高锌饮食通过调控AMPK-Drp1通路对链脲霉素诱导的1型糖尿病小鼠心肌损伤的保护作用

Protective effect of high zinc diet on streptozotocin-induced myocardial injury in type 1 diabetic mice through adenine monophosphate activated protein kinase/dynamin related protein 1 pathway

目的观察补锌治疗对1型糖尿病(T1DM)心肌病小鼠心肌腺苷酸活化蛋白激酶(AMPK)、线粒体动力相关蛋白1(Drp1)及氧化应激水平的变化,初步探讨锌对糖尿病心肌病保护作用的分子机制。方法取8周龄雄性FVB小鼠60只,通过小剂量(50 mg/kg)链脲霉素连续腹腔注射5 d的方法构建T1DM心肌损伤小鼠模型。造模成功后将小鼠通过整群随机抽样方法分为3组,分别为对照组(A组,20只)、糖尿病心肌损伤模型组(B组,18只)和模型+高锌饮食组(C组,18只)。A组和B组给予正常锌饮食(锌含量为30 mg/kg),C组给予高锌饮食(锌含量150 mg/kg),饲养时间均为3个月。测量小鼠体重;检测血清葡萄糖、丙二醛、超氧化物歧化酶水平;心脏彩色超声多普勒测量小鼠心脏功能,测量左室射血分数(EF)、收缩期和舒张期心室充盈早期峰值速度、晚期充盈速度的比值(E/A比值)和左室舒张末期内径(LVDD);观察心肌组织病理学改变,并检测AMPK、磷酸化腺苷酸活化蛋白激酶(pAMPK^(Thr172))、Drp1和磷酸化动力相关蛋白1(pDrp1^(Ser616))的蛋白表达水平,计算pAMPK^(Thr172)/AMPK和pDrp1^(Ser616)/Drp1比值。多组间比较采用单因素方差分析方法。结果A、B、C三组间,体重、血糖、丙二醛和超氧化物歧化酶水平、E/A比值、EF值和LVDD的差异均有统计学意义(P<0.05),其中B组血浆丙二醛水平较A组升高,超氧化物歧化酶水平较A组降低,而C组较B组丙二醛水平降低,超氧化物歧化酶水平较B组升高,差异有统计学意义(P<0.05)。与A组相比,B组小鼠E/A比值和EF值降低,而C组较B组增加,差异有统计学意义(P<0.05)。与A组相比,B组的pAMPK^(Thr172)/AMPK值降低,而C组较B组升高,差异有统计学意义(P<0.05);与A组相比,B组的pDrp1^(Ser616)/Drp1值增加,C组较B组降低,差异有统计学意义(P<0.05)。补锌治疗增加了心肌AMPK蛋白Thr172位点磷酸化水平,减少了Drp1蛋白Ser616位点磷酸化水平(P<0.05)。结论补锌治疗可能通过调控AMPK/Drp1通路来抑制糖尿病心肌损伤的氧化应激及心肌细胞线粒体分裂而达到治疗目的。

Objective To observe the effect of zinc supplementation on the changes of myocardial adenine monophosphate activated protein kinase(AMPK),dynamin related protein 1(Drp1)and oxidative stress in type 1 diabetic mice(T1DM),and to preliminarily explore the mechanism of the protective effect of zinc treatment on diabetic cardiomyopathy.Methods Sixty eight-week-old male FVB mice were selected,and a small dose(50 mg/kg)of streptozocin(STZ)was continuously injected intraperitoneally for five days to construct a T1DM myocardial injury mouse model.After successful modeling,the mice were divided into three groups by block randomization:control group(group A,n=20),diabetic myocardial injury group(group B,n=18)and diabetic myocardial injury model+high zinc diet group(group C,n=18).Groups A and B were given normal zinc diet(the content of zinc is 30 mg/kg)and group C was given a high-zinc diet(the content of zinc was 150 mg/kg)for 3 months.The body weight,serum glucose,malondialdehyde and superoxide dismutase levels were measured;the cardiac function,left ventricular ejection fraction(EF value),the ratio of the early peak and late filling rates of systolic and diastolic ventricles(E/A ratio)and left ventricular end diastolic diameter(LVDD)were also detected;the pathological changes of myocardial tissue were observed.Furthermore,the protein expressions of AMPK,phospho adenine monophosphate activated protein kinase(pAMPK^(Thr172)),Drp1 and phospho dynamin related protein1(pDrp1ser616)were measured by Western blotting,and the ratios of pAMPK^(Thr172)/AMPK and pDrp1^(Ser616)/Drp1 were calculated.One-way analysis of variance(ANOVA)was used for comparison among multiple groups.Results The differences in weight,blood glucose,malondialdehyde and superoxide dismutase levels,E/A ratio,EF value and LVDD among the three groups A,B,and C were statistically significant(P<0.05).The levels of plasma malondialdehyde were higher and superoxide dismutase were lower in group B compared with those of group A,while the levels of malondialdehyde were lower and superoxide dismutase were higher in group C.Compared with group A,the E/A ratio and EF value of group B decreased,while group C increased compared with group B.Compared with group A,the pAMPK^(Thr172)/AMPK value of group B decreased,while group C was higher than group B.The pDrp1^(Ser616)/Drp1 value of group B increased compared with group A,while Group C was lower than group B.All the differences above were statistically significant(P<0.05).Zinc supplementation increased the phosphorylation level of AMPKThr172 and decreased the phosphorylation level of the Drp1Ser616 in the myocardial tissue.Conclusion Zinc supplementation may be effective in inhibiting the oxidative stress of diabetic myocardial injury and myocardial cell mitochondrial division through regulating AMPK/Drp1 pathway.