加味涤痰汤对慢性间歇低氧大鼠心肌铁死亡的影响

王奕锦; 陈沁; 吴润华; 林凡; 陈丽丽; 伍娟; 王春桔; 杨眉峰

doi:10.3969/j.issn.1006-2157.2024.10.013

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加味涤痰汤对慢性间歇低氧大鼠心肌铁死亡的影响

实验研究 | 更新时间：2024-10-29

- 加味涤痰汤对慢性间歇低氧大鼠心肌铁死亡的影响
- Effect of Jiawei Ditan Decoction on cardiomyocyte ferroptosis in rats with chronic intermittent hypoxia
- 北京中医药大学学报 2024年47卷第10期页码：1427-1437
- 作者机构：
  
  1.福建中医药大学中西医结合学院　福州　350122
  2.福建中医药大学临床技能教学中心
- 作者简介：
  
  王奕锦，男，在读硕士生
  #杨眉峰，女，硕士，高级实验师，主要研究方向：中医药防治呼吸系统疾病，E-mail：2007068@fjtcm.edu.cn
- 基金信息：
  
  国家自然科学基金项目(82074192)
- DOI：10.3969/j.issn.1006-2157.2024.10.013
  中图分类号： R285.5
- 收稿：2024-05-09，
  
  纸质出版：2024-10-30
- 稿件说明：
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王奕锦, 陈沁, 吴润华, 等. 加味涤痰汤对慢性间歇低氧大鼠心肌铁死亡的影响[J]. 北京中医药大学学报, 2024,47(10):1427-1437.

WANG Yijin, CHEN Qin, WU Runhua, et al. Effect of Jiawei Ditan Decoction on cardiomyocyte ferroptosis in rats with chronic intermittent hypoxia[J]. Journal of Beijing University of Traditional Chinese Medicine, 2024, 47(10): 1427-1437. DOI: 10.3969/j.issn.1006-2157.2024.10.013.
王奕锦, 陈沁, 吴润华, 等. 加味涤痰汤对慢性间歇低氧大鼠心肌铁死亡的影响[J]. 北京中医药大学学报, 2024,47(10):1427-1437. DOI： 10.3969/j.issn.1006-2157.2024.10.013.

WANG Yijin, CHEN Qin, WU Runhua, et al. Effect of Jiawei Ditan Decoction on cardiomyocyte ferroptosis in rats with chronic intermittent hypoxia[J]. Journal of Beijing University of Traditional Chinese Medicine, 2024, 47(10): 1427-1437. DOI: 10.3969/j.issn.1006-2157.2024.10.013. DOI：

摘要

目的

基于磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路探讨加味涤痰汤对慢性间歇低氧大鼠心肌铁死亡的影响及其机制。

方法

将30只8周龄雄性SD大鼠以随机数字表法分为正常对照组、低氧干预组、加味涤痰汤干预组，每组10只。低氧干预组和加味涤痰汤干预组采用间歇低氧箱造模，每天干预8 h。每日干预前后予以灌胃，加味涤痰汤干预组药物剂量为16.38 g/kg，其余组予生理盐水，持续12周。HE染色观察心肌组织形态学；透射电镜观察心肌细胞线粒体超微结构；生化法检测心肌组织Fe

2＋

、丙二醛(MDA)含量；蛋白质印迹法检测心肌组织磷酸化PI3K(p-PI3K)、PI3K、磷酸化Akt(p-Akt)、Akt、核因子E2相关因子2(Nrf2)、谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)、酰基辅酶A合成酶长链家族成员4(ACSL4)蛋白表达。DMEM高糖培养基培养大鼠心肌细胞(H9c2)；超高效液相色谱检测加味涤痰汤药物成分；CCK－8检测不同质量浓度加味涤痰汤干预下的细胞存活率。将细胞分为常氧组、铁死亡诱导剂组、铁死亡诱导剂＋加味涤痰汤干预组，采用蛋白质印迹法检测p-Akt、GPX4蛋白表达；常氧组、低氧干预12 h组、低氧干预12 h＋740Y-P组检测p-PI3K、GPX4蛋白表达；常氧组、低氧干预6 h组、低氧干预12 h组、低氧干预12 h＋加味涤痰汤低、中、高剂量组(药物质量浓度分别为5、10和20 g/L)，检测p-PI3K、PI3K、p-Akt、Akt、GPX4蛋白表达。

结果

与正常对照组比较，低氧干预组大鼠心肌细胞排列紊乱、肿胀，线粒体排列混乱、膜密度增高、嵴断裂，同时伴有空泡变性；与低氧干预组比较，加味涤痰汤干预组心肌细胞排列较为整齐，未见明显肿胀，线粒体形态较为规则。与正常对照组比较，低氧干预组MDA、Fe

2＋

含量升高，p-PI3K/PI3K、p-Akt/Akt值降低，SLC7A11、GPX4、Nrf2表达减少，ACSL4表达升高(

0.05)；与低氧干预组比较，加味涤痰汤干预组MDA、Fe

2＋

含量降低，p-PI3K/PI3K、p-Akt/Akt值升高，SLC7A11、GPX4、Nrf2表达升高，ACSL4表达降低(

0.05)。超高效液相色谱检测加味涤痰汤含有DL-水苏碱、D-(＋)-苹果酸、腺苷等。与加味涤痰汤0、1.25、2.50、5.00和20.00 g/L组比较，10.00 g/L组的细胞存活率增高(

0.05)。与常氧组比较，铁死亡诱导剂组的p-Akt、GPX4表达降低(

0.05)；与铁死亡诱导剂组比较，铁死亡诱导剂＋加味涤痰汤干预组p-Akt、GPX4表达升高(

0.05)；与常氧组比较，低氧干预12 h组的p-PI3K、GPX4表达减少(

0.05)；与低氧干预12 h组比较，低氧干预12 h＋740Y-P组p-PI3K、GPX4表达升高(

0.05)；与常氧组比较，低氧干预6 h组、低氧干预12 h组的GPX4表达减少，p-Akt/Akt、p-PI3K/PI3K值均降低(

0.05)；与低氧干预6 h组、低氧干预12 h组比较，低氧干预12 h＋加味涤痰汤低、中、高剂量组的p-PI3K/PI3K、p-Akt/Akt值升高，GPX4表达升高(

0.05)。

结论

加味涤痰汤可能通过调节PI3K/Akt信号通路改善大鼠心肌铁死亡，从而对慢性间歇低氧大鼠心脏损伤起到一定的保护作用。

Abstract

Objective

To investigate the effect and mechanism of

Jiawei Ditan

Decoction on cardiomyocyte ferroptosis in rats with chronic intermittent hypoxia based on the phosphoinositide 3-kinase(PI3K)/protein kinase B (Akt) signaling pathway.

Methods

Thirty 8-week-old male Sprague-Dawley rats were randomly divided into normal control

low-oxygen intervention

and

Jiawei Ditan

Decoction intervention groups using the random number table method

with 10 rats per group. The low-oxygen and

Jiawei Ditan

Decoction intervention groups were treated with intermittent hypoxia chamber for modeling

with 8 h of intervention daily. Gavage administration was performed before and after daily intervention. The intervention group received a dosage of 16.38 g/kg

Jiawei Ditan

Decoction

whereas the other two groups received normal saline. The experimental intervention period was 12 weeks. Hematoxylin and eosin staining was used to observe the morphology of myocardial tissue. Transmission electron microscope was used to observe the mitochondrial ultrastructure in cardiomyocytes. Biochemical detection was used to measure the Fe

and malondialdehyde (MDA) content in myocardial tissue. Phosphorylated-phosphoinositide 3-kinase (p-PI3K)

PI3K

phosphorylated-protein kinase B (p-Akt)

Akt

solute carrier family 7 member 11 (SLC7A11)

glutathione peroxidase 4 (GPX4)

nuclear factor erythroid 2-related factor 2 (Nrf2)

and acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) protein expression in myocardial tissue were measured using western blotting. The rat cardiomyocyte cell line

H9c2

was cultured in high glucose Dulbecco′s Modified Eagle Medium

and the ultra-high-performance liquid chromatography was used to detect drug components in

Jiawei Ditan

Decoction. The CCK-8 assay was used to

detect the cell survival rate of cells treated with different concentrations of

Jiawei Ditan

Decoction. The cells were divided into the normoxic

erastin

and erastin +

Jiawei Ditan

Decoction intervention groups

and p-Akt and GPX4 protein expression was measured using western blotting. p-PI3K and GPX4 protein expression were detected in the normoxic

low-oxygen intervention 12 h

and low-oxygen intervention 12 h + 740Y-P groups. The p-PI3K

PI3K

p-Akt

Akt

and GPX4 protein expression was detected in the normoxic

low-oxygen intervention 6 h

low-oxygen intervention 12 h

low-oxygen intervention 12 h +

Jiawei Ditan

Decoction low-dose

low-oxygen intervention 12 h +

Jiawei Ditan

Decoction medium-dose

and low-oxygen intervention 12 h +

Jiawei Ditan

Decoction high-dose groups (drug concentrations of 5

and 20 g/L

respectively).

Results

The myocardial cells in the low-oxygen intervention group were disordered and swollen and the mitochondrial arrangement of myocardial cells was disordered

with increased mitochondrial membrane density

ruptured cristae

and vacudar degeneration compared with those in the normal control group. The myocardial cells in the

Jiawei Ditan

Decoction intervention group were arranged neater

and the morphology of mitochondria was relatively regular than those in the low-oxygen intervention group

and no apparent swelling was observed. Compared with the normal control group

the low-oxygen intervention group showed an increase in the MDA and Fe

content

a decrease in the p-PI3K/PI3K and p-Akt/Akt values

decreased SLC7A11

GPX4

and Nrf2 expression

and increased ACSL4 expression (

0.05). Compared with the low-oxygen intervention group

the

Jiawei Ditan

Decoction intervention group showed a decrease in MDA and Fe

content

an increase in the p-PI3K/PI3K and p-Akt/Akt values

increased SLC7A11

GPX4

and Nrf2 expression

and decreased ACSL4 expression (

0.05).

Jiawei Ditan

Decoction contained DL-stachydrine

D-(+ )-malicacid

adenosine

etc.The cell survival rate of the 10.00 g/L group increased (

0.05) compared to that of the

Jiawei Ditan

Decoction 0

1.25

2.50

5.00

and 20.00 g/L groups. Compared with the normoxic group

p-Akt and GPX4 expression decreased in the erastin group (

0.05); compared with the erastin group

the erastin +

Jiawei Ditan

Decoction intervention group showed increased p-Akt and GPX4 expression (

0.05). Compared with the normoxic group

p-PI3K and GPX4 expression decreased in the low-oxygen intervention 12 h group (

0.01); compared with the low-oxygen intervention 12 h group

the low-oxygen intervention 12 h + 740Y-P group showed increased p-PI3K and GPX4 expression (

0.05). Compared with the normoxic group

GPX4 expression and the p-Akt/Akt and p-PI3K/PI3K were reduced in the low-oxygen intervention 6 and 12 h groups (

0.05); compared with the low-oxygen intervention 6 h and 12 h groups

the p-PI3K/PI3K and p-Akt/Akt values increased in the low-oxygen intervention 12 h +

Jiawei Ditan

Decoction low

medium

and high-dose groups (

0.05). GPX4 expression was also increased (

0.05).

Conclusion

Jiawei Ditan

Decoction may improve cardiomyocyte ferroptosis in rats by regulating the PI3K/Akt signaling pathway

thereby playing a protective role against chronic intermittent hypoxia-induced cardiac injury in rats.

关键词

Keywords

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