199)U、(86.7±6.3)%及(83.0±3.6)%,ROS水平显著下降为(180.0±18.1)%；与治疗组比较,抑制剂组中SIRT3表达、JC-1红色/绿色荧光、钙黄绿素荧光、ATP水平及细胞活力分别显著下降为(69.0±7.8)%、(62.8±6.2)%、(2116±254)U、(72.8±5.8)%及(73.3±4.1)%,ROS水平显著增加为(212.0±18.2)%。结论 虎杖苷可以显著改善LPS介导的肺泡上皮线粒体损伤,其机制可能与激活SIRT3有关。

Abstract:

Objective To investigate the effects and mechanism of polydatin on the lipopolysaccharide-induced mitochondrial injury in alveolar epithelial cells. Methods A549 cells were assigned to four groups. Cells in the control group were treated with 0.1% DMSO for 7h only; those in model group received the pretreatment of 0.1% DMSO for 1 h and were incubated with LPS (5mg/L) for 6h; those in the treatment group received the pretreatment of polydatin (50μmol/L) for 1 h and were incubated with LPS (5mg/L) for 6h; and those in the inhibitor group received the pretreatment of polydatin (50μmol/L) and silent information regulator 2 related enzyme 3(SIRT3) inhibitor 3-TYP (50μmol/L) for 1h, and were incubated with LPS (5mg/l) for 6h. The cells were then assessed for viability by CCK-8, ATP level by fluorescein-luciferase kits, status of mitochondrial permeability transition pore (mPTP) by Calcein-AM-CoCl₂ probe, reactive oxygen species (ROS) level by DCFH-DA fluorescent probe, mitochondrial membrane potential (MMP) by fluorescent probe JC-1, and expression of SIRT3 by Western blotting. SPSS statistics 22.0 was used for data analysis. One-way ANOVA, LSD test or Tamhane′s T2 test was used for comparison among groups. Results Compared with control group, SIRT3 expression in model group decreased to (73.3±4.5)%, JC-1 red/green to (54.0±6.5)%, Calcein fluorescence to (2035±217)U, ATP level to (72.2±4.8)% and cell viability of cells to (73.7±3.7)%, but ROS level increased to (218.0%±21.7)%. Compared with model group, SIRT3 expression in treatment group increased to (86.7±7.6)%, JC-1 red/green [JP+1]to (75.8±7.6)%, Calcein fluorescence to (2571±199)U, ATP level to (86.7±6.3)% and cell viability of cells (83.0±3.6)%, but ROS level decreased to (180.0±18.1)%. Compared with treatment group, SIRT3 expression in inhibitor group decreased to (69.0±7.8)%, JC-1 red/green to (62.8±6.2)%, Calcein fluorescence to (2116±254)U, ATP level to (72.8±5.8)% and cell viability of cells (73.3±4.1)%, but the ROS level increased to (212.0±18.2)%. Conclusion Polydatin alleviates LPS-induced mitochondrial injury in alveolar epithelial cells possibly by the activation of SIRT3.