摘要
In the present study,we aimed to confirm whether NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC)could induce apoptosis of human hepatic stellate cells(HSCs)LX-2 and explore the potential pharmacological mechanism underlying these effects.In this study,LX-2 cells were cultured in vitro,and the experiment was divided into two groups,including the control and PDTC groups.The viability of LX-2 cells was measured by CCK8 assay after the cells were exposed to PDTC.The anti-apoptotic effect of PDTC was detected by AO/EB double assay staining kit.Additionally,the activities of NF-κB,Fas/FasL,apoptosis-related proteins,as well as the cellular localization of AIF,were determined by Western blotting analysis and immunofluorescence staining respectively.After PDTC treatment for 12 and 24 h,AO/EB dual staining showed typical apoptotic changes,such as cell volume reduction,cell shrinkage,nuclear fragmentation,and so on.PDTC at 60μmol/L significantly increased the proliferation inhibition rate and decreased the secretion of collagen I,collagen III,andα-SMA in LX-2 cells.The Western blotting analysis and RT-PCR showed no significant difference in the expression of AIF between the control group and PDTC group,and the expressions of Fas and FasL were not observed in all groups(P>0.05).Further results showed that PDTC could promote the displacement of AIF from mitochondria to the nucleus,activate the apoptotic signaling in the cell nucleus,and possibly participate in the apoptosis process of LX-2 cells.In conclusion,the pharmacological mechanism of PDTC against hepatic fibrosis might be to promote the displacement of AIF from mitochondria to the nucleus,then activate the apoptotic signaling in the cell nucleus,and finally induce the apoptosis of LX-2 cells.Meanwhile,these results also revealed that the Fas/FasL-mediated apoptosis pathway was not involved in the PDTC-induced apoptosis process of LX-2 cells.
本研究旨在探讨NF-κB抑制剂吡咯烷二硫代氨基甲酸酯(PDTC)是否可以诱导人肝星状细胞LX-2凋亡,并分析其潜在药理机制。通过体外培养人肝星状细胞系LX-2细胞,将实验分为空白对照组和PDTC干预组。将LX-2细胞暴露于PDTC后,使用CCK8检测法评估LX-2细胞活力;采用AO/EB双染试剂盒检测PDTC的抗凋亡作用。此外,分别采用Western blotting法和免疫荧光染色法测定NF-κB、Fas/FasL、凋亡相关蛋白的活性和AIF的细胞定位。PDTC处理12和24小时后,AO/EB双染呈现典型的凋亡变化,比如细胞体积缩小、细胞核固缩、核碎裂等。PDTC干预浓度在60μmol/L时可显著提高细胞增殖抑制率,减少LX-2细胞中的胶原蛋白I、胶原蛋白III和α-SMA的分泌。Western blotting和RT-PCR结果显示,对照组与PDTC干预组的AIF表达水平无统计学意义,且各组均未观察到Fas和FasL的表达(P>0.05)。PDTC还可以促进AIF从线粒体向细胞核的移位,激活细胞核内的凋亡信号,并可能参与LX-2细胞的凋亡过程。综上所述,PDTC抗肝纤维化的药理机制可能是促进AIF从线粒体向细胞核移位,进而激活细胞核内的凋亡信号,最终诱导LX-2细胞凋亡。同时,也揭示了Fas/FasL介导的细胞凋亡通路不参与PDTC诱导的LX-2细胞凋亡过程。
基金
supported by the Fujian Provincial Natural Science Foundation,China(Grant No.2019J01611)
the Youth Project of Fujian Provincial Health Commission,Fujian Province,China(Grant No.2017-1-94).
