Polydatin Upregulation The 14-3-3γ Inhibits Ferroptosis To Alleviates Doxorubicin-induced Cardiotoxicity

Objective:To investigate the cardioprotective mechanism of polydatin（PD）in doxorubicin-induced cardiotoxicity by regulating 14-3-3γ.Methods:In vitro investigations were performed on H9c2 cells and grouped as follows:（1）Control group;（2）1μM DOX group;（3）25μM PD+1μM DOX group;（4）25μM PD+p AD/14-3-3γ-sh RNA+1μM DOX group;（5）2μM Fer-1+1μM DOX group;（6）10μM Mito TEMPO+1μM DOX group.The cell viability was measured by CCK-8;the content of iron,malondialdehyde（MDA）,adenine nucleoside triphosphate（ATP）,the activity of lactate dehydrogenase（LDH）,superoxide dismutase（SOD）,catalase（CAT）,glutathione peroxidase（GSH-Px）and the alteration of reduced glutathione/oxidized glutathione（GSH/GSSG）were measured by enzyme-labeled assay.Enzyme-linked immunosorbent assay for the determination of 4-hydroxynonenoic acid（4-HNE）.Flow cytometry measurement of intracellular and mitochondrial reactive oxygen species（ROS）production levels,mitochondrial permeability transition pore（m PTP）opening,and cell apoptosis.Intercellular reactive oxygen species（ROS）and alterations in mitochondrial membrane potential（MMP）observed by fluorescence inverted microscopy.The expression of 14-3-3γ,GPX4,PTGS2,NDUFB8,UQCRC2 was detected by immunoblotting.In vivo investigations were conducted with C57/BL6 male mice at 8 weeks and grouped as follows:（1）Control group;（2）15 mg/kg DOX group;（3）40 mg/kg PD+15 mg/kg DOX group;（4）40 mg/kg PD+p AD/14-3-3γ-sh RNA+15 mg/kg DOX group;（5）2 mg/kg Fer-1+15 mg/kg DOX group.Measurement of ejection fraction（EF）,fraction shortening（FS）,left ventricular end-systolic inner dimension（LVIDs）,left ventricular end-diastolic inner dimension（LVIDd）in mice with small animal echocardiography.The determination of lactate dehydrogenase（LDH）,aspartate transaminase（AST）,creatine kinase（CK）activities in serum,the content of iron and malondialdehyde（MDA）in myocardial tissues by enzyme-labeled assay.Enzyme-linked immunosorbent assay for the content of 4-hydroxynonenoic acid（4-HNE）in serum.HE staining to detect pathological changes in the myocardium.Fluorescence inverted microscopy was used to observe the level of reactive oxygen species（ROS）production in cardiomyocytes and mitochondrial.The expression of14-3-3γ,GPX4 and PTGS2 in myocardium was detected by immunoblotting.Results:In vitro results indicated that PD pretreatment significantly upregulated the expression of 14-3-3γin H9c2 cells,increased the activity of intracellular antioxidant enzymes SOD,CAT,GSH-Px,elevated the ratio of GSH/GSSG,reduced the production of intracellular and mitochondrial ROS,decreased the content of Fe²⁺,MDA,4-HNE,decreased the opening of mitochondrial permeability transition pore,and stabilized the mitochondrial membrane potential,increased the expression of GPX4,NDUFB8,UQCRC2 and decreased the expression of PTGS2,reduced the release of LDH and significantly increased cell survival.The protective effect of PD on H9c2 cells was significantly inhibited after the addition of p AD/14-3-3γ-sh RNA adenovirus.In vivo results showed that PD pretreatment significantly upregulated the expression of 14-3-3γin myocardial tissue,decreased ROS production,reduced the content of Fe²⁺,MDA,and 4-HNE,increased GPX4 and decreased the expression of PTGS2,decreased the activities of LDH,CK,and AST,thereby enhanced cardiac function.Consistent with the findings in vitro,the cardioprotective effect of PD was significantly abrogated after treatment with p AD/14-3-3γ-sh RNA adenovirus.Conclusion:PD provides significant protection against DOX-induced cardiotoxicity by upregulating the expression of 14-3-3γ,thereby reducing excessive oxidative stress,decreasing the accumulation of Fe²⁺and lipid peroxides and improving mitochondrial function.