Effect of Jiawei Ditan Decoction on cardiomyocyte ferroptosis in rats with chronic intermittent hypoxia
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 Fe2+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 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 Fe2+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 Fe2+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 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.
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