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中华细胞与干细胞杂志(电子版) ›› 2022, Vol. 12 ›› Issue (03) : 145 -152. doi: 10.3877/cma.j.issn.2095-1221.2022.03.003

论著

类叶升麻苷通过上调miR-204对缺氧/复氧诱导H9C2细胞凋亡的抑制作用
郭周威1, 屈艳玲1, 李永慧1,()   
  1. 1. 044000 运城,山西省运城市中心医院心血管内二科
  • 收稿日期:2021-02-26 出版日期:2022-06-01
  • 通信作者: 李永慧

Acteoside inhibites hypoxia/reoxygenation-induced apoptosis by up-regulating miR-204 in H9C2 cells

Zhouwei Guo1, Yanling Qu1, Yonghui Li1,()   

  1. 1. The Second Department of Cardiovascular Medicine, Yuncheng Central Hospital, Shanxi 044000, China
  • Received:2021-02-26 Published:2022-06-01
  • Corresponding author: Yonghui Li
引用本文:

郭周威, 屈艳玲, 李永慧. 类叶升麻苷通过上调miR-204对缺氧/复氧诱导H9C2细胞凋亡的抑制作用[J/OL]. 中华细胞与干细胞杂志(电子版), 2022, 12(03): 145-152.

Zhouwei Guo, Yanling Qu, Yonghui Li. Acteoside inhibites hypoxia/reoxygenation-induced apoptosis by up-regulating miR-204 in H9C2 cells[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(03): 145-152.

目的

探讨类叶升麻苷对缺氧/复氧(H/R)处理大鼠心肌细胞(H9C2)损伤的影响及其分子机制。

方法

体外培养H9C2细胞,H/R (4 h/20 h)建立心肌细胞损伤模型,并采用1、10、100 μmol/L类叶升麻苷,转染miR-204模拟物阴性对照(miR-NC)、转染miR-204模拟物(miR-24),100 μmol/L类叶升麻苷干预+转染miR-204抑制剂阴性对照、100 μmol/L类叶升麻苷+转染miR-204抑制剂干预H/R细胞。分别进行RT-qPCR、MTT、流式细胞术、Western blot检测miR-204表达水平、细胞活力、细胞凋亡率和相关蛋白表达,利用相应试剂盒检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)水平,酶联免疫吸附试验(ELISA)检测白细胞介素-6 (IL-6)、白细胞介素-β (IL-β)和肿瘤坏死因子-α (TNF-α)的含量。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。

结果

与心肌损伤模型比较,10、100 μmol/L类叶升麻苷的细胞凋亡率[(25.62±1.96)%比(18.17±1.27)%,(11.24±0.57)%]、Bax(0.71±0.05比0.51±0.04、0.29±0.03)、LDH [(243.16±11.31)比(121.22±4.52),(94.39±2.82)U/g]、MDA [(1.82±0.07)比(1.13±0.04),(0.92±0.04)nmol/mg]、IL-6 [(121.45±6.18)比(87.16±4.53),(47.11± 2.24)pg/mL]、IL-1β [(229.82±8.48)比(175.32±8.73),(113.14±5.63)pg/mL]和TNF-α表达水平[(138.18±6.60)比(92.24±4.04),(61.53±4.17)pg/mL]降低,Bcl-2 (0.18±0.01比0.35± 0.03、0.52±0.04)、SOD [(18.72±1.26)比(38.81±1.51),(45.43±1.29)U/mg]和GSH-Px表达水平[(58.74±2.28)比(89.24±2.82),(94.66±3.05)U/mg]升高(P均< 0.05);与miR-NC比较,转染miR-204的细胞凋亡率[(24.12±1.12)%比(9.26±0.49)%]、Bax (0.62±0.04比0.25±0.02)、LDH [(229.11±8.47)比(86.32±5.92)U/g]、MDA [(1.75±0.08)比(0.85± 0.05)nmol/mg]、IL-6 [(134.47±7.31)比(55.26±2.13)pg/mL]、IL-1β [(211.14±9.70)比(98.11±3.18)pg/mL]和TNF-α表达水平[(152.92±3.49)比(51.34±2.66)pg/mL]降低,Bcl-2 (0.22±0.01比0.57±0.03)、SOD [(20.92±1.38)比(47.68±1.76)U/mg]和GSH-Px表达水平[(62.65±2.76)比(91.13±3.80)U/mg]升高(P < 0.05);10、100 μmol/L类叶升麻苷可提高H/R诱导H9C2细胞中miR-204的表达水平(P < 0.05);且下调miR-204逆转了类叶升麻苷对H/R处理H9C2细胞凋亡、氧化应激和炎症因子的影响。

结论

类叶升麻苷可能通过上调miR-204表达缓解H/R诱导的H9C2细胞损伤。

Objective

To investigate the effect and molecular mechanism of acteoside on injury of rat myocardial cells (H9C2) treated with hypoxia/reoxygenation (H/R) .

Methods

H9C2 cells were cultured in vitro, and H/R (4 h/20 h) was used to establish cardiomyocytes injury model. The H/R-induced cells were treated with 1, 10, 100 μmol/L acteoside, transfected with miR-204 mimics negative control (miR-NC) , miR-204 mimics (miR-204) , treated with 100 μmol/L acteoside plus transfected with miR-204 inhibitor negative control or miR- 204 inhibitor. RT-qPCR, MTT, flow cytometry and Western blot were used to detect miR-204 expression level, cell activity, apoptosis rate and the expression of related proteins, respectively. The corresponding kits were used to detect the expression levels of lactate dehydrogenase (LDH) , malondialdehyde (MDA) , superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) . ELISA was used to evaluate the levels of interleukin-6 (IL-6) , interleukin-β (IL-β) and tumor necrosis factor-α (TNF-α) . Independent sample t test was used for comparison between two groups, one-way analysis of variance (ANOVA) was used for comparison between multiple groups, and SNK-q test was used for pair wise comparison between groups.

Results

Compared with cardiomyocytes injury model, the apoptosis rate [ (25.62±1.96) % vs (18.17±1.27) %, (11.24±0.57) %], the expression levels of Bax (0.71±0.05 vs 0.51±0.04, 0.29±0.03) , LDH [ (243.16±11.31) vs (121.22±4.52) , (94.39±2.82) U/g], MDA [ (1.82±0.07) vs (1.13±0.04) , (0.92±0.04) nmol/mg], IL-6 [ (121.45±6.18) vs (87.16± 4.53) , (47.11±2.24) pg/mL], IL-1β [ (229.82±8.48) vs (175.32±8.73) , (113.14±5.63) pg/mL] and TNF-α [ (138.18±6.60) vs (92.24±4.04) , (61.53±4.17) pg/mL] were significantly decreased in 10 and 100 μmol/L acteoside groups, and the expression levels of Bcl-2 (0.18±0.01 vs 0.35±0.03, 0.52±0.04) , SOD [ (18.72±1.26) vs (38.81±1.51) , (45.43±1.29) U/mg] and GSH-Px [ (58.74±2.28) vs (89.24±2.82) , (94.66±3.05) U/mg] were significantly increased (P < 0.05) . Compared with miR-NC group, apoptosis rate [ (24.12±1.12) %vs (9.26±0.49) %], the expression levels of Bax (0.62±0.04 vs 0.25±0.02) , LDH [ (229.11±8.47) vs (86.32±5.92) U/g], MDA [ (1.75±0.08) vs (0.85±0.05) nmol/mg], IL-6 [ (134.47±7.31) vs (55.26±2.13) pg/mL], IL-1β[ (211.14±9.70) vs (98.11±3.18) pg/mL] and TNF-α[ (152.92±3.49 vs 51.34±2.66) pg/mL] were significantly decreased, and the expression levels of Bcl-2 (0.22±0.01vs 0.57±0.03) , SOD [ (20.92±1.38) vs (47.68±1.76) U/mg] and GSH-Px [ (62.65±2.76) vs (91.13±3.80) U/mg] were significantly increased in miR-204 group (P < 0.05) . 10, 100 μmol/L acteoside, significantly increased the expression level of miR-204 in H/R-induced cells. In addition, down-regulation of miR-204 reversed the effects of acteoside on the apoptosis, oxidative stress and inflammatory factor of H9C2 cells treated with H/R (P < 0.05) .

Conclusion

Acteoside may alleviate H/R-induced H9C2 cell injury by up-regulating the expression of miR-204.

表1 引物序列信息
表2 类叶升麻苷对H9C2细胞活力的影响( ± s
图1 类叶升麻苷对H/R处理H9C2细胞凋亡的影响注:a ~ e图分别为流式细胞术检测对照、模型、1、10、100 μmol/L类叶升麻苷后进行H/R处理的H9C2细胞凋亡率;f图为Western blot检测Bcl-2和Bax的蛋白表达水平
表3 类叶升麻苷对H/R处理H9C2细胞凋亡的影响( ± s
表4 类叶升麻苷对H/R处理H9C2细胞氧化应激和炎症因子的影响( ± sn = 3)
图2 上调miR-204表达对H/R处理H9C2细胞凋亡的影响注:a ~ b图为流式细胞术检测转染miR-NC和miR-204后进行H/R处理的细胞凋亡率;c图为Western blot检测Bcl-2和Bax的蛋白表达水平
表5 上调miR-204表达对H/R处理H9C2细胞凋亡的影响( ± s
表6 上调miR-204表达对H/R处理H9C2细胞氧化应激和炎症因子的影响( ± s
图3 下调miR-204表达逆转了类叶升麻苷对H/R处理H9C2细胞凋亡的影响注:a ~ b图分别为流式细胞术检测转染100 μmol/L类叶升麻苷+anti-miR-NC和100 μmol/L类叶升麻苷+anti-miR-204后进行H/R处理的H9C2细胞凋亡率;c图为Western blot检测Bcl-2和Bax的蛋白表达水平
表7 下调miR-204表达逆转了类叶升麻苷对H/R处理H9C2细胞凋亡的影响( ± s
表8 下调miR-204表达逆转了类叶升麻苷对H/R处理H9C2细胞氧化应激和炎症因子的影响( ± s
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