秋水仙碱通过激动PI3K/AKT/eNOS信号通路对急性心肌梗死大鼠心功能的影响

Colchicine Promotes Cardiac Function in Rats with Acute Myocardial Infarction by Activating PI3K/AKT/eNOS Signaling Pathway

目的:探究秋水仙碱通过调节磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/内皮型一氧化氮合酶(eNOS)信号通路对急性心肌梗死(AMI)大鼠心功能的影响。方法:78只大鼠中随机选取12只作为假手术组,剩余大鼠构建AMI模型,造模成功大鼠分为模型组、秋水仙碱组[4 mg/(kg·d)]、秋水仙碱[4 mg/(kg·d)]+LY294002(20 mg/mL)组、秋水仙碱[4 mg/(kg·d)]+MK-2206(60μg/mL)组、秋水仙碱[4 mg/(kg·d)]+L-NAME(1.6 mg/mL)组,每组12只。超声心动图检测大鼠左心室舒张末期内径(LVEDD)、左心室收缩末期内径(LVESD)、射血分数(EF)及短轴缩短率(FS)。处死大鼠,苏木素-伊红(HE)染色检测大鼠心肌组织石蜡切片病理学变化;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测心肌细胞凋亡率;酶联免疫吸附实验(ELISA)测定大鼠血清肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)、肌酸激酶同工酶(CK-MB)以及心肌组织匀浆中超氧化物歧化酶(SOD)、丙二醛(MDA)、过氧化氢酶(CAT)水平;免疫印迹法(Western Blot)测定大鼠心肌组织PI3K/AKT/eNOS通路蛋白表达。结果:与假手术组相比,模型组大鼠LVESD、LVEDD,血清CK-MB、TNF-α、IL-6水平,心肌匀浆MDA、心肌细胞凋亡率升高,EF、FS水平,心肌匀浆SOD、CAT,心肌组织磷酸化PI3K(p-PI3K)/PI3K、磷酸化AKT(p-AKT)/AKT、磷酸化eNOS(p-eNOS)/eNOS降低(P<0.05);与模型组相比,秋水仙碱组大鼠LVESD、LVEDD,血清CK-MB、TNF-α、IL-6水平,心肌匀浆MDA、心肌细胞凋亡率降低,EF、FS水平,心肌匀浆SOD、CAT,心肌组织p-PI3K/PI3K、p-AKT/AKT、p-eNOS/eNOS升高(P<0.05);与秋水仙碱组相比,秋水仙碱+LY294002组、秋水仙碱+MK-2206组、秋水仙碱+L-NAME组大鼠LVESD、LVEDD,血清CK-MB、TNF-α、IL-6水平,心肌匀浆MDA、心肌细胞凋亡率升高,EF、FS水平,心肌匀浆SOD、CAT,心肌组织p-PI3K/PI3K、p-AKT/AKT、p-eNOS/eNOS降低(P<0.05)。结论:秋水仙碱可能通过激活PI3K/AKT/eNOS信号通路对AMI大鼠发挥心功能保护作用。

Objective:To investigate the effect of colchicine on cardiac function in rats with acute myocardial infarction(AMI)by regulating phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/endothelial nitric oxide synthase(eNOS)signaling pathway.Methods:There were 78 rats,12 were randomly selected as the sham-operated group,and the remaining 66 rats were used to construct an AMI model.Rats with successful modeling were divided into model group,colchicine group[4 mg/(kg·d)],and colchicine[4 mg/(kg·d)]+LY294002(20 mg/mL)group,colchicine[4 mg/(kg·d)])+MK-2206(60μg/mL)group,colchicine[4 mg/(kg·d)]+L-NAME(1.6 mg/mL)group,with 12 rats in each group.Echocardiography was applied to measure left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD),ejection fraction(EF),and short-axis shortening(FS)in rats.Rats were sacrificed,and hematoxylin-eosin(HE)staining was used to measure the pathological changes in rat myocardial paraffin sections.Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL)method was implemented to measure cardiomyocyte apoptosis rate.Enzyme-linked immunosorbent assay(ELISA)method was implemented to measure the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,creatine kinase-MB(CK-MB)in rat serum and superoxide dismutase(SOD),malondialdehyde(MDA),and catalase(CAT)in myocardial tissue homogenate.Western Blot was performed to determine the protein expression of PI3K/AKT/eNOS pathway in rat myocardial tissue.Results:Compared with sham-operated group,the LVESD,LVEDD,serum CK-MB,TNF-α,IL-6 levels,myocardial homogenate MDA,and myocardial cell apoptosis rate in model group increased,and the EF,FS levels,myocardial homogenate SOD,CAT,myocardial tissue p-PI3K/PI3K,p-AKT/AKT,and p-eNOS/eNOS decreased(P<0.05).compared with model group,the LVESD,LVEDD,serum CK-MB,TNF-α,IL-6 levels,myocardial homogenate MDA,and myocardial cell apoptosis rate in colchicine group decreased,and the EF,FS levels,myocardial homogenate SOD,CAT,myocardial tissue p-PI3K/PI3K,p-AKT/AKT,and p-eNOS/eNOS increased(P<0.05).Compared with colchicine group,the LVESD,LVEDD,serum CK-MB,TNF-α,IL-6 levels,myocardial homogenate MDA,and myocardial cell apoptosis rate in colchicine+LY294002 group,colchicine+MK-2206 group,and colchicine+L-NAME group increased,and the EF,FS levels,myocardial homogenate SOD,CAT,myocardial tissue p-PI3K/PI3K,p-AKT/AKT,and p-eNOS/eNOS decreased(P<0.05).Conclusion:Colchicine may exert some protective effect on cardiac function in AMI rats by activating the PI3K/AKT/eNOS pathway.