利拉鲁肽对早期糖尿病大鼠心肌自噬及脂质沉积的影响

Effects of liraglutide on myocardial autophagy and lipid deposition in early diabetic rats

目的观察利拉鲁肽对早期糖尿病(DM)大鼠心肌自噬标记物LC3B、LC3B mRNA及脂质沉积的影响。方法选4~5周龄(l80~200g)健康雄性Wistar大鼠36只,随机数字表法分为正常组(NC组,10只)和造模组(26只)。NC组(10只,予等量生理盐水)予常规饲料;造模组予高糖脂饲料,12周时一次性予链脲佐菌素25 mg/kg造DM模型,共成模25只,分为3组:糖尿病组(DM/NS组,9只,予等量生理盐水)、利拉鲁肽组(DM/LIR组,8只,予利拉鲁肽100μg/kg,2次/d)及利拉鲁肽+氯喹组(DM/LIR+CQ组,8只,予利拉鲁肽100μg/kg 2次/d,氯喹50 mg/kg 1次/2 d)。4周后测血糖,麻醉后取心肌组织,HE染色观察心肌细胞泡沫样变、免疫组化法测LC3B水平、RT-PCR法测LC3B mRNA水平。组间比较用单因素方差分析,两两比较用LSD-t检验,相关性用Pearson相关分析。结果①与NC组比较,DM/NS组及DM/LIR+CQ组泡沫样变细胞/总心肌细胞数升高,LC3B mRNA及LC3B水平降低[DM/NS组:(2.18±0.90)vs(11.79±0.74)、(2.03±0.10)vs(1.85±0.06)、(194.18±10.19)vs(175.99±6.09)(t=25.24、4.69、3.22);DM/LIR+CQ组:(2.18±0.90)vs(11.24±1.29)、(2.03±0.10)vs(1.89±0.08)、(194.18±10.19)vs(176.73±7.82)(t=17.56、4.65、3.99,均P<0.05)];DM/LIR组上述指标无差异(t=2.20、0.28、1.40,均P>0.05)。②与DM/NS组比较,DM/LIR+CQ组泡沫细胞/总心肌细胞数、LC3B mRNA及LC3B表达无差异(t=1.09、1.18、0.22,均P>0.05);DM/LIR组泡沫细胞/总心肌细胞数下降、LC3B mRNA及LC3B表达升高[(11.79±0.74)vs(1.29±0.60)、(1.85±0.06)vs(2.01±0.20)、(175.99±6.09)vs(201.27±11.35)(t=31.86、2.39、5.82,均P<0.05)]。③相关性分析,LC3B mRNA、LC3B水平与泡沫细胞/总心肌细胞数比值显著相关(r=-0.977、-0.986,均P<0.05)。结论利拉鲁肽能增强早期糖尿病大鼠心肌自噬,减少心肌细胞泡沫样变,改善心肌脂质沉积,对心肌结构发挥保护作用。

Objective To investigate the effects of liraglutide on the expressions of autophagy markers LC3B,LC3B mRNA and lipid deposition in myocardial tissue of rats with early diabetes mellitus.Methods A total of 36 healthy male Wistar rats aged 4-5 weeks and weighing(l80-200)g were selected and divided into normal group(group NC,10 rats)and model group(26 rats)according to random number table.Rats in the NC group were fed with routine diet and rats in the model group were given high glucose and high fat diet for 12 weeks.Rats in the model group were injected with streptozotocin into the abdominal cavity in a single dose of 25 mg/kg after molding.Rats in the model group were further divided into three groups:T2DM group(group DM/NS,9 rats,given equal volume of saline),liraglutide intervention group(group DM/LIR,8 rats,injected with 100μg/kg liraglutide twice daily)and Liraglutide and Chloroquine intervention group(group DM/LIR+CQ,8 rats,injected with 100μg/kg liraglutide twice daily,and injected with Chloroquine 50 mg/kg once every two days).Rats in group NC were given equal volume of saline.At the end of 12 weeks,all the rats were tested blood glucose and anaesthetized to collect myocardial tissues to observe myocardial lipid deposition and fiber arrangement under light microscope after HE staining.The expressions of LC3B were detected by immunohistochemical method,and the expressions of LC3B mRNA were detected by real-time fluorescence quantitative polymerase chain reaction method.Differences among groups were compared by one-way analysis of variance,pairwise comparison was conducted by using LSD-t test,correlation was analyzed by Pearson correlation.Results(1)Compared with group NC,the myocardial fibers arranged in disorder,and the ratio of myocardial foam cells/total cardiomyocytes were increased,the level of LC3B mRNA and LC3B were decreased in group DM/NS and DM/LIR+CQ(in group DM/NS:2.18±0.90 vs 11.79±0.74,2.03±0.10 vs 1.85±0.06,194.18±10.19 vs 175.99±6.09,t=25.24,4.69,3.22,respectively;in group DM/LIR+CQ:2.18±0.90 vs 11.24±1.29,2.03±0.10 vs 1.89±0.08,194.18±10.19 vs 176.73±7.82,t=17.56,4.65,3.99,respectively,all P<0.05).There is no difference in above indicators(2.18±0.90 vs 1.29±0.60,2.03±0.10 vs 2.01±0.20,194.18±10.19 vs 201.27±11.35,t=2.20,0.28,1.40,respectively,all P>0.05).(2)Compared with group DM/NS,the ratio of myocardial foam cells/total cardiomyocytes,the level of LC3B mRNA and LC3B were no difference in group DM/LIR+CQ(t=1.09,1.18,0.22,respectively,all P>0.05).The ratio of myocardial foam cells/total cardiomyocytes was decreased,the level of LC3B mRNA and LC3B were increased in group DM/LIR(11.79±0.74 vs 1.29±0.60,1.85±0.06 vs 2.01±0.20,175.99±6.09 vs 201.27±11.35,t=31.86,2.39,5.82,respectively,all P<0.05).(3)The significant negative correlation were observed between the ratio of myocardial foam cells/total cardiomyocytes and LC3B mRNA,LC3B levels(r=-0.977,-0.986,respectively,all P<0.05).Conclusion Liraglutide can protect the myocardial structure in early diabetic rats by increasing myocardial autophagy,reducing the number of myocardial foam cells,and improving the myocardial lipid deposition.