Proanthocyanidins Pretreatment Alleviates Doxorubicin-induced Cardiotoxicity In Aged Mice By Upregulating AMPKα1

Objective:To investigate the effect of proanthocyanidins(PCs)on alleviating doxorubicin-induced cardiotoxicity in aged mice by upregulating AMPKα1 and inhibiting apoptosis.Methods:In vitro,HL-1 cells were treated with D-galactose for 48 h to establish the cell senescence model,and treated with Dox for 24 h to establish the cell injury model.The activity of β-galactosidases was detected by SA-β-gal staining,and the expression of senescence markers p21 and p53 was detected by Western blot to evaluate the degree of senescence of HL-1 cells.Cell viability,the activity of lactate dehydrogenase(LDH),the activity of antioxidant enzymes(SOD,GSH-Px,and CAT),and the content of lipid oxidation products(MDA)were measured by enzyme-labeled assay.The level of apoptosis,intracellular and mitochondrial reactive oxygen species(ROS)production,and mitochondrial function were measured by flow cytometry.The level of apoptosis was also detected by the TUNEL assay.The expression of p-AMPKα,AMPK α1,Bax,Bcl-2,Cyt c,and Cleaved Caspase-3 were detected by Western blot.In vivo experiments were conducted in C57/BL6 mice,including the young(2-3months)and the aged(18-20 months).To establish the overexpression AMPK α1aged mice model by injecting p AD/Flag-AMPK α1 into caudal vein.Dox was injected intraperitoneally to establish the injury model after two weeks.The cardiac function was measured by echocardiography and histopathological analysis.The expressions of AMPK α1,Bax,and Bcl-2 were detected by Western blot.The content of CK and LDH in serum was detected by enzyme-labeled assay.Results:The results of in vitro study showed that PCs pretreatment significantly upregulated the expression of AMPK α1,regulated oxidative stress,stabilized mitochondrial function,regulated the expression of apoptosis-related proteins,and decreased LDH activity,and increased cell viability in D-gal-induced mimetic aging HL-1?cells.The protective effect of PCs on mimetic aging HL-1 cells was significantly inhibited after pretreating with 5 μM Compound C(an AMPK inhibitor).The results of in vivo study showed that PCs pretreatment significantly upregulated the expression of AMPK α1,regulated oxidative stress,decreased the content of CK and LDH in serum,regulated the expression of apoptosis-related proteins,inhibited cell apoptosis,improved cardiac function,and alleviated Dox-induced cardiac injury in aged mice.The cardioprotective effects were similar to PCs pretreatment after p AD/Flag-AMPK α1 adenovirus treatment.Conclusion:Aged mice can have more severe cardiotoxicity after Dox treatment,while PCs pretreatment can reduce oxidative stress,and protect mitochondrial function under stress conditions by upregulating AMPK α1,thereby resisting the cardiomyocyte apoptosis caused by Dox damage and reducing Dox-induced cardiotoxicity in aged mice.