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丹酚酸B对三氧化二砷诱导HepaRG人肝细胞损伤的保护作用
Protective Effect of Salvianolic Acid B on Arsenic Trioxide-induced HepaRG Human Hepatocyte Injury
- 中国实验方剂学杂志 2020年26卷第8期 页码：68-74
- 作者机构：
1.哈尔滨商业大学 药学院，哈尔滨 150076
2.中国医学科学院 药用植物研究所，北京 100193
- 作者简介：
[第一作者] 孙佳富，在读硕士，从事心血管药理学方向研究，E-mail：1002269060@qq.com
*孙晓波，研究员，博士生导师，从事中药药理学方向研究，E-mail：sun-xiaobo@163.com
*孙桂波，研究员，博士生导师，从事中药药理学方向研究，E-mail：sunguibo@126.com；
- 基金信息：国家科技重大专项(2017ZX09301069)
DOI：10.13422/j.cnki.syfjx.20200821
中图分类号： R22; R242; R2-031;R285.5收稿日期：2019-08-26，
网络出版日期：2020-01-07，
纸质出版日期：2020-04-20
- 稿件说明：
移动端阅览
孙佳富, 孙潇, 罗云, 等. 丹酚酸B对三氧化二砷诱导HepaRG人肝细胞损伤的保护作用[J]. 中国实验方剂学杂志, 2020,26(8):68-74.
Jia-fu SUN, Xiao SUN, Yun LUO, et al. Protective Effect of Salvianolic Acid B on Arsenic Trioxide-induced HepaRG Human Hepatocyte Injury[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(8): 68-74.
孙佳富, 孙潇, 罗云, 等. 丹酚酸B对三氧化二砷诱导HepaRG人肝细胞损伤的保护作用[J]. 中国实验方剂学杂志, 2020,26(8):68-74. DOI： 10.13422/j.cnki.syfjx.20200821.
Jia-fu SUN, Xiao SUN, Yun LUO, et al. Protective Effect of Salvianolic Acid B on Arsenic Trioxide-induced HepaRG Human Hepatocyte Injury[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(8): 68-74. DOI： 10.13422/j.cnki.syfjx.20200821.
摘要
目的：
2
探讨丹酚酸B对三氧化二砷(As
2
O
3
)诱导的HepaRG人肝细胞损伤的保护作用及其机制。
方法：
2
采用5 μmol·L
-1
As
2
O
3
孵育24 h建立HepaRG人肝细胞损伤模型。实验设置空白组、模型组(As
2
O
3
5 μmol·L
-1
)，单给药组(丹酚酸B 10 μmol·L
-1
)，丹酚酸B高浓度组(丹酚酸B 10 μmol·L
-1
＋ As
2
O
3
5 μmol·L
-1
)，丹酚酸B中浓度组(丹酚酸B 5 μmol·L
-1
＋As
2
O
3
5 μmol·L
-1
)，丹酚酸B低浓度组(丹酚酸B 2.5 μmol·L
-1
＋ As
2
O
3
5 μmol·L
-1
)。丹酚酸B预孵育2 h，弃去含药培养基，继续用5 μmol·L
-1
As
2
O
3
孵育24 h；实验终止，通过噻唑蓝(MTT)比色法检测细胞存活率，Hoechst33342荧光染色观察细胞凋亡，Annexin V-FITC/碘化丙啶(PI)双染色流式细胞术定量检测细胞凋亡率，JC-1荧光染色观察线粒体膜电位，蛋白免疫印迹法(Western blot)检测相关蛋白B淋巴细胞瘤-2(Bcl-2)，Bcl-2相关X蛋白(Bax)，蛋白激酶B(Akt)，磷酸化Akt(p-Akt)表达对丹酚酸B的肝脏保护作用机制。
结果：
2
As
2
O
3
浓度依赖性的降低HepaRG细胞的存活率(
P
<
0.01)；丹酚酸B单独作用2 h对正常细胞活力无影响；丹酚酸B(5，10 μmol·L
-1
)预孵育2 h可显著增加由As
2
O
3
引起的细胞存活率下降(
P
<
0.01)。As
2
O
3
导致肝细胞凋亡比例显著上升(
P
<
0.01)，丹酚酸B(10 μmol·L
-1
)预孵育可降低细胞凋亡率(
P
<
0.01)；As
2
O
3
孵育24 h引起线粒体膜电位下降，丹酚酸B可维持线粒体膜电位，提示丹酚酸B的抗凋亡作用与其抑制凋亡线粒体通路有关；与As
2
O
3
组比较，丹酚酸B预处理升高了Bcl-2/Bax水平(
P
<
0.01)，增加了p-Akt/Akt水平(
P
<
0.01)。
结论：
2
丹酚酸B对As
2
O
3
诱导的肝细胞损伤具有保护作用，其作用机制与维持线粒体功能、抑制肝细胞凋亡有关。
Abstract
Objective:
2
To investigate the protective effect of salvianolic acid B on HepaRG hepatocyte injury induced by arsenic trioxide (As
2
O
3
) and its mechanism.
Method:
2
HepaRG cells were incubated with 5μmol·L
-1
As
2
O
3
for 24 h to induce hepatocyte injury. The cells were divided into control group
model group
salvianolic acid B 10 μmol·L
-1
group
salvianolic acid B 10 μmol·L
-1
+ As
2
O
3
group
salvianolic acid B 5 μmol·L
-1
+ As
2
O
3
group
and salvianolic acid B 2.5 μmol·L
-1
+ As
2
O
3
group. HepaRG cells were preincubated with salvianolic acid B for 2 h and then incubated with As
2
O
3
for 24 h. At the end of the incubation
cell viability was detected by thiazolyl blue tetrazolium bromide assay
apoptosis was observed by Hoechst33342 fluorescence staining
apoptosis rate was detected by annexin V-FITC/propidium iodide double staining flow cytometry
and mitochondrial membrane was observed by JC-1 fluorescence staining. Western blot was used to detect the protective effect of expressions of relevant proteins Bcl-2
Bax
Akt
p-Akt on salvianolic acid B in the liver.
Result:
2
As
2
O
3
concentration-dependently reduced the survival rate of HepaRG cells(
P
<
0.01)
salvianolic acid B had no effect on normal cell viability for 2 h
pre-incubation with salvianolic acid B(5
10 μmol·L
-1
) for 2 h significantly increased the decreased cell survival rate caused by As
2
O
3
(
P
<
0.01). As
2
O
3
significantly increased hepatocytes apoptosis rate(
P
<
0.01)
while pre-incubation with salvianolic acid B(10 μmol·L
-1
) deceased apoptosis rate(
P
<
0.01). Incubation with As
2
O
3
for 24 h caused decrease of mitochondrial membrane potential
pre-incubation with salvianolic acid B maintained mitochondrial membrane potential
indicating that the anti-apoptotic effect of salvianolic acid B were related to the mitochondrial pathway modulation. Western blot analysis showed that salvianolic acid B promoted the ratio of Bcl-2/Bax and promoted p-Akt/Akt compared with As
2
O
3
group(
P
<
0.01).
Conclusion:
2
Salvianolic acid B has a protective effect on hepatocyte injury induced by As
2
O
3
and its mechanism is related to maintenance of mitochondrial function and inhibition of hepatocyte apoptosis.
关键词
Keywords
references
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