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Irisin Alleviates Inflammatory Injury in Diabetic Cardiomyopathy by Regulating NF-κB Pathway
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摘要:
目的 探究鸢尾素（irisin）在糖尿病心肌病（diabetic cardiomyopathy, DCM）中的保护作用及其机制。 方法 高脂饮食联合链脲佐菌素建立DCM小鼠模型，动物实验设置对照组，DCM组，DCM+低、高剂量irisin组以及DCM+吡咯烷二硫代氨基甲酸（pyrrolidine dithiocarbamate, PDTC）〔核因子（nuclear factor, NF）-κB抑制剂 〕组，成功建模后irisin干预3周。采用HE、Masson染色观察心肌形态学改变；全自动生化分析仪检测血清肌酸激酶（creatine kinase, CK）和肌酸激酶同工酶（creatine kinase isoenzyme, CK-MB）水平。将H9c2细胞分为对照组、高糖/高脂（HG/HL）组、HG/HL+低剂量irisin组、HG/HL+高剂量irisin组以及HG/HL+PDTC组，CCK-8法检测细胞活力。ELISA测定心肌组织和细胞肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）、白细胞介素（interleukin, IL）-1β和IL-6表达水平；Western blot检测心肌组织和细胞中NF-κB p65蛋白核转移情况以及核因子κB抑制蛋白α（nuclear factor-κB inhibitor protein α, IκBα）表达水平。 结果 动物实验结果显示，低、高剂量irisin能不同程度减轻心肌组织病理损伤与纤维化，抑制CK、CK-MB和炎症因子水平，上调IκBα蛋白表达，抑制NF-κB p65核转移；细胞实验结果显示，低、高剂量irisin能不同程度增强H9c2细胞活力，上调IκBα蛋白水平，抑制NF-κB p65核转移和炎症因子水平。DCM+低、高剂量irisin组的上述变化与DCM+PDTC组相似。 结论 irisin可能通过抑制NF-κB p65核转移，减轻DCM小鼠心肌组织和高糖高脂诱导H9c2心肌损伤细胞中的炎症反应，发挥心肌保护作用。 Abstract:Objective To investigate the protective effect of irisin in diabetic cardiomyopathy (DCM) and its mechanism. Methods A mouse model of DCM was established by high-fat diet combined with the injection of streptozotocin. The mice were assigned to a control group, a DCM group, a DCM+low-dose irisin group, a DCM+high-dose irisin group, and a DCM+pyrrolidine dithiocarbamate (PDTC) (nuclear factor [NF]-κB inhibitor) group. Then, the mice received irisin intervention for 3 weeks after successful modeling. Myocardial morphologic changes were observed by hematoxylin and eosin (HE) staining and Masson staining. The levels of serum creatine kinase (CK) and creatine kinase isoenzyme CK-MB were examined by automatic biochemical analyzer. H9c2 cells were divided into the control group, high glucose and high lipid (HG/HL) group, HG/HL+low-dose irisin group, HG/HL+high-dose irisin group, and HG/HL+PDTC group. CCK-8 assay was conducted to determine cell viability. The expression levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 in the myocardial tissue and the cells were determined by ELISA. In addition, nuclear translocation of NF-κB p65 protein and the protein expression level of NF-κB inhibitor protein α (IκBα) in the myocardial tissue and the cells were determined by Western blot. Results According to the results of animal experiment, low and high doses of irisin could alleviate the pathological injury and fibrosis of myocardial tissue to varying degrees. Irisin inhibited the levels of CK, CK-MB, and inflammatory factors, up-regulated IκB protein expression, and diminished NF-κB nuclear translocation. According to the results of cell experiment, low and high doses of irisin could enhance H9c2 cell viability to varying degrees, increase the level of intracellular IκB proteins, and inhibit NF-κB p65 nuclear translocation and inflammatory factor expression. The changes in these aspects in the DCM+low-dose irisin group and the DCM+high-dose irisin group were similar to those in the DCM+PDTC group. Conclusion Through inhibiting NF-κB p65 nuclear translocation, irisin may reduce the inflammatory response in the myocardial tissue of DCM mice and H9c2 cells of myocardial injury induced by high glucose and high fat, thereby exerting a protective effect on myocardium. -
Key words:
- Diabetic cardiomyopathy /
- Irisin /
- H9c2 cells /
- Mice /
- Inflammatory response /
- NF-κB nuclear translocation
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图 1 各组小鼠心脏病理变化以及血清CK、CK-MB的表达水平
Figure 1. Pathological changes of the heart and expression levels of CK and CK-MB in each group
CVF: collagen volume fraction. a: Control group; b: DCM group; c: DCM+low-dose irisin group; d: DCM+high-dose irisin group; e: DCM+PDTC group. * P<0.05, ** P<0.01, *** P<0.001, vs. a group; ### P<0.001, vs. b group. n=3.
图 2 各组H9c2细胞的细胞活力
Figure 2. Cell viability of H9c2 cells in each group
A: Control group; B: HG/HL group; C: HG/HL+low dose irisin group; D: HG/HL+high dose irisin group; E: HG/HL+PDTC group. *** P<0.001, vs. A group; ## P<0.01, ### P<0.001, vs. B group. n=3.
图 3 各组小鼠心肌组织与H9c2细胞炎症因子水平
Figure 3. The contents of inflammatory factors in the H9c2 cells and heart tissues of mice in each group
a-e denote the same as those in Fig 1 (mice); A-E denote the same as those in Fig 2 (cells). * P<0.05, ** P<0.01, *** P<0.001, vs. a or A group; ## P<0.01, ### P<0.001, vs. b or B group. n=3.
图 4 各组小鼠心肌组织与H9c2细胞NF-κB相关蛋白的表达
Figure 4. Expression of NF-κB related proteins in the heart tissue and H9c2 cells of mice in each group
a-e denote the same as those in Fig 1 (mice); A-E denote the same as those in Fig 2 (cells). * P<0.05, ** P<0.01, *** P<0.001, vs. a or A group; # P<0.05, ## P<0.01, ### P<0.001, vs. b or B group. n=3.
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