丹酚酸B对2型糖尿病小鼠心肌病氧化应激的作用及机制

Effect of salvianolic acid B on oxidative stress in mice with type 2 diabetic cardiomyopathy and its mechanism

目的探讨丹酚酸B(Sal B)对2型糖尿病小鼠心肌病(DCM)氧化应激的作用及潜在机制。方法60只C57BL/6J小鼠分为空白组(n=12,Control组,正常饲料,灌胃生理盐水)和高脂高糖(HG)组[n=48,HFG组,链脲佐菌素(STZ)联合HFG饲料构建DCM模型],造模成功的HFG组小鼠分为DCM组(灌胃生理盐水)、Sal B低剂量[1.50 mg/(kg·d)]组(Sal B.L组)、Sal B高剂量[3.00 mg/(kg·d)]组(Sal B.H组)及200 mg/(kg·d)二甲双胍治疗组(Metformin组),灌胃连续8周;麻醉各组小鼠,采用小动物超声仪检测仰卧位时各组小鼠心脏的左室射血分数(LVEF)、缩短分数(FS)、左室收缩末期容积(LVESV)及左室收缩期末期内径(LVIDs);处死各组小鼠,取心脏制作切片、苏木素-伊红(HE)和Masson染色观察心肌组织形态学特征;采用试剂盒检测各组小鼠心肌组织中细胞丙二醛(MDA)、超氧化物歧化酶(SOD)及谷胱甘肽(GSH)的水平,采用Western blot分析氧化应激蛋白Kelch样ECH关联蛋白1(Keap1)、核因子-E2相关因子2(Nrf2)、磷酸化Nrf2(pNrf2)、过氧化物酶1(PRDX1)、血红素加氧酶1(HO-1)及凋亡相关蛋白活化的半胱氨酸蛋白(Cleaved-caspase3)、B淋巴细胞瘤-2蛋白(Bcl2)、Bcl2关联X(bax)蛋白的表达;SD乳鼠心脏提取分离培养原代新生大鼠心肌细胞(PNRCMs)构建DCM体外模型,将PNRCMs分为40 mmol/L甘露醇(Mannitol)组、空白(Control)组、40 mmol/L HG(HG)组、40 mmol/L HG+25μmol/L Sal B(Sal B低剂量,Sal B.L)组、40 mmol/L HG+50μmol/L Sal B(Sal B高剂量,Sal B.H)组、40 mmol/L HG+0.25 mmol/L二甲双胍(Metformin)组,检测各组细胞的活性氧(ROS)、MDA、SOD及GSH水平,采用Western blot检测氧化应激蛋白Keap1、Nrf2、pNrf2、PRDX1、HO-1及凋亡相关蛋白Cleaved-caspase3、bax、Bcl2蛋白的表达;采用实时定量反转录聚合酶链式反应(RT-qPCR)法检测氧化应激相关基因Keap1、Nrf2、PRDX1及HO-1信使RNA(mRNA)的表达;采用Nrf2抑制剂(ML385)和激动剂(甲基巴多索隆)进一步分析氧化应激蛋白Keap1、Nrf2、pNrf2、PRDX1及HO-1的表达。结果与Control组比较,DCM组小鼠的LVEF、FS降低(P<0.01),LVESV、LVIDs升高(P<0.01),心肌组织排列紊乱、间质细胞增多并伴有炎性细胞浸润及胶原沉积,心肌组织中MDA含量增加、SOD和GSH减少(P<0.01),心肌组织中Keap1、Cleaved-caspase3及bax蛋白表达上调,Nrf2、pNrf2、PRDX1、HO-1及Bcl2蛋白表达下调(P<0.01);与Control组比较,HG组心肌细胞中ROS表达增加,SOD表达下降(P<0.05),MDA含量增加、GSH表达下降(P<0.01),心肌细胞中的Keap1、Cleaved-caspase3及bax蛋白表达增加(P<0.01),Nrf2、pNrf2、PRDX1、HO-1及Bcl2蛋白表达下降(P<0.01),心肌细胞中的Keap1 mRNA表达升高(P<0.05),Nrf2、PRDX1及HO-1 mRNA表达降低(P<0.05);与DCM组比,Sal B.L和Sal B.H组小鼠的LVEF、FS增加(P<0.01)、LVESV、LVIDs降低(P<0.01),心肌组织排列整齐、炎性细胞浸润减少、心肌胶原沉积减少,心肌组织中MDA含量减少、SOD和GSH含量增加(P<0.01),心肌组织中Keap1、Cleaved-caspase3及bax表达下调,Nrf2、pNrf2、PRDX1、HO-1及Bcl2蛋白表达上调(P<0.05);与HG组相比,Sal B.L和Sal B.H量组心肌细胞中ROS表达、MDA含量减少,SOD和GSH含量增加(P<0.05),心肌细胞中的Keap1、Cleaved-caspase3及bax蛋白表达减少(P<0.05),Nrf2、pNrf2、PRDX1、HO-1及Bcl2表达增加(P<0.05);心肌细胞中的Keap1 mRNA表达降低(P<0.05),Nrf2、PRDX1和HO-1 mRNA表达升高(P<0.05);进一步采用Nrf2抑制剂ML385和激动剂甲基巴多索隆干预,与HG组相比,Sal B.H组心肌细胞中的Nrf2、pNrf2、PRDX1及HO-1表达增加(P<0.05);与Sal B.H组相比,ML385和Sal B联用组心肌细胞中的Nrf2、pNrf2、PRDX1及HO-1的表达减少(P<0.05)。结论Sal B可降低2型糖尿病小鼠DCM的氧化应激作用,其机制与Keap1/Nrf2信号通路蛋白的表达有关。

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+25μmol/L Sal B),Sal B.H group(40 mmol/L HG+50μmol/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.