重组球形脂联素对糖尿病小鼠心肌细胞自噬和内质网应激水平的影响

Effect of recombinant globular adiponectin on myocardial autophagy and endoplasmic reticulum stress in diabetic mice

目的观察重组球形脂联素对糖尿病小鼠心肌细胞自噬和内质网应激水平的影响。方法48只雄性C57BL/6小鼠随机分为正常对照组(NC组)、糖尿病组(DM组)、糖尿病脂联素低剂量干预组(低剂量组)和糖尿病脂联素高剂量干预组(高剂量组)。采用链脲佐菌素(STZ)制备Ⅰ型糖尿病小鼠模型。低剂量组和高剂量组于造模成功后分别腹腔内注射重组球形脂联素5μg/(kg·d)和15μg/(kg·d)。脂联素干预第8周末小鼠腹主动脉采血留取血标本用于测定血糖、血脂和胰岛素水平,处死动物并迅速取小鼠心脏,用生理盐水冲洗干净并用滤纸吸干水分后称重,计算心脏质量指数。取部分左室心肌组织用于HE染色;其余左室心肌组织迅速置入液氮中,待组织冷冻完全后保存于-70℃冰箱用于检测内质网应激指标及自噬指标。血糖用葡萄糖氧化酶法检测。血脂用酶偶联比色法测定。胰岛素用酶联免疫法测定。实时荧光定量PCR法测定内质网应激指标(GRP78、CHOP、Caspase-12)和自噬指标(LC3、Beclin1、p62)mRNA表达。结果与NC组比较,DM组糖尿病小鼠血糖、血脂、心脏质量指数显著升高(P<0.05),血清胰岛素水平显著降低(P<0.05),心肌病理改变加重。心肌组织GRP78、CHOP、Caspase-12和p62 mRNA表达水平显著增高(P<0.05),LC3和Beclin 1 mRNA表达水平显著降低(P<0.05)。重组球形脂联素治疗后,糖尿病小鼠血糖、血脂、心脏质量指数显著降低(P<0.05),血清胰岛素水平显著升高(P<0.05),心肌组织病理改变明显减轻,心肌GRP78、CHOP、Caspase-12和p62 mRNA表达水平显著降低(P<0.05),LC3和Beclin 1 mRNA表达水平显著升高(P<0.05),且高剂量组效果优于低剂量组。结论糖尿病小鼠心肌细胞自噬水平降低而内质网应激水平升高,重组球形脂联素可能通过激活自噬及减轻内质网应激对糖尿病小鼠心肌组织产生保护作用,且这种保护作用具有剂量依赖性。

Objective To observe the effect of recombinant globular adiponectin on the autophagy and endoplasmic reticulum stress in the heart of diabetic mice.Methods Forty-eight male C57BL/6 mice were randomly divided into normal control group(NC group),diabetic group(DM group),low-dose adiponectin group(low dose group)and high-dose adiponectin group(high dose group).Type I diabetic mice were induced by intraperitoneal injection of streptozotocin(STZ).The mice in low dose group and high dose group were given intraperitoneal injection of 5μg/(kg·d)and 15μg/(kg·d)recombinant globular adiponectin for 8 weeks after induction of diabetes,respectively.Mice were sacrificed at the end of 8 weeks of adiponectin treatment.Blood samples were collected from the abdominal aorta to determine blood glucose,lipid and insulin levels.The heart was quickly removed,washed with normal saline,drained with filter paper and weighed to calculate the ratio of heart weight to body weight.Part of left ventricular myocardial tissue was taken for HE staining.The rest of left ventricular myocardial tissue was quickly put into liquid nitrogen and stored in the refrigerator at-70℃after freezing for detecting endoplasmic reticulum stress and autophagy.Blood glucose was detected by glucose oxidase method.Lipids were determined by enzyme coupled colorimetry.Insulin was determined by enzyme-linked immunoassay.Real-time fluorescence quantitative PCR was used to determine the mRNA expression of myocardial endoplasmic reticulum stress indexes(GRP78,CHOP,Caspase-12)and autophagy indexes(LC3,Beclin1,p62)in mice.Results Compared to NC group,the levels of blood glucose and lipid,and the ratio of heart weight to body weight were significantly increased in DM group(P<0.05),the level of serum insulin was significantly decreased(P<0.05),and the myocardial pathological changes were significantly aggravated.Compared to NC group,the mRNA expression of myocardial GRP78,CHOP,Caspase-12 and p62 were significantly increased in DM group(P<0.05),while the mRNA expression of myocardial LC3 and Beclin 1 were significantly decreased(P<0.05).Compared with DM group,the levels of blood glucose and lipid,and the ratio of heart weight to body weight were significantly decreased in high dose group and low dose group(P<0.05),the level of serum insulin was significantly increased(P<0.05),the myocardial pathological changes were significantly alleviated,the mRNA levels of myocardial GRP78,CHOP,Caspase-12 and p62 were significantly decreased(P<0.05),and the mRNA levels of myocardial LC3 and Beclin 1 were significantly increased(P<0.05).The effect in high-dose group was better than that of low-dose group.Conclusion The level of autophagy is decreased while the level of endoplasmic reticulum stress is increased in the heart of diabetic mice.Recombinant globular adiponectin may produce the protective effect on the heart of diabetic mice by activating autophagy and reducing endoplasmic reticulum stress.