miR-21-5p通过调控TGF-β1减轻2型糖尿病心肌病小鼠的心肌损伤

MiR-21-5p alleviates myocardial injury in type 2 diabetic cardiomyopathy mice by regulating TGF-β1

目的探讨miR-21-5p对2型糖尿病小鼠心肌损伤的影响及潜在的分子机制。方法10只雄性db/m小鼠作为正常对照组(Con组),30只雄性db/db小鼠随机分成模型组(DCM组)、模型+miR-21-5p激动剂处理组(DCM+miR-agomir组)和模型+miR-21-5p拮抗剂处理组(DCM+miR-antagomir组),每组10只。DCM+miR-agomir组和DCM+miR-antagomir组小鼠每3 d分别经尾静脉注射100μl 10 nmol/L的miR-21-5p agomir和miR-21-5p antagomir,共10次。Con组和DCM组小鼠则在相同时间点经尾静脉注射同等剂量的生理盐水。超声心动图检测心脏功能(LVEF、LVFS),试剂盒测定心肌损伤标志物乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)活性,TUNEL染色检测心肌细胞凋亡程度,Western blotting法检测TGF-β1、cleaved Caspase-3的蛋白表达,qRT-PCR法检测心肌组织miR-21-5p、TGF-β1 mRNA的表达。双荧光素酶报告系统验证miR-21-5p与TGF-β1的靶向关系。检测各组小鼠的氧化应激指标和炎症指标。结果与Con组相比,DCM组小鼠心肌miR-21-5p的表达显著降低,TGF-β1表达显著升高(P<0.05);且DCM组小鼠LVEF和LVFS值明显降低,血清LDH及CK-MB含量显著上升,心肌细胞凋亡明显增加(P<0.05);心肌丙二醛(MDA)水平、活性氧(ROS)生成及NOX2和NOX4 mRNA表达显著提高,超氧化物歧化酶(SOD)、谷胱甘肽(GSH)和谷胱甘肽过氧化物酶(GSH-Px)水平明显减低(P<0.05);此外DCM组小鼠心肌肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和IL-6的水平显著增加,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)和cleaved Caspase-1的蛋白表达明显上调(P<0.05)。与DCM组相比,DCM+miR-antagomir组小鼠的心肌损伤(LDH及CK-MB)、心脏功能障碍(LVEF和LVFS)、心肌细胞凋亡、心肌氧化应激(MDA、ROS及NOX2和NOX4 mRNA表达)和炎症(TNF-α、IL-1β、IL-6、NLRP3、ASC和cleaved Caspase-1表达)均进一步加重(P<0.05)。而与DCM组相比,DCM+miR-agomir组小鼠的心肌损伤(LDH及CK-MB)、心脏功能障碍(LVEF和LVFS)、心肌细胞凋亡、心肌氧化应激(MDA、ROS及NOX2和NOX4 mRNA表达)和炎症(TNF-α、IL-1β、IL-6、NLRP3、ASC和cleaved Caspase-1表达)则明显减轻(P<0.05)。双荧光素酶报告系统检测表明miR-21-5p可靶向负调控TGF-β1的表达。结论miR-21-5p可通过靶向调控TGF-β1的表达抑制心肌氧化应激和炎症以减轻心肌细胞凋亡,进而改善2型糖尿病心肌病小鼠的心肌损伤。

Objective To investigate the effect of miR-21-5p on myocardial injury in type 2 diabetic mice and its underlying molecular mechanism.Methods Ten male db/m mice were selected as control group(Con group).Thirty male db/db mice were randomly divided into model group(DCM group),miR-21-5p agomir treatment group(DCM+miR-agomir group)and miR-21-5p antagomir treatment group(DCM+miR-antagomir group),with 10 mice in each group.Mice in DCM+miR-agomir group and DCM+miR-antagomir group were injected with 100μl 10 nmol/L miR-21-5p agomir and miR-21-5p antagomir via tail vein every 3 d for 10 times,respectively.Mice in Con group and DCM group were injected with the same dose of normal saline through tail vein at the same time.Cardiac function(LVEF,LVFS)was detected by echocardiography.Lactate dehydrogenase(LDH)and creatine kinase isoenzyme(CK-MB)levels were detected by special kits.TUNEL staining was used to detect the apoptosis of cardiomyocytes.Western blotting was used to detect the protein expression levels of TGF-β1 and cleaved Caspase-3.RT-qPCR was used to detect the mRNA expression of miR-21-5p and TGF-β1.Dual luciferase reporter system was used to verify the relationship between miR-21-5p and TGF-β1.The indexes of oxidative stress and inflammation were detected.Results Compared with Con group,the expression of miR-21-5p was significantly decreased,while the expression of TGF-β1 was significantly increased in DCM group(all P<0.05).Compared with Con group,LVEF and LVFS values were significantly decreased,serum LDH and CK-MB contents were significantly increased,and cardiomyocyte apoptosis was significantly elevated in DCM group(all P<0.05).Compared with Con group,malondialdehyde(MDA)level,reactive oxygen species(ROS)generation,and NOX2 and NOX4 mRNA levels were significantly increased,while superoxide dismutase(SOD),glutathione(GSH)and glutathione peroxidase(GSH-Px)levels were significantly reduced in DCM group(all P<0.05).Compared with Con group,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 levels were significantly increased,NOD-like receptor thermal protein domain associated protein 3(NLRP3),apoptosis-associated speck-like protein containing a caspase-recruitment domain(ASC)and cleaved Caspase-1 protein expression levels were significantly up-regulated in DCM group(all P<0.05).Compared with DCM group,myocardial injury(LDH and CK-MB contents),cardiac dysfunction(LVEF,LVFS),cardiomyocyte apoptosis,myocardial oxidative stress(MDA,ROS,NOX2 and NOX4 mRNA levels)and inflammation(TNF-α,IL-1β,IL-6,NLRP3,ASC and cleaved Caspase-1 expression levels)were further aggravated in DCM+miR-antagomir group(P<0.05).Compared with DCM group,myocardial injury(LDH and CK-MB contents),cardiac dysfunction(LVEF,LVFS),cardiomyocyte apoptosis,myocardial oxidative stress(MDA,ROS,NOX2 and NOX4 mRNA levels)and inflammation(TNF-α,IL-1β,IL-6,NLRP3,ASC and cleaved Caspase-1 expression levels)were significantly alleviated in DCM+miR-agomir mice group(P<0.05).Dual luciferase reporter system showed that miR-21-5p negatively regulated the expression of TGF-β1.Conclusion MiR-21-5p can inhibit the myocardial oxidative stress and the inflammation,and reduce the cardiomyocyte apoptosis by targeting TGF-β1 expression,and then alleviate the myocardial injury in type 2 diabetic cardiomyopathy mice.