Objective

2

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

2

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

2+

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

10

and 20 g/L

respectively).

Results

2

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

2+

content

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

decreased SLC7A11

GPX4

and Nrf2 expression

and increased ACSL4 expression (

P

<

0.05). Compared with the low-oxygen intervention group

the

Jiawei Ditan

Decoction intervention group showed a decrease in MDA and Fe

2+

content

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

increased SLC7A11

GPX4

and Nrf2 expression

and decreased ACSL4 expression (

P

<

0.05).

Jiawei Ditan

Decoction contained DL-stachydrine

D-(+ )-malicacid

adenosine

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

P

<

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 (

P

<

0.05); compared with the erastin group

the erastin +

Jiawei Ditan

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

P

<

0.05). Compared with the normoxic group

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

P

<

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 (

P

<

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 (

P

<

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 (

P

<

0.05). GPX4 expression was also increased (

P

<

0.05).

Conclusion

2

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.