Mechanism of Trichosanthis Pericarpium Aqueous Extract in Protecting H9c2 Cardiomyocytes from Hypoxia/Reoxygenation Injury via PI3K/Akt/NO Signaling Pathway

Dong-hai CHU; Zhen-qiu ZHANG

doi:10.13422/j.cnki.syfjx.20192104

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Mechanism of Trichosanthis Pericarpium Aqueous Extract in Protecting H9c2 Cardiomyocytes from Hypoxia/Reoxygenation Injury via PI3K/Akt/NO Signaling Pathway

Pharmacology | 更新时间：2021-02-09

- Mechanism of Trichosanthis Pericarpium Aqueous Extract in Protecting H9c2 Cardiomyocytes from Hypoxia/Reoxygenation Injury via PI3K/Akt/NO Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 22, Pages: 42-48(2019)
- 作者机构：
  
  1.辽宁科技学院　生物医药与化学工程学院，辽宁　本溪　 117004；
  2. 　辽宁中医药大学　药学院，辽宁　大连　 116600
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20192104
  CLC： R2-0; R22; R285.5
- Published：20 November 2019，
  
  Published Online：18 July 2019，
  
  Received：03 April 2019，
- 稿件说明：
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Dong-hai CHU, Zhen-qiu ZHANG. Mechanism of Trichosanthis Pericarpium Aqueous Extract in Protecting H9c2 Cardiomyocytes from Hypoxia/Reoxygenation Injury via PI3K/Akt/NO Signaling Pathway. [J]. Chinese Journal of Experimental Traditional Medical Formulae 25(22):42-48(2019)
DOI：

Dong-hai CHU, Zhen-qiu ZHANG. Mechanism of Trichosanthis Pericarpium Aqueous Extract in Protecting H9c2 Cardiomyocytes from Hypoxia/Reoxygenation Injury via PI3K/Akt/NO Signaling Pathway. [J]. Chinese Journal of Experimental Traditional Medical Formulae 25(22):42-48(2019) DOI： 10.13422/j.cnki.syfjx.20192104.

摘要

目的：

研究磷脂酰肌醇3-激酶/蛋白激酶B/一氧化氮(PI3K/Akt/NO)信号通路在瓜蒌皮提取物保护心肌细胞缺氧/复氧损伤中的作用。

方法：

以2.5 mmol·L

－1

诱导心肌细胞建立缺氧/复氧损伤模型，实验分为正常组、模型组、瓜蒌皮提取物组及抑制剂组。采用噻唑蓝(MTT)比色法检测细胞活力；酶联免疫吸附测定(ELISA)检测心肌细胞中一氧化氮(NO)的释放量及内皮型一氧化氮合酶(eNOS)，诱导型一氧化氮合酶(iNOS)的活性水平；实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞中Akt，eNOS，iNOS mRNA表达水平；蛋白免疫印迹法(Western blot)测定Akt，磷酸化Akt(p-Akt)，eNOS，iNOS蛋白表达水平。

结果：

与正常组比较，模型组心肌细胞活力明显下降(

0.01)，心肌细胞NO释放量减少(

0.01)，心肌细胞中p-Akt，Akt及eNOS mRNA和蛋白表达显著降低(

0.01)，而iNOS mRNA和蛋白表达显著升高(

0.01)；与模型组比较，瓜蒌皮提取物预处理24 h后能改善心肌细胞形态，明显提高心肌细胞活力(

0.01)。瓜蒌皮提取物能促进p-Akt，Akt及eNOS mRNA和蛋白表达(

0.01)，抑制iNOS mRNA和蛋白的表达(

0.01)，增加心肌细胞NO的释放量(

0.01)。PI3K抑制剂LY294002与瓜蒌皮提取物联合处理后，逆转了瓜蒌皮提取物对心肌细胞的上述作用，导致细胞存活率下降。

结论：

瓜蒌皮提取物可能通过激活PI3K/Akt信号通路上调eNOS，下调iNOS的表达，增加内源性NO的基础含量水平，发挥抗凋亡作用，从而改善缺氧/复氧损伤诱导的心肌细胞损伤。

Abstract

Objective:

To investigate the effect of Trichosanthis Pericarpium aqueous extract(TPAE)in protecting H9c2 cells from hypoxia/reoxygenation (H/R) injury by activating phosphatidylionsitol-3-kinase/protein kinase B/nitric oxide(PI3K/Akt/NO) signaling pathway.

Method:

The 2.5 mmol·L

-1

was used to induce the model of H9c2 cardiomyocytes H/R injury in the experiments. The cultured H9c2 cardiomyocytes were randomly divided into normal group

H/R group (model group) and inhibition group (LY294002

10 μmol·L

-1

). In the H/R + TPAE group

50 mg·L

-1

TPAE was added to the cultures at 24 h before H/R exposure. Cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay. The amounts of NO

endothelial nitric oxide synthase (eNOS)

and induced nitric oxide synthase (iNOS) were tested by enzyme linked immunosorbent assay (ELISA) kits. Reverse transcription-quantitative real-time polymerase chain reaction (Real-time PCR) was performed to analyze relative mRNA expressions of Akt

eNOS and iNOS. Western blot was used to detect the expressions of Akt

p-Akt (Ser 473)

eNOS

and iNOS.

Result:

Compared with the normal control group

the cell viability significantly decreased in the model control group (

0.01)

the release of NO was obviously down-regulated (

0.01)

the mRNA and protein expressions of p-Akt

Akt

eNOS were remarkably decreased (

0.01)

while those of iNOS were up-regulated (

0.01). Compared with the H/R group

the pretreatment with TPAE remarkably improved the morphological lesion of cardiomyocytes

enhanced cell viability(

0.01)

increased the expressions of Akt

p-Akt (Ser 473) and eNOS(

0.01)

decreased the expression of iNOS(

0.01)

and increased the release of NO(

0.01). The PI3K inhibitor LY294002 was used. Obviously

cardioprotection of Trichosanthis Pericarpium aqueous extract was blocked by co-treatment LY294002

and cell viability was correspondingly reversed.

Conclusion:

TPAE can protect H9c2 cardiomyocytes from H/R injury by activating PI3K/Akt signaling pathway

which might be related to the up-regulation of the mRNA and protein expressions of eNOS

the down-regulation of the level of iNOS

and the increase of the production of physiological amounts of NO.

关键词

瓜蒌皮提取物缺氧/复氧损伤心肌细胞磷脂酰肌醇3-激酶/蛋白激酶B/一氧化氮(PI3K/Akt/NO)

Keywords

Trichosanthis Pericarpium aqueous extracthypoxia/reoxygenation injurycardiomyocytesphosphatidylionsitol-3-kinase/protein kinase B/nitric oxide(PI3K/Akt/NO)

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