稳定缺氧诱导因子-1α表达对1型糖尿病心肌缺血再灌注损伤大鼠心肌细胞线粒体自噬的影响

Effect of hypoxia-inducible factor-1α on mitochondrial autophagy in myocardial ischemia-reperfusion injury rats with type 1 diabetes

目的观察1型糖尿病心肌缺血再灌注损伤(ischemia-reperfusion injury, IRI)大鼠稳定缺氧诱导因子-1α(hypoxia-inducible factor-1α, HIF-1α)表达情况,探讨其对心肌细胞线粒体自噬的影响及可能机制。方法 SD大鼠32只,随机分为假手术组、IRI组、稳定HIF-1α组、自噬抑制组各8只。4组大鼠均腹腔注射质量分数1%链脲佐菌素柠檬酸盐缓冲液60 mg/kg制备1型糖尿病模型。造模成功后8周,IRI组、稳定HIF-1α组、自噬抑制剂组结扎左前降支30 min后松开结扎线再灌注2 h,制备IRI模型;假手术组只穿线不结扎。再灌注前1 h,稳定HIF-1α组腹腔注射HIF-1α表达稳定剂二甲基草酰甘氨酸40 mg/kg,自噬抑制剂组腹腔注射二甲基草酰甘氨酸40 mg/kg+自噬抑制剂3-甲基腺苷10 mg/kg,假手术组、IRI组腹腔注射等量生理盐水。再灌注2 h,采用ELISA法检测4组血清肌酸激酶同工酶(creatine kinase-MB, CK-MB)、乳酸脱氢酶(lactate dehydrogenase, LDH)水平;处死大鼠,采用TTC法检测心肌梗死面积,HE染色观察心肌组织病理改变,采用Western blot法检测心肌组织微管相关蛋白B轻链3-Ⅱ(microtubule-associated protein 1 light chain 3-Ⅱ, LC3-Ⅱ)、Bcl-2相互作用蛋白3(Bcl-2 interacting protein 3, BNIP3)、HIF-1α蛋白相对表达量。结果再灌注2 h,IRI组、稳定HIF-1α组、自噬抑制组心肌梗死面积[(43.92±7.34)%、(25.71±4.65)%、(39.24±6.43)%]、血清CK-MB水平[(801.32±86.54)、(512.61±52.32)、(721.42±77.32)u/L]及LDH水平[(621.12±69.13)、(400.23±39.45)、(599.32±55.45)u/L]均高于假手术组[0、(452.12±49.34)u/L、(350.43±37.24)u/L](P<0.05),且IRI组、自噬抑制组高于稳定HIF-1α组(P<0.05),IRI组高于自噬抑制组(P<0.05)。再灌注2 h,假手术组心肌结构正常,纤维组织无肿胀、紊乱,间质少量炎症细胞浸润;IRI组、稳定HIF-1α组、自噬抑制组心肌纤维间质组织肿胀,下突肌松散,大量炎症细胞浸润,IRI组病变最严重,自噬抑制组次之,稳定HIF-1α组较轻。再灌注2 h,IRI组、稳定HIF-1α组、自噬抑制组心肌组织HIF-1α蛋白相对表达量(1.53±0.13、2.50±0.23、1.73±0.14)、LC3-Ⅱ相对表达量(1.48±0.12、2.43±0.21、1.65±0.15)、BNIP3蛋白相对表达量(1.40±0.09、2.32±0.19、1.67±0.13)均高于假手术组(1.00±0.00、1.00±0.00、1.00±0.00)(P<0.05),稳定HIF-1α组、自噬抑制组高于IRI组(P<0.05),稳定HIF-1α组高于自噬抑制组(P<0.05)。结论稳定HIF-1α表达可能通过促进心肌组织LC3-Ⅱ、BNIP3表达增强心肌细胞线粒体自噬,发挥对1型糖尿病大鼠缺血IRI心肌的保护作用。

Objective To investigate the expression of hypoxia-inducible factor-1α(HIF-1α) in rats with type 1 diabetic myocardial ischemia-reperfusion injury(IRI) and its influence on the level of mitochondrial autophagy in cardiomyocytes and the potential mechanism. Methods Thirty-two SD rats were randomly divided into sham-operation group, IRI group, stable HIF-1α group and mitochondrial autophagy group, with 8 rats in each group, and were established type 1 diabetes models by intraperitoneally injecting 1% STZ 60 mg/kg. All but sham-operation group were established IRI models by ligating left anterior descending coronary artery for 30 min, followed by reperfusion for 2 h. Stable HIF-1α group was intraperitoneally injected with 40 mg/kg DMOG, mitochondrial autophagy group was intraperitoneally injected with 40 mg/kg DMOG and 10 mg/kg 3-methyladenine, and sham-operation group and IRI group were intraperitoneally injected with equivalent volume of normal saline 1 h before reperfusion. After 2 h of reperfusion, ELISA was used to detect the levels of serum creatine kinase-MB(CK-MB) and lactate dehydrogenase(LDH);TTC was used to detect the myocardial infarction areas;HE staining was used to observe the myocardial pathological changes;Western blot was used to detect the relative expressions of microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ), Bcl-2 interacting protein 3 (BNIP3)and HIF-1αproteins.Results After 2 hof reperfusion,the myocardial infarction area((43.92±7.34)%,(39.24±6.43)%,(25.71±4.65)%,0),serum CK-MB level((801.32±86.54),(721.42±77.32),(512.61±52.32),(452.12±49.34)u/L)and LDH level((621.12±69.13),(599.32±55.45),(400.23±39.45),(350.43±37.24)u/L)reduced gradually in turn in IRI group,mitochondrial autophagy group,stable HIF-1αgroup and sham-operation group(P<0.05).The myocardial structure was normal,the fibrous tissue was not swollen or disordered,and a small amount of interstitial inflammatory cells infiltrated in sham-operation group;the myocardial fibrous interstitial tissue was swollen,the inferior process was loose,and a large amount of inflammatory cells infiltrated in the other three groups,which was the most severe in IRI group,followed by mitochondrial autophagy group and stable HIF-1αgroup in turn.The relative expressions of HIF-1α (2.50±0.23,1.73±0.14,1.53±0.13,1.00±0.00),LC3-Ⅱ(2.43±0.21,1.65±0.15,1.48±0.12,1.00±0.00)and BNIP3(2.32±0.19,1.67±0.13,1.40±0.09,1.00±0.00)decreased gradually in turn in stable HIF-1αgroup,mitochondrial autophagy group,IRI group and sham-operation group(P<0.05).Conclusion Stable HIF-1αexpression can reduce the degree of myocardial IRI in rats with type 1 diabetes probably by enhancing mitochondrial autophagy through increasing the expressions of LC3-Ⅱ and BNIP3 in rat myocardial cells.