AIM2基因沉默对糖尿病大鼠心肌损伤的影响机制

Mechanism of AIM2 gene silencing alleviating myocardial damage in diabetic rats

目的 探究AIM2基因沉默通过抑制炎性小体信号通路减轻糖尿病大鼠心肌损伤的机制。方法将60只Sprague-Dawley大鼠随机分为对照组、模型组、模型+sh AIM2组和模型+sh NC组,对照组接受基础饮食、其他3组接受高脂肪(16%脂肪和0.30%胆固醇)饮食4周,60只SD大鼠随机均分为对照组、模型组、模型+sh AIM2组和模型+sh NC组,对照组接受基础饮食、其他3组接受高脂肪(16%脂肪和0.30%胆固醇)饮食4周,后3组大鼠腹腔注射链脲佐菌素(STZ)制作糖尿病模型,并于造模后第8周,心肌分别转染空质粒、AIM2-sh RNA(序列为5’-GGTCACCAGTTCCTCAGAG-3’)、sh RNA阴性对照NC-shRN (序列为5’-TTCTCCGAACGTGTCACGT-3’);于实验第20周检测4组大鼠的空腹血糖、总胆固醇、甘油三酯、胰岛素含量及胰岛素敏感性指数,于转染后4周采用Vevo770成像系统测量大鼠的左心室舒张末期尺寸(LVEDD)、左心室射血分数(LVEF)、峰E与峰A之比(E/A)、早期(e’)至晚期(a’)、舒张期速度比(e/a)、峰值E与早期(e’)之比(E/e’)及射血分数(FS);实验结束时取心肌组织切片染色观察纤维化程度、蛋白质印迹分析大鼠心肌的AIM2、胶原蛋白Ⅰ、胶原蛋白Ⅲ、MMP2和MMP9蛋白的表达。结果 糖尿病模型构建20周后,糖尿病大鼠的空腹血糖、总胆固醇、甘油三酯水平、胰岛素含量和胰岛素敏感性指数提高(P<0.05);AIM2抑制对总胆固醇、甘油三酯、空腹血糖、胰岛素敏感性指数和实验结束时胰岛素含量无影响(P>0.05);与对照组相比,模型组大鼠LVEF、FS、E/A比和e’/a’比降低(P<0.05),LVEDd和E/e’比增加(P<0.05);与对照组相比,模型组大鼠的AIM2蛋白水平增加(P<0.05);与对照组相比,模型组呈现偏心室肥大和较高的心肌细胞宽度,AIM2-shRNA治疗后缓解;与对照组相比,模型组心肌细胞外基质沉积增加,但通过sh RNA处理得以缓解(P<0.05);与对照相比,模型组表现出胶原蛋白Ⅰ和胶原蛋白Ⅲ的过表达,但被AIM2抑制下调(P<0.05);与对照组相比,模型组大鼠中通过sh RNA-AIM2转染抑制MMP2和MMP9的升高(P<0.05);模型组中AIM2炎性小体、caspase-1和GSDMD表达升高,但被AIM2抑制下调(P<0.05);模型组心脏组织中TUNEL阳性细胞增加,但被AIM2抑制下调(P<0.05)。结论 沉默AIM2基因抑制了炎症小体caspase-1的表达,降低炎症水平,可通过抑制炎性小体信号通路来减轻糖尿病大鼠心肌的损伤。

Objective To investigate the mechanism of AIM2 gene silencing alleviating myocardial damage in diabetic rats by inhibiting the inflammasome signal pathway.Methods 60 Sprague-Dawley rats were randomly divided into control group,model group,model + sh AIM2 group and model + shNC group.The control group received basic diet and the other three groups received high fat diet(16% fat and 0.30% cholesterol) for 4 weeks.The other three groups received intraperitoneal injection of STZ for diabetic modeling.At the 8 thweek after modeling,the myocardium was transfected with empty plasmid,AIM2-shRNA(sequence: 5’-ggtcaccagtcctcacagg-3’),and shRNA negative control NCshRNA(sequence: 5’-ttctccgaacgtgtgcacgt-3’).Fasting plasma glucose,total cholesterol,triglyceride,insulin content and insulin sensitivity index were measured at the 20 thweek.The left ventricular end diastolic dimension(LVEDD),left ventricular ejection fraction(LVEF),ratio of peak e to peak a(E/A),early(e’) to late(a’),diastolic velocity ratio(e/a),peak E to early(e’) ratio(E/e’),and ejection fraction(FS) were measured by Vevo770 imaging system 4 weeks after transfection.At the end of the experiment,myocardial tissue sections were taken to be stained and observe the degree of fibrosis.Western blot was used to analyze the expression of AIM2,collagen I,collagen III,MMP2,and MMP9.Results After 20 weeks of modeling, the levels of fasting blood glucose, total cholesterol,triglyceride,insulin content and insulin sensitivity index of diabetic rats were increased(P<0.05).AIM2 inhibition had no effect on index of total cholesterol,triglyceride,fasting blood glucose,insulin sensitivity,and insulin content at the end of the experiment(P<0.05).Compared with the control group,LVEF,FS,E/A ratio,and e’/a’ ratio were decreased in the model group(P<0.05),while LVEDd and E/e’ ratio were increased(P<0.05).Compared with the control group,the AIM2 protein level in the model group was increased(P<0.05).Compared with the control group,the model group showed eccentric ventricular hypertrophy and higher myocardial cell width,which was relieved after AIM2-shRNA treatment.Compared with the control group,the deposition of extracellular matrix increased in the model group,but was relieved by shRNA treatment(P<0.05).Compared with the control group,the model group showed over-expression of collagen I and collagen III,which were inhibited and down-regulated by AIM2(P<0.05).Compared with the control group,the expression of MMP2 and MMP9 was inhibited by sh RNA-AIM2 transfection in the model group(P<0.05).In the model group,the expression of inflammatory corpuscles,caspase-1,and GSDMD were increased,but were inhibited and down-regulated by AIM2(P<0.05).In the model group,TUNEL positive cells were increased,but down-regulated by AIM2(P<0.05).Conclusion Silencing AIM2 gene inhibits the expression of inflammasome caspase-1 and reduces the level of inflammation.Clinically,inhibition of inflammasome signal pathways can reduce myocardial damage in diabetic rats.