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胡黄连苦苷Ⅱ通过下调microRNA-1表达抑制H2O2诱导的心肌细胞凋亡
Effect of Picroside Ⅱ on Expression of microRNA-1 in H2O2-induced H9c2 Cardiomyocytes Damage
- 中国实验方剂学杂志 2019年25卷第8期 页码：77-82
- 作者机构：
广西中医药大学 附属瑞康医院，南宁 530011
- 作者简介：
邵庆瑞，硕士，主管药师，从事心血管药理学研究，Tel: 0771-2239133，E-mail: shaoqingrui123@126.com
李健哲，博士，副主任药师，从事心血管药理学研究，Tel: 0771-2239133，E-mail: lijianzhe20035@126.com
- 基金信息：国家自然科学基金项目(81460613);广西自然科学基金项目(2015GXNSFAA139115);广西卫计委资助项目(GZZC14-36)
DOI：10.13422/j.cnki.syfjx.20190802
中图分类号： R2-0;R22;R285.5收稿日期：2018-10-10，
网络出版日期：2019-01-04，
纸质出版日期：2019-04-20
- 稿件说明：
移动端阅览
邵庆瑞, 零伟德, 李健哲. 胡黄连苦苷Ⅱ通过下调microRNA-1表达抑制H2O2诱导的心肌细胞凋亡[J]. 中国实验方剂学杂志, 2019,25(8):77-82.
Qing-rui SHAO, Wei-de LING, Jian-zhe LI. Effect of Picroside Ⅱ on Expression of microRNA-1 in H2O2-induced H9c2 Cardiomyocytes Damage[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(8): 77-82.
邵庆瑞, 零伟德, 李健哲. 胡黄连苦苷Ⅱ通过下调microRNA-1表达抑制H2O2诱导的心肌细胞凋亡[J]. 中国实验方剂学杂志, 2019,25(8):77-82. DOI： 10.13422/j.cnki.syfjx.20190802.
Qing-rui SHAO, Wei-de LING, Jian-zhe LI. Effect of Picroside Ⅱ on Expression of microRNA-1 in H2O2-induced H9c2 Cardiomyocytes Damage[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(8): 77-82. DOI： 10.13422/j.cnki.syfjx.20190802.
摘要
目的：
2
研究胡黄连苦苷Ⅱ(picroside Ⅱ)对过氧化氢(H
2
O
2
)诱导的大鼠H9c2心肌细胞凋亡中微小RNA-1 (microRNA-1
miR-1)及下游靶基因抗凋亡因子B淋巴细胞瘤-2(B-cell lymphoma-2
Bcl-2)表达的影响，探讨胡黄连苦苷Ⅱ的心肌保护作用及其机制。
方法：
2
将H9c2心肌细胞随机分为正常组，模型组(H
2
O
2
，200 μmol·L
-1
)，胡黄连苦苷Ⅱ (50
100
200 μmol·L
-1
)+H
2
O
2
(200 μmol·L
-1
)组，胡黄连苦苷Ⅱ 200 μmol·L
-1
组。胡黄连苦苷Ⅱ与心肌细胞孵育6 h后，加入H
2
O
2
继续培养2 h。药物处理结束后，噻唑蓝(methyl thiazolyl tetrazolium
MTT)比色法检测细胞存活率，比色法测定细胞培养液中乳酸脱氢酶(lactate dehydrogenase
LDH)活性，4′，6-二脒基-2-苯基吲哚(destination access point identifier
DAPI)染色和细胞凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶-3 (cysteinyl aspartate specific proteinase-3，Caspase-3)检测评估细胞凋亡，实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞中Caspase-3，Bcl-2，miR-1 mRNA的表达，蛋白免疫印迹法(Western blot)检测细胞抗凋亡因子Bcl-2蛋白的表达。
结果：
2
与正常组相比，H
2
O
2
能显著降低细胞生存率，增加细胞凋亡率，且Caspase-3活性和mRNA表达均增加(
P
<
0.01)，同时伴有miR-1 mRNA表达水平的上调及其下游靶基因抗凋亡因子Bcl-2 mRNA表达的下调(
P
<
0.01)。与模型组比较，胡黄连苦苷Ⅱ预处理可明显升高细胞存活率，明显降低LDH活性，降低细胞凋亡率，明显降低Caspase-3活性和mRNA表达(
P
<
0.05，
P
<
0.01)，并使Bcl-2 mRNA表达升高(
P
<
0.05，
P
<
0.01)，Western blot结果表明，胡黄连苦苷Ⅱ能明显下调其下游靶基因抗凋亡因子Bcl-2蛋白表达(
P
<
0.05，
P
<
0.01)。
结论：
2
胡黄连苦苷Ⅱ对H
2
O
2
诱导的H9c2心肌细胞凋亡具有保护作用，其机制与下调 miR-1的表达进而上调其靶基因Bcl-2的表达有关。
Abstract
Objective:
2
To study the effect of picroside Ⅱ on the expression of microRNA-1 (miR-1) in the H
2
O
2
-induced H9c2 cardiomyocytes damage
in order to explore the mechanism of picroside Ⅱ in protecting H9c2 cardiomyocytes from oxidative stress.
Method:
2
H9c2 cardiomyocytes were divided into 6 groups: control group
model group (H
2
O
2
200 μmol·L
-1
)
picroside Ⅱ (50
100
200 μmol·L
-1
)+ H
2
O
2
(200 μmol·L
-1
) group and picroside Ⅱ (200 μmol·L
-1
) group. Picroside Ⅱ group was incubated with picroside Ⅱ for 6 h and then cultured with H
2
O
2
for 2 h. At the end of drugs treatment
the cell viability and the cellular damage of cardiomyocytes were respectively assessed by 3-(4
5-dimethylthiazol-2-yl)-2
5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. 4′
6-diamidino-2-phenylindole (DAPI) staining and cysteinyl aspartate specific proteinase-3 (Caspase-3) test were used to evaluate cell apoptosis. The mRNA expressions of Caspase-3
B-cell lymphoma-2 (Bcl-2) and miR-1 were measured by Real-time polymerase chain reaction (Real-time PCR). The protein expression of Bcl-2 was detected by Western blot.
Result:
2
Compared with the control group
H
2
O
2
could significantly decrease the cell viability and increase the rate of apoptosis
up-regulate mRNA expression of Caspase-3 and miR-1
and down-regulate expression of Bcl-2 in H9c2 cells (
P
<
0.01). Compared with the model group
the cell survival rate was significantly increased by pretreating with Picroside Ⅱ for 6 h (
P
<
0.05
P
<
0.01). Picroside Ⅱ not only decreased the rate of apoptosis and up-regulated mRNA expression of Bcl-2
but also down-regulated mRNA expressions of Caspase-3 and miR-1 (
P
<
0.05
P
<
0.01). Western blot showed that picroside Ⅱ can significantly enhance the expression of Bcl-2 (
P
<
0.05
P
<
0.01).
Conclusion:
2
Picroside Ⅱ has a protective effect on H9c2 cells from H
2
O
2
-induced cardiomyocyte injury by down-regulating mRNA-1 expression and up-regulating the expression of the downstream Bcl-2.
关键词
Keywords
references
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