砷酸氢钠对人肝星状细胞凋亡和自噬的影响

惠瑜; 袁艳杰; 孙立; 吴顺华

doi:10.13213/j.cnki.jeom.2020.19835

砷酸氢钠对人肝星状细胞凋亡和自噬的影响

Effects of sodium arsenate on apoptosis and autophagy of human hepatic stellate cells

摘要

摘要:
背景肝星状细胞是肝脏主要的效应细胞，其活化被认为是促进肝纤维化发展的主要机制之一。自噬和凋亡在肝星状细胞的激活中起关键作用。
目的探讨砷酸氢钠（Na₂HAsO₄）对人肝星状细胞（LX-2细胞）凋亡和自噬的影响。
方法体外培养LX-2细胞株，使用红色荧光蛋白（RFP）-绿色荧光蛋白（GFP）-微管相关蛋白轻链3（LC3）慢病毒稳定感染LX-2细胞，采用流式细胞仪进行筛选和感染率的测定。用不同浓度Na₂HAsO₄（0、0.6、6、60 μmol·L^-1）孵育慢病毒稳定感染的LX-2细胞，同时设立未经慢病毒感染正常培养的LX-2细胞为空白组。采用CCK-8法检测LX-2细胞活性；采用流式细胞仪检测LX-2细胞凋亡；采用实时荧光定量PCR和Western blotting检测自噬指标LC3、重组人自噬效应蛋白（Beclin-1）、泛素结合蛋白P62（SQSTM-1/P62）以及凋亡指标B淋巴细胞瘤-2（BCL-2）。
结果 RFP-GFP-LC3慢病毒稳定感染后，LX-2细胞慢病毒感染率为70%，在激光共聚焦下观察RFP和GFP的荧光强度无明显差异。Na₂HAsO₄处理LX-2细胞24、48、72 h后，与Na₂HAsO₄（0 μmol·L^-1）比较，除LC3+Na₂HAsO₄（0 μmol·L^-1）组外，其余各剂量组细胞抑制率均升高（P < 0.05）。随着Na₂HAsO₄染毒剂量的增加，凋亡率呈上升趋势，且与Na₂HAsO₄（0 μmol·L^-1）组相比，差异均有统计学意义（P < 0.05）。各组间LC3（F=5.64，340.66）、Beclin-1（F=20.59，87.70）、SQSTM-1/P62（F=15.91，107.24）、BCL-2（F=113.29，9.41）mRNA和蛋白表达水平差异均具有统计学意义（P < 0.05）。
结论 Na₂HAsO₄可诱导LX-2细胞发生细胞凋亡和自噬，凋亡和自噬之间可能存在拮抗作用。

Abstract:
Background Hepatic stellate cells are the main effector cells of the liver, and their activation is considered to be one of the main mechanisms that promote the development of liver fibrosis. Autophagy and apoptosis have been reported to play a key role in the activation of hepatic stellate cells.
Objective This experiment explores the effects of sodium arsenate on the apoptosis and autophagy of human hepatic stellate cells (LX-2 cells).
Methods A LX-2 cell line was cultured in vitro, and stably infected with the red fluorescent protein (RFP)-green fluorescent protein (GFP)-microtubule-associated protein light chain 3 (LC3) lentivirus. Flow cytometry was used to screen and determine the infection rate. LX-2 cells infected with lentivirus were treated with different concentrations of Na₂HAsO₄ (0, 0.6, 6, and 60 μmol·L^-1), and LX-2 cells without lentivirus infection were used as blank control group. CCK-8 method was used to detect the activity of LX-2 cells, flow cytometry for the apoptosis of LX-2 cells, and real-time fluorescence quantitative PCR (RT-PCR) and Western blotting for autophagy indicatorsLC3, recombinant human autophagy effector protein (Beclin-1), and sequestosome 1/P62 (SQSTM-1/P62) and apoptosis indicatorB-cell lymphoma-2 (BCL-2).
Results After the RFP-GFP-LC3 lentivirus infection, the lentivirus infection rate of the LX-2 cells was 70%. The fluorescence expression intensity was not different between RFP and GFP under laser confocal microscope. After designed Na₂HAsO₄ treatment for 24, 48, and 72 h, compared with the Na₂HAsO₄ (0 μmol·L^-1) group, the cell inhibition rate of the other Na₂HAsO₄ dose groups all increased (P < 0.05), except the LC3+Na₂HAsO₄ (0 μmol·L^-1) group. With the increase of Na₂HAsO₄ dose, the apoptosis rate showed an upward trend, and was significantly higher than that of the Na₂HAsO₄ (0 μmol·L^-1) group (P < 0.05). The mRNA and protein expression levels of LC3 (F=5.64, 340.66), Beclin-1 (F=20.59, 87.70), SQSTM-1/P62 (F=15.91, 107.24), and BCL-2 (F=113.29, 9.41) were different among the designed groups (P < 0.05).
Conclusion Na₂HAsO₄ can induce apoptosis and autophagy in LX-2 cells, and there may be an antagonistic effect between apoptosis and autophagy.

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