Effect of salvianolic acid B on oxidative stress in mice with type 2 diabetic cardiomyopathy and its mechanism
Objective To investigate the effect of Salvianolic acid B(Sal B)on oxidative stress in mice with type 2 diabetic cardiomyopathy(DCM)and its potential mechanism.Methods Sixty C57BL/6J mice were divided into blank group(n=12,normal diet,normal saline)and HFG group(high-fat,high-glucose(HG),n=48,Streptozotocin(STZ)combined with HFG to establish DCM model).The mice in HFG group were classified into DCM group(physiological saline),Sal B.L group(low-dose Sal B,1.50 mg/(kg·d),Sal B.H group[high-dose Sal B,3.00 mg/(kg·d)],and Metformin group(200 mg/kg·d).The mice were intragastrically administrated for continuous 8 weeks.The mice in each group were anesthetized.Small animal ultrasound was used to detect the left ventricular ejection fraction(LVEF),fractional shortening(FS),left ventricular end-systolic volume(LVESV),and left ventricular internal dimension in systole(LVIDs)when the mice were in the supine position.The mice were sacrificed.Mouse hearts were sectioned for hematoxylin-eosin(HE)and Masson staining to observe the morphological characteristics of myocardial tissues.The reagent kits were used to detect the levels of cellular malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione(GSH)in mouse myocardial tissues in each group.Western blot was used to detect the protein expressions of oxidative stress proteins such as Kelch-like ECH associated protein 1(Keap1),nuclear factor-E2 associated factor 2(Nrf2),phosphorylated Nrf2(pNrf2),peroxidase 1(PRDX1),heme oxygenase 1(HO-1),apoptosis-associated proteins such as activated cysteine protein(Cleaved-caspase3),B-cell lymphoma-2 protein(Bcl2),and Bcl2-associated X(bax)protein.Primary neonatal rat cardiomyocytes(PNRCMs)were isolated from SD rat neonatal hearts for establishing in vitro DCM model.PNRCMs were divided into mannitol group(40 mmol/L),blank group,HG group(40 mmol/L),Sal B.L group(40 mmol/L HG+25 μmol/L Sal B),Sal B.H group(40 mmol/L HG+50 μmol/L Sal B),and metformin group(40 mmol/L HG+0.25 mmol/L metformin).The levels of reactive oxygen species(ROS),MDA,SOD,and GSH were detected in each group of cells.Western blot was used to detect the expressions of oxidative stress-related proteins Keap1,Nrf2,pNrf2,PRDX1,and HO-1 as well as apoptosis-related proteins Cleaved-caspase3,bax,and Bcl2.Real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the messenger RNA(mRNA)expressions of oxidative stress-related genes Keap1,Nrf2,PRDX1,and HO-1.Further analysis was conducted on the expressions of oxidative stress-related proteins Keap1,Nrf2,pNrf2,PRDX1,and HO-1 using Nrf2 inhibitor(ML385)and agonist(Bardoxolone methyl).Results When compared to control group,LVEF and FS were decreased in DCM group(P＜0.01),while LVESV and LVIDs were increased in DCM group(P＜0.01).The arrangement of myocardial tissue was disordered.Interstitial cells were increased accompanied by inflammatory cell infiltration and collagen deposition.MDA content in myocardial tissue was increased,while SOD and GSH were decreased(P＜0.01).The protein expressions of Keap1,cleaved-caspase3,and bax were upregulated in myocardial tissues,while the protein expressions of Nrf2,pNrf2,PRDX1,HO-1,and Bcl2 were downregulated(P＜0.01).When compared with control group,myocardial cells in the HG group showed an increase in ROS and a decrease in SOD expression in myocardial cells(P＜0.05),an increase in MDA content and a decrease in GSH expression(P＜0.01),the increased protein expressions of Keap1,Cleaved-caspase3,and bax(P＜0.01),the decreased protein expressions of Nrf2,pNrf2,PRDX1,HO-1,and Bcl2(P＜0.01),the increased Keap1 mRNA(P＜0.05),the decreased mRNA expressions of Nrf2,PRDX1,and HO-1(P＜0.05).When compared with DCM group,the mice in Sal B.L and Sal B.H groups had increased LVEF and FS(P＜0.01),decreased LVESV and LVIDs(P＜0.01),well-arranged myocardial tissues,reduced inflammatory cell infiltration,reduced myocardial collagen deposition,decreased MDA content,increased contents of SOD and GSH in myocardial tissues(P＜0.01),the downregulated expressions of Keap1,cleaved-caspase3 and bax,the upregulated protein expressions of Nrf2,pNrf2,PRDX1,HO-1,and Bcl2 in myocardial tissues(P＜0.05).When compared with HG group,myocardial cells in Sal B.L and Sal B.H groups had decreased ROS and MDA contents,increased SOD and GSH contents(P＜0.05),decreased protein expressions of Keap1,Cleaved-caspase3,and bax(P＜0.05),increased protein expressions of Nrf2,pNrf2,PRDX1,HO-1,and Bcl2(P＜0.05),decreased Keap1 mRNA expression in cardiomyocytes(P＜0.05),increased mRNA expressions of Nrf2,PRDX1,and HO-1(P＜0.05).After the intervention with Nrf2 inhibitor ML385 and agonist Bardoxolone methyl,when compared with HG group,the expressions of Nrf2,pNrf2,PRDX1,and HO-1 in cardiomyocytes were increased in Sal B.H group(P＜0.05).When compared with Sal B.H group,ML385 and Sal B combined group showed decreased expressions of Nrf2,pNrf2,PRDX1,and HO-1 in cardiomyocytes(P＜0.05).Conclusion Sal B can reduce oxidative stress in mice with type 2 diabetic cardiomyopathy(DCM),and its mechanism is related to the protein expressions of Keap1/Nrf2 signaling pathway.
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