熊果苷对糖尿病心肌病小鼠心肌损伤的抑制作用及其机制

Effect of arbutin on myocardial injury in diabetic cardiomyopathy mice and its mechanism

目的观察熊果苷(AR)对糖尿病心肌病(DCM)小鼠心肌损伤的影响,探讨其心肌保护作用是否与沉默信息调节因子1(SIRT1)有关。方法将60只雄性C57BL/6小鼠随机均分为Con组、DCM组、DCM+AR组、DCM+AR+EX527组,每组15只。除Con组外均采用腹腔注射链脲佐菌素联合高糖高脂饮食法建立DCM小鼠模型,DCM+AR组和DCM+AR+EX527组建模成功后给予AR 50 mg/(kg·d)灌胃12周,同时DCM+AR+EX527组建模后即给予SIRT1特异性抑制剂EX52720 mg/kg腹腔注射,1周/次,共注射11次。各组小鼠进行心脏超声检查,计算左心室射血分数(LVEF)和左心室短轴缩短率(LVFS);处死后观察心肌组织纤维化程度和胶原沉积情况,比较各组心肌组织超氧化物歧化酶(SOD)、丙二醛(MDA)及活性氧(ROS)表达,免疫荧光染色观察心肌组织NF-κB表达,ELISA法检测SIRT1去乙酰化活性,实时荧光定量PCR法检测Collagen1、Collagen3、烟酰胺腺嘌呤二核苷酸磷酸氧化酶2(NOX2)、烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)mRNA表达,Western blotting法检测SIRT1及细胞焦亡相关因子NOD样受体热蛋白结构域相关蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、白细胞介素1β(IL-1β)、白细胞介素18(IL-18)和裂解的半胱氨酸蛋白酶1(cleaved Caspase-1)蛋白表达。结果Con组心肌组织无明显蓝色胶原纤维及红色胶原着色,即无显著纤维化及胶原沉积;与Con组相比,DCM+AR组、DCM组、DCM+AR+EX527组小鼠均存在心肌组织纤维化程度增加、胶原沉积增多,以DCM组、DCM+AR+EX527组变化更明显。与Con组比较,DCM+AR组、DCM组、DCM+AR+EX527组小鼠LVEF、LVFS及心肌组织SIRT1去乙酰化活性、SOD表达均降低,NF-κB、MDA、ROS及IL-6、TNF-α、Collagen1、Collagen3、NOX2、NOX4 mRNA相对表达量均升高,以DCM组、DCM+AR+EX527组变化更明显(P均<0.05)。与Con组和DCM+AR组比较,DCM组和DCM+AR+EX527组小鼠SIRT1蛋白相对表达量降低,NLRP3、ASC、IL-1β、IL-18、cleaved Caspase-1蛋白相对表达量均升高(P均<0.05)。结论AR可通过减轻心肌组织氧化应激损伤和炎症反应,进而抑制炎症小体形成和心肌细胞焦亡,最终减轻DCM小鼠的心肌损伤,其机制可能与激活SIRT1信号通路有关。

Objective To observe the effect of arbutin(AR)on myocardial injury in diabetic cardiomyopathy(DCM)mice,and to explore whether the protective action of AR is related to silencing information regulator 1(SIRT1).Methods Sixty male C57BL/6 mice were randomly divided into the Con group,DCM group,DCM+AR group and DCM+AR+EX527 group,with 15 mice in each group.Except the Con group,the DCM mouse models were established by intra⁃peritoneal injection of streptozotocin combined with high-glucose-high-fat diet in the other groups.AR of 50 mg/(kg·d)was given in the DCM+AR group and DCM+AR+EX527 group through intragastric administration for 12 weeks.At the same time,EX527(20 mg/kg)was given in the DCM+AR+EX527 group through intraperitoneal injection,once a week,with a total of 11 injections.The left ventricular ejection fraction(LVEF)and left ventricular fraction shortening(LVFS)were calculated,the degree of myocardial fibrosis and collagen deposition were observed,and the expression levels of su⁃peroxide dismutase(SOD),malondialdehyde(MDA)and reactive oxygen species(ROS)were compared.The expression of NF-κB in the myocardial tissue was observed by immunofluorescence staining,and the deacetylation activity of SIRT1 was detected by ELISA kit.Real-time quantitative PCR was used to detect the mRNA expression levels of Collagen1,Col⁃lagen3,nicotinamide adenine dinucleotide phosphate oxidase 2(NOX2),nicotinamide adenine dinucleotide phosphate ox⁃idase 4(NOX4),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α).Western blotting was used to detect protein expression levels of SIRT1 and pyroptosis-associated molecules including Nod-like receptor thermal protein domain associ⁃ated protein 3(NLRP3),apoptosis-associated speck-like protein(ASC),interleukin-1β(IL-1β),interleukin-18(IL-18),and cleaved Caspase-1.Results There were no significant fibrosis and collagen deposition in the Con group,that was,there were no obvious blue collagen fibers and red collagen staining in the myocardial tissues.Compared with the Con group,the degree of myocardial fibrosis and collagen deposition in the DCM+AR group,DCM group and DCM+AR+EX527 group increased,with more significant changes in the DCM group and DCM+AR+EX527 group.Compared with the Con group,LVEF,LVFS,SOD expression and SIRT1 deacetylation activity decreased,but the relative expression levels of NF-κB,MDA,ROS,and the mRNA levels of IL-6,TNF-α,Collagen1,Collagen3,NOX2,and NOX4 increased in the DCM+AR group,DCM group and DCM+AR+EX527 group,with more significant changes in the DCM group and DCM+AR+EX527 group(all P<0.05).Compared with the Con group and DCM+AR group,the protein expression levels of SIRT1 significantly decreased,while the protein expression levels of NLRP3,ASC,IL-1β,IL-18,and cleaved Cas⁃pase-1 significantly increased in DCM group and DCM+AR+EX527 group(all P<0.05).Conclusions AR can mitigate the oxidative stress and inflammatory response in the myocardial tissues,and then inhibit the inflammasome formation and cardiomyocyte pyroptosis,thus finally alleviating the myocardial injury of DCM mice.The mechanism may be related to the activation of SIRT1 signaling pathway.